Use of a diterpene aldehyde inhibitor in the control of tmv virus

Abstract

The application discloses application of a strigolactone inhibitor in prevention and treatment of TMV viruses and relates to the technical field of molecular biology. The application inoculates TMV after treating Nicotiana attenuata with a strigolactone (SL) hormone, performs transcriptome sequencing, analyzes gene conditions of difference changes, and performs qPCR verification on differentially expressed genes; research on SL-induced promotion of Nicotiana attenuata to TMV infection is carried out, and it is clear that the SL has the ability to inhibit tobacco resistance to tobacco mosaic virus (TMV); it is determined that the SL is a plant hormone for inhibiting plant defense reaction, and the SL inhibitor Tis108 has the ability to enhance tobacco resistance to TMV, and it is determined that the Tis108 is a plant hormone inhibitor for promoting plant defense reaction. The application lays a foundation for further research and utilization of the SL hormone, development of a new type of plant elicitor, safe and efficient prevention and treatment of plant diseases.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and more specifically to the application of styracin inhibitors in the prevention and treatment of TMV virus. Background Technology

[0002] Plant hormones are naturally occurring compounds within plants that regulate plant growth, development, and responses to environmental factors. When pathogens invade, plant hormones can influence a plant's resistance and response mechanisms, thus helping it fight disease. Besides combating pathogens, plant hormones also regulate plant growth and development, enhancing its ability to adapt to its environment. This regulatory mechanism enables plants to respond quickly to disease attacks and minimize the extent of damage. Strigolactone (SL) is a recently discovered endogenous plant hormone derived from carotenoids. It regulates stem branching, root structure, axillary bud root formation, secondary growth, and leaf senescence within plants, as well as the plant's response to nutrient stress, particularly phosphorus (P) deficiency. Recent findings also indicate its involvement in responses to abiotic stresses such as drought and salt stress.

[0003] Plant hormones play important roles in viral disease infection. For example, spraying abscisic acid (ABA) inhibits the infection of BaMV, TMV, TNV, and TMV-cg, and enhances plant resistance; spraying ethylene (ET) inhibits the infection of TMV, TNV, and ChiVMV, but promotes the infection of TMV-cg, CaMV, TuMV, RDV, and CMV; auxin inhibits the infection of RDV and TVCV, but promotes the infection of RBSDV; brassinolides (BRs) inhibit the infection of ORMV, CMV, TCV, and RSV, but promote the infection of RBSDV; SA and JA both inhibit the infection of RSV and TBSV, etc.

[0004] However, to date, there have been no reports of SLs participating in the infection of viral diseases, especially the model virus TMV. Summary of the Invention

[0005] In view of this, the present invention provides the application of styracil lactone inhibitors in the prevention and treatment of TMV virus.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of suppressing or deleting the expression levels of key genes involved in stromal lactone synthesis in tobacco anti-TMV virus.

[0008] As a preferred technical solution, the accession numbers of the key genes for the synthesis of styrax lactone are Niben101Scf00878g02006.1 and Niben101Scf01611g07010.1.

[0009] Another object of the present invention is to provide the application of biomaterials that reduce the expression levels of key genes in strigolactone synthesis in tobacco anti-TMV virus, wherein the biomaterials are any one of the following:

[0010] A. An expression cassette that causes the gene to be deleted or suppressed;

[0011] B. An expression carrier containing the expression cassette described in A;

[0012] C. A cloning vector containing the expression cassette described in A;

[0013] D. Engineered bacteria containing the expression cassette described in A and / or the expression vector described in B and / or the cloning vector described in C.

[0014] As a preferred embodiment, the accession numbers for the key genes for the synthesis of styrax lactone are Niben101Scf00878g02006.1 and Niben101Scf01611g07010.1.

[0015] Another object of the present invention is to provide the application of styracil lactone inhibitors in tobacco anti-TMV virus.

[0016] As a preferred technical solution, the unicornulide inhibitor is Tis108.

[0017] Another objective of this invention is to provide a method for improving tobacco resistance to TMV virus by applying an exogenous strigolactone inhibitor or reducing the expression level of key genes involved in strigolactone synthesis.

[0018] As a preferred technical solution, the styrax lactone inhibitor is Tis108, and the accession numbers of the key genes for styrax lactone synthesis are Niben101Scf00878g02006.1 and Niben101Scf01611g07010.1.

[0019] More preferably, the preparation method of the unicorn lactone inhibitor Tis108 is to dissolve Tis108 in DMSO to obtain a mother liquor, then dilute the mother liquor with water and add Tween 20 to obtain a working solution.

[0020] The reduction of expression levels of key genes for styrax lactone synthesis was achieved by inhibiting the expression of Niben101Scf00878g02006.1 and Niben101Scf01611g07010.1 using VIGS technology.

[0021] Another objective of this invention is to provide a method for preparing a tobacco mosaic virus infection model, achieved by enhancing the expression level of key genes involved in the synthesis of styrax lactones.

[0022] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of styracin inhibitors in the prevention and treatment of TMV virus.

[0023] This invention involves treating native tobacco with SL hormone and then inoculating it with TMV. Transcriptome sequencing was performed to analyze differentially expressed genes, and qPCR was used to verify the differentially expressed genes. The invention also investigated the effect of SL-induced native tobacco on TMV infection, clarifying that SL has the ability to inhibit tobacco's resistance to TMV. Furthermore, SL was identified as a plant hormone that inhibits plant defense responses, while the SL inhibitor Tis108 enhanced tobacco's resistance to TMV, thus identifying Tis108 as a plant hormone inhibitor that promotes plant defense responses.

[0024] This invention analyzes the transcriptome of tobacco treated with SL (soil extractant) to reduce resistance to TMV, identifying key pathways and related genes involved in this process. Furthermore, VIGS inhibition of CCD7 and CCD8, key genes in the SL synthesis pathway that exhibit altered expression levels in the transcriptome, enhanced plant resistance to TMV. This demonstrates the important function of SL hormones in regulating the plant immune system and their significant research and development potential. This invention lays the foundation for further research and utilization of SL hormones, the development of novel plant inducers, and the safe and efficient control of plant diseases. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 Images of tobacco TMV resistance performance in SL treatment, SL inhibitor Tis108 treatment, and control treatment are provided for embodiments of the present invention.

[0027] Figure 2 Images showing the infection results of tobacco plants in SL treatment and control treatment 8 days after inoculation with TMV-GFP, as provided in this embodiment of the invention.

[0028] Figure 3 Images showing the infection results of tobacco plants in SL treatment and control treatment 9 days after inoculation with TMV-GFP, as provided in this embodiment of the invention.

[0029] Figure 4 These are quantitative fluorescence images of tobacco infected with TMV-GFP 8 days after inoculation with SL-treated and control tobacco, as provided in this embodiment of the invention.

[0030] Figure 5 These are quantitative fluorescence images of tobacco infected with TMV-GFP 9 days after inoculation with SL-treated and control tobacco, as provided in this embodiment of the invention.

[0031] Figure 6 Images showing the TMV content detection of tobacco after inoculation with TMV in the SL treatment and control treatments provided in this embodiment of the invention.

[0032] Figure 7 Images showing the infection results of tobacco treated with Tis108 and the control group 10 days after inoculation with TMV-GFP, as provided in this embodiment of the invention.

[0033] Figure 8 Images showing the infection results of tobacco treated with Tis108 and the control group 11 days after inoculation with TMV-GFP, as provided in this embodiment of the invention.

[0034] Figure 9 These are quantitative fluorescence images of tobacco infected with TMV-GFP 10 days after inoculation with Tis108-treated and control tobacco, provided in this embodiment of the invention.

[0035] Figure 10 These are quantitative fluorescence images of tobacco infected with TMV-GFP 11 days after inoculation with Tis108-treated and control tobacco, provided in this embodiment of the invention.

[0036] Figure 11 This is a summary of the differentially expressed gene data from transcriptome sequencing of tobacco plants after inoculation with TMV in the SL treatment and control treatments provided in this embodiment of the invention.

[0037] Figure 12 The images show pathways enriched by differentially expressed genes (GO) obtained from transcriptome sequencing of tobacco plants inoculated with TMV after SL treatment and control treatment, as provided in this embodiment of the invention.

[0038] Figure 13 The images show pathways enriched by KEGG in differentially expressed genes obtained from transcriptome sequencing after tobacco inoculation with TMV in SL-treated and control-treated tobacco, as provided in this embodiment of the invention.

[0039] Figure 14 The images show a comparison of the expression levels of relevant genes in tobacco plants treated with SL and those treated with TMV, as provided in this embodiment of the invention.

[0040] Figure 15This is a diagram illustrating the resistance identification of TMV after inoculation by inhibiting the expression of NtCCD7 and NtCCD8 genes in the SLs pathway using VIGS, as provided in an embodiment of the present invention.

[0041] In the above results, * represents a significant difference p-value < 0.05, and ** represents a significant difference p-value < 0.01. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Source of biomaterials in this invention embodiment:

[0044] The construction methods of the VIGS vectors TRV1 and TRV2 used in this invention are referenced in the literature (Chen, LJ, Zou, WS, Wu, G., Lin, HH, and Xi, DH 2018. Tobacco alpha-expansin EXPA4 plays a role in Nicotiana benthamiana defence against Tobacco mosaic virus. Planta 247:355-368.).

[0045] The tobacco used in this invention is commercially available, and all reagents used in the experimental examples of this invention are also commercially available, with no specific limitation on their source. For example, SL powder can be purchased from Yuanye Biotechnology (molecular weight: 298.29 g / m), and Tis108 powder can be purchased from Ron (molecular weight: 335.4 g / m).

[0046] The transcriptome sequencing service used in this invention was provided by Ouyi Bio.

[0047] Example 1

[0048] Effects of SL hormone and its inhibitor Tis108 on TMV-infected tobacco

[0049] 1. Preparation and spraying methods of SL and Tis108

[0050] SL stock solution: Dissolve 1 mg of SL powder in 3.352 mL of methanol until the powder is no longer visible, to obtain a 1 mM / L stock solution (store at 4°C).

[0051] SL working solution: Dilute the SL stock solution 1000 times with sterile ddH2O, and then add Tween 20 at 0.015% (v / v) to obtain the SL working solution.

[0052] Preparation method of SL working solution control solution: Dilute methanol 1000 times with sterile ddH2O, and then add Tween 20 at 0.015% (v / v) to obtain SL working solution control solution.

[0053] Tis108 stock solution: Dissolve 1 mg of Tis108 powder in 2.98 mL of DMSO until the powder is no longer visible, to obtain a stock solution of 1 mM / L.

[0054] Tis108 working solution: Dilute the stock solution 1000 times with sterile ddH2O, and then add Tween 20 at 0.015% (v / v) to obtain Tis108 working solution.

[0055] Preparation method of Tis108 working solution control solution: Dilute DMSO with sterile ddH2O 1000 times, and then add Tween 20 at 0.015% (v / v) to obtain Tis108 working solution control solution.

[0056] Spraying method:

[0057] Healthy, 4-5 leaf-stage native tobacco plants were randomly divided into two large groups, each further divided into two smaller groups. SL working solution, Tis108 working solution, and a control solution were sprayed evenly from approximately 10 cm above the leaves, until the leaves were fully wetted and droplets began to form. After spraying, the plants were placed in a dark environment at approximately 28 degrees Celsius for 24 hours.

[0058] 2. Inoculation of TMV-GFP strain into Tobacco Benedictine burmannii

[0059] The TMV-GFP used in this invention is a TMV strain of GFP fluorescent protein transformed into the vector pRK415-GFP, which is a clone transformed into Agrobacterium. For specific methods, please refer to the literature (Zhu, F., Zhang, P., Meng, YF, Xu, F., Zhang, DW, Cheng, J., Lin, HH, and Xi, DH 2013. Alpha-momorcharin, a RIP produced by bitter melon, enhances defense response into bacco plants against diverse plant viruses and shows antifungal activity in vivo. Planta 237:77-88.).

[0060] Activation: The TMV-GFP strain stored at -70°C was inoculated into LB solution and cultured at 28°C and 200 rpm for 2 days until OD500 was reached. 600 The value is around 0.6-0.8.

[0061] Inoculation: The tobacco plants sprayed with SL working solution and its control solution, as well as Tis108 working solution and its control solution in step 1, were each divided into two equal portions. One portion was inoculated with Agrobacterium tumefaciens infection clones containing TMV-GFP, and the other portion was inoculated with LB control solution. (The inoculation method was to use a 1ml syringe to take 1ml of TMV-GFP or LB solution and, holding the leaf against the underside of the first and second fully unfolded leaves from top to bottom, slowly push the liquid into the tobacco leaves to allow it to spread). After injection, the plants were placed in a dark environment at approximately 28 degrees Celsius for 24 hours, and then moved to a light environment (alternating between light and dark for 16 / 8 hours) for further cultivation.

[0062] 3. Results Survey and Statistics

[0063] Five days after inoculation, the tobacco was photographed under fluorescent light to observe the infection status. [The specific processing method was as follows: after opening the image to be processed in the ImageJ web version (https: / / cnij.imjoy.io / ), a single channel was extracted (Image-Color-Split Channels); the green fluorescence channel was selected and the grayscale threshold was adjusted to determine the detection area (Image-Adjust-Threshold). To avoid large errors in the image processing process, the minimum and maximum grayscale values ​​were fixed at 40 and 255, respectively, and Dark Background was checked; the parameters to be measured were set (Analyze-SetMeasurements), and Mean gray value and Limit to threshold were checked to measure the average fluorescence intensity of the selected area; the detection results were displayed after clicking Measure (Analyze-Measure).] Based on this method, the fluorescence area of ​​the fluorescent photographs taken on the 8th and 9th days after TMV-GFP inoculation was calculated, and the fluorescence area of ​​the fluorescent photographs taken on the 10th and 11th days after TMV-GFP inoculation was calculated after spraying Tis108. By day 14, the leaves showed symptoms of TMV infection. The leaves were photographed for investigation, and RNA was quantitatively extracted from the leaves, reverse transcribed into cDNA, and then detected by qPCR using universal primers for the virus.

[0064] The results showed that, through investigation of leaves 14 days after infection, the disease was more pronounced after SL spraying, while the disease was less pronounced after spraying with the inhibitor Tis108 (see appendix). Figure 1); Through observation of the infection process after inoculation with fluorescent virus strains, it was found that on the 8th day after inoculation (see Appendix) Figure 2 ) and day 9 (see appendix) Figure 3 It was observed that the fluorescent infection area of ​​the SL treatment was significantly larger than that of the CK; Quantitative analysis of all inoculated plants using the aforementioned fluorescence conversion method revealed that on day 8 post-inoculation (see Appendix...), the fluorescence infection area was significantly larger than that of the CK. Figure 4 ) and day 9 (see appendix) Figure 5 The fluorescent infection area of ​​the SL-treated plants was significantly greater than that of the CK-treated plants. Quantitative RNA extraction and quantitative PCR analysis of leaves 14 days after disease onset revealed that the viral load of the SL-treated plants was significantly higher than that of the CK-treated plants (see Appendix). Figure 6 On the 10th day after vaccination (see appendix) Figure 7 ) and day 11 (see appendix) Figure 8 It was observed that the fluorescent infection area of ​​the Tis108-treated plants was significantly smaller than that of the control (CK). Quantitative analysis was performed on all inoculated plants using the fluorescence conversion method described above. The results showed that on day 10 post-inoculation (see Appendix...), the fluorescence infection area was significantly smaller than that of the control (CK). Figure 9 ) and day 11 (see appendix) Figure 10 The fluorescence-stained area of ​​the Tis108-treated group was significantly smaller than that of the control group (CK). This indicates that the SL treatment promoted TMV staining of tobacco, while the Tis108 treatment inhibited TMV staining.

[0065] Example 2

[0066] 1. RNA-seq sequencing

[0067] In this invention, 24 hours after inoculation with the TMV-GFP strain, tobacco samples treated with SL control solution and inoculated with TMV control solution are referred to as CK; samples sprayed with SL and inoculated with TMV control solution are referred to as SL; samples sprayed with SL control solution and then inoculated with TMV-GFP are referred to as TMV; and samples sprayed with SL first and then inoculated with TMV-GFP are referred to as SL-TMV.

[0068] Samples above the inoculated leaf were quickly removed, flash-frozen in liquid nitrogen, and then transferred to dry ice before being sent to the company (EasyBio) for sequencing. This analysis completed reference transcriptome sequencing of 12 samples, yielding a total of 81.22 G of Clean Data. The effective data volume for each sample ranged from 6.13 to 7.37 G, the Q30 base distribution ranged from 95.2% to 95.99%, and the average GC content was 43.90%. Using known reference gene sequences and annotation files as a database, sequence similarity alignment was used to identify the expression abundance of each protein-coding gene in each sample. The htseq-count software was used to obtain the number of reads aligned to protein-coding genes in each sample. By aligning the reads to the reference genome, the genome alignment results for each sample were obtained, with an alignment rate of 97.19%–98.0%. Based on the alignment results, protein-coding gene expression analysis was performed. Based on the expression levels of protein-coding genes in different samples, differential expression screening was performed, resulting in five differential expression groups. The number of differentially expressed genes detected in these groups were 964, 604, 1405, 979, and 2299, respectively (see appendix). Figure 11 ).

[0069] 2. RNA-seq data analysis

[0070] GO enrichment analysis:

[0071] After obtaining the differentially expressed genes, GO enrichment analysis was performed on them to describe their functions (combined with GO annotation results). The method for GO functional enrichment analysis was as follows: the number of differentially expressed genes included in each GO entry was counted, and the significance of differential gene enrichment in each GO entry was calculated using the hypergeometric distribution algorithm. The calculation results returned a p-value for enrichment significance (calculated using Fisher's exact test for each term in BP, CC, and MF); the lower the value, the more statistically significant it was. GO analysis was performed on TMV / CK, SL / CK, SL-TMV / TMV, and SL-TMV / SL respectively to obtain the entries enriched in the three modules of cellular_component, biological_process, and molecular_function (see appendix). Figure 12 ).

[0072] The results showed that the TMV / CK, SL / CK, SLTMV / TMV, and SLTMV / SL samples were enriched with the most cellular processes and metabolic processes.

[0073] KEGG Analysis:

[0074] KEGG is a major public database for pathway analysis. This study utilizes the KEGG database to perform pathway analysis on differentially expressed protein-coding genes (combined with KEGG annotation results), and uses the hypergeometric distribution test to calculate the significance of differential gene enrichment in each pathway entry. Using the database and analysis methods, the top 20 enriched pathways for TMV / CK, SL / CK, SL-TMV / TMV, and SL-TMV / SL were obtained (selected from pathways with more than 2 differentially expressed genes, sorted in descending order of their corresponding -log10 p-values). The bubble structure is shown in the attached image. Figure 13 .

[0075] Example 3

[0076] In this invention, after inoculating with the TMV-GFP strain, 24 hours later, tobacco samples treated with SL control solution and inoculated with TMV control solution are respectively called CK; samples sprayed with SL and inoculated with TMV control solution are called SL; samples sprayed with SL control solution and then inoculated with TMV-GFP are called TMV; samples sprayed with SL first and then inoculated with TMV-GFP are called SL-TMV; samples are quickly removed and placed in liquid nitrogen for flash freezing, and then RNA extraction is performed.

[0077] In this invention, total RNA extraction from tobacco was performed using the TRIzol (Invitrogen) extraction method. The leaves were placed in a mortar pre-cooled with liquid nitrogen and ground into an extremely fine powder. Approximately 100 mg of the powder was added to 1 ml of Trizol, shaken well, and incubated at room temperature for 10 minutes. The mixture was then incubated at 4°C with a concentration of 1.0 × 10⁻⁶. 4 Centrifuge at rpm for 10 min. Transfer the supernatant to another centrifuge tube, add 250 μl of chloroform, and shake vigorously 60 times; incubate at room temperature for 15 min until separation occurs; centrifuge at 4℃, 1.2 × 10⁻⁶. 4 Centrifuge at rpm for 15 min; transfer the supernatant to a centrifuge tube containing twice the volume of 95% ethanol, gently agitate several times, and incubate at -20℃ for at least 2 h; at 4℃, centrifuge at 1.2 × 10⁻⁶ rpm for 15 min. 4 Centrifuge at rpm for 15 min, discard the supernatant, rinse the RNA precipitate with 500 μl of 75% ethanol (prepared with DEPC water), centrifuge at 8000 rpm for 5 min at 4 °C, repeat this step once; discard the supernatant, remove the residual ethanol with a pipette tip, and blow dry the RNA precipitate on a clean bench (10 min). Finally, add 25 μl of DEPC water to each tube, dissolve in a 65 °C water bath for 10 min, and store at -70 °C.

[0078] The concentration of extracted total RNA from rice was determined, and approximately 5 μg was used for reverse transcription experiments. 1 μl of Dnase I (Invitrogen) and buffer were added, and the mixture was incubated at 37°C for 15 min to degrade any DNA possibly mixed with RNA. 1 μl of 150 ng / μl Oligo(dT)15 and 1 μl of 25 mM EDTA were added, and the mixture was incubated at 75°C for 10 min. The mixture was then placed on ice for 5 min. 2 μl of 10 mM dNTP, 1.5 μl of 0.1 M DTT, 4 μl of 5× M-MLV buffer, and 1 μl of M-MLV reverse transcriptase were added sequentially, and the mixture was incubated at 37°C for 1 h. Finally, the mixture was inactivated at 85°C for 10 min. The reverse transcription product was diluted to 200 μl with sterile double-distilled water and stored. 5 μl was used for PCR reactions. The Realtime-PCR reaction analysis kit used in this invention is [missing information]. The Green PCRMasterMix (Takara, Japan) was used, and the instrument was an ABI 7500 Real-Time PCR system (Applied Biosystems, USA). The internal reference gene used to measure expression levels and homogenize sample RNA content was the tobacco endogenous actin encoding gene (NtActin). Three technical replicates were performed for each sample, and the qPCR primers used are shown in Table 1. The detection results are attached. Figure 14 The results showed that the expression levels of 11 genes verified by qPCR were basically consistent with the trends observed by RNA-seq.

[0079] Table 1. Specific primers for qPCR verification of relevant genes and VIGS repression sites of NtCCD7 and NtCCD8.

[0080]

[0081]

[0082] Example 4

[0083] VIGS inhibits the expression of NtCCD7 and NtCCD8.

[0084] TRV2-NtCCD7 and TRV2-NtCCD8 vectors were constructed as follows: A 302 bp fragment of NtCCD7 (Niben101Scf00878g02006.1) and a 355 bp fragment of NtCCD8 (Niben101Scf01611g07010.1) were amplified using primers VIGSCCD7F and VIGSCCD7R, and VIGSCCD8F and VIGSCCD8R, respectively. The fragments were then ligated into the TRV2 vector, which had been digested with Xba1 and BamH1, using T4 ligase. Sequencing was confirmed to be correct. The plasmids were then transformed into Agrobacterium GV3101.

[0085] VIGS expression inhibition: TRV1 and TRV2-NtCCD7 / TRV2-NtCCD8 Agrobacterium clones were infected into LB cells and cultured overnight at 28°C in a shaker. TRV1 and TRV2-NtCCD7 / TRV2-NtCCD8 Agrobacterium were centrifuged at 5000 rpm for 1 min. The clones were resuspended in 1×PBS to their original volume. TRV1 and TRV2 were mixed in a 1:1 ratio, and leaf compression was used to infect the clones, followed by dark culture overnight. The expression levels of TRV2-NtCCD7 / TRV2-NtCCD8 were measured in infected leaves 48 h and later to verify the inhibitory effect. The control and VIGS groups were then inoculated with TMV-GFP and investigated using the method described in Example 1.

[0086] The results are attached. Figure 15 As shown, the expression of TRV2-NtCCD7 / TRV2-NtCCD8 was suppressed, and the fluorescence range of the suppressed plants was reduced after inoculation with TMV-GFP. This proves that suppressing the expression of TRV2-NtCCD7 / TRV2-NtCCD8 enhances the plant's resistance to TMV.

[0087] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0088] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of inhibiting or deleting key genes for strigolactone synthesis in improving tobacco resistance to TMV virus, characterized in that, The accession numbers for the key genes involved in the synthesis of styrax lactones are Niben101Scf00878g02006.1 or Niben101Scf01611g07010.

1.

2. The application of biomaterials that reduce the expression levels of key genes in strigolactone synthesis in improving tobacco resistance to TMV virus, characterized in that, The accession number for the key gene for the synthesis of unicornulide is Niben101Scf00878g02006.1 or Niben101Scf01611g07010.1; The biomaterial is any one of the following: Expression cassettes that cause the gene to be deleted or suppressed; An expression carrier containing the expression cassette described in A; Cloning vectors containing the expression cassette described in A; Engineered bacteria containing the expression cassette described in A and / or the expression vector described in B and / or the cloning vector described in C.

3. The application of styracil lactone inhibitors in enhancing tobacco's resistance to TMV virus, characterized in that, The unicornulide inhibitor is Tis108.

4. A method for improving tobacco's resistance to TMV virus, characterized in that, This can be achieved by applying an exogenous inhibitor of strigolactone or reducing the expression of key genes involved in strigolactone synthesis. The styrax lactone inhibitor is Tis108, and the accession number of the key gene for styrax lactone synthesis is Niben101Scf00878g02006.1 or Niben101Scf01611g07010.

1.

5. The method for improving tobacco's resistance to TMV virus according to claim 4, characterized in that, The preparation method of the unicorn lactone inhibitor Tis108 is to dissolve Tis108 in DMSO to obtain a mother liquor, then dilute the mother liquor with water and add Tween 20 to obtain a working solution. The reduction of expression levels of key genes for styrax lactone synthesis was achieved by inhibiting the expression of Niben101Scf00878g02006.1 or Niben101Scf01611g07010.1 using VIGS technology.

Images