Infectious clone of Pinot Grigio grape virus variant and its construction method and application

Abstract

The present invention discloses an infectious clone of a Pinot Gris grape virus variant, a construction method and an application thereof. The infectious clone of the Pinot Gris grape virus variant is the infectious clones GP6 and GP18 of the Pinot Gris grape virus; the infectious clones GP6 and GP18 of the Pinot Gris grape virus are plant expression vectors containing the full-length cDNA sequence of the Pinot Gris grape virus GP6 or GP18 genome shown in sequence 1 or sequence 2, respectively. Experiments have shown that the infectious clones GP6 and GP18 of the Pinot Gris grape virus constructed by the present invention can be stably and efficiently expressed in the Agrobacterium EHA105 strain, and can successfully infect western tobacco and grape hosts, and have obvious differences in pathogenicity on the hosts, which not only makes the research on GPGV easy to operate at the DNA level, but also provides a powerful tool and important support for the subsequent biological characteristics and pathogenicity research of GPGV.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an infectious clone of a Pinot Gris grape virus variant and a construction method and application thereof. Background Art

[0002] Pinot Gris grape virus (GPGV) is a member of the Betafiloviridae family ( Betaflexiviridae ) of the genus Ciliophore virus ( Trichovirus ) was first discovered on Pinot Grigio grapes in northern Italy in 2012 and is considered to be the causative agent of grapevine leaf mottling and deformation (GLMD). GPGV can cause mottled, wrinkled and deformed grape leaves, reduced grape bunches, reduced berry weight, delayed ripening and increased acidity. Currently, GPGV has been reported in 58 countries around the world. In 2016, the virus was first reported in my country, and it was preliminarily shown that GPGV was associated with the occurrence of grape chlorotic mosaic symptoms, and that Beda grapes, which are often used as rootstocks, were more sensitive to GPGV. In 2018, 195 grape samples from 15 provinces, autonomous regions and municipalities in my country were tested, and the results showed that the GPGV detection rate was 28.2%. GPGV can be transmitted by grape gall mites, and the virus has herbaceous hosts in the field. Some researchers believe that Pinot Grigio grape virus is more harmful than grape fan leaf virus. As a grape virus that is widespread and causes serious harm, it is necessary to conduct in-depth research on its pathogenicity to provide a theoretical basis for its harmfulness and comprehensive prevention and control technology research. Conducting research on virus pathogenicity is crucial for the prevention and control of viral diseases in the future.

[0003] At present, the study of GPGV pathogenicity is a hot topic in grape virology research at home and abroad. GPGV is a +ssRNA virus with a genome length of about 7259 bp and consists of three overlapping open reading frames, encoding replicase (RP), movement protein (MP) and coat protein (CP), respectively. The research results of Saldarelli et al. showed that some GPGV isolates had SNP mutations at the 3' end of the MP gene, resulting in premature termination of the gene encoding the MP protein, and GPGV isolates encoding shorter MP protein genes were generally highly pathogenic and could cause obvious symptoms. However, the results of other researchers later did not fully support this view. When the inventors of this application conducted a genetic variation study on GPGV isolates in my country in 2018, they found that the GPGV isolate BJ-Cas1 had an MP termination codon UAA, encoding a shorter MP, but the sample did not show obvious symptoms. Therefore, the correspondence between the SNP mutations on the GPGV MP gene and the occurrence of severe symptoms needs to be further clarified.

[0004] As a technical means of reverse genetics research, infectious clones are convenient for studying the pathogenicity of viruses and the relationship between viruses and diseases at the DNA level. Therefore, constructing infectious clones of important viruses is very important in the study of plant virus pathogenicity. The construction of viral infectious clones is to insert the full-length cDNA fragment of the virus into the downstream of the 35S strong promoter of the cauliflower mosaic virus (CaMV), thereby initiating the transcription of viral cDNA in eukaryotic plants and obtaining infectious viral RNA. The use of infectious clones to inoculate virus-free plants can clarify the relationship between viruses and plant diseases. Previous studies have reported that the pathogenicity of GPGV may be related to the GPGV titer and the resistance of different varieties.

[0005] Although Giulia Tarquini et al. first constructed two GPGV infectious cDNA clones in 2019 (reference Agroinoculation of Grapevine Pinot Gris Virus into bacco and grapevineprovides insights on viral pathogenesis, PLoS One, 2019, Vol. 14, No. 3), the two GPGV isolates fvg-Is12 (accession MH087443) and fvg-Is15 (accession MH087446) used by Giulia Tarquini et al. to construct infectious clones have similar sequences, with a homology of 96.95%, and are located in the same phylogenetic branch, with a close relationship. Although fvg-Is12 and fvg-Is15 were amplified from symptomatic and asymptomatic grapes, respectively, the two infectious clones pRI::GPGV-vir and pRI::GPGV-lat produced similar symptoms after inoculation of grapes. In addition, Giulia Tarquini et al. did not obtain the complete GPGV genome sequence when constructing GPGV, which may have a certain impact on the true pathogenicity of GPGV. It can be seen that the GPGV infectious clones pRI::GPGV-vir and pRI::GPGV-lat constructed by Giulia Tarquini et al. do not have pathogenicity differentiation, and it is speculated that the SNP at the 3' end of the MP gene may not necessarily be related to the strength of pathogenicity. At present, no studies have reported the pathogenicity differences between GPGV variants with large homology differences and located in different evolutionary branches. Summary of the invention

[0006] The purpose of the present invention is to provide an infectious clone of a Pinot Gris grape virus variant with a large difference in pathogenicity, a construction method and an application thereof.

[0007] In order to achieve the above object, the present invention first provides an infectious full-length cDNA clone of Pinot Gris grape virus.

[0008] The infectious full-length cDNA clone of Pinot Gris virus provided by the present invention is a plant expression vector containing the full-length cDNA sequence of Pinot Gris virus genome; the full-length cDNA sequence of Pinot Gris virus genome is shown in sequence 2 or sequence 1 in the sequence table.

[0009] In the above-mentioned Pinot Gris virus infectious full-length cDNA clone, the plant expression vector further comprises a promoter, and the function of the promoter is to initiate the expression of the Pinot Gris virus genome full-length cDNA sequence.

[0010] In the above-mentioned Pinot Gris grape virus infectious full-length cDNA clone, the plant expression vector also includes a ribozyme (HDV-RZ) gene, and the function of the ribozyme gene is to cut the Pinot Gris grape virus genome at a suitable position when the Pinot Gris grape virus infectious full-length cDNA clone is expressed in the plant.

[0011] In one embodiment of the present invention, the plant expression vector is pCB301-2x35S-HDV RZ -NOS carrier.

[0012] In another embodiment of the present invention, the plant expression vector is a pCB301 vector.

[0013] In a preferred embodiment of the present invention, the infectious full-length cDNA clone of Pinot Gris virus is pCB301-2x35S-HDV containing the full-length cDNA sequence of the Pinot Gris virus genome shown in SEQ ID NO:1. RZ -NOS carrier.

[0014] In another preferred embodiment of the present invention, the infectious full-length cDNA clone of Pinot Gris virus is a pCB301 vector containing the full-length cDNA sequence of the Pinot Gris virus genome shown in SEQ ID NO:2.

[0015] In order to achieve the above object, the present invention further provides a Pinot Gris grape virus infectious full-length cDNA clone composition.

[0016] The infectious full-length cDNA clone composition of Pinot Gris grape virus provided by the present invention comprises the infectious full-length cDNA clone of Pinot Gris grape virus GP6 and the infectious full-length cDNA clone of Pinot Gris grape virus GP18;

[0017] The infectious full-length cDNA clone of Pinot Gris Grape Virus GP6 is a plant expression vector containing the full-length cDNA sequence of the Pinot Gris Grape Virus GP6 genome; the full-length cDNA sequence of the Pinot Gris Grape Virus GP6 genome is shown in Sequence 1 in the sequence list;

[0018] The infectious full-length cDNA clone of Pinot Gris virus GP18 is a plant expression vector containing the full-length cDNA sequence of the Pinot Gris virus GP18 genome; the full-length cDNA sequence of the Pinot Gris virus GP18 genome is shown in Sequence 2 in the sequence list.

[0019] In the above-mentioned Pinot Gris virus infectious full-length cDNA clone composition, the plant expression vector also includes a promoter, and the function of the promoter is to promote the expression of the Pinot Gris virus genome full-length cDNA sequence.

[0020] In the above-mentioned Pinot Gris grape virus infectious full-length cDNA clone composition, the plant expression vector also includes a ribozyme (HDV-RZ) gene, and the function of the ribozyme gene is to cut the Pinot Gris grape virus genome at a suitable position when the Pinot Gris grape virus infectious full-length cDNA clone is expressed in the plant.

[0021] In one embodiment of the present invention, the plant expression vector is pCB301-2x35S-HDV RZ -NOS carrier.

[0022] In another embodiment of the present invention, the plant expression vector is a pCB301 vector.

[0023] In a preferred embodiment of the present invention, the infectious full-length cDNA clone of Pinot Gris virus GP6 is pCB301-2x35S-HDV containing the full-length cDNA sequence of the Pinot Gris virus genome shown in SEQ ID NO:1. RZ -NOS carrier.

[0024] In another preferred embodiment of the present invention, the infectious full-length cDNA clone of Pinot Gris virus GP18 is a pCB301 vector containing the full-length cDNA sequence of the Pinot Gris virus genome shown in SEQ ID NO:2.

[0025] In order to achieve the above object, the present invention also provides a method for preparing the above Pinot Gris virus infectious full-length cDNA clone or the above Pinot Gris virus infectious full-length cDNA clone composition.

[0026] The infectious full-length cDNA clone of Pinot Gris virus provided by the present invention comprises the following steps: connecting the full-length cDNA sequence of the Pinot Gris virus genome to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the Pinot Gris virus genome; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris virus; the full-length cDNA sequence of the Pinot Gris virus genome is shown in sequence 2 or sequence 1 in the sequence table.

[0027] The method for preparing the above-mentioned Pinot Gris virus infectious full-length cDNA clone composition provided by the present invention comprises the following steps: preparing Pinot Gris virus GP6 infectious full-length cDNA clone and Pinot Gris virus GP18 infectious full-length cDNA clone;

[0028] The method for preparing the infectious full-length cDNA clone of Pinot Gris grape virus GP6 comprises the following steps: connecting the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris grape virus GP6; the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome is shown in sequence 1 in the sequence list;

[0029] The method for preparing the infectious full-length cDNA clone of Pinot Gris grape virus GP18 comprises the following steps: connecting the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris grape virus GP18; the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome is shown in sequence 2 in the sequence list.

[0030] In the above method, the full-length cDNA sequence of the Pinot Gris virus genome is connected to the plant expression vector by seamless cloning, which is to connect the full-length cDNA sequence of the Pinot Gris virus GP6 genome to the plant expression vector by seamless cloning or to connect the full-length cDNA sequence of the Pinot Gris virus GP18 genome to the plant expression vector by seamless cloning.

[0031] In one embodiment of the present invention, the method for connecting the full-length cDNA sequence of the Pinot Gris virus GP6 genome to a plant expression vector by seamless cloning comprises the following steps: using the cDNA of the Pinot Gris virus GP6 as a template, obtaining the full-length cDNA sequence of the Pinot Gris virus GP6 genome by long-fragment one-step amplification, and then connecting the full-length cDNA sequence of the Pinot Gris virus GP6 genome to a plant expression vector by a seamless cloning kit.

[0032] In a preferred embodiment of the present invention, the method of connecting the full-length cDNA sequence of the Pinot Gris GP6 genome to a plant expression vector by seamless cloning comprises the following steps:

[0033] 1) Using the cDNA of Pinot Gris virus GP6 as a template, PCR amplification was performed using the DNA molecules shown in SEQ ID NO: 3 and SEQ ID NO: 4 to obtain the full-length genome sequence of GP6;

[0034] 2) pCB301-2x35S-HDV RZ -NOS vector was used as a template, and the DNA molecule shown in sequence 6 and the DNA molecule shown in sequence 8 were used for linear amplification to obtain a linearized vector with a size of 4623 bp;

[0035] 3) Using a seamless cloning kit, the linearized vector with a size of 4623 bp was connected to the full-length sequence of the GP6 genome to obtain the recombinant expression vector.

[0036] In another embodiment of the present invention, the method for connecting the full-length cDNA sequence of the Pinot Gris virus GP18 genome to a plant expression vector by seamless cloning comprises the following steps: using the cDNA of the Pinot Gris virus GP18 as a template, obtaining the full-length cDNA sequence of the Pinot Gris virus GP18 genome by long-fragment one-step amplification, and then connecting the full-length cDNA sequence of the Pinot Gris virus GP18 genome to a plant expression vector by a seamless cloning kit.

[0037] In another preferred embodiment of the present invention, the method of connecting the full-length cDNA sequence of the Pinot Gris virus GP18 genome to a plant expression vector by seamless cloning may include the following steps:

[0038] 1) Using the cDNA of Pinot Gris grape virus GP18 as a template, PCR amplification was performed using the DNA molecules shown in SEQ ID NO: 3 and SEQ ID NO: 5 to obtain the full-length genome sequence of GP18;

[0039] 2) pCB301-2x35S-HDV RZ-NOS vector was used as a template, and the DNA molecule shown in sequence 7 and the DNA molecule shown in sequence 8 were used for linear amplification to obtain a linearized vector with a size of 4178 bp;

[0040] 3) Using a seamless cloning kit, the linearized vector with a size of 4178 bp was connected to the full-length sequence of the GP18 genome to obtain the recombinant expression vector.

[0041] The Pinot Gris virus infectious full-length cDNA clone prepared according to the above method or the Pinot Gris virus infectious full-length cDNA clone composition prepared according to the above method also belongs to the protection scope of the present invention.

[0042] In order to achieve the above-mentioned object, the present invention also provides a recombinant bacterium or a recombinant bacterium composition;

[0043] The recombinant bacteria are recombinant bacteria containing the above-mentioned Pinot Gris virus infectious full-length cDNA clone, specifically, recombinant bacteria containing the above-mentioned Pinot Gris virus GP6 infectious full-length cDNA clone or recombinant bacteria containing the above-mentioned Pinot Gris virus GP18 infectious full-length cDNA clone.

[0044] The recombinant bacterial composition includes a recombinant bacterium containing the above-mentioned Pinot Gris grape virus GP6 infectious full-length cDNA clone and a recombinant bacterium containing the above-mentioned Pinot Gris grape virus GP18 infectious full-length cDNA clone, specifically consisting of a recombinant bacterium containing the above-mentioned Pinot Gris grape virus GP6 infectious full-length cDNA clone and a recombinant bacterium containing the above-mentioned Pinot Gris grape virus GP18 infectious full-length cDNA clone.

[0045] In one embodiment of the present invention, the recombinant bacteria containing the infectious full-length cDNA clone of Pinot Gris virus GP6 is Agrobacterium EHA105 containing the infectious full-length cDNA clone of Pinot Gris virus GP6.

[0046] In another embodiment of the present invention, the recombinant bacteria containing the infectious full-length cDNA clone of Pinot Gris virus GP18 is Agrobacterium EHA105 containing the infectious full-length cDNA clone of Pinot Gris virus GP18.

[0047] In order to achieve the above-mentioned purpose, the present invention also provides new uses of the above-mentioned Pinot Gris virus infectious clone or the above-mentioned Pinot Gris virus infectious clone composition, or the Pinot Gris virus infectious full-length cDNA clone prepared according to the above-mentioned method, or the Pinot Gris virus infectious full-length cDNA clone composition prepared according to the above-mentioned method, or the above-mentioned recombinant bacteria or recombinant bacteria composition.

[0048] The present invention provides the use of the above-mentioned Pinot Gris virus infectious clone or the above-mentioned Pinot Gris virus infectious clone composition or the Pinot Gris virus infectious full-length cDNA clone prepared according to the above-mentioned method or the Pinot Gris virus infectious full-length cDNA clone composition prepared according to the above-mentioned method or the above-mentioned recombinant bacteria or the recombinant bacteria composition in any one of the following R1)-R8):

[0049] R1) Prepare products for constructing single virus infection plant models;

[0050] R2) Construct a single virus infection plant model;

[0051] R3) Prepare products for constructing infected plant models with different or significant pathogenicity;

[0052] R4) Constructing plant infection models with different or significant pathogenicity;

[0053] R5) Preparation of products for studying the biological characteristics and / or pathogenicity and / or pathogenesis of Pinot Grigio virus;

[0054] R6) Study the biological characteristics and / or pathogenicity and / or pathogenesis of Pinot Grigio virus;

[0055] R7) Preparation of products for studying the interaction between Pinot Gris virus and host plants;

[0056] R8) Study the interaction between Pinot Gris virus and host plants;

[0057] The single virus is the Pinot Gris virus.

[0058] The pathogenicity is different or significantly different as shown in the following T1) or T2):

[0059] T1) GP6 infectious clones showed mild chlorosis and mottled symptoms on tobacco leaves, while GP18 infectious clones showed necrosis symptoms on tobacco leaves;

[0060] T2) After the GP6 infectious clone infects the grapes, the leaves show mild chlorosis and mottled symptoms, while the GP18 infectious clone infects the grapes, resulting in dwarf plants, terminal bud dieback, and severe chlorosis and mottled symptoms on the leaves.

[0061] Any of the plants or host plants described above is tobacco (such as Nicotiana tabacum) or grape (such as Vitis vinifera).

[0062] The complete genome sequence of the Pinot Gris virus shown in Sequence 1 or Sequence 2 and the primer pair used to amplify the complete genome sequence of the Pinot Gris virus shown in Sequence 1 or Sequence 2 also fall within the protection scope of the present invention.

[0063] The primer pair used to amplify the full genome sequence of the Pinot Gris virus shown in the above sequence 1 consists of the DNA molecule shown in sequence 3 and the DNA molecule shown in sequence 4.

[0064] The primer pair used to amplify the full genome sequence of the Pinot Gris virus shown in the above sequence 2 consists of the DNA molecule shown in sequence 3 and the DNA molecule shown in sequence 5.

[0065] In order to achieve the above objectives, the present invention finally provides the full genome sequence of the Pinot Gris virus shown in the above sequence 1 or sequence 2 and the use of the primer pair for amplifying the full genome sequence of the Pinot Gris virus shown in the above sequence 1 or sequence 2 in any one of the following S1)-S8):

[0066] S1) preparing products for the detection and / or identification of Pinot Grigio viruses;

[0067] S2) Detection and / or identification of viruses in Pinot Grigio grapes;

[0068] S3) preparing products for detecting or assisting in detecting whether the virus to be detected is Pinot Gris virus;

[0069] S4) Detecting or assisting in detecting whether the virus to be tested is Pinot Gris virus;

[0070] S5) preparing products for detecting or assisting in detecting whether a sample is infected with or carries Pinot Gris virus;

[0071] S6) Detecting or assisting in detecting whether the sample to be tested is infected with or carries Pinot Gris virus;

[0072] S7) Preparation of products for evolutionary analysis and / or molecular epidemiological studies of Pinot Grigio viruses;

[0073] S8) Evolutionary analysis and / or molecular epidemiology of Pinot Grigio viruses.

[0074] Beneficial effects of the present invention: The present invention obtains accurate full-length genome sequences of two GPGV variants GP6 and GP18 with large sequence differences through PCR and RACE amplification. Through comparison, it is found that the homology between them is low, only 82.89%, and they are located on different evolutionary branches. More importantly, the present invention uses direct long fragments to amplify the whole genome and seamlessly clone it into an infectious cloning vector, successfully constructs infectious clones of GP6 and GP18, and proves that GP6 and GP18 have obvious differences in pathogenicity by inoculating western tobacco and grapes respectively. The present invention discloses for the first time the full-length cDNA sequences of the genomes of Pinot Gris grape virus GP6 and GP18, and uses infectious clone inoculation to prove that different GPGV variants GP6 and GP18 have large differences in pathogenicity, which is of great significance for the study of the biological characteristics, pathogenicity, pathogenicity mechanism, hazard laws and comprehensive prevention and control technologies of Pinot Gris grape virus.

[0075] In order to study the relationship between different variants of GPGV and the occurrence of symptoms, the present invention obtains the full-length genome sequences of two GPGV isolates GP6 and GP18 with large variations by PCR amplification. At the same time, in order to further study the biological characteristics and pathogenicity of the two differential variants, the infectious full-length cDNA clones of GPGV isolates GP6 and GP18 are quickly obtained by artificially constructing the infectious cloning method of the virus. The infectious full-length cDNA clones are recombinant expression vectors obtained by connecting the full-length genome fragments of two Pinot Gris grape virus variants GP16 and GP18 to a binary vector containing a 35S promoter through seamless cloning. Next, the recombinant expression vector is transferred into Agrobacterium EHA105 competent cells by freeze-thaw method, and its infectivity is verified by inoculating western tobacco and grapes with Agrobacterium, and finally the infectious clones of two GPGV variants GP6 and GP18 with large sequence differences are successfully obtained. Experiments have shown that the GPGV infectious clones GP6 and GP18 constructed in the present invention can be stably and efficiently expressed in the Agrobacterium EHA105 strain, and can successfully infect western tobacco and grape hosts, and have obvious differences in pathogenicity on the hosts. This not only makes the study of GPGV easy to operate at the DNA level, but also provides a powerful tool and important support for the subsequent research on the biological characteristics and pathogenicity of GPGV. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Amplification of the full-length genome sequences of GPGV isolates GP6 and GP18. 1: GP6; 2: GP18.

[0077] Figure 2 Symptoms of inoculation of western tobacco with GP6 (left) and GP18 (right) infectious clones.

[0078] Figure 3PCR verification of the infectious clone of GPGV isolate GP6 inoculated into tobacco.

[0079] Figure 4 PCR verification of the infectious clone of GPGV isolate GP18 inoculated into tobacco.

[0080] Figure 5 Electron microscopic observation of GPGV particles in symptomatic western tobacco after GP6 and GP18 inoculation.

[0081] Figure 6 Symptoms of Vitis vinifera inoculated with the GP6 and GP18 infectious clones.

[0082] Figure 7 PCR verification of the infectious clone of GPGV isolate GP6 inoculated with grapes.

[0083] Figure 8 PCR verification of the infectious clone of GPGV isolate GP18 inoculated with grapes.

[0084] Fig. 9 Phylogenetic tree analysis of GP6 and GP18 and other GPGV full-length genome sequences. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0086] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0087] pCB301-2x35S-HDV in the following examples RZ -NOS vector contains 2×35S promoter and ribozyme (HDV-RZ), which is recorded in the document "Yao Min, Zhang Tianqi, Tian Zhichao, Wang Yuanchao, Tao Xiaorong; Agrobacterium-mediated CMV infectious cloning and 2b deletion mutant construction; Chinese Agricultural Science; 2011, 44 (14): 3060-3068". The public can obtain it from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0088] The pCB301 vector in the following examples is recorded in the document “Hongguang Cui and Aiming Wang. Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection, 2016, 90(10): 5119-5131”, which is available to the public from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0089] Example 1. Obtaining the full-length genome sequences of GPGV isolates GP6 and GP18

[0090] 1. Sources of GPGV isolates GP6 and GP18

[0091] GPGV isolate GP6 was derived from leaves of Beda grape plants showing slight chlorotic spots and identified as infected with GPGV, while GPGV isolate GP18 was derived from leaves of Cabernet Gernischt grape plants showing chlorotic necrotic spots and identified as infected with GPGV. Both sample pieces were stored in the toxin source preservation nursery of the Fruit Research Institute of the Chinese Academy of Agricultural Sciences.

[0092] 2. Total RNA Extraction and Reverse Transcription

[0093] 1. Total RNA Extraction

[0094] Take about 50 mg of the grape leaf samples in step 1, extract total RNA using the improved adsorption column method, and store at -80°C.

[0095] 2. Reverse transcription

[0096] The total RNA obtained in step 1 was used as a template for reverse transcription to obtain cDNA. The specific steps of reverse transcription are as follows: in a 1.5 mL sterile centrifuge tube, 7.0 µL of sterile pure water, 1.0 µL of oligodT18 (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.), and 2.0 µL of total RNA were added in sequence, centrifuged and mixed, incubated at 95°C for 5 min, and placed on ice for 2 min; 5.0 µL of 5×M-MLV buffer, 1.5 µL of 10 mmol / L dNTPs, 0.8 µL of 200 U / mL M-MLV reverse transcriptase, and 2.7 µL of sterile pure water were added, and the mixture was incubated at 37°C for 10 min, 42°C for 50 min, and 70°C for 5 min; the synthesized cDNA was immediately subjected to PCR amplification or stored in a -20°C refrigerator.

[0097] 3. Segmental amplification of the full-length sequence of GP6 and GP18 genomes and terminal sequence amplification

[0098] 1. Segmental amplification of the full-length sequence of GP6 genome and terminal sequence amplification

[0099] The high-fidelity amplification kit of NEB (Beijing Adlai Biotechnology Co., Ltd.) was used to amplify the full-length sequence of the GP6 genome using the cDNA obtained in step 2 as a template and four pairs of primers (GP6-1F / GP6-1R, GP6-2F / GP6-2R, GP6-3F / GP6-3R, GP6-4F / GP6-4R) to obtain amplified products. The primer sequences are shown in Table 1.

[0100] Table 1. Primers for amplifying the full-length genome sequences of GPGV isolates GP6 and GP18

[0101]

[0102] PCR reaction system (25 μL): 5.0 μL 5×Phution buffer, 1 μL forward and reverse primer mixture (10 μM each), 0.5 μL dNTPs (10 mM), 0.3 μL Phusion DNA polymerase (2 U / μL), 2 μL cDNA first-strand synthesis product and 15.2 μL ddH 2 O.

[0103] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 40 s, annealing at 60°C for 40 s, extension at 72°C for 1 min30 s, 35 cycles; final reaction at 72°C for 10 min.

[0104] The 5' and 3' end sequences of the GP6 genome were determined using the SMARTer® RACE 5′ / 3′Kit (TaKaRa) of Takara Biotech. All amplified products were purified and recovered using an agarose recovery kit (Beijing Adelaide Biotechnology Co., Ltd.) and ligated to the pTOPO-Blunt vector. The positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The determined sequences were aligned and spliced ​​to obtain the full-length genome sequence of the GPGV isolate GP6. The full-length genome sequence of the GPGV isolate GP6 is shown in Sequence 1 in the sequence table.

[0105] 2. Segmental amplification of the full-length sequence of GP18 genome and amplification of the terminal sequence

[0106] The high-fidelity amplification kit from NEB (Beijing Adlai Biotechnology Co., Ltd.) was used to amplify the full-length sequence of the GP18 genome using the cDNA obtained in step 2 as a template and three pairs of primers (GP18-1F / GP18-1R, GP18-2F / GP18-2R, GP18-3F / GP18-3R). The primer sequences are shown in Table 1. The PCR reaction system and PCR reaction procedure were the same as in step 1.

[0107] The 5' and 3' end sequences of the GP18 genome were determined using the SMARTer® RACE 5′ / 3′ Kit (TaKaRa) of Takara Biotech. All amplified products were purified and recovered using an agarose recovery kit (Beijing Adelaide Biotechnology Co., Ltd.) and ligated to the pTOPO-Blunt vector. Positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The determined sequences were aligned and spliced ​​to obtain the full-length genome sequence of the GPGV isolate GP18. The full-length genome sequence of the GPGV isolate GP18 is shown in Sequence 2 in the sequence table.

[0108] Example 2: Construction of infectious cloning vectors for GPGV isolates GP6 and GP18

[0109] 1. Amplification of the full-length genome sequences of GP6 and GP18

[0110] Using the cDNA obtained in step 2 of Example 1 as a template, PCR amplification was performed using primers GPGVcommon_fwd / GP6_rev and GPGVcommon_fwd / GP18-rev (sequences overlapping with the vector were added to the ends of the primers, represented by lowercase letters) to obtain the full-length genome sequences of GP6 and GP18, respectively ( Figure 1 ). The primer sequences are shown in Table 2.

[0111] Table 2. Primers for constructing GP6 and GP18 infectious clones

[0112]

[0113] Note: “T (40) " represents the number of base T is 40, "T (40) ” represents the number of base T is 20.

[0114] The PCR reaction system (50 μL) was as follows: 25.0 μL 2 × Phanta Max Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL each of forward and reverse primers (10 μM), 2 μL cDNA first-strand synthesis product, and 21 μL ddHO. 2 O.

[0115] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 7 min30 s, 35 cycles; final reaction at 72°C for 10 min.

[0116] The amplified products were separated and recovered by 1% agarose gel electrophoresis and purified using a recovery kit.

[0117] 2. Vector Linearization

[0118] The pCB301-2x35S-RZ-NOS vector was linearized and amplified using PCB1_fwd and PCBcommon_rev to obtain a linearized vector with a size of 4623 bp. The linearized vector was used to connect with the amplified fragment of the full-length GP6 genome sequence.

[0119] The pCB301 vector was linearized and amplified using PCB2_fwd and PCBcommon_rev to obtain a linearized vector with a size of 4178 bp. The linearized vector was used to connect with the amplified fragment of the full-length sequence of the GP18 genome.

[0120] The amplified products were separated and recovered by 1% agarose gel electrophoresis and purified using a recovery kit.

[0121] 3. Connection of the full-length viral genome to the vector

[0122] The linearized vector was connected to the amplified fragments of the GP6 and GP18 full genome sequences at 50°C for 1 h using a seamless cloning kit (Adlai). After the connection was completed, the connection product was transformed into Escherichia coli DH5ɑ by heat shock method, spread on a plate containing kan antibiotics, and cultured at 37°C to screen the vector containing the full-length sequence of the GPGV genome.

[0123] 4. Identification of positive clones

[0124] The positive clone bacterial suspension was identified by PCR (PCR identification used the two pairs of primers in Table 3), and the plasmids identified as positive clones were sent to Bio-Tech Biotech (Shanghai) Engineering Co., Ltd. for KBseq sequencing. The sequencing results were analyzed to finally determine whether the vector construction was accurate.

[0125] Table 3. Primers for amplifying GPGV isolates GP6 and GP18

[0126]

[0127] The results showed that GP6 and GP18 could amplify the target fragments through PCR verification. Further plasmid sequencing showed that GP6 was inserted correctly without base deletion, while GP18 was missing a base T at base 229. The missing T of GP18 was supplemented by the point mutation kit Q5® Site-Directed Mutagenesis Kit (NEB, Beijing), and confirmed by sequencing, and finally the positive recombinant plasmids GP6 and GP18 were obtained.

[0128] The positive recombinant plasmid GP6 was pCB301-2x35S-HDV RZ -NOS carrier Stu I and Sma The fragment between the restriction sites was replaced with the full-length cDNA sequence of the GP6 genome shown in sequence 1, and pCB301-2x35S-HDV was maintained. RZ -The plasmid obtained after the other sequences of the NOS vector remain unchanged.

[0129] The positive recombinant plasmid GP18 is a plasmid obtained by replacing the fragment between the Stu I and Pst I restriction sites of the pCB301 vector with the full-length cDNA sequence of the GP18 genome shown in Sequence 2, while keeping the other sequences of the pCB301 vector unchanged.

[0130] 5. Transformation of positive clone plasmid into Agrobacterium EHA105

[0131] Take the competent Agrobacterium EHA105 stored at -80℃ and melt it on ice. Add 1μg plasmid (positive recombinant plasmid GP6 or GP18) to every 100μL competent state and mix well. Incubate on ice for 10 minutes, in liquid nitrogen for 5 minutes, at 28℃ for 5 minutes, and in ice bath for 5 minutes. Add 700μL LB liquid medium without antibiotics and shake culture at 28℃ for 2-3 hours. Centrifuge at 5000 rpm for 1 min to collect bacteria, take about 100μL of supernatant, gently blow and resuspend the bacteria block, and spread it on the LB plate containing 50μg / mL Km and 50μg / mL Rif, and invert it in a 28℃ incubator for 2-3 days. Using the colony as a template, the positive clone plasmid identified by PCR is sequenced by KBseq to confirm that the inserted sequence is correct, and finally obtain positive monoclonal GP6 and GP18.

[0132] Example 3: Inoculation of western tobacco with infectious clones of Pinot Gris virus and verification of its infectivity

[0133] 1. Inoculation and identification of infectious clones of Pinot Gris virus in tobacco

[0134] The positive monoclonal clones (positive monoclonal clone GP6, positive monoclonal clone GP18) were inoculated into LB liquid medium containing 50 μg / mL Km and 50 μg / mL Rif, and cultured on a shaker at 28°C and 200 rpm until the OD 600nm The concentration was about 1.0, and the mixture was centrifuged at 8000 rpm for 10 min to collect the precipitate. 2 , 10 mM MES, 100 μM acetosyringone 1 / 2MS medium to dissolve the precipitate and adjust the OD 600nm To 1.0, to obtain the inoculum. After standing for 4-5 hours, the inoculum was inoculated into the leaves of the 4-leaf tobacco seedlings through a syringe, so that the inoculum filled the entire leaf, and two leaves were inoculated for each tobacco plant. After inoculation, the tobacco seedlings were placed in the dark for 12 hours, and then cultured in an isolated greenhouse at 25°C. After inoculation, the disease was observed every day, and the inoculated tobacco was identified by PCR (PCR identification used the two pairs of primers in Table 3). Electron microscopy was used to determine that the infectious clones could form virus particles after inoculation.

[0135] 2. Infectivity Verification

[0136] It was observed that 9 days after the GP6 infectious clone constructed by Agrobacterium injection was inoculated into western tobacco, the systemic leaves of western tobacco showed slight chlorosis and mottled symptoms, while 9 days after the GP18 infectious clone constructed by Agrobacterium injection was inoculated into western tobacco, the systemic leaves of western tobacco showed obvious necrosis symptoms ( Figure 2 To further confirm whether the two GPGV variants of infectious clones were successfully infected, PCR identification was performed on 10 symptomatic western tobacco plants using the primers in Table 3. The results showed that positive amplification bands were obtained in all inoculated tobacco plants ( Figure 3 and Figure 4 ).

[0137] In order to verify whether GP6 and GP18 infectious clones form virus particles after infection, negative staining method was used to observe the symptomatic tobacco inoculated with GP6 under electron microscopy. The results showed that a small amount of GPGV virus particles could be observed in the symptomatic tobacco ( Figure 5 A). In order to more easily observe the virus particles after inoculation, the virus particles were extracted from the tobacco inoculated with GP18, and the extracted virus suspension was observed under an electron microscope. As a result, a large number of hair-like virus particles were observed ( Figure 5 B). The above experiments show that the GP6 and GP18 infectious clones constructed by the present invention have infectious activity on tobacco, and there is a large difference in the pathogenicity of the GP6 and GP18 infectious clones on tobacco. Compared with the GP6 infectious clone, the GP18 infectious clone has stronger pathogenicity and causes necrosis of tobacco leaf tissue.

[0138] Example 4: Inoculation of Pinot Gris virus-infectious clones into Beda grapes and verification of their infectivity

[0139] 1. Inoculation and identification of infectious clones of Pinot Gris virus in Beda grapes

[0140] The positive monoclonal clones (positive monoclonal clone GP6, positive monoclonal clone GP18) were inoculated into LB liquid medium containing 50 μg / mL Km and 50 μg / mL Rif, and cultured in a shaker at 28°C and 200 rpm until OD 600nm The OD value was about 1.0, and the mixture was centrifuged at 8000 rpm for 10 min to collect the precipitate. The precipitate was dissolved in 1 / 2MS medium containing 100 μM acetosyringone (AS), 1 mg / L BA, and 0.5 mg / L NAA, and the OD value was adjusted. 600nm to 1.0 to obtain the inoculum. The inoculum was dispensed into test tubes on the clean bench, each test tube was about 20 mL. The roots of the tissue culture seedlings of Beida grape were pierced with an inoculation needle, each root was pierced at two locations, and the roots were fixed in the test tubes with filter paper so that the roots were completely immersed in the liquid and placed in the light culture room. After 10 days, it was transferred to fresh MS liquid culture medium and cultured for another 15 days. After that, it was transferred to fresh MS solid culture medium containing 500 mg / L cefotaxime and placed in the light culture room for culture to obtain grape seedlings. The inoculated grape seedlings were identified by PCR (PCR identification used the two pairs of primers in Table 3), and the tissue culture seedlings identified as GINV positive by PCR were transplanted to the field. The disease was observed every day after transplanting, and GPGV was detected by RT-PCR (RT-PCR detection primers were the primers in Table 3).

[0141] 2. Infectivity Verification

[0142] The phenotype of the constructed GP6 infectious clone inoculated with Beda grapes one year after infection with the roots of tissue culture seedlings was as follows: Figure 6 As shown in A, some of the grapes showed mild chlorosis and mottled symptoms, and some of the Beda grapes were infected with GPGV by RT-PCR ( Figure 7 ), while no specific bands of GPGV were amplified in the symptom-free plants. The phenotype of the constructed GP18 infectious clone inoculated with Beda grapes one year later by Agrobacterium infection of tissue culture seedling roots is shown in the figure below. Figure 6 As shown in B, after observation, some plants showed severe symptoms such as dwarfing, terminal bud dieback, and severe chlorosis and mottled spots. The RT-PCR test results showed that the grapes inoculated with GP18 and showing symptoms could amplify GPGV-specific fragments, while the plants without symptoms could not amplify GPGV-specific fragments ( Figure 8). The above experiments show that the GP6 and GP18 infectious clones constructed by the present invention have infectious activity on grapes, and there is a large difference in pathogenicity between GP6 and GP18 infectious clones on grapes. GP6 only shows weak chlorosis and ring spot symptoms. Compared with the GP6 infectious clone, the GP18 infectious clone has stronger pathogenicity and causes the grape plants to be short, the terminal buds to be necrotic, and the leaves to show severe chlorosis and mottled symptoms.

[0143] Example 5. Phylogenetic tree analysis of GPGV variants GP6, GP18 and other GPGV genome full-length sequences

[0144] MEGA11 software was used to perform phylogenetic tree analysis on GP6, GP18 and 186 other GPGV full-length genome sequences registered in NCBI, with GINV LN BETA RS as the outgroup.

[0145] The results are as follows Fig. 9 As shown, the results showed that the evolutionary tree was divided into 4 groups, of which group 4 was further divided into 5 subgroups, namely 4a-4e, GP18 (represented as 18 in the figure) was located in group 1, GP6 (represented as 6 in the figure) was located in subgroup 4e, and the GPGV isolates fvg-Is12 and fvg-ls15 used in the two infectious clones constructed in the literature "Agroinoculation of Grapevine Pinot Gris Virus intobacco and grapevineprovides insights onviral pathogenesis, PLoS One, 2019, Vol. 14, No. 3" were both located in group 4, of which fvg-Is12 was located in subgroup 4c, and fvg-ls15 was located in subgroup 4d.

Claims

1. A full-length cDNA clone of Pinot Gris virus, wherein the full-length cDNA clone of Pinot Gris virus is a plant expression vector containing a full-length cDNA sequence of the Pinot Gris virus GP6 genome or a full-length cDNA sequence of the Pinot Gris virus GP18 genome; The full-length cDNA sequence of the Pinot Gris virus GP6 genome is shown in SEQ ID No. 1 in the sequence list; The full-length cDNA sequence of the Pinot Gris virus GP18 genome is shown in SEQ ID No. 2 in the sequence list.

2. A Pinot Gris virus infectious full-length cDNA clone composition, comprising a Pinot Gris virus GP6 infectious full-length cDNA clone and a Pinot Gris virus GP18 infectious full-length cDNA clone; The infectious full-length cDNA clone of Pinot Gris Grape Virus GP6 is a plant expression vector containing the full-length cDNA sequence of the Pinot Gris Grape Virus GP6 genome; the full-length cDNA sequence of the Pinot Gris Grape Virus GP6 genome is shown in SEQ ID No. 1 in the sequence list; The infectious full-length cDNA clone of Pinot Gris virus GP18 is a plant expression vector containing the full-length cDNA sequence of the Pinot Gris virus GP18 genome; the full-length cDNA sequence of the Pinot Gris virus GP18 genome is shown in SEQ ID No. 2 in the sequence list.

3. The method for preparing the infectious full-length cDNA clone of Pinot Gris virus according to claim 1, comprising the following steps: connecting the full-length cDNA sequence of the GP6 genome of Pinot Gris virus or the full-length cDNA sequence of the GP18 genome of Pinot Gris virus to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the GP6 genome of Pinot Gris virus or the full-length cDNA sequence of the GP18 genome of Pinot Gris virus; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris virus; The full-length cDNA sequence of the Pinot Gris virus GP6 genome is shown in SEQ ID No. 1 in the sequence list; The full-length cDNA sequence of the Pinot Gris virus GP18 genome is shown in SEQ ID No. 2 in the sequence list.

4. A method for preparing a composition of infectious full-length cDNA clones of Pinot Gris grape virus, comprising the following steps: preparing a Pinot Gris grape virus GP6 infectious full-length cDNA clone and a Pinot Gris grape virus GP18 infectious full-length cDNA clone; The method for preparing the infectious full-length cDNA clone of Pinot Gris grape virus GP6 comprises the following steps: connecting the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris grape virus GP6; the full-length cDNA sequence of the Pinot Gris grape virus GP6 genome is shown in SEQ ID No. 1 in the sequence list; The method for preparing the infectious full-length cDNA clone of Pinot Gris grape virus GP18 comprises the following steps: connecting the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome to a plant expression vector by seamless cloning to obtain a recombinant expression vector containing the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome; the recombinant expression vector is the infectious full-length cDNA clone of Pinot Gris grape virus GP18; the full-length cDNA sequence of the Pinot Gris grape virus GP18 genome is shown in SEQ ID No. 2 in the sequence list.

5. A recombinant bacterium comprising the infectious full-length cDNA clone of the Pinot Gris virus according to claim 1.

6. A recombinant bacterial composition; the recombinant bacterial composition comprises a recombinant bacterium containing the infectious full-length cDNA clone of Pinot Gris virus GP6 described in claim 2 and a recombinant bacterium containing the infectious full-length cDNA clone of Pinot Gris virus GP18 described in claim 2.

7. Use of the infectious clone of Pinot Gris virus according to claim 1 or the infectious clone composition of Pinot Gris virus according to claim 2, or the infectious full-length cDNA clone of Pinot Gris virus prepared according to the method of claim 3, or the infectious full-length cDNA clone composition of Pinot Gris virus prepared according to the method of claim 4, or the recombinant bacteria according to claim 5, or the recombinant bacteria composition according to claim 6 in any of the following R1) to R8): R1) Prepare products for constructing single virus infection plant models; R2) Construct a single virus infection plant model; R3) Prepare products for constructing infected plant models with different or significant pathogenicity; R4) Constructing plant infection models with different or significant pathogenicity; R5) Preparation of products for studying the biological characteristics and / or pathogenicity and / or pathogenesis of Pinot Grigio virus; R6) Study the biological characteristics and / or pathogenicity and / or pathogenesis of Pinot Grigio virus; R7) Preparation of products for studying the interaction between Pinot Gris virus and host plants; R8) Study the interaction between Pinot Gris virus and host plants; The single virus is the Pinot Gris virus.

8. A DNA molecule, the nucleotide sequence of which is shown as SEQ ID No.1 or SEQ ID No.2.

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