WO2025067527A1 - Cultivation method for herbicide-resistant, insect-resistant, and stress-tolerant soybeans jingdou 625 and jingdou 626, and use thereof - Google Patents

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

This application claims the priority of 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

The present application belongs to the field of biotechnology, and in particular relates to a breeding method and application of herbicide-resistant, insect-resistant and stress-resistant soybeans Jingdou 625 and Jingdou 626.

Background Art

Soybean is not only the main oil crop and source of vegetable protein for human beings, but also an important industrial raw material. In the process of soybean cultivation, weeds, insect pests and abiotic stress are important factors affecting crop production. In field management, weed control mainly relies on chemical weed control, supplemented by mechanical and manual weeding. The use of traditional herbicides has caused widespread weed damage, soil environmental deterioration and serious crop damage. The prevention and control of pests and diseases usually requires spraying a large amount of pesticides to control pests. In addition, drought and salinity are also one of the main factors affecting crop production.

As an emerging biotechnology, plant transgenic technology has obvious advantages in improving crop resistance, yield, breeding, etc. At present, there is a transgenic silencing phenomenon in soybean transgenics. When transgenic soybeans, it is necessary not only to detect the integration of exogenous genes, but also to make the exogenous genes stable and expressed at a high level as much as possible. In addition, the plant regeneration rate of soybean tissue culture is not high, and the regenerated plants obtained by genetic transformation using cotyledon nodes as explants are mostly chimeras, which affects the genetic transformation of soybeans.

Summary of the invention

The present application provides a method for cultivating soybeans with herbicide-resistant, insect-resistant and reversal-resistant genes, wherein a plant recombinant expression vector pGR20 is introduced into an explant of a recipient soybean, Qiqihar small-grain bean; the plant recombinant expression vector pGR20 comprises a glyphosate-resistant gene g2m-epsps, an insect-resistant gene cry1C, and a stress-resistant gene GmNFYB1.

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

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 of chromosome 6 of the recipient soybean Qiqihar small-grain bean genome.

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.

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.

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

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.

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.

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 perform PCR amplification on the genomic DNA of the soybean to be tested, and if the corresponding band is amplified, it is Jingdou 626 or its derivative material.

The present application provides a method for cultivating transgenic soybeans with composite traits of herbicide resistance, insect resistance and stress resistance, 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 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 a method for identifying Jingdou 625 and its derivatives, The present invention describes the application of the method for identifying Jingdou 626 and its derivative materials in soybean breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: PCR detection of exogenous genes g2m-epsps, cry1C and GmNFYB1 in T2 plants of soybean transgenic lines; M: DNA marker, lane 1: no template control, lane 2: plasmid positive control; lane 3: non-transgenic receptor 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;

Figure 2: Glyphosate resistance identification of T3 generation Jingdou 625 and Jingdou 626;

Figure 3: Glyphosate resistance identification of T4 generation Jingdou 625 and Jingdou 626;

Figure 4: Identification of insect resistance of T3 generation Jingdou 625 and Jingdou 626;

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

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

Figure 7: Evaluation of drought tolerance of Jingdou 625 and Jingdou 626 at the seedling stage in T4 generation;

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

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

Figure 10: Insertion position of foreign genes in Jingdou 625;

Figure 11: Insertion position of foreign genes in Jingdou 626;

Figure 12: Specific qualitative PCR detection of Jingdou 625 transgenic soybean;

Figure 13: Specific qualitative PCR detection of Jingdou 626 transgenic soybean;

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

Figure 15: Specific PCR detection of T3-T4 generation materials of transgenic soybean Jingdou 626.

DETAILED DESCRIPTION

The present application provides a method for cultivating soybeans with herbicide-resistant, insect-resistant and reversible genes, wherein a plant recombinant expression vector pGR20 is introduced into an explant of a recipient soybean, Qiqihar small-grain bean; the plant recombinant expression vector pGR20 includes a glyphosate-resistant gene g2m-epsps, an insect-resistant gene cry1C and a stress-resistant gene GmNFYB1, and the stress resistance is drought resistance and salt-alkali resistance. By introducing herbicide-resistant, insect-resistant and stress-resistant genes into recipient soybeans through plant vectors, transgenic soybeans with complex traits can be cultivated, which can effectively prevent and control weeds and insect pests and cope with drought and salt-alkali adversities, and have good application prospects.

The glyphosate-resistant gene is a 5-enolpyruvyl-3-phosphate synthase (epsps) gene, which comes from Pseudomonas fluorescens, a bacterium that can grow in a high-concentration glyphosate environment, and the isolated g2m-epsps enzyme is insensitive to glyphosate. In transgenic plants, plant cells over-synthesize the g2m-epsps enzyme, which can replace the epsps enzyme bound by glyphosate in the plant body in the presence of glyphosate to perform functions, thereby protecting the plant from glyphosate toxicity.

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 pesticides, it has less impact on the environment and is safer. Bt protein synthesizes toxin precursors during spore formation, is activated under the alkaline environmental conditions of the pest intestine, and then the activated protein molecules are inserted into the midgut epithelial cell membrane to form a cation channel on the cell membrane, breaking the osmotic balance of the cell membrane, causing the midgut cells to disintegrate, and ultimately causing the death of the insect. The cry1C gene encodes the insecticidal crystal protein cry1C protein (Bt protein) that is toxic to lepidopteran insects, making transgenic soybeans resistant to lepidopteran pests and reducing the losses caused by insect pests.

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

The nucleotide sequence of the glyphosate-resistant gene g2m-epsps described 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.

The present application also provides a method for constructing the plant recombinant expression vector pGR20, comprising the following steps:

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;

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;

Using the pBI121-crylC vector as a template, using specific primers with KpnI and HindIII restriction sites at both ends, amplifying a fragment of the insect-resistant gene expression frame, and connecting it with the p3301-NF vector cut with KpnI and HindIII to construct a p3301-NF-cry1C plant expression vector;

The g2m-epsps fragment containing the 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 the herbicide-resistant gene expression frame.

In the optional construction of the p3301-NF vector, the reaction system of double enzyme digestion is preferably: template 10 μL, 10×Buffer 2 μL, BglII 1 μL, BstEII 1 μL, and ddH ₂ O is supplemented to a total system of 20 μL. The reaction conditions of the double enzyme digestion are preferably 37°C overnight. The connection is preferably performed using T4 ligase. The connection conditions are preferably 16°C for 4 hours. After the connection, the recombinant vector is preferably verified. The present application has no special restrictions on the verification method, and the verification method well known in the art can be used, such as transformation and cultivation in Escherichia coli, extraction of plasmids, and colony PCR amplification or sequencing. The present 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 the present application was purchased from Cambia.

In the constructed pBI121-crylC vector, the double enzyme digestion reaction system is: template 10μL, 10×Buffer 2μL, BamHI 1μL, SacI 1μL, and ddH ₂ O is supplemented to a total system of 20μL. The reaction conditions of the double enzyme digestion are preferably 37°C 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. The present 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, extraction of plasmids, and colony PCR amplification or sequencing. The present 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 the present application was purchased from Clontech.

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 restriction enzyme digestion, the connection condition is preferably 16°C for 4 hours. After the connection, the recombinant vector is preferably verified. The present application has no special restrictions on the verification method, and the verification method well known in the art can be used, such as transformation and cultivation in Escherichia coli, extraction of plasmids, and colony PCR amplification or sequencing.

In the present application, the g2m-epsps fragment containing the 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 frame. The present application has no special restrictions on the sequence synthesis of the g2m-epsps fragment containing the XhoI restriction site, and the sequence synthesis technology known in the art can be used. The g2m-epsps fragment containing the XhoI restriction site described in the examples of the present application was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

In the present application, the T-DNA region of the plant recombinant expression vector pGR20 contains only the expression frames of the g2m-epsps, cry1C and GmNFYB1 genes, and the target gene is driven by the 35S promoter derived from the cauliflower mosaic virus. It is particularly pointed out that there is no antibiotic marker gene or reporter gene in the inserted sequence, and the vector is non-pathogenic and has no possibility of evolving into pathogenicity.

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 cotyledon node transformation method, and the Agrobacterium strain is Ag10.

The present application uses normally germinated and pollution-free recipient soybean seedlings of Qiqihar small beans to prepare explants. In one embodiment, the soybean is cut along the hypocotyl with a scalpel in a clean bench, retaining 3-4 mm 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 axial incisions are made at the junction of the cotyledon and the cotyledon hypocotyl, and the incision is about 3-4 mm long. Each explant is composed of a cotyledon connected to a section of the 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: 1/10 B5+30 g/L sucrose+3.9 g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67 mg/L 6-BA+39 mg/L acetosyringone+0.25 mg/L gibberellin (GA ₃ ).

The present application takes out the low-temperature preserved Agrobacterium strain from the ultra-low temperature refrigerator and freezes and thaws it on ice. In one embodiment, a small amount of the strain is dipped with an inoculation loop or a sterile pipette tip and inoculated on an LB plate, and inverted and cultured at a constant temperature of 25-30°C for 1-2 days to obtain a single clone. In one embodiment, the culture is carried out at a constant temperature of 26-28°C for 2 days; after the culture is completed, a single clone is picked and inoculated into a YEP culture medium for shaking culture. In one embodiment, the culture conditions are activation culture at 200-230rpm and 25-30°C for 10-14h. In another embodiment, the activation culture is carried out at 210-220rpm and 26-28°C. 11-12h; when the bacterial solution is activated to saturation for the first time, the bacterial solution is extracted and inoculated into the YEP culture solution, the volume ratio of the bacterial solution to the YEP culture solution is 1: (90-110), in one embodiment, the volume ratio of the bacterial solution to the YEP culture solution is 1: (98-105), and the second activation is carried out at 26-28°C and 210-220rpm. When Agrobacterium is fully activated to _OD600 = 0.9-1.1, the bacterial solution is centrifuged at 4°C. In one embodiment, the centrifugation parameters are 3800-4200rpm and centrifugation for 8-12min. In one embodiment, the centrifugation parameters are 4000-4100rpm and centrifugation for 9-10min. The supernatant is discarded to collect the precipitate, and the bacteria precipitated at the bottom of the tube are suspended with an equal volume of co-culture solution, and the _OD600 is adjusted to 0.5-0.8 for standby use. In the present application, the LB solid culture medium contains 50 mg/L kanamycin, and the YEP culture medium contains 50 mg/L kanamycin.

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 bacterial solution is added to each flask, and the bacterial solution needs to immerse the explant. Co-infect for 30-35 minutes under dark or weak light conditions, and shake the triangular flask once every 5 minutes to allow Agrobacterium and explant to fully contact. After the infection is completed, the excess Agrobacterium bacterial solution is carefully poured out, and a layer of sterile filter paper is laid flat on the co-cultivation medium. The explant after the infection is laid flat on the filter paper with the axis side facing down, and co-cultivated for 2-6 days at 22-30°C and dark conditions. It can be implemented to co-cultivate for 3-5 days at 26-28°C and dark conditions. The co-culture medium is: 1/10B5+30g/L sucrose+3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg/L gibberellin (GA ₃ )+1mmol/L dithiothreitol+1mmol/L sodium thiosulfate+1mmol/L cysteine+5g/L agar powder, pH 5.4.

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, rooting, etc. to obtain regenerated plants; the cluster bud induction medium is: B5+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine) ethanesulfonic acid (MES)+1.67mg/L 6-BA+150mg/L thiophanate-methyl sulfonate+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-morpholine) ethanesulfonic acid (MES)+50mg/L aspartic acid+50mg/L glutamine+0.3mg/L indole-3-acetic acid (IAA)+0.5mg/L gibberellin (GA ₃ )+150mg/L thiophanate-methyl+400mg/L carbenicillin+0.1mg/L zeatin (Ze)+5mg/L glyphosate, pH5.7; the rooting medium is: MS+B5+30g/L sucrose+8g/L agar powder+0.6g/L 2-(N-morpholine) ethanesulfonic acid (MES)+50mg/L aspartic acid+50mg/L glutamine, pH5.7.

In the present application, the explants in the cluster bud induction stage and the elongation bud induction stage are subcultured once every two weeks, and a new incision is prepared on the back of the explant during the subculture so that the explant can better absorb nutrients. In the present application, when the elongation buds are elongated to 4-6 cm, roots are induced, and after rooting, the sealing film on the culture dish is opened to harden the seedlings for 1-3 days, and then transplanted to potted plants or fields for growth to obtain transgenic plants.

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

Most regenerated plants obtained by genetic transformation using cotyledon nodes as explants are chimeras. Somatic embryogenesis induced by immature cotyledons of soybeans provides a possible way to solve this problem, but the somatic embryos cannot proliferate in succession. This low transformation efficiency also affects the genetic transformation of soybeans to a certain extent. The present application introduces multiple genes into the receptor for genetic transformation at the same time, and obtains transgenic plants with multiple stable genetic traits through tissue culture, which is conducive to accelerating the improvement of multiple traits of soybeans and the cultivation of new varieties.

In the present application, MS and B5 dry powder culture medium and acetosyringone are purchased from Sigma, 2-(N-morpholino)ethanesulfonic acid (MES), cefotaxime, carbenicillin, agar powder, zeatin, aspartic acid, glutamine, gibberellin (GA ₃ ) and 6-benzylaminoadenine (6-BA) are products of Biodee, and sucrose is a domestic reagent.

The present application provides a transgenic soybean Jingdou 625, in which 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.

In the present application, 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.

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 of chromosome 6 of the recipient soybean Qiqihar small-grain bean genome.

In the present application, the 5' 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' 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.

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.

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.

The present application provides an identification method for Jingdou 625 and its derivative materials, comprising: extracting genomic DNA of soybeans to be tested; using the primer combination for identifying Jingdou 625 and its derivative materials to perform PCR amplification on the genomic DNA of the soybeans to be tested. Through the PCR reaction, observe whether the sample to be tested can amplify a band. If the corresponding band can be amplified, it means that the sample to be tested is Jingdou 625 or its derivative materials. If the band cannot be amplified, it means that the sample to be tested is not Jingdou 625 or its derivative materials.

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.

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.

The present application provides a method for identifying Jingdou 626 and its derivatives, comprising: extracting genomic DNA of soybeans to be tested; using the primer combination for identifying Jingdou 626 and its derivatives to perform PCR amplification on the genomic DNA of the soybeans to be tested. Through the PCR reaction, observe whether the sample to be tested can amplify a band. If the corresponding band can be amplified, it indicates that the sample to be tested is Jingdou 626 or its derivatives. If the band cannot be amplified, it indicates that the sample to be tested is not Jingdou 626 or its derivatives.

The present application provides a method for cultivating transgenic soybeans with composite traits of herbicide resistance, insect resistance and stress resistance, 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 an application of the method for identifying Jingdou 626 and its derivatives in soybean breeding.

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

This application uses bioengineering technology to cultivate new varieties of transgenic soybeans with complex traits, providing a better solution for the effective prevention and control of weeds and pests in soybean fields and for coping with drought and salinity adversity. In transgenic plants, plant cells over-synthesize g2m-epsps enzymes, which can replace the epsps enzymes bound by glyphosate in the plant body in the presence of glyphosate to perform functions, protecting the plant from glyphosate toxicity; 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 pests; over-expression of the GmNFYB1 gene in transgenic soybeans can participate in regulating downstream gene expression and enhance the drought and salinity tolerance of the plant.

In the present application, unless otherwise specified, the chemical reagents used are all conventional commercially available reagents, and the technical means used are all conventional technical means well known to those skilled in the art.

The technical solutions provided by the present application are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present application.

Example 1

Construction of plant expression vector pGR20

(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: 10 μL template, 2 μL 10× Buffer, 1 μL BglII, 1 μL BstEII, and 20 μL ddH ₂ O to make up the total system. The enzyme digestion was carried out at 37°C overnight.

The GmNFYB1 gene fragment double-digested with BglII and BstEII and the commercial vector pCAMBIA3301 were ligated at 16°C for 4 hours using T4 ligase to construct the p3301-NF vector;

(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, ddH ₂ O to make up the total system 20 μL, and the enzyme digestion was carried out at 37°C overnight;

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

(3) Using the pBI121-crylC vector as a template, amplify the DNA using the specific primers shown in SEQ ID NO: 14 and SEQ ID NO: 15. The fragment of the insect-resistant gene expression frame was amplified and connected with the p3301-NF vector cut with KpnI and HindIII at 16°C for 4 hours to construct the p3301-NF-cry1C plant expression vector

(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 frame.

Example 2

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

1. Prepare culture medium and culture fluid:

1) Co-culture solution: 1/10B5+30g/L sucrose+3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg/L gibberellin (GA ₃ );

2) Co-culture medium: 1/10B5+30g/L sucrose+3.9g/L 2-(N-morpholine)ethanesulfonic acid (MES)+1.67mg/L 6-BA+39mg/L acetosyringone+0.25mg/L gibberellin (GA ₃ )+1mmol/L dithiothreitol+1mmol/L sodium thiosulfate+1mmol/L cysteine+5g/L agar powder, pH 5.4;

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

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

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;

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

2. Select normal germination and pollution-free recipient soybean seedlings, cut the soybean along the hypocotyl with a scalpel in a clean bench, keep 3-4mm of the hypocotyl, and place it in a sterile culture dish. Add 5mL of the co-culture solution prepared in step 1 to the culture dish, and then cut the hypocotyl vertically along the cotyledon hypocotyl, remove the clean true leaf tissue, and make 5-7 axial incisions at the junction of the cotyledon and the cotyledon hypocotyl. The incision is about 3-4mm long. Each explant consists of a cotyledon connected to a hypocotyl, and one seed can form two explants;

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 in YEP culture medium (containing 50 mg/L kanamycin), and activate it at 28°C and 220 rpm for 12 hours; extract 1 mL of bacterial solution and inoculate it into 100 mL YEP culture medium (containing 50 mg/L kanamycin), and activate it at 28°C and 220 rpm. When the Agrobacterium is fully activated to _OD600 = 1.0, centrifuge the bacterial solution at 4000 rpm for 10 minutes at 4°C, discard the supernatant, collect the precipitate, and suspend the bacterial bodies precipitated at the bottom of the tube with an equal volume of the co-culture solution prepared in step 1, adjust the _OD600 to 0.7, and set it aside;

4. Place 50 explants prepared in step 2 in a conical flask, add 50 mL of the Agrobacterium solution resuspended in step 3, and infect for 35 min in the dark. Gently shake the conical flask every 5 min to ensure full contact between the Agrobacterium and the explants. After the infection is completed, carefully pour out the excess Agrobacterium solution, spread a layer of sterile filter paper on the co-cultivation medium prepared in step 1, and 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.

5. After the co-cultivation, the explant is placed in the clustered bud induction medium prepared in step 1. The explant is subcultured once every two weeks, and a new incision is prepared on the back of the explant during the subculture. After three subcultures, the explant is placed in the bud elongation medium prepared in step 1, and the explant is subcultured once every two weeks. During the subculture, a new incision is prepared on the back of the explant. When the elongated buds are elongated to 4-6cm, the explant is placed in the rooting medium prepared in step 1 to induce rooting. After rooting, the sealing film on the culture dish is opened to harden the seedlings for 1-3 days, and then transplanted to the field for growth to obtain transgenic plants.

Example 3

Detection of target gene in transgenic lines obtained in Example 2

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

2. Using the DNA extracted in step 1 as a template, specific primers were used to detect the target genes of 10 soybean transgenic lines. The specific primers are shown in Table 1. The reaction system is: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up the total system to 20μL; the reaction conditions are: 94℃ denaturation for 3min, 94℃ denaturation for 30s, 61-63℃ annealing for 30s, 72℃ extension 30s, 35 cycles, and 72℃ extension for 8min after the cycle. Take 10μl of the PCR amplification product, and detect it by 1.5% agarose gel electrophoresis (containing ethidium bromide), 5V/cm electrophoresis for 40min, and observe and take pictures under ultraviolet light; the results are shown in Figure 1.

Table 1 List of primers for transgenic detection

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

Example 4

Identification of target traits of soybean transgenic plants

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;

2. Glyphosate resistance identification of T3-T4 generation Jingdou 625 and Jingdou 626 transgenic soybeans and recipient Qiqihar small-grained beans: During the first to third compound leaf stage of transgenic soybeans, 41% glyphosate solution (glyphosate isopropylamine salt solution) was sprayed with a handheld pressure sprayer from Hudson Company, USA, at a spraying rate of 6L/hectare, and the pesticide damage was investigated after 2 weeks. The results are shown in Figures 2 and 3;

The results showed that under the above spraying dose, the recipient Qiqihar small-grain bean had no resistance and the plant died after 2 weeks; the growth of the homozygous transgenic soybean Jingdou 625 and Jingdou 626 lines in the T3 generation was not inhibited, the leaves did not turn green, did not shrink, and there was no glyphosate injury such as yellowing of new leaves. This shows that the transgenic soybeans Jingdou 625 and Jingdou 626 were highly resistant to glyphosate (Figure 2). Similarly, the homozygous transgenic soybeans Jingdou 625 and Jingdou 626 lines in the T4 generation also showed uninhibited growth, no green chlorosis, no shrinkage of leaves, and no glyphosate injury such as yellowing of new leaves (Figure 3). This shows that the glyphosate resistance in the transgenic soybeans Jingdou 625 and Jingdou 626 can be stably inherited in different generations.

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 uniform unfolded leaves from T3 transgenic soybean plants, punch the leaves into 2.5 cm discs with a hole puncher, select 5 leaves and put them into a sponge-covered culture dish, moisturize with 13 mL sterile water, inoculate 2nd-instar Spodoptera litura larvae at a ratio of 1 head/dish, investigate the leaf damage every day after inoculation, and investigate 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 condition was investigated 2 weeks after inoculation, and the leaf biting condition was graded according to the leaf biting condition (Table 2), and the average insect susceptibility index was calculated; the resistance was graded according to Table 3; the results are shown in Figures 4 and 5;

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

Table 2. Leaf damage identification and grading standards

Table 3. Classification of resistance of leaf-feeding pests

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.

4. Stress tolerance was identified for the T3-T4 generation Jingdou 625 and Jingdou 626 transgenic soybeans and the recipient Qiqihar small-grain soybean: For drought tolerance identification at the bud stage, water treatment control and 17.5% PEG treatment were set up respectively, and the germination of soybean seeds was observed 5 days after germination; for drought tolerance identification at the seedling stage, the soybean cotyledons were subjected to drought treatment for 2 weeks after expansion, and the recovery of soybean seedlings was observed after rehydration. The results are shown in Figures 6 and 7.

According to the drought tolerance identification results at the bud and seedling stages, the drought tolerance of the T3 generation transgenic soybeans Jingdou 625 and Jingdou 626 at the bud stage was higher than that of the recipient Qiqihar small-grain bean (Figure 6), and the drought tolerance of the T4 generation Jingdou 625 and 332 at the seedling stage was significantly higher than that of the recipient Qiqihar small-grain bean (Figure 7).

5. Analysis of exogenous gene expression: RNA was extracted from the roots, stems, leaves and seeds of transgenic materials, and RT-PCR was performed to detect g2m-epsps, cry1C and GmNFYB1 genes using ACTIN gene as internal reference after reverse transcription. The results showed that g2m-epsps and cry1C were not expressed in the recipient Qiqihar small-grain bean, and the expression level of GmNFYB1 gene was extremely low, while it was expressed in various tissues of Jingdou 625 and Jingdou 626 transgenic materials (Figure 8-Figure 9). This shows that exogenous genes can be stably expressed in transgenic materials.

Example 5

Identification of foreign gene insertion sites in Jingdou 625

1. Size and structure of the insert sequence

The foreign gene in the Jingdou 625 soybean plant was inserted into chromosome 13. The DNA of the leaves of the Jingdou 625 soybean plant was extracted, 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: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up the total system to 20μL; the reaction conditions were: 94℃ denaturation for 3min, 94℃ denaturation for 30s, 61-63℃ annealing for 30s, 72℃ extension for 30s, 35 cycles, and 72℃ extension for 8min after the cycle; the results are shown in Figure 10.

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

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 was 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 was SEQ ID NO:5. The full-length sequence of the Jingdou 625 insertion site was shown in SEQ ID NO:16. The results showed that the insertion site of the exogenous gene 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.

2. Determination of specific PCR detection method

Primer JD625P-1 derived from the soybean genome at the 5' insertion site of Jingdou 625 and primer GR20LB-R derived from an exogenous gene were used to form a primer pair that can specifically detect the 5' insertion position of Jingdou 625 soybean, and the size of the amplified product is 637bp.

The PCR reaction system was as follows: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up to a total system of 20μL; PCR reaction conditions: 94℃ denaturation for 4min, 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 35 cycles.

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

Example 6

Identification of foreign gene insertion sites in Jingdou 626

1. Size and structure of the insert sequence

The foreign gene in the Jingdou 626 soybean plant was inserted into chromosome 6. The DNA of the leaves of the Jingdou 626 soybean plant was extracted, 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: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up the total system to 20μL; the reaction conditions were: 94℃ denaturation for 3min, 94℃ denaturation for 30s, 61-63℃ annealing for 30s, 72℃ extension for 30s, 35 cycles, and 72℃ extension for 8min after the cycle; the results are shown in Figure 11.

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

The 5’ flanking sequence of Jingdou 626 soybean obtained by sequencing included 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 was SEQ ID NO:6; the 3’ flanking sequence included 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 was 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 insertion site of the exogenous gene was between positions 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.

2. Determination of specific PCR detection method

Primer JD626P-1 derived from the soybean genome at the 5' insertion site of Jingdou 626 and 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, and the size of the amplified product was 543 bp;

The PCR reaction system was as follows: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up to a total system of 20μL; PCR reaction conditions: 94℃ denaturation for 4min, 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, 35 cycles.

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

Example 7

Application of specific PCR detection method

DNA from the T3-T4 transgenic soybean leaves of Jingdou 625 and Jingdou 626 was extracted, and PCR detection was performed on T3-T4 Jingdou 625 and Jingdou 626 using primers JD625P-1 derived from the soybean genome at the 5' insertion site of Jingdou 625 and primers GR20LB-R derived from the exogenous gene, as well as primers JD626P-1 derived from the soybean genome at the 5' insertion site of Jingdou 626 and primers GR20LB-R derived from the exogenous gene. The PCR reaction system was: 10×Buffer 2μL, dNTP 1μL, upstream and downstream primers 0.5μL each, Taq enzyme 0.5μL, template 1μL, ddH ₂ O to make up the total system to 20μL; the reaction conditions were: 94℃ denaturation for 3min, 94℃ denaturation for 30s, 61-63℃ annealing for 30s, 72℃ extension for 30s, 35 cycles, and 72℃ extension for 8min after the cycle.

The results of agarose gel electrophoresis showed that the transgenic plants of Jingdou 625 and Jingdou 626 of each generation were able to amplify the target size fragments (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.

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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (20)

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 is introduced into the explants of the recipient soybean Qiqihar small-grain bean, and regenerated plants are obtained through the stages of resistant cluster bud induction, elongation bud induction and rooting; The plant recombinant expression vector pGR20 comprises a glyphosate-resistant gene g2m-epsps, an insect-resistant gene cry1C and a stress-resistant gene GmNFYB1. The cultivation method according to claim 1 is characterized in that the stress tolerance is drought tolerance and saline-alkali tolerance. The breeding method according to claim 1, characterized in that the nucleotide sequence of the glyphosate-resistant gene g2m-epsps is as shown in SEQ ID NO: 1, the nucleotide sequence of the insect-resistant gene cry1C is as shown in SEQ ID NO: 2, and the nucleotide sequence of the stress-resistant gene GmNFYB1 is as shown in SEQ ID NO: 3, The breeding method according to claim 1 is characterized in that the T-DNA region of the plant recombinant expression vector pGR20 contains expression frames of the g2m-epsps, cry1C and GmNFYB1 genes. The cultivation method according to claim 1 is characterized in that the method for transferring the plant recombinant expression vector pGR20 is the Agrobacterium cotyledon node transformation method. The cultivation method according to claim 5, characterized in that the Agrobacterium strain is Ag10. The cultivation method according to claim 1 is characterized in that the cluster bud induction medium used for inducing the resistant cluster buds is: B5+30g/L sucrose+8g/L agar powder+0.6g/L MES+1.67mg/L6-BA+150mg/L thiophanate-methyl+400mg/L carbenicillin+15mg/L glyphosate. The cultivation method according to claim 1 is characterized in that the bud elongation medium used for inducing elongated buds 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. The cultivation method according to claim 1 is 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. A transgenic 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. The transgenic soybean Jingdou 625 according to claim 10 is 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. A transgenic 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. The transgenic soybean Jingdou 626 according to claim 12 is characterized in that the 5' 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' 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. A primer combination for identifying Jingdou 625 and its derivative materials, characterized by comprising 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. A kit for identifying Jingdou 625 and its derivative materials, characterized by comprising the primer combination according to claim 14. A method for identifying Jingdou 625 and its derivative materials, characterized by comprising: extracting genomic DNA of the soybean to be tested; using the primer combination of claim 14 to perform PCR amplification on the genomic DNA of the soybean to be tested, and if a corresponding band is amplified, it is Jingdou 625 or its derivative material. A primer combination for identifying Jingdou 626 and its derivative materials, characterized by comprising 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. A kit for identifying Jingdou 626 and its derivative materials, characterized in that it comprises the primer combination according to claim 17. A method for identifying Jingdou 626 and its derivative materials, characterized by comprising: extracting genomic DNA of the soybean to be tested; using the primer combination of claim 17 to perform PCR amplification on the genomic DNA of the soybean to be tested, and if a corresponding band is amplified, it is Jingdou 626 or its derivative material. 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.