Soybean GmSUC6 gene and application of protein encoded by soybean GmSUC6 gene in regulation and control of protein content of soybean seeds

Abstract

The invention relates to the technical field of gene engineering, and discloses application of a soybean GmSUC6 gene and an encoded protein thereof in regulating and controlling the protein content of soybean seeds. The invention reveals that the soybean GmSUC6 gene and the encoded protein thereof have the function of regulating and controlling the protein content of soybean seeds for the first time, and the protein content of soybeans can be effectively improved by mutating the encoded protein of the GmSUC6 gene in plants. The GmSUC6 gene and the encoded protein thereof provided by the invention can be used for breeding high-protein soybean varieties, and have a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to the application of soybean GmSUC6 gene and its encoded protein in regulating the protein content of soybean seeds. Background Art

[0002] Soybean is an important food and oil crop, and its seed protein content is one of the key indicators to measure soybean quality. Soy protein is one of the few high-quality proteins containing all essential amino acids, especially rich in lysine, and is suitable as a high-quality source of dietary protein. For vegetarians or people who limit their intake of animal protein, soy protein is an important nutritional supplement. Soy protein has a wide range of uses and is used in animal feed and human food in a variety of ways. Soy protein is widely used in the food industry and can be used to make soy milk, tofu, vegetarian meat, protein powder and other products. Its functional properties (such as emulsification and gelation) make it an important raw material in food processing. Soy protein is also rich in a variety of ingredients that are beneficial to the human body, such as bioactive peptides and isoflavones, which are beneficial to reducing the risk of chronic diseases such as cardiovascular disease, diabetes, and immune disorders. Therefore, improving the protein content of soybean seeds is one of the important directions for high-quality soybean breeding. Summary of the invention

[0003] The purpose of the present invention is to provide application of soybean GmSUC6 gene and its encoded protein in regulating the protein content of soybean seeds.

[0004] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of soybean GmSUC6 gene and its encoded protein in regulating the protein content of soybean seeds.

[0005] The soybean GmSUC6 gene is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; or (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 1.

[0006] The CDS sequence of gene GmSUC6 is shown in SEQ ID NO:2.

[0007] In a second aspect, the present invention provides a method for increasing the protein content of soybean seeds. GmSUC6 The gene is modified to make the gene function ineffective, thereby increasing the protein content of soybean seeds.

[0008] Furthermore, the method includes: taking the soybean GmSUC6 gene as a target, designing a gRNA sequence based on CRISPR-Cas9, connecting a DNA fragment encoding the gRNA sequence to a vector carrying CRISPR-Cas9, transforming soybeans, and thereby obtaining transgenic soybeans with a functional loss of the gene. Specifically, taking the soybean GmSUC6 gene as a target, designing a knockout target sequence based on CRISPR-Cas9, connecting an oligonucleotide sequence encoding the knockout target sequence to a pBlu gRNA intermediate vector to construct a gRNA, then enzymatically cutting the constructed gRNA, connecting the enzymatically cut gRNA to a CRISPR-Cas9 vector, and then transforming soybeans, thereby obtaining transgenic soybeans with a functional loss of the gene.

[0009] Accordingly, the nucleotide sequence of the gRNA knockout target is shown in SEQ ID NO:5.

[0010] In a third aspect, the present invention provides soybean GmSUC6 gene mutants, including mutants gmsuc6-1 and gmsuc6-2 , among which the mutant gmsuc6-1 for: I) the nucleotide sequence shown in SEQ ID NO:3; II) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence expresses a protein with the same function; III) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:3 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or IV) is a nucleotide sequence that has more than 90% homology with the nucleotide sequence of I), II) or III) and expresses a protein with the same function.

[0011] Mutants gmsuc6-2 for: i) the nucleotide sequence shown in SEQ ID NO:4; ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence expresses a protein with the same function; iii) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses a protein with the same function.

[0012] In a fourth aspect, the present invention provides the use of the soybean GmSUC6 gene mutant or a biological material containing the gene mutant in regulating the protein content of soybean seeds.

[0013] In a fifth aspect, the present invention provides the use of the soybean GmSUC6 gene mutant or a biological material containing the gene mutant in preparing transgenic plants.

[0014] In a sixth aspect, the present invention provides a method for increasing the protein content of soybean seeds, the method comprising: gmsuc6-1 or gmsuc6-2 It is introduced into soybean through plasmid or integrated into soybean chromosome through genetic engineering.

[0015] In a seventh aspect, the present invention provides the use of transgenic soybeans obtained according to the method in plant breeding.

[0016] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention discloses for the first time that the soybean GmSUC6 gene and its encoded protein have the function of regulating the protein content of soybean seeds, and the protein content of soybeans can be effectively increased by mutating the encoded protein of the GmSUC6 gene in plants. The GmSUC6 gene and its encoded protein provided by the present invention can be used for the breeding of high-protein soybean varieties, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a partial genomic sequence of the soybean GmSUC6 gene provided in Example 1 of the present invention; wherein bold indicates exon sequences, italics indicate intron sequences, and ATG indicates the start codon.

[0019] Figure 2 This is a partial genomic sequence of the soybean GmSUC6 gene provided in Example 1 of the present invention; wherein bold indicates exon sequences, italics indicate intron sequences, and TGA indicates a stop codon.

[0020] Figure 3 This is a diagram of the intermediate vector used to construct the GmSUC6 gene CRISPR-Cas9 knockout plant provided in Example 2 of the present invention.

[0021] Figure 4 A diagram of the CRISPR-Cas9 vector used to construct a GmSUC6 gene CRISPR-Cas9 knockout plant provided in Example 2 of the present invention.

[0022] Figure 5 Two mutants produced by knocking out the GmSUC6 gene provided in Example 2 of the present invention gmsuc6-1 , gmsuc6-2 The mutation pattern diagram.

[0023] Figure 6 This is a schematic diagram of the protein content of the plants expressing the GmSUC6 gene mutant and the wild-type Williams 82 soybean seeds provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] The present invention aims to provide application of soybean GmSUC6 gene and its encoded protein in regulating the protein content of soybean seeds.

[0025] The present invention adopts the following technical solution: The present invention provides a GmSUC6 protein, comprising: an amino acid sequence as shown in SEQ ID NO:1.

[0026] The GmSUC6 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2.

[0027] The present invention also provides a nucleic acid, which is used to encode the GmSUC6 protein.

[0028] The nucleic acid includes genomic DNA, cDNA, recombinant DNA, mRNA, hnRNA encoding the GmSUC6 protein; or a nucleic acid molecule that is reverse complementary to the above DNA, cDNA, recombinant DNA or mRNA.

[0029] The present invention also provides two GmSUC6 gene mutants ( gmsuc6-1 and gmsuc6-2 ), comprising: as shown in SEQ ID NO: 3 gmsuc6-1 Nucleotide sequence, as shown in SEQ ID NO:4 gmsuc6-2 Nucleotide sequence.

[0030] The present invention also provides a biomaterial, which comprises the nucleic acid or the GmSUC6 Gene mutants; the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0031] The present invention also provides a kit, comprising one or more of the GmSUC6 protein, the nucleic acid, the GmSUC6 gene mutant, or the biological material.

[0032] The present invention further provides the use of the GmSUC6 protein, or the nucleic acid, or the GmSUC6 gene mutant, or the biological material, or the kit in increasing the protein content of soybean seeds.

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0034] Example 1 This embodiment provides a method for isolating the GmSUC6 gene in soybean and performing structural analysis thereof, which specifically includes the following process: 1. Isolation of GmSUC6 gene The present invention extracts total RNA from soybean variety Williams 82, uses the total RNA as a template, and uses oligonucleotide sequence (T)17 as a primer to synthesize the first chain of cDNA. Using the first chain of cDNA as a template, PCR amplification is performed using a forward primer (5'-ATGGAGCCTCTCTCTTCCACC -3') and a reverse primer (5'-TTAGTGGAATCCTCCTCCGGC -3'). A 1566 bp long cDNA is obtained. GmSUC6 Gene cDNA fragment, and the fragment was pEasy - Blunt The vector (Kangrun Biotechnology Co., Ltd.) was connected and named Blunt- GmSUC6 .

[0035] The CDS sequence of the obtained GmSUC6 gene is shown in SEQ ID NO: 2, and the full length of the CDS of the GmSUC6 gene is 1566 bp; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, which has 522 amino acids in total.

[0036] 2. Analysis of GmSUC6 gene structure DNA was extracted from young leaves of soybean variety Williams 82, and the genomic DNA was used as a template to amplify the GmSUC6 genomic fragment. The GmSUC6 genomic sequence is shown in Figure 1 and Figure 2 As shown, the total length is 3677 bp, including 3 exons and 2 introns.

[0037] Example 2 The present invention constructs a GmSUC6 mutant ( gmsuc6-1 , gmsuc6-2 ), the specific process is as follows: 1. Design of GmSUC6 gene knockout target sequence Search the GmSUC6 gene in the Arabidopsis database (www.arabidopsis.org) to obtain its amino acid sequence. Use the BLAST tool (https: / / phytozome-next.jgi.doe.gov / blast-search) to find the corresponding homologous gene in the Glycine max (soybean) genome. Use the CRISPR design tool (http: / / cfans-pmorrell.oit.umn.edu / CRISPR / ) to input the DNA sequence of GmSUC6. Design and synthesize the target oligonucleotide sequence (oligo) with sticky ends.

[0038] Oligonucleotide sequence: F:5'-gattGCTGGGGATTCCCCACACTT-3'; R:5'-aaacAAGTTGTGGGGAATCCCCAGC-3'.

[0039] 2. gRNA cloning The oligonucleotide sequence (oligo) of the synthetic target was diluted and annealed (50°C, 6 hours), and the pBlu gRNA (https: / / stuparlab.cfans.umn.edu / protocols / crisprcas9-glycine-max) vector was digested with BbsI ( Figure 3 ). The digestion products were separated by gel electrophoresis, and a 3500 bp vector fragment was extracted. The annealed oligonucleotide sequence (oligo) was ligated to the digested pBlu gRNA vector (using T4 ligase, 16°C overnight). The ligation product was transformed into Escherichia coli (DH5α) and positive clones were screened. The plasmid was extracted and sequenced (using T3 primer 5'-aattaaccctcactaaaggg-3') to confirm that the gRNA sequence was correct. The nucleotide sequence of the gRNA knockout target is shown in SEQ ID NO:5.

[0040] 3. Insert gRNA into Cas9 vector Use EcoRI to digest the verified pBlu gRNA vector and target Cas9 vector. Separate the digestion products by gel electrophoresis and extract the 557bp gRNA fragment. Mix the gRNA fragment with the digested Cas9 vector (https: / / stuparlab.cfans.umn.edu / protocols / crisprcas9-glycine-max) Figure 4 ) ligated (using T4 ligase, 16°C overnight), the ligated product was transformed into Escherichia coli (DH5α), and positive clones were screened. Colony PCR was used to screen positive clones to confirm that the gRNA fragment was correctly inserted. The plasmid was extracted and sequenced to ensure that the vector was constructed correctly.

[0041] 4. Transform soybean and screen positive plants The constructed CRISPR-Cas9 vector was introduced into Williams 82 soybean callus by Agrobacterium-mediated transformation. The positive transformed plants were screened using the screening marker Bar cassette. The genomic DNA of the transformed plants was extracted, and the knockout of the target gene was verified by PCR and sequencing to confirm whether it was a homozygous knockout plant. The knockout results were as follows: Figure 5 shown.

[0042] Example 3 GmSUC6 mutant ( gmsuc6-1 , gmsuc6-2 ) and Williams 82 seeds protein content determination, with Williams 82 seeds protein content as the control, the specific method is as follows: The protein content of mature soybean seeds was determined using a Thermo Fisher Antaris II FT-NIR analyzer (Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Key Laboratory of Soybean Molecular Design, Chinese Academy of Sciences). At least 30 seeds were prepared for each plant, and the determination was repeated 3 times. The average value of the 3 replicates was taken as the soybean seed protein content of the plant.

[0043] Williams 82, gmsuc6-1 and gmsuc6-2 Ten individual plants were harvested and the protein content of the seeds of each individual plant was measured. The average of the measured results of all individual plants of each variety was taken as the protein content of soybean seeds of each variety. The measurement results are shown in Figure 6 shown.

[0044] From the above experimental results, we can see that compared with the control group (Williams 82), gmsuc6-1 and gmsuc6-2The protein content was significant, indicating that GmSUC6 protein negatively regulated the protein content of soybeans. Therefore, mutation of GmSUC6 can effectively increase the protein content of soybeans, and GmSUC6 can be used as a potential target for breeding soybeans with high protein content.

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of soybean GmSUC6 gene and its encoded protein in regulating soybean seed protein content; The soybean GmSUC6 gene is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The regulation is negative regulation.

3. A method for increasing the protein content of soybean seeds, characterized in that: The soybean GmSUC6 gene is modified to make the gene function deficient, thereby increasing the protein content of soybean seeds; wherein the soybean GmSUC6 gene is the same as that described in claim 1.

4. The method according to claim 3, characterized in that The method comprises: taking the soybean GmSUC6 gene as a target, designing a gRNA sequence based on CRISPR-Cas9, connecting a DNA fragment containing the encoding gRNA sequence to a vector carrying CRISPR-Cas9, transforming soybean, and thereby obtaining transgenic soybean with the gene function missing.

5. The method according to claim 4, characterized in that The nucleotide sequence of the gRNA action site is shown in SEQ ID NO:

5.

6. A soybean GmSUC6 gene mutant, characterized in that: Including mutants gmsuc6-1 and gmsuc6-2 , among which the mutant gmsuc6-1 for: I) the nucleotide sequence shown in SEQ ID NO:3; II) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence expresses a protein with the same function; III) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:3 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS, and the membrane is washed with the solution; or IV) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of I), II) or III) and expresses a protein with the same function; Mutants gmsuc6-2 for: i) the nucleotide sequence shown in SEQ ID NO:4; ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 4 and the nucleotide sequence expresses a protein with the same function; iii) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses a protein with the same function.

7. Use of the soybean GmSUC6 gene mutant or a biological material containing the gene mutant according to claim 6 in regulating the protein content of soybean seeds.

8. Use of the soybean GmSUC6 gene mutant or a biological material containing the gene mutant according to claim 6 in preparing transgenic plants.

9. A method for increasing the protein content of soybean seeds, characterized in that: The method comprises: gmsuc6-1 or gmsuc6-2 It is introduced into soybean through plasmid or integrated into soybean chromosome through genetic engineering.

10. Use of the transgenic soybean obtained according to the method of any one of claims 3 to 5 or 9 in plant breeding.

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