A GmCNL1 gene for enhancing resistance to Phytophthora in soybean and its application

Abstract

This invention discloses a GmCNL1 gene for enhancing soybean resistance to Phytophthora solanum and its applications. The soybean plants transgenic with the GmCNL1 gene disclosed in this invention exhibit enhanced resistance to Phytophthora solanum. Specifically, the phenotype, lesion area, survival rate, and Phytophthora accumulation of the soybean cultivar "Williams 82" under Phytophthora stress, as well as other results, demonstrate that GmCNL1 plays a crucial positive regulatory role in improving plant resistance to Phytophthora solanum. Genetic engineering improvement of plants against Phytophthora root rot using this transformant revealed that this gene has a certain effect on breeding plant varieties with higher resistance to Phytophthora root rot, and can contribute to improving plant yield and quality by enhancing plant resistance to Phytophthora solanum.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the application of the gene GmCNL1, 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 (P. sojae). It has been reported globally and can affect soybeans at all growth stages, severely impacting yield and quality and 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 soybean transformants carrying the GmCNL1 gene in the prevention of Phytophthora root rot, thereby enhancing the resistance of soybeans to Phytophthora soybeanis by enhancing soybean plants expressing this gene, thus improving soybean varieties.

[0004] One of the objectives of this invention is to provide a gene, GmCNL1, that enhances resistance to Phytophthora in soybean.

[0005] The second objective of this invention is to provide a recombinant expression vector containing the gene GmCNL1 and its transformant.

[0006] The third objective of this invention is to provide applications for the gene GmCNL1.

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

[0008] This invention provides a gene GmCNL1 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 GmCNL1 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. 2 and SEQ ID No. 3.

[0010] The present invention also provides a recombinant expression vector containing the gene GmCNL1 described in the present invention.

[0011] The recombinant expression vector can be a commonly used expression plasmid in plant genetic transformation. In a specific example, the gene GmCNL1 is ligated into the pTF101.1 vector using Spe I and Xba I enzymes to form a recombinant expression vector, namely the plant expression vector pTF101.1-GmCNL1.

[0012] In recombinant expression vectors, a tag sequence encoding a commercial antibody can be added to the 3' or 5' end of GmCNL1 to form a fusion protein, which is beneficial for detecting GmCNL1 protein expression in transgenic plants.

[0013] The present invention also provides a transformant comprising a recombinant expression vector, obtained by introducing the recombinant expression vector into a host cell, wherein the host cell is preferably an *E. coli* cell or an *Agrobacterium* cell. In one embodiment, the host bacterium containing the pTF101.1-GmCNL1 encoding gene is obtained by transforming the GmCNL1 gene into the *Agrobacterium tumefaciens* strain EHA101.

[0014] This invention also provides a stable genetically inherited soybean plant containing the GmCNL1 gene. An Agrobacterium EHA101 transformant containing the pTF101.1-GmCNL1 recombinant expression vector was introduced into soybean cells using the host bacterium EHA101. After herbicide screening and propagation, stable genetically inherited GmCNL1-transgenic soybean plants were obtained. Phenotypic identification yielded transgenic soybean plants with altered disease resistance.

[0015] This invention also provides the gene GmCNL1, amplification primer pairs, a recombinant expression vector containing the gene GmCNL1, and the application of host cells in improving soybean resistance to diseases caused by Phytophthora soybeanis or improving resistance to Phytophthora soybeanis. Specifically, it involves overexpressing the gene GmCNL1 in soybean.

[0016] The present invention also provides a method for improving the resistance of soybean to diseases caused by Phytophthora in soybean or improving the resistance of soybean to Phytophthora in soybean, specifically by overexpressing the gene GmCNL1 in soybean.

[0017] This invention also provides the gene GmCNL1, amplification primer pairs, a recombinant expression vector containing the gene GmCNL1, and the application of host cells in cultivating soybean varieties resistant to diseases caused by Phytophthora sojae or with enhanced resistance to Phytophthora sojae. Specifically, it involves overexpressing the gene GmCNL1 in soybeans.

[0018] The present invention also provides a method for breeding soybean varieties resistant to diseases caused by Phytophthora sojae or with improved resistance to Phytophthora sojae, specifically by overexpressing the gene GmCNL1 in soybeans.

[0019] The present invention also provides the application of recombinant expression vectors containing the gene GmCNL1, host cells or soybean plants in varieties that have been introduced into soybeans to obtain significant resistance to fungal diseases.

[0020] The diseases caused by Phytophthora in soybean as described in this invention include, but are not limited to, Phytophthora root rot of soybean.

[0021] Beneficial effects of the present invention

[0022] This invention utilizes an enzyme digestion and ligation method to construct GmCNL1 onto pTF101.1, transforms it into Escherichia coli DH5α and Agrobacterium tumefaciens EHA101, and introduces it into soybean “Williams 82” via Agrobacterium tumefaciens. After propagation to the T3 generation, it is used for phenotypic identification.

[0023] The phenotype, lesion size, and Phytophthora accumulation in soybean "Williams 82" transgenic with the GmCNL1 gene under Phytophthora stress indicate that GmCNL1 plays a crucial regulatory role in enhancing plant resistance to Phytophthora. Genetic engineering modification of soybeans using this gene to combat Phytophthora root rot can serve as an indicator for breeding soybean varieties with higher resistance to this disease. Providing genetically modified soybean plants with enhanced resistance to Phytophthora can contribute to improving soybean yield and quality. Attached Figure Description

[0024] Figure 1 A schematic diagram of a plant expression vector containing GmCNL1;

[0025] Figure 2 Screening of transgenic elements at the transcriptional level in roots, stems and leaves of transgenic soybean plants;

[0026] Figure 3 Screening of herbicide resistance in genetically modified soybean leaves;

[0027] Figure 4 Phenotype of genetically modified soybean leaves after inoculation with Phytophthora soybeanis;

[0028] Figure 5 Leaf lesion area and Phytophthora accumulation in genetically modified soybean leaves inoculated with Phytophthora soybeanis;

[0029] Figure 6 Phenotypic characteristics of transgenic soybean etiolated seedlings after inoculation with Phytophthora soybeanis;

[0030] Figure 7 Phytophthora accumulation in genetically modified soybean etiolated seedlings inoculated with Phytophthora soybeanis. Detailed Implementation

[0031] 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.

[0032] Unless otherwise specified, the soybean Phytophthora in the following examples is P7076, which is kept in the applicant's own possession and the applicant promises to make it permanently available to the public.

[0033] Example 1

[0034] (1) The GmCNL1 vector was constructed using homologous recombination. Using soybean "Williams 82" as a template, GmCNL1 (SEQ ID No. 1) was amplified using primers SEQ ID No. 2 and SEQ ID No. 3, and then ligated into the pTF101.1 vector digested with Spe I and Xba I to form the recombinant vector. Figure 1 The above recombinant vector was transformed into Escherichia coli DH5α and Agrobacterium EHA101, and then transformed into soybean variety "Williams 82" using the cotyledon transformation method.

[0035] (2) Identification of positive GmCNL1 transgenic plants: RNA was extracted from the roots, stems, and leaves of the transgenic plants. Using the cDNA obtained after inversion as a template, the Bar gene fragment was amplified using primers shown in SEQ ID No. 4 and SEQ ID No. 5. Compared with the wild type, the transgenic positive plants could amplify the band ( Figure 2 Applying a 200 mg / L glufosinate-ammonium aqueous solution to the leaves of 7-day-old plants and observing them 3 days later, the leaves of transgenic positive plants did not turn yellow. Figure 3 ).

[0036] (3) Method for identifying the resistance of GmCNL1 transgenic plants from detached leaves: After the first true leaf of soybean unfolds (about 8 days), select leaves of uniform growth (≥6 leaves) for inoculation with zoospores of Phytophthora soybeanis. The zoospore preparation process is as follows: Phytophthora soybeanis is cultured in 10% V8 vegetable juice medium containing 1.5% agar for 4 days. The outer edge of the mycelium is chopped and transferred to 10% V8 vegetable juice medium for 3 days. Finally, it is washed 1-3 times with sterile tap water until no medium residue remains. It is then cultured overnight in the dark at 25°C. The zoospore solution is obtained by filtration (≈1×10⁻⁶). 4 / mL). 50 μL of zoospore solution was dropped onto one side of the leaf, and simultaneously, 50 μL of sterile tap water was added to a leaf of the same size as a control. After 24 h, photos were taken using a Canon camera, and the area of ​​the lesion was calculated using ImageJ online software (https: / / imagej.nih.gov / ij / ). Samples were collected, flash-frozen in liquid nitrogen, and stored at -80℃. The samples were used for nucleic acid extraction, and the accumulation of *Phytophthora indicum* was detected using qPCR. The ratio of the expression levels of soybean internal control GmCons 4 and *Phytophthora indicum* internal control PsTEF represented the accumulation of *Phytophthora indicum*. Primer pairs SEQ ID No. 6 and SEQ ID No. 7 were used to amplify GmCons 4, and primer pairs SEQ ID No. 8 and SEQ ID No. 9 were used to amplify PsTEF.

[0037] The results are as follows Figure 4 and Figure 5 As shown, compared with the empty vector, the lesion area and Phytophthora inoculation of GmCNL1 overexpression lines with leaves were significantly reduced and the amount of Phytophthora inoculation was significantly reduced, indicating that GmCNL1 overexpression lines showed higher resistance to Phytophthora inoculation.

[0038] (4) Identification of resistance in GmCNL1 plants from etiolated seedlings: Soybean seedlings germinated in darkness. After 4 days, approximately 10 seedlings with uniform growth were inoculated with zoospores of Phytophthora. 50 μL of zoospore solution was dropped onto the hypocotyl. Typical phenotypes were observed and photographed after 24 hours. Samples were collected, flash-frozen in liquid nitrogen, and stored at -80℃. The samples were used for nucleic acid extraction. The accumulation of Phytophthora was detected by qPCR. The ratio of the expression levels of soybean internal control GmCons 4 and the internal control PsTEF of Phytophthora soybean represented the accumulation of Phytophthora. Primer pairs SEQ ID No. 6 and SEQ ID No. 7 were used to amplify GmCons 4, and primer pairs SEQ ID No. 8 and SEQ ID No. 9 were used to amplify PsTEF.

[0039] The results are as follows Figure 6 and Figure 7 As shown, compared with the empty vector, the susceptibility of yellow seedlings of GmCNL1 overexpression lines to Phytophthora soybean was reduced after inoculation with Phytophthora soybean, and the accumulation of Phytophthora soybean was significantly reduced, which also indicates that the GmCNL1 overexpression lines exhibited higher resistance to Phytophthora soybean.

Claims

1. Overexpression of the gene shown in SEQ ID No. 1 GmCNL1 Application in improving soybean resistance to Phytophthora sojae.

2. The application according to claim 1, characterized in that, By including the gene shown in SEQ ID No. 1 GmCNL1 The recombinant expression vector was overexpressed.

3. A method for breeding soybean varieties with improved resistance to Phytophthora indica, characterized in that, Overexpression of the gene shown in SEQ ID No. 1 in soybean GmCNL1 .

4. Overexpression of the gene shown in SEQ ID No. 1 GmCNL1 Application in improving soybean resistance to diseases caused by mold.

5. A method for cultivating soybean varieties that are more resistant to diseases caused by antifungal fungi, characterized in that, Overexpression of the gene shown in SEQ ID No. 1 in soybean GmCNL1 .

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