Patent Application
20250115925
The invention relates generally to the field of molecular biology. Provided herein are isolated polynucleotides or polypeptides that lead to improved plant oil yields.
Edible plant oil obtained from seeds, pulps, fruits and plumules is primarily made up of triacylglycerols (TAGs, familiar to most people as vegetable oils) that comprises glycerol and fatty acids (FAs) linked by ester bonds. TAG can serve as the energy and carbon source to support seed germination and seedling development. Edible plant oils are essential for human diet and nutrition, and have been widely used in daily cooking, cosmetics and health supplements. In addition, plant oils have broad industrial applications, including serving as resource for biomaterials and fuels. Approximately 100 billion kilograms of plant oil, worth about US$120 billion, was produced in 2011. Global demand for vegetable oils is increasing rapidly. The rising demand of vegetable oils cannot be met by traditional production methods. Hence, there is a need to develop new methods for producing vegetable oils.
Accordingly, it is generally desirable to overcome or ameliorate one or more of the aforementioned difficulties.
Disclosed herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 74 as set forth in SEQ ID NO: 1.
Disclosed herein is a polypeptide encoded by an isolated polynucleotide as defined herein.
Disclosed herein is a construct comprising an isolated polynucleotide as defined herein.
Disclosed herein is a vector comprising a construct as defined herein.
Disclosed herein is a method of preparing a transgenic plant cell, the method comprising transforming a plant cell with a construct as defined herein or a vector as defined herein.
Disclosed herein is a transgenic plant, plant cell or plant seed comprising a polynucleotide as defined herein or a construct as defined herein.
Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:
The present specification teaches an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 (WRI1) polypeptide by at least one amino acid substitution in the wild-type WRI1 amino acid sequence.
The isolated polynucleotide may encode a polypeptide that is distinguished from a wild-type Wrinkled1 (WRI1) polypeptide by at least one amino acid substitution in the wild-type WRI1 amino acid sequence, wherein the at least one amino acid substitution is at a position corresponding to position 74 of the wild-type Wrinkled1 (WRI1) polypeptide.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 74 as set forth in SEQ ID NO: 1.
WRI1 is a transcription factor that can increase the synthesis of proteins involved in oil synthesis. Modification of plants to express increased levels of stabilized WRI1 transcription factors can further increase the oil content of seeds and non-seed tissues (e.g., leaves, stalks and roots) in a variety of transgenic plants. Without being bound by theory, the inventors have engineered the WRI1 protein by one single residue mutation leading to higher affinity for its target DNA as well as oil production. Plants can be generated as described herein to include WRI1 nucleic acids that encode engineered WRI transcription factors. Plants are especially desirable when the WRI1 nucleic acids are operably linked to control sequences capable of WRI1 expression in a multitude of plant tissues, or in selected tissues and during selected parts of the plant life cycle to optimize the synthesis of oil. Such control sequences are typically heterologous to the coding region of the WRI1 nucleic acids.
The term “polynucleotide” or “nucleic acid” is used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.
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. The isolated nucleic acid or the isolated polypeptide can also be a nucleic acid or protein that is modified but has been introduced into a cell where it is or was naturally present. Thus, a modified isolated nucleic acid or an isolated polypeptide expressed from a modified isolated nucleic acid can be present in a cell along with a wild copy of the (unmodified) natural nucleic acid and along with wild type copies of the (natural) polypeptide.
As used with reference to polypeptides, the term “wild-type” refers to any polypeptide having an amino acid sequence present in a polypeptide from a naturally occurring organism, regardless of the source of the molecule; i.e., the term “wild-type” refers to sequence characteristics, regardless of whether the molecule is purified from a natural source; expressed recombinantly, followed by purification; or synthesized.
An “isolated polynucleotide” or “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
An isolated nucleic acid may be operably linked to one or more expression control sequences.
The term “encoding” or “encodes” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e. rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription of a gene and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylation, acetylation, phosphorylation and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.
The term “substitution” as used herein refers to the presence of an amino acid residue at a certain position of the derivative sequence which is different from the amino acid residue which is present or absent at the corresponding position in the reference sequence.
The term “at least one substitution” may refer to one, two, three, four, five, six, seven, eight, nine, ten or more substitutions.
The polypeptide as defined herein may, for example, have one or more “conservative amino acid substitution”. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:
The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
The term “at least 70%” as used herein includes at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
The polypeptide as referred to herein may be distinguished from the WRI1 polypeptide by at least one amino acid substitution in the AP2 region of the wild-type WRI1 amino acid sequence.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 1 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to the AP2 region (positions 55-255) of SEQ ID NO: 1.
Related WRI1 sequences can be isolated from a variety of plant types such as alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rapeseed, rice, rutabaga, sorghum, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and wheat
The wild-type WRI1 polypeptide may, for example, be from Arabidopsis thaliana, Brassica napus, Glycine max, Elaeis guineensis, Zea mays or Camelina sativa.
The wild-type WRI1 polypeptide may be from Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Camelina sativa, Glycine max, Elaeis guineensis, Jatropha curcas or Zea mays.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 7 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 8 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 9 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 10 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 11 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 12 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 13 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 14 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 15 or a fragment thereof.
In one embodiment, the wild-type WRI1 polypeptide has an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 16 or a fragment thereof.
In one embodiment, the at least one amino acid substitution is at a position corresponding to position 74 of the wild-type Wrinkled1 (WRI1) polypeptide. The at least one amino acid substitution may comprise an amino acid substitution at a position corresponding to position 74 as set forth in SEQ ID NO: 1. The amino acid substitution corresponding to position 74 may, for example, be a substitution to a positively charged residue. The positively charged residue may be arginine or lysine.
In one embodiment, the polypeptide is distinguished from a wild-type Wrinkled1 (WRI1) polypeptide by at least one amino acid substitution in the wild-type WRI1 amino acid sequence, wherein the wild-type WRI1 amino acid sequence is an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 1, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or a fragment thereof. The at least one amino acid substitution may comprise an amino acid substitution at a position corresponding to position 74 as set forth in SEQ ID NO: 1.
The polypeptide may have an amino acid sequence having at least 70% (or 80%, 90%, 95% or 99%) sequence identity to any one of SEQ ID NO: 2 or SEQ ID NO: 17-26, or a fragment thereof.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 7, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 71 as set forth in SEQ ID NO: 7.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 73 as set forth in SEQ ID NO: 8.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 75 as set forth in SEQ ID NO: 9.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 92 as set forth in SEQ ID NO: 10.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 11, 12 or 13, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 76 as set forth in SEQ ID NO: 11, 12 or 13.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 14, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 92 as set forth in SEQ ID NO: 14.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 15, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 65 as set forth in SEQ ID NO: 15.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the wild-type Wrinkled1 amino acid sequence has an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 16, and wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to position 63 as set forth in SEQ ID NO: 16.
Disclosed herein is an isolated polynucleotide encoding a polypeptide comprising a positively charged amino acid residue at position 74 corresponding to a wild-type Wrinkled1 (WRI1) polypeptide (such as SEQ ID NO: 1).
Provided herein is an isolated polynucleotide encoding a polypeptide comprising a positively charged amino acid residue at a position corresponding to position 74 as set forth in SEQ ID NO: 1.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 90 as set forth in SEQ ID NO: 1. The amino acid substitution at position 90 may be an amino acid substitution to a glutamic or aspartic acid.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 91 as set forth in SEQ ID NO: 1. The amino acid substitution at position 91 may be an amino acid substitution to glycine.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 92 as set forth in SEQ ID NO: 1. The amino acid substitution at position 92 may be an amino acid substitution to a positively charged amino acid residue such as lysine or arginine.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 174 as set forth in SEQ ID NO: 1. The amino acid substitution at position 174 may be an amino acid substitution to a glutamine.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 175 as set forth in SEQ ID NO: 1. The amino acid substitution at position 175 may be an amino acid substitution to a glutamine.
The at least one amino acid substitution may further comprise an amino acid substitution at a position corresponding to position 176 as set forth in SEQ ID NO: 1. The amino acid substitution at position 176 may be an amino acid substitution to a lysine.
Provided herein is an isolated polynucleotide encoding a polypeptide that is distinguished from a wild-type Wrinkled1 polypeptide by at least one amino acid substitution in the wild-type Wrinkled1 amino acid sequence, wherein the at least one amino acid substitution comprises an amino acid substitution at a position corresponding to positions 74, 90, 91, 92, 174, 175 and 176 as set forth in SEQ ID NO: 1.
The at least one amino acid substitution may comprise amino acid substitutions at positions 74, 90, 91 and 92 as set forth in SEQ ID NO: 1.
The at least one amino acid substitution may comprise amino acid substitutions at positions 74, 174, 175 and 176 as set forth in SEQ ID NO: 1.
Disclosed herein is a polypeptide encoded by an isolated polynucleotide as defined herein.
In order to engineer plants with increased vegetable oil content that, one of skill in the art can introduce nucleic acids encoding the WRI1 proteins described herein into the plants to promote the production of oils.
For example, one of skill in the art can generate genetically-modified plants that contain nucleic acids encoding WRI1 proteins within their somatic and/or germ cells. Such genetic modification 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 WRI1 proteins. Plant cells can be transformed by the expression cassette or expression vector, and whole plants (and their seeds) can be generated from the plant cells that were successfully transformed with the WRI1 nucleic acids. Some procedures for making such genetically modified plants and their seeds are described below.
Disclosed herein is a construct comprising an isolated polynucleotide as defined herein. The term “construct” refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors.
An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions, and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.
The isolated polynucleotide may be operably linked to a control element.
By “control element” or “control sequence” is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
The WRI1 nucleic acids can be operably linked to a promoter, which provides for expression of an mRNA expressed from the WRI1 nucleic acids. The promoter can be a promoter functional in plants and/or seeds, and/or it can be a promoter functional during plant growth and development or in a mature plant. The promoter can be a heterologous promoter. As used herein, “heterologous” when used in reference to a gene or nucleic acid refers to a gene or nucleic acid that has been manipulated in some way. For example, a heterologous promoter is a promoter that contains sequences that are not naturally linked to an associated coding region.
A WRI1 nucleic acid is operably linked to the promoter when it is located downstream from the promoter, thereby forming a key portion of an expression cassette. Any of the nucleic acids encoding WRI1 proteins can have an ATG start codon, either naturally or as an added codon, for example between the promoter and the 5′ end of the WRI1 coding region.
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 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 homologous DNA.
Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, 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. Promoters can also provide for tissue specific or developmental regulation. 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. In some embodiments, the promoter is an inducible promoter and/or a tissue-specific promoter.
Examples of promoters that can be used include, but are not limited to, the CaMV 35S promoter, or others such as CaMV 19S, nos, Adh1, sucrose synthase, α-tubulin, ubiquitin, actin, cab, PEPCase, the CCR (cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.44) promoter sequence isolated from Lollium perenne, (or a perennial ryegrass) and/or those associated with the R gene complex. 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 and the actin promoter from rice. Seed specific promoters, such as the phaseolin promoter from beans, may also be used. Other promoters useful in the practice of the invention are known to those of skill in the art.
Alternatively, novel tissue specific promoter sequences may be employed in the practice of the present invention. cDNA clones from a particular tissue are isolated and those clones which are expressed specifically in that tissue are identified, for example, using Northern blotting. Preferably, 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 localized using techniques well known to those of skill in the art.
For example, the promoter can be an inducible promoter. Such inducible promoters can be activated by agents such as chemicals, hormones, sugars, metabolites, or by the age or developmental stage of the plant. For example, the promoter can be an ethanol-inducible promoter, a sugar-inducible promoter, a senescence-induced promoter or any promoter activated in vegetative tissues of dicots and monocots. One example of a sugar-inducible promoter is a patatin B33 promoter. Such a patatin B33 promoter can, for example, be used in tuber crops such as cassava, potato, rutabaga, sugar beet, and the like.
Disclosed herein is a vector comprising a construct as defined herein.
By the term “vector”, as used herein, is meant any plasmid or virus encoding an exogenous nucleic acid. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like. The vector may be a viral vector which is suitable as a delivery vehicle for delivery of a nucleic acid that encodes a polypeptide of the present invention to the patient, or the vector may be a non-viral vector which is suitable for the same purpose. Examples of viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746). Examples of viral vectors include, but are not limited to, a lentiviral vector, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like. Examples of non-viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
Disclosed herein is a method of preparing a transgenic plant cell, the method comprising transforming a plant cell with a construct as defined herein or a vector as defined herein.
Disclosed herein is a transgenic plant, plant cell or plant seed comprising a polynucleotide as defined herein or a construct as defined herein.
In one embodiment, there is provided a transgenic plant comprising a polynucleotide as defined herein or a construct as defined herein.
In one embodiment, there is provided a transgenic plant cell comprising a polynucleotide as defined herein or a construct as defined herein.
In one embodiment, there is provided a transgenic plant seed comprising a polynucleotide as defined herein or a construct as defined herein.
The term “transgenic” when used in reference to a plant or leaf or vegetative tissue or seed for example a “transgenic plant,” transgenic leaf,” “transgenic vegetative tissue,” “transgenic seed,” or a “transgenic host cell” refers to a plant or leaf or tissue or seed that contains at least one heterologous or foreign gene in one or more of its cells. The term “transgenic plant material” refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells.
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), ornamental or decorative, crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, herb plant, woody plant, flower plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g. microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g. volvox) or a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, et cetera.
The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds, and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
As used herein, the term “plant part” as used herein refers to a plant structure or a plant tissue, for example, pollen, an ovule, a tissue, a pod, a seed, a leaf and a cell. Plant parts may comprise one or more of a tiller, plug, rhizome, sprig, stolen, meristem, crown, and the like. In some instances, the plant part can include vegetative tissues of the plant.
Vegetative tissues or vegetative plant parts do not include plant seeds, and instead include non-seed tissues or parts of a plant. The vegetative tissues can include reproductive tissues of a plant, but not the mature seeds.
The term “seed” refers to a ripened ovule, consisting of the embryo and a casing.
The term “propagation” refers to the process of producing new plants, either by vegetative means involving the rooting or grafting of pieces of a plant, or by sowing seeds. The terms “vegetative propagation” and “asexual reproduction” refer to the ability of plants to reproduce without sexual reproduction, by producing new plants from existing vegetative structures that are clones, i.e., plants that are identical in all attributes to the mother plant and to one another. For example, the division of a clump, rooting of proliferations, or cutting of mature crowns can produce a new plant.
The term “heterologous” when used in reference to a nucleic acid refers to a nucleic acid that has been manipulated in some way. For example, a heterologous nucleic acid includes a nucleic acid from one species introduced into another species. A heterologous nucleic acid also includes a nucleic acid native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous nucleic acids can include cDNA forms of a nucleic acid; the cDNA 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). For example, heterologous nucleic acids can be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are typically joined to nucleic acids comprising regulatory elements such as promoters that are not found naturally associated with the natural gene for the protein encoded by the heterologous gene. Heterologous nucleic acids can also be distinguished from endogenous plant nucleic acids in that the heterologous nucleic acids are in an unnatural chromosomal location, or are associated with portions of the chromosome not found in nature (e.g., the heterologous nucleic acids are expressed in tissues where the gene is not normally expressed).
The WRI1 nucleic acid can be introduced into a recipient cell to create a transformed cell by available methods. The frequency of occurrence of cells taking up exogenous (foreign) DNA can be low, and it is 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, cells from virtually any dicot or monocot species can be stably transformed, and those cells can be regenerated into transgenic plants, for example, through the application of the techniques disclosed herein.
Another aspect of the invention is a plant species with increased vegetative tissue oil content, wherein the plant has an introduced WRI1 nucleic acid. The plant 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. Another aspect of the invention includes transgenic seeds from which transgenic plants can be grown. The plants, cells and seeds can be either monocotyledons or dicotyledons. 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.
Examples of plants, seeds, and/or plant cells that can be modified as described herein to express the WRI1 transcription factors proteins include alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, corn, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and wheat. In some embodiments, the plant is a Brassicaceae or other Solanaceae species.
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; direct DNA transfer to plant cells by PEG precipitation; direct DNA transfer to plant cells by microprojectile bombardment 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. 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. For example, embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been previously described.
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 lack functions for disease induction.
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.
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.
After effecting delivery of one or more WRI1 nucleic acid(s) to recipient cells by any of the methods discussed above (e.g., in an expression vector), the transformed cells can be identified for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the WRI1 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. Alternatively, the introduced (e.g., transgenic) nucleic acids can be detected and/or characterized by use of a nucleic acid probe to detect the presence of an expression cassette and/or expressed RNA. The introduced nucleic acids can also be detected and/or evaluated by sequencing.
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 soilless plant growth mix, and hardened. 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. 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 express the desired trait(s). 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 WRI1 nucleic acid 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 WRI1 nucleic acid, the plant is self-pollinated at least once in order to produce a homozygous backcross converted inbred containing the WRI1 nucleic acid. Progeny of these plants are true breeding.
Alternatively, seed from transformed 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 be evaluated for the presence and/or expression of the WRI1 nucleic acid (or the WRI1 protein product). Transgenic plant and/or seed tissue can be analyzed for WRI1 expression using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a WRI1 protein.
Once a transgenic seed expressing the WRINKLED transcription factor and having an increase in oil in the plant tissue is identified, the seed can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants with an increase in the percent of oil in the plant tissues while still maintaining other desirable functional agronomic traits. Adding the trait of increased oil/decreased carbohydrate production to the plant can be accomplished by back-crossing with this trait and with plants that do not exhibit these traits and 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 an increased percent of oil in the plant. The resulting progeny are then crossed back to the parent that expresses the increased oil/decreased carbohydrate trait. The progeny from this cross will also segregate so that some of the progeny carry the traits and some do not. This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait involving an increase in oil and/or a decrease in carbohydrate in the vegetative tissues of the plant. Such expression of the increased percentage of oil or decreased percentage of carbohydrate in plant tissues can be expressed in a dominant fashion.
Subsequent to back-crossing, the new transgenic plants can be evaluated for an increase in the weight percent of oil (TAG) incorporated into vegetative tissues of the plant. This can be done, for example, by thin layer chromatography (TLC), gas chromatography, gas chromatography-flame ionization detector (GC-FID), electrospray ionization mass spectrometry (ESI-MS), mass spectroscopy, nuclear magnetic resonance (NMR), high pressure liquid chromatography (HPLC), and/or infrared spectral analysis of plant tissue or by other available methods of detecting and quantifying oils in harvested plant tissues. The new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as lodging, kernel hardness, yield, resistance to disease, resistance to insect pests, drought resistance, and/or herbicide resistance.
Plants that can be generated by these methods include but are not limited to oil and/or starch plants (canola, potatoes, cassava, lupins, rape, rapeseed, soybean, sunflower and cottonseed), forage plants (alfalfa, clover and fescue), 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). Examples of plants and/or plant cells that can be modified as described herein include alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, corn, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and wheat.
To confirm the presence of the WRI1 nucleic acid 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. In some embodiments, the amount of oil in plant tissues is quantified. Such a quantified oil content can be compared to a control plant, for example, a control plant of the same species that has not be modified to express the WRI1 transcription factor protein.
Provided herein is a method of generating oil comprising isolating plant tissue, cell, or seed as defined herein and extracting oil from the plant tissue, cell, or seed.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.
Throughout this specification and the statements which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
The coding region of AtWRI158-307 was codon optimized, synthesized and subcloned into pOPT370 MBP vector encoding Hexahistidine-Maltose binding protein (MBP) tag. The mutagenesis was finished by site-directed mutagenesis kit (New England Biolabs). All the insertions and mutagenesis were confirmed by DNA sequencing. For plant expression constructs, the coding sequence (CDS) of WRI1s (native form and variants) was amplified by PCR and cloned into pENTR4 (Thermo Fisher) or synthesized to pTwist ENTR by Twist Bioscience to obtain entry constructs. WRI1s entry constructs were introduced to pEarleyGate binary vectors (pEarleyGate100) by LR Clonase II (Thermo Fisher). To obtain proAtWRI1:AtWRI1 construct, CaMV 35S promoter was replaced by proAtWRI1 (2 kb upstream of the start codon).
The plasmid encoding AtWRI158-307 was transformed into E. coli BL21 (DE3). A single colony was picked and inoculated into 10 mL 2×YT medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl) supplemented with a final concentration of 100 μg/mL ampicillin for overnight growth at 37° C. The bacteria were then inoculated into 1 litter 2×YT medium and grown until OD600 reached ˜0.6, the cells were then cooled down at cold room for one hour before adding the final concentration of 0.2 mM IPTG for protein expression at 16° C. overnight. The bacteria were harvested by centrifugation (Beckman Coulter Avanti-26, JA8.1, 4, 000 rpm) for 10 minutes at 4° C., then resuspended in lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl). Cells were lysed by sonication then crude lysate clarified by centrifugation (Beckman Coulter Avanti-26, JA25.5, 20,000 rpm,) for 1 hour at 4° C. The supernatant was incubated with 5 mL Nickel-NTA beads (Biobasic, Singapore) for 1 hour at 4° C. Then the beads were collected by centrifugation, washed by Buffer-A (25 mM Tris pH 7.5, 150 mM NaCl, 30 mM Imidazole. The protein was eluted by Buffer-B (25 mM Tris pH 7.5, 150 mM NaCl, 200 mM Imidazole). The eluted protein was incubated with Tobacco Etch Virus (TEV) protease to cut off the Hexahistidine-MBP tag for overnight at 4° C. The cut protein was separated by anion ion exchange, collected, concentrated, and loaded to size-exclusion chromatography using Hiload Superdex 200 16/60 pre-equilibrated with buffer (25 mM Tris pH 7.5, 150 mM NaCl). The fractions eluted from size-exclusion chromatography were collected, concentrated to 20 mg/mL and snap frozen, stored at −80° C. for future use. The selenomethionine (Se-Met) substituted WRI1 and the WRI1 mutants were purified similarly.
Analytical gel filtration chromatography was performed using Superdex 200 Increase 10/300 GL (GE Healthcare) in buffer (25 mM Tris pH 7.5, 150 mM NaCl) for the following samples: WRI1, WRI1 and dsDNA in 1:1 ratio and dsDNA. WRI1 and its binding dsDNA were mixed and incubated on ice for 1 hour prior to loading.
ITC measurement was performed using MicroCal PEAQ-ITC (Malvern Instruments) at 20° C. 200 μM WRI1 binding ds-DNA (5′-TACTTCCTCGGTTTCATCGTCCAC (SEQ ID NO: 5)) was injected into the sample cell containing 20 μM WRI1 or its mutants in a total of 19 injections. Injection time was 4 s, with each 2 μL injection spaced across 150 s intervals. A titration of DNA to the buffer was performed using the same procedures. The raw data were processed and analyzed using MicroCal PEAQ-ITC analysis software.
WRI1 (7.5 mg/mL) was mixed with equimolar WRI1 binding ds-DNA BCCP2(−34/−11) (5′-TACTTCCTCGGTTTCATCGTCCAC (SEQ ID NO: 5) and incubated at 4° C. for 1 hour. Crystallization screening was performed with Crystal Screen™ (CS), Index, PEG Rx and PEG/Ion Screen (Hampton Research) using the sitting drop vapor diffusion method at 20° C. on Intelli-Plate 96-3 LVR (Art Robbins Instruments). Crystal appeared in crystal screening A9 condition (0.2 M Ammonium acetate, 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 30% w/v Polyethylene glycol 4,000). The final optimized condition for crystal growth is (0.2 M Ammonium acetate, 0.1 M MES pH 6.5, 28% w/v PEG 4,000). The native dataset of WRI1-DNA crystal was collected in Swiss Light Source (SLS). SAD datasets for Se-Met labeled WRI1-DNA crystals were collected at an inflection wavelength of 0.9795 Å at Australian light source (ALS) and Swiss Light Source (SLS). All the datasets were processed using XDS. The Phenix AutoSol program was used for phasing, and 4 selenium atoms were found in the substructure solution (figure of merit: 0.23). The resultant structure served as a template to solve native WRI1-DNA structures by molecular replacement. The models were built and refined using Phenix and Coot. All the structure related figures were generated by PyMol.
Arabidopsis and N. benthamiana plants were grown in a growth chamber at 23° C. with a photoperiod of 16 h light (100-150 μmol m−2 se−1 illumination)/8 h dark. Arabidopsis wild-type (Columbia ecotype) and wri1-1 were used in this study. Arabidopsis transformation, seed sterilization and germination followed the methods described previously.
Transient Expression in N. benthamiana
Agrobacterium tumefaciens-mediated transient expression in N. benthamiana leaves was conducted as previously described.
Fatty acid analysis of Arabidopsis seeds and N. benthamiana leaves was done as previously described.
The biosynthesis of TAG starts in plastids where the imported pyruvate and glycolytic intermediates are converted to acetyl-coA by pyruvate kinase (PK) and plastidial pyruvate dehydrogenase (PDH), Then, the acetyl-coA is converted into malonyl-coA by acetyl-coA carboxylase (ACCase) in the first committed step of FA synthesis. FAs are assembled on acyl carrier proteins (ACPs) in 7 cycles that add 2 carbons per cycle, following which a fraction of the FAs are further extended and desaturated. Unsaturated and saturated FAs are released from ACPs by acyl-ACP thioesterase A (FATA) and acyl-ACP thioesterase B (FATB), respectively. Free FAs (FFAs) are exported to the ER, where they are sequentially added to glycerol-3-phosphate (G3P) to form TAG. Finally, synthesized TAGs are stored into spherical oil bodies with 0.2 to 0.25 μm in diameter.
Various genes involved in FA de novo biosynthesis (e.g. genes encoding PK and ACCase), are transcriptionally controlled by a ‘master regulator’ WRI1. Arabidopsis WRI1 (AtWRI1) loss-of-function mutant shows significant decrease (˜80%) of seed oil content compared to wild-type (WT). WRI1 encodes an APETALA2 (AP2) transcription factor that and displays high expression level in developing seeds. WRI1 binds to a conserved element denoted the AW(ASML1/WRI1)-box—CnTnG(n)7CG, where n represents any nucleotide. Overexpression of WRI1 in Arabidopsis and other plant species (e.g. maize and soybean) leads to increased seed oil content, which validates its significant role in mediating oil production.
The inventors have engineered the wild-type WRI1 polypeptide by a single amino acid substitution (at position 74) to have higher affinity for DNA binding (by over 10 folds), leading to an increase in oil production by over 60% as compared to wild-type polypeptide (see
To further verify the effect of W74R of AtWRI1 in plant cells, stable transgenic wri1-1 expressing AtWRI1 and AtWRI1W74R (driven by the native promoter of AtWRI1 (proAtWRI1)) were generated. Compared to transgenic wri1-1 expressing AtWRI1, the seed oil content from multiple transgenic lines was significant enhanced in transgenic wri1-1 expressing AtWRI1W74R (
Given the well-known conservation of WRI1's function, the above variants and the combination for the orthologs in other economic crops, including Arabidopsis thaliana (At), Brachypodium distachyon (Bd), Brassica napus (Bn), soybean (Glycine max, Gm), Jatropha curcas (Jc), maize (Zea mays, Zm) and camelina (Camelina sativa, Cs) is shown in
An amino acid substitution at a position corresponding to position 74 as set forth in SEQ ID NO: 1 (i.e. WRI1 from Arabidopsis thaliana (At)) can also be made in the orthologs as shown in bold and underlined below. The amino acid substitution can be an amino acid substitution to a positively charged amino acid.
AP2 region of WRI1 functions as DNA binding domain of WRI1 transcription factor. AP2 region of WRI1 binds to the cis-element, AW-box, in promoters of WRI1 target genes involved in fatty acid biosynthesis. Enhancing binding affinity of AP2 of AtWRI1 (i.e., AtWRI155-255) leads to oil yield increase. The AP2 region of WRI1 is conserved across AtWRI1 and WRI1 orthologs identified in other plant species. Therefore, the invention also includes WRI1 orthologs from other plant species with at least one amino acid substitution. For example, the WRI1 orthologs comprise an AP2 region having amino acid sequence having at least 70% sequence identity to the AP2 region of wild-type WRI1 polypeptide (i.e. AtWRI155-255)
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202110937Q | Sep 2021 | SG | national |
| 10202250129P | Jun 2022 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2022/050683 | 9/22/2022 | WO |
