JP5910702B2 - Gene for improving substance productivity in seed and method for using the same - Google Patents

Description

  Plants are cultivated for the purpose of producing a part of the tissue itself (seed, root, leaf stem, etc.) or for the purpose of producing various substances such as fats and oils. For example, as fats and oils produced by plants, soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn oil, bean flower oil, palm oil, rapeseed oil, etc. have been known since ancient times. Widely used in industrial applications. In addition, oils and fats produced by plants are also used as raw materials for biodiesel fuel and bioplastics, and their applicability is expanding as petroleum alternative energy.

  Under such circumstances, in order to industrially succeed in producing fats and oils using plants, it is necessary to improve productivity per unit cultivated land area. Here, assuming that the number of cultivated individuals per unit cultivated land area is constant, it can be seen that it is necessary to improve the amount of oil production per individual. When recovering fats and oils from seeds collected from plants, the oil yield per individual can be improved by techniques such as improving the seed yield per individual and improving the fat content in the seeds. It is expected.

  Techniques for increasing the oil production of plant seeds can be broadly divided into improvements in cultivation methods and development of varieties that increase oil production. The methods for developing oil-and-fat increased varieties are broadly divided into conventional breeding methods centering on mating technology and molecular breeding methods by genetic recombination. The production of fats and oils by genetic recombination includes A) a technique that modifies the synthesis system of seed triacylglycerol (TAG), the main component of vegetable fats and oils, and B) the morphogenesis and metabolism of plants and the expression of genes related to them. There are known techniques for altering various regulatory genes that control the regulation.

  As the method of A), as a method of increasing the synthesis amount of TAG synthesized using sugar produced by photosynthesis as a raw material, (1) enhancing the synthesis activity from fatty acids that are constituents of TAG or glycerol sugar Method (2) A method of enhancing the reaction of synthesizing TAG from glycerol and fatty acid is conceivable. Regarding these, the following techniques have been reported as techniques using genetic engineering methods. As an example of (1), there is a report (Non-patent Document 1) that the oil content of seeds is improved by 5% by overexpressing Arabidopsis cytosolic acetyl-coenzyme A carboxylase (ACCase) in rapeseed plastids. Can be mentioned. Further, as an example of (2), there is a report on fat and oil production technology by overexpression of DGAT (diacylglycerol acyltransferase) that transfers an acyl group to the sn-3 position of diacylglycerol (Non-patent Document 2). In the method of Non-Patent Document 2, it has been reported that the fat content and seed weight increase as the expression level of DGAT increases, and the number of seeds per individual may increase. The seed oil content of Arabidopsis to which this method was applied increased by 46%, and the amount of oil per individual increased by about 125% at the maximum.

  On the other hand, as the method B), a method of controlling the expression of a transcription factor gene involved in the control of the expression of a biosynthetic enzyme gene can be considered. An example of this is Patent Document 1. In this Patent Document 1, a technique is employed in which a gene that enhances the oil content of seeds is selected after a recombinant plant in which transcription factors are overexpressed or knocked out comprehensively is produced. According to Patent Document 1, it is described that the oil content of seeds increased by 23% due to overexpression of the ERF subfamily B-4 transcription factor gene. However, Patent Document 1 does not describe the increase or decrease in the fat content per individual. Non-Patent Document 3 describes that the oil content of seeds is improved by overexpressing WRINKLED1, which is a transcription factor having an AP2 / EREB domain.

  On the other hand, when saccharifying a hydrocarbon component such as cellulose contained in a plant body and then producing alcohol by fermentation, the fat component contained in the plant becomes an impurity and may cause a decrease in saccharification efficiency in the saccharification step. It is done. Therefore, if the fat and oil content can be reduced, the saccharification efficiency in the saccharification step can be improved, and as a result, improvement in alcohol productivity can be expected. For example, Non-Patent Document 3 discloses that a WRI1 / ASML1 (AP2 family transcription factor, AGI-code: AT3g54320) -deficient strain has wrinkled seeds and a reduced fat content. Patent Document 2 discloses that the oil content of seeds is reduced by 13% by overexpressing AT3g23250 (MYB15), the oil content of seeds is reduced by 12% by overexpressing AT1g04550 (IAA12), and AT1g66390 ( Overexpression of MYB90) is disclosed to reduce the oil content of seeds by 16%.

  However, despite the development of the above-described molecular breeding methods aimed at improving various traits, a technology that achieves an increase or decrease in fat productivity has not reached the practical range.

  The reason for this is thought to be that a truly excellent gene has not yet been discovered, and that a new recombinant variety that is effective at the test stage cannot exhibit the expected effect in various natural environments at the practical stage. In addition, the quantitative traits such as the productivity of the target substance involve many genes in various steps from the control system to the metabolic system, and discover and develop truly excellent useful genes that improve the quantitative traits. It was difficult. In order to solve these problems, it is a challenge to find a new gene having a dramatically high effect, and to develop a gene that exhibits an effect under practical environmental conditions even if the effect level is the same.

WO01 / 36597 WO01 / 35727

Plant Physiology (1997) Vol. 11, pp. 75-81 Plant Physiology (2001), Vol. 126, pp. 861-874 Plant J. (2004) 40, 575-585

  Then, in view of the above-mentioned actual condition, it aims at searching the gene which has a novel function which can increase or decrease substance productivity, and providing the technique which can improve these characteristics in a plant body.

  As a result of intensive studies by the present inventors to achieve the above-mentioned object, a specific transcription factor and a functional peptide that converts an arbitrary transcription factor into a transcription repressor (hereinafter sometimes referred to as a repressor domain) The inventors have found that various quantitative traits can be improved by expressing a chimeric protein fused with and, in particular, the substance productivity can be increased or decreased, and the present invention has been completed.

The plant according to the present invention comprises a chimeric protein obtained by fusing a transcription factor comprising any of the following proteins (a) to (c) with a functional peptide that converts an arbitrary transcription factor into a transcriptional repressor. It has been expressed.
(A) a protein comprising an amino acid sequence represented by any even number among SEQ ID NOs: 1 to 158 (b) one or more amino acids in the amino acid sequence represented by any even number among SEQ ID NOs: 1 to 158 A protein having a deleted, substituted, added or inserted amino acid sequence and having a transcription promoting activity (c) consisting of a base sequence complementary to the base sequence indicated by the odd number in any one of SEQ ID NOs: 1 to 158 A protein encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide and having transcription-promoting activity In the plant according to the present invention, transcription of a predetermined transcription factor by fusing a functional peptide It is preferable that the activity, particularly the transcription promoting activity is suppressed. Here, examples of the functional peptide include the following formulas (1) to (8).

(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)

(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
(5) α1-Leu-β1-Leu-γ1-Leu
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(In the formulas (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn. , Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys or Asp, and γ2 represents Gln , Asn, Thr, Ser, His, Lys or Asp.)

  Further, in the plant according to the present invention, the substance productivity per individual, in particular, the productivity of fats and oils contained in seeds is significantly improved or decreased. A seed can be mentioned as a specific tissue. Here, “significant” means that the substance productivity is increased or decreased with a statistically significant difference when compared with the substance productivity in a plant that does not express the chimeric protein.

  On the other hand, according to the present invention, it is possible to provide the above-described chimeric protein, a gene encoding the chimeric protein, an expression vector containing the gene, and a transformant containing the gene.

  The plant according to the present invention has an improved or reduced substance productivity per individual, in particular, the amount of fats and oils contained in seeds. Therefore, by using the plant according to the present invention, it is possible to improve the productivity when the oil derived from the plant is intended, or to reduce the fat as impurities, for example, production of bioalcohol etc. using the plant Excellent efficiency can be achieved.

Hereinafter, the present invention will be described in detail.
The plant according to the present invention expresses a chimeric protein in which a predetermined transcription factor is fused with a functional peptide that converts an arbitrary transcription factor into a transcription repressor, and is compared with a wild-type plant. Thus, the substance productivity per individual, in particular, the productivity of fats and oils contained in seeds is significantly improved or decreased. That is, the plant according to the present invention is a plant in which a transcription factor is expressed as a chimeric protein with the functional peptide so as to significantly improve or decrease the substance productivity of the desired plant. It is.

  In particular, in the plant according to the present invention, it is preferable that the transcription promoting activity in the transcription factor is suppressed by fusing with the functional peptide. In other words, in the plant according to the present invention, as a result of expressing a chimeric protein in which the functional peptide is fused to a transcription factor, the transcription repressing effect due to the functional peptide appears as a dominant trait. It is preferable to have.

  Here, per-individual substance productivity means the content per unit volume about the various substances which a plant produces | generates. The substance is not particularly limited, and may be a substance that is originally produced by a plant, or a substance that is not originally produced by a plant but can be produced by a genetic manipulation technique or the like. Also good.

  In particular, if the content of the target product per tissue is high, the refining cost and the transportation cost can be reduced, which is industrially useful. In particular, the target product may be lignocellulose that occupies most of the weight of the plant, or may be vegetable oil that is industrially used as seed oil. The vegetable oil may be a simple lipid that is an ester of a fatty acid and an alcohol, may be a complex lipid containing phosphorus, sugar, nitrogen, or the like, or may be a fatty acid itself. The simple lipid alcohol may be a higher alcohol having a high molecular weight or a polyhydric alcohol such as glycerol (glycerin). The fatty acid of the simple lipid may be a saturated fatty acid, an unsaturated fatty acid, or a special fatty acid containing a hydroxyl group or an epoxy group. The simple lipid that is an ester of glycerol and a fatty acid may be monoacylglycerol, diacylglycerol, or triacylglycerol.

  On the other hand, depending on the use of the plant body, a predetermined substance contained in the plant body becomes an impurity. Therefore, if the productivity of a predetermined substance is lowered, the impurity content is reduced, and the industrial utility is high. For example, when lignocellulose contained in a plant body is saccharified, the fat and oil component contained in the plant body may adversely affect saccharification efficiency as an impurity. Therefore, if the productivity of fats and oils becomes low, the efficiency of a saccharification process can be improved among manufacturing processes, such as bioalcohol using a plant body.

  In the following description, fats and oils will be exemplified and described as substances that improve or reduce productivity, but the technical scope of the present invention is not limited thereto. The present invention is similarly applied to substances other than fats and oils as substances produced by plants.

  Here, the plant body is not particularly limited, and any plant can be targeted. In particular, it is preferable to target plants conventionally used for the production of fats and oils. Examples of the target plant include soybean, sesame, olive oil, eggplant, rice, cotton, sunflower, corn, sugarcane, jatropha, palm palm, tobacco, beni flower and rapeseed. Moreover, Arabidopsis thaliana, which is widely used as a model organism in gene analysis of plants and has established a method for gene expression analysis, can also be used as a target plant.

  In addition, transcriptional repression that the transcription factor chimeric protein has as an activity is to recognize the cis sequence recognized by the transcription factor and cis sequences in other transcription factors similar to the cis sequence, and actively promote downstream gene expression. It can also be called a transcriptional repressing factor. The method for suppressing transcription that the chimeric protein of transcription factor has as an activity is not particularly limited, but the method of constructing a chimeric protein (fusion protein) to which a repressor domain sequence or SRDX sequence is added is most preferable.

  In this technique, the repressor domain sequence is an amino acid sequence that constitutes a peptide that converts an arbitrary transcription factor into a transcription repressor, and various sequences found by the present inventors. Regarding the method using the repressor domain sequence, for example, JP 2001-269177 A, JP 2001-269178 A, JP 2001-292767 A, JP 2001-292777 A, JP 2001-269176 A, JP 2001-269179, International Publication No. WO03 / 055903, Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. and Ohme-Takagi, M., The Plant Cell, Vol.13, 1959 -1968, August, 2001 and Hiratsu, K., Ohta, M., Matsui, K., Ohme-Takagi, M., FEBS Letters 514 (2002) 351-354. The repressor domain sequence is excised from Class II ERF (Ethylene Responsive Element Binding Factor) protein and plant zinc finger protein (Zinc Finger Protein, such as Arabidopsis SUPERMAN protein), and has a very simple structure. .

  As shown in Tables 1 and 2, examples of transcription factors expressed as chimeric proteins include transcription factors specified by the AGI code in Arabidopsis thaliana. In addition, when the transcription factor shown in Table 1 is expressed in a plant body as a chimeric protein with a repressor domain, the fat content in seeds is significantly improved. On the other hand, when the transcription factor shown in Table 2 is expressed in a plant as a chimeric protein with a repressor domain, it significantly reduces the oil content in seeds.

  In addition, the transcription factor to be a target of the chimeric protein is not limited to the amino acid sequences shown in Tables 1 and 2 (even numbers of SEQ ID NOs: 1 to 158). The amino acid sequence may include a deleted, substituted, added or inserted amino acid sequence, and may have transcription promoting activity. Here, as the plurality of amino acids, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, particularly preferably 1 to 3 are used. means. In addition, amino acid deletion, substitution, or addition can be performed by modifying the base sequence encoding the transcription factor by a technique known in the art. Mutation can be introduced into a base sequence by a known method such as Kunkel method or Gapped duplex method or a method according thereto, for example, a mutation introduction kit (for example, Mutant-) using site-directed mutagenesis. Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA Bio) or the like, or using LA PCR in vitro Mutagenesis series kits (trade name, manufactured by TAKARA Bio). In addition, as a method for introducing mutations, EMS (ethyl methanesulfonic acid), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N'-nitro-N nitrosoguanidine, and other carcinogenic compounds are representative. A method using a chemical mutagen such as that described above may be used, or a method using radiation treatment or ultraviolet treatment represented by X-rays, alpha rays, beta rays, gamma rays and ion beams may be used.

  Furthermore, the transcription factor to be a target of the chimeric protein is not limited to the transcription factor in Arabidopsis thaliana shown in Tables 1 and 2, but a transcription factor having the same function in plants other than Arabidopsis thaliana (for example, the above-mentioned plants) (hereinafter referred to as homologous transcription). (Referred to as factors). These homologous transcription factors can be searched from the plant genome information to be searched based on the amino acid sequences shown in Tables 1 and 2 or the base sequence of each gene if the plant genome information is known. At this time, the homologous transcription factor has a homology of, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with respect to the amino acid sequences shown in Tables 1 and 2. The amino acid sequence is searched. Here, the homology value means a value obtained by default setting using a computer program in which the blast algorithm is implemented and a database storing gene sequence information.

  If plant genome information is not clear, extract the genome from the target plant or construct a cDNA library of the target plant and encode the transcription factors shown in Tables 1 and 2 A homologous gene can be identified by isolating a genomic region or cDNA that hybridizes under stringent conditions to at least a part of the gene. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

  The plant according to the present invention exhibits the characteristic that the production amount of oil and fat in seeds significantly fluctuates (improves or decreases) by expressing the chimeric protein of the transcription factor and the functional peptide as described above. In particular, by using a chimeric protein, the target transcription factor is expressed in a state in which the transcription promoting activity is suppressed, and the cis sequence that is homologous to the cis sequence recognized by the target transcription factor is recognized. Oil production in the seeds significantly fluctuates (improves or decreases) when expressed as transcriptional repression activity, or by changing the affinity specificity of other transcription factors, nucleic acids, lipids and carbohydrates. It shows the characteristics. At this time, in the above plant body, an endogenous transcription factor may be modified to produce the chimeric protein, but a gene encoding the chimeric protein may be introduced to express the gene.

  As an example, a gene encoding a chimeric protein (fusion protein) obtained by fusing a transcription factor as described above and a functional peptide that converts an arbitrary transcription factor into a transcription repressor is introduced into the target plant, A technique for expressing a chimeric protein (fusion protein) in a plant is preferred.

  The “transcription factor with suppressed transcription promoting activity” described in the present specification is not particularly limited, and a transcription factor having a significantly reduced transcription promoting activity inherent in the transcription factor. It means that. In addition, “functional peptide that converts an arbitrary transcription factor into a transcriptional repressor” refers to a transcriptional promoter inherent in the transcription factor when fused to an arbitrary transcription factor to form a chimeric protein. It means that the peptide has a function of becoming a transcription factor with significantly reduced activity (sometimes referred to as a transcription repressor conversion peptide). Such a “functional peptide that converts an arbitrary transcription factor into a transcriptional repressor” is not particularly limited, but is preferably a peptide consisting of an amino acid sequence known as a repressor domain sequence or SRDX sequence. About this transcription repression conversion peptide, it describes in full detail in Unexamined-Japanese-Patent No. 2005-204657, All can be used for the thing indicated by the said gazette.

Examples of the transcription repressing conversion peptide include amino acid sequences represented by any of the following formulas (1) to (8).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)

(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
(5) α1-Leu-β1-Leu-γ1-Leu
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(In the formulas (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn. , Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys or Asp, and γ2 represents Gln , Asn, Thr, Ser, His, Lys or Asp.)

Transcriptional Repression Conversion Peptide of Formula (1) In the transcriptional repression conversion peptide of the above formula (1), the number of amino acid residues represented by X1 may be in the range of 0 to 10. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X1 is not specifically limited, What kind of thing may be sufficient. The amino acid residue represented by X1 is preferably as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (1). Specifically, the number of amino acid residues represented by X1 is preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (1), the number of amino acid residues represented by X3 may be at least 6. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X3 is not specifically limited, What kind of thing may be sufficient.

Transcriptional Repression Converting Peptide of Formula (2) In the transcriptional repression converting peptide of the above formula (2), the number of amino acid residues represented by Y1 is 0, similar to X1 of the transcriptional repression converting peptide of the above formula (1). It may be in the range of -10. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Y1 is not specifically limited, What kind of thing may be sufficient. Specifically, the number of amino acid residues represented by Y1 is preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (2), the number of amino acid residues represented by Y3 may be at least 6 as in the case of X3 of the transcription repressing converting peptide of the above formula (1). . Moreover, the specific kind of amino acid which comprises the amino acid residue represented by Y3 is not specifically limited, What kind of thing may be sufficient.

Transcriptional Repression Conversion Peptide of Formula (3) In the transcriptional repression conversion peptide of the above formula (3), the amino acid residue represented by Z1 contains Leu within a range of 1 to 3. The case of 1 amino acid is Leu, the case of 2 amino acids is Asp-Leu, and the case of 3 amino acids is Leu-Asp-Leu.

  On the other hand, in the transcriptional repression converting peptide of the above formula (3), the number of amino acid residues represented by Z3 may be in the range of 0-10. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Z3 is not specifically limited, What kind of thing may be sufficient. Specifically, the number of amino acid residues represented by Z3 is more preferably 5 or less. Specific examples of the amino acid residue represented by Z3 include Gly, Gly-Phe-Phe, Gly-Phe-Ala, Gly-Tyr-Tyr, Ala-Ala-Ala and the like. It is not limited.

  In addition, the number of amino acid residues in the entire transcriptional repressor conversion peptide represented by the formula (3) is not particularly limited, but from the viewpoint of ease of synthesis, it may be 20 amino acids or less. preferable.

Transcriptional Repression Conversion Peptide of Formula (4) The transcriptional repression conversion peptide of the above formula (4) is a hexamer (6mer) composed of 6 amino acid residues. The amino acid sequence in the case where the amino acid residue represented by Z4 is Glu in the transcriptional repression converting peptide of the above formula (4) corresponds to the 196th to 201st amino acid sequence of Arabidopsis SUPERMAN protein (SUP protein). .

  The various transcription repressor converting peptides described above can be modified with the transcription factors described above to form chimeric proteins (fusion proteins). Specifically, a transcription factor can be modified into a transcriptional repression factor or a negative transcription coupling factor by fusing with the transcription factor described above to form a chimeric protein (fusion protein). Furthermore, a transcriptional repressing factor that is not dominant can be used as a dominant type transcriptional repressing factor.

  Moreover, a chimeric protein (fusion protein) can be produced by obtaining a fusion gene with a gene encoding a transcription factor using the polynucleotide encoding the transcription repressor conversion peptide. Specifically, a fusion gene is constructed by linking a polynucleotide encoding the transcription repressing conversion peptide (referred to as a transcription repressing conversion polynucleotide) and a gene encoding the transcription factor, and introducing the gene into a plant cell. . Thereby, a chimeric protein (fusion protein) can be produced. The specific base sequence of the transcriptional repression conversion polynucleotide is not particularly limited, and may include a base sequence corresponding to the amino acid sequence of the transcriptional repression conversion peptide based on the genetic code. In addition, if necessary, the transcriptional repression conversion polynucleotide may include a base sequence serving as a linking site for linking with a transcription factor gene. Furthermore, when the amino acid reading frame of the transcription repressor conversion polynucleotide does not match the reading frame of the transcription factor gene, an additional base sequence for matching them may be included. Furthermore, a polypeptide having a linker function for connecting between a transcription factor and a transcription repressor conversion peptide, a polypeptide for epitope-labeling a chimeric protein (fusion protein) such as His, Myc, Flag, etc. Various additional polypeptides may be included. Furthermore, the chimeric protein (fusion protein) may contain a structure other than the polypeptide, such as a sugar chain or an isoprenoid group, as necessary.

  The method for producing a plant body is not particularly limited as long as it includes a process for producing a chimeric protein of the above-described transcription factor and transcription repressor conversion peptide in the plant body. It can mention as a manufacturing method method including processes, such as a conversion process and a selection process. Hereinafter, each step will be specifically described.

Expression Vector Construction Step The expression vector construction step is not particularly limited as long as it is a step for constructing a recombinant expression vector containing the above-described gene encoding a transcription factor, a transcriptional repression conversion polynucleotide, and a promoter. Various vectors known in the art can be used as a base vector for the recombinant expression vector. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI vectors. In particular, when the method for introducing a vector into a plant is a method using Agrobacterium, it is preferable to use a pBI binary vector. Specific examples of the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.

  The promoter is not particularly limited as long as it is a promoter capable of expressing a gene in a plant body, and a known promoter can be suitably used. Examples of such promoters include cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase Oxidase small subunit gene promoter, napin gene promoter, oleosin gene promoter and the like. Among these, cauliflower mosaic virus 35S promoter, actin gene promoter, or ubiquitin gene promoter can be more preferably used. When each of the above promoters is used, any gene can be strongly expressed when introduced into a plant cell. The promoter may be linked so that it can express a fusion gene in which a gene encoding a transcription factor or transcription coupling factor and a transcriptional repression-converting polynucleotide are linked, and introduced into the vector. The specific structure is not particularly limited.

  The recombinant expression vector may further contain other DNA segments in addition to the promoter and the fusion gene. The other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency. The recombinant expression vector may further have a T-DNA region. The T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the recombinant expression vector into a plant body.

  The transcription terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthase gene, the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used. Of these, the Nos terminator can be more preferably used. In the above recombinant vector, by placing the transcription terminator at an appropriate position, after being introduced into the plant cell, an unnecessarily long transcript is synthesized, or a strong promoter reduces the copy number of the plasmid. Occurrence of such a phenomenon can be prevented.

  As a transformant selection marker, for example, a drug resistance gene can be used. Specific examples of such drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body growing in the medium containing the antibiotic.

  Examples of the base sequence for increasing the translation efficiency include an omega sequence derived from tobacco mosaic virus. By placing this omega sequence in the untranslated region (5′UTR) of the promoter, the translation efficiency of the fusion gene can be increased. As described above, the recombinant expression vector can contain various DNA segments depending on the purpose.

  The method for constructing the recombinant expression vector is not particularly limited, and the promoter, the gene encoding the transcription factor, the transcription repressor conversion polynucleotide, and, if necessary, the above-described vector as a base vector appropriately selected. Other DNA segments may be introduced in a predetermined order. For example, a fusion gene is constructed by linking a gene encoding a transcription factor and a transcription repressor conversion polynucleotide, and then the fusion gene and a promoter (such as a transcription terminator, if necessary) are linked to an expression cassette. May be constructed and introduced into a vector.

  In the construction of the chimeric gene (fusion gene) and the expression cassette, for example, the cleavage site of each DNA segment is set as a complementary protruding end and the order of the DNA segment is defined by reacting with a ligation enzyme. Is possible. When the terminator is included in the expression cassette, the promoter, the chimeric gene, and the terminator may be in order from the upstream. Further, the types of reagents for constructing the recombinant expression vector, that is, the types of restriction enzymes and ligation enzymes are not particularly limited, and commercially available ones may be appropriately selected and used.

  Moreover, the propagation method (production method) of the recombinant expression vector is not particularly limited, and a conventionally known method can be used. In general, Escherichia coli may be used as a host and propagated in the E. coli. At this time, a preferred E. coli type may be selected according to the type of vector.

Transformation step The transformation step performed in the present invention is a step of introducing a plant cell using the above-described recombinant expression vector so as to express the above-described fusion gene. A method (transformation method) for introducing a recombinant expression vector into a plant cell is not particularly limited, and any conventionally known method suitable for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of directly introducing into plant cells can be used. Examples of methods using Agrobacterium include Bechtold, E., Ellis, J. and Pelletier, G. (1993) In Planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis plants. CR Acad. Sci. Paris Sci. Vie, 316, 1194-1199. Or Zyprian E, Kado Cl, Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.Plant Molecular Biology, 1990, 15 (2), The method described in 245-256 can be used.

  Examples of methods for directly introducing a recombinant expression vector and DNA containing the gene of interest into plant cells include microinjection, electroporation (electroporation), polyethylene glycol, particle gun, and protoplast fusion. Method, calcium phosphate method and the like can be used.

  In addition, if the method of introducing DNA directly into plant cells is adopted, a transcription unit necessary for expression of the target gene, such as a promoter or transcription terminator, and DNA containing the target gene are sufficient, and the vector function Is not mandatory. Furthermore, even if it is DNA which contains only the protein coding region of the gene of interest which does not have a transcription unit, it can be integrated into the transcription unit of the host to express the gene of interest.

  Examples of plant cells into which the above recombinant expression vector and the DNA containing the gene of interest and the DNA containing the gene DNA of interest and not containing the expression vector are introduced include, for example, plant organs such as flowers, leaves, and roots Cell, callus, suspension culture cell and the like of each tissue. Here, in the method for producing a plant according to the present invention, the recombinant expression vector may be appropriately constructed according to the type of plant to be produced. An expression vector may be constructed in advance and introduced into plant cells. That is, the method for producing a plant according to the present invention may or may not include a transformation DNA construction step using the above-described recombinant expression vector.

Other Steps and Other Methods In the method for producing a plant according to the present invention, it is sufficient if the transformation step is included, and further, a step for constructing a transformation DNA using the recombinant expression vector is included. However, other steps may be included. Specifically, the selection process etc. which select an appropriate transformant from the plant body after transformation can be mentioned.

  The selection method is not particularly limited. For example, selection may be performed based on drug resistance such as hygromycin resistance, and after growing the transformant, the seed of the plant is collected, and the fat and oil in the seed is collected. You may select based on content. For example, as an example of selection based on the fat and oil content, a method of collecting plant seeds, measuring the fat and oil content in the seeds according to a conventional method, and comparing with the fat and oil content in the seeds of non-transformed plant bodies. (See the examples below).

  In the method for producing a plant according to the present invention, since the fusion gene is introduced into the plant, it is possible to obtain offspring from which the oil content is significantly improved by sexual reproduction or asexual reproduction. Become. It is also possible to obtain propagation materials such as plant cells, seeds, fruits, strains, calluses, tubers, cut ears, lumps, etc. from the plants and their progeny, and mass-produce the plants based on these. . Therefore, the method for producing a plant according to the present invention may include a breeding process (mass production process) for breeding the selected plant.

  The plant body in the present invention includes at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed. That is, in this invention, if it is a state which can be made to grow finally to a plant individual, all will be considered as a plant body. The plant cells include various forms of plant cells. Such plant cells include, for example, suspension culture cells, protoplasts, leaf sections and the like. Plants can be obtained by growing and differentiating these plant cells. In addition, the reproduction | regeneration of the plant body from a plant cell can be performed using a conventionally well-known method according to the kind of plant cell. Therefore, the method for producing a plant according to the present invention may include a regeneration step for regenerating the plant from plant cells or the like.

  Moreover, the production method of the plant body which concerns on this invention is not limited to the method of transforming with a recombinant expression vector, You may use another method. Specifically, for example, the chimeric protein (fusion protein) itself may be administered to a plant body. In this case, the chimeric protein (fusion protein) may be administered to the young plant so that the fat content can be improved at the site of the plant finally used. Also, the administration method of the chimeric protein (fusion protein) is not particularly limited, and various known methods may be used.

  As described above, according to the present invention, by expressing a chimeric protein of a predetermined transcription factor and the above functional peptide, substance productivity varies (improves or decreases) compared to a wild-type plant body. ) Plant bodies can be provided. When the chimeric protein is expressed in a plant body, the transcription promoting activity of the target transcription factor may be suppressed, or the transcriptional inhibitory effect on the homologous sequence of the cis sequence recognized by the target transcription factor may be exhibited. is there. Furthermore, the chimeric protein may act to change the affinity specificity for other factors, DNA, RNA, lipids or carbohydrates that have affinity for the transcription factor or transcription coupling factor of interest. In some cases, it may act to improve the affinity for a substance having no affinity for the transcription factor of interest. In the plant according to the present invention, a transcription factor that is a target of the chimeric protein, a transcription factor that recognizes a cis sequence that is homologous to the cis sequence recognized by the transcription factor, and a homology with the transcription factor that is the target of the chimeric protein Although other transcription factors and other factors having affinity for the target transcription factor of the chimeric protein are also expressed in the plant body, the target is controlled to be dominant negative due to the effect of the chimeric protein described above. Gene expression can be suppressed. Thereby, in the plant body according to the present invention, the expression level of the gene group related to the growth of the plant plant and the expression level of the gene group related to the fat production and / or the gene group related to the degradation of the produced fat. As a result, it is considered that the fat content changes significantly.

  Here, the fat and oil content varies significantly, but there is no change in the seed mass per grain compared to the wild type, but the seed mass per grain compared to the wild type when the fat content is improved. When it becomes significantly large or small and the amount of fats and oils is improved, it means either when the fats and oils content in seeds is improved or decreased as compared with the wild type. In either case, the amount of oil and fat produced by a single plant varies.

  More specifically, when the chimeric protein of the transcription factor shown in Table 1 is expressed, the plant body has an improved fat content. Among the plants according to the present invention, those having an increased fat content can be used in a plant-derived oily production method. For example, fats and oils can be produced by growing a plant according to the present invention, collecting seeds, and collecting oil and fat components from the collected seeds. In particular, the method for producing fats and oils using the plant according to the present invention can be said to be a method with excellent productivity because the fat content in a single plant is high. In other words, assuming that the number of cultivated individuals per unit cultivated land area is constant, the amount of fats and oils produced from per unit cultivated land area is greatly improved by using the plant according to the present invention. Therefore, the manufacturing cost required for oil production can be significantly reduced by using the plant according to the present invention.

  Furthermore, it can be said that the method for producing fats and oils using the plant according to the present invention is a method that is excellent in productivity because the fat content in seeds per unit weight is high. In the method for producing fats and oils using the plant according to the present invention, the fats and oils to be produced are not particularly limited. For example, soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn Examples thereof include oils derived from plants such as oil, benflower oil and rapeseed oil. Moreover, the manufactured fats and oils can be widely used for household use and industrial use, and can also be used as a raw material for biodiesel fuel. That is, by using the plant body according to the present invention, the above-described fats and oils for home use or industrial use, biodiesel fuel, and the like can be produced at low cost.

  On the other hand, when the chimeric protein of the transcription factor shown in Table 2 is expressed, the plant body has a reduced fat content. Among the plants according to the present invention, those having a reduced fat content can be used in a method for producing a bioalcohol using lignocellulose contained in a plant. That is, since the fat component which becomes an impurity in the process of saccharifying lignocellulose is low, a bioalcohol having excellent saccharification efficiency and low impurity content can be produced.

As described above for At5g22380, the transcription factor shown in Table 1 significantly improves the oil content in seeds when expressed in a plant as a chimeric protein with a repressor domain. The transcription factor shown in Table 2 When expressed in a plant as a chimeric protein with a repressor domain, the oil content in seeds is significantly reduced. Therefore, when the transcription factor shown in Table 1 is introduced into a plant body in an original form having no repressor domain, there is a high probability of significantly reducing the fat content in seeds. Moreover, when it introduce | transduces into a plant body in the original form which does not have the repressor domain shown in Table 2, the probability that the fats and oils content in a seed will improve significantly is high. Here, the technology described in the columns of “expression vector construction step”, “transformation step” and “other steps and other methods” described above may be applied to each transcription factor.

  In particular, among the transcription factors shown in Table 2, At5g22380 is significantly reduced in oil content in seeds when expressed in a plant as a chimeric protein with a repressor domain, as demonstrated in Examples described later. When expressed in plants as an original transcription factor that does not have a repressor domain, it exhibits characteristic properties such as significantly improving the oil content in seeds. That is, by expressing the Arabidopsis transcription factor At5g22380 in a plant body, the productivity of fats and oils contained in seeds can be improved.

  In order to express the transcription factor At5g22380 in a plant body, the techniques described in the above-mentioned sections of “expression vector construction step”, “transformation step” and “other steps and other methods” may be applied. Moreover, the transcription factor to be expressed in order to improve the oil content in seeds is not limited to the Arabidopsis derived transcription factor At5g22380, and may be a homologous transcription factor defined as described above. The homologous transcription factor can be searched from the plant genome information to be searched based on the amino acid sequence of At5g22380 shown in SEQ ID NO: 156 or the base sequence of the At5g22380 gene shown in SEQ ID NO: 155. At this time, the homologous transcription factor has a homology of, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with respect to the amino acid sequence shown in SEQ ID NO: 156. Searched as amino acid sequence. Here, the homology value means a value obtained by default setting using a computer program in which the blast algorithm is implemented and a database storing gene sequence information.

  If the plant genome information is not clear, the genome is extracted from the target plant or the cDNA library of the target plant is constructed, and the gene consisting of the base sequence shown in SEQ ID NO: 155 A homologous gene can be identified by isolating a genomic region or cDNA that hybridizes under stringent conditions to at least a part. Here, stringent conditions are synonymous with the various conditions described above.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to these Examples.
[Example 1]
Amplification of transcription factor gene From the cDNA library of Arabidopsis thaliana, transcription factors At1g01010, At1g01250, At1g09540, At1g10200, At1g12260, At1g12890, At1g12980, At1g14510, At1g17520, At1g18 490At1g183570 , At1g22985, At1g24260, At1g24590, At1g25470, At1g25580, At1g27360, At1g27370, At1g27730, At1g28160, At1g28520, At1g30210, At1g30810, At1g32770, At1g33760, At1g34190, At1g36060, At1g43160, At1g43640, At1g44830, At1g49120, At1g52150, At1g52880, At1g52890, At1g53230, At1g56010 , At1g56650, At1g60240, At1g61110, At1g62700, At1g63040, At1g63910, At1g64380, At1g67260, At1g67780, At1g68800, At1g69120, At1g69490, At1g71030, At1g71450, At1g71692, At1g72360, At1g72570, At1g74840, At1g74930, At1g76580, At1g77200, At1g77450, At1g78080, At1g79180, At1g80580 , At2g02070, At2g02450, At2g17040, At2g18060, At2g22200, At2g23290, At2g23760, At2g26060, At2g28550, At2g30420, At2g30470, At2g31230, At2g33480, At2g33710, At2g35700, At2g40220, At2g41710, At2g42400, At2g42830, At2g44840, At2g44940, At2g45650, At2g45680, At2g46310, At2g46590, At2g47520, At3g01530, At3g02150, At3g02310, At3g04060, At3g04070, At3g04420, At3g05760, At3g09600, At3g10490, At3g11280, At3g14230, At3g15500, At3g15510, At3g18550, At3g20770, At3g23220, At3g23230, At3g23240, At3g25890, At3g27920, At3g28910, At3g29035, At3g45150, At3g49850, At3g54320, At3g61910, At4g01060, At4g01550, At4g18390, At4g18450, At4g23750, At4g26150, At4g27950, At4g28140, At4g28530, At4g31060, At4g31270, At4g32800, At4g34410, At4g35580, At4g36160, At4g37750, At4g38620, At4g39780, At5g02460, At5g06100, At5g07310, At5g07580, At5g07680, At5g07690, At5g08070, At5g08790, At5g09330, At5g13180, At5g13330, At5g13910, At5g14000, At5g18270, At5g18560, At5g18830, At5g22290, At5g22380, At5g23260, At5g24520, At5g 24590, At5g25190, At5g25390, At5g25810, At5g35550, At5g39610, At5g40330, At5g41030, At5g43270, At5g47220, At5g47230, At5g47390, At5g51190, At5g52020, At5g630, At5g630, At5g630, At5g6900 A DNA fragment in the coding region excluding the start codon of At5g67580 was amplified by PCR. For At5g22380, the DNA fragment in the region containing the stop codon was amplified in the same manner. PCR conditions were 94 ° C for 1 minute, 47 ° C for 2 minutes, and extension reaction at 74 ° C for 1 minute for 25 cycles. Next, PCR products were separated and collected by agarose gel electrophoresis.

Preparation of Improved Transcription Factor To add a repressor domain sequence to the 3 ′ end of the transcription factor gene encoded by the above DNA fragment, a SmaI site and a repressor domain (amino acid sequence: GLDLDLELRLGFA (SEQ ID NO: 519) are downstream of the CaMV35S promoter. )) P35SSXG, a vector having a sequence, was used. In order to link the transcription factor gene sequence and the repressor domain sequence, this vector was cut with SmaI, and PCR amplified fragments encoding the above transcription factors were inserted to produce 180 types of vectors (p35SSXG (TFs)). did. In addition, although the vector was described as p35SSXG (TFs), the AGI code of a transcription factor is described in the TFs part in the description. For example, a vector having a transcription factor specified by At3g04070 is p35SSXG (At3g04070). In the following description, the notation of TFs is also used in the notation of vector and the like.

Construction of an improved transcription factor expression vector pBCKH was used as a binary vector for gene transfer into plants by Agrobacterium. In this vector, the cassette of the Gateway vector conversion system (Invitrogen) is incorporated into the HindIII site of pBIG (Hygr) (Nucleic Acids Res. 18, 203 (1990)). In order to incorporate the improved transcription factor gene sequence into this vector, this vector and 180 types of p35SSXG (TFs) were mixed, and recombination reaction was performed using GATEWAY LR clonase (Invitrogen). 180 types of vectors (pBCKH) -p35SSXG (TFs)).

Introduction of Improved Transcription Factor Gene Expression Vector into Plants Arabidopsis thaliana, Columbia (Col-0) was used as a plant into which improved transcription factors were introduced. The gene transfer method was in accordance with Transformation of Arabidopsis thaliana by vacuum infiltration (http://www.bch.msu.edu/pamgreen/protocol.htm). However, in order to infect, no vacuum treatment was performed, and it was only immersed in Agrobacterium solution. Specifically, the improved transcription factor expression vector pBCKH-p35SSXG (TFs) was introduced into the soil bacterium Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Schell 1986) by electroporation. The introduced bacteria were cultured in a YEP medium containing 1 liter of antibiotics (kanamycin (Km) 50 μg / ml, gentamicin (Gm) 25 μg / ml, rifampicillin (Rif) 50 μg / ml) until OD600 was 1. Next, the cells were collected from the culture solution, and 1 liter of infectious medium (Infiltration medium, 2.2 g of MS salt, 1X B5 vitamins, 50 g of sucrose, 0.5 g of MES, 0.044 μM benzylaminopurine, 400 μl per liter) Of Silwet, suspended in pH 5.7).

After immersing Arabidopsis thaliana grown for 14 days in this solution for 1 minute and infecting, cultivation was continued again and fruited. The seeds (T1 seeds) were sterilized with 50% bleach and 0.02% Triton X-100 solution for 7 minutes, rinsed with sterile water three times, and sterilized hygromycin selective medium (4.3 g / l MS salts, 0.5 % Sucrose, 0.5 g / l MES, pH 5.7, 0.8% agar, 30 mg / l hygromycin, 250 mg / l Vancomycin). Transformed plants (T1 plants) that grew on the hygromycin plate were selected from 5 to 10 lines for each improved transcription gene and transplanted to a 50 mm diameter pot containing vermiculite mixed soil. This was cultivated at 22 ° C., 16 hours light period 8 hours dark period, and light intensity of about 60-80 μE / cm ² to obtain seeds (T2 seeds).

T2 seed analysis
Oil content analysis of T2 seeds of 5 to 10 lines of transformants into which 180 kinds of TFs-SRDX were introduced was performed. For quantitative analysis of fats and oils, 2 to 10 mg of Arabidopsis seeds were measured using MARAN-23 (Resonance Instruments Ltd., UK) H-NMR and analysis software RI-NMR Ver. 2.0. A calibration curve was prepared using olive oil as the standard substance for fats and oils, and the fat content (wt%) in the seeds was determined.
Tables 4-6 summarize the results of analysis of the oil content of the transformant T2 seeds into which each TFs-SRDX gene was introduced.

  The oil content in the seeds seeded in 33 control WT (Col-0) individuals not transfected with the gene was 34.9 ± 3.8%. On the other hand, the average oil and fat content of the transformant T2 seeds overexpressing the chimeric protein in which SRDX was added to each of 180 types of transcription factors was 25.2% to 41.3%. In order to compare the average value obtained with the average oil content of wild strains, t-test was performed. As a result (P <0.05), the oil content increased significantly in 15 lines (8.3% of the transcription factors analyzed). ) Expression of chimeric protein T2 seeds. On the other hand, the fat content decreased significantly (P <0.05) in T2 seeds expressing the chimeric protein of 70 lines (38.9% of the analyzed transcription factors). About 47.2% of the chimeric protein expression increased or decreased the seed fat content. In other words, about half or more of the transcription factors used in this experiment hardly affect the oil content in seeds when expressed as a chimeric protein with a repressor domain (for example, At5g40330, At4g23750, At5g18270, etc.).

  According to this example, as a transcription factor having a function of improving oil content in seeds by expressing it as a chimeric protein with a repressor domain added, At5g47230, At1g22985, At1g80580, At1g25470, At1g67260, At4g36160, At5g64750, At4g01550, At1g24260, Eleven kinds of transcription factors, At5g09330 and At2g31230, were newly identified. Further, according to this example, as a transcription factor having a function of reducing oil content in seeds by expressing as a chimeric protein with a repressor domain added, At2g17040, At5g07690, At3g15500, At2g30420, At3g09600, At1g36060, At1g01250, At1g25580, At3g20770, At1g12890, At2g18060, At4g18390, At5g08070, At1g76580, At4g28140, At5g60970, At2g42830, At1g30210, At1g71450, At1g09540, At3g10490, At1g62700, At1g49120, At1g44830, At1g30810, At1g74840, At5g18830, At1g72360, At1g32770, At5g14000, At2g23290, At2g02450, At1g27360, At1g33760, At3g27920, At3g18550, At1g52880, At5g07310, At4g26150, At1g19490, At1g52150, At3g04060, At4g32800, At5g66300, At5g13180, At1g71692, At1g27730, At3g49850, At3g02150, At5g47220, At5g43270, At5g52020, At1g69490, At4g38620, At2g45650, At5g02460, At1g12260, At5g13330, At4g01060, At2g46590, At1g69120, At1g77450, At2g23760, At2g02070, At1g226 68 new transcription factors, 40, At5g22380 and At5g62380 were newly identified.

  As described above, according to the present example, it was revealed that the fat content in seeds can be significantly modified by expressing a specific transcription factor by fusing with a repressor domain.

Preparation of At5g22380-expressing strain and analysis of fat and oil content As described above, for At5g22380, a DNA fragment was amplified so as to include a stop codon. In the same manner as described above, the DNA fragment was ligated downstream of the 35S promoter and introduced into Arabidopsis thaliana. That is, in this experiment, a transgenic Arabidopsis thaliana that expresses the original At5g22380 with no SRDX added under the control of a constitutive expression promoter was prepared. And the fat and oil content of T2 seed of this transformed Arabidopsis thaliana was measured in the same manner as described above. As a result, the fat content of T2 seeds of individuals expressing At5g22380 was 37.6 ± 1.8%. On the other hand, the oil and fat content of the wild strain cultivated at the same time was 36.3 ± 0.4%. The t-test significantly increased the fat content (P <0.05). The strain with the highest fat content was 40.0%, an increase of 10.3% compared to the wild type.

  From this result, in the experiment described above, the transcription factor that was found to be able to significantly reduce the oil content in seeds by being fused and expressed with the repressor domain is expressed in its original form without the repressor domain ( For example, it was strongly suggested that the fat and oil content in seeds can be significantly improved by controlling the expression with a constant expression promoter. In addition, in the above-described experiment, a transcription factor that has been found to be able to significantly improve oil content in seeds by being fused and expressed with a repressor domain is expressed in an original form having no repressor domain (for example, It was strongly suggested that the oil and fat content in seeds can be significantly reduced by controlling the expression with a constant expression promoter.

Claims (8)

By expressing in a plant a chimeric protein in which a transcription factor consisting of any of the following proteins (a) to (c) and a functional peptide that converts any transcription factor into a transcriptional repression factor are fused: A method of significantly improving fat productivity per individual.
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (b) comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2, and having transcription promoting activity Protein (c) a protein encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and having transcription promoting activity   The method according to claim 1, wherein the transcription promoting activity of the transcription factor is suppressed.   The method according to claim 1, wherein the chimeric protein has transcriptional repressor activity. The functional peptide is represented by the following formulas (1) to (8):
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(In the formula, X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Wherein, Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Wherein, Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(However, in the formula, Z4 represents Glu, Gln or Asp.)
(5) α1-Leu-β1-Leu-γ1-Leu
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(In the formulas (5) to (8), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn. , Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, γ1 represents Arg, Gln, Asn, Thr, Ser, His, Lys or Asp, and γ2 represents Gln , Asn, Thr, Ser, His, Lys or Asp.)
The method according to claim 1, wherein the method has an amino acid sequence represented by any one of the following:   The method according to claim 1, wherein the plant is an angiosperm.   The method according to claim 1, wherein the plant is a dicotyledonous plant.   The method according to claim 1, wherein the plant is a cruciferous plant.   The method according to claim 1, wherein the plant is Arabidopsis thaliana.