Method for inserting GFP large protein into pCB301-ChiVMV and application of method in disease resistance identification of pepper vein mottle virus

Abstract

The invention relates to the field of disease resistance identification of pepper vein mottle viruses, and particularly discloses a method for inserting a GFP large protein into pCB301-ChiVMV and application of the method in disease resistance identification of the pepper vein mottle viruses, and a constructed fusion vector has the advantages that (1) the fusion vector has infectivity, and the infectivity of the ChiVMV on a host is not influenced after a GFP foreign protein sequence is introduced; and (2) the GFP protein can be stably expressed in nicotiana benthamiana and pepper. On the basis, the method can be used for inoculating a pepper material, and the resistance of the pepper to be detected can be evaluated by observing fluorescence and calculating the morbidity, disease index and resistance index. The efficient infection and expression of ChiVMV and GFP proteins in the pepper are realized, and the step of purifying the virus for identification is simplified; the resistance condition of a host to a single virus can be accurately identified; the method can accurately observe the morbidity of the pepper through green fluorescence, is suitable for batch development of pepper resistance identification, saves manpower and material resources, and improves the accuracy and reliability of resistance identification.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering and pepper disease resistance screening, in particular to a method for inserting a large GFP protein into pCB301-ChiVMV and its application in disease resistance identification of pepper vein mottle virus. Background Art

[0002] Chilli veinal mottle virus (ChiVMV) is a dominant virus that harms Solanaceae plants. It belongs to the Potyvirus family and the Potyvirus genus. Under natural conditions, it can infect a variety of crops in the Solanaceae family, such as pepper, tobacco, tomato, and eggplant. Currently, ChiVMV has become one of the main viruses that grow peppers, causing serious impacts on the product and quality of peppers, and even leading to crop failure.

[0003] According to the survey, ChiVMV is common in many pepper varieties in Sichuan, and the incidence of diseased peppers is as high as 100%. Since screening and using disease-resistant varieties is the most important means to prevent virus damage, given the large number of pepper varieties, it is necessary to establish an efficient identification method for pepper resistance to vein mottle virus, promote resistance screening of pepper varieties and materials, and then promote the application of pepper disease-resistant varieties in production.

[0004] It should be noted that the ChiVMV genome encodes a polyprotein that produces 11 mature proteins through self-cleavage or frameshifting, including P1, HC-Pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP. Virus infectious cloning is to fuse the viral genome into a plant binary expression vector to achieve viral replication and transmission in the host.

[0005] Therefore, by introducing the corresponding sequence during the construction of the viral infectious clone, once the sequence is introduced, even slight sequence changes or errors may cause the virus to lose or weaken its pathogenicity.

[0006] In patent application number 202311595457.7, we (the inventor team) successfully constructed an infectious clone of ChiVMV, and based on this, created a recombinant infectious clone pCB301-ChiVMV:NIb-Myc fused with the small protein Myc. This clone can stably express NIb-Myc fusion protein. This method takes advantage of the characteristics of the virus's own protein expression and uses the method of adding a tail to NIb to achieve the expression of Myc protein.

[0007] In terms of host resistance to virus identification, the current means of crop resistance to virus identification is usually to inoculate susceptible plants into intermediate hosts, such as ordinary tobacco, Sansheng tobacco and other plants for virus purification, but because ChiVMV often has multiple viruses in production, such as potato virus Y (PVY), tobacco mosaic virus (TMV), etc., and the hosts of these viruses have a great overlap with the hosts of ChiVMV, the conventional purification method cannot determine that only ChiVMV virus exists in the host, so it is necessary to construct an infectious clone of ChiVMV, and inoculate a single virus after replication in the host to improve the accuracy of identifying specific viruses. At the same time, after the conventional virus infectious clone is inoculated into the host plant, the virus is generally detected by methods such as RT-PCR, Dot-ELISA, and Western blot, but it is time-consuming and laborious, and if quantitative comparison is required, it is even more difficult and laborious. Therefore, it is urgent to obtain an efficient vector for identifying virus infection, skip the steps of nucleic acid and protein detection, and directly and quickly identify the host's resistance to the virus.

[0008] At present, there is no method for identifying the resistance of pepper varieties to ChiVMV. The previous literature report (Deng Wenqiao) identified the resistance of pepper to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) by using the corresponding infected tobacco new leaves as the medium, rubbing the peppers at the 6-leaf stage, and grading the individual plants according to the mosaic, necrotic spots, leaf deformity, wrinkling, and dwarfing of the new leaves, which were recorded as 0, 1, 3, 5, 7, and 9, respectively, and calculating the disease index to determine the resistance of the plants. Since ChiVMV usually produces similar symptoms such as leaf wrinkling, veining, and plant dwarfing after infection of peppers, it is difficult to quickly distinguish peppers from ChiVMV using the original grading method. Patent application No. 201311820244.X published a method and application for identifying tobacco resistance to pepper vein mottle virus, which also graded the tobacco into 6 levels according to yellow spots, leaf necrosis, mosaic, and plant dwarfing after virus infection, which is also not applicable to the identification of pepper resistance to ChiVMV.

[0009] Therefore, the inventor team hopes to establish an efficient identification method for pepper's resistance to pepper vein mottle virus. By constructing a ChiVMV infectious clone fused with green fluorescent protein GFP, while clarifying the single virus infection host, the ChiVMV infection of pepper can be clearly understood under handheld ultraviolet light observation. Through the distribution of leaf GFP fluorescence, a 4-level grading standard (immune, mild, obvious, severe) is established, and then the resistance of pepper to ChiVMV is judged by the disease index. The difficulties that may be encountered are: due to the small viral genome and different replication and coding strategies of its own proteins, it is very difficult to stably express the GFP large protein after it is introduced into the viral infectious clone (which is also a problem that plagues the industry).

[0010] Based on this, the inventor team introduced the GFP protein sequence between the NIb and CP proteins of ChiVMV, and stably expressed it in a certain way, thereby achieving the pathogenicity of the virus to the hosts Nicotiana benthamiana and pepper, and being able to stably express GFP protein in Nicotiana benthamiana and pepper, thereby providing a powerful tool for realizing the application of ChiVMV infectious cloning. Summary of the invention

[0011] The purpose of the present invention is to overcome the shortcomings of the prior art, provide a method for inserting a large GFP protein into pCB301-ChiVMV and an application of the method in identifying the disease resistance of pepper vein mottle virus, solve the problem that the large GFP protein cannot be stably expressed when inserted into pCB301-ChiVMV, and also solve the problem of how to quickly identify the resistance of pepper to pepper vein mottle virus. By fusing the GFP protein sequence into the ChiVMV infectious clone, a recombinant infectious clone pCB301-ChiVMV-GFP is constructed, and Agrobacterium-mediated inoculation confirms that the vector can systemically infect Nicotiana benthamiana and stably express green fluorescent protein GFP; using tobacco samples containing ChiVMV-GFP as a medium, after friction transfer of peppers, GFP fluorescence is stably expressed in new leaves of peppers, and by establishing a single-plant grading standard for inoculated peppers, the resistance of different pepper varieties to ChiVMV is distinguished.

[0012] The purpose of the present invention is achieved through the following technical solutions: In the first aspect, a method for inserting a large GFP protein into pCB301-ChiVMV is provided, comprising the following steps:

[0013] S1: Construction of the ChiVMV full genome infection clone pCB301-ChiVMV;

[0014] S2: Amplify and obtain a fragment containing GPF and a suitable conserved cleavage site of viral protein, specifically:

[0015] Using GFP-insert-1F / 3-GFP-insert-1R as primers and the plasmid containing the GFP fragment as a template, a high-fidelity DNA polymerase was used to amplify the fragment containing GFP;

[0016] Using 3-GFP-insert-2F / GFP-insert-2R as primers and pCB301-ChiVMV plasmid as template, a high-fidelity DNA polymerase was used to amplify the sequence containing the appropriate conserved cleavage site of viral proteins;

[0017] Among them, the nucleotide sequences of GFP-insert-1F / 3-GFP-insert-1R and 3-GFP-insert-2F / GFP-insert-2R are shown in SEQ ID: No.1-4;

[0018] S3: constructing a recombinant infectious clone pCB301-ChiVMV-GFP containing the ChiVMV genome fused with the GFP fragment, and adding a nucleotide sequence containing Pos I; wherein the nucleotide sequence of Pos I is shown in SEQ ID: No. 5;

[0019] Specifically, two specific enzymes, BamHI and SmaI, are used to perform double enzyme digestion on the infectious clone of pCB301-ChiVMV to obtain the double enzyme digestion product; the double enzyme digestion product and the GFP-containing fragment obtained in S2 and the sequence containing the appropriate conserved cleavage site of the viral protein are gel-recovered and purified, and then transformed into Escherichia coli after a homologous recombination reaction, and then after positive clone screening and sequencing verification, a recombinant infectious clone pCB301-ChiVMV-GFP having the complete nucleotide sequence of the ChiVMV genome, a fused GFP fragment, and an inserted sequence of the appropriate conserved cleavage site of the viral protein is obtained, that is, the GFP fragment is inserted between the NIb protein and the CP protein in pCB301-ChiVMV.

[0020] Furthermore, a method for inserting a large GFP protein into pCB301-ChiVMV was used to identify disease resistance to pepper vein mottle virus.

[0021] In a third aspect, a method for inserting a large GFP protein into pCB301-ChiVMV is provided for use in identifying resistance to pepper vein mottle virus, comprising the following steps:

[0022] Step 1) inserting green fluorescent protein-GFP between the NIb protein and the CP protein in pCB301-ChiVMV using the method of claim 1, thereby constructing a pCB301-ChiVMV-GFP recombinant infectious clone;

[0023] Step 2) introducing the pCB301-ChiVMV-GFP plasmid into Agrobacterium, inoculating the host Nicotiana benthamiana with the Agrobacterium to infect the Nicotiana benthamiana, and allowing the Nicotiana benthamiana to stably express green fluorescent protein after infection;

[0024] Step 3) raising seedlings of the pepper material to be tested and the susceptible control material, and inoculating them when the cotyledons are fully expanded and the first pair of true leaves are just born, using Nicotiana benthamiana that produces green fluorescence as a medium, and frictionally transferring them to the cotyledons of the peppers. After inoculation, they are cultured at 25-28° C. with a photoperiod of 16:8 h. On the 21st day after inoculation, the virus infection of the peppers is observed by handheld fluorescence to investigate the disease level of individual plants;

[0025] Step 4) Calculate the morbidity and disease index according to the following formula;

[0026] Incidence rate = (number of infected plants / total number of plants surveyed) × 100%

[0027] Disease index (DI) = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100

[0028] When the incidence of the infected control exceeds 80% and the disease index (DI) is greater than 50, the test results are valid. The evaluation criteria are as follows:

[0029] DI=0 means immunity, 0>DI≤15 means resistance; 15>DI≤50 means susceptible to disease; 50>DI means highly susceptible to disease.

[0030] Furthermore, in the step 2), the OD of Agrobacterium inoculation is 600 The value is 0.5-1.0.

[0031] Furthermore, in the step 3), around the 10th day after injection inoculation of Nicotiana benthamiana, the expression of green fluorescence in the new systemic leaves of Nicotiana benthamiana was checked by a handheld fluorescent lamp, leaves producing green fluorescence were selected, and an inoculum was prepared at a concentration of 1 g / (5-10 mL), which was transferred to a pair of cotyledons of pepper by friction, and the disease status of individual pepper plants was observed by a handheld fluorescent lamp on the 21st day.

[0032] Furthermore, in the step 3), when the pepper cotyledons are fully expanded and the first pair of true leaves are just born, a small amount of 600-800 mesh quartz sand is sprayed on the two cotyledons, and the fingers wearing sterilized gloves are dipped in the grinding inoculation solution, and the bottom of the pepper cotyledons are dragged with one hand, and the other hand is used to gently wipe and rub in a fixed direction for 5 times, and no obvious scratches are produced on the surface of the pepper leaves.

[0033] Furthermore, in step 4), the grading standards of peppers are as follows:

[0034] The severity is graded according to the green fluorescence expression of leaves of individual plants:

[0035] Level 0: immune, no green fluorescence expression;

[0036] Level 1: Slight, pepper true leaves have sporadic green fluorescence, and the green fluorescence area accounts for less than 10% of the entire leaf area;

[0037] Level 2: Obvious, the green fluorescence area accounts for 10% to 50% of the entire leaf area;

[0038] Level 3: Severe, the green fluorescent area accounts for more than 50% of the entire leaf area.

[0039] For ease of understanding, the core design points of this solution are explained:

[0040] This scheme intends to insert the green fluorescent protein GFP into the genome of pepper vein mottle virus (to be precise, the pCB301-ChiVMV genome previously developed and designed by the inventor's team), so as to obtain the disease resistance of peppers through fluorescence observation; the problem is that since the green fluorescent protein GFP is a large protein, it cannot be stably expressed when inserted into pCB301-ChiVMV, which is also a problem in the current industry;

[0041] The reasons for the inability to express stably are: due to the small size of the viral genome, protein expression is affected by promoter activity, etc. If exogenous sequences are added artificially, the pressure to start protein expression will increase, especially after the introduction of large proteins, the limit of viral protein expression will be exceeded, making the virus unable to replicate and move in the host, thus losing its pathogenicity; the ChiVMV genome encodes a polyprotein, which forms 10 proteins through self-cutting. Although each protein is closely connected, it performs its own unique function. If exogenous sequences are directly inserted, it will inevitably destroy the balance of viral protein replication and expression, affecting the function of the virus;

[0042] Therefore, in the present invention, by deeply analyzing the characteristics of protein cleavage expression, the specific gene sequence Pos I is artificially introduced to simulate the environment of protein cleavage, avoid the redundancy of non-protein gene sequences after cleavage, and cleverly use the original virus protein promoter to drive the expression of foreign proteins, so that the virus can stably express foreign proteins during the host replication and infection process, and the virus's own proteins and the exogenous introduced proteins do not affect each other;

[0043] Specifically, in the present invention: for pCB301-ChiVMV; a. using the genome-specific restriction site (BamHI and SmaI) fragment cutting and recombination method, convenient construction of recombinant clones is achieved, avoiding the problems of difficulty in large fragment recombination and vector construction; b. by designing specific primers containing Pos I, amplifying a fragment containing GFP and a sequence containing a suitable conservative cleavage site for viral proteins (the suitable cleavage site refers to a position between NIb and CP) through these primers; c. then the fragment containing GFP, the sequence containing a suitable conservative cleavage site for viral proteins, and the product of pCB301-ChiVMV digested with BamHI and SmaI in a are recombined to form pCB301-ChiVMV-GFP; d. in the pCB301-ChiVMV-GFP, due to the introduction of the specific gene sequence Pos I, the mutual influence of the replication of the virus's own protein and the exogenous introduced protein is avoided, and the infectivity of the pCB301-ChiVMV-GFP recombinant infectious clone is guaranteed.

[0044] It can be said that the stable systemic expression of exogenous GFP large protein and ChiVMV in the host has been achieved. When the host is infected with the virus, it can visually display obvious green fluorescence under ultraviolet light, and it continues to produce green fluorescence as the plant grows; the plants producing green fluorescence can be transferred to the original host pepper of ChiVMV by friction transfer, and can stably express green fluorescent protein; a disease grading system based on green fluorescence expression has been established, and the grading is clear and concise, avoiding the problem of confusion in disease grading and improving the accuracy of identification results; the pCB301-ChiVMV-GFP virus-infectious clone infects the host, avoiding the disadvantage that it is difficult to obtain a single virus by purifying the transferred host.

[0045] The present invention has the following advantages:

[0046] (1) In this scheme, the ChiVMV genome sequence obtained from infected peppers was used to insert the GFP sequence between the NIb and CP proteins in the ChiVMV infectious clone, making it highly infectious and stably expressing the GFP protein. The fusion vector pCB301-ChiVMV-GFP was constructed. The characteristics and advantages of pCB301-ChiVMV-GFP are as follows: a. It is infectious, and the introduction of the GFP exogenous protein sequence does not affect the infectiousness of ChiVMV to the host; b. It can stably express the GFP protein in Nicotiana benthamiana and pepper;

[0047] (2) Based on the advantages of a and b in (1), it can be used for inoculation of pepper materials. After 21 days of inoculation, the disease condition of pepper can be accurately observed by handheld fluorescent lamp. By calculating the disease incidence, disease index and resistance index, the resistance evaluation of the pepper to be tested can be achieved.

[0048] (3) Therefore, the vector designed in this scheme achieves efficient infection and expression of ChiVMV and GFP proteins in peppers, simplifies the steps of purifying the virus for identification, can accurately identify the host's resistance to a single virus, and can accurately observe the disease status of peppers through green fluorescence. It is suitable for batch resistance identification of peppers, saving manpower and material resources and improving the accuracy and reliability of resistance identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the construction of the recombinant infectious clone of pCB301-ChiVMV-GFP; A: Schematic diagram of the ChiVMV genome in pCB301-ChiVMV, with each color representing a virus-encoded protein; B: Schematic diagram of the insertion of exogenous GFP and other fragments, which are divided into Fragment I and Fragment II fragments, and the Pos I sequence indicated by the triangle in the two fragments is the same; C: The nucleotide sequence represented by PosI in B.

[0050] Figure 2 Phenotype and virus detection after pCB301-ChiVMV-GFP infection of Nicotiana benthamiana; A: Phenotypes of Nicotiana benthamiana in the control and pCB301-ChiVMV-GFP treated groups under white light and ultraviolet light; B: Bands of each sample amplified using ChiVMV virus-specific detection primers; C: Expression of GFP protein in each Nicotiana benthamiana sample was detected using GFP antibody.

[0051] Figure 3 Phenotype and virus detection of peppers transferred by friction with infected Nicotiana benthamiana samples; A: Phenotypes of peppers in the control and pCB301-ChiVMV-GFP treated groups under white light and ultraviolet light; B: Bands of each sample amplified using ChiVMV virus-specific detection primers; C: Expression of GFP protein in each pepper sample was detected using GFP antibody.

[0052] Figure 4 Disease grading chart for different pepper materials; Level 0, immune, no green fluorescence expression; Level 1: Mild, the pepper true leaves have sporadic green fluorescence, and the green fluorescence area accounts for less than 10% of the total leaf area; Level 2: Obvious, the green fluorescence area accounts for 10% to 50% of the total leaf area; Level 3: Severe, the green fluorescence area accounts for more than 50% of the total leaf area. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following description.

[0054] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In the first aspect, the present invention discloses a method for inserting a large GFP protein into pCB301-ChiVMV. The method constructs different ChiVMV-GFP recombinant infectious clones based on the whole genome infectious clone of ChiVMV according to the methods of direct insertion of foreign proteins, introduction of polyprotein cleavage sites, and introduction of different cleavage sites, and comprises the following steps:

[0056] S1: Construction of the ChiVMV full genome infectious clone pCB301-ChiVMV.

[0057] The whole genome infectious clone pCB301-ChiVMV of ChiVMV was constructed according to the patent method with application number 202311595457.7: the whole gene sequence of ChiVMV was obtained from pepper, and the cDNA of the pepper sample infected with ChiVMV was used as a template, and the bands containing the whole genome sequence of ChiVMV (Chi-1F / 1R, Chi-2F / 2R, and Chi-3F / 3R) were amplified in three sections using high-fidelity DNA polymerase. hi-2F / 2R, Chi-3F / 3R are the corresponding primers in application number 202311595457.7); then, StuI and SmaI were used to double-digest the linearized pCB301 vector, and the double-digested product and the three-segment amplified bands containing the ChiVMV full genome sequence were gel-recovered and purified, and transformed into Escherichia coli after homologous recombination reaction. After positive clone screening and sequencing verification, the clone pCB301-ChiVMV with the correct ChiVMV genome nucleotide sequence was obtained.

[0058] S2: Amplify and obtain a fragment containing GPF and a suitable conserved cleavage site of viral proteins.

[0059] Using GFP-insert-1F / 3-GFP-insert-1R as primers and the plasmid containing the GFP fragment as a template, a high-fidelity DNA polymerase was used to amplify the fragment containing GFP. Using 3-GFP-insert-2F / GFP-insert-2R as primers and the plasmid containing pCB301-ChiVMV as a template, a high-fidelity DNA polymerase was used to amplify the sequence containing the appropriate conserved cleavage site of the viral protein.

[0060] It should be noted that GFP-insert-1F / 3-GFP-insert-1R and 3-GFP-insert-2F / GFP-insert-2R are specially designed primers. These primers contain the nucleotide sequence of Pos I - as shown in SEQ ID: No.5, which is the core point of implementing this solution.

[0061] It should be noted that the plasmid containing the GFP fragment and the plasmid containing pCB301-ChiVMV are artificially screened and can be expressed in plants, such as peppers, and bacteria, such as Escherichia coli. The screening method and method of this plasmid is a very common means in biology.

[0062] S3: Construction of the recombinant infectious clone pCB301-ChiVMV-GFP in which the ChiVMV genome was fused with the GFP fragment. After recombination, a nucleotide sequence containing Pos I was added to the infectious clone pCB301-ChiVMV-GFP.

[0063] The pCB301-ChiVMV infectious clone obtained from S1 was double-digested by using two specific enzymes, BamHI and SmaI, to obtain a double-digested product; the double-digested product and the GFP-containing fragment obtained from S2 and a sequence containing a suitable conservative cleavage site for viral proteins were gel-recovered and purified, and then transformed into Escherichia coli after a homologous recombination reaction, and then after positive clone screening and sequencing verification, a recombinant infectious clone pCB301-ChiVMV-GFP having the complete nucleotide sequence of the ChiVMV genome, a fused GFP fragment, and an inserted suitable conservative cleavage site sequence for viral proteins was obtained, that is, the GFP fragment was inserted between the NIb protein and the CP protein in pCB301-ChiVMV.

[0064] In order to understand the method of inserting the above-mentioned large GFP protein into pCB301-ChiVMV, Example 1 is further illustrated below.

[0065] Example 1

[0066] This embodiment discloses a method for inserting a large GFP protein into pCB301-ChiVMV, which is performed by referring to the method of the first aspect described above.

[0067] (1) Figure 1 As shown in A, according to the composition of the ChiVMV genome, BamHI and SmaI were selected for insert fusion and construction on the pCB301-ChiVMV infectious cloning plasmid.

[0068] There are four build methods:

[0069] The first design: directly insert the GFP sequence at the C-terminus of NIb, design GFP-insert-1F / 1-GFP-insert-1R, 1-GFP-insert-2F / GFP-insert-2R primers (as shown in SEQ ID: No.1, 6, 7, 4), and obtain pCB301-ChiVMV-GFP1 by homologous recombination of the amplified fragment and the vector.

[0070] The second design: The 5 amino acids at the junction of NIb and CP were selected as the cleavage site, and GFP-insert-1F / 2-GFP-insert-1R and 2-GFP-insert-2F / GFP-insert-2R primers were designed (as shown in SEQ ID: No.1, 8, 9, 4), and the amplified fragment and the vector were subjected to homologous recombination to obtain pCB301-ChiVMV-GFP2.

[0071] The third design method: introduce the 8 amino acids at the junction of NIb and CP as the cleavage site as the protein cleavage sequence, design GFP-insert-1F / 3-GFP-insert-1R, 3-GFP-insert-2F / GFP-insert-2R primers (as shown in SEQ ID: No.1-4), and obtain pCB301-ChiVMV-GFP3 by homologous recombination of the amplified fragment and the vector.

[0072] The fourth design: The fourth method introduces the 12 amino acids at the junction of NIb and CP as the cleavage site as the protein cleavage sequence, designs GFP-insert-1F / 4-GFP-insert-1R, 4-GFP-insert-2F / GFP-insert-2R primers (as shown in SEQ ID: No.1, 10, 11, 4), and obtains pCB301-ChiVMV-GFP4 by homologous recombination of the amplified fragment and the vector.

[0073] The above primers were used as templates for plasmids containing GFP fragments and pCB301-ChiVMV plasmids, respectively, and amplified using high-fidelity DNA polymerase. The PCR amplification system was: 10μL 5×TransStart FastPfu Buffer, 4μL 2.5mMdNTPs, 1μL each of upstream and downstream primers (10μM), 1μL cDNA template, 1μL TransStart FastPfu DNAPolymerase, 32μL ddH2O, and the total reaction system was 50μL; the amplification reaction conditions were: 95℃3min; 95℃20s, 55℃30s, 72℃2min, 35 cycles; 72℃8min. The fragment containing GFP and protein conservative cleavage sites amplified was named Fragment I, and the fragment containing ChiVMV genome was named Fragment II ( Figure 1 B) The protease cleavage site in Fragment I and Fragment II recognizes the amino acid sequence and nucleotide sequence Pos I, such as Figure 1 As shown in C.

[0074] (2) BamHI and SmaI were used to double-digest the pCB301-ChiVMV infectious clone, and the product and the band described in step (1) were purified by gel recovery, and then the vector and the differently treated fragments were homologously recombined and connected. IIOne Step Cloning Kit (Nanjing Novogene Biotechnology Co., Ltd.) was used for recombination, and a 10 μL system reaction was used, including 2 μL of 5×CEⅡBuffer, 20 ng of the linearized cloning vector pCB301-ChiVMV cut with StuI and SmaI, 50 ng each of Insert1 and Insert2, 1 μL of ExnaseⅡ, and ddH2O to make up the system to 10 μL; the recombination reaction conditions were 37°C and 30 min. The ligation product was transferred into Escherichia coli, and the virus-specific detection primers CHi-F / R were used to screen positive clones. Two positive clones were screened for culture (cultured in a shaking incubator at 28°C and 220 rpm for 14 h), plasmids were extracted and sequenced, and the correct clones were verified to be inserted. They were named pCB301-ChiVMV-GFP1, pCB301-ChiVMV-GFP2, pCB301-ChiVMV-GFP3 and pCB301-ChiVMV-GFP4. 200 ng of plasmid was added to 100 μL EHA105 Agrobacterium competent cells respectively, and the plasmids were transferred into Agrobacterium EHA105 by electroporation to obtain Agrobacterium strains for plant infiltration pathogenicity testing.

[0075] In a second aspect, the present invention also discloses a method for inserting a large GFP protein into pCB301-ChiVMV and its application in identifying the resistance of pepper vein mottle virus, comprising the following steps:

[0076] Step 1) The green fluorescent protein-GFP is inserted between the NIb protein and the CP protein in pCB301-ChiVMV using the above-mentioned "a method for inserting a large GFP protein into pCB301-ChiVMV", thereby constructing a pCB301-ChiVMV-GFP recombinant infectious clone.

[0077] Step 2) The pCB301-ChiVMV-GFP plasmid is introduced into Agrobacterium, and the host Nicotiana benthamiana is inoculated with Agrobacterium to infect the host Nicotiana benthamiana, so that the Nicotiana benthamiana can stably express green fluorescent protein. 600 The value is 0.5-1.0.

[0078] Step 3) The pepper material to be tested and the susceptible control are grown as seedlings, and the cotyledons are fully expanded and the first pair of true leaves are just born. Nicotiana benthamiana that produces green fluorescence is used as a medium, and the mixture is rubbed into the cotyledons of the pepper. After inoculation, the pepper material is cultured at 25-28° C. with a photoperiod of 16:8 h. On the 21st day after inoculation, the virus infection of the pepper is observed by handheld fluorescence to investigate the disease level of individual plants.

[0079] It should be noted that around the 10th day after injection inoculation of Nicotiana benthamiana, the expression of green fluorescence in the new systemic leaves of Nicotiana benthamiana was checked by a handheld fluorescent lamp, and the leaves producing green fluorescence were selected, and the inoculation solution was prepared at a concentration of 1g / (5-10mL). The inoculation solution was transferred to a pair of cotyledons of pepper by friction, and the disease status of individual pepper plants was observed by a handheld fluorescent lamp on the 21st day.

[0080] It should also be noted that when the cotyledons of the pepper are fully expanded and the first pair of true leaves are just born, spray a small amount of 600-800 mesh quartz sand on the two cotyledons, dip the fingers wearing sterilized gloves into the grinding inoculation solution, drag the bottom of the pepper cotyledons with one hand, and gently rub them in a fixed direction with the other hand for 5 times. No obvious scratches will appear on the surface of the pepper leaves.

[0081] Step 4) Calculate the morbidity and disease index according to the following formula;

[0082] Incidence rate = (number of infected plants / total number of plants surveyed) × 100%

[0083] Disease index (DI) = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100

[0084] When the incidence of the infected control exceeds 80% and the disease index (DI) is greater than 50, the test results are valid. The evaluation criteria are as follows:

[0085] DI=0 means immunity, 0>DI≤15 means resistance; 15>DI≤50 means susceptible to disease; 50>DI means highly susceptible to disease.

[0086] It should be noted that in step 4), the grading standards for peppers are as follows:

[0087] The severity is graded according to the green fluorescence expression of leaves of individual plants:

[0088] Level 0: immune, no green fluorescence expression;

[0089] Level 1: Slight, pepper true leaves have sporadic green fluorescence, and the green fluorescence area accounts for less than 10% of the entire leaf area;

[0090] Level 2: Obvious, the green fluorescence area accounts for 10% to 50% of the entire leaf area;

[0091] Level 3: Severe, the green fluorescent area accounts for more than 50% of the entire leaf area.

[0092] In order to understand the application of the above-mentioned method of inserting a large GFP protein into pCB301-ChiVMV in the identification of disease resistance to pepper vein mottle virus, Example 2 is further illustrated below.

[0093] Example 2

[0094] Inoculation and infectivity assay of pCB301-ChiVMV-GFP infectious clones.

[0095] Single colonies of pCB301-ChiVMV-GFP1, 2, 3, and 4 (EHA105) transformed by electroporation were picked and inoculated into LB medium containing kanamycin (50 mg / L) and rifampicin (50 mg / L) resistance and cultured at 28°C with shaking at 240 rpm until OD 600 The bacterial cells were collected by centrifugation at 4000 rpm for 10 min, and resuspended in infiltration buffer (final concentration of 10 mM MgCl2, 10 mM MES (pH 5.6), 200 μM acetosyringone (Acs)) to adjust the bacterial solution concentration to OD 600 1.0; Place in dark place at room temperature for 2-3 hours. Use a 1mL syringe without a needle to infiltrate about 400μL of bacterial suspension into the back of the two upper leaves of 4-5 leaves of Nicotiana benthamiana. Treat 10 plants with each bacterial concentration. Place the plants in a growth room at 25℃ for cultivation with a photoperiod of 16:8h.

[0096] From the 6th day after inoculation, the plants were docked and observed, and the virus infection and GFP protein expression were checked by hand-held fluorescent lamp, and the observation was continued until the 12th day. Comparing the clones constructed by the 4 methods in Example 1, it was found that among the 10 inoculated Nicotiana benthamiana, only 9 of the pCB301-ChiVMV-GFP3 plants were sick on the 6th day; all the test seedlings inoculated with pCB301-ChiVMV-GFP3 were sick on the 8th day, while only half (5) of the test seedlings of pCB301-ChiVMV-GFP4 were sick; 7 of the test seedlings of pCB301-ChiVMV-GFP4 were sick on the 10th day, and the number did not increase after continued observation; the test seedlings treated with pCB301-ChiVMV-GFP1 and pCB301-ChiVMV-GFP2 did not get sick (Table 1). It shows that the third method in Example 1, that is, the introduction of the 8 amino acids at the junction of NIb and CP as the cleavage site as the protein cleavage sequence, is the best for maintaining the infectivity of the clone to the host. After the clone infected Nicotiana benthamiana, the leaves of Nicotiana benthamiana showed curling and shrinking symptoms of virus infection ( Figure 2 A), under ultraviolet light, the new systemic leaves can be seen to produce obvious green fluorescence ( Figure 2 A). The control sample showed no corresponding virus symptoms and fluorescence ( Figure 2 A). RT-PCR was used to test the samples of plants with virus infection symptoms and the control samples, indicating that the plants with symptoms were ChiVMV positive, while the control samples were ChiVMV negative ( Figure 2B) Total protein was extracted from the green fluorescent N. benthamiana leaves and the control leaves that did not produce green fluorescence, and analyzed by Western blot. Figure 2 As shown in C, the presence of GFP protein in leaves of Nicotiana benthamiana treated with the pCB301-ChiVMV-GFP vector was determined.

[0097] Table 1 Statistics of disease incidence after inoculation of different clones of Nicotiana benthamiana (10 strains)

[0098]

[0099] Example 3

[0100] Comparison of inoculation at different inoculum concentrations.

[0101] The pCB301-ChiVMV-GFP3 clone was used as the experimental object, and the concentration of the inoculated Agrobacterium solution was set to OD 600 The inoculation test and statistical observation of Nicotiana benthamiana were carried out with the values ​​of 0.1, 0.5, 1.0 and 1.5. On the 6th day of inoculation, OD 600 The incidence of the test seedlings was 75%-90% at 0.5-1.5, and all the test seedlings were sick on the 10th day. 600 When OD is 0.1 or 0.3, the number of cases is low in the early stage, and not all cases are diagnosed on the 10th day. 600 When the inoculation concentration was 1.5, some plants wilted on the 14th day due to the high inoculation concentration (Table 2). 600 An inoculation concentration of 0.5-1.0 is more conducive to the infection of the host by clones.

[0102] Table 2 Infection of Nicotiana benthamiana at various inoculation concentrations

[0103]

[0104] Example 4

[0105] The Nicotiana benthamiana leaves infected by the virus and expressing GFP in Example 2 were taken for friction transfer peppers, 0.5 g of the infected Nicotiana benthamiana leaves were taken, 5 mL of PBS buffer was added and ground into juice, and the juice was rubbed and inoculated into peppers. Since the green fluorescence expression of the true leaves of the peppers needs to be observed later, in order to avoid scratching the leaves and affecting the observation statistics, the present invention adopts the method of rubbing the pepper cotyledons, and the number of frictions and the effect are counted, a small amount of 600-800 mesh quartz sand is sprayed on the two cotyledons, and the fingers with sterilized gloves are dipped in the grinding inoculation liquid, one hand drags the bottom of the pepper cotyledons, and the other hand gently wipes and rubs in a fixed direction, five test groups are set, and they are rubbed 1 time, 3 times, 5 times, 7 times, and 10 times, respectively. 20 strains are treated in each test group, and the incidence of peppers is observed with a handheld fluorescent lamp at different stages after inoculation.

[0106] Observation of friction-inoculated peppers revealed slight shrinkage and veining symptoms of virus infection in the new leaves of the inoculated peppers ( Figure 3 A), under ultraviolet light, the new leaves of pepper can be seen to produce green fluorescence ( Figure 3 A). The control sample showed no corresponding virus symptoms and fluorescence ( Figure 3 A). RT-PCR testing of plants with virus infection symptoms and control samples showed that plants with symptoms were ChiVMV positive, while control samples were ChiVMV negative ( Figure 3 B) Total protein was extracted from the new system leaves of pepper that produced green fluorescence and the control leaves that did not produce green fluorescence, and analyzed by Western blot. Figure 3 C, the presence of GFP protein in the leaves of Nicotiana benthamiana treated with the pCB301-ChiVMV-GFP vector was determined. Statistics of pepper samples with different friction times showed that when the cotyledons were rubbed once and three times, the morbidity of the inoculated peppers on the 14th day was only 60% and 80%, while when the friction times were greater than or equal to 5 times, the morbidity of the peppers was 100% (Table 3), indicating that increasing the number of frictions can increase the morbidity of the inoculated peppers, but an increase in the number of frictions means an increase in the workload, so it is recommended that the number of frictions be selected as 5 times.

[0107] Table 3 Disease incidence of pepper samples at different friction times

[0108]

[0109]

[0110] Example 5

[0111] Identification of disease resistance of different pepper materials.

[0112] The method of rubbing five times in Example 4 was adopted, and screw pepper was used as the disease control. Spicy Hangzhou Pepper No. 1, Spicy Girl 8819 Line Pepper, Thin Pepper King, Extra Large Horn Pepper, Qiemen Big Sweet Pepper, Original Spicy Chaotian Pepper, Colorful Pepper and other pepper varieties were inoculated. The peppers were identified for disease resistance after the disease was stable on the 21st day after inoculation. Disease resistance was identified based on the expression of green fluorescence in the leaves of the inoculated peppers, and the disease level classification standard was established as follows:

[0113] The severity is graded according to the green fluorescence expression of leaves of individual plants:

[0114] Level 0: immune, no green fluorescence expression;

[0115] Level 1: Slight, pepper true leaves have sporadic green fluorescence, and the green fluorescence area accounts for less than 10% of the entire leaf area;

[0116] Level 2: Obvious, the green fluorescence area accounts for 10% to 50% of the entire leaf area;

[0117] Level 3: Severe, the green fluorescent area accounts for more than 50% of the entire leaf area.

[0118] The incidence rate and disease index were calculated according to the following formula;

[0119] Incidence rate = (number of infected plants / total number of plants surveyed) × 100%

[0120] Disease index (DI) = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100

[0121] When the incidence of the infected control exceeds 80% and the disease index (DI) is greater than 50, the test results are valid. The evaluation criteria are as follows:

[0122] DI=0 means immunity, 0>DI≤15 means resistance; 15>DI≤50 means susceptible to disease; 50>DI means highly susceptible to disease.

[0123] The test results show that the morbidity of the infected control screw pepper is 100%, and the morbidity of the tested pepper varieties is between 60% and 100%. The degree of disease in individual pepper plants also varies. Plants without disease show no green fluorescence expression ( Figure 4 A), some pepper plants had mild disease, with sporadic green fluorescence on the true leaves, accounting for less than 10% of the total leaf area ( Figure 4 B), some plants were obviously diseased, and the fluorescent area accounted for 10% to 50% of the entire leaf area ( Figure 4 C), some plants were seriously ill, with the green fluorescence area accounting for more than 50% of the entire leaf area ( Figure 4D) The disease index was calculated according to the survey results of each test group to evaluate the resistance of each pepper material to ChiVMV. The test was repeated twice. The results are shown in Table 4.

[0124] Table 4 Resistance of tested pepper varieties to ChiVMV

[0125]

[0126] The above embodiments only express preferred implementation modes, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A method for inserting a large GFP protein into pCB301-ChiVMV, characterized in that: The following steps are involved: S1: Construction of the ChiVMV full genome infection clone pCB301-ChiVMV; S2: Amplify and obtain a fragment containing GPF and a suitable conserved cleavage site of viral protein, specifically: Using GFP-insert-1F / 3-GFP-insert-1R as primers and the plasmid containing the GFP fragment as a template, a high-fidelity DNA polymerase was used to amplify the fragment containing GFP; Using 3-GFP-insert-2F / GFP-insert-2R as primers and pCB301-ChiVMV plasmid as template, a high-fidelity DNA polymerase was used to amplify the sequence containing the appropriate conserved cleavage site of viral proteins; Among them, the nucleotide sequences of GFP-insert-1F / 3-GFP-insert-1R and 3-GFP-insert-2F / GFP-insert-2R are shown in SEQ ID: No.1-4; S3: constructing a recombinant infectious clone pCB301-ChiVMV-GFP containing the ChiVMV genome fused with the GFP fragment, and adding a nucleotide sequence containing Pos I; wherein the nucleotide sequence of Pos I is shown in SEQ ID: No. 5; Specifically, two specific enzymes, BamHI and SmaI, are used to perform double enzyme digestion on the infectious clone of pCB301-ChiVMV to obtain the double enzyme digestion product; the double enzyme digestion product and the GFP-containing fragment obtained in S2 and the sequence containing the appropriate conserved cleavage site of the viral protein are gel-recovered and purified, and then transformed into Escherichia coli after a homologous recombination reaction, and then after positive clone screening and sequencing verification, a recombinant infectious clone pCB301-ChiVMV-GFP having the complete nucleotide sequence of the ChiVMV genome, a fused GFP fragment, and an inserted sequence of the appropriate conserved cleavage site of the viral protein is obtained, that is, the GFP fragment is inserted between the NIb protein and the CP protein in pCB301-ChiVMV.

2. The method for inserting a large GFP protein into pCB301-ChiVMV according to claim 1, characterized in that: Applied to the identification of resistance to pepper vein mottle virus.

3. Application of the method of inserting a large GFP protein into pCB301-ChiVMV as claimed in claim 1 or 2 in the identification of disease resistance of pepper vein mottle virus, characterized in that: The following steps are involved: Step 1) inserting green fluorescent protein-GFP between the NIb protein and the CP protein in pCB301-ChiVMV using the method of claim 1, thereby constructing a pCB301-ChiVMV-GFP recombinant infectious clone; Step 2) introducing the pCB301-ChiVMV-GFP plasmid into Agrobacterium, inoculating the host Nicotiana benthamiana with the Agrobacterium to infect the Nicotiana benthamiana, and allowing the Nicotiana benthamiana to stably express green fluorescent protein after infection; Step 3) raising seedlings of the pepper material to be tested and the susceptible control material, and inoculating them when the cotyledons are fully expanded and the first pair of true leaves are just born, using Nicotiana benthamiana that produces green fluorescence as a medium, and frictionally transferring them to the cotyledons of the peppers. After inoculation, they are cultured at 25-28° C. with a photoperiod of 16:8 h. On the 21st day after inoculation, the virus infection of the peppers is observed by handheld fluorescence to investigate the disease level of individual plants; Step 4) Calculate the morbidity and disease index according to the following formula; Incidence rate = (number of infected plants / total number of plants surveyed) × 100% Disease index (DI) = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100 When the incidence of the infected control exceeds 80% and the disease index (DI) is greater than 50, the test results are valid. The evaluation criteria are as follows: DI=0 means immunity, 0>DI≤15 means resistance; 15>DI≤50 means susceptible to disease; 50>DI means highly susceptible to disease.

4. Application of the method of inserting a large GFP protein into pCB301-ChiVMV according to claim 3 in the identification of disease resistance of pepper vein mottle virus, characterized in that: In the step 2), the OD of Agrobacterium inoculation is 600 The value is 0.5-1.

0.

5. Application of the method of inserting a large GFP protein into pCB301-ChiVMV according to claim 4 in the identification of disease resistance to pepper vein mottle virus, characterized in that: In the step 3), around the 10th day after injection inoculation of Nicotiana benthamiana, the expression of green fluorescence in the new systemic leaves of Nicotiana benthamiana was checked by a handheld fluorescent lamp, leaves producing green fluorescence were selected, and an inoculum was prepared at a concentration of 1 g / (5-10 mL), which was transferred to a pair of cotyledons of pepper by friction, and the disease condition of a single pepper plant was observed by a handheld fluorescent lamp on the 21st day.

6. Application of the method of inserting a large GFP protein into pCB301-ChiVMV according to claim 5 in the identification of disease resistance to pepper vein mottle virus, characterized in that: In the step 3), When the pepper cotyledons are fully expanded and the first pair of true leaves are just born, spray a small amount of 600-800 mesh quartz sand on the two cotyledons, dip the fingers wearing sterilized gloves into the grinding inoculation solution, drag the bottom of the pepper cotyledons with one hand, and gently rub in a fixed direction with the other hand for 5 times. No obvious scratches will be produced on the surface of the pepper leaves.

7. Application of the method of inserting a large GFP protein into pCB301-ChiVMV according to claim 6 in the identification of disease resistance to pepper vein mottle virus, characterized in that: In the step 4), the grading standards of peppers are as follows: The severity is graded according to the green fluorescence expression of leaves of individual plants: Level 0: immune, no green fluorescence expression; Level 1: Slight, pepper true leaves have sporadic green fluorescence, and the green fluorescence area accounts for less than 10% of the entire leaf area; Level 2: Obvious, the green fluorescence area occupies 10% to 50% of the entire leaf area; Level 3: Severe, the green fluorescent area accounts for more than 50% of the entire leaf area.

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