This application claims benefit of priority to the filing date of U.S. Provisional Application Ser. No. 61/928,559, filed Jan. 17, 2014, the contents of which are specifically incorporated herein by reference in their entity.
This invention was made with government support under DE-FCO2-07ER64494, DE-FG02-91ER20021, DE-FG02-98ER20305 awarded by the U.S. Department of Energy and FA9550-11-1-0264 awarded by the U.S. Air Force Office of Scientific Research. The government has certain rights in the invention.
Oil produced by plants is one of the most energy-rich forms of reduced carbon available from nature. As crude oil supply declined, plant oils are gaining increasing interest as substitutes for petroleum-derived and non-renewable fuels. As one example, Hu et al., describes the use of microalgal triacylglycerols (TAGs) as feedstocks for biofuel production (Hu et al., The Plant J., 54(4):621-639 (2008).
One of the biggest challenges in using plant oils is the limited growing areas available for plants whose oil is designated for the biofuel industry along with finite supplies of fertilizers, pesticides, and resources for growing these plants and processing their oils. Thus, compositions and methods are needed for increasing the amount of harvestable amount of plant oils from plants per acre for decreasing the cost per liter of plant oil. Further, compositions and methods are needed for growing oil crop plants for increasing the amount of harvestable oil in plants destined as sources of biofuel.
As described herein, expression of Diacylglycerol Acyltransferase Type Two (DGTT) enzymes from nucleic acid constructs can alter acyl carbon partitioning in plant vegetative tissues. Such expression can increase acyl-CoA-dependent triacylglycerol synthesis and thereby increase the lipid content and nutritional value of leaves and other vegetative tissues. For example, the DGTT enzymes can be DGTT2 or DGTT3 enzymes.
Thus, plant cells, plant seeds, and plants are described herein that have a nucleic acid construct that includes a nucleic acid encoding a polypeptide with at least 80% sequence identity with an amino acid sequence selected from the group of SEQ ID NO:1, 3 5, and 7, that is operably linked to a heterologous promoter operable in at least one plant tissue.
Also described herein is method that includes:
(a) transfecting a plant tissue or plant cell with a nucleic acid construct that includes a nucleic acid encoding a polypeptide with at least 40% sequence identity with an amino acid sequence selected from the group of SEQ ID NO:1, 3 5, and 7, that is operably linked to a heterologous promoter operable in at least one plant tissue; and
(b) growing the tissue or the cell into a whole plant.
In addition a method is described herein that includes:
(a) growing a plant from a seed with a nucleic acid construct that includes a nucleic acid encoding a polypeptide with at least 40% sequence identity with an amino acid sequence selected from the group of SEQ ID NO:1, 3 5, and 7, that is operably linked to a heterologous promoter operable in at least one plant tissue; and (b) harvesting lipid or oil from the plant or a portion of the plant.
As described herein, algal Diacylglycerol Acyltransferase Type Two (DGTT) enzymes can alter acyl carbon partitioning in plant vegetative tissues. Expression of such enzymes can increase acyl-CoA-dependent triacylglycerol synthesis and thereby increase the lipid content and nutritional value of the plants' tissues. For example, the lipid content of plant tissues such as leaves can be increased by expression of heterologous nucleic acids that express algal Diacylglycerol Acyltransferase Type Two (DGTT) enzymes.
Diacylglycerol Acyltransferase Type Two (DGTT)
Diacylglycerol acyltransferase type two (DGTT) enzymes can catalyze the transfer of fatty acyl group and formation of triacylglycerols (TAGs) from diacylglycerol and acyl-CoA substrates.
As illustrated herein, expression of DGTT enzymes from the photosynthetic green alga Chlamydomonas reinhardtii in Arabidopsis plants increased TAG content in vegetative tissues by altering the acyl carbon partitioning through a broad range of acyl-CoA substrates. The gain in energy density of the transgenic leaves was evident, for example, from the gain in weight of caterpillar larvae that were allowed to feed on them.
Sequences for diacylglycerol acyltransferase enzymes and nucleic acids encoding such enzymes are available from the National Center for Biotechnology Information (NCBI) database (see, e.g., the website at ncbi.nlm.nih.gov).
For example, a sequence of a protein from Chlamydomonas reinhardtii that the inventors have now identified as being a DGTT1 enzyme is available from the NCBI database as accession number XP_001702848.1 (GI:159489727), and is provided below as SEQ ID NO:1.
A nucleic acid encoding the Chlamydomonas reinhardtii DGTT1 enzyme with SEQ ID NO:1 is available from the NCBI database with accession number XM_001702796.1 (GI:159489726), and provided below as SEQ ID NO:2.
One example of a sequence of a protein from Chlamydomonas reinhardtii that the inventors have now identified as being a DGTT2 enzyme is available from the NCBI database as accession number XP_001694904.1 (GI:159473565), and provided below as SEQ ID NO:3.
A nucleic acid encoding the Chlamydomonas reinhardtii DGTT2 enzyme with SEQ ID NO:3 is available from the NCBI database with accession number XM_001694852.1 (GI:159473564) with the following sequence (SEQ ID NO:4).
One example of a sequence of a protein from Chlamydomonas reinhardtii that the inventors have now identified as being a DGTT3 enzyme is available from the NCBI database as accession number XP_001691447.1 (GI:159466500), and provided below as SEQ ID NO:5.
A nucleic acid encoding the Chlamydomonas reinhardtii DGTT3 enzyme with SEQ ID NO:5 is available from the NCBI database with accession number XM_001691395.1 GI:159466499) with the following sequence (SEQ ID NO:6).
One example of a sequence of a protein from Chlamydomonas reinhardtii that the inventors have now identified as being a DGTT4 enzyme is provided below as SEQ ID NO:7.
Although the DGTT enzymes can be any of the DGTT enzymes described herein, in some embodiments the DGTT enzymes can be DGTT2 or DGTT3 enzymes.
Related Nucleic Acids or Polypeptides
The nucleic acids, polypeptides, plants, and seeds can also have sequences related to any of the sequences described herein. For example, related nucleic acids can be isolated and identified by mutation of any of the SEQ ID NO:1-7 sequences and/or by hybridization to DNA and/or RNA isolated from other plant species using any of SEQ ID NO:2, 4, or 6 nucleic acids (or portions thereof) as probes.
In some embodiments, the related nucleic acids and proteins are identified by hybridization of any of SEQ ID NO: 2, 4, or 6 nucleic acids (or portions thereof) as probes under stringent hybridization conditions. The terms “stringent conditions” or “stringent hybridization conditions” include conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are somewhat sequence-dependent and can vary in different circ*mstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified with up to 100% complementarity to the probe (hom*ologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of sequence similarity are detected (heterologous probing). The probe can be approximately 20-500 nucleotides in length, but can vary greatly in length from about 18 nucleotides to equal to the entire length of the target sequence. In some embodiments, the probe is about 10-50 nucleotides in length, or about 18-25 nucleotides in length, or about 18-50 nucleotides in length, or about 18-100 nucleotides in length.
Typically, stringent conditions will be those where the salt concentration is less than about 1.5 M Na ion (or other salts), typically about 0.01 to 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3 and the temperature is at least about 30° C. for shorter probes (e.g., 10 to 50 nucleotides), and at least about 60° C. for longer probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's solution. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1×SSC to 2×SSC (where 20×SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.5×SSC to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution.
For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal Biochem 138:267-84 (1984)):
Tm=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% formamide)−500/L
where M is the molarity of monovalent cations; % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about 1° C. for each 1% of mismatching. Thus, the Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than or equal to 90% sequence identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can include hybridization and/or a wash at 1, 2, 3 or 4° C. lower than the thermal melting point (Tm). Moderately stringent conditions can include hybridization and/or a wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm). Low stringency conditions can include hybridization and/or a wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and a desired Tm, those of ordinary skill can identify and isolate nucleic acids with sequences related to any of SEQ ID NO:2, 4, or 6 nucleic acids.
Those of skill in the art also understand how to vary the hybridization and/or wash solutions to isolate desirable nucleic acids. For example, if the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used.
An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, part 1, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (1993); and in Current Protocols in Molecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995).
For example, high stringency can be defined as hybridization in 4×SSC, 5×Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and a wash in 0.1×SSC, 0.1% SDS at 65° C. However, the stringency of hybridization is actually determined by the wash conditions. Thus, wash conditions in 0.1×SSC, 0.1% SDS at 65° C. are a sufficient definition of stringent hybridization conditions.
Such selective hybridization substantially excludes non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, at least about 50% sequence identity, at least 55% sequence identity, at least about 60% sequence identity, at least 70% sequence identity, at least about 80% sequence identity, at least 90% sequence identity, at least about 95% sequence identity, or 40-95% sequence identity, or 50-95% sequence identity, or 60-90% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 98-99% sequence identity, or 100% sequence identity or complementarity with any of the SEQ ID NO:2, 4, or 6 nucleic acids.
The nucleic acids of the invention include those with about 500 of the same nucleotides as any of SEQ ID NO:2, 4, or 6 nucleic acids, or about 600 of the same nucleotides, or about 700 of the same nucleotides, or about 800 of the same nucleotides, or about 900 of the same nucleotides, or about 1000 of the same nucleotides, or about 1100 of the same nucleotides, or about 1200 of the same nucleotides, or about 1300 of the same nucleotides, or about 500-1325 of the same nucleotides. The identical nucleotides or amino acids can be distributed throughout the nucleic acid, and need not be contiguous.
The polypeptides of the invention include those with about 50 of the same amino acids as any of SEQ ID NO:1, 3, 5, or 7 polypeptides, or about 60 of the same amino acids, or about 70 of the same amino acids, or about 80 of the same amino acids, or about 90 of the same amino acids, or about 100 of the same amino acids, or about 110 of the same amino acids, or about 120 of the same amino acids, or about 130 of the same amino acids, or about 140 of the same amino acids, or about 150 of the same amino acids, or about 50-80 of the same amino acids, or about 150-325 of the same amino acids as any of any of SEQ ID NO:1, 3, 5, or 7 polypeptides. The identical amino acids can be distributed throughout the nucleic acid, and need not be contiguous.
The polypeptides have about at least 40% sequence identity, at least about 50% sequence identity, at least 50% sequence identity, at least about 60% sequence identity, at least 70% sequence identity, at least about 80% sequence identity, at least 90% sequence identity, at least about 95% sequence identity, or 40-95% sequence identity, or 50-95% sequence identity, or 60-90% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 98-99% sequence identity, or 100% sequence identity with any of the SEQ ID NO:1, 3, 5, or 7 polypeptides.
Note that if a value of a variable that is necessarily an integer, e.g., the number of nucleotides or amino acids in a nucleic acid or protein, is described as a range, e.g., or 90-99% sequence identity, what is meant is that the value can be any integer between 90 and 99 inclusive, i.e., 90-99% sequence identity means any of 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity.
For example, the DGTT enzymes can be DGTT2 or DGTT3 enzymes, with DGTT2 sequences SEQ ID NO:3 and 4 (or at least 40% sequence identity therewith), or with DGTT3 sequences SEQ ID NO: 5 and 6 (or at least 40% sequence identity therewith).
Modified Plants Having Diacylglycerol Acyltransferase Type Two (DGTT)
In order to engineer plants with desired quantities of triacylglycerols, one of skill in the art can introduce diacylglycerol acyltransferase type two (DGTT) enzymes or nucleic acids encoding such enzymes into the plants. Any of the diacylglycerol acyltransferase type two (DGTT) nucleic acids, and related nucleic acid sequences, described herein can be incorporated into the expression cassettes, plants and seeds described herein.
In some embodiments, one of skill in the art could inject diacylglycerol acyltransferase type two (DGTT) enzymes or nucleic acids encoding such diacylglycerol acyltransferase type two (DGTT) enzymes into young plants, or into selected regions of plants. Alternatively, one of skill in the art can generate genetically-modified plants that contain nucleic acids encoding diacylglycerol acyltransferase type two (DGTT) within their somatic and/or germ cells. For example, any of the diacylglycerol acyltransferase type two (DGTT) nucleic acids described herein can be operably linked to a selected promoter (e.g., a heterologous promoter), to generate an expression cassette that can be used to generate transgenic plants and/or seeds. Examples of diacylglycerol acyltransferase type two (DGTT) coding regions that can be used in such expression cassettes include any of the following SEQ ID NOs: 1, 3, 5, 7, or any combination thereof. The expression cassettes can be introduced into plants to increase the triacylglycerols content of the plant's tissues.
To facilitate expression of a coding region of interest, an expression cassette can be made that encodes any of the diacylglycerol acyltransferase type two (DGTT) or related enzymes described herein. The genetic modifications involved can be accomplished by procedures available in the art. For example, one of skill in the art can prepare an expression cassette or expression vector that can express one or more encoded enzymes. In general, a nucleic acid segment encoding a diacylglycerol acyltransferase type two (DGTT) enzyme can be operably linked to a promoter, for example, by inserting the promoter nucleic acid segment upstream of a selected enzyme coding region.
Plant cells can be transformed by the expression cassettes or expression vector, and whole plants (and their seeds) can be generated from the plant cells that were successfully transformed with the diacylglycerol acyltransferase type two (DGTT) nucleic acids. Some procedures for making such genetically modified plants and their seeds are described in more detail below.
Heterologous Promoters: The diacylglycerol acyltransferase type two (DGTT) (or related) nucleic acids can be operably linked to a promoter, such as a heterologous promoter, which provides for expression of mRNA encoding the diacylglycerol acyltransferase type two (DGTT) enzymes. The heterologous promoter employed is typically a promoter functional in plants and/or seeds, and can be a promoter functional during plant growth and development. The heterologous promoter is a promoter that is not operably linked to diacylglycerol acyltransferase type two (DGTT) nucleic acids in nature. A diacylglycerol acyltransferase type two (DGTT) nucleic acid is operably linked to the promoter when the diacylglycerol acyltransferase type two (DGTT) nucleic acid is located downstream from the promoter, so that the promoter is configured to express the diacylglycerol acyltransferase type two (DGTT) enzymes.
Promoters regulate gene expression. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences can also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs that is a DNA different from the native or hom*ologous DNA generally associated with the promoter.
Promoter sequences can be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, expression can be stimulated from an inducible promoter by factors such as alcohol, acetaldehyde, antibiotics (e.g., tetracycline), steroids, metals and other compounds. An environmentally inducible promoter can induce expression of a gene in response to environmental stimuli such as drought, cold, heat, longer exposure to light, or shorter exposure to light. A bacterial promoter such as the Ptac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Steroid inducible promoters have also been employed in plants. Dexamethasone-inducible promoters are activated by introduction of dexamethasone to a cell, tissue, cell culture, or tissue culture. The alc promoter system from the filamentous fungi Aspergillus nidulans can be induced by alcohol (e.g., ethanol) or acetaldehyde (see, e.g., Schaarschmidt et al., Plant & Cell Physiol 45(11): 1566-77 (2004). The nopaline synthase (nos) promoter is inducible by hydrogen peroxide and/or methyl jasmonate (see, e.g., Sai & An, Plant Physiol. 109(4): 1191-97 (1995)).
Promoters can also provide for tissue specific or developmental regulation. In some embodiments, an isolated promoter sequence that is a strong promoter for heterologous DNAs is advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
Expression cassettes encoding a diacylglycerol acyltransferase type two (DGTT) enzyme can include, but are not limited to, a plant promoter such as the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adh1 (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin, actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)), GAL4/UAS (Brand & Perrimon, Development 118: 401-15 (1993); and/or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)). Further suitable promoters include the poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actin promoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)). Seed specific promoters, such as the phaseolin promoter from beans, may also be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324 (1985). Other promoters useful in the practice of the invention are available to those of skill in the art.
Alternatively, novel tissue specific promoter sequences may be employed for the expression of the diacylglycerol acyltransferase type two (DGTT) enzyme(s). cDNA clones from a particular tissue can be isolated and those clones that are expressed specifically in a tissue of interest are identified, for example, using Northern blotting, quantitative PCR and other available methods. In some embodiments, the gene isolated is not present in a high copy number, but is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones can then be identified, isolated and utilized using techniques well known to those of skill in the art.
A diacylglycerol acyltransferase type two (DGTT) nucleic acid can be combined with a selected promoter by standard methods to yield an expression cassette, for example, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Second Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (1989); Molecular Cloning: A Laboratory Manual. Third Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)). Briefly, a plasmid containing a promoter such as the 35S CaMV promoter can be constructed as described in Jefferson (Plant Molecular Biology Reporter 5:387-405 (1987)) or obtained from Clontech Lab in Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, these plasmids are constructed to have multiple cloning sites having specificity for different restriction enzymes downstream from the promoter. The diacylglycerol acyltransferase type two (DGTT) nucleic acids can be subcloned downstream from the promoter using restriction enzymes and positioned to ensure that the diacylglycerol acyltransferase type two (DGTT) DNA is inserted in proper orientation with respect to the promoter so that the DNA can be expressed. The diacylglycerol acyltransferase type two (DGTT) nucleic acid operably linked to a promoter can form an expression cassette, which can be subcloned into a plasmid or other vector (e.g., an expression vector).
In some embodiments, a cDNA encoding a protein with at least 60% sequence identity to any of SEQ ID NO:1, 3, 5, or 7 is isolated from a selected plant species, and operably linked to a heterologous promoter. The a cDNA encoding a protein can, for example, be a cDNA from an oleaginous plant, an Arabidopsis plant, corn, sugar beets, soybean, sugar cane, potato, grasses (e.g., miscanthus, switchgrass, and the like), rice, Diatoms, Nannochloropsis, a fresh water algal species, a marine algal species, a beet species, a grape species, an Arabidopsis species, a Brassica species, a Brassica napus plant, an algae species, an oilseed rape species, a sunflower, a soybean species, a flax species, an olive species, an alfalfa species, an oat species, a poplar, an aspen, a willow, and similar plant species. Species of algae from which the cDNA can be obtained from Archaeplastidia, Rhizaria, Excavate, Chromista, Alveolata, Hetrokonts, Cryptophyta, Dinoflagellates, Haptophyta, and any combination thereof.
In other embodiments, cDNA from other species that encode a diacylglycerol acyltransferase type two (DGTT) enzyme is isolated from selected plant tissues, or a nucleic acid encoding a mutant or modified diacylglycerol acyltransferase type two (DGTT) enzyme is prepared by available methods or as described herein. For example, the nucleic acid encoding a mutant or modified diacylglycerol acyltransferase type two (DGTT) protein can be any nucleic acid with a coding region that hybridizes to SEQ ID NO:1, 3, 5, or 7 that can promote expression of a diacylglycerol acyltransferase type two (DGTT) enzyme. Using restriction endonucleases, the entire coding sequence for the diacylglycerol acyltransferase type two (DGTT) can be subcloned downstream of the promoter in a 5′ to 3′ sense orientation.
Targeting Sequences: Additionally, expression cassettes can be constructed and employed to target the diacylglycerol acyltransferase type two (DGTT) to intracellular compartments within plant cells, or to target the diacylglycerol acyltransferase type two (DGTT) for extracellular secretion.
In general, the diacylglycerol acyltransferase type two (DGTT) operate intracellularly. Therefore, the diacylglycerol acyltransferase type two (DGTT) is preferably not directed to the extracellular environment. However, there may be instances where is it desirable to secrete or sequester the diacylglycerol acyltransferase type two (DGTT) within organelles or storage vesicles (e.g., to facilitate isolation and/or purification of the diacylglycerol acyltransferase type two (DGTT) enzyme. Therefore, the invention contemplates targeting the diacylglycerol acyltransferase type two (DGTT) enzymes to various intracellular and extracellular locations.
A nuclear localization signal or sequence is an amino acid sequences that ‘tags’ a protein for import into the cell nucleus by nuclear transport. A localization signal or sequence can be operably linked to the diacylglycerol acyltransferase type two (DGTT) sequence. Transit peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. Polypeptides of interest can be operably linked to nuclear localization signals/sequences, to transit peptides or to signal peptides.
Targeting to selected intracellular regions can generally be achieved by joining a DNA sequence encoding a nuclear localization sequence, or a transit peptide or a signal peptide sequence to the coding sequence of the diacylglycerol acyltransferase type two (DGTT) or the related polypeptide of interest. The resultant nuclear localization sequence (or transit, or signal, peptide) will transport the diacylglycerol acyltransferase type two (DGTT) enzyme to a particular intracellular (or extracellular) destination. Such sequences (nuclear localization sequences, transit peptides or signal peptides) may be post-translationally removed by cellular enzymes. By facilitating transport of the protein into compartments inside or outside the cell, these sequences can increase the accumulation of a particular gene product in a particular location.
3′ Sequences: The expression cassette can also optionally include 3′ nontranslated plant regulatory DNA sequences that act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA. The 3′ nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequences. For example, 3′ elements that can be used include those derived from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., Nucleic Acid Research. 11:369-385 (1983)), or the terminator sequences for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and/or the 3′ end of the protease inhibitor I or II genes from potato or tomato. Other 3′ elements known to those of skill in the art can also be employed. These 3′ nontranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3′ nontranslated regulatory sequences are already present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif. The 3′ nontranslated regulatory sequences can be operably linked to the 3′ terminus of the diacylglycerol acyltransferase type two (DGTT) nucleic acids by standard methods.
Selectable and Screenable Marker Sequences: In order to improve identification of transformants, a selectable or screenable marker gene can be employed with the expressible diacylglycerol acyltransferase type two (DGTT) nucleic acids. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ‘select’ for the marker by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether marker is simply a trait that one can identify through observation or testing, i.e., by ‘screening’ (e.g., the R-locus trait). Many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.
Included within the terms selectable or screenable marker genes are also genes which encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
With regard to selectable secretable markers, the use of a gene that encodes a polypeptide that becomes sequestered in the cell wall, where the polypeptide includes a unique epitope may be advantageous. Such a secreted antigen marker can employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that imparts efficient expression and targeting across the plasma membrane, and can produce protein that is bound in the cell wall and yet is accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy such requirements.
Examples of marker proteins suitable for modification in this manner include extensin or hydroxyproline rich glycoprotein (HPRG). For example, the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is well characterized in terms of molecular biology, expression, and protein structure and therefore can readily be employed. However, any one of a variety of extensins and/or glycine-rich wall proteins (Keller et al., EMBO J. 8:1309-1314 (1989)) could be modified by the addition of an antigenic site to create a screenable marker.
Numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to the one set forth herein. Therefore, it will be understood that the following discussion is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques that are known in the art, the present invention readily allows the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant cell, e.g., a monocot cell or dicot cell.
Possible selectable markers for use in connection with expression cassettes include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycin resistance and can be selected for using kanamycin, G418, and the like; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922 (1988)) thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154,204 (1985)); a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0 218 571 (1987)).
Another selectable marker gene capable of being used in for selection of transformants is the gene that encodes the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twell et al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulation of ammonia and cell death. The success in using this selective system in conjunction with monocots was surprising because of the major difficulties that have been reported in transformation of cereals (Potrykus, Trends Biotech. 7:269-273 (1989)).
Screenable markers that may be employed include, but are not limited to, a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, J. P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282 (1988)); a β-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA. 75:3737-3741 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA. 80:1101 (1983)) which encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Bio/technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science. 234:856-859.1986), which allows for bioluminescence detection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may be employed in calcium-sensitive bioluminescence detection, or a green or yellow fluorescent protein gene (Niedz et al., Plant Cell Reports. 14:403 (1995).
For example, genes from the maize R gene complex can be used as screenable markers. The R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue. Maize strains can have one, or as many as four, R alleles that combine to regulate pigmentation in a developmental and tissue specific manner. A gene from the R gene complex does not harm the transformed cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector. If a maize line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation. Exemplary lines include Wisconsin 22 that contains the rg-Stadler allele and TR112, a K55 derivative that is r-g, b, Pl. Alternatively any genotype of maize can be utilized if the Cl and R alleles are introduced together.
The R gene regulatory regions can be employed in chimeric constructs in order to provide mechanisms for controlling the expression of chimeric genes. More diversity of phenotypic expression is known at the R locus than at any other locus (Coe et al., in Corn and Corn Improvement, eds. Sprague, G. F. & Dudley, J. W. (Am. Soc. Agron., Madison, Wis.), pp. 81-258 (1988)). It is contemplated that regulatory regions obtained from regions 5′ to the structural R gene can be useful in directing the expression of genes, e.g., insect resistance, drought resistance, herbicide tolerance or other protein coding regions. For the purposes of the present invention, it is believed that any of the various R gene family members may be successfully employed (e.g., P, S, Lc, etc.). However, one that can be used is Sn (particularly Sn:bol3). Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R.
A further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for population screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
Other Optional Sequences: An expression cassette of the invention can also further comprise plasmid DNA. Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes (e.g., antibiotic or herbicide resistance), unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette and sequences that enhance transformation of prokaryotic and eukaryotic cells.
Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An (Methods in Enzymology. 153:292 (1987)) and is available from Dr. An. This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as rice cells. The binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colE1 replication of origin and a wide host range replicon. The binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells, but is preferably used to transform dicot plant cells.
In Vitro Screening of Expression Cassettes: Once the expression cassette is constructed and subcloned into a suitable plasmid, it can be screened for the ability to express the diacylglycerol acyltransferase type two (DGTT) of interest. For example, an expression cassette encoding a diacylglycerol acyltransferase type two (DGTT) can be screened to ascertain whether it can promote expression of a diacylglycerol acyltransferase type two enzyme by methods described herein or other available methods for detecting triacylglycerols. An expression cassette encoding other enzymes of interest can be screened to ascertain whether it can promote expression of the enzyme, for example, by immunological detection of the enzyme of interest, by detection of the activity of the enzyme, by hybridization or PCR detection of transcripts encoding the enzyme, or by other procedures available to those of skill in the art.
DNA Delivery of the DNA Molecules into Host Cells: The diacylglycerol acyltransferase type two (DGTT) nucleic acids can be introduced into host cells by a variety of methods. For example, a preselected cDNA encoding the selected diacylglycerol acyltransferase type two (DGTT) can be introduced into a recipient cell to create a transformed cell by available procedures. The frequency of occurrence of cells taking up exogenous (foreign) DNA may be low. Moreover, it is most likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell wherein the DNA is stably integrated into the plant genome and/or expressed. Some may show only initial and transient gene expression. However, certain cells from virtually any dicot or monocot species may be stably transformed, and these cells can be regenerated into transgenic plants, through the application of the techniques disclosed herein.
Another aspect of the invention is an isolated plant or plant cell that has at least one of the diacylglycerol acyltransferase type two (DGTT) nucleic acids, or related nucleic acids, introduced into the cell. The plant or plant cell can be a monocotyledon or a dicotyledon. Another aspect of the invention includes plant cells (e.g., embryonic cells or other cell lines) that can regenerate fertile transgenic plants and/or seeds. The cells can be derived from either monocotyledons or dicotyledons.
Suitable examples of plant species for the plant cells, plants and seeds include an oleaginous plant, an Arabidopsis plant, corn, sugar beets, soybean, sugar cane, potato, grasses (e.g., miscanthus, switchgrass, and the like), rice, a beet species, a grape species, an Arabidopsis species, a Brassica species, a Brassica napus plant, an algae species, an oilseed rape species, a sunflower, a soybean species, a flax species, an olive species, an alfalfa species, an oat species, a wheat species, a poplar, an aspen, a willow, and similar plant species. In some embodiments, the plant or cell is a monocotyledon plant or cell. For example, the plant or cell can be a maize plant or cell. The cell(s) may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus.
Transformation of the cells of the plant tissue source can be conducted by any one of a number of methods known to those of skill in the art. Examples are: Transformation by direct DNA transfer into plant cells by electroporation (U.S. Pat. Nos. 5,384,253 and 5,472,869, Dekeyser et al., The Plant Cell. 2:591-602 (1990)); direct DNA transfer to plant cells by PEG precipitation (Hayashimoto et al., Plant Physiol. 93:857-863 (1990)); direct DNA transfer to plant cells by microprojectile bombardment (McCabe et al., Bio/Technology. 6:923-926 (1988); Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990); U.S. Pat. Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer to plant cells via infection with Agrobacterium. Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.
One method for dicot transformation, for example, involves infection of plant cells with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et al., Science 227:1229-1231 (1985). Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase-containing enzyme (U.S. Pat. Nos. 5,384,253; 5,472,869). For example, embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment as described by Gordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Pat. Nos. 5,489,520; 5,538,877 and 5,538,880, cited above. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0 604 662, 1994) and Saito et al. (European Patent 0 672 752, 1995).
Methods such as microprojectile bombardment or electroporation are carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but eliminate functions for disease induction.
The choice of plant tissue source for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells. Type I or Type II embryonic maize callus and immature embryos are preferred Zea mays tissue sources. Selection of tissue sources for transformation of monocots is described in detail in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.
The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA or RNA carrying the diacylglycerol acyltransferase type two (DGTT) nucleic acids for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-3 day co-cultivation in the presence of plasmid-bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
Electroporation: Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253) may be advantageous. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells can be made more susceptible to transformation, by mechanical wounding.
To effect transformation by electroporation, one may employ either friable tissues such as a suspension cell cultures, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. The cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin-degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner. Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.
Microprojectile Bombardment: A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, microparticles may be coated with DNA and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. For example, non-embryogenic Black Mexican Sweet maize cells can be bombarded with intact cells of the bacteria E. coli or Agrobacterium tumefaciens containing plasmids with either the β-glucuronidase or bar gene engineered for expression in maize. Bacteria can be inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the β-glucuronidase gene may be observed 24-48 hours following DNA delivery. In addition, stable transformants containing the bar gene can be recovered following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. The particles may increase the level of DNA delivery but may not be, in and of themselves, necessary to introduce DNA into plant cells.
An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming monocots, is that the isolation of protoplasts (Christou et al., PNAS. 84:3962-3966 (1987)), the formation of partially degraded cells, or the susceptibility to Agrobacterium infection is not required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990)). The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing damage inflicted on the recipient cells by an aggregated projectile.
For bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of such techniques one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from about 1 to 10 and average about 1 to 3.
In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the path and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA.
One may wish to adjust various bombardment parameters in small scale studies to fully optimize the conditions and/or to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Execution of such routine adjustments will be known to those of skill in the art.
An Example of Production and Characterization of Stable Transgenic Maize: After effecting delivery of a diacylglycerol acyltransferase type two (DGTT) nucleic acid into recipient cells by any of the methods discussed above, the transformed cells can be identified for further culturing and plant regeneration. As mentioned above, in order to improve the ability to identify transformants, one may employ a selectable or screenable marker gene in addition to the diacylglycerol acyltransferase type two (DGTT) nucleic acids. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.
Selection: An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells that have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
To use the bar-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/l bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media. The R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the Cl and B genes will result in pigmented cells and/or tissues.
The enzyme luciferase is also useful as a screenable marker in the context of the present invention. In the presence of the substrate luciferin, cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.
It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells. For example, selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone. In an illustrative embodiment embryogenic Type II callus of Zea mays L. can be selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified.
Regeneration and Seed Production: Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants. One example of a growth regulator that can be used for such purposes is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA+2,4-D or perhaps even picloram. Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators.
The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants. Developing plantlets are transferred to soil-less plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins/sec·m2 of light. Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con™. Regenerating plants can be grown at about 19° C. to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
Mature plants are then obtained from cell lines that are known to express the trait. In some embodiments, the regenerated plants are self-pollinated. In addition, pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants. The trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of particular importance if the traits are to be commercially useful.
Regenerated plants can be repeatedly crossed to inbred plants in order to introgress the diacylglycerol acyltransferase type two (DGTT) nucleic acids into the genome of the inbred plants. This process is referred to as backcross conversion. When a sufficient number of crosses to the recurrent inbred parent have been completed in order to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced diacylglycerol acyltransferase type two (DGTT) nucleic acids, the plant is self-pollinated at least once in order to produce a hom*ozygous backcross converted inbred containing the diacylglycerol acyltransferase type two (DGTT) nucleic acids. Progeny of these plants are true breeding.
Alternatively, seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants.
Seed from the fertile transgenic plants can then be evaluated for the presence and/or expression of the diacylglycerol acyltransferase type two (DGTT) nucleic acids (or the encoded enzyme). Transgenic plant and/or seed tissue can be analyzed for diacylglycerol acyltransferase type two (DGTT) expression using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a product of the diacylglycerol acyltransferase type two (DGTT) activity (e.g., increased triacylglycerols or heightened expression of a the diacylglycerol acyltransferase type two (DGTT) enzyme).
Once a transgenic seed expressing the diacylglycerol acyltransferase type two (DGTT) is identified, the seed can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants that express the diacylglycerol acyltransferase type two (DGTT) or related enzymes described herein, while still maintaining other desirable functional agronomic traits. Adding the trait of increased diacylglycerol acyltransferase type two (DGTT) expression to the plant can be accomplished by back-crossing with this trait with plants that do not exhibit this trait and by studying the pattern of inheritance in segregating generations. Those plants expressing the target trait in a dominant fashion are preferably selected. Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of expression of the diacylglycerol acyltransferase type two (DGTT) in the plant. The resulting progeny are then crossed back to the parent that expresses the trait. The progeny from this cross will also segregate so that some of the progeny carry the trait and some do not. This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the desired trait within the plant. Such expression of the increased expression of the diacylglycerol acyltransferase type two (DGTT) in plant can be expressed in a dominant fashion.
Subsequent to back-crossing, the new transgenic plants can be evaluated for expression of the diacylglycerol acyltransferase type two (DGTT) enzyme or mRNA. For example, when the diacylglycerol acyltransferase type two (DGTT) is expressed the weight percent of triacylglycerols within the plant or within selected tissues of the plant is increased. Detection of increased triacylglycerols can be done, for example, by staining plant tissues for triacylglycerols or by observing whether the plant tissue oil content is increased relative to a plant that does not contain the exogenously added diacylglycerol acyltransferase type two (DGTT). The new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as lodging, kernel hardness, yield, resistance to disease and insect pests, drought resistance, and/or herbicide resistance.
Plants that may be improved by these methods (incorporation of nucleic acids encoding the diacylglycerol acyltransferase type two (DGTT) enzymes) include but are not limited to fiber-containing plants, trees, flax, grains (maize, wheat, barley, oats, rice, sorghum, millet and rye), grasses (switchgrass, prairie grass, wheat grass, sudangrass, sorghum, straw-producing plants), softwood, hardwood and other woody plants (e.g., those used for paper production such as poplar species, pine species, and eucalyptus), oil and/or starch plants (canola, potatoes, lupins, sunflower and cottonseed), and forage plants (alfalfa, clover and fescue). In some embodiments the plant is a gymnosperm. Examples of plants useful for pulp and paper production include most pine species such as loblolly pine, Jack pine, Southern pine, Radiata pine, spruce, Douglas fir and others. Hardwoods that can be modified as described herein include aspen, poplar, eucalyptus, and others. Plants useful for making biofuels and ethanol include corn, grasses (e.g., miscanthus, switchgrass, and the like), as well as trees such as poplar, aspen, willow, and the like. Plants useful for generating dairy forage include legumes such as alfalfa, as well as forage grasses such as bromegrass, and bluestem.
Determination of Stably Transformed Plant Tissues: To confirm the presence of the diacylglycerol acyltransferase type two (DGTT) nucleic acids in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues. PCR techniques may also be used for detection and quantification of RNA produced from introduced diacylglycerol acyltransferase type two (DGTT) nucleic acids. PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified through the use of conventional PCR techniques. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.
While Southern blotting and PCR may be used to detect the diacylglycerol acyltransferase type two (DGTT) nucleic acid in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced diacylglycerol acyltransferase type two (DGTT) nucleic acids or evaluating the phenotypic changes brought about by their expression.
Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the diacylglycerol acyltransferase type two (DGTT) enzyme such as evaluation by amino acid sequencing following purification. The Examples of this application also provide assay procedures for detecting and quantifying the diacylglycerol acyltransferase type two (DGTT) or its enzyme activities. Other procedures may be additionally used.
The expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant.
Biofuel Manufacture
Lipids can be isolated from plants or plant tissues transformed by nucleic acids or nucleic acid constructs encoding diacylglycerol acyltransferase type two (DGTT) enzymes. Such lipids are useful for manufacture of biofuels.
Manufacture of biofuel can involve one or more steps such as tissue crushing, tissue sonication, tissue fractionation, cellular lysis, filtering, water removal, solvent extraction, removal of volatile components, titration to ascertain lipid (e.g, fatty acid) content, transesterification (e.g., conversion of acid moieties to alkyl esters), separation of desired lipid components from undesired lipid components, lipid fractionation, and combinations of such steps. Such steps can be performed in the order listed, or in any other order. For example, water and/or volatiles can be removed before and/or after transesterification.
A filtering step can involve particle removal. This process can include warming the lipid extract to maintain the lipid in liquid form and/or to reduce its viscosity. After warming up the liquid, it can be filtered. The filter can have pore sizes smaller than the particles to be removed but sufficient to pass the lipid(s).
Water can be removed by heating the lipid(s) and/or by use of one or more desiccants. For example, water can be removed by boiling the lipid(s) at 100° C.
Titration can be used to assess the amount of acid to be converted to ester. This process can involve dissolving the lipids in an alkanol solution containing an indicator. This solution can then titrated with alkali (e.g., KOH or NAOH) until the indicator shows that the pH of the solution is approximately neutral.
Transesterification can involve mixing the lipid(s) with sodium alkoxide (e.g., sodium methoxide). To generate sodium alkoxide (e.g., sodium methoxide), the selected alkanol (e.g., methanol) is mixed with sodium hydroxide to produce sodium alkoxide (e.g., sodium methoxide). In some instances, the quantity of alkanol used can be 20 percent of the lipid volume. The transesterification mixture can be heated and mixed to facilitate reaction. For example, the mixture can be heated to about 40 to 60° C., or about 45 to 55° C., with mixing.
The lipids can then be cooled, and separated if desired. After the cooling process, the biofuel is generally floating at the top while a heavier glycerin layer can be found at the bottom. The glycerin can be easily separated by allowing it to drain out from the bottom. The process can generate pure biofuel which can be used for various purposes. For example, pure purified lipids can be used as a fuel. Alternatively, the lipids can be mixed with petroleum (e.g., diesel, or heating oil), alcohol, or other fuel product and then used as a fuel (e.g., B20 fuel).
Definitions
As used herein, the terms “crop” and “crop plant” are used herein its broadest sense. The term includes, but is not limited to, any species of plant or alga edible by humans or used as a feed for animals or fish or marine animals, or consumed by humans, or used by humans, or viewed by humans (flowers) or any plant or alga used in industry or commerce or education, such as vegetable crop plants, fruit crop plants, fodder crop plants, fiber crop plants, wood-generating plants, and turf grass plants.
As used herein, the term “exogenous promoter” refers to a promoter in operable combination with a coding region wherein the promoter is not the promoter naturally associated with the coding region in the genome of an organism. The promoter which is naturally associated or linked to a coding region in the genome is referred to as the “endogenous promoter” for that coding region.
As used herein, the term “expression” when used in reference to a nucleic acid sequence, such as a coding region or protein, refers to the process of converting genetic information encoded in a coding region into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of a gene or expression cassette (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (as when a coding region encodes a protein), through “translation” of mRNA. Gene expression can be regulated at many stages in the process. “Up-regulation” or “activation” or “increased expression” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” or “decreased expression” refers to regulation that decreases production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation can also be called “activators” and “repressors,” respectively.
As used herein, the term “heterologous” when used in reference to a gene, promoter, or nucleic acid refers to a gene, promoter, or nucleic acid that has been manipulated in some way. For example, a heterologous nucleic acid or a heterologous promoter includes a nucleic acid or promoter from one species that is introduced into another species. A heterologous nucleic acid or promoter also includes a nucleic acid or promoter that is native to an organism but that has been altered in some way (e.g., placed in a different chromosomal location, mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous coding regions can be distinguished from endogenous plant coding regions, for example, when the heterologous coding regions are joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the coding region, or when the heterologous coding regions are associated with portions of a chromosome not found in nature (e.g., genes expressed in loci where the protein encoded by the coding region is not normally expressed). Similarly, heterologous promoters can be promoters that at linked to a coding region to which they are not linked in nature.
As used herein, “isolated” means a nucleic acid or polypeptide has been removed from its natural or native cell. Thus, the nucleic acid or polypeptide can be physically isolated from the cell or the nucleic acid or polypeptide can be present or maintained in another cell where it is not naturally present or synthesized.
As used herein, the terms “leaf” and “leaves” refer to a usually flat, green structure of a plant where photosynthesis and transpiration take place and attached to a stem or branch.
As used herein, a “native” nucleic acid or polypeptide means a DNA, RNA or amino acid sequence or segment that has not been manipulated in vitro, i.e., has not been isolated, purified, and/or amplified.
As used herein, the term “naturally linked” or “naturally located” when used in reference to the relative positions of nucleic acid sequences means that the nucleic acid sequences exist in nature in those positions.
As used herein, the terms “operably linked” or “in operable combination” or “in operable order” refers to the linkage of nucleic acids in such a manner that a nucleic acid molecule capable of directing the transcription of a given coding region and/or the synthesis of a desired protein molecule is produced. As used herein, the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
As used herein, the term “plant” is used in its broadest sense. It includes, but is not limited to, any species of grass (e.g. turf grass), sedge, rush, ornamental or decorative, crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, woody, flower or tree. It is not meant to limit a plant to any particular structure. Such structures include, but are not limited to, stomata, a seed, a tiller, a sprig, a stolon, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, etc.
As used herein, the terms “protein,” “polypeptide,” “peptide,” “encoded product,” “amino acid sequence,” are used interchangeably to refer to compounds comprising amino acids joined via peptide bonds and. A “protein” encoded by a gene is not limited to the amino acid sequence encoded by the gene, but includes post-translational modifications of the protein. Where the term “amino acid sequence” is recited herein to refer to an amino acid sequence of a protein molecule, the term “amino acid sequence” and like terms, such as “polypeptide” and “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. The deduced amino acid sequence from a coding nucleic acid sequence includes sequences which are derived from the deduced amino acid sequence and modified by post-translational processing, where modifications include but not limited to glycosylation, hydroxylations, phosphorylations, and amino acid deletions, substitutions, and additions. Thus, an amino acid sequence comprising a deduced amino acid sequence can include post-translational modifications of the encoded and deduced amino acid sequence.
As used herein, “seed” refers to a ripened ovule, consisting of the embryo and a casing.
As used herein, “stem” refers to a main ascending axis of a plant.
As used herein, the term “transfection” refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment), Agrobacterium infection, and the like. Methods of transfection are described herein.
As used herein, the term “transgene” refers to a foreign gene (e.g., an expression cassette) that is placed into an organism by the process of transfection.
As used herein, the term “vector” refers to nucleic acid molecules that transfer DNA segment(s). Transfer can be into a cell, cell-to-cell, etc.
As used herein, the term “wild-type” when made in reference to a nucleic acid or gene refers to a functional nucleic acid or gene common throughout an outbred population. As used herein, the term “wild-type” when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
The following non-limiting Examples illustrate how aspects of the invention have been developed and can be made and used.
This Example describes some of the materials and methods used in the development of the invention.
Phylogenetic Analysis The protein sequences for the Chlamydomonas DGTT1-5 genes were retrieved by sequence comparison with Arabidopsis DGAT2 using BLAST (Altschul et al., Nucleic Acids Res 25, 3389-3402 (1997)). Sequences of DGTT1-5 and other DGAT2 proteins were aligned using the ClustalW software in MEGA5 (Tamura et al., Mol Biol Evol 28, 2731-2739 (2011)). The alignment was then used to construct a phylogenetic tree using the Neighbor-Joining method in MEGA5, with the tree being tested by bootstrapping with 1000 replicates.
Plasmid Construction
Chlamydomonas strain dw15.1 (cw15, nitl, mt+), provided by Arthur Grossman, was grown under continuous light (˜80 μm/m2/s) and at 22° C. in liquid TAP media (Harris, E. H., T
DGTT2 cDNA was amplified from pYES2-DGTT2 by PCR using gene specific primers (see Table 1). A fragment of 975 by containing the complete open reading frame was digested with BamHI and EcoRI. This fragment was then placed between the CaMV 35S promoter and OCS terminator of vector p9-35S-OCS (DNA Cloning Service, Hamburg, Germany) to form 35S:DGTT2.
Yeast Expression
Yeast strain H1266 (are2Δlro1Δdga1Δ) (Sandager et al., J Biol Chem 277, 6478-6482 (2002)) was grown to mid-log phase in YPD media (Sherman, Methods in Enzymology 350, 3-41 (2002)) and transformed with the DGTT constructs, along with an empty pYES2 vector as a negative control, according to Gietz et al. (Yeast 11, 355-360 (1995)). The transformants were selected on SC media (Sherman, Methods in Enzymology 350, 3-41 (2002)) with 2% glucose and the uracil omitted (SC-U). Colonies were picked and grown overnight in SC-U+2% glucose, before being transferred to SC-U+2% galactose, 1% raffinose. After 48 hours, 30 mL of the cultures were collected and pelleted by centrifugation for 5 min at 3000×g. Lipids were extracted and analyzed as described below.
Microsome DGTT Assays
Transformed yeast colonies were picked and grown in SC-U+2% glucose overnight, and then transferred to SC-U+2% galactose, 1% raffinose. The cells were harvested after 12 hours, and the microsomes were prepared as described in Milcamps et al. (J Biol Chem 280, 5370-5377 (2005)). The total protein concentration was measured using Bio-Rad Protein Assay Dye Reagent (see website at bio-rad.com), by adding 900 μL of the reagent to 20 μL of BSA standards or microsome samples. 50 ng of microsome were added to a mix containing 100 mM Tris, 8 mM MgCl2, 1 mg/mL BSA, 20% glycerol, 0.25 mg/mL DAG, and 1.725 nmol[1-14C]-16:0-acyl-CoA or [1-14C]-18:1-acyl-CoA (Moravek Biochemicals, see website at moravek.com/). The reaction was incubated at room temperature for one hour. The lipids were extracted with chloroform:methanol (1:1 v/v) and phase separated with 0.2 M H3PO4 and 1 M KCl. The organic layer was extracted and separated on a silica TLC plate using 80:20:1 (v/v/v) petroleum ether:ethyl ether:acetic acid as the solvent. The TLC plate was exposed to film for 72 hours to visualize the radiolabeled lipids.
For the competition assay, 1.725 nmol unlabeled 16:0-acyl-CoA, 18:1-acyl-CoA, or 22:1-acyl-CoA were added. The separated TAG bands were scraped from the plate, and counted in a scintillation counter to quantify the amount of radioactivity incorporated into the lipid.
Plant Material and Generation of Arabidopsis Transgenic Plants
Arabidopsis wild-type (Col2) seeds or transgenic seeds were surface sterilized and grown on ½ Murashige and Skoog (MS) (Physiol. Plant. 15, 473-497 (1962)) agar plates containing 1% sucrose in a growth chamber adjusted to 16 h light/8 h dark (100 μm/m2/s) at 22° C. after 3 days of stratification at 4° C. Fifteen-day-old wild-type and transgenic plants were transferred onto soil and grown in a growth chamber at 16 h light/8 h dark (100 μm/m2/s) and 22° C. The plants were harvested at six weeks for lipid and metabolic assays. Starch analysis was performed using a Megazyme kit as per the manufacturer's instructions (Megazyme, Wicklow, Ireland). For insect assays, transgenic and wild type were grown in a short-day growth chamber at 12 h light/12 h dark (100 μm/m2/s) at 22° C. The binary vector containing DGTT2 was introduced into Agrobacterium strain GV3101 by electroporation. Arabidopsis was transformed using the flower-dip method (see, Clough & Bent, Plant J 16: 735-743 (1998). Transgenic plants (T1) were selected on ½ MS agar plates containing 1% sucrose and 100 mg/L kanamycin. Wild type and hom*ozygous transgenic seedlings were grown on the same shelf of the growth chamber when used for lipid, metabolite, and insect assays.
Lipid Isolation and Quantification
Soil grown 6-week-old and 15-day-old agar-grown (grown on ½ MS agar plates containing 1% sucrose) wild-type and transgenic Arabidopsis plants were freeze-dried. Neutral lipids were extracted from dried samples using chloroform:methanol (1:1, v/v) with 100 μM internal standard tri15:0 TAG and separated on a silica thin layer chromatography (TLC) plate, using a mixture of solvents consisting of petroleum ether:ethyl ether:acetic acid (80:20:1, by volume). TAG bands were isolated from the TLC plate after separation, dissolved in toluene with 10 μM tri13:0 TAG internal standard and assayed using ESI-MS as described by Durrett et al., Proc Natl Acad Sci USA 107, 9464-9469 (2010). To quantify the amount of TAG accumulating in yeast expressing the DGTT constructs, neutral lipids were extracted from the yeast pellets and submitted for ESI-MS, following the method described by Durrett et al. (2010).
Distinct lipid and TAG bands were scraped from the TLC plates and used to prepare fatty acid methyl esters (FAMEs) by acid-catalyzed transmethylation. Identification and quantification of FAMEs was performed as described by Xu et al., EMBO J 22, 2370-2379 (2003). The amounts of lipids were calculated based on the content of fatty acids measured by gas chromatography using C15:0 as an internal standard.
Microscopy
For oil droplet visualization and TEM, the leaf samples from 6-week-old soil grown transgenic and wild-type plants were used. Whole leaf samples for TEM were fixed in a mixture of 2.5% glutaraldehyde and 2.5% paraformaldehyde in 0.1 M cacodylate buffer at 4° C. for 24 hours, post-fixed in 1% osmium tetroxide and dehydrated in a graded acetone series. Samples were infiltrated and embedded in Spurr resin (Polysciences, see website at polysciences.com). Thin sections were imaged using a JEOL 100CX Transmission Electron Microscope (Japan) at a 100 kV accelerating voltage. Freshly harvested leaf samples were used for oil droplet visualization by confocal microscopy as described by Sanjaya et al. (Plant Biotechnol J 9: 874-883 (2011)). For SEM, leaves from 6-week-old transgenic line 57 and wild-type plants were plunge frozen in liquid nitrogen, freeze dried with an EMS750X Turbo Freeze Drier (Electron Microscopy Sciences, Hatfield, Pa., USA), and mounted on aluminum stubs using carbon tape (Ted Pella, Inc., Redding, Calif., USA). These samples were coated with gold (about 20 nm thickness) for 3.5 minutes in an Emscope Sputter Coater model SC 500 (Ashford, Kent, England) purged with argon gas and coated with osmium (about 10 nm thickness) in an NEOC-AT osmium coater (Meiwafosis Co., Ltd., Osaka, Japan) for 35 seconds. The samples were examined in a JEOL JSM-6400V (Lanthanum Hexaboride electron emitter) scanning electron microscope (JEOL Ltd, Tokyo, Japan). Digital images were acquired using Analysis Pro software version 3.2 (Olympus Soft Imaging Solution Crop., Munster, Germany) at the Center for Advanced Microscopy, Michigan State University.
Analysis and Quantification of Sphingolipid Classes
Leaves of soil-grown (6-week-old) plants were used for the extraction of sphingolipids, using methods described by Markham et al. (J Biol Chem 281, 22684-22694 (2006)). Sphingolipid identification and quantification from wild type and line 57 samples was performed by ESI-high resolution/accurate mass spectrometry operating in positive ion mode. All solvents used were high performance liquid chromatography (HPLC) grade, or the highest grade available. Isopropanol, methanol, and chloroform were purchased from Macron Chemical (St. Louis, Mo., USA). Hexane and HPLC water were from Fisher (Hampton, N.H., USA). Ammonium formate was purchased from Alfa Aesar (Ward Hill, Mass., USA). The sphingolipid internal standard mixture used for mass spectrometry analysis was Avanti Sphingolipid Mix II (Avanti Polar Lipids, Alabaster, Ala., USA). Around 30 μl aliquots of total lipid extract were dried under nitrogen and subjected to mild alkaline hydrolysis of glycerolipids by the addition of 500 μl of methanol, 250 μl of chloroform, and 75 μl of 1 M KOH. Samples were incubated for two hours at 37 degrees Celsius in a shaking water bath, then neutralized with 6 μl of glacial acetic acid (Merrill et al., Methods 36, 207-224 (2005)). For ceramide (Cer) and glucosylceramide (GlcCer) analysis, samples were re-extracted by a modified Folch method (Busik et al., Methods Mol Biol 579, 33-70 (2009)) and resuspended in 300 μl of isopropanol/methanol/chloroform (4:2:1 v/v/v). For glycosylinositol-phosphoceramide (GIPC) lipid analysis, dried samples were extracted into 300 μl of isopropanol/hexane/water (3:1:1 v/v/v) (Markham et al., J Biol Chem 281, 22684-22694 (2006)), centrifuged to remove particulates, and the supernatants saved for analysis. Samples were further diluted 30-fold in isopropanol/methanol/chloroform (4:2:1 v/v/v, for ceramide and glucosylceramide analysis) or isopropanol/hexane/water (3:1:1 v/v/v, for GIPC analysis) containing a final concentration of 20 mM ammonium formate and the sphingolipid internal standard mixture diluted to 100 nM of each sphingolipid species. Samples were centrifuged and loaded into Whatman Multichem 96-well plates (Sigma Aldrich, St. Louis, Mo., USA) sealed with Teflon UltraThin Sealing Tape (Analytical Sales and Services, Prompton Plains, N.J., USA).
Samples were directly infused into a Thermo Scientific model LTQ Orbitrap Velos high resolution/accurate mass spectrometer (San Jose, Calif.) using an Advion Triversa Nanomate nano-electrospray ionization (nESI) source (Advion Ithaca, N.Y.) with a spray voltage of 1.4 kV and a gas pressure of 0.3 psi. The ion source interface settings (inlet temperature of 100° C. and S-Lens value of 50%) were optimized to maximize the sensitivity for precursor ions while minimizing in-source fragmentation. High resolution mass spectra were acquired using the FT analyzer operating at 100,000 resolving power, across the range of m/z from 400-2000, and were signal averaged for 2 minutes. All mass spectra were recalibrated offline using XCalibur software (Thermo Scientific, San Jose, Calif., USA) and the exact masses of the sphingolipid internal standards d18:1/12:0 ceramide, d18:1/12:0 glucosyl/galactosyl ceramide, d18:1/12:0 sphingomyelin, and d18:1/12:0 lactosyl ceramide. Automated peak finding, correction for 13C isotope effects, and quantitation of lipid molecular species were performed using Lipid Mass Spectrum Analysis (LIMSA) software version 1.0 (Haimi et al., Analytical chemistry 78, 8324-8331 (2006)) linear fit algorithm. Sphingolipids were quantitated as the sum of their [M+H]+ and [M+NH4]+ (when present) ion abundances against the d18:1/12:0 lactosyl ceramide internal standard. As no attempts were made to correct for differences in ionization efficiency among individual molecular species of ceramide, glucosyl ceramide, and GIPC lipids, as well as the lack of an ideal internal standard for quantitation of GIPC lipids, sphingolipid molecular species are presented only as a fraction of the total normalized abundance of each lipid class.
Surface Wax and Cutin Analysis
Rosette leaves of 6-week-old soil grown transgenic and wild-type plants were used for cutin and wax analysis, using slight modifications of methods described in Molina et al., Phytochemistry 67, 2597-2610 (2006); Kosma et al., Plant Physiol 151, 1918-1929 (2009). Briefly, surface waxes were extracted from leaves by a 30 second submersion in hexane. Internal standards pentadecanoic acid (C15:0), tricosanol (C23:0), and octacosane (C28:0) were added to the hexane extracts and the solvent was removed by evaporation under nitrogen gas. Dried extracts were derivatized with 100 μL each of pyridine and N, O-bis (trimethylsilyl trifluoroacetamide) (BSTFA). Excess pyridine and BSTFA were removed by evaporation with N2 and samples were dissolved in heptane:toluene (1:1 v/v) for GC-MS analysis.
Thoroughly delipidated ground leaf tissues were used for cutin monomer analysis. Methyl heptadecanoate and pentadecalactone were used as internal standards. Base catalyzed transmethylation reactions (NaOMe/MeOH) consisted of 4.5% sodium methoxide (NaOMe) and 7.5% methyl acetate in methanol in a total volume of 6 mL.
Reaction mixtures were heated overnight (˜16 h) at 60° C. then allowed to cool to room temperature. Reaction mixtures were acidified with glacial acetic acid to pH 4-5, 2-3 mL saline solution were added (0.5 M NaCl), and FAMEs were extracted with 7 mL CH2Cl2. The organic phase was washed twice with dilute saline solution (0.9% NaCl, w/v) and dried over anhydrous Na2SO4. Extracts were evaporated to dryness under N2 and the product silylated to convert hydroxyl groups to their trimethylsilyl ethers using BSTFA and pyridine. Excess pyridine and BSTFA were evaporated under N2 and samples were dissolved in heptane:toluene (1:1, v/v) for GC-MS analysis. GC-MS temperature programs were as follows: Wax: inlet 350° C., detector 320° C., oven 130° C. for 3 min then temperature increased at 5° C./min to 325° C., 325° C. for 10 min. Cutin: inlet 330° C., detector 320° C., oven 140° C. for 3 min then temperature increased at 5° C./min to 310° C. then held at 310° C. for 10 min.
Elemental Analysis
Rosette leaves of 6-week-old soil grown transgenic line 57 and wild-type plants were harvested at the end of the day and thoroughly freeze dried, ground into fine powder in a Retsch Mill at a frequency of 25 for 2 min and used to measure carbon, hydrogen, sulfur, nitrogen and ash content employing a commercial service (Elemental Analysis Inc., Lexington, Ky., USA). Protocols are listed on the website at elementalanalysis.com. Calculation of heating value (MJ/kg DW)=0.3491XC+1.1783XH+0.1005XS−0.0151XN−0.1034X0−0.0211Xash; X's entered as mass percentages (Guar & Reed, An atlas of thermal data for biomass and other fuels. NREL/TB-433-7965, UC Category: 1310, DE95009212, National Renewable laboratory, Golden Colo., USA (1995)).
Insect Feeding Assay and JA-Ile Quantification Using Mass Spectrometry
Insect feeding trials and JA-Ile analysis by LC-MS/MS were performed as described by Koo et al. (Front Plant Sci 3: 19 (2011)). S. exigua eggs (Benzon Research, see website at benzonresearch.com/) were hatched at 30° C. on artificial insect diet (Southland Products INC., Lake Village, Ark.) for 2 days and used for feeding experiments. Newly hatched neonates were allowed to grow on artificial diet for another 2 days before being transferred to 6-week-old plants. Ten larvae were reared per single pot, each of which contained two plants of the same genotype. A total of 14 pots were used for each genotype. Larvae were caged in each pot using an inverted clear plastic cup with an opening on the top covered with miracloth for air exchange. Plants with insects were grown in a growth chamber maintained at 2° C. under a 12-h light (100 μm/m2/s) and 12-h dark cycle. Fresh weight of individual larvae was determined 14 days after the start of the feeding trial. For JA-Ile measurements, approximately 350 mg of leaf tissue was harvested from insect damaged and undamaged control plants at the end of the trial. Jasmonate extraction and JA-Ile quantification by LC-MS/MS were performed as previously described (Koo et al., Front Plant Sci 3: 19 (2011)).
Microarray and Data Analysis
Leaves from 6-week-old plants grown on soil at 22° C. under 16 h light/8 h dark (100 μm/m2/s) were used for isolating total RNAs with the RNeasy plant mini kit (Qiagen). Subsequently, the total RNA samples were pretreated with RNase-free DNase I and cleaned with a Plant Total RNA isolation kit (Qiagen). Four biologically independent RNA samples from DGTT2 line 57 and wild type plants each were used for the microarray experiments. Probe preparation, hybridization to the 4-plex GeneChip Arabidopsis ATH1 Genome Arrays (NimbleGen, see website at nimblegen.com), and subsequent processing steps were performed according to the manufacturer's instructions (Gene Expression Center, University of Wisconsin). Arrays were scanned at 5 μm on an Axon4000B Scanner (Molecular Dynamics, Sunnyvale, Calif., USA). Microarray data were analyzed using Partek Genomics Suite software (2009; see website at partek.com). Microarray data were analyzed using Partek Genomics Suite software (2009; see website at partek.com. RMA normalization (Irizarry et al., Nucleic Acids Res 31, el5 (2003); Irizarry et al., Biostatistics 4, 249-264 (2003)) was performed by Partek upon data import, producing log 2-transformed intensity values. Probe set log-intensities had a bimodal distribution with a valley at about 9 (not shown). Such bimodality appears to be a common feature of oligonucleotide arrays, and the lower mode is thought to correspond to unexpressed genes (Irizarry et al., Biostatistics 4, 249-264 (2003); see website at stat.ethz.ch/pipermail/bioconductor/2006-June/013255.html). Therefore, probe sets whose maximal values across all arrays were less than 9 were taken to target genes that were not expressed at detectable levels in any of the samples and were therefore removed from further analysis. This decreased the number of probe sets in the analysis from 30,361 to 18,221.
The PCA plot of the filtered dataset did not show clear separation between wild type and transgenic plants (data not shown), indicating that the transgene expression did not have a strong effect on the global gene expression profile of the plants. Since the experiment was performed on 2 NimbleGen chips, with 4 arrays per chip (2 wild type and 2 transgenic each), two factor mixed model ANOVA analysis was employed to account for the effects of genotype and chip, the latter being a random effect. P values were calculated for the wild type vs. line 57 contrast. Multiple testing correction using the step-up FDR method identified only one gene with adjusted p value<0.05, encoding a transposable element. Therefore, putative hits were selected using the unadjusted p value threshold of 0.002 and fold change threshold on >2 or <−2. This identified 15 genes (see Table 2), several of which could be expected to be false positives.
Q-RT PCR
Total RNA was extracted from 15-day-old and 6-week-old DGTT2 transgenic and wild-type plants. cDNA synthesis, q-RT PCR with gene specific primers (see Table 1) and data analysis was performed as described by Sanjaya et al., Plant Biotechnol J 9: 874-883 (2011).
Accession Numbers
The protein sequences used in the phylogenetic analysis were as follows DGTT1, XP_001702848.1; DGTT2, XP_001694904.1; DGTT3, XP_001691447.1; DGTT4, XP_001693189.1; DGTT5, XP_001701667.1; Arabidopsis DGAT2, NP_566952.1; VfDGAT2, DQ356682.1; RcDGAT2, DQ923084.1; ScDGA1, NP_014888.1; and OtDGAT2B, XP_003083539.1.
The microarray data set is deposited into GEO (see website at ncbi.nlm.nih.gov/geo) with accession number GSE38898.
Amino acid sequence similarity to Arabidopsis DGAT2 was used to identify potential DGAT orthologs, using BLAST against Version 3 of the Chlamydomonas genome. The BLAST search results returned five gene models with high sequence similarity to the Arabidopsis type-2 DGAT. These five candidates were named Diacylglycerol Acyltransferase Type Two (DGTT) 1-5 and compared to type-2 DGATs from Arabidopsis thaliana, Ostreococcus tauri, yeast (Saccharomyces cerevisiae), castor bean (Ricinus communis), and tung tree (Vernicia fordii).
When analyzed with MEGA5 (Tamura et al., Mol Biol Evol 28, 2731-2739 (2011)), DGTT1 was the most closely related to Sc-DGA1. DGTT2, DGTT3 and DGTT5 formed their own clade, as did the three land plant DGATs. Ot-DGAT2B and DGTT4 were both more similar to the land plant DGATs than to the other Chlamydomonas DGTTs (
Structural analysis of the gene model-translated protein sequences was performed in silico using the TMHMM Server 2.0 (see website at cbs.dtu.dk/services/TMHMM/) to predict transmembrane sequences. All five candidates had 1-3 transmembrane domains in the N-terminal half of the protein, consistent with other type-2 DGATs. SignalP V3.0 (see website at cbs.dtu.dk/services/SignalP/) and TargetP V1.1 (website at cbs.dtu.dk/services/TargetP/) failed to identify potential cellular localization signals. Searching against the NCBI Conserved Domain Database revealed a DAGAT domain in the C-terminal half of all five candidates, although DGTT5 had an apparent disruption in its domain (
To test the function of the predicted DGTT genes, the coding sequences of DGTT2-5 were isolated and expressed in yeast strain H1266, a triple knockout mutant for DGA1, LRO1, and ARE2 (Sandager et al., J Biol Chem 277, 6478-6482 (2002)). This strain has very little native DGAT activity, providing a suitable background for testing the Chlamydomonas DGAT2 candidates. The inventors were unable to isolate a DGTT1 cDNA based on the available gene model. The presence of the DGTT2-5 proteins was confirmed in yeast by immunoblotting. To quantify the amount of TAG produced, electrospray ionization mass spectrometry (ESI-MS) was performed on lipid extracts from the transgenic yeast (
The substrate preference of the DGTT proteins was estimated in an in vitro reaction with isolated microsomes. DGTT-containing microsomes were incubated with radiolabeled palmitoyl-CoA (16:0) and oleoyl-CoA (18:1), along with dioleoyl DAG. The level of activity was estimated from their incorporation of radiolabel into isolated TAGs. Comparison of the results using the two different substrate acyl-CoAs suggested an apparent difference in substrate specificity, with DGTT2 and DGTT3 preferring 16:0-CoA and DGTT4 preferring 18:1-CoA (
Although DGTT2 and DGTT3 belong to the same clade, following nitrogen-deprivation the level of DGTT2 transcripts remained unchanged, whereas DGTT3 transcript levels increased (Miller et al. Plant Physiol 154, 1737-1752 (2010); Boyle et al., J Biol Chem 287(19): 15811-15825 (2012)). When expressed in yeast, DGTT2 activity in vitro was higher than that of the other isoforms.
Based on the observed activity levels, the inventors focused on the characterization of the DGTT2 enzyme as a potential tool for the manipulation of cytosolic acyl-CoAs in vegetative tissues of plants, the primary goal of this study. A microsome-based competition assay was used to further probe the substrate specificity of DGTT2. When incubated with equimolar amounts of 14C-labeled and unlabeled 16:0-CoA, the radioactivity in the TAG band was reduced to ˜50% compared to the reactions with only 14C-labeled 16:0-CoA (
Together, these data indicated that DGTT2 is able to incorporate varying acyl-CoA species into TAG, perhaps preferring very long chain acyl groups, which is relevant for the interpretation of lipid data for the transgenic plants described below.
High-throughput transcript profiling of Chlamydomonas under nitrogen-deprivation showed that the expression levels and patterns of the five putative DGAT2-encoding genes varied considerably. DGTT1 was the most highly regulated, being strongly up-regulated following nitrogen-deprivation. Based on the sequence analysis, DGTT2 and DGTT3 are the most similar. Additionally, they showed near-identical results in yeast assays. Although DGTT3 did show a change in expression during nitrogen-deprivation, the overall change was less than two-fold, which was statistically insignificant. For these reasons, it was assumed that their activity in Arabidopsis would also be similar. The results for either one of the two isoforms were representative of both.
Structural analysis of the DGAT2 candidates indicates that they are similar but not identical to previously identified DGAT2 proteins. The absence of predicted signal or targeting sequences may be explained by the fact that the programs were trained on land plant signal sequences, which could hinder the detection of such sequences in Chlamydomonas. Given the absence of obvious organellar targeting sequences, the most likely location is the endoplasmic reticulum (ER) membrane, which is the main site of TAG synthesis in plants (Lung and Weselake, Lipids 41, 1073-1088 (2006)). Recently, however, the existence of a separate TAG assembly pathway in chloroplasts has been suggested (Fan et al., 2011; Goodson et al., 2011). It seems possible that some of the DGAT2 proteins are preferentially associated with the endoplasmic reticulum, whereas other DGAT2 proteins are targeted preferentially to the chloroplast envelope by mechanisms that would not require classic targeting sequences.
Biochemically, DGTT2 possesses broad substrate specificity and is able to incorporate varying acyl-CoA species into TAG in vitro. However, it is unclear what the natural substrate of DGTT2 may be in Chlamydomonas. Both Ostreococcus DGAT2B and yeast Dga1 also have broad substrate specificity, in contrast to the more substrate specific type 2 DGATs found in land plants (Wagner et al., 2010). These, along with the relative abundance of type 2 DGATs in other green algae, suggest a more distinct role for these enzymes in algae, compared to plants. This difference may be related to the different roles and regulation of TAG synthesis, as microalgae produce oil in response to environmental stresses, whereas land plants produce oil primarily during seed development.
To explore DGTT2 as a tool to manipulate acyl-CoA pools in plants and to engineer TAGs in vegetative tissues, the full length DGTT2 coding sequence was expressed in Arabidopsis under the control of the heterologous constitutive 35S cauliflower mosaic virus (CaMV) promoter, 35S:DGTT2. Five hom*ozygous lines, 14, 22, 46, 52 and 57, were carried forward to the T4 generation and used for detailed analysis.
The hypocotyls of the DGTT2 transgenic 10-to-12-day-old seedlings grown on MS agar plates supplemented with 1% sucrose were strikingly more elongated than those of control plants (
To determine the effect of DGTT2 production on the accumulation of TAG, 15-day-old whole seedlings were analyzed by ESI-MS. The TAG levels in 15-day seedlings of transgenic lines were increased up to 22-fold and 25-fold (
ESI-MS was also used to analyze the TAG content in the leaves of 6-week-old soil-grown plants before bolting. As shown in
Leaves of 6-week-old TAG-accumulating line 57 and of wild type were compared by confocal microscopy following Nile Red staining and by transmission electron microscopy (TEM). Oil droplets were abundant in line 57 and were distributed in the proximity of the chloroplasts (
The inventors used TEM to analyze the location of oil droplets in leaf sections. In line 57, large and distinct electron dense oil droplets were observed outside the chloroplast in mesophyll cells, most likely associated with the ER (
The inventors suspected that the group of lipids specifically affected by the ectopic accumulation of TAGs with VLCFAs in leaves might be epidermal surface lipids. To test this hypothesis, leaf epidermal cell sections of DGTT2 transgenic line 57 and wild-type plants were examined by TEM. Plants of line 57 had leaves with a noticeably less osmium-dense cuticle than wild type plants (
To observe quantitative and qualitative changes in wax composition, the rosette leaf waxes of lines 14, 22 and 57 and wild type (6-week-old) were subjected to GC-MS analysis. A considerable change in the wax composition was observed. More specifically a decrease in nonacosane (C29 alkane) was detected that was the most abundant leaf wax constituent, and triacontanol (C30 1-alcohol) in the DGTT2 transgenic lines (
Changes in cutin and cutin-associated features were observed in the transgenic lines. Alteration of cutin content and composition is often associated with changes in the cuticular ledge ultrastructure of guard cells (Kosma & Jenks, Eco-physiological and molecular-genetic determinants of plant cuticle function in drought and salt tolerant crops. In Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops., M. A. Jenks, P. M. Hasegawa, and S. M. Jain, eds (Netherlands: Springer), pp. 91-120 (2007); Li et al., Proc Natl Acad Sci USA 104, 18339-18344 (2007). Stomata of leaves of line 57 and wild type were therefore examined using SEM and TEM. SEM showed that leaves of line 57 had an altered stomatal structure, with wider stomatal openings (
DGTT2 accepts a broad range of acyl-CoA substrates. However, expression of DGTT2 could result in reduced sphingolipid content in the leaves. The inventors analyzed sphingolipid content in wild type and transgenic lines using ESI-high resolution/accurate mass spectrometry to identify glycosyl inositolphosphoceramide (GIPC) lipids. Normalization of GIPC lipid abundances against an internal standard (d18:1/12:0 lactosyl ceramide) demonstrated that in wild-type plants the t18:1/h24:0 and t18:1/h24:1 molecular species were the most abundant (
To determine whether DGTT2-dependent modulation of sphingolipid 24:1 and 24:0 fatty acid levels was specific to GIPC lipids or distributed across other sphingolipid classes, the less abundant glucosylceramide (GlcCer) and ceramide (Cer) lipids were also analyzed. As shown in
To evaluate possible compensatory mechanisms in the transgenic lines, microarray experiments were conducted with RNA extracts from the leaves of 6-week-old soil-grown plants. A two factor mixed model ANOVA analysis was employed to account for the effects of genotype and chip, and P values were calculated for the wild type vs. line 57. Since no meaningful hits could be identified after a false discovery rate correction, 15 genes were selected using the unadjusted p value threshold of <0.002 and fold change>2 or <−2 (Table 4).
Of the 15 genes selected from the microarray study, 10 were up-regulated and 5 down-regulated (Table 5).
Two up-regulated genes were predicted to encode enzymes with a known involvement, or a possible involvement, in TAG biosynthesis, including a bifunctional enzyme WSD1 (wax ester synthase and diacylglycerol acyltransferase), At5g37300 (Li et al., Plant Physiol 148, 97-107 (2008) and a gene encoding an HXXXD-type acyl-transferase, At1g65450. The inventors also identified changes in expression of the qua-quine starch (QQS) regulatory gene, At3g30720, and cytosolic beta-amylase encoding gene, At4g15210. Both proteins are involved in starch metabolism (Li et al., Plant J 58, 485-498 (2009). One of the down-regulated genes with unknown function was predicted to encode a mini zinc finger protein, At3g28917, and another a UDP-glycosyltransferase, superfamily protein, At2g23210.
To validate the microarray analysis, the transcript levels of the four genes were quantified relevant to oil biosynthesis and starch metabolism, using q-RT-PCR. For this experiment RNA was isolated from an independent set of wild-type and line 57 plants. When normalized to wild-type values, the transcript levels for the WSD1-encoding gene were 3.5-fold higher, for the HXXXD-type acyl-transferase protein-encoding gene 4.4-fold, for the QQS gene 4.3-fold, and for the cytosolic beta-amylase-encoding gene 3.0-fold elevated in line 57, thus confirming the microarray results (see
As explained above, most of the DGTT2-producing Arabidopsis lines examined developed distinct seedling phenotypes, such as accelerated hypocotyl elongation and pale green cotyledons. The increased hypocotyl length may be due to excess storage compounds such as TAG in the seeds that may accelerate cell elongation compared to wild type. The reduction in chlorophyll content in the DGTT2 seedlings may be related to the reduction in the major plastid lipids, limiting the extent of the photosynthetic membrane. The fact that this phenotype is only visible at the seedling stages, when leaves need to rapidly expand and when demands on membrane lipid biosynthesis are high, supports this hypothesis. The acyl-group supply may be limited and TAG accumulation in leaves diverts acyl groups away from other pathways, such as plastid glycerolipid biosynthesis, without a compensatory increase in acyl synthesis. This hypothesis is supported by the microarray results, which revealed no significant induction of genes encoding enzymes necessary for fatty acid biosynthesis (Tables 4 and 5).
In general, plants accumulate TAG in seeds as stored energy that can be subsequently used to fuel germination and seedling establishment. As demonstrated herein accumulation of TAG with VLCFA, such as C20:0 and longer, in green seedlings and leaves of soil grown plants can be achieved by expression of DGTT2, even though VLCFA-containing glycerolipids do not normally accumulate in green tissues of Arabidopsis. This observation was unexpected because Chlamydomonas TAG primarily contains C16:0 and C18:1 fatty acids, but no TAG species containing VLCFAs (Fan et al., 2011). Following the ectopic production of DGTT2 in the leaves of Arabidopsis, most likely the availability of particular acyl-CoA species as well as the substrate preference of DGTT2 for VLCFAs leads to the observed composition of the TAG produced in the transgenic lines. It is interesting to note that the amount of TAG accumulated in DGTT2 transgenic seedlings was higher than that of soil grown plants (
The dry weight of the aerial parts of soil-grown transgenic line 57 and wild-type plants was measured on a weekly basis. The overall morphology of the leaves and shoots from the DGTT2 transgenic line was unchanged. Transgenic line 57 gained a considerably higher biomass than wild type (105.6 and 87.6 mg DW per seedling, p<0.001, n=3) during the first 5 weeks after germination and DW values continued to be higher until the end of the experiment (
The increased transcript levels of the QQS gene and cytosolic beta-amylase encoding gene in transgenic line 57 from the microarray experiments led us to investigate the starch content of DGTT2-transgenic plants. Contrary to expectation, given the increase in the expression of the genes encoding QQS and beta-amylase, six-week-old plants of line 57 accumulated 18.7% more starch than wild-type plants (58.0 and 47.1 μg Glu per mg DW (p<0.01, n=5), respectively) (
Increased TAG accumulation or decreased wax and cutin in vegetative tissue may result in an unusual diet for phytophagous organisms and may perhaps affect the nutritional value of the plants. To test this hypothesis, the inventors conducted a “no choice” insect-feeding assay, in which the generalist lepidopteran herbivore Spodoptera exigua was reared on wild type and transgenic lines 22 and 57 (
Active plant defense responses to insect herbivory are controlled by wound-induced production of the 18:3-derived phytohormone, jasmonoyl-L-isoleucine (JA-Ile) (Koo & Howe, Proc Natl Acad Sci USA 108, 9298-9303(2012). The altered lipid content of DGTT2-producing plants raised the possibility that increased larvae performance was caused by reduced levels of JA-Ile in the transgenic lines. To test this idea, JA-Ile levels were quantified in leaf tissue from insect-fed and control undamaged plants (
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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The following statements of the invention are intended to summarize features of the invention.
Statements:
- 1. A construct comprising a nucleic acid encoding a polypeptide with at least 40% sequence identity with an amino acid sequence selected from the group of SEQ ID NO:1, 3, 5, and 7, that is operably linked to a heterologous promoter operable in at least one plant tissue.
- 2. The construct of Statement 1, wherein the polypeptide has at least about 50% sequence identity, at least 55% sequence identity, at least about 60% sequence identity, at least 70% sequence identity, at least about 80% sequence identity, at least 90% sequence identity, at least about 95% sequence identity, or 40-95% sequence identity, or 50-95% sequence identity, or 60-90% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 98-99% sequence identity, or 100% sequence identity with an amino acid sequence selected from the group of SEQ ID NO:1, 3, 5, and 7.
- 3. The construct of Statement 1 or 2, wherein the nucleic acid has at least about 50% sequence identity or complementarity with any of the SEQ ID NO:2, 4, or 6.
- 4. The construct of any of Statements 1-3, wherein the nucleic acid has at least about 50% sequence identity, at least 55% sequence identity, at least about 60% sequence identity, at least 70% sequence identity, at least about 80% sequence identity, at least 90% sequence identity, at least about 95% sequence identity, or 40-95% sequence identity, or 50-95% sequence identity, or 60-90% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 98-99% sequence identity, or 100% sequence identity or complementarity with any of the SEQ ID NO:2, 4, or 6.
- 5. The construct of any of Statements 1-4, wherein the polypeptide is a Diacylglycerol Acyltransferase Type Two enzyme.
- 6. The construct of any of Statements 1-5, wherein the polypeptide is a Diacylglycerol Acyltransferase Type Two enzyme selected from the group of DGTT1, DGTT2, DGTT3 and DGTT4.
- 7. The construct of any of Statements 1-6, wherein the polypeptide is a Diacylglycerol Acyltransferase Type Two enzyme is DGTT2 or DGTT3.
- 8. The construct of any of Statements 1-7, wherein the polypeptide is a Diacylglycerol Acyltransferase Type Two enzyme with at least 50% of DGTT2 or DGTT3 activity as measured in an assay of microsomal protein conversion of labeled monoacyl-CoA to labeled triacylglycerols.
- 9. The construct of any of Statements 1-8, wherein the polypeptide increases expression of a protein selected from the group of wax ester synthase (WS), diacylglycerol acyltransferase (DGAT), Qua-Quine Starch (QQS), Cytosolic beta-amylase, HXXXD-type acyl-transferase, Sodium potassium root defective 2, Peroxidase superfamily protein, PARI protein, Nodulin Mt-N21/EamA-like transporter, and any combination thereof.
- 10. The construct of any of Statements 1-9, wherein the polypeptide decreases expression of mini zinc finger 2, UDP-Glycosyltransferase superfamily protein, or a combination thereof.
- 11. The construct of any of Statements 1-10, wherein the promoter is a constitutive promoter, a developmental promoter, or an inducible promoter.
- 12. The construct of any of Statements 1-11, wherein the promoter is selected from the group of CaMV 35S promoter, a CaMV 19S promoter, a nos promoter, an Adh1 promoter, a sucrose synthase promoter, an α-tubulin promoter, a ubiquitin promoter, an actin promoter, a PEPCase promoter, a GAL4/UAS promoter, an R gene promoter, a poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, a cauliflower mosaic virus promoter, an OLE (Oleosin) promoter, a Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, a light inducible promoter derived from the pea rbcS gene, an actin promoter from rice, a phaseolin promoter, or any combination thereof.
- 13. A plant cell comprising the construct of any of Statements 1-12.
- 14. A plant seed comprising the construct of any of Statements 1-12.
- 15. A plant comprising the construct of any of Statements 1-12.
- 16. A reproductively competent plant comprising the construct of any of Statements 1-12.
- 17. A plant comprising the construct of any of Statements 1-12, wherein the plant has lipid in its tissues or seeds.
- 18. A plant of Statement 15, 16, or 17, or comprising the construct of any of Statements 1-12, wherein the plant has lipid in its vegetative tissues.
- 19. The plant of any of Statements 15-18, wherein the plant has lipid comprising triacylglycerol.
- 20. A plant of any of Statements 15-18, or 19, or comprising the construct of any of Statements 1-12, wherein the plant has at least 1% lipid per dry weight of tissue.
- 21. A plant of any of Statements 15-18, or 19, or comprising the construct of any of Statements 1-12, wherein the plant has at least 1% lipid, or at least 2% lipid, or at least 3% lipid, or at least 4% lipid, or at least 5% lipid, or at least 6% lipid, or at least 7% lipid, or at least 8% lipid, or at least 9% lipid, or at least 10% lipid, or at least 11% lipid, or at least 12% lipid, or at least 13% lipid, or at least 14% lipid, or at least 15% lipid, or at least 16% lipid, or at least 17% lipid, or at least 18% lipid, or at least 19% lipid, or at least 20% lipid per dry weight of tissue.
- 22. The plant of any of Statements 15-18, or 19, or comprising the construct of any of Statements 1-12, wherein the plant has about 1% to about 30% lipid per dry weight of tissue, or about 5% to about 25% lipid per dry weight of tissue, or about 10% to about 20% lipid per dry weight of tissue.
- 23. The plant of any of Statements 15-22, wherein the plant is an oleaginous plant.
- 24. The plant of any of Statements 15-23, wherein the plant is selected from the group of an oleaginous plant, an Arabidopsis plant, corn, sugar beets, soybean, sugar cane, potato, grasses (e.g., miscanthus, switchgrass, and the like), rice, a beet species, a grape species, an Arabidopsis species, a Brassica species, a Brassica napus plant, an algae species, an oilseed rape species, a sunflower, a soybean species, a flax species, an olive species, an alfalfa species, an oat species, a wheat species, a poplar, an aspen, a willow, and similar plant species.
- 25. The plant of any of Statements 15-24, wherein the plant has less cutin and more fatty acids than an wild type plant of the same species.
- 26. An isolated lipid from tissues of the plant of any of Statements 1-12, 15-24, or 25.
- 27. An isolated lipid from vegetative tissues of the plant of any of Statements 1-12, 15-24, or 25.
- 28. The isolated lipid of Statement 26 or 27, containing trace amounts of the construct of any of Statements 1-12.
- 29. A method, comprising transfecting at least one plant cell with the construct of any of Statements 1-12 and growing the at least one plant cell into a population of plant cells.
- 30. A method, comprising transfecting a plant tissue with the construct of any of Statements 1-12 and growing the tissue into a whole plant.
- 31. The method of Statement 29 or 30, wherein the population of plant cells or tissues of the whole plant has at least 1% lipid per dry weight of cell population or per dry weight of tissue.
- 32. A method of any of Statements 29-31, wherein the population of plant cells or tissues of the whole plant has at least 1% lipid, or at least 2% lipid, or at least 3% lipid, or at least 4% lipid, or at least 5% lipid, or at least 6% lipid, or at least 7% lipid, or at least 8% lipid, or at least 9% lipid, or at least 10% lipid, or at least 11% lipid, or at least 12% lipid, or at least 13% lipid, or at least 14% lipid, or at least 15% lipid, or at least 16% lipid, or at least 17% lipid, or at least 18% lipid, or at least 19% lipid, or at least 20% lipid per dry weight of tissue or of cell population.
- 33. The method of any of Statements 29-32, wherein the population of plant cells or tissues of the whole plant has at about 1% to about 30% lipid per dry weight of tissue, or about 5% to about 25% lipid per dry weight of tissue, or about 10% to about 20% lipid per dry weight of tissue.
- 34. A method, comprising,
a) providing at least one plant cell that comprises the construct of any of Statements 1-12;
b) culturing the at least one plant cell in media that supports regeneration of plant tissue, to thereby generate at least one transformed plant tissue;
c) generating at least one plant from the at least one transformed plant tissue to thereby generate at least one transformed plant; and
d) growing the at least one transformed plant.
- 35. The method of statement 34, wherein the at least one transformed plant is a reproductively competent plant.
- 36. The method of Statement 34 or 35, further comprising growing the at least one transformed plant to generate at least one transformed plant with seeds.
- 37. The method of any of Statements 34-36, wherein the at least one transformed plant has transformed seeds comprising the construct of any of Statements 1-12.
- 38. The method of any of Statement 34-37, further comprising isolating one or more seeds from the least one transformed plant with seeds.
- 39. The method of any of Statements 34-37, wherein the at least one transformed plant has at least 1% lipid per dry weight of tissue.
- 40. The method of any of Statements 34-39, wherein the at least one transformed plant has at least 1% lipid per dry weight of vegetative tissue.
- 41. The method of any of Statements 34-40, wherein the at least one transformed plant has at least 1% lipid, or at least 2% lipid, or at least 3% lipid, or at least 4% lipid, or at least 5% lipid, or at least 6% lipid, or at least 7% lipid, or at least 8% lipid, or at least 9% lipid, or at least 10% lipid, or at least 11% lipid, or at least 12% lipid, or at least 13% lipid, or at least 14% lipid, or at least 15% lipid, or at least 16% lipid, or at least 17% lipid, or at least 18% lipid, or at least 19% lipid, or at least 20% lipid per dry weight of cell population or per dry weight of tissue.
- 42. The method of any of Statements 34-41, wherein the at least one transformed plant has at about 1% to about 30% lipid per dry weight of tissue, or about 5% to about 25% lipid per dry weight of tissue, or about 10% to about 20% lipid per dry weight of tissue.
- 43. The method of any of Statements 34-42, further comprising isolating increased lipid from the tissues or seeds of the plant.
- 44. A method, comprising,
a) providing a plant tissue that comprises the construct of any of Statements 1-12;
b) growing the tissue into a whole plant; and
c) isolating lipid from the tissues or seeds of the plant.
- 45. The method of statement 44, wherein the plant is a reproductively competent plant.
- 46. The method of any of Statements 31-33, 39-44, or 45, wherein the lipid comprises triacylglycerol.
- 47. The method of any of Statements 31-33, 39-45, or 46, wherein the lipid is vegetable lipid.
- 48. The method of any of Statements 31-33, 39-44, or 47, or 47, wherein the lipid is isolated from a tissue is selected from the group of a plant leaf, plant stem, plant stalk, or a combination thereof.
- 49. The method of Statement 48, wherein the tissue is from an oleaginous plant.
- 50. The method of Statement 48 or 49, wherein the tissue is from a plant selected from the group of an oleaginous plant, an Arabidopsis plant, corn, sugar beets, soybean, sugar cane, potato, grasses (e.g., miscanthus, switchgrass, and the like), rice, a beet species, a grape species, an Arabidopsis species, a Brassica species, a Brassica napus plant, an algae species, an oilseed rape species, a sunflower, a soybean species, a flax species, an olive species, an alfalfa species, an oat species, a wheat species, a poplar, an aspen, a willow, or a combination thereof.
- 51. The method of any of Statements 48-50, wherein the tissue is from a plant selected form the group of a rice plant, a Miscanthus plant, oilseed rape plant, sunflower plant, soybean plant, flax plant, olive plant, alfalfa plant, oat plant, a switchgrass plant.
- 52. The method of any of Statements 29-51, further comprising manufacturing a biofuel from a lipid in the plant cells or the plant.
- 53. A biofuel comprising the lipid of any of Statements 26-28.
The specific methods, plants, seeds, and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” or “a nucleic acid” or “a polypeptide” includes a plurality of such plants, nucleic acids or polypeptides (for example, a series of plants, a solution of nucleic acids or polypeptides, or a series of nucleic acid or polypeptide preparations), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
Under no circ*mstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circ*mstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
