CN117305320A - Application, preparation, mutant and vector of GmLBP3 protein and gene - Google Patents
Application, preparation, mutant and vector of GmLBP3 protein and gene Download PDFInfo
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Abstract
The invention provides application, preparation, mutant and carrier of GmLBP3 protein and gene, which belong to the technical field of molecular biology, wherein the application of GmLBP3 protein and gene in regulating and controlling the protein and grease content of soybean seeds comprises at least one of the following: gmLBP3 protein; proteins with the same or similar functions as the GmLBP3 protein by substituting, deleting or adding one or more amino acids in the amino acid sequence of the GmLBP3 protein; nucleic acid molecules encoding the above proteins; a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of the above nucleic acid molecule and which is capable of encoding the same or a similar functional protein; a substance capable of modulating the level or activity of at least one of the foregoing substances. The application provided by the invention can improve the soybean quality and reduce the production cost, and lays a foundation for cultivating high-protein or high-fat soybean varieties.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to application, a preparation, a mutant and a carrier of GmLBP3 protein and gene.
Background
Soybeans are the most important grain, oil and feed crops in the world, play an important role in the production and life of people in China, and can provide about 71% of protein and 29% of grease for the world diet. Soy protein is considered to be the most desirable protein for humans and animals and is more preferred for soybean oil in the traditional diet of our country. With the rapid population growth and rapid breeding industry development, the soybean yield in China is seriously insufficient, the import dependence is as high as more than 85%, and the contradiction between the soybean yield and the soybean demand is serious. The breeding of new soybean varieties with high protein, high grease and environmental protection has become a great challenge.
The soybean seeds contain about 40% protein and 20% oil. The protein and oil content in soybean seeds is a complex quantitative polygenic property, and the protein and oil content show a negative correlation. However, there are many undeveloped current mechanisms for regulating soybean protein content and lipid content.
Disclosure of Invention
The technical problem solved by the invention is how to provide a gene for regulating and controlling the protein and grease content in soybean seeds and application of the coded protein thereof in soybeans.
In order to solve the problems, the invention provides application of GmLBP3 protein and gene in regulating and controlling protein and grease content of soybean seeds, wherein the GmLBP3 protein and gene comprises at least one of the following I-V:
i: gmLBP3 protein;
II: proteins with the same or similar functions as the GmLBP3 protein by substituting, deleting or adding one or more amino acids in the amino acid sequence of the GmLBP3 protein;
III: nucleic acid molecules encoding the proteins described in I or II;
IV: a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of said nucleic acid molecule in III and which is capable of encoding the same or a similar functional protein;
v: a substance capable of modulating the level or activity of at least one of I to V;
wherein the amino acid sequence of the GmLBP3 protein is shown as SEQ ID NO. 1.
Preferably, the nucleic acid molecule encoding the GmLBP3 protein comprises genomic DNA, cDNA, recombinant DNA, mRNA or hnRNA encoding the GmLBP3 protein; or a nucleic acid molecule which is reverse complementary to said DNA, said cDNA, said recombinant DNA or said mRNA.
Preferably, the nucleic acid molecule encoding the GmLBP3 protein comprises a GmLBP3 gene, and the nucleotide sequence of the GmLBP3 gene is shown in SEQ ID No. 2.
The invention takes a potential gene for encoding the soybean lipopolysaccharide binding protein as a novel entry point, and discovers that the GmLBP3 protein and a nucleic acid molecule for encoding the GmLBP3 protein are related to the protein and grease content in soybean seeds by analyzing the interaction process of the potential soybean lipopolysaccharide binding protein and rhizobia lipopolysaccharide, so that the gene can be applied to regulating and controlling the protein and grease content in the soybean seeds. The application provided by the invention can improve the soybean quality and reduce the production cost, and lays a foundation for cultivating high-protein or high-fat soybean varieties.
The invention also provides a preparation for reducing the protein content and increasing the grease content in soybean seeds, which comprises at least one of the following i-vi:
i: knocking out or knocking down an expression vector of the GmLBP3 gene;
ii: a recombinant host comprising i;
iii: a nucleic acid molecule that interferes with expression of the GmLBP3 gene;
iv: a terminator of the GmLBP3 gene expression;
v: an agent that inhibits expression of the GmLBP3 gene;
vi: an inhibitor of the activity of GmLBP3 protein.
According to the invention, the level and/or activity of the GmLBP3 protein can be reduced by knocking out or knocking down, interfering and stopping the expression of the GmLBP3 gene or inhibiting the activity of the GmLBP3 protein, so that the effects of reducing the protein content in soybean seeds and improving the grease content are achieved.
The invention also provides a gene mutant, which is deleted at 31 st-37 th positions in the nucleotide sequence of the GmLBP3 gene.
The invention also provides another gene mutant, wherein the 156 th site of the nucleotide sequence of the GmLBP3 gene is provided with 26 nucleotide sequence insertions, and 157-789 deletions.
Compared with the prior art, the gene mutant provided by the invention has the beneficial effects that the gene mutant is the same as a preparation for reducing the protein content in soybean seeds and improving the oil content, and is not described in detail herein.
The invention provides a method for constructing a gene mutant, which is used for constructing the gene mutant, and a target sequence in a gene knockout expression vector is shown as SEQ ID NO.5 or SEQ ID NO. 6.
Compared with the prior art, the method for constructing the gene mutant has the beneficial effects that the method is the same as a preparation for reducing the protein content in soybean seeds and improving the oil content, and is not repeated here.
The present invention also provides a preparation for increasing protein content and decreasing fat content in soybean seeds, comprising at least one of the following (1) to (6):
(1) the method comprises the following steps A GmLBP3 gene overexpression vector;
(2) the method comprises the following steps A recombinant host comprising (1);
(3) the method comprises the following steps A nucleic acid molecule that enhances expression of the GmLBP3 gene;
(4) the method comprises the following steps An enhancer of the GmLBP3 gene expression;
(5) the method comprises the following steps A formulation that enhances expression of the GmLBP3 gene;
(6) the method comprises the following steps An activity enhancer of GmLBP3 protein.
According to the invention, the level and/or activity of the GmLBP3 protein can be improved by enhancing the expression of the GmLBP3 gene or enhancing the activity of the GmLBP3 protein, so that the effects of improving the protein content in soybean seeds and reducing the oil content are achieved.
The invention also provides a GmLBP3 gene overexpression vector, which comprises a vector for overexpressing the GmLBP3 gene driven by a tobacco mosaic virus 35S promoter.
Compared with the prior art, the GmLBP3 gene overexpression vector has the beneficial effects that the GmLBP3 gene overexpression vector is the same as a preparation for improving the protein content in soybean seeds and reducing the oil content, and is not repeated here.
The invention also provides a method for regulating and controlling the protein and grease content in plants, which is to introduce the gene fragment of the GmLBP3 into plant cells, wherein the plants do not contain the GmLBP3 gene.
The invention provides a method for regulating and controlling the protein and grease content in a fabric without GmLBP3 gene, which can regulate and control the protein and grease content in the plant by utilizing an exogenous GmLBP3 gene segment by introducing the GmLBP3 gene segment into the plant cell.
Drawings
FIG. 1 is a diagram of the gene structure of GmLBP3 in an embodiment of the present invention;
FIG. 2 is a diagram of the gene structure of the lbp3-1 mutant according to an embodiment of the present invention;
FIG. 3 is a diagram of the gene structure of the lbp3-2 mutant according to an embodiment of the present invention;
FIG. 4 is a graph showing the comparative analysis of the lipid and protein content of different types of soybean seeds in the examples of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. In addition, the terms "comprising," "including," "containing," "having" and their derivatives are not limiting, as other steps and other ingredients not affecting the result may be added. Materials, equipment, reagents are commercially available unless otherwise specified.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, mass fractions, and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of embodiments of the invention will be rendered by reference to specific embodiments thereof.
The embodiment of the invention provides application of GmLBP3 protein and gene in regulating and controlling protein and grease content of soybean seeds, wherein the GmLBP3 protein and gene comprises at least one of the following I-V:
i: gmLBP3 protein;
II: proteins with the same or similar functions as the GmLBP3 protein by substituting, deleting or adding one or more amino acids in the amino acid sequence of the GmLBP3 protein;
III: nucleic acid molecules encoding the proteins described in I or II;
IV: a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of said nucleic acid molecule in III and which is capable of encoding the same or a similar functional protein;
v: a substance capable of modulating the level or activity of at least one of I to V;
wherein the amino acid sequence of the GmLBP3 protein is shown as SEQ ID NO. 1.
According to the embodiment of the invention, the potential gene for encoding the soybean lipopolysaccharide binding protein is taken as a novel entry point, and the processes of interaction of the potential soybean lipopolysaccharide binding protein and rhizobia lipopolysaccharide are analyzed, so that the GmLBP3 protein and the nucleic acid molecule for encoding the GmLBP3 protein are found to be related to the protein and oil content in soybean seeds, and can be applied to regulating and controlling the protein and oil content in the soybean seeds. The application provided by the embodiment of the invention can improve the soybean quality and reduce the production cost, and lays a foundation for cultivating high-protein or high-fat soybean varieties.
The amino acid sequence of the GmLBP3 protein is shown in SEQ ID NO.1, and total 494 amino acids (comprising terminator).
Wherein the nucleic acid molecule encoding the GmLBP3 protein comprises genomic DNA, cDNA, recombinant DNA, mRNA or hnRNA encoding the GmLBP3 protein; or a nucleic acid molecule which is reverse complementary to said DNA, said cDNA, said recombinant DNA or said mRNA.
It should be noted that, the above nucleic acid molecule encoding GmLBP3 protein may be modified or optimized according to actual needs, so that gene expression is more efficient. For example: (a) The codon can be altered to conform to the preferences of the recipient plant while maintaining its coding amino acid sequence, depending on the codon preferred by the recipient plant; (b) Modifying the gene sequence adjacent to the initiation methionine to effect initiation of translation, such as by using sequences known to be effective in plants; (c) Promoters linked to the expression of various plants to facilitate their expression in plants may include constitutive, inducible, chronoregulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters, the choice of which will vary with the time and space of expression needs and depends on the target species; (d) Enhancer sequences such as intron sequences (derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV) are introduced.
Specifically, the nucleic acid molecule encoding the GmLBP3 protein comprises a GmLBP3 gene (i.e., genomic DNA encoding the GmLBP3 protein), and the nucleotide sequence of the GmLBP3 gene is shown in SEQ ID No. 2. As shown in FIG. 1, the total length of the GmLBP3 gene is 5184bp, and comprises 6 exons and 5 introns.
The embodiment of the invention also provides a preparation for reducing the protein content and improving the grease content in soybean seeds, which comprises at least one of the following i-vi:
i: knocking out or knocking down an expression vector of the GmLBP3 gene;
ii: a recombinant host comprising i;
iii: a nucleic acid molecule that interferes with expression of the GmLBP3 gene;
iv: a terminator of the GmLBP3 gene expression;
v: an agent that inhibits expression of the GmLBP3 gene;
vi: an inhibitor of the activity of GmLBP3 protein.
According to the embodiment of the invention, the level and/or activity of the GmLBP3 protein can be reduced by knocking out or knocking down, interfering and stopping the expression of the GmLBP3 gene or inhibiting the activity of the GmLBP3 protein, so that the effects of reducing the protein content in soybean seeds and improving the grease content are achieved.
It should be noted that, in the embodiment of the present invention, the vector may be a plasmid, a cosmid, a phage, or a viral vector, and the host may be a fungus, a bacterium, an alga, or a cell.
The embodiment of the invention provides a gene mutant, wherein 31-37 th nucleotide sequence of GmLBP3 gene in one gene mutant lbp3-1 is deleted, and 156 th nucleotide sequence of GmLBP3 gene in another gene mutant lbp3-2 is inserted, and 157-789 th nucleotide sequence is deleted.
Specifically, the nucleotide sequence of lbp3-1 is shown as SEQ ID NO.3, as shown in FIG. 2, the 31 st to 37 th positions of the nucleotide sequence of GmLBP3 gene are deleted (the dark grey shaded part in FIG. 2 represents the deleted nucleotide sequence); the nucleotide sequence of lbp3-2 is shown as SEQ ID NO.4, as shown in FIG. 3, 26 nucleotide sequence insertions are made at position 156 and 633 nucleotide deletions are made at positions 157-789 in the nucleotide sequence of the GmLBP3 gene (the underlined parts in FIG. 3 indicate the inserted nucleotide sequences, and the dark grey shaded parts indicate the deleted nucleotide sequences).
The expression level of the GmLBP3 protein in the gene mutant is inhibited, so that the effects of reducing the protein content in soybean seeds and improving the oil content are achieved.
Further, when constructing the gene part variant as described above, the target sequence in the gene expression vector is shown as SEQ ID NO.5 or SEQ ID NO. 6.
SEQ ID NO.5 has the sequence 5'-GTCTCTACTACCCCTTCTTC-3', SEQ ID NO.6 as 5'-GAATTCTGCCCAAGTCCCTT-3'. Wherein, when the target sequence in the gene expression vector is SEQ ID NO.5, the method is used for constructing lbp3-1, and when the target sequence in the gene expression vector is SEQ ID NO.6, the method is used for constructing lbp3-2.
The embodiment of the invention also provides a preparation for increasing the protein content and reducing the grease content in soybean seeds, which comprises at least one of the following (1) - (6):
(1) the method comprises the following steps A GmLBP3 gene overexpression vector;
(2) the method comprises the following steps A recombinant host comprising (1);
(3) the method comprises the following steps A nucleic acid molecule that enhances expression of the GmLBP3 gene;
(4) the method comprises the following steps An enhancer of the GmLBP3 gene expression;
(5) the method comprises the following steps A formulation that enhances expression of the GmLBP3 gene;
(6) the method comprises the following steps An activity enhancer of GmLBP3 protein.
According to the embodiment of the invention, the level and/or activity of the GmLBP3 protein can be improved by enhancing the expression of the GmLBP3 gene or enhancing the activity of the GmLBP3 protein, so that the effects of improving the protein content in soybean seeds and reducing the oil content are achieved.
The embodiment of the invention also provides a GmLBP3 gene overexpression vector, which comprises a vector for overexpressing the GmLBP3 gene driven by a tobacco mosaic virus 35S promoter.
The GmLBP3 gene over-expression vector is transferred into soybean, so that the over-expression of the GmLBP3 gene can be caused, the level and/or activity of GmLBP3 protein can be improved, and the effects of improving the protein content in soybean seeds and reducing the oil content can be achieved.
The embodiment of the invention also provides a method for regulating and controlling the protein and grease content in plants, which is to introduce the gene fragment of the GmLBP3 into plant cells, wherein the plants do not contain the GmLBP3 gene.
For example, for plants not containing the GmLBP3 gene, the gene fragment of GmLBP3 may be introduced into plant cells by chemical methods, bird gun methods, microinjection, electroporation, or the like, or the gene fragment of GmLBP3 may be introduced into plant cells by homologous recombination, zinc finger nucleases, TALENs, CRISPR, or the like.
The embodiment of the invention provides a method for regulating and controlling the protein and grease content in a fabric without a GmLBP3 gene, which can regulate and control the protein and grease content in the plant by utilizing an exogenous GmLBP3 gene segment by introducing the GmLBP3 gene segment into the plant cell.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, which do not address specific conditions in the following examples, are generally in accordance with the conditions recommended by the manufacturer.
Example 1 isolation and structural analysis of the GmLBP3 Gene in Soybean
(1) Isolation of the GmLBP3 Gene
Extracting total RNA from soybean variety Williams 82, and synthesizing cDNA first strand by using total RNA as a template and Oligo (T) 17 as a primer;
the first strand of the cDNA was used as a template, and PCR amplification was performed using a forward primer (5'-ATGGCACCCTCAATTGTTTTG-3') and a reverse primer (5'-TTATGAAACATAAGCTAACTGTTGACC-3') to obtain a cDNA fragment of the GmLBP3 gene of 1482bp in length, which was then combined with a pEasy-Blunt vector (full-size gold Co.) and designated as Blunt-GmLBP3.
The nucleotide sequence of the obtained GmLBP3 gene is shown as SEQ ID NO.2, and the total CDS full length of the GmLBP3 gene is 1482bp; the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO.1, and total amino acids are 494 (comprising a terminator).
(2) GmLBP3 Gene structural analysis
Extracting genome DNA in young leaves of soybean variety Williams 82, and amplifying to obtain GmLBP3 genome fragment by using the genome DNA as a template, wherein the GmLBP3 genome sequence is shown as SEQ ID NO.2, and the total length is 5184bp, and the genome fragment comprises 6 exons and 5 introns (the gray part in the figure 1 represents the exons, and the rest represents the introns).
Example 2 construction of Soybean GmLBP3 Gene knockout vector and overexpression vector
(1) Construction of soybean GmLBP3 gene knockout vector
Constructing a CRISPR/Cas9 vector which drives a gRNA and GmUbi3 promoter system to drive Cas9 protein by using a GmU promoter, introducing a recombinant plasmid into wild soybean Williams 82 by using an agrobacterium-mediated genetic transformation system, knocking out a target gene GmLBP3, and constructing a knocking-out vector of the GmLBP3 gene to transform the wild soybean Williams 82.
The example provides two mutants of soybean GmLBP3 gene, wherein the lbp3-1 is characterized in that 31 st to 37 th nucleotide is deleted in the nucleotide sequence of the GmLBP3 gene (shown in figure 2), the nucleotide sequence is shown as SEQ ID NO.3, the lbp3-2 is characterized in that the 156 th nucleotide of the GmLBP3 gene is inserted into 26 th nucleotide, the 157 th to 789 th nucleotide is deleted (shown as figure 3), and the nucleotide sequence is shown as SEQ ID NO. 4.
In order to construct the lbp3-1 and lbp3-2 gene mutants respectively, the target site sequences in the gene knockout expression vector are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively, and the sequence of SEQ ID NO.5 is 5'-GTCTCTACTACCCCTTCTTC-3', SEQ ID NO.6 and 5'-GAATTCTGCCCAAGTCCCTT-3'. .
(2) Construction of soybean GmLBP3 gene overexpression vector
Constructing an over-expression GmLBP3 vector driven by a tobacco mosaic virus 35S promoter, introducing a recombinant plasmid into wild soybean Williams 82 through an agrobacterium-mediated genetic transformation system, over-expressing a target gene GmLBP3, constructing an over-expression GmLBP3 gene vector to transform the wild soybean Williams 82, and respectively constructing two over-expression GmLBP3 transgenic soybeans of GmLBP3-1 and GmLBP 3-2.
EXAMPLE 3 Agrobacterium-mediated transformation of leguminous plants
In this example, an explant of wild type soybean Williams 82 containing the GmLBP3 knockout vector was obtained by agrobacterium-mediated transformation of soybean cotyledonary nodes.
(1) Acquisition of soybean explants
Selecting wild soybean Williams 82 mature soybean seeds with smooth surfaces, no damage, no disease spots and no cracks, and sterilizing for 14h by a chlorine method; ventilating the sterilized seeds on an ultra-clean bench to volatilize chlorine completely, and germinating in a germination culture medium for 6 hours; the soybean 1/2 hypocotyl was removed, the soybean was slit longitudinally along the hypocotyl, and the remaining hypocotyl was used as a receptor material for agrobacterium-mediated transformation.
(2) Conversion of soybean
The agrobacterium-mediated method employs a secondary agrobacterium infection (employing the gene knockout vector and the overexpression vector obtained in example 2), and dark culture is performed on a co-culture medium at 22 ℃ for 5d; culturing in SI-I culture medium under strong light for 7d; cutting off large buds of explants (wild soybean Williams 82), and culturing in SI-II culture medium under strong light for 14d; cutting off cotyledons and hypocotyls of the explants, and carrying out secondary culture once every 14d in SE culture medium; cutting off cluster buds about 3cm, and putting the cluster buds into a rooting culture medium for rooting; transferring plants with developed root growth into soil in RM rooting culture medium for planting; 30 resistant plants are obtained through screening by Bar resistance detection; the pod begins to mature after 5 months of cultivation in a greenhouse, and the seed collection is completed after 6 months.
(3) Obtaining genetically modified plants
The harvested T2 generation seeds were planted in a plant cultivation room to obtain soybean seeds, and the protein content and the oil content in the soybean seeds were analyzed, and the results are shown in FIG. 4.
FIG. 4 (A) is a graph showing the comparison of the oil content in the soybean seeds of different types, and FIG. 4 (B) is a graph showing the comparison of the protein content in the soybean seeds of different types. The abscissas of (A) and (B) in FIG. 4 represent different soybean types, wherein WT represents wild-type soybean Williams 82, lbp3-1 and lbp3-2 represent gene mutants obtained after transformation of a gene knockout vector, gmLBP3-1 and GmLBP3-2 represent gene overexpressing plants obtained after transformation of a gene overexpressing vector, the ordinate of (A) in FIG. 4 represents lipid content, and the ordinate of (B) in FIG. 4 represents protein content. In fig. 4, P < 0.01 is shown with significant differences relative to WT.
As can be seen from fig. 4, after the GmLBP3 knockout vector is transferred into wild soybean Williams 82, the seed protein content is significantly reduced, and the oil content is significantly improved; and after the over-expression GmLBP3 vector is transferred into wild soybean Williams 82, the protein content of seeds is obviously improved, and the grease content is obviously reduced.
Although the invention is disclosed above, the scope of the invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications will fall within the scope of the invention.
Claims (10)
- Application of GmLBP3 protein and gene in regulating and controlling protein and grease content of soybean seeds, which is characterized in that the GmLBP3 protein and gene comprises at least one of the following I-V:i: gmLBP3 protein;II: proteins with the same or similar functions as the GmLBP3 protein by substituting, deleting or adding one or more amino acids in the amino acid sequence of the GmLBP3 protein;III: nucleic acid molecules encoding the proteins described in I or II;IV: a nucleic acid molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence of said nucleic acid molecule in III and which is capable of encoding the same or a similar functional protein;v: a substance capable of modulating the level or activity of at least one of I to V;wherein the amino acid sequence of the GmLBP3 protein is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein the nucleic acid molecule encoding the GmLBP3 protein comprises genomic DNA, cDNA, recombinant DNA, mRNA or hnRNA encoding the GmLBP3 protein; or a nucleic acid molecule which is reverse complementary to said DNA, said cDNA, said recombinant DNA or said mRNA.
- 3. The use according to claim 2, wherein the nucleic acid molecule encoding the GmLBP3 protein comprises a GmLBP3 gene, the nucleotide sequence of the GmLBP3 gene being shown in SEQ ID No. 2.
- 4. A formulation for reducing protein content and increasing lipid content in soybean seeds, comprising at least one of the following i-vi:i: knocking out or knocking down an expression vector of the GmLBP3 gene;ii: a recombinant host comprising i;iii: a nucleic acid molecule that interferes with expression of the GmLBP3 gene;iv: a terminator of the GmLBP3 gene expression;v: an agent that inhibits expression of the GmLBP3 gene;vi: an inhibitor of the activity of GmLBP3 protein.
- 5. A gene mutant, which is characterized in that 31-37 bits of a nucleotide sequence of a GmLBP3 gene are deleted.
- 6. A gene mutant is characterized in that 26 nucleotide sequence insertions are arranged at 156 th position in the nucleotide sequence of GmLBP3 gene, and 157-789 are deleted.
- 7. A method for constructing a gene mutant according to claim 5 or 6, wherein the target sequence in the gene knockout expression vector is shown in SEQ ID No.5 or SEQ ID No. 6.
- 8. A formulation for increasing protein content and decreasing lipid content in soybean seeds, comprising at least one of the following (1) to (6):(1) the method comprises the following steps A GmLBP3 gene overexpression vector;(2) the method comprises the following steps A recombinant host comprising (1);(3) the method comprises the following steps A nucleic acid molecule that enhances expression of the GmLBP3 gene;(4) the method comprises the following steps An enhancer of the GmLBP3 gene expression;(5) the method comprises the following steps A formulation that enhances expression of the GmLBP3 gene;(6) the method comprises the following steps An activity enhancer of GmLBP3 protein.
- 9. The GmLBP3 gene over-expression vector is characterized by comprising a vector for over-expressing the GmLBP3 gene driven by a tobacco mosaic virus 35S promoter.
- 10. A method for regulating protein and grease content in a plant, which is characterized in that a gene fragment of GmLBP3 is introduced into a plant cell, wherein the plant does not contain the GmLBP3 gene.
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