Gene gmsrc2 for improving resistance to phytophthora sojae and application thereof

Abstract

The application discloses a GmSRC2 gene for improving resistance of soybean phytophthora and application, and the soybean plant with the GmSRC2 gene disclosed by the application enhances the resistance of the soybean to the soybean phytophthora, that is, the phenotype, disease spot area, phytophthora accumulation amount and hydrogen peroxide and other physiological indexes of the soybean cultivar "Jack" with overexpressed GmSRC2 under the stress of the phytophthora are detected, and the results show that the GmSRC2 plays an important positive regulation role in improving the resistance of the plant to the phytophthora. Through the gene engineering improvement of the plant resistance to the phytophthora root rot of the transformant, it is found that the gene has a certain effect on cultivating the plant variety with high resistance to the phytophthora root rot, and can improve the resistance of the plant to the phytophthora, thereby serving the improvement of the yield and quality of the plant.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application of the gene GmSRC2, which enhances resistance to Phytophthora in soybean. Technical Background

[0002] Soybean root rot (PRR) is one of the most destructive diseases caused by Phytophthora sojae. It has been reported globally, severely impacting soybean yield and quality, causing significant economic losses. In addition to conventional control measures, improving soybean resistance through breeding is one of the most effective control strategies. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the soybean GmSRC2 gene in the prevention of Phytophthora root rot in soybeans. Introducing this gene into plants can improve the resistance of soybeans to Phytophthora sojae, thereby improving soybean varieties.

[0004] One of the objectives of this invention is to provide a gene, GmSRC2, that enhances resistance to Phytophthora in soybean.

[0005] The second objective of this invention is to provide a recombinant expression vector containing the gene GmSRC2 and its transformants.

[0006] The third objective of this invention is to provide applications for the gene GmSRC2.

[0007] The specific details of this invention are as follows:

[0008] This invention provides a gene GmSRC2 that enhances resistance to Phytophthora in soybean, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0009] The present invention also provides primer pairs for extending the GmSRC2 gene. These primer pairs can be designed according to conventional methods in the art, and in one specific example, the primer pairs are shown as SEQ ID No. 6 and SEQ ID No. 7.

[0010] The present invention also provides a recombinant expression vector containing the gene GmSRC2 described in the present invention.

[0011] The recombinant expression vector is selected from commonly used expression plasmids in plant genetic transformation. In a specific example, the gene GmSRC2 is ligated into the pTF101.1 vector using Sma I and Sac I enzymes to form a recombinant expression vector, namely the plant expression vector pTF101.1-GmSRC2.

[0012] This invention also provides a transformant obtained by introducing the recombinant expression vector into host cells, preferably *E. coli* cells or *Agrobacterium* cells. The host bacterium containing the pTF101.1-GmSRC2 encoding gene is obtained by transforming the GmSRC2 gene into *Agrobacterium tumefaciens* strain EHA101. Subsequently, pTF101.1-GmSRC2 is introduced into soybean cells using the host bacterium EHA101. After herbicide selection and propagation, soybean plants with stably heritable GmSRC2 gene transgenic genes are obtained. Phenotypic identification yields transgenic soybean plants with altered disease resistance.

[0013] The present invention also provides the application of the gene GmSRC2 or its amplification primers or recombinant expression vectors or transformants in improving resistance to Phytophthora in soybean.

[0014] The present invention also provides the application of the gene GmSRC2 or its amplification primers or recombinant expression vectors or transformants in improving the resistance of soybean to diseases caused by Phytophthora in soybean.

[0015] The present invention also provides the application of the gene GmSRC2 or its amplification primers or recombinant expression vectors or transformants in soybean breeding.

[0016] The present invention also provides the application of the gene GmSRC2 or its amplification primers or recombinant expression vectors or transformants in soybean varieties that have been introduced to obtain significant resistance to fungal diseases.

[0017] Beneficial effects of the present invention

[0018] Tissue expression analysis of GmSRC2 in this invention showed its expression in the roots, stems, leaves, and cotyledons of soybean. Induced expression analysis of GmSRC2 indicated its involvement in the responses of *Phytophthora sojae*, salicylic acid (SA), methyl jasmonate (MeJA), and bacterial flagellin (Flg22). Phenotypic results, lesion size, *Phytophthora* accumulation, and physiological indicators of soybean Jack transgenic with the GmSRC2 gene under *Phytophthora sojae* stress indicated that GmSRC2 plays a crucial regulatory role in enhancing plant resistance to *Phytophthora sojae*. Genetic engineering improvement of soybean against *Phytophthora sojae* root rot using this gene can provide an indicator for breeding soybean varieties with higher resistance to this disease. Providing genetically modified soybean plants with enhanced resistance to *Phytophthora sojae* can contribute to improving soybean yield and quality. Attached Figure Description

[0019] Figure 1 Tissue expression analysis of GmSRC2;

[0020] Figure 2 Analysis of GmSRC2 induced expression;

[0021] Figure 3 GmSRC2 phylogenetic analysis;

[0022] Figure 4 A schematic diagram of a plant expression vector containing GmSRC2;

[0023] Figure 5 PCR screening of the genome of transgenic soybean plants (where M is DL2000 DNA Marker, 1 is wild-type Jack, and 2-3 are OE-GmSRC2-1 and OE-GmSRC2-2).

[0024] Figure 6 Expression level of GmSRC2 in transgenic soybean plants;

[0025] Figure 7 Phenotypic characteristics of transgenic soybean seedlings inoculated with Phytophthora soybean P6497 in the cotyledons;

[0026] Figure 8 Phytophthora accumulation in cotyledons of transgenic soybean seedlings inoculated with Phytophthora p6497;

[0027] Figure 9 Phenotypic characteristics of transgenic soybean seedlings inoculated with Phytophthora soybean P6497;

[0028] Figure 10 The area of ​​lesions and the amount of Phytophthora p6497 accumulated on the leaves of transgenic soybean seedlings. Detailed Implementation

[0029] The present invention will be further described in conjunction with the embodiments to facilitate understanding. The specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Any simple improvements to the preparation method of the present invention under the premise of the present invention are within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out according to well-known means in the art.

[0030] Unless otherwise specified, the soybean Phytophthora in the following examples is P6497, which is kept in the applicant's possession and the applicant promises to make it permanently available to the public.

[0031] The GmSRC2 phylogenetic tree analysis involved in the following examples is as follows: Figure 3 As shown.

[0032] Example 1

[0033] (1) Expression pattern of GmSRC2: Root, stem, leaf and cotyledon samples were taken from 7-day-old soybean "Williams 82"; 7-day-old soybean "Williams 82" was inoculated with Phytophthora soybeani P6497 using the hypocotyl wound inoculation method; 4-day-old Phytophthora soybeani was cultured on 10% V8 vegetable medium containing 1.5% agar, and samples were taken at 0h, 1h, 2h, 3h and 6h of treatment; 7-day-old soybean "Williams 82" seedlings were treated with 10μM Flg22, and samples were taken at 0min, 5min, 15min, 30min, 45min, 60min, 90min and 120min of treatment; 7-day-old soybean "Williams 82" was treated with 1 / 2 Hoagland nutrient solution containing 200mM salicylic acid (SA) and 200mM methyl jasmonate (MeJA). 82” seedlings were sampled at 0h, 3h, 6h, 9h, 12h, and 24h after treatment. All samples were used to extract RNA, and the reverse-engineered cDNA was used as a template for gene expression level detection. Figure 1 and Figure 2 A partial fragment of GmSRC2 was amplified using primers shown in SEQ ID No. 2 and SEQ ID No. 3, and a partial fragment of the soybean internal reference gene GmCons 4 was amplified using primers shown in SEQ ID No. 4 and SEQ ID No. 5. The amplification was performed using a 2- ΔΔCT Calculate its relative expression level.

[0034] (2) The GmSRC2 vector was constructed using homologous recombination. The cDNA of soybean “Williams 82” was used as a template. The full-length GmSRC2 (SEQ ID No. 1) was amplified using the primer pairs shown in SEQ ID No. 6 and SEQ ID No. 7, and then ligated into the pTF101.1 vector digested with Sma I and Sac I to form the recombinant vector. Figure 4 The above recombinant vector was transformed into Escherichia coli DH5α and Agrobacterium EHA101, and then transformed into the soybean variety "Jack" using the cotyledon transformation method.

[0035] (3) Identification of positive GmSRC2 transgenic plants: DNA was extracted from the leaves of transgenic plants as templates. The CaMV35S, Bar, and internal control GmActin11 gene fragments were amplified using primer pairs SEQ ID No. 8 and SEQ ID No. 9, primer pairs SEQ ID No. 10 and SEQ ID No. 11, and primer pairs SEQ ID No. 12 and SEQ ID No. 13, respectively. Compared with the wild type, the positive transgenic plants showed amplified bands (…). Figure 5The expression level of GmSRC2 in transgenic positive plants was detected using the gene expression detection method described in step (1). Figure 6 ).

[0036] (4) Four-day-old soybean seedlings with cotyledons were used for inoculation. A hole punch was used to create a surface wound in the center of the soybean cotyledon. 50 μL of *Phytophthora sojae* zoospore solution was dripped into the wound. The zoospore preparation process was as follows: *Phytophthora sojae* was cultured for 4 days in a 10% V8 vegetable juice medium containing 1.5% agar. The outer edge of the mycelium was then chopped and transferred to a 10% V8 vegetable juice medium for 3 days. Finally, the mycelium was washed 1–3 times with sterile tap water until no medium residue remained. The mixture was then cultured overnight at 25°C in the dark. The resulting zoospore solution (≈1 × 10⁻⁶) was obtained by filtration. 4 / mL). 50μL of sterile tap water was added to cotyledons with uniform growth as a control. Typical phenotypes were observed and photographed after 24h. Samples were taken, flash-frozen in liquid nitrogen, and stored at -80℃. The samples were used for nucleic acid extraction. Gene expression levels were detected using a gene expression detection method similar to that described in step (1). The ratio of the expression levels of soybean internal reference GmCons 4 and soybean Phytophthora indicum internal reference PsTEF represented the amount of Phytophthora indicum accumulation. Primer pairs SEQ ID No.4 and SEQ ID No.5 were used to amplify a partial fragment of soybean internal reference GmCons 4, and primer pairs SEQ ID No.14 and SEQ ID No.15 were used to amplify a partial fragment of Phytophthora indicum internal reference PsTEF.

[0037] The results are as follows Figure 7 and Figure 8 As shown, compared with the wild-type control, the GmSRC2 overexpression lines had lower disease severity and significantly reduced Phytophthora accumulation, indicating that GmSRC2 overexpression has a positive regulatory effect on improving soybean resistance to Phytophthora soybeanis.

[0038] (5) After the first true leaf of soybean unfolds (about 8 days), select leaves of uniform growth (6 or more leaves) for inoculation with zoospores of Phytophthora soybeanis. Drop 50 μL of zoospore solution onto one side of the leaf, and at the same time, drop 50 μL of sterile tap water onto a leaf of uniform size as a control. After 24 hours, take pictures with a Canon camera and calculate the size of the lesion area using ImageJ online software (https: / / imagej.nih.gov / ij / ). Take samples, quick-freeze in liquid nitrogen, and store at -80℃. The samples are used for nucleic acid extraction. The gene expression level is detected using a gene expression detection method similar to that described in Example (1). The ratio of the expression levels of soybean internal reference GmCons 4 and Phytophthora soybeanis internal reference PsTEF indicates the amount of Phytophthora soybeanis accumulation. Primer pairs SEQ ID No. 4 and SEQ ID No. 5 are used to amplify a partial fragment of soybean internal reference GmCons 4, and primer pairs SEQ ID No. 14 and SEQ ID No. 15 are used to amplify a partial fragment of Phytophthora soybeanis internal reference PsTEF.

[0039] The results are as follows Figure 9 and Figure 10 As shown, compared with the wild-type control, the lesion area and Phytophthora accumulation of the GmSRC2 overexpression line were significantly reduced, indicating that GmSRC2 overexpression has a positive regulatory effect on improving soybean resistance to Phytophthora beannulata.

Claims

1. The gene shown in SEQ ID No. 1 GmSRC2 The primer pair shown in SEQ ID No. 6 and SEQ ID No. 7 contains the gene shown in SEQ ID No.

1. GmSRC2 The application of recombinant expression vectors or transformants in improving resistance to Phytophthora in soybean is characterized by, Gene overexpression in soybeans GmSRC2 .

2. A method for breeding soybean varieties with improved resistance to Phytophthora sojae, characterized in that, Overexpression of the gene of claim 1 in soybean GmSRC2 .

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