CN116555284A - 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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- CN116555284A CN116555284A CN202310447471.6A CN202310447471A CN116555284A CN 116555284 A CN116555284 A CN 116555284A CN 202310447471 A CN202310447471 A CN 202310447471A CN 116555284 A CN116555284 A CN 116555284A
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- soybean
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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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- 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
- 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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- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
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- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
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Abstract
The invention discloses an application of GmBBX4 gene in regulating and controlling soybean isoflavone synthesis. The nucleotide sequence of the soybean GmBBX4 gene is shown as SEQ ID NO.1 in a sequence table; the amino acid sequence of the protein coded by the soybean 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. The method finds that the content of secondary metabolites in soybean with over-expressed GmBBX4 gene is obviously reduced, so that the functional deletion of GmBBX4 gene in plants is an effective way for improving plant quality, and has important production and theoretical research significance.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to application of GmBBX4 gene in regulating and controlling soybean isoflavone synthesis
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.
So far, the research of soybean GmBBX4 gene has not been reported. Therefore, the cloning and functional research of the GmBBX4 gene are of great significance.
Disclosure of Invention
The invention aims to provide an application of a soybean GmBBX4 gene in regulating and controlling plant isoflavone synthesis.
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.
Editing the coding gene of the GmBBX4 by using a CRISPR-Cas9 system. 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 as 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.
The recombinant plant expression vector for over-expressing GmBBX4 is transferred into soybean cultivar Williams 82 (hereinafter referred to as Wm 82) by a transgenic technology, so that the expression level of GmBBX4 is improved in soybean. After the overexpression of GmBBX4, the content of soybean isoflavone in the grains is obviously reduced.
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.
The invention has the remarkable advantages that:
the invention provides an application of a protein coded by a B-box family gene GmBBX4 gene in regulating and controlling soybean isoflavone accumulation for the first time. Therefore, the invention can obtain plant lines with high soybean isoflavone and low soybean isoflavone and has important application value.
Drawings
FIG. 1 is a graph showing the detection of the overexpression expression level of the soybean GmBBX4 mutant gene-edited form (1A) and YFP-GmBBX4 transgenic line of the present invention (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.
Detailed Description
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.
The soybean (Glycine max) variety Wm82 is provided by the national center for soybean improvement (National Center for Soybean Improvement).
The gene editing vector used in the invention is JRH0912, and the over-expression vector is JRH0641, which are 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：
AATGTGCCACCACATGGATTGAGTGTACGGACTCGAAGAAGTTTTAGAGCTAGAAATA
GCAA
GmBBX4-2+sgRNA-F：
AATGTGCCACCACATGGATTGGACGGGGATGCGCTCGTGGGTTTTAGAGCTAGAAATA
GCAA
(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 dNTP, 25mM 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 vector JRH0951 and 34. Mu.L of ddH 2 O composition.
Vector JRH0951 is described in the following documents: sun X, hu Z, chen R, jiang Q, song G, zhang H, xi Y. (2015) Targeted 845mutagenesis 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 10s at 68 ℃ and 35 cycles; preserving at 12 ℃.
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 dNTP, 25mM MgSO 4 1.5. Mu.L of primer U6-Xbal-F aqueous solution (concentration: 10. Mu.M), 1.5. Mu.L of primer U6-R aqueous solution (concentration: 10. Mu.M), 1. Mu.L of vector JRH0951 and 34. Mu.L of 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 10s at 68 ℃ and 35 cycles; preserving at 12 ℃.
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 dNTP, 25mM 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 10s at 68 ℃ and 35 cycles; preserving at 12 ℃.
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, 25mM MgSO 4 1.5. Mu.L of aqueous solution of the primer Dual-U6-F1 (concentration: 10. Mu.M), 1.5. Mu.L of aqueous solution of the Dual-U6-R1 primer (concentration: 10. Mu.M), 1. Mu. L U6-gRNA1 fragment and 34. Mu.L of 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, 25mM 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 15s at 68 ℃ and 35 cycles; preserving at 12 DEG C
5. The vector JRH0912 was digested with the restriction enzymes XbaI and BglII, and the vector backbone of about 14000bp was recovered.
Vector JRH0912 is described in the following documents: meng, Y., 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 trunk. Plant Cell Rep 36 (2): 371-374. In the literature the name is pFGC5941-Cas9. The vector JRH0912 contains 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. Inoculating the positive monoclonal to an LB liquid culture medium, and culturing to obtain bacterial liquid; then extracting plasmid, i.e. recombinant plasmid GmBBX4-gRNA from the bacterial liquid.
According to the sequencing result, the structure of the recombinant plasmid GmBBX4-gRNA is described as follows: the DNA fragment between the recognition sequences of the restriction enzymes xbai and bglii of vector JRH0912 was replaced with SEQ ID No:3 to obtain 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 dNTP, 25mM 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 the vector pEargateway 104 and 34. Mu.L of ddH 2 O composition
Vector pEargateway 104 is described in the following literature: 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 30s at 68 ℃ and 35 cycles; preserving at 12 DEG C
Acquisition of GmBBX4 CDS 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 dNTP, 25mM MgSO 4 1.5. Mu.L of an aqueous solution (10. Mu.M concentration) of the primer JRH0641-GmBBX4-F, 1.5. Mu.L of an aqueous solution (10. Mu.M concentration) of the primer JRH0641-GmBBX4-R) 1 μl of soybean cDNA and 34 μl 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 ℃ for 2min; 15s at 94 ℃, 30s at 57 ℃, 30s at 68 ℃ and 35 cycles; preserving at 12 DEG C
3. The linearized vector was recovered by double digestion of the vector JRH0641 with restriction enzymes XhoI and SpeI.
Vector JRH0641 is 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). GmCRY1sModulate Gibberellin Metabolism to Regulate Soybean Shade Avoidance in Response to Reduced Blue light.
3. Obtaining of ligation products
(1) mu.L of YFP fragment, 1. Mu.L of GmBBX4 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. Clone containing 1740bp of YFP-GmBBX4 target fragment is sent to bacterial liquid for sequencing, and positive clone is obtained.
5. Inoculating the positive monoclonal to an LB liquid culture medium, and culturing to obtain bacterial liquid; then extracting plasmid from bacterial liquid, namely recombinant plasmid YFP-GmBBX4.
According to the sequencing result, the structure of the recombinant plasmid YFP-GmBBX4 is described as follows: the DNA fragment between the recognition sequences of the restriction enzymes Xho i and Spe i of vector JRH0641 was replaced with a DNA fragment encoding SEQ ID No:4 to obtain the 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 with 15mL of concentrated hydrochloric acid and 100mL of chlorine gas reacted with sodium hypochlorite for 2.5-3 hours. 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 10min 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 cut off, kept for 3-4mm only, 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 4-5cm, 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 3 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 the CRISPR knockout vector, we detected its basta resistance gene and Cas9 protein. And performing PCR amplification according to the positions of the gRNAs, and sequencing.
For the transgenic line of overexpressing GmBBX4, we detected its basta resistance gene and based on the recombinant vector, designed specific primers to detect the insertion of T-DNA in the 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 GmBBX4.
After Sanger sequencing, gmbbx4 plants were obtained, which did not contain Cas9 protein. The edited form is shown in fig. 1A.
And obtaining YFP-GmBBX4#1 and YFP-GmBBX4#3 plants through transcription and protein level identification of the transgenic lines, as shown in figures 1B and 1C.
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) 0.02g of soybean powder was weighed and placed in a 2mL centrifuge tube. (2) Adding 1.0mL of 80% chromatographic grade methanol solution (feed-liquid ratio is 1:50), swirling for 30s, placing on a floating plate, performing ultrasonic (frequency 40kHz, power 300W) auxiliary extraction at 50 ℃ for 1h, taking out every ten minutes, and mixing uniformly upside down. (3) centrifugation at 12000rpm at 4℃for 10min. (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 20deg.C 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 detection wavelength is 254nm, and the chromatography column is DIONEX Ultimate 3000: ACQUITY UPLC HSS T3 1.8 μm,2.1mm 100 mmcolumn). Sample injection amount: 2 μl, mobile phase: a:0.5% acetic acid (analytically pure), B:100% acetonitrile (Merck). Mobile phase flow rate: 0.4mL/min, gradient elution is: 0-16min 15% -26% B (v/v), 16-16.1min 26% -15% B,16.1-18min 15% B.
The total isoflavone content in soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmBBX4 transgenic soybean seed is shown in fig. 2. The total isoflavone content in gmbbx4 was 1.32 times the wild type content. The total isoflavone content in YFP-GmBBX4 transgenic soybean seed is 0.78 times and 0.81 times that of wild type seed.
The genistin content in soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmBBX4 transgenic soybean seed is shown in fig. 3. The genistin content in gmbbx4 was 2.01 times the wild type content. The genistin content in YFP-GmBBX4 transgenic soybean seeds was 0.63 times and 0.85 times that of the wild type.
The Huang Douhuang glycoside content of soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmbBBX 4 transgenic soybean seed is shown in FIG. 4. The daidzein content of gmbbx4 is 1.27 times the wild type content. The content of daidzein in YFP-GmBBX4 transgenic soybean seeds is 0.96 times and 0.86 times that of wild type.
The malonyl genistin content of soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmBBX4 transgenic soybean seed is shown in fig. 5. The malonyl genistin content in gmbbx4 was 1.72 times the wild type content. The malonyl genistin content in YFP-GmBBX4 transgenic soybean seeds was 0.61 times and 0.86 times the wild type content, respectively.
The malonyl daidzein content of soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmBBX4 transgenic soybean seed is shown in figure 6. The malonyl daidzein content of gmbbx4 is 1.22 times the wild type content. The malonyl daidzein content in YFP-GmBBX4 transgenic soybean seeds was 0.83 times and 0.86 times that of the wild type.
The malonyl daidzin content of soybean Wm82 seed, gmBBX4 mutant seed and YFP-GmBBX4 transgenic soybean seed is shown in fig. 7. The malonyl soyase content in gmbbx4 was 1.41 times the wild type content. The malonyl-daidzin content in YFP-GmBBX4 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 (10)
1. The application of any one of the following substances in regulating and controlling the synthesis of 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.
2. The application of any one of the following substances in preparing a product for regulating and controlling the synthesis of 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.
3. Use of any 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.
4. Use of any of the following in 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.
5. The use in plant breeding of any of the following:
(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.
6. The use according to any one of claims 1 to 5, wherein the soybean B-box transcription factor GmBBX4 gene has the nucleotide sequence set forth in SEQ ID No.1 of the sequence listing.
7. The use according to any one of claims 1 to 5, wherein the plant is a monocotyledonous plant or a dicotyledonous plant; preferably, the dicotyledonous plant is a plant of the family Leguminosae.
8. The use according to any one of claims 1 to 5, wherein the isoflavone is selected from one or more of genistin, daidzein, malonyl genistin, malonyl daidzein, and malonyl daidzein.
9. A method for controlling the synthesis of plant isoflavones, which is selected from the group consisting of (B1) and (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;
preferably, 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;
more preferably, the nucleotide sequence of the gRNA targeting the GmBBX4 gene in the CRISPR-Cas9 system is: gmBBX4-gRNA1: GAGTGTACGGACTCGAAGAACGG;
GmBBX4-gRNA2:GCTGACGTCAGCATCGGCGTCGG；
(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.
10. The method for regulating and controlling plant isoflavone synthesis according to claim 9, wherein the improvement 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; preferably, 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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