A gene GmCDK8 for negatively regulating soybean resistance to Phytophthora spp. and its application

Abstract

The present invention discloses a gene, GmCDK8, that negatively regulates soybean resistance to Phytophthora spp., and its applications. The disclosed GmCDK8 knockout soybean plants exhibit enhanced resistance to the fungus. Specifically, the results of the soybean cultivar "Williams 82," which lacks GmCDK8, under Phytophthora stress, including root lesion length, leaf lesion area, Phytophthora accumulation, and infected necrotic area on leaves, indicate that knocking out GmCDK8 significantly improves plant resistance to Phytophthora spp. Genetic engineering of this gene for plant resistance to Phytophthora root rot not only provides an important genetic resource for plant molecular breeding but also offers an effective means for achieving high plant yields.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and in particular relates to an application of a gene GmCDK8 for negatively regulating resistance to soybean Phytophthora. Background Art

[0002] Phytophthora root rot (PRR) is a disease caused by Phytophthora Phytophthora sojae ) is one of the devastating diseases caused by soybean phytophthora root rot. Soybean phytophthora root rot can continuously infect from the seedling stage to the adult stage, causing seedling sudden death, rhizome rot and plant wilting. In the rainy season or when there is water in the field, the disease spreads very quickly, and the yield loss can reach more than 30%, or even lead to a total crop failure, seriously endangering soybean production and causing billions of dollars in economic losses to the world each year. High humidity, rainy weather and heavy soil conditions promote the formation of a large number of sporangia and the spread of zoospores, leading to large-scale outbreaks and spread of diseases during the growing season. The thick-walled spores formed on the mycelium or diseased residues have stronger environmental tolerance and can survive for a long time under adverse conditions, allowing soybean phytophthora to survive in the soil for a long time and spread rapidly during the growing season, thereby causing a continuous disease cycle. Gene editing technology forms a double-strand break in the DNA by cutting the target site of the target gene, and uses the receptor's own DNA repair mechanism to achieve mutations in the target gene. Among them, CRISPR / CAS editing technology has the advantages of simple experimental operation process, high editing efficiency and low cost, making it the currently mainly used gene editing system. Therefore, on the basis of conventional prevention and control, using molecular breeding methods such as gene editing to improve soybean disease resistance is one of the most economical and effective prevention and control measures. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of the soybean GmCDK8 gene in resistance to Phytophthora root rot. Knocking out the gene in plants can improve the resistance of soybeans to Phytophthora sojae, thereby improving soybean varieties.

[0004] One of the objectives of the present invention is to provide a gene GmCDK8 that regulates resistance to soybean Phytophthora.

[0005] A second object of the present invention is to provide a recombinant knockout vector targeting GmCDK8.

[0006] The third object of the present invention is to provide breeding applications of the gene GmCDK8.

[0007] The purpose of the present invention is mainly achieved through the following technical solutions:

[0008] The present invention provides a gene GmCDK8 or a specific fragment thereof for inhibiting soybean resistance to Phytophthora. The nucleotide sequence of the gene GmCDK8 is shown in SEQ ID No. 1; the specific fragment sequence of the gene GmCDK8 is shown in SEQ ID No. 14; and the CDS fragment is shown in SEQ ID No. 15.

[0009] The present invention also provides a primer pair for amplifying the gene GmCDK8. The primer pair can be designed according to conventional methods in the art. In a specific example, the primer pair is shown as SEQ ID No. 6 and SEQ ID No. 7.

[0010] The present invention also provides a primer pair for amplifying a specific fragment of the gene GmCDK8. The primer pair can be designed according to conventional methods in the art. In a specific example, the primer pair is shown as SEQ ID No. 2 and SEQ ID No. 3.

[0011] The present invention also provides a recombinant knockout vector for knocking out the gene GmCDK8 or a specific fragment thereof.

[0012] The recombinant knockout vector of the present invention can be selected from expression plasmids commonly used in plant genetic transformation. In a specific example, the gene GmCDK8 or a specific fragment thereof is used as a template, and the vector recombinant sequence containing sgRNA is amplified using SEQ ID No. 8 and SEQ ID No. 9, and then ligated to the pGES201 vector digested with BsaI to form the pGES201-GmCDK8 recombinant knockout vector. In one embodiment, the recombinant sequence containing the sgRNA vector of the recombinant knockout vector is shown in SEQ ID No. 8 and SEQ ID No. 9.

[0013] In order to facilitate the identification and screening of transgenic knockout plants, the plant expression vector used can be processed to add genes encoding herbicide-resistant proteins expressed in plants (bar protein), antibiotic resistance markers (kanamycin markers), etc.

[0014] The present invention also provides a transformant obtained by introducing the recombinant knockout vector into a host cell. The host cell can be a host cell commonly used in the art, preferably an Escherichia coli cell or an Agrobacterium cell. For example, the host strain can be obtained by transforming the recombinant knockout vector pGES201-GmCDK8 into the Agrobacterium tumefaciens strain EHA105.

[0015] The present invention can introduce the transformant into soybeans to produce transgenic soybeans with the GmCDK8 gene knocked out. For example, pGES201-GmCDK8 can be introduced into soybean cells using the host bacterium EHA105. After herbicide screening, genome sequencing, and multi-generational propagation, soybean plants with a stable GmCDK8 gene knockout can be obtained. Phenotypic identification can also yield genetically modified soybean plants with enhanced disease resistance.

[0016] The present invention also provides the use of the gene GmCDK8 or its specific fragment, or the recombinant knockout vector or transformant in improving resistance to Phytophthora sojae, specifically knocking out the gene GmCDK8 or its specific fragment in soybean.

[0017] The present invention also provides the use of the gene GmCDK8 or its specific fragment, or recombinant knockout vector or transformant in improving soybean resistance to diseases caused by Phytophthora sojae, specifically knocking out or silencing the gene GmCDK8 or its specific fragment in soybean.

[0018] The present invention also provides the use of the gene GmCDK8 or its specific fragment, or recombinant knockout vector or transformant in breeding soybean varieties resistant to diseases caused by soybean Phytophthora, specifically knocking out or silencing the gene GmCDK8 or its specific fragment in soybean.

[0019] The present invention also provides the use of the gene GmCDK8 or its specific fragment, or recombinant knockout vector or transformant in soybean varieties that obtain significant resistance to Phytophthora diseases after introduction, specifically knocking out or silencing the gene GmCDK8 or its specific fragment in soybeans.

[0020] Beneficial effects of the present invention:

[0021] The expression pattern analysis of GmCDK8 in the present invention showed that it was expressed in the roots, stems, leaves and cotyledons of soybeans and could participate in the growth of Phytophthora sojae ( Phytophthora sojae ), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and bacterial flagellin (Flg22);

[0022] In soybean Williams82, where the GmCDK8 gene was knocked out, various resistance identification methods, such as leaf lesion area and root phytophthora extension length, showed that knocking out GmCDK8 was beneficial to improving plant resistance to phytophthora.

[0023] By using this gene for genetic engineering improvement of soybean resistance to Phytophthora root rot, or directly using the homozygous mutant as a disease-resistant parent for backcrossing, soybean varieties with improved resistance to Phytophthora can be further created, thereby comprehensively improving soybean yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Expression pattern of GmCDK8.

[0025] Figure 2 Gene knockout detection of soybean gmcdk8 mutant.

[0026] Figure 3 Resistance identification of soybean gmcdk8 mutants. DETAILED DESCRIPTION

[0027] The present invention will be further described with reference to the examples to facilitate understanding. The specific examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention. Any simple modification of the preparation method of the present invention based on the concepts of the present invention falls within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed in accordance with known methods in the art.

[0028] Unless otherwise specified, the soybean Phytophthora used in the following examples are P6497, RFP-labeled P6497, and P7076, which are preserved by the applicant and are permanently available to the public.

[0029] Example 1

[0030] GmCDK8 expression pattern:

[0031] Root, stem, leaf and cotyledon samples were collected from 7-day-old soybean “Williams 82”.

[0032] Seven-day-old soybean "Williams 82" were inoculated with P6497 (4-day-old P6497 was cultured on 10% V8 vegetable medium containing 1.5% agar for 3 days) using the hypocotyl wound inoculation method. Samples were taken at different time points after treatment.

[0033] Soybean plants grown to 7 days old were sprayed with 10 μM bacterial flagellin (Flg22) and 100 μM abscisic acid (ABA), and leaves were sampled.

[0034] Seven-day-old soybean "Williams 82" seedlings grown in 1 / 2 Hoagland nutrient solution were taken, 200 mM salicylic acid (SA) or 200 mM methyl jasmonate (MEJA) was added to the nutrient solution, and tissue samples were taken 2 cm from the root tip.

[0035] The above samples were used to extract RNA, and the reversed cDNA was used as a template to detect the expression level of the gene GmCDK8.

[0036] The specific fragment of GmCDK8 (SEQ ID No. 14) was amplified using the primers shown in SEQ ID No. 2 and SEQ ID No. 3, and the partial fragment of the soybean internal reference gene GmActin11 was amplified using the primers shown in SEQ ID No. 4 and SEQ ID No. 5. -ΔΔCT Calculate the relative expression levels.

[0037] The results are as follows Figure 1 As shown, GmCDK8 is expressed in roots, stems, leaves, and cotyledons, with the highest expression in cotyledons, followed by leaves and roots, and the lowest expression in stems (Figure 1A). Phytophthora sojae After inoculation with P6497, GmCDK8 expression showed a significant downregulation (Figure 1, B). Furthermore, treatment with the plant immunity inducer flg22 significantly downregulated GmCDK8 expression (Figure 1, C). Furthermore, exogenous plant hormone treatments demonstrated that ABA, JA, and SA significantly regulated GmCDK8 expression (Figure 1, D–F).

[0038] Example 2

[0039] (1) Construction of recombinant knockout vector

[0040] The GmCDK8 vector was constructed using homologous recombination. Using soybean "Williams 82" cDNA as a template, the full-length GmCDK8 gene (SEQ ID No. 1) was amplified using the primer pair shown in SEQ ID Nos. 6 and 7. The recombinant sequence containing the sgRNA was then amplified using SEQ ID Nos. 8 and 9 and ligated into the BsaI-digested pGES201 vector to construct the pGES201-GmCDK8 recombinant vector. This recombinant vector was transformed into Escherichia coli DH5α and Agrobacterium tumefaciens EHA105, and then into soybean "Williams 82" via the cotyledonary transformation method.

[0041] (2) Identification of GmCDK8 knockout plants

[0042] DNA was extracted from the leaves of the knockout plants and PCR amplified using primers for the genomic target sites of SEQ ID No. 10 and SEQ ID No. 11. After sequencing, the genome GmCDK8 gene and the encoded protein were identified to determine whether they were changed.

[0043] The results are as follows Figure 2As shown, a knockout recombinant vector was used to genetically transform the GmCDK8 genome for targeted knockout, resulting in several tissue culture seedlings. After screening, two stably inherited gmcdk8 mutants were obtained. gmcdk-1 exhibited 1-bp and 4-bp deletions in the GmCDK8 genomic region, while gmcdk-2 exhibited a 7-bp deletion. These deletions resulted in a frameshift in the GmCDK8 coding region and triggered premature stop codons, ultimately producing a truncated, non-functional protein.

[0044] (3) Resistance identification of GmCDK8 knockout plants through leaves

[0045] After the first true leaf of soybean unfolds (about 10 days), leaves with consistent growth are selected for inoculation of zoospores of soybean Phytophthora P7076. 50 μL of zoospore liquid is dropped on one side of the leaf, and 50 μL of sterilized tap water is added to the other side of the leaf as a control. Pictures are taken after 48 hours and the size of the lesion area is calculated using ImageJ software. Quickly freeze in liquid nitrogen and store at -80°C for RNA extraction and gene expression detection. The ratio of the expression levels of soybean internal reference GmAct11 and soybean Phytophthora internal reference PsActin represents the amount of Phytophthora accumulation. Primer pair SEQ ID No. 4 and SEQ ID No. 5 is used to amplify a partial fragment of soybean internal reference GmAct11, and primer pair SEQ ID No. 12 and SEQ ID No. 13 is used to amplify a partial fragment of Phytophthora internal reference GmAct11.

[0046] The results showed that the lesion area of ​​the GmCDK8 knockout strain was significantly reduced compared with the wild-type control ( Figure 3 A and Figure 3 B in the figure), the accumulation of Phytophthora was significantly reduced ( Figure 3 E in Figure ), indicating that knocking out GmCDK8 has a positive regulatory effect on improving soybean resistance to Phytophthora sojae.

[0047] (4) Resistance identification of GmCDK8 knockout plants was performed through root analysis.

[0048] Use RFP-labeled P6497, which expresses RFP protein (red fluorescence). Zoospore production is as follows: Cultivate RFP-labeled P6497 in 10% V8 vegetable juice medium containing 1.5% agar for 5 days. Cut the outer edges of the fresh mycelium and transfer it to 10% V8 vegetable juice liquid medium for 3 days. Wash several times with sterile tap water until the liquid medium is clean. Incubate overnight in the dark at 25°C. Filter the bacterial mass to obtain a zoospore solution (≈1× 10 4 / mL).

[0049] Soybean Phytophthora was cultured in 10% V8 vegetable juice medium containing 1.5% agar at 25°C in the dark for 5 d. The outer edges of fresh mycelia were cut into blocks of uniform size (3 mm × 3 mm). The mycelial surface was placed on the roots of 7-day-old soybean seedlings and kept moist. After 48 hours, the inoculated site was observed under a fluorescence microscope and pictures were taken. The extension speed of Phytophthora was determined by the length of the fluorescent lesions, and the lesion length was calculated using ImageJ software.

[0050] The results are as follows Figure 3 C and Figure 3 As shown in Figure 5D, the root lesion length of the GmCDK8 knockout line was significantly reduced compared with the wild-type control, indicating that the knockout of GmCDK8 had a positive regulatory effect on soybean resistance to Phytophthora sojae.

Claims

1. Application of reducing the expression of gene GmCDK8 in improving resistance to soybean Phytophthora. The CDS of gene GmCDK8 is shown in SEQ ID No.

15.

2. The use according to claim 1, characterized in that Reducing the expression of the gene GmCDK8 involves knocking out the gene GmCDK8 using a CRISPR / CAS9 recombination knockout vector.

3. Application of reducing the expression of gene GmCDK8 in improving soybean resistance to diseases caused by Phytophthora sojae. The CDS of gene GmCDK8 is shown in SEQ ID No.

15.

4. The use according to claim 3, characterized in that Reducing the expression of the gene GmCDK8 involves knocking out the gene GmCDK8 using a CRISPR / CAS9 recombination knockout vector.

5. Application of reducing the expression of gene GmCDK8 in breeding soybean varieties resistant to diseases caused by Phytophthora sojae. The CDS of gene GmCDK8 is shown in SEQ ID No.

15.

6. The use according to claim 5, characterized in that Reducing the expression of the gene GmCDK8 involves knocking out the gene GmCDK8 using a CRISPR / CAS9 recombination knockout vector.

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