Application of soybean GmSH2 gene in improvement of soybean sugar content

Abstract

The invention discloses an application of a soybean GmSH2 gene in improving the sugar content of soybeans. The GmSH2 gene comprises a GmSH2a gene with a nucleotide sequence as shown in SEQ ID NO: 1 and a GmSH2b gene with a nucleotide sequence as shown in SEQ ID NO: 3. Through deletion of the soybean GmSH2 gene in the soybean, the sugar content of the soybean can be remarkably increased, and the soybean GmSH2 gene participates in sugar content regulation and control. The discovery of new functions of the soybean GmSH2 gene provides new gene targets and resources for genetic breeding of high-sugar-content soybeans, and can improve the utilization value of vegetable soybeans.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of crop genetic breeding, and particularly relates to application of a soybean GmSH2 gene in improving sugar content of soybean. BACKGROUND

[0002] Soybean (Glycine max, grain soybean) is an important economic crop in the world, mainly planted as raw material for edible oil, protein and biodiesel, and its whole bean can also be processed into tofu, soy milk and fermented food, etc.

[0003] In East Asia and the world, a kind of vegetable soybean called "hair bean" (China) or "branch bean" (Japan) gradually rises as a high-value vegetable crop. This kind of soybean is mainly sold in the form of fresh or frozen pods, and its popularity is due to its unique sensory and nutritional characteristics: sweet and soft flavor, plump seeds, bright pod color, and high content of protein, soluble sugar, vitamins, minerals, dietary fiber and isoflavones, etc. Among them, the taste quality composed of sweetness, umami, texture and aroma directly affects consumer preference, and the sweetness dominated by soluble sugar content is the most critical evaluation index, and sucrose accounts for more than 80% of the total soluble sugar content of vegetable soybean. Therefore, it is of great significance to analyze the regulation mechanism of soluble sugar biosynthesis for directional improvement breeding.

[0004] The existing research on the regulation of soluble sugar in soybean is mainly based on genome-wide association analysis (GWAS) and quantitative trait locus (QTL) positioning to screen candidate genes. For example, 7 potential QTL sites related to sucrose content in grain soybean were identified by GWAS (19), and the interval of 19,496,314-19,698,413 bp on chromosome 8 was located as a candidate interval affecting seed sucrose content in vegetable soybean.

[0005] However, the functional verification research of these candidate genes is still insufficient, and it is necessary to further explore the genes involved in the regulation of soluble sugar in soybean. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a gene that can improve the sugar content of vegetable soybean.

[0007] The specific technical scheme for achieving the above-mentioned purpose of the present application includes the following.

[0008] In a first aspect of the present application, the application of soybean GmSH2 gene in improving the sugar content of soybean is provided, and the GmSH2 gene includes a GmSH2a gene with a nucleotide sequence as shown in SEQ ID NO: 1 and a GmSH2b gene with a nucleotide sequence as shown in SEQ ID NO: 3.

[0009] In a second aspect of the present application, the use of a protein encoded by a soybean GmSH2 gene in increasing the sugar content of soybeans is provided, wherein the protein encoded by the GmSH2 gene comprises a protein encoded by a GmSH2a gene with an amino acid sequence as shown in SEQ ID NO: 2 and a protein encoded by a GmSH2b gene with an amino acid sequence as shown in SEQ ID NO: 4.

[0010] In a third aspect of the present application, the use of a knockout vector of a soybean GmSH2 gene in increasing the sugar content of soybeans is provided, wherein the knockout vector comprises a CRISPR target site with a sequence as shown in SEQ ID NO: 7.

[0011] In a fourth aspect of the present application, the use of a recombinant engineering bacterium transformed with a knockout vector of a soybean GmSH2 gene in increasing the sugar content of soybeans is provided, wherein the knockout vector comprises a CRISPR target site with a sequence as shown in SEQ ID NO: 7.

[0012] In a fifth aspect of the present application, a method for increasing the sugar content of soybeans is provided, which comprises knocking out a soybean GmSH2 gene in a plant, wherein the GmSH2 gene comprises a GmSH2a gene with a nucleotide sequence as shown in SEQ ID NO: 1 and a GmSH2b gene with a nucleotide sequence as shown in SEQ ID NO: 3.

[0013] The present inventors have found that the deletion of a soybean GmSH2 gene in soybeans can significantly increase the contents of soluble sugar, sucrose and total sugar in the leaves, fresh beans and seeds of vegetable soybeans, proving that the soybean GmSH2 gene is involved in the regulation of the sugar content of soybeans. The discovery of the new function of the soybean GmSH2 gene provides a new gene target and resource for the genetic breeding of high-sugar vegetable soybeans, and can improve the value of vegetable soybeans. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 FIG. 1 is a structural diagram of the CRISPR vector before digestion with the restriction enzyme XbaI in Example 1 of the present application.

[0015] Figure 2 FIG. 4 is the target sequencing result of the GmSH2 gene knockout mutant strain in Example 2 of the present application.

[0016] Figure 3 FIG. 6 is the sugar content result of the leaves of the GmSH2 gene knockout mutant in Example 3 of the present application.

[0017] Figure 4 FIG. 7 is the sugar content result of the fresh beans of the GmSH2 gene knockout mutant in Example 3 of the present application.

[0018] Figure 5Sugar content results of the seeds of the GmSH2 gene knockout mutant in Example 3 of the present application. DETAILED DESCRIPTION

[0019] For the purpose of promoting the understanding of the present application, the present application will be described in further detail below. The present application can be realized in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the present application to those skilled in the art.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0021] Unless otherwise indicated, the examples were carried out in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning: A Laboratory Manual (2013), or as suggested by the manufacturer's instructions.

[0022] In some embodiments of the present application, the use of soybean GmSH2 gene in improving the sugar content of soybean is disclosed, wherein the GmSH2 gene includes GmSH2a gene with nucleotide sequence as shown in SEQ ID NO: 1 and GmSH2b gene with nucleotide sequence as shown in SEQ ID NO: 3.

[0023] In one embodiment, the improvement of the sugar content of soybean is achieved by knocking out the soybean GmSH2 gene. The two homologous genes of soybean GmSH2 gene, GmSH2a gene and GmSH2b gene, are simultaneously knocked out, so as to cause the loss of the function of the whole gene.

[0024] In one embodiment, the soybean is vegetable soybean.

[0025] In one embodiment, the soybean is vegetable soybean.

[0026] In some embodiments of the present application, the use of the encoded protein of soybean GmSH2 gene in improving the sugar content of soybean is disclosed, wherein the encoded protein of GmSH2 gene includes the encoded protein of GmSH2a gene with amino acid sequence as shown in SEQ ID NO: 2 and the encoded protein of GmSH2b gene with amino acid sequence as shown in SEQ ID NO: 4.

[0027] In some embodiments of the present application, the application discloses a use of a knock-out vector of a soybean GmSH2 gene in improving sugar content of soybean, wherein the knock-out vector comprises a CRISPR target site as shown in SEQ ID NO: 7.

[0028] In one embodiment, the knock-out vector is constructed by the following steps:

[0029] (1) amplifying a GmU6 promoter fragment using soybean leaf DNA as a template and SEQ ID NO: 5 and SEQ ID NO: 6 as primers;

[0030] (2) performing PCR amplification using a CRISPR target site as shown in SEQ ID NO: 7 as a template and SEQ ID NO: 8 and SEQ ID NO: 9 as primers;

[0031] (3) performing PCR amplification using the GmU6 promoter fragment obtained in step (1) and the PCR amplification product obtained in step (2) as templates and SEQ ID NO: 5 and SEQ ID NO: 9 as primers;

[0032] (4) connecting the PCR amplification product of step (3) with a CRISPR vector digested by a restriction enzyme XbaI, and obtaining the knock-out vector.

[0033] In some embodiments of the present application, the application discloses a use of a recombinant engineering bacterium transformed with a knock-out vector of a soybean GmSH2 gene in improving sugar content of soybean, wherein the knock-out vector comprises a CRISPR target site as shown in SEQ ID NO: 7.

[0034] In some embodiments of the present application, the application discloses a method for improving sugar content of soybean, comprising: knocking out a soybean GmSH2 gene in a plant, wherein the GmSH2 gene comprises a GmSH2a gene as shown in SEQ ID NO: 1 and a GmSH2b gene as shown in SEQ ID NO: 3.

[0035] In one embodiment, the soybean is vegetable soybean.

[0036] In one embodiment, the soybean is green soybean.

[0037] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] Example 1: Construction of a knock-out vector of a soybean GmSH2 gene

[0039] The present embodiment utilizes the Infusion system (Novozyme) to construct a soybean GmSH2 gene knockout vector, which comprises the following steps:

[0040] 1. Wild-type HC6 soybean seeds were planted in nutrient soil and grown under long-day conditions for 15 days. The first trifoliolate leaves of the soybean were harvested and the DNA was extracted.

[0041] 2. The GmU6 promoter fragment was amplified using soybean leaf DNA as the template and GmU6-XbaI-F (SEQ ID NO: 5) and GmU6-R (SEQ ID NO: 6) as primers. The PCR amplification reaction system and reaction program are shown in Table 1 and Table 2.

[0042] GmU6-XbaI-F (SEQ ID NO: 5):

[0043] GGAAGCTTAGGCCTTCTAGA AAAATAAATGGTAAAATGTC

[0044] GmU6-R (SEQ ID NO: 6): CAATCCATGTGGTGGCACAT

[0045] Table 1

[0046]

[0047] Table 2

[0048]

[0049] 3. The SgRNA sequence (SEQ ID NO: 7) which simultaneously targets the GmSH2a gene (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) and the GmSH2b gene (nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 4) was used as the template, and GmSH2-F (SEQ ID NO: 8, wherein the underlined sequence is the target site sequence of GmSH2a and GmSH2b genes) and sgRNA-XbaI-R (SEQ ID NO: 9) were used as primers for PCR amplification. The PCR amplification reaction system and reaction program are shown in Table 1 (except for the different templates, the others are the same) and Table 2.

[0050] sgRNA (SEQ ID NO: 7): CTAGAATCGAGCTTCGTTC

[0051] GmSH2-F (SEQ ID NO: 8):

[0052] TGTGCCACCACATGGATTGCAGAAGGGAAGGTTCCAAT GTTTTAGAGCTAGAAATAGC

[0053] sgRNA-XbaI-R (SEQ ID NO: 9):

[0054] GGCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGTGCCAC

[0055] The GmU6 promoter fragment obtained in step 2 and the PCR product obtained in step 3 were used as templates, GmU6-XbaI-F (SEQ ID NO: 5) and sgRNA-XbaI-R (SEQ ID NO: 9) were used as primers, and a GmU6-SgRNA fragment was amplified. The PCR amplification reaction system and reaction procedure are shown in Table 1 (except that the templates are different, other conditions are the same) and Table 2.

[0056] 3, digest the CRISPR vector (structure as shown in Figure 1 Table 3) with restriction enzyme XbaI.

[0057] Table 3

[0058]

[0059] 4, perform gel electrophoresis on the PCR product of step 4 and the vector after enzyme digestion of step 5, determine the size of the target band and cut it off (use Axygen gel recovery kit to recover various corresponding products, detailed experimental steps are operated according to the instructions of Axygen company). Place the target band in a 1.5 mL centrifuge tube, connect at 37°C for 30 min (the connection reaction is shown in Table 4), obtain the CRISPR vector for knocking out soybean GmSH2 gene (named CRISPR-GmSH2), and store at 4°C.

[0060] Table 4

[0061]

[0062]

[0063] Nucleotide sequence of GmSH2a (SEQ ID NO: 1, Glyma.04G030300):

[0064] ATGGATTCAACTTGTGCAATCCTGAGTGGCCGCAATCTAGCTAAAGTTTGTGAGGGAATT

[0065] GGAAGAAACAGAAGAAGTGGCTTCTGGGGTGAGAGTACGAGGCGAAGTGTGAACACA

[0066] AGGTTTTTGAGTGTTCAATCATGGAAGACTTCACGTACCAGTAGGAATCTTAGAAACTCC

[0067] AAGCCTGGAAGTGGAATTGCACACGCTGTTCTCACATCAGACATTAACGAAGATTCGAT

[0068] GGCATTTCAAGGGGTACCCACTTTTGAAAAACCCGAAGTGGACCCAAAAAGTGTGGCT

[0069] TCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAAGAGC

[0070] CAAGCCTGCGGTTCCAATTGGCGGGTGTTATAGACTCATAGATATCCCCATGAGCAATTG

[0071] CATCAATAGTGGCATCAGAAAAATATTCATTTTGACGCAGTTCAATTCTTTCTCTCTCAAC

[0072] CGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGAATTACTTTTGGAGATGGGTTTGTG

[0073] GAGGTCTTGGCAGCTACTCAAACACCTGGAGAGGCCGGGAAGAAGTGGTTCCAAGGGA

[0074] CCGCCGATGCTGTAAGACAATTTATATGGGTTTTTGAGGATGCCAAGAACAAGAATGTTG

[0075] AGCATATATTGATACTTTCTGGTGATCATCTTTACCGAATGGACTACATGAACTTTGTACA

[0076] GAGACATGTTGACACAAATGCTGATATCACAGTTTCATGTGTACCCATGGATGACAGTCG

[0077] GGCATCAGACTATGGACTGATGAAAATTGACAAAACAGGACGGATTATACAGTTTGCAG

[0078] AAAAACCTAAGGGATCAGATCTAAAGGCAATGCGTGTTGACACCACTCTTCTAGGGTTA

[0079] TCGCCACAAGAAGCAGAAAAATATCCTTATATTGCATCCATGGGTGTCTATGTGTTTAGA

[0080] ACTGAAACCCTGCTGCAACTATTAAGATGGAATGGTTCTTCATGCAATGACTTTGGATCT

[0081] GAAATTATCCCATCTGCTGTGAATGAGCACAATGTCCAGGCATATTTATTCAATGACTACT

[0082] GGGAAGATATTGGAACTATAAAGTCCTTCTTTGATGCAAATCTTGCCCTAACAGAACAAC

[0083] CTCCTAAATTTGAATTCTATGATCCAAAGACTCCTTTCTTCACTTCCCCAAGATTCCTACC

[0084] ACCTACCAAAGTAGAAAAATGCAAGATTGTGGATGCAATTATATCTCATGGTTGCTTCTT

[0085] GAGGGAGTGTAGCATTCAACATTCCATTGTAGGAGTACGCTCACGTTTGGAGTCTGGTGT

[0086] AGAGCTTCAGGATACGATGATGATGGGTGCCGACTACTATCAAACTGAGTATGAAATTGC

[0087] ATCTCTGCTGGCAGAAGGGAAGGTTCCAATTGGTGTTGGGGAAAATACTAAAATTAGGA

[0088] ATTGCATAATCGACAAGAATGCCAAGATAGGAAGAAATGTGGTCATAGAAAACATCGAT

[0089] GGTGTTCAAGAAGCTGACAGGGCAAAGGAAGGATTCTACATTAGATCGGGCATCACAAT

[0090] TACATTAAAAAATGCAACAATTAAAGATGGAACAGTTATATGA

[0091] Amino acid sequence of GmSH2a (SEQ ID NO: 2):

[0092] MDSTCAILSGRNLAKVCEGIGRNRRSGFWGESTRRSVNTRFLSVQSWKTSRTSRNLRNSKP

[0093] GSGIAHAVLTSDINEDSMAFQGVPTFEKPEVDPKSVASIILGGGAGTRLFPLTGRRAKPAVPIG

[0094] GCYRLIDIPMSNCINSGIRKIFILTQFNSFSLNRHLSRAYSFGNGITFGDGFVEVLAATQTPGE

[0095] AGKKWFQGTADAVRQFIWVFEDAKNKNVEHILILSGDHLYRMDYMNFVQRHVDTNADIT

[0096] VSCVPMDDSRASDYGLMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLSPQEAEKYPYIA

[0097] SMGVYVFRTETLLQLLRWNGSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDAN

[0098] LALTEQPPKFEFYDPKTPFFTSPRFLPPTKVEKCKIVDAIISHGCFLRECSIQHSIVGVRSRLES

[0099] GVELQDTMMMGADYYQTEYEIASLLAEGKVPIGVGENTKIRNCIIDKNAKIGRNVVIENIDGVQEADRAKEGFYIRSGITITLKNATIKDGTVI*

[0100] Nucleotide sequence of GmSH2b (SEQ ID NO: 3, Glyma.06G030400):

[0101] ATGGATTCAGCTTGTGCAACCCTGAATGGCCGCCATCTAGCCAAAGTTAGTGAGGGAAT

[0102] TGGAAGAAACAGAACAAGTGGCTTCTGGGGTGAGAGTACGAGGGGAAGTGTGAACAC

[0103] AAAAAGGTTTTTGAGTGTTCAATCATGCAAGACTTCACGAACCAATAGGAATCTTAGAA

[0104] ACTCCAAGCCTGGAAGTGGAATTGCACGCGCTGTTCTCACATCAGACATCGACGAAGAT

[0105] TCCATGGCATTTCAAGGGGTACCCACTTTTGAGAAACCTGAAGTGGACCCAAAAAGTGT

[0106] GGCTTCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAA

[0107] GAGCCAAGCCAGCGGTTCCAATTGGAGGGTGTTATAGACTCATAGATATCCCCATGAGCA

[0108] ATTGCATCAATAGTGGCATCAGAAAAATTTTCATCTTGACGCAGTTCAATTCTTTCTCTCT

[0109] CAACCGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGCATGACTTTTGGAGATGGGT

[0110] TTGTGGAGGT CTTGGCAGCT ACTCAAACAC CGGGTGAGGC TGGGAAGAAG TGGTTCCA

[0111] AGGGACAGCT GATGCTGTAA GACAATTTAT ATGGGTTTTT GAGGATGCCC AAGAACAAGA

[0112] ATGTTGAGCA TATATTGATA CTTTCTGGCG ATCATCTTTA CCGTATGGAC TATATGGAC TT

[0113] GTACAGAGAC ATGTTGACAC AAATGCCGAT ATCACAGTTT CATGTGTACC CATGGATGAC

[0114] AGTCGGGCAT CAGACTATGG ACTGATGAAA ATTGATAAAA CAGGACGGAT TATACAGTTT

[0115] GCAGAAAAAC CTAAGGGATC AGATCTAAAG GCAATGCGTG TTGACACCACT CTTTAGG

[0116] GTTATTGCCA CAAGAAGCAG AAAACATCCT TATATTGCAT CCATGGGTGT CTACGTGTT

[0117] TAGAACTGAA ACCTTGCTGC AACTATTAAG ATGGAAATGT TCTTCATGCA ATGACTTTGG

[0118] ATCTGAAATT ATCCCATCTG CTGTGAATGA GCACAATGTCC AGGCATATTT GTTCAATGAC

[0119] TACTGGGAAG ATATTGGAAA CTAAGAATTA TTTTGGATGC AATCTTGCTC TAACAGAA

[0120] CAGCCTCCTA AATTTGAATT CTATGATCCA AAGACACCTT TCTTCACTTC CCCAGATTCC

[0121] TACCACCTACCAAAGTAGAAAAATGCAAGATTGTGGATGCAATTATATCTCATGGTTGCT

[0122] TCTTGAGGGAGTGCAGCGTTCAACATTCTATTGTTGGAGTACGCTCACGTTTGGAGTCTG

[0123] GTGTGGAGCTTCAGGATACGATGATGATGGGTGCTGACTATTATCAAACTGAGTATGAAA

[0124] TTGCATCTCTGGTGGCAGAAGGGAAGGTTCCAATTGGTGTCGGGGCAAATACTAAAATC

[0125] AGGAATTGCATAATCGACAAGAATGCCAAGATAGGAAGAAATGTGATCATAGCAAACAC

[0126] CGATGGTGTTCAAGAAGCTGACAGGGCAAAGGAAGGATTCTACATTAGGTCGGGCATCA

[0127] CAGTTACATTAAAAAATGCAACAATCAAAGATGGAACAGTTATATGA

[0128] Amino acid sequence of GmSH2b (SEQ ID NO: 4):

[0129] MDSACATLNGRHLAKVSEGIGRNRTSGFWGESTRGSVNTKRFLSVQSCKTSRTNRNLRNSKPGSG

[0130] IARAVLTSDIDEDSMAFQGVPTFEKPEVDPKSVASIILGGGAGTRLFPLTGRRAKPAVPIGGCYRLID

[0131] IPMSNCINSGIRKIFILTQFNSFSLNRHLSRAYSFGNGMTFGDGFVEVLAATQTPGEAGKKWFQGTA

[0132] DAVRQFIWVFEDAKNKNVEHILILSGDHLYRMDYMDFVQRHVDTNADITVSCVPMDDSRASDYG

[0133] LMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLLPQEAEKHPYIASMGVYVFRTETLLQLLRWK

[0134] CSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDANLALTEQPPKFEFYDPKTPFFTSPRF

[0135] LPPTKVEKCKIVDAIISHGCFLRECSVQHSIVGVRSRLESGVELQDTMMMGADYYQTEYEIASLVA

[0136] EGKVPIGVGANTKIRNCIIDKNAKIGRNVIIANTDGVQEADRAKEGFYIRSGITVTLKNATIKDGTVI*

[0137] Obtaining of soybean GmSH2 gene knockout plant in Example 2

[0138] In this example, the soybean GmSH2 gene knockout vector CRISPR-GmSH2 constructed in Example 1 was transformed into soybean cotyledon node by Agrobacterium EHA105 mediated genetic transformation method to obtain soybean GmSH2 gene knockout plant. The specific steps include the following:

[0139] 1. Preparation of Agrobacterium competence

[0140] Pick single colonies of Agrobacterium tumefaciens EHA105 and place them in 5 mL of LB liquid medium containing the corresponding antibiotic. The EHA105 resistance is 100 μg / mL of rifampicin (Rif). Incubate at 28°C overnight; inoculate 500 μL of overnight culture into 50 mL of LB liquid medium containing the corresponding antibiotic, and incubate at 28°C until the OD 600 is about 0.5; place on ice for 30 min; centrifuge at 5,000 rpm for 10 min at 4°C; resuspend the Agrobacterium cells with 15 mL of pre-cooled 10 mM CaCl2, centrifuge at 5,000 rpm for 10 min at 4°C; resuspend the precipitate with 2 mL of pre-cooled 10 mM CaCl2, divide 100 μL / tube on ice, freeze in liquid nitrogen, and store at -80°C.

[0141] 2. Electroporation of Agrobacterium

[0142] In 50 μL of Agrobacterium competence, 3 μL of plasmid (i.e. soybean GmSH2 gene knockout vector CRISPR-GmSH2 constructed in Example 1) was added, and the mixture was mixed and homogenized by a sterile gun head, and was placed on ice for 30 min. During this period, the super-clean workbench of the electroporation cup was dried and pre-cooled. The competence was transferred to the electroporation cup on ice, and the electroporation transformation was prepared. The Bacterial electroporation transformation was performed using a BIO-RAD transformation instrument (Gene PulserXcell Electroporation System). After electroporation, 700 μL of antibiotic-free LB liquid medium was added to the super-clean bench to recover the bacterial liquid. The bacterial liquid was recovered at 28°C on a shaker at 200 rpm for 2 h. The bacterial liquid was collected by centrifugation and plated. After 2-3 days of culture, the Agrobacterium was grown, and the Agrobacterium monoclonal was screened according to the method of screening positive E. coli monoclonal.

[0143] 3. Agrobacterium infection of soybean cotyledon node

[0144] The co-culture solid / liquid medium, the induction solid / liquid medium, the elongation solid medium, and the rooting medium in this step are all conventional commercially available media.

[0145] (1) The positive Agrobacterium monoclonal screened in step 2 was cultured in a 10 mL test tube for overnight.

[0146] (2) 100 mL of Bleach + 4 mL of concentrated hydrochloric acid was used to sterilize the soybean in a sealed container for 16-18 hours. The soybean was placed in a sterilized super-clean bench for 30 minutes, and the chlorine gas was blown away.

[0147] (3) The co-culture solid / liquid medium, water, filter paper, and conical flask were prepared and sterilized at 121°C for 20 min. The sterilized water was used to soak the beans for about 8 hours (in general, the beans were placed in a 28°C dark incubator for about 8 hours at 6-10 o'clock in the evening, and the beans were not soaked for too long). Meanwhile, 50 mL of Agrobacterium was shaken in a conical flask for infection.

[0148] (4) The soaked beans were cut in half with a scalpel, and the excess hypocotyl part was removed from 3-4 mm of the hypocotyl. The hypocotyl node was gently scraped 5-7 times with a scalpel at a depth of 0.5 mm. After cutting, the bean pieces were placed in a conical flask with water.

[0149] (5) When the OD 600 of Agrobacterium was 0.5-0.7, the bacterial liquid was collected by centrifugation at 4,000 rpm for 10 min, the supernatant was removed, and an appropriate amount of co-culture liquid medium was added and vortexed to mix, so that the OD 600 of the bacterial liquid was 0.6.

[0150] (6) After the beans are cut, the water in the conical flask is removed, and the bacteria solution prepared with co-culture liquid medium is added. Shake 2-3 times, and infect for 30 minutes, with appropriate shaking in between. Meanwhile, sterilized filter paper is laid on co-culture solid medium, one dish of medium corresponding to one piece of filter paper. After the infection is completed, the bacteria solution is removed, and the infected beans are placed on an empty culture dish containing several layers of filter paper, and the excess bacteria solution is absorbed. Then, the beans are evenly placed on the co-culture solid medium on which the filter paper has been laid, and cultured in the dark at 28°C for 3 days. 20-30 beans can be laid on one dish of co-culture solid medium. Too many beans laid on one dish of co-culture solid medium can cause poor growth of the beans and cross contamination.

[0151] (7) Prepare the induction solid / liquid medium, water, filter paper and conical flask, and sterilize at 121°C for 20 min. The bean embryos that have been co-cultured for 3 days and the excess hypocotyls are removed, and the hypocotyls are retained for 3-4 mm, and placed in a conical flask containing water. Wash 4-5 times with sterilized water and 4-5 times with the induction liquid medium, and place on an empty culture dish containing filter paper, and absorb the water. Then, the bean hypocotyls are inserted into the induction solid medium with the hypocotyls upward, and cultured under light for 10 days.

[0152] (8) After 10 days, the original bean embryos at the base are removed, and 5-7 cuts are made at the hypocotyls. The bean hypocotyls are inserted into new induction solid medium with the hypocotyls upward, and cultured under light for 10 days. Repeat twice.

[0153] (9) Prepare the elongation solid medium, and sterilize at 121°C for 20 min. After 3 times of induction culture, most of the beans grow callus with embryos, and the beans are removed, and the callus is retained. The black tissue on the surface of the callus is removed, and the remaining callus is placed in the elongation medium, and cultured under light for 10 days. Repeat three times.

[0154] (10) During the replacement of the elongation medium, when the embryo of the callus is elongated to a length of more than 3 cm, it is cut from the callus, and placed in the rooting medium, and cultured under light for 10-14 days. After the seedlings grow roots, they are transplanted into soil, and hardened for about 7 days, to obtain the T0 generation of transgenic seedlings.

[0155] 4. Identification of soybean GmSH2 gene knockout lines

[0156] The leaves of T0 and T1 generation transgenic seedlings were taken, DNA was extracted, and primer pairs GmSH2a-F (SEQ ID NO: 10, GAGGAAAGAAGAGAAAGGGT) and GmSH2a-R (SEQ ID NO: 11, ATCTTGGCATTCTTGTCGAAC), GmSH2b-F (SEQ ID NO: 12, CACGACGCTCTTCCGATCTTGAGCACATCAACATCATCACC TC) and GmSH2b-R (SEQ ID NO: 13, CGACAACTAAATTGGTAAGCATG) were used for PCR amplification, and the reaction volume was 50 μL (the reaction system is shown in Table 1, and the reaction procedure is shown in Table 2). After PCR was completed, 5 μL was taken for gel electrophoresis to detect whether specific bands were present, and the remaining 45 μL sample was sent to Genesee Biologics Co., Ltd. for first-generation sequencing and comparison with wild-type genomic DNA to determine whether the soybean GmSH2 gene knockout strain was a homozygous mutant.

[0157] Two homozygous mutant soybean GmSH2 gene knockout strains (mutants) were finally obtained, designated as gmsh2-1 and gmsh2-2 (the mutant forms of the mutants are shown in Table 3). Figure 2

[0158] The nucleotide sequence of the GmSH2a gene in the mutant gmsh2-1 is shown in SEQ ID NO: 14 (a base is deleted at 1400 bp), and the amino acid sequence is shown in SEQ ID NO: 15, the nucleotide sequence of the GmSH2b gene is shown in SEQ ID NO: 16 (8 bases are deleted at 1397 bp-1404 bp), and the amino acid sequence is shown in SEQ ID NO: 17.

[0159] SEQ ID NO: 14 (GmSH2a Mutant CDS in gmsh2-1)

[0160] ATGGATTCAACTTGTGCAATCCTGAGTGGCCGCAATCTAGCTAAAGTTTGTGAGGGAATT

[0161] GGAAGAAACAGAAGAAGTGGCTTCTGGGGTGAGAGTACGAGGCGAAGTGTGAACACA

[0162] AGGTTTTTGAGTGTTCAATCATGGAAGACTTCACGTACCAGTAGGAATCTTAGAAACTCC

[0163] ​AAGCCTGGAAGTGGAATTGCACACGCTGTTCTCACATCAGACATTAACGAAGATTCGAT

[0164] GGCATTTCAAGGGGTACCCACTTTTGAAAAACCCGAAGTGGACCCAAAAAGTGTGGCT

[0165] TCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAAGAGC

[0166] CAAGCCTGCGGTTCCAATTGGCGGGTGTTATAGACTCATAGATATCCCCATGAGCAATTG

[0167] CATCAATAGTGGCATCAGAAAAATATTCATTTTGACGCAGTTCAATTCTTTCTCTCTCAAC

[0168] CGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGAATTACTTTTGGAGATGGGTTTGTG

[0169] GAGGTCTTGGCAGCTACTCAAACACCTGGAGAGGCCGGGAAGAAGTGGTTCCAAGGGA

[0170] CCGCCGATGCTGTAAGACAATTTATATGGGTTTTTGAGGATGCCAAGAACAAGAATGTTG

[0171] AGCATATATTGATACTTTCTGGTGATCATCTTTACCGAATGGACTACATGAACTTTGTACA

[0172] GAGACATGTTGACACAAATGCTGATATCACAGTTTCATGTGTACCCATGGATGACAGTCG

[0173] GGCATCAGACTATGGACTGATGAAAATTGACAAAACAGGACGGATTATACAGTTTGCAG

[0174] AAAAACCTAAGGGATCAGATCTAAAGGCAATGCGTGTTGACACCACTCTTCTAGGGTTA

[0175] TCGCCACAAGAAGCAGAAAAATATCCTTATATTGCATCCATGGGTGTCTATGTGTTTAGA

[0176] ACTGAAACCCTGCTGCAACTATTAAGATGGAATGGTTCTTCATGCAATGACTTTGGATCT

[0177] GAAATTATCCCATCTGCTGTGAATGAGCACAATGTCCAGGCATATTTATTCAATGACTACT

[0178] GGGAAGATATTGGAACTATAAAGTCCTTCTTTGATGCAAATCTTGCCCTAACAGAACAAC

[0179] CTCCTAAATTTGAATTCTATGATCCAAAGACTCCTTTCTTCACTTCCCCAAGATTCCTACC

[0180] ACCTACCAAAGTAGAAAAATGCAAGATTGTGGATGCAATTATATCTCATGGTTGCTTCTT

[0181] GAGGGAGTGTAGCATTCAACATTCCATTGTAGGAGTACGCTCACGTTTGGAGTCTGGTGT

[0182] AGAGCTTCAGGATACGATGATGATGGGTGCCGACTACTATCAAACTGAGTATGAAATTGC

[0183] ATCTCTGCTGGCAGAAGGGAAGGTTCAATTGGTGTTGGGGAAAATACTAAAATTAGGAA

[0184] TTGCATAATCGACAAGAATGCCAAGATAGGAAGAAATGTGGTCATAGAAAACATCGATG

[0185] GTGTTCAAGAAGCTGACAGGGCAAAGGAAGGATTCTACATTAGATCGGGCATCACAATT

[0186] ACATTAAAAAATGCAACAATTAAAGATGGAACAGTTATATGA

[0187] SEQ ID NO: 15 (GmSH2a Mutant protein in gmsh2-1)

[0188] MDSTCAILSGRNLAKVCEGIGRNRRSGFWGESTRRSVNTRFLSVQSWKTSRTSRNLRNSKP

[0189] GSGIAHAVLTSDINEDSMAFQGVPTFEKPEVDPKSVASIILGGGAGTRLFPLTGRRAKPAVPIG

[0190] GCYRLIDIPMSNCINSGIRKIFILTQFNSFSLNRHLSRAYSFGNGITFGDGFVEVLAATQTPGE

[0191] AGKKWFQGTADAVRQFIWVFEDAKNKNVEHILILSGDHLYRMDYMNFVQRHVDTNADIT

[0192] VSCVPMDDSRASDYGLMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLSPQEAEKYPYIA

[0193] SMGVYVFRTETLLQLLRWNGSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDAN

[0194] LALTEQPPKFEFYDPKTPFFTSPRFLPPTKVEKCKIVDAIISHGCFLRECSIQHSIVGVRSRLESGVELQDTMMMGADYYQTEYEIASLLAEGKVQLVLGKILKLGIA*

[0195] SEQ ID NO: 16 (GmSH2b Mutant CDS in gmsh2-1)

[0196] ATGGATTCAGCTTGTGCAACCCTGAATGGCCGCCATCTAGCCAAAGTTAGTGAGGGAAT

[0197] TGGAAGAAACAGAACAAGTGGCTTCTGGGGTGAGAGTACGAGGGGAAGTGTGAACAC

[0198] AAAAAGGTTTTTGAGTGTTCAATCATGCAAGACTTCACGAACCAATAGGAATCTTAGAA

[0199] ACTCCAAGCCTGGAAGTGGAATTGCACGCGCTGTTCTCACATCAGACATCGACGAAGAT

[0200] TCCATGGCATTTCAAGGGGTACCCACTTTTGAGAAACCTGAAGTGGACCCAAAAAGTGT

[0201] GGCTTCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAA

[0202] GAGCCAAGCCAGCGGTTCCAATTGGAGGGTGTTATAGACTCATAGATATCCCCATGAGCA

[0203] ATTGCATCAATAGTGGCATCAGAAAAATTTTCATCTTGACGCAGTTCAATTCTTTCTCTCT

[0204] CAACCGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGCATGACTTTTGGAGATGGGT

[0205] TTGTGGAGGTCTTGGCAGCTACTCAAACACCGGGTGAGGCTGGGAAGAAGTGGTTCCA

[0206] AGGGACAGCTGATGCTGTAAGACAATTTATATGGGTTTTTGAGGATGCCAAGAACAAGA

[0207] ATGTTGAGCATATATTGATACTTTCTGGCGATCATCTTTACCGTATGGACTATATGGACTTT

[0208] GTACAGAGACATGTTGACACAAATGCCGATATCACAGTTTCATGTGTACCCATGGATGAC

[0209] AGTCGGGCATCAGACTATGGACTGATGAAAATTGATAAAACAGGACGGATTATACAGTTT

[0210] GCAGAAAAACCTAAGGGATCAGATCTAAAGGCAATGCGTGTTGACACCACTCTTTTAGG

[0211] GTTATTGCCACAAGAAGCAGAAAAACATCCTTATATTGCATCCATGGGTGTCTACGTGTT

[0212] TAGAACTGAAACCTTGCTGCAACTATTAAGATGGAAATGTTCTTCATGCAATGACTTTGG

[0213] ATCTGAAATTATCCCATCTGCTGTGAATGAGCACAATGTCCAGGCATATTTGTTCAATGAC

[0214] TACTGGGAAGATATTGGAACTATAAAGTCCTTCTTTGATGCAAATCTTGCTCTAACAGAA

[0215] CAGCCTCCTAAATTTGAATTCTATGATCCAAAGACACCTTTCTTCACTTCCCCCAGATTCC

[0216] TACCACCTACCAAAGTAGAAAAATGCAAGATTGTGGATGCAATTATATCTCATGGTTGCT

[0217] TCTTGAGGGAGTGCAGCGTTCAACATTCTATTGTTGGAGTACGCTCACGTTTGGAGTCTG

[0218] GTGTGGAGCTTCAGGATACGATGATGATGGGTGCTGACTATTATCAAACTGAGTATGAAA

[0219] TTGCATCTCTGGTGGCAGAAGGGAATTGGTGTCGGGGCAAATACTAAAATCAGGAATTG

[0220] CATAATCGACAAGAATGCCAAGATAGGAAGAAATGTGATCATAGCAAACACCGATGGTG

[0221] TTCAAGAAGCTGACAGGGCAAAGGAAGGATTCTACATTAGGTCGGGCATCACAGTTACA

[0222] TTAAAAAATGCAACAATCAAAGATGGAACAGTTATATGA

[0223] SEQ ID NO: 17 (GmSH2b Mutant protein in gmsh2-1)

[0224] MDSACATLNGRHLAKVSEGIGRNRTSGFWGESTRGSVNTKRFLSVQSCKTSRTNRNLRNSK

[0225] PGSGIARAVLTSDIDEDSMAFQGVPTFEKPEVDPKSVASIILGGGAGTRLFPLTGRRAKPAVPI

[0226] GGCYRLIDIPMSNCINSGIRKIFILTQFNSFSLNRHLSRAYSFGNGMTFGDGFVEVLAATQTP

[0227] GEAGKKWFQGTADAVRQFIWVFEDAKNKNVEHILILSGDHLYRMDYMDFVQRHVDTNAD

[0228] ITVSCVPMDDSRASDYGLMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLLPQEAEKHPYI

[0229] ASMGVYVFRTETLLQLLRWKCSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDA

[0230] NLALTEQPPKFEFYDPKTPFFTSPRFLPPTKVEKCKIVDAIISHGCFLRECSVQHSIVGVRSRLESGVELQDTMMMGADYYQTEYEIASLVAEGNWCRGKY*

[0231] The nucleotide sequence of the GmSH2a gene in the mutant gmsh2-2 is shown in SEQ ID NO: 18 (7 bases are deleted at 1394bp-1400bp), and the amino acid sequence is shown in SEQ ID NO: 19, the nucleotide sequence of the GmSH2b gene is shown in SEQ ID NO: 20 (8 bases are deleted at 1397bp-1404bp), and the amino acid sequence is shown in SEQ ID NO: 21. SEQ ID NO: 18 (GmSH2a Mutant CDS in gmsh2-2)

[0232] ATGGATTCAACTTGTGCAATCCTGAGTGGCCGCAATCTAGCTAAAGTTTGTGAGGGAATT

[0233] GGAAGAAACAGAAGAAGTGGCTTCTGGGGTGAGAGTACGAGGCGAAGTGTGAACACA

[0234] AGGTTTTTGAGTGTTCAATCATGGAAGACTTCACGTACCAGTAGGAATCTTAGAAACTCC

[0235] AAGCCTGGAAGTGGAATTGCACACGCTGTTCTCACATCAGACATTAACGAAGATTCGAT

[0236] GGCATTTCAAGGGGTACCCACTTTTGAAAAACCCGAAGTGGACCCAAAAAGTGTGGCT

[0237] TCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAAGAGC

[0238] CAAGCCTGCGGTTCCAATTGGCGGGTGTTATAGACTCATAGATATCCCCATGAGCAATTG

[0239] CATCAATAGTGGCATCAGAAAAATATTCATTTTGACGCAGTTCAATTCTTTCTCTCTCAAC

[0240] CGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGAATTACTTTTGGAGATGGGTTTGTG

[0241] GAGGTCTTGGCAGCTACTCAAACACCTGGAGAGGCCGGGAAGAAGTGGTTCCAAGGGA

[0242] CCGCCGATGCTGTAAGACAATTTATATGGGTTTTTGAGGATGCCAAGAACAAGAATGTTG

[0243] AGCATATATTGATACTTTCTGGTGATCATCTTTACCGAATGGACTACATGAACTTTGTACA

[0244] GAGACATGTTGACACAAATGCTGATATCACAGTTTCATGTGTACCCATGGATGACAGTCG

[0245] GGCATCAGACTATGGACTGATGAAAATTGACAAAACAGGACGGATTATACAGTTTGCAG

[0246] AAAAACCTAAGGGATCAGATCTAAAGGCAATGCGTGTTGACACCACTCTTCTAGGGTTA

[0247] TCGCCACAAGAAGCAGAAAAATATCCTTATATTGCATCCATGGGTGTCTATGTGTTTAGA

[0248] ACTGAAACCCTGCTGCAACTATTAAGATGGAATGGTTCTTCATGCAATGACTTTGGATCT

[0249] GAAATTATCCCATCTGCTGTGAATGAGCACAATGTCCAGGCATATTTATTCAATGACTACT

[0250] GGGAAGATATTGGAACTATAAAGTCCTTCTTTGATGCAAATCTTGCCCTAACAGAACAAC

[0251] CTCCTAAATTTGAATTCTATGATCCAAAGACTCCTTTCTTCACTTCCCCAAGATTCCTACC

[0252] GAGGGAGTGT AGCATTCAAC ATTCCATTGT AGGAGTACGC TCACGTTTGG AGTCTGGTGT

[0253] GAGGGAGTGT AGCATTCAAC ATTCCATTGT AGGAGTACGC TCACGTTTGG AGTCTGGTGT

[0254] AGAGCTTCAG GATACGATGA TGATGGGTGC CGACTACTAT CAAACTGAGT ATGAAATTGC

[0255] ATCTCTGCTG GCAGAAGGGA AATTGGTGTT GGGGAAAATA CTAAAATTAG GAATTGCAT

[0256] AATCGACAAG AATGCCAAGA TAGGAAGAAA TGTGGTCATA GAAAACATCG ATGGTGTTC

[0257] AAGAAGCTGA CAGGGCAAAG GAAGGATTCT ACATTAGATC GGGCATCACA ATTACATTA

[0258] AAAAATGCA ACAATTAAAG ATGGAACAGT TATATGA

[0259] SEQ ID NO: 19 (GmSH2a Mutant protein in gmsh2-2)

[0260] MDSTCAILSG RNLAKVCEGI GRNRRSGFWG ESTRRSVNTR FLSVQSWKTS RTRNLRNSKP

[0261] GSGIAHAVLT SDINEDSMAF QGVPTFEKPE VDPKSVASII LGGGAGTRLF PLTGRRAKPA VPIG

[0262] GCYRLIDIPM SNCINSGIRK IFILTQFNSF SLNRHLSRAYS FGNGITFGDG FVEVLAATQ TPGE

[0263] AGKKWFQGT ADAVRQFIWV FEDAKNKNVE HILILSGDHL YRMDYMNFVQ RHVDTNADIT

[0264] VSCVPMDDSRASDYGLMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLSPQEAEKYPYIA

[0265] SMGVYVFRTETLLQLLRWNGSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDAN

[0266] LALTEQPPKFEFYDPKTPFFTSPRFLPPTKVEKCKIVDAIISHGCFLRECSIQHSIVGVRSRLESGVELQDTMMMGADYYQTEYEIASLLAEGKLVLGKILKLGIA*

[0267] SEQ ID NO: 20 (GmSH2b Mutant CDS in gmsh2-2)

[0268] ATGGATTCAGCTTGTGCAACCCTGAATGGCCGCCATCTAGCCAAAGTTAGTGAGGGAAT

[0269] TGGAAGAAACAGAACAAGTGGCTTCTGGGGTGAGAGTACGAGGGGAAGTGTGAACAC

[0270] AAAAAGGTTTTTGAGTGTTCAATCATGCAAGACTTCACGAACCAATAGGAATCTTAGAA

[0271] ACTCCAAGCCTGGAAGTGGAATTGCACGCGCTGTTCTCACATCAGACATCGACGAAGAT

[0272] TCCATGGCATTTCAAGGGGTACCCACTTTTGAGAAACCTGAAGTGGACCCAAAAAGTGT

[0273] GGCTTCCATCATATTGGGTGGAGGTGCAGGAACTCGACTCTTTCCTCTTACTGGCAGAA

[0274] GAGCCAAGCCAGCGGTTCCAATTGGAGGGTGTTATAGACTCATAGATATCCCCATGAGCA

[0275] TTGTGGAGGTCTTGGCAGCTACTCAAACACCGGGTGAGGCTGGGAAGAAGTGGTTCCA

[0276] CAACCGCCACCTGTCCCGTGCATACAGCTTCGGAAATGGCATGACTTTTGGAGATGGGT

[0277] TTGTGGAGGTCTTGGCAGCTACTCAAACACCGGGTGAGGCTGGGAAGAAGTGGTTCCA

[0278] AGGGACAGCTGATGCTGTAAGACAATTTATATGGGTTTTTGAGGATGCCAAGAACAAGA

[0279] ATGTTGAGCATATATTGATACTTTCTGGCGATCATCTTTACCGTATGGACTATATGGACTTT

[0280] GTACAGAGACATGTTGACACAAATGCCGATATCACAGTTTCATGTGTACCCATGGATGAC

[0281] AGTCGGGCATCAGACTATGGACTGATGAAAATTGATAAAACAGGACGGATTATACAGTTT

[0282] GCAGAAAAACCTAAGGGATCAGATCTAAAGGCAATGCGTGTTGACACCACTCTTTTAGG

[0283] GTTATTGCCACAAGAAGCAGAAAAACATCCTTATATTGCATCCATGGGTGTCTACGTGTT

[0284] TAGAACTGAAACCTTGCTGCAACTATTAAGATGGAAATGTTCTTCATGCAATGACTTTGG

[0285] ATCTGAAATTATCCCATCTGCTGTGAATGAGCACAATGTCCAGGCATATTTGTTCAATGAC

[0286] TACTGGGAAGATATTGGAACTATAAAGTCCTTCTTTGATGCAAATCTTGCTCTAACAGAA

[0287] CAGCCTCCTAAATTTGAATTCTATGATCCAAAGACACCTTTCTTCACTTCCCCCAGATTCC

[0288] TACCACCTACCAAAGTAGAAAAATGCAAGATTGTGGATGCAATTATATCTCATGGTTGCT

[0289] TCTTGAGGGAGTGCAGCGTTCAACATTCTATTGTTGGAGTACGCTCACGTTTGGAGTCTG

[0290] GTGTGGAGCTTCAGGATACGATGATGATGGGTGCTGACTATTATCAAACTGAGTATGAAA

[0291] TTGCATCTCTGGTGGCAGAAGGGAATTGGTGTCGGGGCAAATACTAAAATCAGGAATTG

[0292] CATAATCGACAAGAATGCCAAGATAGGAAGAAATGTGATCATAGCAAACACCGATGGTG

[0293] TTCAAGAAGCTGACAGGGCAAAGGAAGGATTCTACATTAGGTCGGGCATCACAGTTACA

[0294] TTAAAAAATGCAACAATCAAAGATGGAACAGTTATATGA

[0295] SEQ ID NO:21 (GmSH2b Mutant protein in gmsh2-2)

[0296] MDSACATLNGRHLAKVSEGIGRNRTSGFWGESTRGSVNTKRFLSVQSCKTSRTNRNLRNSK

[0297] PGSGIARAVLTSDIDEDSMAFQGVPTFEKPEVDPKSVASIILGGGAGTRLFPLTGRRAKPAVPI

[0298] GGCYRLIDIPMSNCINSGIRKIFILTQFNSFSLNRHLSRAYSFGNGMTFGDGFVEVLAATQTP

[0299] GEAGKKWFQGTADAVRQFIWVFEDAKNKNVEHILILSGDHLYRMDYMDFVQRHVDTNAD

[0300] ITVSCVPMDDSRASDYGLMKIDKTGRIIQFAEKPKGSDLKAMRVDTTLLGLLPQEAEKHPYI

[0301] ASMGVYVFRTETLLQLLRWKCSSCNDFGSEIIPSAVNEHNVQAYLFNDYWEDIGTIKSFFDA

[0302] NLALTEQPPKFEFYDPKTPFFTSPRFLPPTKVEKCKIVDAIISHGCFLRECSVQHSIVGVRSRLESGVELQDTMMMGADYYQTEYEIASLVAEGNWCRGKY*

[0303] Effect of soybean GmSH2 gene on sugar content of soybean

[0304] The seeds of HC6, gmsh2-1 and gmsh2-2 mutants with full grains and uniform size were selected and planted in soil, and then harvested after maturation. The sugar content in mature leaves, fresh beans and grains was determined by a conventional method.

[0305] The results of sugar content in leaves, fresh beans and grains are shown in Figures 3 to 5 , and it can be seen from Figures 3 to 5 that, compared with the control HC6, the sucrose and total sugar contents in leaves of gmsh2-1 and gmsh2-2 mutants were significantly increased, the soluble sugar and total sugar contents in fresh beans were significantly increased, and the soluble sugar, sucrose and total sugar contents in grains were all significantly increased.

[0306] The results of this example show that the knockout of soybean gene GmSH2 can significantly improve the sugar content of vegetable soybean.

[0307] Each technical feature of the above-described embodiments can be combined arbitrarily, and to make the description simple, each technical feature in the above-described embodiments is not described in all possible combinations, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0308] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Application of soybean GmSH2 gene in increasing sugar content of soybean, characterized in that, The GmSH2 gene comprises a GmSH2a gene with a nucleotide sequence as shown in SEQ ID NO: 1 and a GmSH2b gene with a nucleotide sequence as shown in SEQ ID NO:

3.

2. Use according to claim 1, characterized in that, The sugar content of soybean is improved by knocking out the GmSH2 gene of soybean.

3. Use according to claim 1, characterized in that, The soybean is vegetable soybean.

4. Use according to claim 3, characterized in that, The soybean is hairless soybean.

5. The use of the coding protein of soybean GmSH2 gene in increasing the sugar content of soybean, characterized in that, The encoded protein of the GmSH2 gene comprises an encoded protein of a GmSH2a gene with an amino acid sequence as shown in SEQ ID NO: 2 and an encoded protein of a GmSH2b gene with an amino acid sequence as shown in SEQ ID NO:

4.

6. Application of a knock-out vector of soybean GmSH2 gene in improving sugar content of soybean, characterized in that, The knocking-out vector comprises a CRISPR target site with a sequence as shown in SEQ ID NO:

7.

7. Use according to claim 6, characterized in that, The gene knocking-out vector is constructed by the following steps: (1) using soybean leaf DNA as a template, and using SEQ ID NO: 5 and SEQ ID NO: 6 as primers, a GmU6 promoter fragment is amplified; (2) using a CRISPR target site with a sequence as shown in SEQ ID NO: 7 as a template, and using SEQ ID NO: 8 and SEQ ID NO: 9 as primers, PCR amplification is performed; (3) using the GmU6 promoter fragment obtained in step (1) and the PCR amplification product obtained in step (2) as templates, and using SEQ ID NO: 5 and SEQ ID NO: 9 as primers, PCR amplification is performed; (4) the PCR amplification product of step (3) is connected with a CRISPR vector digested by a restriction enzyme XbaI, and the knocking-out vector is obtained.

8. The application of the recombination engineering bacteria transformed with the knock-out vector of the soybean GmSH2 gene in improving the sugar content of soybean, characterized in that, The knocking-out vector comprises a CRISPR target site with a sequence as shown in SEQ ID NO:

7.

9. A method of increasing sugar content in soybean, characterized by, The method comprises knocking out the GmSH2 gene of soybean in a plant, wherein the GmSH2 gene comprises a GmSH2a gene with a nucleotide sequence as shown in SEQ ID NO: 1 and a GmSH2b gene with a nucleotide sequence as shown in SEQ ID NO:

3.

10. The method of increasing sugar content of soybean according to claim 9, wherein, The soybean is vegetable soybean, preferably hairless soybean.

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