Application of bacterial quorum sensing molecule AHL in inducing activation of GmWRKY33 pathway to prevent early warning against soybean mosaic virus disease, detection and new strain identification method

Abstract

The application discloses a soybean GmWRKY33-Chr18-1 gene and application thereof in resisting a mosaic virus disease through a bacterial quorum sensing signal molecule AHL (Acyl-homoserine Lactone) induction, and belongs to the technical field of plant genetic engineering, in particular relates to a soybean WRKY transcription factor gene GmWRKY33-Chr18-1 and application thereof in resisting a mosaic virus disease through induction, and the application obtains a key gene GmWRKY33-Chr18-1 of responding to AHL defense warning and promoting soybean to resist a mosaic virus disease through a genetic engineering method, and introduces the key gene GmWRKY33-Chr18-1 into a soybean protoplast through gene editing and overexpression, and a GmWRKY33-Chr18-1 gene editing seedling obtained by deletion loses response to AHL, loses an AHL-induced phenotype of resisting a mosaic virus disease, and meanwhile, overexpression of the GmWRKY33-Chr18-1 transformation seedling significantly enhances AHL-induced resistance to a mosaic virus disease. Therefore, the key gene GmWRKY33-Chr18-1 can be applied to green control of a soybean mosaic virus disease, and has important application value in application of the soybean mosaic virus disease prevention and control and cultivation of a resistant variety.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to the application, detection and identification of new varieties of a bacterial quorum sensing molecule AHL in inducing and activating the GmWRKY33 pathway for defense and early warning resistance against soybean mosaic virus disease. Specifically, it relates to a soybean WRKY-like transcription factor gene GmWRKY33-Chr18-1 and its application in inducing soybean defense and early warning resistance against soybean mosaic virus disease. Background Technology

[0002] Soybeans are an important dual-purpose crop in my country, used for grain, oil, and feed, and are also a significant source of plant protein for humans. Soybean mosaic virus (SMV), one of the major viral diseases affecting soybean production in my country, is prevalent in every major soybean-producing area from north to south. It not only severely impacts soybean yield but also causes seed coat mottling, resulting in brown spots that affect soybean quality. The pathogen of soybean mosaic virus is SMV. Currently, control measures mainly involve removing virus-carrying seeds and spraying chemical control agents. These methods are time-consuming, labor-intensive, and pose risks of non-point source pollution and pesticide residues, and they cannot effectively and comprehensively prevent the spread of the virus.

[0003] Therefore, researching control strategies for soybean mosaic virus and developing environmentally friendly, green biological pesticide formulations are of great theoretical and practical significance and will become a key development direction for the biological control of soybean mosaic virus in the future. At the same time, discovering SMV resistance genes and exploring their molecular mechanisms of resistance will provide important theoretical basis and candidate gene resources for the breeding of soybean varieties resistant to soybean mosaic virus. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an application and detection method for the bacterial quorum sensing molecule AHL in inducing and activating the GmWRKY33 pathway to defend against and warn against soybean mosaic virus disease. By inducing soybean defense and warning through the bacterial quorum sensing signal molecule AHL, the resistance to soybean mosaic virus disease is improved.

[0005] The specific technical solution adopted in this invention is as follows: The application of a bacterial quorum sensing molecule, AHL, in inducing and activating the GmWRKY33 pathway to defend against soybean mosaic virus disease, the application comprising the following steps: S1. Application of AHL signaling molecules: Prepare a 5mM AHL stock solution and apply it to the rhizosphere of soybean plants; S2. WRKY33 pathway activation: The expression of the soybean GmWRKY33-Chr18-1 gene was upregulated by the administration of AHL signaling molecules in step S1. S3. Verification of disease resistance: Soybean plants treated with soybean mosaic virus were inoculated, and resistance was confirmed by detecting the disease index and viral load.

[0006] In step S1, the AHL mother liquor is prepared using 3OC8-HSL.

[0007] The molecular structure of AHL is shown in the following formula: ; The molecular structure of 3OC8-HSL is shown in the following formula: ; In step S1, the AHL mother liquor is diluted 1000 times to obtain a diluted solution with an effective concentration of 5 μM. 30 mL of the diluted solution is applied to the roots of each plant for root irrigation treatment, and the treatment is repeated once every 3 days for a total of 2 applications.

[0008] The nucleotide sequence of the GmWRKY33-Chr18-1 gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] The disease index DI < 30 indicates that the viral load is reduced by at least two orders of magnitude compared to the control group.

[0010] Defense against and early warning of mosaic virus disease is achieved through the following molecular mechanisms. a. Infection with soybean mosaic virus SMV SC3 can induce H3K4me3 enrichment in the promoter region of the PR1 gene; b. The administration of AHL signaling molecules induces activation of the GmWRKY33-Chr18-1 gene, which in turn activates the MAPK signaling pathway; c. The salicylic acid content increases significantly, initiating the disease resistance process and enhancing resistance to mosaic virus.

[0011] A method for detecting the role of bacterial quorum sensing molecule AHL in inducing and activating the GmWRKY33 pathway to provide early warning and defense against soybean mosaic virus disease was developed. The method detected the H3K4me3 modification level of the target gene PR1, which is regulated by GmWRKY33-Chr18-1. The H3K4me3 modification level of the experimental group was significantly higher than that of the control group after AHL induction, indicating that the experimental group was in a state of enhanced resistance to soybean mosaic virus.

[0012] A method for identifying new soybean mosaic virus (SMV) strains by inducing activation of the GmWRKY33 pathway through bacterial quorum sensing molecule AHL. A new strain of transgenic seedlings overexpressing the GmWRKY33-Chr18-1 gene was constructed. The primers for identifying the new strain were F33-18-1(+) and F33-18-1(-), where F33-18-1(+) is shown in SEQ ID NO.3 and F33-18-1(-) is shown in SEQ ID NO.4.

[0013] The beneficial effects of this invention are: This invention identifies the GmWRKY33-Chr18-1 gene as a core regulatory factor for soybean resistance to mosaic virus. Through N-acyl homoserine lactone (AHL), a regulatory factor mediated by bacterial quorum sensing, it can cross-speciesly regulate resistance to the virus. In particular, its expression is specifically induced by 3OC8-HSL, which can significantly reduce the incidence of soybean mosaic virus in different varieties, and significantly reduce leaf wrinkling and lesions. Compared with traditional random control methods, the disease resistance effect is more precise and stable. Attached Figure Description

[0014] Figure 1 A comparative diagram showing the resistance of soybean to mosaic virus after 3OC8-HSL induction; Figure 2 A comparison of CP levels in soybean leaves after 3OC8-HSL induction; Figure 3 To induce the expression of soybean-specific differentially expressed genes using 3OC8-HSL; Figure 4 A comparison of 3OC8-HSL-induced expression of GmWRKY33 and GmPAD4; Figure 5 A comparative graph showing the changes in salicylic acid content after AHL application; Figure 6 ChIP-qPCR results after AHL administration; Detailed Implementation The present invention will be further described below with reference to the accompanying drawings and specific embodiments: Specific Implementation Example 1, This application uses potted and hydroponically grown soybean variety Nannong 1138-2 as an example. Soybean roots were pretreated with a 5 μM 3OC8-HSL solution, repeated once every 3 days, for a total of 2 applications. Two to 3 days after application, soybean mosaic virus (SMV) SC3 was inoculated onto the third leaf. Virus load in the soybean leaves was measured 24-72 hours later, and the area of ​​lesions was also measured. Figure 1 The results showed that treatment with 3OC8-HSL solution significantly reduced the incidence of soybean mosaic virus, and the leaves of pretreated soybean plants showed a significant reduction in leaf wrinkling and lesions. Figure 2 q-PCR results showed that the copy number of the SMV SC3 coat protein gene CP in pretreated leaves was significantly lower than that in the untreated control, indicating that 3OC8-HSL pretreatment significantly reduced virus replication in soybean leaves.

[0015] Specific embodiment 2, Transcriptome sequencing analysis was performed on AHL pretreated and untreated, and SMV SC3 inoculated and uninoculated individuals. The results showed that 3OC8-HSL pretreatment alone affected 732 differentially expressed genes, of which 385 were upregulated and 347 were downregulated. SMV SC3 inoculation alone induced the expression of 4977 differentially expressed genes, of which 2349 were upregulated and 2628 were downregulated. SMV SC3 inoculation 48 hours after 3OC8-HSL pretreatment induced the expression of 2803 differentially expressed genes, of which 1441 were upregulated and 1362 were downregulated.

[0016] Table 1. RNA-seq analysis of the number of differentially expressed genes in SMV SC3 pretreated with 3OC8-HSL: The three treatments shared 37 differentially expressed genes, but only 3OC8-HSL pretreatment specifically induced the expression of 356 genes. Figure 3 Analysis of differentially expressed genes revealed that among the 356 genes specifically induced by 3OC8-HSL, 45 were transcription factors, indicating that 3OC8-HSL pretreatment significantly affected the expression of transcription factors. 3OC8-HSL significantly induced changes in many pathway marker genes involved in AHL defense and early warning systems, such as GmHsp90, GmPAD4, GmPR2, GmWRKY5, GmWRKY33, and GmATX2 (Table 2).

[0017] Table 2. Significantly differentially expressed genes induced by 3OC8-HSL: After inoculation with soybean mosaic virus, the expression of 221 genes was significantly regulated in both pretreated and untreated soybean leaves. Of these 221 genes, only 4 showed the same expression trend, while 217 showed opposite trends. Combined with pathological examination results, many signals in the 3OC8-HSL-induced defense warning system were found to have changed. These results indicate that the 3OC8-HSL-induced disease resistance response in soybean is a defense warning mechanism. Among these, GmWRKY33-Chr18-1 in the GmWRKY33 pathway was one of the transcription factors that showed significant changes. Significant differences were also observed in related genes in both Nannong 1138-2 and Shidou 936 soybean varieties. Further analysis revealed that GmWRKY33-Chr18-1 in the GmWRKY33 pathway was significantly induced by 3OC8-HSL. Figure 4 Therefore, GmWRKY33-Chr18-1 may be involved in the regulation of AHLs’ disease resistance immunity in plants and play a role in signal transduction.

[0018] Specific embodiment 3, To verify how AHL forms "immune memory" through histone methylation, ChIP-qPCR / ChIP-seq experiments were used to detect changes in histone methylation markers H3K4me3 and H3K27me3 in the GmWRKY33-Chr18-1 disease resistance-related target gene in different genetic materials (Nanong 1138-2, GmWRKY33-OE, and Gmwrky33) after AHL treatment. H3K4me3 was the activation marker, and H3K27me3 was the repression marker.

[0019] Among them, the untreated Nannong 1138-2 served as the wild-type blank control group; GmWRKY33-OE was the overexpression transgenic vector with a FLAG tag constructed by cloning the GmWRKY33-Chr18-1 gene and producing overexpression transgenic plants; Gmwrky33 was the gene-edited plant material produced by using the gene editing tool CRISPR / Cas12, namely, functionally deficient plants.

[0020] The H3K4me3 methylation level of the GmWRKY33-Chr18-1 gene in soybean was detected by ChIP-PCR under 3OC8-HSL pretreatment and untreated conditions, as well as after stress from soybean mosaic virus disease. The whole-genome H3K4me3 methylation level after virus inoculation with 3OC8-HSL pretreatment and untreated soybean was detected at the genome level using ChIP-seq technology.

[0021] The results showed that the binding peak of GmWRKY33-Chr18-1 and the enrichment peak of H3K4me3 highly overlapped on the genome, especially in the promoter region of defense genes activated by AHL.

[0022] In the control group GmWRKY33-OE, without AHL induction, the H3K4me3 methylation level was significantly higher than that of the wild-type blank control group, proving that simply enhancing the activity of GmWRKY33-Chr18-1 is sufficient to increase the H3K4me3 modification level at the target gene site, that is, GmWRKY33-Chr18-1 is a sufficient condition for establishing this modification.

[0023] In the control group, AHL induction significantly increased H3K4me3 methylation levels at GmWRKY33-Chr18-1-related sites, while in the GmWRKY33 group, AHL treatment failed to induce a significant increase in H3K4me3 at these sites. This demonstrates that the presence of GmWRKY33-Chr18-1 is a necessary condition for AHL-induced H3K4me3 deposition at these sites.

[0024] Therefore, it can be concluded that the increased H3K4me3 modification level at specific defense gene loci during AHL-induced soybean mosaic virus resistance is indeed driven primarily by GmWRKY33-Chr18-1. Thus, by detecting the H3K4me3 modification level of the target gene regulated by GmWRKY33-Chr18-1 in soybean plants, if the H3K4me3 modification level in the experimental group is significantly higher than that in the control group after AHL induction, and the enrichment fold measured by ChIP-qPCR increases by at least 1.5 times, it indicates that the experimental group is in a state of enhanced resistance to soybean mosaic virus.

[0025] Defense against and early warning of mosaic virus disease is achieved through the following molecular mechanisms. a. Infection with soybean mosaic virus SMV SC3 can induce H3K4me3 enrichment in the promoter region of the PR1 gene; b. The administration of AHL signaling molecules induces activation of the GmWRKY33-Chr18-1 gene, which in turn activates the MAPK signaling pathway; c. After treatment with AHL signaling molecules, the salicylic acid content increased significantly, such as... Figure 5 As shown, after applying AHL(3OC8-HSL) + SC3, the free salicylic acid in the AHL-treated plants was 3.54 times that of the untreated plants, and the total salicylic acid was 2.82 times that of the untreated plants after 6 hours. This indicates that the soybean plants initiate a disease resistance process, enhancing their resistance to soybean mosaic virus.

[0026] Specific embodiment 4, A method for detecting the role of bacterial quorum sensing molecule AHL in inducing and activating the GmWRKY33 pathway to provide early warning and defense against soybean mosaic virus disease was developed. The method detected the H3K4me3 modification level of the target gene PR1, which is regulated by GmWRKY33-Chr18-1. The H3K4me3 modification level of the experimental group was significantly higher than that of the control group after AHL induction, indicating that the experimental group was in a state of enhanced resistance to soybean mosaic virus.

[0027] according to Figure 6 As shown, the ChIP-qPCR results indicated extremely low enrichment signal in the IgG negative control, confirming the specificity and reliability of the ChIP experiment. Compared to the blank CK group, SMV SC3 alone altered the abundance of H3K4me3 modification in the GmWRKY33-Chr18-1 promoter region (P < 0.01); AHL (3OC8-HSL) treatment alone only slightly upregulated H3K4me3 levels (P < 0.05), indicating limited effect of single action. When AHL pretreatment was followed by SC3 inoculation, the enrichment of H3K4me3 in the GmWRKY33-Chr18-1 promoter was significantly higher than that of SC3 alone (P < 0.001). The results showed that AHL pretreatment significantly amplified the SC3-induced activation of histone modifications in the GmWRKY33-Chr18-1 promoter, and that AHL pretreatment enhanced the epigenetic response of transcription factor GmWRKY33-Chr18-1 to soybean mosaic virus SC3. Specific Implementation Example 5 A method for identifying a new soybean mosaic virus (SMV) strain by inducing and activating the GmWRKY33 pathway through a bacterial quorum sensing molecule (AHL) to provide early warning and defense against SMV was developed. Transformed seedlings overexpressing the GmWRKY33-Chr18-1 gene were constructed. The primers for identifying the new strain were F33-18-1(+) and F33-18-1(-), as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The PCR system and procedure are shown in Table 3 below.

[0029] 3. PCR system and procedure program After the PCR program is completed, electrophoresis is performed on a 1% agarose gel at 5V / cm for 20 minutes. The obtained electrophoretic bands are compared with the positive control group. If the transformed seedling sample to be tested shows a band at the corresponding position of the marker that corresponds to the positive control group, and the negative control shows no band, then the transformed seedling can be identified as a new line.

Claims

1. The application of a bacterial quorum sensing molecule, AHL, in inducing and activating the GmWRKY33 pathway to defend against and warn against soybean mosaic virus disease, characterized in that: The application includes the following steps: S1. Application of AHL signaling molecules: Prepare a 5mM AHL stock solution and apply it to the rhizosphere of soybean plants; S2. WRKY33 pathway activation: The expression of the soybean GmWRKY33-Chr18-1 gene was upregulated by the administration of AHL signaling molecules in step S1. S3. Verification of disease resistance: Soybean plants treated with soybean mosaic virus were inoculated, and resistance was confirmed by detecting the disease index and viral load.

2. The application according to claim 1, characterized in that: In step S1, the AHL mother liquor is prepared using 3OC8-HSL.

3. The application according to claim 1, characterized in that: In step S1, the AHL mother liquor is diluted 1000 times to obtain a diluted solution with an effective concentration of 5 μM. 30 mL of the diluted solution is applied to the roots of each plant for root irrigation treatment, and the treatment is repeated once every 3 days for a total of 2 applications.

4. The application according to claim 1, characterized in that: The nucleotide sequence of the GmWRKY33-Chr18-1 gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2.

5. The application according to claim 1, characterized in that: The disease index DI < 30 indicates that the viral load is reduced by at least two orders of magnitude compared to the control group.

6. The application according to claim 1, characterized in that: Defense against and early warning of mosaic virus disease is achieved through the following molecular mechanisms. a. Infection with soybean mosaic virus SMV SC3 can induce H3K4me3 enrichment in the promoter region of the PR1 gene; b. The administration of AHL signaling molecules induces activation of the GmWRKY33-Chr18-1 gene, which in turn activates the MAPK signaling pathway; c. The salicylic acid content increases significantly, initiating the disease resistance process and enhancing resistance to mosaic virus.

7. A method for detecting the bacterial quorum sensing molecule AHL inducing and activating the GmWRKY33 pathway to defend against and warn against soybean mosaic virus disease, based on the application described in claim 1, characterized in that: The H3K4me3 modification level of the target gene PR1 regulated by GmWRKY33-Chr18-1 was detected. The H3K4me3 modification level of the experimental group was significantly higher than that of the control group after AHL induction, indicating that the experimental group was in a state of defense and early warning against enhanced resistance to mosaic virus.

8. A method for identifying new soybean mosaic virus (SMV) strains by inducing and activating the GmWRKY33 pathway in response to early warning of bacterial quorum sensing molecule AHL, based on the application described in claim 1, characterized in that: A new seedling line overexpressing the GmWRKY33-Chr18-1 gene was constructed. The identification primers for the new line were F33-18-1(+) and F33-18-1(-), where F33-18-1(+) is shown in SEQ ID NO.3 and F33-18-1(-) is shown in SEQ ID NO.4.

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