CN116555284B - Application of GmBBX4 gene in regulating and controlling soybean isoflavone synthesis - Google Patents
Application of GmBBX4 gene in regulating and controlling soybean isoflavone synthesis Download PDFInfo
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- CN116555284B CN116555284B CN202310447471.6A CN202310447471A CN116555284B CN 116555284 B CN116555284 B CN 116555284B CN 202310447471 A CN202310447471 A CN 202310447471A CN 116555284 B CN116555284 B CN 116555284B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
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- Gastroenterology & Hepatology (AREA)
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Abstract
The invention discloses an application of GmBBX4 gene in regulating and controlling soybean isoflavone synthesis. Wherein the method comprises the steps ofGmBBX4The nucleotide sequence of the gene is shown as SEQ ID NO.1 in the sequence table; the amino acid sequence of the protein coded by the GmBBX4 gene is shown as SEQ ID NO.2 in a sequence table. The invention creates mutants and creates an over-expression transgenic soybean strain by utilizing CRISPR-Cas9 gene editing technology on GmBBX4 genes, and discovers that the content of secondary metabolites in the soybean of the GmBBX4 gene editing mutants is obviously improved, such as genistin, daidzin, malonyl genistin, malonyl daidzin and malonyl daidzin, so as to improve the quality of the soybean. Thus, in plantsGmBBX4The functional deletion of the gene is an effective way for improving plant quality, and has important production and theoretical research significance.
Description
Technical Field
The invention belongs to the field of biotechnology, in particular toGmBBX4The application of the gene in regulating and controlling the synthesis of soybean isoflavone.
Background
Soybean is one of important commercial crops in China, is rich in protein, grease and various substances with biological activity, wherein soybean isoflavone is a flavonoid substance mainly existing in leguminous plants, and is rich in soybean seeds. The soybean isoflavone has the effects of improving the capability of resisting abiotic stress and biotic stress on plants, and preventing osteoporosis, cardiovascular and cerebrovascular diseases and the like on human health. Among soybeans, isoflavones are mainly classified into 3 types, namely genistins (Daidzingroups), glycitin glycosides (glycitin roups), and daidzins (genistins). All exist in 4 main forms of free form, glucoside form, acetyl glucoside form and malonyl glucoside form.
Soy isoflavones are derived from the well-established metabolic pathways of phenylpropane in plants and their synthesis is affected by a variety of factors, including the environment and the activity of various catalytic enzymes in their synthetic pathways. Uncertainty in the field environment makes it important to study the process of the soybean itself affecting isoflavone accumulation. Soy isoflavones accumulate to varying degrees in each tissue of the soybean, with the most abundant accumulation in mature kernels. Therefore, it is important to study the process of affecting isoflavone accumulation during the maturation of soybean seeds.
The expression of various catalytic enzymes in the phenylpropane metabolic pathway is regulated by transcription factors. At present, some MYB family transcription factors have also been identified sequentially to promote soybean isoflavone accumulation. But the related research is still relatively deficient. B-box family proteins are a family of transcription factors that have one or two B-box domains and are widely found in higher plants. Numerous studies, whether in the model plant arabidopsis thaliana, or in the horticultural crops apple, pear and tomato, have shown that: BBX proteins regulate the accumulation of flavonoids in plants, usually by affecting the transcriptional activation activity of HY5 or by being able to directly regulate the expression of various flavonoid synthetic genes.
To date, soybeansGmBBX4Gene studies have not been reported. Thus, forGmBBX4The cloning and functional research of the gene are of great significance.
Disclosure of Invention
The purpose of the present invention is to provide soybeansGmBBX4The application of the gene in regulating and controlling the synthesis of plant isoflavone.
The aim of the invention can be achieved by the following technical scheme.
In a first aspect, the invention provides the use of any one of the following for modulating plant isoflavone synthesis:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is as follows (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;
(A2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.2 in the sequence table.
In a second aspect, the invention provides the use of any one of the following for the preparation of a product for modulating the synthesis of a plant isoflavone:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is as follows (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;
(A2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.2 in the sequence table.
In a third aspect, the invention provides the use of any one of the following for growing high/low isoflavone plants:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is as follows (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;
(A2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.2 in the sequence table.
In a fourth aspect, the invention provides the use of any one of the following for the preparation of a product for growing high/low isoflavone plants:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is as follows (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;
(A2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.2 in the sequence table.
In a fifth aspect, the invention provides the use of any one of the following in plant breeding:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is as follows (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;
(A2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.2 in the sequence table.
Using a CRISPR-Cas9 system for the describedGmBBX4Is edited. After the mutation of GmBBX4, the content of soybean isoflavone in the grains is obviously increased. In contrast, after overexpression of GmBBX4, the soybean isoflavone content in the grain was significantly reduced.
In a specific embodiment, the soybean B-box transcription factor GmBBX4 gene has a nucleotide sequence shown in SEQ ID NO.1 of the sequence Listing.
In specific embodiments, the plant is a monocot or dicot.
Preferably, the dicotyledonous plant is a plant of the family Leguminosae.
In a specific embodiment, the isoflavone is selected from one or more of genistin, daidzein, malonyl genistin, malonyl daidzein, and malonyl daidzein.
In a sixth aspect, the present invention provides a method for increasing soy isoflavone content comprising:
the method comprises reducing the content and/or activity of the transcription factor GmBBX4;
preferably, the reduction of the content and/or activity of the transcription factor GmBBX4 described above is achieved by editing the encoding gene of the GmBBX4 using a CRISPR-Cas9 system.
The nucleotide sequence of the gRNA of the GmBBX4 coding gene in the CRISPR-Cas9 system is as follows: gmBBX4-gRNA1: GAGTGTACGGACTCGAAGAACGG and GmBBX4-gRNA2: GCTGACGTCAGCATCGGCGTCGG.
In a seventh aspect, the present invention provides a method of inhibiting soy isoflavone content:
the method comprises increasing the content and/or activity of the transcription factor GmBBX4;
the content and/or activity of the transcription factor GmBBX4 are improved by improving the expression level of the transcription factor GmBBX4 gene.
In a specific embodiment, the improvement of the expression level of the transcription factor GmBBX4 gene is realized by transferring a recombinant plant expression vector for over-expressing GmBBX4 into a plant.
Overexpression by transgenic techniquesGmBBX4Is transferred into soybean cultivar Williams 82 (hereinafter abbreviated as Wm 82) to increase in soybeanGmBBX4Is a factor (B) of the expression level of (C). Discovery ofGmBBX4After over-expression, the content of soy isoflavone in the grain is significantly reduced.
The recombinant plant expression vector is specifically constructed and obtained by the following method: will beGmBBX4Gene fragments are constructed by homologous recombinationJRH0641The vector thus obtained is a recombinant plant expression vector.
The invention has the remarkable advantages that:
the invention provides B-box family genes for the first timeGmBBX4The application of the protein coded by the gene in regulating and controlling the accumulation of soybean isoflavone. Thus, high soy isoflavones and low soy isoflavones can be obtained by the present inventionHas important application value.
Drawings
FIG. 1 shows a soybean of the present inventiongmbbx4Mutant Gene editing form (1A)YFP-GmBBX4The overexpression expression level of the transgenic lines is schematically detected (1B, 1C).
FIG. 2 shows the results of the determination of total isoflavone content after mutation and after overexpression of soybean GmBBX4 according to the invention.
FIG. 3 shows the results of the measurement of genistin content after mutation and after overexpression of soybean GmBBX4 according to the invention.
FIG. 4 shows the results of soybean xanthosine content measurement after mutation and after overexpression of soybean GmBBX4 of the present invention.
FIG. 5 shows the results of the measurement of malonyl genistin content after mutation and after overexpression of soybean GmBBX4 according to the invention.
FIG. 6 shows the results of the measurement of malonyl-glycitin content after mutation and after overexpression of soybean GmBBX4 according to the invention.
FIG. 7 shows the results of the assay of malonyl-soybean glycoside content after mutation and after overexpression of soybean GmBBX4 according to the invention.
Description of the embodiments
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The sequencing of the vectors in the examples below was determined by sequencing by conventional sequencing companies.
Soybean(Glycine max) Variety Wm82 was supplied by the national soybean improvement center (National Center for Soybean Improvement).
The gene editing vector used in the present invention isJRH0912The over-expression vector isJRH0641，Are all provided by the national soybean improvement center (National Center for Soybean Improvement).
Example 1 construction of soybean GmBBX4 gene mutant plant expression vector.
The invention provides primers for constructing an expression vector, comprising primers for amplifying a sequence comprising guide RNA:
GmBBX4-1+sgRNA-F：AATGTGCCACCACATGGATTGAGTGTACGGACTCGAAGAAGTTTTAGAGCTAGAAATAGCAA
GmBBX4-2+sgRNA-F：AATGTGCCACCACATGGATTGGACGGGGATGCGCTCGTGGGTTTTAGAGCTAGAAATAGCAA
(gRNA+sgRNA)-R: GCTCGGCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGT
primers for amplifying sequences of U6 fragments
U6-Xbal-F: GGAAGCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTC
U6-R: CAATCCATGTGGTGGCACAT
Adaptor primer for double-target connection
Dual-U6-F1:GCCTTCTAGAGAGACCAAAATAAATGGTAAAATGTCAA
Dual-U6-R1:TCTCGAATTCGAGACCAAAAAAAGCACCGACTCGGTGC
Dual-U6-F2:GGTCTCGAATTCGAGACCAAAATAAATGGTAAAATGTC
Dual-U6-R2:TTGTAGATCTGAGACCAAAAAAAGCACCGACTCGGTGC
Construction of recombinant plasmid GmBBX4-gRNA
KOD Plus is a product of TOYOBO Co.
Acquisition of gRNA fragments
(1) Reaction system 1 was prepared. The reaction system 1 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of primer GmBBX4-1+sgRNA-F aqueous solution (concentration: 10. Mu.M), 1.5. Mu.L of primer (gRNA+sgRNA) -R aqueous solution (concentration: 10. Mu.M), 1. Mu.L of vectorJRH0951And 34. Mu.L ddH 2 O composition.
Carrier bodyJRH0951Are described in the following documents: sun X, hu Z, chen R, jiang Q, song G, zhang H, xi Y. (2015) Targeted 845 mutagenesis in soybean using the CRISPR-Cas9 system Sci. Rep. 5 (1), 1-10.
(2) After the step (1) is completed, the reaction system 1 is taken for PCR amplification, and the gRNA fragment is recovered by using an agarose gel recovery kit (Zhuang Mengguo, biological gene technologies Co., ltd.).
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 10 s at 68℃,35 cycles; 12. preserving at the temperature.
Acquisition of the U6 promoter fragment
(1) Reaction system 2 was prepared. The reaction system 2 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of an aqueous solution of primer U6-Xbal-F (concentration: 10. Mu.M), 1.5. Mu.L of an aqueous solution of primer U6-R (concentration: 10. Mu.M) and 1. Mu.L of a carrierJRH0951And 34. Mu.L ddH 2 O composition.
(2) After the completion of the step (1), the reaction system 2 was used for PCR amplification, and the U6 promoter fragment was recovered by using an agarose gel recovery kit (Zhuang Mengguo, biological gene technologies Co., ltd.).
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 10 s at 68℃,35 cycles; 12. preserving at the temperature.
Obtaining of the U6-gRNA fragment
(1) Reaction system 3 was prepared. The reaction system 3 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of an aqueous solution of primer U6-Xbal-F (concentration: 10. Mu.M), 1.5. Mu.L of an aqueous solution of primer (gRNA+sgRNA) -R (concentration: 10. Mu.M) 0.5. Mu.L of a gRNA fragment, 0.5. Mu. L U6 fragment and 34. Mu.L of ddH 2 O composition. .
(2) After the step (1) is completed, the reaction system 3 is taken for PCR amplification, and the U6-gRNA fragment recovered by an agarose gel recovery kit (Zhuang Mengguo, biological gene technologies Co., ltd.) is used.
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 10 s at 68℃,35 cycles; 12. preserving at the temperature.
4. Preparing fragments of U6-gRNA1 and U6-gRNA2,
(1) Reaction systems 4 and 5 were prepared.
The reaction system 4 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of aqueous Dual-U6-F1 primer solution (10. Mu.M concentration), 1.5. Mu.L of aqueous Dual-U6-R1 primer solution (10. Mu.M concentration) 1. Mu. L U6-gRNA1 fragment and 34. Mu.L ddH 2 O composition.
The reaction system 5 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of aqueous Dual-U6-F2 primer (concentration: 10. Mu.M), 1.5. Mu.L of aqueous Dual-U6-R2 primer (concentration: 10. Mu.M), 1. Mu. L U6-gRNA2 fragment and 34. Mu.L of ddH 2 O composition.
(2) After the completion of the step (1), the reaction systems 4 and 5 were taken for PCR amplification, and the Dual-U6-gRNA1 and Dual-U6-gRNA2 fragments were recovered using an agarose gel recovery kit (Zhuang Mengguo, biological Gene technologies Co., ltd.).
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 15 s at 68℃,35 cycles; 12. preserving at a temperature of
5. Double digestion of vectors with restriction enzymes XbaI and BglIIJRH0912The vector backbone of about 14000bp was recovered.
Carrier bodyJRH0912Are described in the following documents: meng, Y., hou, H, wang, R, ji, B, liu, J, wen, L, niu and H, lin (2017), targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula Plant Cell Rep 36 (2): 371-374, named pFGC5941-Cas9 in the literature. Carrier bodyJRH0912Contains Cas9 protein.
6. Obtaining of ligation products
(1) mu.L of Dual-U6-gRNA1, 1. Mu.L of Dual-U6-gRNA2 fragment (about 100 ng), 0.5. Mu.L of the vector backbone recovered in step 5 (50 ng) and 2.5. Mu.L of 2 XClonExpress Mix (Vazyme, C115) were mixed to give a ligation system.
(2) Taking the connecting system, and reacting for 45min at 50 ℃ to obtain a connecting product.
7. The ligation products were transformed into E.coli DH 5. Alpha. Competent cells (ipecac) to obtain several monoclonal antibodies.
8. PCR amplification was performed using each of the individual monoclonal as a template and a primer pair consisting of primers 912-F5'-acatttaatacgcgatagaaaac-3' and 912-R5'-cgtgctccaccatgttgacggatc-3'. Contains clone with target fragment size of 1000bp, and sends bacterial liquid to sequence to obtain positive clone.
9. Positive listCloning and inoculating to LB liquid culture medium, culturing to obtain bacterial liquid; then extracting plasmid from bacterial liquid, i.e. recombinant plasmidGmBBX4-gRNA。
Based on the sequencing results, recombinant plasmidsGmBBX4-gRNAThe structure is described as follows: the carrier is put intoJRH0912The DNA small fragment between the recognition sequences of restriction enzymes XbaI and BglII is replaced by the DNA molecule shown in SEQ ID No. 3 to obtain the recombinant plasmid.
EXAMPLE 2 construction of Soybean GmBBX4 overexpression vector
Total RNA of tender leaves of soybean Wm82 seedlings is extracted, and cDNA is synthesized by using a reverse transcription kit.
The invention provides primers for constructing an over-expression vector, which comprises primers for amplifying a target fragment and primers for fusing labels
JRH0641-EYFP-F: GGAGAGCCACCATGCTCGAGATGGGCAAGGGCGAGGAG
JRH0641-EYFP-GmBBX4-R：GTCGCAGAGCTTGGAAGCCATGAAGCCTGCTTTTTTGTACAAAC
JRH0641-GmBBX4-F：GTTTGTACAAAAAAGCAGGCTTCATGGCTTCCAAGCTCTGCGAC
JRH0641-GmBBX4-R：CTGGGGGAGGACCACTAGTACACGACGGAACGACGCCGTA
1. Fusion tag YFP fragment acquisition
(1) Reaction system 1 was prepared. The reaction system 1 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of an aqueous solution of the primer JRH0641-EYFP-F (concentration: 10. Mu.M), 1.5. Mu.L of an aqueous solution of the primer JRH0641-EYFP-GmBBX4-R (concentration: 10. Mu.M), 1. Mu.L of a vectorpEargateway 104And 34. Mu.L ddH 2 O composition
Carrier bodypEargateway 104Are described in the following documents: katzen F (2007) Gateway recombinational cloning: a biological operating system. Expert Opin Drug discovery 4:571-589.
(2) After the step (1) is completed, the reaction system 1 is taken for PCR amplification, and the gRNA fragment is recovered by using an agarose gel recovery kit (Zhuang Mengguo, biological gene technologies Co., ltd.).
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 30s at 68℃,35 cycles; 12. preserving at a temperature of
2.GmBBX4 Acquisition of CDS fragments
(1) Reaction system 2 was prepared. The reaction system 2 was 50. Mu.L composed of 1. Mu.L KOD Plus, 5. Mu.L 10 XPCR Buffer, 5. Mu.L dNTPs, 25 mM MgSO 4 1.5. Mu.L of an aqueous solution of the primer JRH0641-GmBBX4-F (concentration: 10. Mu.M), 1.5. Mu.L of an aqueous solution of the primer JRH0641-GmBBX4-R (concentration: 10. Mu.M), 1. Mu.L of soybean cDNA and 34. Mu.L of ddH 2 O composition
(2) After the step (1) is completed, the reaction system 1 is taken for PCR amplification, and the gRNA fragment is recovered by using an agarose gel recovery kit (Zhuang Mengguo, biological gene technologies Co., ltd.).
The reaction procedure is: 94. 2 min at the temperature; 94. 15 s at C, 30s at 57℃, 30s at 68℃,35 cycles; 12. preserving at a temperature of
3. Double cleavage of the vector with restriction enzymes XhoI and SpeIJRH0641And (5) recovering the linearization carrier.
Carrier bodyJRH0641Are described in the following documents: lyu, x, cheng, q, qin, c, li, y, xu, x, ji, r, mu, r, li, h, zhao, t, liu, j, zhou, y, li, h, yang, g, chen, q, and Liu, b (2020). GmCRY1s Modulate Gibberellin Metabolism to Regulate Soybean Shade Avoidance in Response to Reduced Blue light.
3. Obtaining of ligation products
(1) Will 1 μlYFPFragment, 1. Mu.LGmBBX4The fragment, 0.5. Mu.L of the vector backbone recovered in step 3 (50 ng) and 2.5. Mu.L of 2 XClonExpress Mix (Vazyme, C115) were mixed to obtain a ligation system.
(2) Taking the connecting system, and reacting for 30min at 50 ℃ to obtain a connecting product.
4. The ligation products were transformed into E.coli DH 5. Alpha. Competent cells (ipecac) to obtain several monoclonal antibodies.
And respectively taking each monoclonal as a template, and performing bacterial liquid PCR amplification detection. ComprisesYFP-GmBBX4Cloning the target fragment with the size of 1740bp, and sequencing by using a bacterial liquid to obtain positive cloning.
5. Inoculating positive monoclonal toLB liquid culture medium, culturing to obtain bacterial liquid; then extracting plasmid from bacterial liquid, i.e. recombinant plasmidYFP-GmBBX4。
Based on the sequencing results, recombinant plasmidsYFP-GmBBX4The structure is described as follows: the carrier is put intoJRH0641The restriction enzyme XhoI and SpeI, and the amino acid sequence shown in SEQ ID No. 4 to obtain a recombinant plasmid.
EXAMPLE 3 obtaining transgenic Soybean plants
1. And (3) transforming the constructed recombinant plasmid into agrobacterium EHA105, and then performing soybean transformation.
2. The recombinant plasmid is transformed into soybean variety Wm82, which is called soybean for short, and the T0 generation quasi-transgenic soybean plant is obtained after screening, differentiation and rooting. The specific steps are as follows:
(1) The soybeans were sterilized 2.5 h-3 h with chlorine gas from the reaction of 15 mL concentrated hydrochloric acid and 100 mL sodium hypochlorite. And taking out, and drying the chlorine in an ultra clean bench.
(2) Accelerating germination of seeds: uniformly sowing soybean in germination culture medium, and about 20-30 grains per dish.
(3) Infection with agrobacterium: cutting soybean in half, removing part of embryo tip, and dividing wound in the meristematic region to obtain the infection object: placing soybean explant in recombinant agrobacterium tumefaciens bacterial solution with OD600 nm of about 0.6, and oscillating for 30min at room temperature; taking out the explant, blowing for 10 min under aseptic condition, spreading on co-culture medium, and culturing in dark for 5 days.
(4) Washing 4-5 times with sterile water and hormone-added liquid induction medium, respectively, to ensure cleaning of Agrobacterium. The elongated embryo was excised and only 3-4 mm remained, the embryo was inserted obliquely downward into solid bud induction medium and placed in a 16h light/8h dark,25℃incubator for 2 weeks.
(5) The explants, which were left after 15 days, began to sprout, and the sprouts were excised from the stakes and transferred to new solid bud induction medium without throwing away the long buds, and continued to be cultivated in the greenhouse under light.
(6) After 15 days, the long buds were subcultured into a new solid bud induction medium, and the bean cotyledons without long buds were thrown away and cultured in a greenhouse for 15 days. The explants were co-cultured in solid bud induction medium for 30 days.
(7) Separating the grown callus from the bean cotyledon, throwing away the explant, scraping off the black surface of the callus, transferring to solid bud elongation culture medium, replacing the solid elongation culture medium every 15 days, and repeating the steps for 4-5 times, for 60-80 days. The callus is also screened during elongation culture, and seedlings can grow out during screening.
(8) When the seedlings grow to about 4-5 cm, they are excised from the calli and transferred to rooting medium.
(9) Culturing in rooting culture medium for 20-30 days, transferring the seedling into disposable cup, hardening off with weak light, covering the seedling with another disposable plastic cup, and hardening off for 5 days.
(10) After several days of adaptation, the disposable cup used for moisturizing was removed when a significantly growing root was observed. Transferring the plant into a large basin containing nutrient soil, and continuing culturing.
Example 4 acquisition of transgenic Soybean Positive plants and molecular characterization of mutant types
In order to determine transgenic positive plants, T0 generation fresh and tender leaves are taken, DNA extraction is carried out, and PCR detection is carried out.
For CRISPR knockout vectors, we tested for thembastaResistance genes and Cas9 proteins. And performing PCR amplification according to the positions of the gRNAs, and sequencing.
For overexpression ofGmBBX4Transgenic lines, we detected their basta resistance gene and designed specific primers based on recombinant vectors to detect T-DNA insertion in T0 generation plants. The positive plants were identified as homozygous after two generations of selfing and were tested for both transcription level and protein level expression of GmBBX 4.
After Sanger sequencing, obtaingmbbx4A plant that does not contain Cas9 protein. The edited form is shown in fig. 1A.
Through transcription and protein level identification of transgenic lines, the obtained product is obtainedYFP-GmBBX4#1 and YFP-GmBBX4#3Plants, as shown in FIGS. 1B and 1CShown.
EXAMPLE 5 determination of isoflavone content in mutant soybeans and transgenic soybeans
The experiment was repeated three times to average the values, and the procedure for each repetition was as follows:
after soybeans (Wm 82, gmbx 4-1 and gmbx 4-2) to be detected in the greenhouse are ripe, seeds are harvested and placed in a 37 ℃ oven to be dried to constant weight. 50 seeds were randomly selected, and the isoflavone content was determined in duplicate for every 5 seeds.
The procedure for isoflavone extraction was as follows: (1) 0.02. 0.02 g soy flour was weighed into a2 mL centrifuge tube. (2) Adding 80% chromatographic grade methanol solution 1.0 mL (feed-liquid ratio is 1:50), swirling for 30s, placing on a floating plate, extracting for 1 h with ultrasonic wave (frequency 40 kHz, power 300W) at 50deg.C, taking out every ten minutes, and mixing. (3) centrifugation at 12000 rpm at 4℃for 10 min. (4) The supernatant was filtered through a 0.22 μm organic phase needle filter and injected into an Agilent autosampler vial (2 mL), and stored at 20 ℃ for on-machine detection.
The isoflavone content is measured by ultra high performance liquid chromatography (UPLC) method, wherein the column temperature is 40 deg.C, the DAD detector is used for detecting the isoflavone content by DIONEX Ultimate 3000, the wavelength is 254 and nm, and the chromatographic column is used for: ACQUITY UPLC HSS T3 1.8 μm, 2.1 mm*100 mm column). Sample injection amount: 2. mu L, mobile phase: a:0.5% acetic acid (analytically pure), B:100% acetonitrile (Merck). Mobile phase flow rate: 0.4 mL/min, gradient elution is: 0-16 min 15% -26% B (v/v), 16-16.1 min 26% -15% B, 16.1-18 min 15% B.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The total isoflavone content in the transgenic soybean seeds is shown in figure 2.gmbbx4The total isoflavone content was 1.32 times the wild type content.YFP-GmBBX4The total isoflavone content in the transgenic soybean seeds is 0.78 times and 0.81 times that of the wild type.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The genistin content in the transgenic soybean seeds is shown in fig. 3.gmbbx4The genistin content in the medium was 2.01 times as high as that in the wild type.YFP-GmBBX4The genistin content in the transgenic soybean seeds is 0.63 times of the wild type content respectivelyAnd 0.85 times.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The Huang Douhuang glycoside content of the transgenic soybean seeds is shown in fig. 4.gmbbx4The content of daidzein is 1.27 times of that of wild type.YFP-GmBBX4The content of daidzein in the transgenic soybean seeds is 0.96 times and 0.86 times that of the wild type.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The malonyl genistin content of the transgenic soybean seeds is shown in figure 5.gmbbx4The malonyl genistin content is 1.72 times the wild type content.YFP- GmBBX4The malonyl genistin content in the transgenic soybean seeds was 0.61 times and 0.86 times the wild type content, respectively.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The malonyl daidzein content of the transgenic soybean seeds is shown in figure 6.gmbbx4The malonyl daidzein content is 1.22 times the wild type content.YFP- GmBBX4The content of malonyl daidzein in the transgenic soybean seeds is 0.83 times and 0.86 times that of the wild type.
The seeds of soybean Wm82 are prepared,gmbbx4mutant seedYFP-GmBBX4The malonyl-soybean glycoside content of the transgenic soybean seeds is shown in fig. 7.gmbbx4The malonyl soyase content is 1.41 times that of the wild type.YFP-GmBBX4The malonyl-soyase content in the transgenic soybean seeds was 0.83 times and 0.84 times that of the wild type.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
Claims (11)
1. The application of any one of the following substances in regulating and controlling the synthesis of soybean isoflavone:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is the amino acid sequence described by SEQ ID NO.2 in the sequence table;
the soybean B-box transcription factor GmBBX4 gene is a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.
2. The application of any one of the following substances in preparing products for regulating and controlling the synthesis of soybean isoflavone:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is the amino acid sequence described by SEQ ID NO.2 in the sequence table;
the soybean B-box transcription factor GmBBX4 gene is a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.
3. Use of any of the following for the cultivation of high/low isoflavone soybeans:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is the amino acid sequence described by SEQ ID NO.2 in the sequence table;
the soybean B-box transcription factor GmBBX4 gene is a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.
4. Use of any of the following in the preparation of a product for cultivating high/low isoflavone soybeans:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is the amino acid sequence described by SEQ ID NO.2 in the sequence table;
the soybean B-box transcription factor GmBBX4 gene is a nucleotide sequence shown in SEQ ID NO.1 in the sequence table.
5. The application of any one of the following substances in soybean breeding:
(1) The soybean B-box transcription factor GmBBX4 gene;
(2) Soybean B-box transcription factor GmBBX4;
(3) Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing soybean B-box transcription factor GmBBX4 genes;
the amino acid sequence of the transcription factor GmBBX4 is the amino acid sequence described by SEQ ID NO.2 in the sequence table;
the soybean B-box transcription factor GmBBX4 gene is a nucleotide sequence shown in SEQ ID NO.1 in the sequence table;
the purpose of the soybean breeding is to obtain high/low isoflavone soybeans.
6. The use according to any one of claims 1 to 5, wherein the isoflavone is selected from one or more of genistin, daidzein and malonyl daidzein.
7. The use according to claim 6, wherein the genistin is malonyl genistin and the daidzein is malonyl daidzein.
8. A method for controlling soybean isoflavone synthesis, which is characterized by being selected from the following (B1) or (B2)
(B1) The method for improving the isoflavone content of the soybean comprises the following steps:
the method comprises reducing the content and/or activity of the transcription factor GmBBX4 of claim 1;
the reduction of the content and/or activity of the transcription factor GmBBX4 of claim 1 is achieved by editing the encoding gene of the GmBBX4 with a CRISPR-Cas9 system;
(B2) A method for inhibiting the isoflavone content of soybeans, which comprises the following steps:
the method comprises increasing the content and/or activity of the transcription factor GmBBX4 of claim 1;
the improvement of the content and/or activity of the transcription factor GmBBX4 of claim 1 is achieved by increasing the expression level of the transcription factor GmBBX4 gene.
9. The method of claim 8, wherein the CRISPR-Cas9 system comprises the nucleotide sequence of a gRNA targeting the GmBBX4 gene:
GmBBX4-gRNA1: GAGTGTACGGACTCGAAGAACGG；
GmBBX4-gRNA2: GCTGACGTCAGCATCGGCGTCGG。
10. the method for controlling soybean isoflavone synthesis according to claim 8, wherein the increasing of the expression level of the transcription factor GmBBX4 gene is achieved by transferring a recombinant plant expression vector that overexpresses GmBBX4 into a plant.
11. The method for regulating and controlling soybean isoflavone synthesis according to claim 10, wherein the recombinant plant expression vector is specifically constructed and obtained by the following method: the GmBBX4 gene fragment is constructed to a JRH0641 vector by a homologous recombination method so as to obtain a recombinant plant expression vector.
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