Cultivation method for herbicide-resistant, insect-resistant, and stress-tolerant soybeans jingdou 625 and jingdou 626, and use thereof

Abstract

Provided is a cultivation method for herbicide-resistant, insect-resistant, and stress-tolerant transgenic soybeans, which belongs to the technical field of biology, and specifically relates to a cultivation method for herbicide-resistant, insect-resistant, and stress-tolerant soybeans Jingdou 625 and Jingdou 626 and the use of said method. A plant recombinant vector comprising herbicide resistance, insect resistance, and stress tolerance genes is utilized to introduce the glyphosate resistance gene g2m-epsps, the insect resistance gene cyr1C, and the stress tolerance gene GmNFYB1 into receptor a Qiqihar small seed soybean, and a herbicide-resistant, insect-resistant, and stress-tolerant transgenic soybean is cultivated.

Description

Breeding method and application of herbicide-resistant, insect-resistant and stress-resistant soybeans Jingdou 625 and Jingdou 626

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 28, 2023, with application number 202311272424.9 and invention name “Cultivation method and application of herbicide-resistant, insect-resistant and stress-resistant soybeans Jingdou 625 and Jingdou 626”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of biotechnology, and in particular relates to a method for cultivating herbicide-resistant, insect-resistant and stress-resistant soybeans Jingdou 625 and Jingdou 626 and their applications. Background Art

[0003] Soybeans are not only a major oil crop and source of plant protein, but also a crucial industrial raw material. During soybean cultivation, weed infestation, insect pests, and abiotic stress are significant factors impacting crop production. Field management primarily relies on chemical weed control, supplemented by mechanical and manual weeding. The use of traditional herbicides has led to widespread weed infestation, soil degradation, and severe crop damage. Pest and disease control typically requires the spraying of large quantities of pesticides. Drought and salinity are also major factors impacting crop production.

[0004] Plant genetic modification, as an emerging biotechnology tool, offers significant advantages in improving crop resistance, yield, and breeding. Currently, soybean genetic modification suffers from transgene silencing. When genetically modifying soybeans, it's crucial not only to detect the integration of exogenous genes but also to ensure stable and high-level expression of the exogenous genes. Furthermore, the plant regeneration rate from soybean tissue culture is low, and regenerated plants from genetic transformation using cotyledonary nodes as explants often exhibit chimeras, hindering soybean genetic transformation efforts.

[0005] Summary of the Invention

[0006] The present application provides a method for cultivating soybeans with herbicide-resistant, insect-resistant and reversal-resistant genes, which includes introducing a plant recombinant expression vector pGR20 into the explants of recipient soybeans (Qiqihar small-grain beans); the plant recombinant expression vector pGR20 includes the glyphosate-resistant gene g2m-epsps, the insect-resistant gene cry1C, and the stress-resistant gene GmNFYB1.

[0007] The present application provides a transgenic soybean Jingdou 625, in which the T-DNA region of the exogenous plant recombinant expression vector pGR20 is inserted between 10864177 and 10864529 on chromosome 13 of the recipient soybean Qiqihar small-grain bean genome.

[0008] The present application provides a transgenic soybean Jingdou 626, in which the T-DNA region insertion site of the plant recombinant expression vector pGR20 is between 8915537-8915590 on chromosome 6 of the recipient soybean Qiqihar small-grain bean genome.

[0009] The present application provides a primer combination for identifying Jingdou 625 and its derivative materials, including a primer pair JD625P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 625 soybean, and a primer pair GR20RB-F and JD625P-2 for specifically detecting the 3' insertion position; the nucleotide sequence of the JD625P-1 is shown in SEQ ID NO: 8, the nucleotide sequence of the GR20LB-R is shown in SEQ ID NO: 9, the nucleotide sequence of the GR20RB-F is shown in SEQ ID NO: 10, and the nucleotide sequence of the JD625P-2 is shown in SEQ ID NO: 11.

[0010] The present application provides a kit for identifying Jingdou 625 and its derivative materials, including the primer combination for identifying Jingdou 625 and its derivative materials.

[0011] The present application provides a method for identifying Jingdou 625 and its derivatives, comprising: extracting genomic DNA of the soybean to be tested; using the primer combination for identifying Jingdou 625 and its derivatives to perform PCR amplification on the genomic DNA of the soybean to be tested; if the corresponding band is amplified, it is Jingdou 625 or its derivatives.

[0012] The present application provides a primer combination for identifying Jingdou 626 and its derivative materials, including a primer pair JD626P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 626 soybean, and a primer pair GR20RB-F and JD626P-2 for specifically detecting the 3' insertion position; the nucleotide sequence of JD626P-1 is shown in SEQ ID NO: 12, and the nucleotide sequence of JD626P-2 is shown in SEQ ID NO: 13.

[0013] The present application provides a kit for identifying Jingdou 626 and its derivative materials, including the primer combination for identifying Jingdou 626 and its derivative materials.

[0014] The present application provides an identification method for Jingdou 626 and its derivative materials, comprising: extracting genomic DNA of the soybean to be tested; using the primer combination for identifying Jingdou 626 and its derivative materials to PCR amplify the genomic DNA of the soybean to be tested, and if the corresponding band is amplified, it is Jingdou 626 or its derivative material.

[0015] The present application provides a method for cultivating transgenic soybeans with composite traits of herbicide resistance, insect resistance and stress tolerance, a primer combination for identifying Jingdou 625 and its derivatives, a kit for identifying Jingdou 625 and its derivatives, a method for identifying Jingdou 625 and its derivatives, a primer combination for identifying Jingdou 626 and its derivatives, a kit for identifying Jingdou 626 and its derivatives, or the application of the method for identifying Jingdou 626 and its derivatives in soybean breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1: PCR detection of exogenous genes g2m-epsps, cry1C, and GmNFYB1 in T2 plants of transgenic soybean lines; M: DNA marker, lane 1: no template control, lane 2: plasmid positive control; lane 3: non-transgenic recipient control; lane 4: Jingdou 621; lane 5: Jingdou 622; lane 6: Jingdou 623; lane 7: Jingdou 624; lane 8: Jingdou 625; lane 9: Jingdou 626; lane 10: Jingdou 627; lane 11: Jingdou 628; lane 12: Jingdou 629; lane 13: Jingdou 630;

[0017] Figure 2: Glyphosate resistance evaluation of T3 generation Jingdou 625 and Jingdou 626;

[0018] Figure 3: Glyphosate resistance evaluation of T4 generation Jingdou 625 and Jingdou 626;

[0019] Figure 4: Insect resistance evaluation of T3 generation Jingdou 625 and Jingdou 626;

[0020] Figure 5: Identification of insect resistance of T4 generation Jingdou 625 and Jingdou 626;

[0021] Figure 6: Evaluation of drought tolerance of Jingdou 625 and Jingdou 626 at the budding stage in the T3 generation;

[0022] Figure 7: Drought tolerance evaluation of Jingdou 625 and Jingdou 626 at the seedling stage in the T4 generation;

[0023] Figure 8: Expression of exogenous genes in various tissues of Jingdou 625 and Jingdou 626 T3 plants;

[0024] Figure 9: Expression of exogenous genes in various tissues of Jingdou 625 and Jingdou 626 T4 plants;

[0025] Figure 10: Insertion location of foreign genes in Jingdou 625;

[0026] Figure 11: Insertion location of foreign genes in Jingdou 626;

[0027] Figure 12: Specific qualitative PCR detection of Jingdou 625 genetically modified soybean;

[0028] Figure 13: Specific qualitative PCR detection of Jingdou 626 genetically modified soybean;

[0029] Figure 14: Specific PCR detection of T3-T4 generation materials of transgenic soybean Jingdou 625;

[0030] Figure 15: Specific PCR detection of T3-T4 generation materials of transgenic soybean Jingdou 626. DETAILED DESCRIPTION

[0031] This application provides a method for cultivating soybeans with herbicide-tolerant, insect-resistant, and reversible gene resistance, by introducing a plant recombinant expression vector pGR20 into explants of a recipient soybean (Qiqihar small-grain bean). The plant recombinant expression vector pGR20 contains the glyphosate-tolerant gene g2m-epsps, the insect-resistant gene cry1C, and the stress-tolerant gene GmNFYB1, and the stress-tolerant genes are drought-tolerant and saline-alkali-tolerant. By introducing the herbicide-tolerant, insect-resistant, and reversible gene into the recipient soybean via a plant vector, transgenic soybeans with complex traits can be cultivated, effectively controlling weeds and insect pests, and coping with drought and saline-alkali stresses, showing promising application prospects.

[0032] The glyphosate-tolerant gene is the 5-enolpyruvylshikimoyl-3-phosphate synthase (EPSPS) gene, derived from Pseudomonas fluorescens, a bacterium that can grow in high glyphosate concentrations. The isolated G2M-EPSPS enzyme is insensitive to glyphosate. In transgenic plants, the G2M-EPSPS enzyme is overproduced in plant cells. In the presence of glyphosate, it can replace the glyphosate-bound EPSPS enzyme in the plant, protecting the plant from glyphosate toxicity.

[0033] The cry gene encodes Bt protein (Cry protein), which has insecticidal activity against certain specific pests. Using Bt protein to selectively act on specific pests is an effective way to control pests. In addition, Bt protein is highly specialized and has no effect on mammals and other animals. Compared with chemical synthetic insecticides, it has less impact on the environment and is safer. Bt protein synthesizes toxin precursors during spore formation and is activated under the alkaline environmental conditions of the pest's intestine. The activated protein molecules then insert into the midgut epithelial cell membrane, forming cation channels on the cell membrane, disrupting the osmotic balance of the cell membrane, causing the midgut cells to disintegrate, and ultimately leading to the death of the insect. The cry1C gene encodes the insecticidal crystal protein crylC protein (Bt protein) that is toxic to lepidopteran insects, making transgenic soybeans resistant to lepidopteran pests and reducing losses caused by insect pests.

[0034] The GmNFYB1 nuclear factor-Y gene is a transcription factor that regulates the expression of downstream genes by interacting with regulatory factors. It participates in multiple biological stress response pathways such as plant drought resistance and salinity, and promotes flower development under long-day conditions. Its transfer into recipient plants can significantly improve the plants' drought and salinity tolerance. Overexpression of the GmNFYB1 gene in transgenic soybeans can participate in regulating the expression of downstream genes, thereby enhancing the plants' drought and salinity tolerance.

[0035] The nucleotide sequence of the glyphosate-resistant gene g2m-epsps in the present application is shown in SEQ ID NO: 1, the nucleotide sequence of the insect-resistant gene cry1C is shown in SEQ ID NO: 2, and the nucleotide sequence of the stress-resistant gene GmNFYB1 is shown in SEQ ID NO: 3.

[0036] This application also provides a method for constructing the plant recombinant expression vector pGR20, comprising the following steps:

[0037] The GmNFYB1 gene fragment containing BglII and BstEII restriction sites and the commercial vector pCAMBIA3301 were double-digested with BglII and BstEII, respectively, and then ligated to construct the p3301-NF vector;

[0038] The insect-resistant gene cry1C containing BamHI and SacI double restriction sites and the commercial vector pBI121 were double-digested with BamHI and SacI respectively and then ligated to construct the pBI121-crylC vector;

[0039] Using the pBI121-crylC vector as a template, specific primers with KpnI and HindIII restriction sites at both ends were used to amplify the insect-resistant gene expression cassette fragment, which was then ligated with the p3301-NF vector digested with KpnI and HindIII to construct the p3301-NF-cry1C plant expression vector;

[0040] The g2m-epsps fragment containing an XhoI restriction site synthesized by sequence and the p3301-NF-cry1C vector are digested with XhoI and then connected to form a plant recombinant expression vector pGR20 containing a herbicide-resistant gene expression cassette.

[0041] In the optional construction of the p3301-NF vector, the reaction system for double enzyme digestion is preferably: 10 μL template, 2 μL 10×Buffer, 1 μL BglII, 1 μL BstEII, and ddH2O is added to a total system of 20 μL. The reaction conditions for the double enzyme digestion are preferably 37°C overnight. The ligation is preferably performed using T4 ligase. The ligation conditions are preferably 16°C for 4 hours. After the ligation, the recombinant vector is preferably verified. This application has no special restrictions on the verification method, and verification methods well known in the art can be used, such as transformation and cultivation in Escherichia coli, plasmid extraction, colony PCR amplification or sequencing. This application has no special restrictions on the source of the commercial vector pCAMBIA3301, and the source of the commercial vector pCAMBIA3301 well known in the art can be used. The commercial vector pCAMBIA3301 described in the examples of this application was purchased from Cambia.

[0042] In the constructed pBI121-crylC vector, the double enzyme digestion reaction system is: template 10μL, 10×Buffer 2μL, BamHI 1μL, SacI 1μL, and ddH2O is supplemented to a total system of 20μL. The reaction conditions for the double enzyme digestion are preferably 37°C enzyme digestion overnight. The ligation is preferably performed using T4 ligase. The conditions for the ligation are preferably 16°C for 4 hours. After the ligation, the recombinant vector is preferably verified. This application has no special restrictions on the verification method, and verification methods well known in the art can be used, such as transformation and culture in Escherichia coli, plasmid extraction, colony PCR amplification or sequencing. This application has no special restrictions on the source of the commercial vector pBI121, and the source of the commercial vector pBI121 well known in the art can be used. The commercial vector pBI121 described in the examples of this application was purchased from Clontech.

[0043] In the construction of the p3301-NF-cry1C plant expression vector, the specific primers with KpnI and HindIII restriction sites at both ends are shown in SEQ ID NO: 14 and SEQ ID NO: 15. When the fragment of the insect-resistant gene expression frame obtained by amplification is connected to the p3301-NF vector after KpnI and HindIII digestion, the connection conditions are preferably 16°C for 4 hours. After the connection, the recombinant vector is preferably verified. The present application does not have any special restrictions on the verification method, and verification methods well known in the art can be used, such as transformation and cultivation in Escherichia coli, plasmid extraction, colony PCR amplification or sequencing.

[0044] In this application, a sequence-synthesized g2m-epsps fragment containing an XhoI restriction site was digested with XhoI and ligated with the p3301-NF-cry1C vector to form the plant recombinant expression vector pGR20 containing the herbicide-tolerant gene expression cassette. This application does not specifically limit the sequence synthesis of the g2m-epsps fragment containing the XhoI restriction site; sequence synthesis techniques well known in the art can be employed. The g2m-epsps fragment containing the XhoI restriction site described in the examples of this application was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0045] In this application, the T-DNA region of the plant recombinant expression vector pGR20 contains only the expression cassettes for the g2m-epsps, cry1C, and GmNFYB1 genes. The target gene is driven by the 35S promoter derived from the cauliflower mosaic virus. Specifically, the inserted sequence contains no antibiotic marker or reporter genes, and the vector is non-pathogenic and has no potential to become pathogenic.

[0046] The present application introduces the plant recombinant expression vector pGR20 into recipient soybeans to obtain transgenic soybeans. As an practicable method, the present application adopts the Agrobacterium-mediated soybean cotyledonary node transformation method, and the Agrobacterium strain is Ag10.

[0047] The present application uses normally germinated and pollution-free recipient soybean Qiqihar small bean seedlings to prepare explants. In one embodiment, the soybean is cut along the hypocotyl with a scalpel in a clean bench, retaining 3-4mm of the hypocotyl, and placed in a sterile culture dish. A co-culture solution is added to the culture dish to facilitate the peeling of the seed coat, and then the hypocotyl is cut vertically along the cotyledon hypocotyl, and the clean true leaf tissue is removed. 5-7 incisions are made axially at the junction of the cotyledon and the cotyledon hypocotyl, and the incision is about 3-4mm long. Each explant is composed of a cotyledon connected to a section of hypocotyl, and one seed can form two explants. In one embodiment, the co-culture solution added in the culture dish is based on submerging the hypocotyl. In one embodiment, the co-culture solution is: 1 / 10B5+30g / L sucrose+3.9g / L 2-(N-morpholino)ethanesulfonic acid (MES)+1.67mg / L 6-BA+39mg / L acetosyringone+0.25mg / L gibberellin (GA3).

[0048] In the present application, the cryopreserved Agrobacterium strain was taken out from the ultra-low temperature freezer and frozen and thawed on ice. In one embodiment, a small amount of the strain was inoculated on an LB plate using an inoculating loop or a sterile pipette tip, and incubated upside down at a constant temperature of 25-30°C for 1-2 days to obtain a single clone. In one embodiment, the culture was carried out at a constant temperature of 26-28°C for 2 days. After the culture was completed, a single clone was picked and inoculated into a YEP culture medium for shaking culture. In one embodiment, the culture conditions were 200-230 rpm, 25-30°C, and 100 mL of culture medium. Activation culture was performed for 10-14 hours. In another embodiment, activation culture was performed at 210-220 rpm and 26-28°C for 11-12 hours. When the bacterial solution was activated to saturation for the first time, bacterial solution was extracted and inoculated into YEP culture solution. The volume ratio of the bacterial solution to the YEP culture solution was 1:(90-110). In one embodiment, the volume ratio of the bacterial solution to the YEP culture solution was 1:(98-105). The second activation was performed at 26-28°C and 210-220 rpm. When Agrobacterium was fully activated to OD 600 =0.9-1.1, the bacterial solution was centrifuged at 4°C. In one embodiment, the centrifugation parameters were 3800-4200 rpm for 8-12 min. In another embodiment, the centrifugation parameters were 4000-4100 rpm for 9-10 min. The supernatant was discarded and the precipitate was collected. The bacterial cells at the bottom of the tube were suspended with an equal volume of co-culture medium and the OD was adjusted. 600 is 0.5-0.8, for standby use; in this application, the LB solid culture medium contains 50 mg / L kanamycin, and the YEP culture medium contains 50 mg / L kanamycin.

[0049] In this application, the explant is co-cultured with Agrobacterium. When the explant is prepared, every 40-60 explants are placed in a triangular flask, and 50 mL of resuspended Agrobacterium liquid is added to each flask, and the liquid needs to immerse the explant. Co-infection is carried out for 30-35 minutes under dark or low light conditions, and the triangular flask is gently shaken once every 5 minutes to allow Agrobacterium and the explant to fully contact. After the infection is completed, the excess Agrobacterium liquid is carefully poured out, a layer of sterile filter paper is spread on the co-cultivation medium, and the infected explant is spread on the filter paper with the axis side facing down, and co-cultivated for 2-6 days at 22-30 ° C in the dark. It can be implemented to co-cultivate for 3-5 days at 26-28 ° C in the dark. The co-culture medium is: 1 / 10B5+30g / L sucrose+3.9g / L 2-(N-morpholino)ethanesulfonic acid (MES)+1.67mg / L 6-BA+39mg / L acetosyringone+0.25mg / L gibberellin (GA3)+1mmol / L dithiothreitol+1mmol / L sodium thiosulfate+1mmol / L cysteine+5g / L agar powder, pH 5.4.

[0050] In the present application, after the explants are co-cultured with Agrobacterium, the explants are subjected to the stages of resistance cluster bud induction, elongation bud induction, and rooting to obtain regenerated plants; the cluster bud induction medium is: B5 + 30g / L sucrose + 8g / L agar powder + 0.6g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 1.67mg / L 6-BA + 150mg / L cefotaxime + 400mg / L carbenicillin + 15mg / L glyphosate, pH 5.7; the bud elongation medium is: MS + B5 + 30g / L sucrose + 8g / L agar powder + 0.6g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 1.67mg / L 6-BA + 150mg / L cefotaxime + 400mg / L carbenicillin + 15mg / L glyphosate, pH 5.7; MES + 50 mg / L aspartic acid + 50 mg / L glutamine + 0.3 mg / L indole-3-acetic acid (IAA) + 0.5 mg / L gibberellin (GA3) + 150 mg / L thiophanate-methylpropional + 400 mg / L carbenicillin + 0.1 mg / L zeatin (Ze) + 5 mg / L glyphosate, pH 5.7; the rooting medium is: MS + B5 + 30 g / L sucrose + 8 g / L agar powder + 0.6 g / L 2-(N-morpholino) ethanesulfonic acid (MES) + 50 mg / L aspartic acid + 50 mg / L glutamine, pH 5.7.

[0051] In the present application, the explants in the cluster bud induction stage and the elongation bud induction stage are subcultured every two weeks. During the subculture, a new incision is made on the back of the explant to enable the explant to better absorb nutrients. In the present application, when the elongation buds extend to 4-6 cm, rooting is induced. After rooting, the sealing film on the culture dish is removed and the seedlings are hardened for 1-3 days. The seedlings are then transplanted to pots or grown in the field to obtain transgenic plants.

[0052] In the present application, the culture medium and culture solution are sterilized at 121° C. for 15-20 min.

[0053] Regenerated plants obtained through genetic transformation using cotyledonary nodes as explants are often chimeras. Somatic embryogenesis induced from immature soybean cotyledons offers a potential solution to this problem, but the inability of these somatic embryos to proliferate in successive generations has, to a certain extent, hindered soybean genetic transformation efforts. This application simultaneously introduces multiple genes into recipients for genetic transformation, and through tissue culture, produces transgenic plants with multiple stable genetic traits, which will accelerate the improvement of multiple soybean traits and the development of new varieties.

[0054] In this application, MS and B5 dry powder culture medium and acetosyringone are products purchased from Sigma, 2-(N-morpholino)ethanesulfonic acid (MES), cefotaxime, carbenicillin, agar powder, zeatin, aspartic acid, glutamine, gibberellin (GA3) and 6-benzylaminoadenine (6-BA) are products of Biodee, and sucrose is a domestic reagent.

[0055] The present application provides a transgenic soybean Jingdou 625, in which the T-DNA region of the plant recombinant expression vector pGR20 is inserted between 10864177 and 10864529 on chromosome 13 of the recipient soybean Qiqihar small-grain bean genome.

[0056] In the present application, the 5'-end flanking sequence of Jingdou 625 includes positions 10863795 to 10864177 of chromosome 13 of the recipient soybean genome and positions 6203 to 6530 of the pGR20 vector, and the nucleotide sequence is shown in SEQ ID NO: 4; the 3'-end flanking sequence includes positions 13165 to 13477 of the pGR20 vector and positions 10864529 to 10864795 of chromosome 13 of the recipient soybean genome, and the nucleotide sequence is shown in SEQ ID NO: 5.

[0057] The present application provides a transgenic soybean Jingdou 626, in which the T-DNA region insertion site of the plant recombinant expression vector pGR20 is between 8915537-8915590 on chromosome 6 of the recipient soybean Qiqihar small-grain bean genome.

[0058] In the present application, the 5'-end flanking sequence of Jingdou 626 includes positions 8915080 to 8915537 of chromosome 6 of the recipient soybean genome and positions 6175 to 6530 of the pGR20 vector, and the nucleotide sequence is shown in SEQ ID NO: 6; the 3'-end flanking sequence includes positions 13165 to 13487 of the pGR20 vector and positions 8915590 to 8916080 of chromosome 6 of the recipient soybean genome, and the nucleotide sequence is shown in SEQ ID NO: 7.

[0059] The present application provides a primer combination for identifying Jingdou 625 and its derivative materials, including a primer pair JD625P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 625 soybean, and a primer pair GR20RB-F and JD625P-2 for specifically detecting the 3' insertion position; the nucleotide sequence of the JD625P-1 is shown in SEQ ID NO: 8, the nucleotide sequence of the GR20LB-R is shown in SEQ ID NO: 9, the nucleotide sequence of the GR20RB-F is shown in SEQ ID NO: 10, and the nucleotide sequence of the JD625P-2 is shown in SEQ ID NO: 11.

[0060] The present application provides a kit for identifying Jingdou 625 and its derivative materials, including the primer combination for identifying Jingdou 625 and its derivative materials.

[0061] The present application provides a method for identifying Jingdou 625 and its derivatives, comprising: extracting genomic DNA from a soybean to be tested; and performing PCR amplification on the genomic DNA of the soybean to be tested using a primer combination for identifying Jingdou 625 and its derivatives. The PCR reaction is used to observe whether a band can be amplified from the sample to be tested. If a corresponding band is amplified, it indicates that the sample to be tested is Jingdou 625 or its derivatives; if no band is amplified, it indicates that the sample to be tested is not Jingdou 625 or its derivatives.

[0062] The present application provides a primer combination for identifying Jingdou 626 and its derivative materials, including a primer pair JD626P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 626 soybean, and a primer pair GR20RB-F and JD626P-2 for specifically detecting the 3' insertion position, for performing a PCR reaction; the nucleotide sequence of the JD626P-1 is shown in SEQ ID NO: 12, and the nucleotide sequence of the JD626P-2 is shown in SEQ ID NO: 13.

[0063] The present application provides a kit for identifying Jingdou 626 and its derivative materials, including the primer combination for identifying Jingdou 626 and its derivative materials.

[0064] The present application provides a method for identifying Jingdou 626 and its derivatives, comprising: extracting genomic DNA from a soybean to be tested; and performing PCR amplification on the genomic DNA of the soybean to be tested using a primer combination for identifying Jingdou 626 and its derivatives. The PCR reaction is used to observe whether a band can be amplified from the sample to be tested. If a corresponding band is amplified, it indicates that the sample to be tested is Jingdou 626 or its derivatives; if no band is amplified, it indicates that the sample to be tested is not Jingdou 626 or its derivatives.

[0065] The present application provides a method for cultivating transgenic soybeans with composite traits of herbicide resistance, insect resistance and stress tolerance, a primer combination for identifying Jingdou 625 and its derivatives, a kit for identifying Jingdou 625 and its derivatives, a method for identifying Jingdou 625 and its derivatives, a primer combination for identifying Jingdou 626 and its derivatives, a kit for identifying Jingdou 626 and its derivatives, or the application of the method for identifying Jingdou 626 and its derivatives in soybean breeding.

[0066] Compared with the prior art, this application has the following beneficial effects:

[0067] This application utilizes bioengineering technology to cultivate new transgenic soybean varieties with complex traits, providing a better solution for effectively controlling weeds and pests in soybean fields, as well as coping with drought and saline-alkali stress. In transgenic plants, plant cells overproduce the G2m-EPSPS enzyme, which, in the presence of glyphosate, can replace the glyphosate-bound EPSPS enzyme in the plant body, protecting the plant from glyphosate toxicity. The cry1C gene encodes the insecticidal crystal protein crylC (Bt protein), which is toxic to lepidopteran insects. This makes the transgenic soybeans resistant to lepidopteran pests and reduces losses caused by insect pests. Overexpression of the GmNFYB1 gene in transgenic soybeans can regulate the expression of downstream genes, enhancing the plant's drought and saline-alkali tolerance.

[0068] In this application, unless otherwise specified, the chemical reagents used are conventional commercial reagents, and the technical means used are conventional technical means well known to those skilled in the art.

[0069] The technical solutions provided in this application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of this application.

[0070] Example 1

[0071] Construction of plant expression vector pGR20

[0072] (1) The commercial vector pCAMBIA3301 and the GmNFYB1 gene fragment containing BglII and BstEII restriction sites were used as templates, respectively. The reaction system was as follows: 10 μL template, 2 μL 10× Buffer, 1 μL BglII, 1 μL BstEII, and ddH2O to make up the total system to 20 μL. The enzyme digestion was carried out at 37°C overnight.

[0073] The GmNFYB1 gene fragment, which was double-digested with BglII and BstEII, and the commercial vector pCAMBIA3301 were ligated using T4 ligase at 16°C for 4 hours to construct the p3301-NF vector.

[0074] (2) The commercial vector pBI121 and the insect-resistant gene cry1C gene fragment containing BamHI and SacI double restriction sites were used as templates, respectively. The reaction system was: template 10 μL, 10× Buffer 2 μL, BamHI 1 μL, SacI 1 μL, ddH2O to make up the total system 20 μL, and enzyme digestion was carried out at 37°C overnight.

[0075] The cry1C gene fragment was double-digested with BamHI and SacI, and then ligated with the commercial vector pBI121 using T4 ligase at 16°C for 4 hours to construct the pBI121-crylC vector.

[0076] (3) Using the pBI121-crylC vector as a template, the specific primers shown in SEQ ID NO: 14 and SEQ ID NO: 15 were used to amplify the insect-resistant gene expression cassette fragment, which was then ligated with the p3301-NF vector digested with KpnI and HindIII at 16°C for 4 hours to construct the p3301-NF-cry1C plant expression vector.

[0077] (4) The g2m-epsps fragment containing the XhoI restriction site synthesized by Suzhou Hongxun Biotechnology Co., Ltd. and the p3301-NF-cry1C vector were digested with XhoI and then ligated to form a plant recombinant expression vector pGR20 containing the herbicide-resistant gene expression cassette.

[0078] Example 2

[0079] Cultivation of transgenic soybeans using the plant recombinant expression vector pGR20 in Example 1

[0080] 1. Prepare culture medium and culture fluid:

[0081] 1) Co-culture medium: 1 / 10 B5 + 30 g / L sucrose + 3.9 g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 1.67 mg / L 6-BA + 39 mg / L acetosyringone + 0.25 mg / L gibberellin (GA3);

[0082] 2) Co-culture medium: 1 / 10 B5 + 30 g / L sucrose + 3.9 g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 1.67 mg / L 6-BA + 39 mg / L acetosyringone + 0.25 mg / L gibberellin (GA3) + 1 mmol / L dithiothreitol + 1 mmol / L sodium thiosulfate + 1 mmol / L cysteine ​​+ 5 g / L agar powder, pH 5.4;

[0083] 3) Cluster shoot induction medium: B5 + 30 g / L sucrose + 8 g / L agar powder + 0.6 g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 1.67 mg / L 6-BA + 150 mg / L cefotaxime + 400 mg / L carbenicillin + 15 mg / L glyphosate, pH 5.7;

[0084] 4) Shoot elongation medium: MS + B5 organic + 30 g / L sucrose + 8 g / L agar powder + 0.6 g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 50 mg / L aspartic acid + 50 mg / L glutamine + 0.3 mg / L indole-3-acetic acid (IAA) + 0.5 mg / L gibberellin (GA3) + 150 mg / L thiazolin + 400 mg / L carbenicillin + 0.1 mg / L zeatin (Ze) + 5 mg / L glyphosate, pH 5.7;

[0085] 5) Rooting medium: MS + B5 organic + 30 g / L sucrose + 8 g / L agar powder + 0.6 g / L 2-(N-morpholino)ethanesulfonic acid (MES) + 50 mg / L aspartic acid + 50 mg / L glutamine, pH 5.7;

[0086] The culture medium and culture solution were sterilized at 121°C for 15 min;

[0087] 2. Select normal germination and pollution-free recipient soybean seedlings from Qiqihar small beans. Use a scalpel to cut the soybean along the hypocotyl in a clean bench, leaving 3-4mm of the hypocotyl. Place it in a sterile culture dish. Add 5mL of the co-culture solution prepared in step 1 to the culture dish. Then cut the hypocotyl vertically along the cotyledon hypocotyl, remove the true leaf tissue, and make 5-7 axial incisions at the junction of the cotyledon and the hypocotyl. The incision is about 3-4mm long. Each explant consists of a cotyledon connected to a hypocotyl. One seed can form two explants.

[0088] 3. Take out the low-temperature preserved Agrobacterium strain and freeze-thaw it on ice. Use an inoculation loop to take a small amount of bacteria and inoculate it on LB medium (containing 50 mg / L kanamycin). Invert and culture it at a constant temperature of 28°C for 2 days to obtain a single clone. Pick a single clone and inoculate it into YEP culture medium (containing 50 mg / L kanamycin). Shake and activate it at 28°C and 220 rpm for 12 hours. Take 1 mL of bacterial liquid and inoculate it into 100 mL YEP culture medium (containing 50 mg / L kanamycin). Shake and activate it at 28°C and 220 rpm. Wait until the Agrobacterium is fully activated to OD 600 =1.0, centrifuge the bacterial solution at 4000 rpm for 10 min at 4°C, discard the supernatant and collect the precipitate, and suspend the bacterial cells at the bottom of the tube with an equal volume of the co-culture solution prepared in step 1 to adjust the OD 600 is 0.7, for standby use;

[0089] 4. Place 50 explants prepared in step 2 in a flask, add 50 mL of the Agrobacterium suspension resuspended in step 3, and infect in the dark for 35 minutes. Gently shake the flask every 5 minutes to ensure full contact between the Agrobacterium and the explants. After infection, carefully pour off the excess Agrobacterium suspension. Spread a layer of sterile filter paper on the co-cultivation medium prepared in step 1, spread the infected explants on the filter paper with the axis side facing down, and co-cultivate at 24°C in the dark for 3 days.

[0090] 5. After the co-cultivation, the explant is placed in the bud induction medium prepared in step 1. The explant is subcultured once every two weeks. A new incision is prepared again on the back side of the explant during subculture. After 3 subcultures, the explant is placed in the bud elongation medium prepared in step 1. The explant is subcultured once every two weeks. A new incision is prepared again on the back side of the explant during subculture. When the elongated buds extend to 4-6cm, the explant is placed in the rooting medium prepared in step 1 to induce rooting. After taking root, the sealing film on the culture dish is opened and the seedlings are hardened for 1-3 days. Then, the seedlings are transplanted to the field to grow and obtain transgenic plants.

[0091] Example 3

[0092] Detection of target genes in the transgenic lines obtained in Example 2

[0093] 1. Extract DNA from leaves of T2 plants of 10 transgenic soybean lines;

[0094] 2. Using the DNA extracted in step 1 as a template, specific primers were used to detect the target gene in 10 transgenic soybean lines. The specific primers are listed in Table 1. The reaction system consisted of 2 μL of 10× Buffer, 1 μL of dNTPs, 0.5 μL of each upstream and downstream primer, 0.5 μL of Taq enzyme, 1 μL of template, and ddH₂O to a total of 20 μL. The reaction conditions were: denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 61-63°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 8 min. 10 μL of the PCR amplification product was analyzed by electrophoresis on a 1.5% agarose gel (containing ethidium bromide) at 5 V / cm for 40 min and observed and photographed under UV light. The results are shown in Figure 1.

[0095] Table 1 List of primers for transgenic detection

[0096] The results showed that the g2m-epsps, cry1C and GmNFYB1 target genes could be detected in the transgenic materials, indicating that the exogenous genes had been stably integrated into the soybean genome.

[0097] Example 4

[0098] Identification of target traits of transgenic soybean plants

[0099] 1. The PCR-positive plants obtained in Example 3 were propagated in a greenhouse to form transgenic plants of different generations from T1 to T4;

[0100] 2. Glyphosate resistance was assessed in T3-T4 generation Jingdou 625 and Jingdou 626 transgenic soybeans and the recipient Qiqihar small-grain bean. From the first to third leaf stage, transgenic soybeans were sprayed with a 41% aqueous glyphosate solution (glyphosate isopropylamine salt solution) at a rate of 6 L / hectare using a Hudson handheld pressure sprayer. Two weeks later, damage was assessed. Results are shown in Figures 2 and 3.

[0101] The results showed that at the above spraying dose, the recipient Qiqihar small-grain bean plant did not develop resistance and died after two weeks. However, the T3 generation homozygous transgenic soybean lines Jingdou 625 and Jingdou 626 showed no growth inhibition, and their leaves did not turn green or shrink, nor did they show signs of glyphosate damage such as yellowing of new leaves. This indicates that the transgenic soybean lines Jingdou 625 and Jingdou 626 exhibited high resistance to glyphosate (Figure 2). Similarly, the T4 generation homozygous transgenic soybean lines Jingdou 625 and Jingdou 626 also showed no growth inhibition, nor did their leaves turn green or shrink, nor did they show signs of glyphosate damage such as yellowing of new leaves (Figure 3). This indicates that glyphosate resistance in the transgenic soybean lines Jingdou 625 and Jingdou 626 can be stably inherited across generations.

[0102] 3. Identification of insect pest resistance of T3-T4 generation Jingdou 625 and Jingdou 626 transgenic soybeans and recipient Qiqihar small-grain beans: Collect healthy, fresh and uniformly tender unfolded leaves from T3 transgenic soybean plants, punch the leaves into 2.5 cm discs with a hole punch, select 5 discs and place them in a sponge-lined culture dish, moisturize with 13 mL sterile water, and inoculate 2nd instar Spodoptera litura larvae at a ratio of 1 head / dish. After inoculation, investigate the leaf damage every day, and analyze the results after 7 days. T4 transgenic soybean plants were identified for resistance to Spodoptera litura in a net room. When the soybeans grew to 3 trifoliate leaves, 10 2nd instar Spodoptera litura larvae were inoculated on each soybean plant. The leaf biting situation was investigated 2 weeks after inoculation, and the leaf biting situation was graded according to Table 2. The average insect susceptibility index was calculated; the resistance was graded according to Table 3; the results are shown in Figures 4 and 5;

[0103] Average insect susceptibility index (%) = Σ(level value × number of leaves of corresponding level) / total number of leaves surveyed × highest level value × 100;

[0104] Table 2. Leaf damage identification and grading standards

[0105] Table 3. Resistance classification standards for leaf-feeding pests

[0106] The results showed that the leaves of the recipient Qiqihar small-grain bean were severely damaged, with an insect susceptibility index of 100, indicating that it was susceptible to insects; while the average insect susceptibility index of Jingdou 625 was 0, and the average insect susceptibility index of Jingdou 626 was 7.62, both of which were highly resistant to Spodoptera litura (Figure 4). The results of the Spodoptera litura resistance identification of T4 transgenic soybean plants in a net house also showed that Jingdou 625 and Jingdou 626 were highly resistant to Spodoptera litura (Figure 5), indicating that the insect resistance of Jingdou 625 and Jingdou 626 transgenic soybeans can be stably inherited in different generations.

[0107] 4. Stress tolerance was assessed for the T3-T4 generation transgenic soybeans Jingdou 625 and Jingdou 626 and the recipient Qiqihar small-grain soybean. For drought tolerance assessment at the bud stage, water treatment and 17.5% PEG treatment were used as controls, and seed germination was observed 5 days after germination. For drought tolerance assessment at the seedling stage, the soybeans were subjected to drought treatment for 2 weeks after the cotyledons unfolded, and the recovery of the soybean seedlings was observed after rehydration. The results are shown in Figures 6 and 7.

[0108] According to the drought tolerance evaluation results at the bud and seedling stages, the T3 generation transgenic soybeans Jingdou 625 and Jingdou 626 had higher drought tolerance at the bud stage than the recipient Qiqihar small-grain bean (Figure 6). The T4 generation Jingdou 625 and 332 had significantly higher drought tolerance at the seedling stage than the recipient Qiqihar small-grain bean (Figure 7).

[0109] 5. Analysis of Exogenous Gene Expression: RNA was extracted from the roots, stems, leaves, and seeds of the transgenic materials. After reverse transcription, RT-PCR was performed to detect the g2m-epsps, cry1C, and GmNFYB1 genes using the ACTIN gene as an internal reference. The results showed that g2m-epsps and cry1C were not expressed in the recipient Qiqihar small-grain bean, and GmNFYB1 was expressed at very low levels. However, it was expressed in all tissues of the transgenic Jingdou 625 and Jingdou 626 materials (Figures 8-9). This indicates that exogenous genes can be stably expressed in transgenic materials.

[0110] Example 5

[0111] Identification of foreign gene insertion sites in Jingdou 625

[0112] 1. Size and structure of the insert sequence

[0113] The exogenous gene was inserted into chromosome 13 in the Jingdou 625 soybean plant. DNA was extracted from the leaves of the Jingdou 625 soybean plant, and specific primers were designed for PCR detection and sequencing analysis of the insertion site. The specific primers are shown in Table 4. The reaction system was: 2 μL of 10× Buffer, 1 μL of dNTP, 0.5 μL of upstream and downstream primers, 0.5 μL of Taq enzyme, 1 μL of template, and ddH2O to make up the total system to 20 μL. The reaction conditions were: denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 61-63°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 8 min after the cycle. The results are shown in Figure 10.

[0114] Table 4. PCR primers and product information for specific identification of Jingdou 625 soybean

[0115] The 5' flanking sequence of Jingdou 625 soybean obtained by sequencing included positions 10863795 to 10864177 of chromosome 13 of the recipient soybean genome and positions 6203 to 6530 of the pGR20 vector, and the specific sequence is SEQ ID NO: 4; the 3' flanking sequence included positions 13165 to 13477 of the pGR20 vector and positions 10864529 to 10864795 of chromosome 13 of the recipient soybean genome, and the specific sequence is SEQ ID NO: 5. The full-length sequence of the Jingdou 625 insertion site is shown in SEQ ID NO: 16; the results showed that the exogenous gene insertion site was between 10864177 and 10864529 of chromosome 13 of the soybean genome (Figure 10), and the arrangement order of the T-DNA sequence in the transgenic soybean Jingdou 625 was consistent with the corresponding genetic elements of its plasmid vector pGR20.

[0116] 2. Determination of specific PCR detection method

[0117] The primer JD625P-1 derived from the soybean genome 5' of the Jingdou 625 insertion site and the primer GR20LB-R derived from the exogenous gene were used to form a primer pair that can specifically detect the 5' insertion position of Jingdou 625 soybean. The amplified product size was 637bp.

[0118] The PCR reaction system was as follows: 2 μL of 10× Buffer, 1 μL of dNTPs, 0.5 μL of upstream and downstream primers, 0.5 μL of Taq enzyme, 1 μL of template, and ddH2O to a total of 20 μL. The PCR reaction conditions were: denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for 35 cycles.

[0119] The leaf DNA of Qiqihar small-grain bean without template and the recipient was used as the control group, and the DNA of Jingdou 625 prepared in step 1 was used as the experimental group. The results of PCR detection are shown in Figure 12: No PCR products were produced in the no-template control (lane 1) and the recipient (lane 2), while the target band was amplified in Jingdou 625 (lane 3) using the primer pair located at the 5' end of the insertion site (Figure 12).

[0120] Example 6

[0121] Identification of foreign gene insertion sites in Jingdou 626

[0122] 1. Size and structure of the insert sequence

[0123] The exogenous gene in the Jingdou 626 soybean plant was inserted into chromosome 6. DNA was extracted from the leaves of the Jingdou 626 soybean plant, and specific primers were designed for PCR detection and sequencing analysis of the insertion site. The specific primers are shown in Table 5. The reaction system was: 2 μL of 10× Buffer, 1 μL of dNTP, 0.5 μL of upstream and downstream primers, 0.5 μL of Taq enzyme, 1 μL of template, and ddH2O to make up the total system to 20 μL; the reaction conditions were: denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 61-63°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 8 min after the cycle; the results are shown in Figure 11.

[0124] Table 5. PCR primers and product information for specific identification of Jingdou 626 soybean

[0125] The 5' flanking sequence of Jingdou 626 soybean obtained by sequencing includes positions 8915080 to 8915537 of chromosome 6 of the recipient soybean genome and positions 6175 to 6530 of the pGR20 vector, and the specific sequence is SEQ ID NO: 6; the 3' flanking sequence includes positions 13165 to 13487 of the pGR20 vector and positions 8915590 to 8916080 of chromosome 6 of the recipient soybean genome, and the specific sequence is SEQ ID NO: 7. The full-length sequence of the Jingdou 626 insertion site is shown in SEQ ID NO: 17; the results showed that the exogenous gene insertion site was between 8915537 and 8915590 of chromosome 6 of the soybean genome (Figure 11), and the arrangement order of the T-DNA sequence in the transgenic soybean Jingdou 626 was consistent with the corresponding genetic elements of its plasmid vector pGR20.

[0126] 2. Determination of specific PCR detection method

[0127] The primer JD626P-1, derived from the soybean genome 5' of the Jingdou 626 insertion site, and the primer GR20LB-R, derived from an exogenous gene, were used to form a primer pair that can specifically detect the 5' insertion site of Jingdou 626 soybean. The amplified product was 543 bp in size.

[0128] The PCR reaction system was as follows: 2 μL of 10× Buffer, 1 μL of dNTPs, 0.5 μL of upstream and downstream primers, 0.5 μL of Taq enzyme, 1 μL of template, and ddH2O to a total of 20 μL. The PCR reaction conditions were: denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for 35 cycles.

[0129] The leaf DNA of Qiqihar small-grain bean without template and the receptor was used as the control group, and the DNA of Jingdou 626 in step 1 was used as the experimental group. The results of PCR detection are shown in Figure 13: No PCR products were produced in the no-template control (lane 1) and the receptor (lane 2), and the target band was amplified in Jingdou 625 (lane 3) using the primer pair located at the 5' end of the insertion site (Figure 13).

[0130] Example 7

[0131] Application of specific PCR detection method

[0132] DNA was extracted from T3-T4 transgenic soybean leaves of Jingdou 625 and Jingdou 626. PCR detection was performed on T3-T4 generations of Jingdou 625 and Jingdou 626 using primers JD625P-1, derived from the soybean genome 5' to the Jingdou 625 insertion site, and GR20LB-R, derived from the exogenous gene. The PCR reaction system consisted of 2 μL of 10× buffer, 1 μL of dNTPs, 0.5 μL of each upstream and downstream primer, 0.5 μL of Taq enzyme, 1 μL of template, and ddH2O to a total of 20 μL. The reaction conditions were: denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 61-63°C for 30 s, and extension at 72°C for 30 s for 35 cycles, followed by extension at 72°C for 8 min.

[0133] The results of agarose gel electrophoresis showed that the target size fragments could be amplified in all generations of transgenic plants of Jingdou 625 and Jingdou 626 (Figures 14-15), while no bands were amplified in the untemplated and non-transgenic recipients, indicating that the insertion site of the exogenous gene can be stably inherited in different generations.

[0134] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for cultivating transgenic soybeans with composite traits of herbicide resistance, insect resistance and stress resistance, characterized in that: The plant recombinant expression vector pGR20 was introduced into the explants of the recipient soybean Qiqihar small-grain bean, and regenerated plants were obtained through the resistance cluster bud induction, elongation bud induction and rooting stages. The plant recombinant expression vector pGR20 comprises a glyphosate-resistant gene g2m-epsps, an insect-resistant gene cry1C and a stress-resistant gene GmNFYB1.

2. The cultivation method according to claim 1, characterized in that The stress tolerance is drought tolerance and salinity tolerance.

3. The cultivation method according to claim 1, characterized in that The nucleotide sequence of the glyphosate-resistant gene g2m-epsps is shown in SEQ ID NO: 1, the nucleotide sequence of the insect-resistant gene cry1C is shown in SEQ ID NO: 2, and the nucleotide sequence of the stress-resistant gene GmNFYB1 is shown in SEQ ID NO:

3.

4. The cultivation method according to claim 1, characterized in that The T-DNA region of the plant recombinant expression vector pGR20 contains the expression frames of the g2m-epsps, cry1C and GmNFYB1 genes.

5. The cultivation method according to claim 1, characterized in that: The method for introducing the plant recombinant expression vector pGR20 is the Agrobacterium cotyledon node transformation method.

6. The cultivation method according to claim 5, characterized in that: The Agrobacterium strain is Ag10.

7. The cultivation method according to claim 1, characterized in that: The clustered bud induction medium used for inducing the resistant clustered buds is: B5+30g / L sucrose+8g / L agar powder+0.6g / L MES+1.67mg / L 6-BA+150mg / L thiophanate-methyl benzylpenicillin+400mg / L carbenicillin+15mg / L glyphosate.

8. The cultivation method according to claim 1, characterized in that: The bud elongation medium used for the elongated bud induction is: MS+B5+30g / L sucrose+8g / L agar powder+0.6g / L MES+50mg / L aspartic acid+50mg / L glutamine+0.3mg / L indole-3-acetic acid+0.5mg / L gibberellin+150mg / L thiophanate-methyl benzylpenicillin+400mg / L carbenicillin+0.1mg / L zeatin+5mg / L glyphosate.

9. The cultivation method according to claim 1, characterized in that: The rooting medium used in the rooting stage is: MS+B5+30g / L sucrose+8g / L agar powder+0.6g / L MES+50mg / L aspartic acid+50mg / L glutamine.

10. A genetically modified soybean Jingdou 625, characterized in that: In the transgenic soybean Jingdou 625, the T-DNA region insertion site of the plant recombinant expression vector pGR20 is between 10864177-10864529 of chromosome 13 of the recipient soybean Qiqihar small-grain bean genome.

11. The transgenic soybean Jingdou 625 according to claim 10, characterized in that: The 5' flanking sequence of Jingdou 625 includes positions 10863795 to 10864177 of chromosome 13 of the recipient soybean genome and positions 6203 to 6530 of the pGR20 vector, and the nucleotide sequence is shown in SEQ ID NO:4; the 3' flanking sequence includes positions 13165 to 13477 of the pGR20 vector and positions 10864529 to 10864795 of chromosome 13 of the recipient soybean genome, and the nucleotide sequence is shown in SEQ ID NO:

5.

12. A genetically modified soybean Jingdou 626, characterized in that: In the transgenic soybean Jingdou 626, the T-DNA region insertion site of the plant recombinant expression vector pGR20 is between 8915537-8915590 of chromosome 6 of the recipient soybean Qiqihar small-grain bean genome.

13. The transgenic soybean Jingdou 626 according to claim 12, characterized in that: The 5' flanking sequence of the Jingdou 626 includes positions 8915080 to 8915537 of chromosome 6 of the recipient soybean genome and positions 6175 to 6530 of the pGR20 vector, and the nucleotide sequence is shown in SEQ ID NO:6; the 3' flanking sequence includes positions 13165 to 13487 of the pGR20 vector and positions 8915590 to 8916080 of chromosome 6 of the recipient soybean genome, and the nucleotide sequence is shown in SEQ ID NO:

7.

14. A primer combination for identifying Jingdou 625 and its derivative materials, characterized in that: It includes a primer pair JD625P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 625 soybean, and a primer pair GR20RB-F and JD625P-2 for specifically detecting the 3' insertion position; The nucleotide sequence of the JD625P-1 is shown in SEQ ID NO:8, the nucleotide sequence of the GR20LB-R is shown in SEQ ID NO:9, the nucleotide sequence of the GR20RB-F is shown in SEQ ID NO:10, and the nucleotide sequence of the JD625P-2 is shown in SEQ ID NO:

11.

15. A kit for identifying Jingdou 625 and its derivative materials, characterized in that: Comprising the primer combination of claim 14.

16. A method for identifying Jingdou 625 and its derivative materials, characterized in that: include: Extracting genomic DNA of soybean to be tested; The genomic DNA of the soybean to be tested is amplified by PCR using the primer combination described in claim 14. If a corresponding band is amplified, it is Jingdou 625 or its derivative material.

17. A primer combination for identifying Jingdou 626 and its derivative materials, characterized in that: It includes a primer pair JD626P-1 and GR20LB-R for specifically detecting the 5' insertion position in Jingdou 626 soybean, and a primer pair GR20RB-F and JD626P-2 for specifically detecting the 3' insertion position; The nucleotide sequence of JD626P-1 is shown in SEQ ID NO: 12, and the nucleotide sequence of JD626P-2 is shown in SEQ ID NO: 13 shown.

18. A kit for identifying Jingdou 626 and its derivative materials, characterized in that: Comprising the primer combination of claim 17.

19. A method for identifying Jingdou 626 and its derivative materials, characterized in that: include: Extracting genomic DNA of soybean to be tested; The genomic DNA of the soybean to be tested is amplified by PCR using the primer combination described in claim 17. If a corresponding band is amplified, it is Jingdou 626 or its derivative material.

20. Use of the breeding method according to any one of claims 1 to 9, the primer combination according to claim 14, the kit according to claim 15, the method according to claim 16, the primer combination according to claim 17, the kit according to claim 18 or the method according to claim 19 in soybean breeding.

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