Application of NbJAR1 gene in resistance to tomato spotted wilt virus

Abstract

The application of NbJAR1 gene in resisting tomato spotted wilt virus belongs to the field of biotechnology, and particularly relates to the antiviral application of NbJAR1 gene. The application enhances the disease resistance of plants to tomato spotted wilt virus by increasing the expression amount of NbJAR1 gene in the plants, and specifically inserts the NbJAR1 gene shown in SEQ ID NO. 1 into a pCAMBIA1301-35SN vector to construct an overexpression vector, and then introduces the vector into target plants by means of Agrobacterium GV3101. Experiments prove that the virus accumulation amount in tobacco plants overexpressing the NbJAR1 gene is significantly reduced, and the disease resistance is greatly improved; and the disease resistance of tobacco is significantly reduced when the gene is knocked down. The application can quickly create solanaceous crop germplasm resistant to tomato spotted wilt virus, and is particularly suitable for tobacco, has a short cycle and high efficiency, and provides new gene resources and theoretical support for green prevention and control of crop disease resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the antiviral application of the NbJAR1 gene. Background Technology

[0002] Common tobacco diseases include viral diseases and tobacco black shank, among which Tomato Spotted Wilt Virus (TSWV), transmitted by thrips, is particularly harmful. TSWV belongs to the family Tomato Spotted Wiltviridae, genus *Orthotomatologica*, and its host range covers over 1000 plant species in 85 families. It can cause leaf necrosis, ring spots, stunted growth, and even crop failure. In recent years, with climate change and the spread of thrips, the harm caused by TSWV has intensified globally, seriously threatening the sustainable development of agriculture. Current control methods mainly rely on chemical pesticides, but their overuse easily leads to environmental pollution, increased pathogen resistance, and excessive residues in agricultural products. Developing resistant varieties should be the fundamental solution, but traditional breeding cycles are long and lack effective resistance gene resources against TSWV.

[0003] The jasmonic acid signaling pathway is a core pathway for plant antiviral activity. JAR1, as a key enzyme in the synthesis of jasmonic acid isoleucine, directly determines the synthesis of the active jasmonic acid isoleucine (JA-Ile) molecule and regulates downstream disease resistance responses. However, current technologies have not disclosed the specific function of JAR1 in TSWV resistance, nor have they revealed that increasing its expression level can significantly alter plant antiviral capabilities. Summary of the Invention

[0004] The purpose of this invention is to utilize the JAR1 key enzyme gene to create highly efficient antiviral germplasm resources and solve the problem of TSWV harm.

[0005] Application of the NbJAR1 gene in improving plant resistance to tomato spotted wilt virus.

[0006] The application of the NbJAR1 gene in improving plant resistance to tomato spotted wilt virus: By increasing the expression level of the NbJAR1 gene in plants, the plant's resistance to tomato spotted wilt virus is enhanced.

[0007] To increase the expression level of the NbJAR1 gene, an overexpression vector of the tobacco NbJAR1 gene was constructed and introduced into the target plant using Agrobacterium. Specifically, the NbJAR1 gene was inserted into the plant expression vector pCAMBIA1301-35SN to obtain the overexpression vector, which was then introduced into the plant using Agrobacterium GV3101.

[0008] Overexpression of the NbJAR1 gene in this invention can significantly reduce the accumulation of TSWV virus in tobacco and significantly improve disease resistance. It can be used to breed and cultivate tobacco varieties resistant to tomato spotted wilt virus. It can rapidly create disease-resistant materials with a short cycle, high efficiency, and strong stability, providing new gene resources and theoretical basis for broad-spectrum antiviral protection in solanaceous crops, which is in line with the development direction of green prevention and control. Attached Figure Description

[0009] Figure 1 This is a graph showing the JARI gene expression levels in plants overexpressing the NbJAR1 gene and wild plants in Example 1.

[0010] Figure 2 This is a graph showing the expression levels of the TSWV N gene in plants overexpressing the NbJAR1 gene and wild plants infected with tomato spotted wilt virus, as shown in Example 1.

[0011] Figure 3 This is a graph showing the expression levels of TSWV N protein in plants overexpressing the NbJAR1 gene and wild plants infected with tomato spotted wilt virus, as shown in Example 1.

[0012] Figure 4 This is an electron microscopy diagram showing the changes in viral particles in plants overexpressing the NbJAR1 gene and wild plants after infection with tomato spotted wilt virus, as observed in Example 1.

[0013] Figure 5 The graph shows the expression levels of the JARI gene in plants with the NbJAR1 gene knocked down and wild plants, as shown in Comparative Example 1.

[0014] Figure 6 The graph shows the expression levels of the TSWV N gene in plants with knocked-down NbJAR1 gene and wild plants after infection with tomato spotted wilt virus, as shown in Comparative Example 1.

[0015] Figure 7 The graph shows the expression levels of TSWV N protein in plants with knocked-down NbJAR1 gene and wild plants after infection with tomato spotted wilt virus, as shown in Comparative Example 1.

[0016] Figure 8 The image shows the changes in viral particles after infection with tomato spotted wilt virus in plants with knocked-down NbJAR1 gene and wild plants, as observed by electron microscopy in Comparative Example 1.

[0017] Figure 9 This is a comparison of the virus infection status between plants overexpressing the NbJAR1 gene and wild-type plants in Example 1.

[0018] Figure 10 This is a comparison of virus infection in plants with knocked-down NbJAR1 gene and wild-type plants, as shown in Figure 1. Detailed Implementation

[0019] Example 1: Application of NbJAR1 gene in resistance to tomato spotted wilt virus. By increasing the expression level of NbJAR1 gene in tobacco, the resistance of tobacco to tomato spotted wilt virus can be improved.

[0020] The specific methods to increase the expression level of the NbJAR1 gene in tobacco include the following steps:

[0021] Construction of S1, recombinant plasmid 35SN:JAR1;

[0022] S1-1, Total RNA was extracted from leaves of Nicotiana benthamiana and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed using a primer pair consisting of upstream primer F: 5'- GGACTAGTATG AAGAT GGTGGTAGAGAGATTGAG - 3' and downstream primer R: 5'- ACGCGTCGACACAGAAGG CACTACTAACAT AGGTCT - 3' to obtain the amplified product.

[0023] S1-2, recover approximately 1750bp DNA fragment from the PCR amplification product;

[0024] S1-3, the recovered DNA fragments were ligated with Blunt-zero to obtain an intermediate vector. The intermediate vector and the overexpression vector pCAMBIA1301-35SN were then double-digested using the SpeⅠ and SalⅠ restriction enzyme sites and recovered.

[0025] S1-4: The pCAMBIA1301-35SN vector backbone and NbJAR1 gene fragment recovered after enzyme digestion in step S1-3 are ligated using T4 DNA ligase to obtain the recombinant plasmid 35SN:JAR1.

[0026] Sequencing of the recombinant plasmid 35SN:JAR1 confirmed that the recombinant plasmid inserted the DNA sequence shown in SEQ ID NO.1; the vector can be transcribed and translated in plant cells to produce the JAR1 protein shown in SEQ ID NO.2.

[0027] S2, Obtaining tobacco plants overexpressing the NbJAR1 gene;

[0028] S2-1, the recombinant plasmid 35SN:JAR1 was introduced into Agrobacterium tumefaciens GV3101 by chemical transformation. The recombinant Agrobacterium GV3101 / 35SN:JAR1 was successfully transformed into NbJAR1 by screening on YEP solid medium containing 50 mg / mL kanamycin and 25 mg / mL rifampin.

[0029] S2-2, GV3101 / 35SN:JAR1 was transformed into cultivated tobacco plants with 5-6 leaves using Agrobacterium-mediated genetic transformation to obtain tobacco plants OE-JAR1 transgenic with the NbJAR1 gene.

[0030] Real-time quantitative PCR was performed on the obtained tobacco plants OE-JAR1 and wild tobacco plants WT. Specifically:

[0031] Total RNA was extracted from leaves of two different tobacco plants and then reverse transcribed to obtain cDNA. Using the cDNA as a template, the amount of the NbJAR1 gene was detected by quantitative real-time PCR, and the primers for the NbJAR1 gene were identified as 5'-GAACTCTTCCCGAATACA-3' and 5'-TAACTCCAACCCATCCTTC-3'.

[0032] The expression level of the NbJAR1 gene is as follows Figure 1 As shown in the figure, the results indicate that the black bar (WT) represents the expression level of the JAR1 gene in wild tobacco plants, which is about 25; the gray bar (OE-JAR1) represents transgenic tobacco plants that overexpress JAR1, and its expression level is significantly increased to about 80, which is more than 3 times that of WT. The expression level of the JAR1 gene in the cDNA of transgenic tobacco plants OE-JAR1 is significantly increased.

[0033] The obtained tobacco plants OE-JAR1 and wild tobacco plants WT were subjected to tomato spotted wilt virus resistance tests, specifically:

[0034] Tomato spotted wilt virus (TSWV) was inoculated into two types of tobacco plants using the friction inoculation method. The plants were cultured under daylight at 28°C for 14 hours and then under darkness at 25°C for 10 hours. Four days later, total RNA was extracted from the leaves of the infected tobacco plants and reverse transcribed to obtain cDNA. Using the cDNA as a template, the replication level of the TSWV N gene was detected by quantitative real-time PCR, and the primers for the TSWV N gene were identified as 5'-TCGACAAGCCTGACCCTGAT-3' and 5'-TCAGACAGGATTGGAGCCAC-3'.

[0035] The expression level of the N gene in TSWV is as follows: Figure 2 As shown, the results indicate that the black bars (OE-JAR1) represent tobacco plants overexpressing the JAR1 gene, with extremely low TSWV N gene expression levels, almost close to 0. The gray bars (WT) represent wild-type tobacco plants, with TSWV N gene expression levels of approximately 130,000, significantly higher than the OE-JAR1 group.

[0036] Western blotting was used to detect the expression of OE-JAR1 in tobacco plants and TSWV N protein in wild-type tobacco plants (WT) after infection with tomato spotted wilt virus. Specifically:

[0037] Total protein was extracted from leaves of two types of tobacco plants infected with TSWV virus. Protein loading buffer was added according to the specified ratio, and the samples were boiled in water for 10 min to ensure complete protein denaturation. 10 μL of total plant protein was loaded and subjected to SDS-PAGE gel electrophoresis. The expression of TSWV N protein in the plants was detected using a TSWV N protein antibody. Western blotting results are shown below. Figure 3 As shown, compared with wild plants, the expression of TSWV N protein in OE-JAR1 plants was significantly reduced after infection with tomato spotted wilt virus.

[0038] Electron microscopy was used to observe changes in virus particles in tobacco plants OE-JAR1 and wild tobacco plants WT after infection with tomato spotted wilt virus. Specifically:

[0039] Leaf samples from two different tobacco plants infected with TSWV virus were collected and prepared using negative staining. The characteristics of virus particles and cell structure were observed under electron microscopy to determine the degree of viral infection. The results are as follows: Figure 4 As shown, the viral particles (Vp) and vesicles (Ve) of TSWV in the OE-JAR1 plant were significantly lower than those in the wild plant.

[0040] The above results indicate that the OE-JAR1 plant exhibits significantly enhanced resistance to tomato spotted wilt virus compared to wild tobacco plants.

[0041] Comparative Example 1: Detection of resistance to tomato spotted wilt virus in tobacco plants with knocked-down NbJAR1 gene.

[0042] The specific method for knocking down the NbJAR1 gene includes the following steps:

[0043] S1, Construction of recombinant plasmid pTRV-PTV00-JAR1;

[0044] S1-1, Total RNA was extracted from leaves of Nicotiana benthamiana and amplified by PCR using a primer pair consisting of upstream primer F: 5'- CCC AAGCTT GGT GGT AGAGAA GAT TGA GAC GAA G- 3' and downstream primer R: 5'- CGC GGATCC CTA AAT CAT TGT GAG TGACAA TAG GG- 3' to obtain the amplified product;

[0045] S1-2, recover approximately 200bp DNA fragments from the PCR amplification product;

[0046] S1-3, the recovered DNA fragments were ligated with Blunt-zero to obtain an intermediate vector. The intermediate vector and the pTRV-PTV00 vector were then double-digested using HindIII and BamHI restriction sites and recovered.

[0047] S1-4, the pTRV-PTV00 vector backbone and NbJAR1 gene fragment recovered after enzyme digestion in step S1-3 are ligated using T4 DNA ligase to obtain the recombinant plasmid pTRV-PTV00-JAR1.

[0048] Sequencing of the recombinant plasmid pTRV-PTV00-JAR1 confirmed that the recombinant plasmid contained the DNA sequence shown in SEQ ID NO.3.

[0049] S2, Obtaining tobacco plants with NbJAR1 gene knockdown;

[0050] S2-1, the recombinant plasmid pTRV-PTV00-JAR1 was introduced into Agrobacterium tumefaciens GV3101 by chemical transformation. The recombinant Agrobacterium GV3101 / pTRV-PTV00-JAR1 was successfully transformed into pTRV-PTV00-JAR1 by screening on YEP solid medium containing 50 mg / mL kanamycin and 25 mg / mL rifampin.

[0051] S2-2, GV3101 / pTRV-PTV00-JAR1 was transformed into cultivated tobacco plants with 5-6 leaves using Agrobacterium-mediated genetic transformation, resulting in tobacco plants with knocked-down NbJAR1 gene, namely TRV-JAR1.

[0052] Real-time quantitative PCR was performed on the obtained tobacco plant TRV-JAR1 and wild tobacco plant WT. Specifically:

[0053] Total RNA was extracted from leaves of two different tobacco plants, and then reverse transcribed to obtain cDNA. Using the cDNA as a template, the NbJAR1 gene abundance was detected by quantitative real-time PCR. Figure 5 As shown, the results indicate that the expression level of the NbJAR1 gene in the cDNA of the tobacco plant TRV-JAR1 is significantly reduced.

[0054] Resistance to tomato spotted wilt virus was tested in tobacco plants with knocked-down NbJAR1 gene (TRV-JAR1) and wild-type tobacco plants (WT). Specifically:

[0055] Tobacco spotted wilt virus (TSWV) was inoculated into tobacco plants using the friction inoculation method. The plants were cultured under daylight conditions at 28°C for 14 hours, followed by nighttime in darkness at 25°C for 10 hours. After 4 days, total RNA was extracted from the leaves of the tested tobacco plants, and then reverse transcribed to obtain cDNA. Using the cDNA as a template, the TSWV N gene replication level was detected by quantitative real-time PCR. The expression level of the TSWV N gene was as follows: Figure 6 As shown, the results indicate that the virus enters a rapid replication phase 5-7 days after inoculation, and its expression level reaches its peak. The differences between the TRV-JAR1 and WT groups are extremely significant. Compared with the wild-type WT plant, the expression level of the TSWV N gene in the cDNA of the tobacco plant TRV-JAR1 is significantly increased. That is, after inhibiting the expression level of the NbJAR1 gene, the plant's resistance to tomato spotted wilt virus is significantly reduced.

[0056] Western blotting was used to detect TRV-JAR1 expression in tobacco plants with NbJAR1 gene knockdown and TSWV N protein expression in wild-type tobacco plants (WT) infected with tomato spotted wilt virus. Specifically:

[0057] Total protein was extracted from leaves of two tobacco plants infected with TSWV virus. Protein loading buffer was added at an appropriate ratio, and the samples were boiled in water for 10 minutes to fully denature the proteins. 10 μL of total plant protein was loaded and subjected to SDS-PAGE gel electrophoresis. The expression of TSWV N protein in the plants was detected using a TSWV N protein antibody. Western blotting results are shown below. Figure 7 As shown, compared with wild-type plants, tobacco plants with knocked-down NbJAR1 genes showed significantly increased TSWV N protein expression after infection with tomato spotted wilt virus.

[0058] Electron microscopy was used to observe changes in viral particles in tobacco plants with knocked-down NbJAR1 gene (TRV-JAR) and in wild-type tobacco plants (WT) infected with tomato spotted wilt virus. Specifically:

[0059] Leaf samples from two tobacco plants infected with TSWV virus were collected and prepared using negative staining. The characteristics of virus particles and cell structure were observed under an electron microscope to determine the degree of viral infection. The results are as follows: Figure 8 As shown, the viral particles (Vp) and vesicles (Ve) of TSWV in the TRV-JAR1 plant were significantly higher than those in the wild-type plant.

[0060] The above results indicate that, compared with wild-type tobacco plants, the resistance of tobacco plants TRV-JAR1 to tomato spotted wilt virus was significantly reduced.

[0061] In both Example 1 and Comparative Example 1 above, the quantitative experiments were performed in triplicate, and the results were averaged. The pCAMBIA1301-35SN vector was purchased from Fenghui Biotechnology, pTRV-PTV00 was owned by the inventor, the Blunt-zero cloning vector was purchased from Vazyme, and Agrobacterium GV3101 was purchased from Qingke Biotechnology.

Claims

1. Application of the NbJAR1 gene in improving plant resistance to tomato spotted wilt virus.

2. The application of the NbJAR1 gene as described in claim 1 in improving plant resistance to tomato spotted wilt virus, characterized in that, By increasing the expression level of the NbJAR1 gene in plants, the plant's resistance to tomato spotted wilt virus can be enhanced.

3. The application of the NbJAR1 gene as described in claim 1 in improving plant resistance to tomato spotted wilt virus, characterized in that, The plant in question is a member of the Solanaceae family, preferably tobacco.

4. The application of the NbJAR1 gene as described in claim 2 in improving plant resistance to tomato spotted wilt virus, characterized in that, One way to increase the expression level of the NbJAR1 gene in plants is to construct an overexpression vector for the NbJAR1 gene and introduce the overexpression vector into the target plant.

5. The application of the NbJAR1 gene as described in claim 4 in improving plant resistance to tomato spotted wilt virus, characterized in that, The overexpression vector was obtained by inserting the NbJAR1 gene shown in SEQ ID NO.1 into the plant expression vector pCAMBIA1301-35SN vector as the backbone.

6. The application of the NbJAR1 gene as described in claim 4 or 5 in improving plant resistance to tomato spotted wilt virus, characterized in that, The overexpression vector was introduced into the target plant using Agrobacterium-mediated genetic transformation, wherein the Agrobacterium was Agrobacterium tumefaciens GV3101.

7. Application of the NbJAR1 gene, as shown in SEQ ID NO.1, in the breeding of transgenic plant varieties resistant to tomato spotted wilt virus.

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