CN109385448B - Pumpkin mosaic virus infectious cloning vector and construction method thereof - Google Patents

Abstract

The invention relates to a pumpkin mosaic virus infectious clone vector and a construction method thereof, wherein a pumpkin mosaic virus is taken as a material, 5.8Kb RNA1 and 3.3Kb RNA2 of a genome are constructed into a plant expression vector pXT1, and virus vectors pSqMV-RNA1 and pSqMV-RNA2 which have high-efficiency infection capacity on host plants mediated by agrobacterium are obtained. The invention lays a foundation for researching the genome of the virus, the structure and the function of each gene and the interaction between the gene and a host, and provides a new method for disclosing the virus seed transmission mechanism.

Description

Pumpkin mosaic virus infectious cloning vector and construction method thereof

Technical Field

The invention relates to a pumpkin mosaic virus infectious cloning vector and a construction method thereof, belonging to the field of genetic engineering.

Background

Squash mosaic virus (SqMV) belongs to the order of picornaviridae, is associated with members of the comovirus family, the comovirus subfamily, the genus comovirus, and is an important seed-borne virus on cucurbitaceae crops. The main hosts of the virus are cucurbitaceae crops such as melons, watermelons, cucumbers, winter squash, pumpkins and cucurbita pepo, but also infect Chenopodium plants such as chenopodium album and the like. The virus can be divided into two groups, I and II, according to whether different strains can infect the watermelon and the severity of symptoms induced on the melon and the pumpkin is different. Wherein, the symptoms of the group I strains on the melon are more serious than the symptoms of the melon, a part of strains can infect the watermelon, the group II strains can not infect the watermelon, and the symptoms of the group I strains on the melon are more serious than the symptoms of the melon. The virus is transmitted in nature primarily by seeds and vector insects. Seed transmission rates are typically from 0.1% to 10%, with commercial and test seeds yielding approximately 10% infected seedlings. The cucumovirus is reported in continents of the world, but has not been reported in many countries. Since the virus can be spread through seeds and the seed transmission rate is high, the virus is likely to be distributed in the growing area of cucurbits along with the increasing frequency of seed trade. On one hand, the virus can be transmitted from an occurred area to an unexecuted new area through seeds to become a new virus disease of a region, on the other hand, the virus is transmitted from the previous growing season to the next growing season through the seeds to become an important initial infection source of the virus disease, and even under the condition of low seed transmission rate, the virus disease is easily caused to occur in a large area through a transmission medium, agricultural operation and the like. Therefore, the research on the structure and the function of the viral genome and the pathogenic mechanism thereof has important significance.

Disclosure of Invention

The invention provides a pumpkin mosaic virus infectious clone vector and a construction method thereof, constructs the pumpkin mosaic virus infectious clone vector, lays a foundation for researching the structure and the function of a virus genome and a pathogenic mechanism thereof, and solves the problems.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a pumpkin mosaic virus infectious clone vector comprises a clone vector with the preservation number of CGMCC No.16296 and a clone vector with the preservation number of CGMCC No. 16297, is classified and named as pumpkin mosaic virus, is preserved in the general microorganism center of China general microbiological culture Collection center in 2018, 9 and 10 days, and the detailed address is No.3 of West Lu No.1 of Xingyang district, Beijing.

The invention also provides a construction method of the pumpkin mosaic virus infectious cloning vector, which comprises the following steps:

(1) extracting plant total RNA from pumpkin leaves infected with pumpkin mosaic virus;

(2) obtaining a full-length sequence of the pumpkin mosaic virus by using the total RNA of the plant;

(3) based on the obtained sequence and combined with the plant expression vector pXT1 sequence, three pairs of primers of two components of RNA1 and RNA2 of the pumpkin mosaic virus are respectively designed,

HF-RNA 1-1F: as shown in SEQ ID NO.3, RNA 1-2597R: as shown in SEQ ID NO. 4; RNA 1-2576F: as shown in SEQ ID NO. 5; RNA 1-4632R: as shown in SEQ ID NO. 6; RNA 1-4613F: as shown in SEQ ID NO. 7; HF-RNA 1-5858R: as shown in SEQ ID NO.8,

the primer sequences of component RNA2 are as follows: HF-RNA 2-1F: as shown in SEQ ID NO. 9; RNA 2-939R: as shown in SEQ ID NO.10, RNA 2-921F: as shown in SEQ ID NO. 11; RNA 2-2362R: as shown in SEQ ID NO. 12; RNA 2-2339F: as shown in SEQ ID NO. 13; HF-RNA2-3386R is shown as SEQ ID NO. 14;

(4) amplifying the component RNA1 by using primers HF-RNA1-1F/RNA1-2597R, RNA1-2576F/RNA1-4632R and RNA1-4613F/HF-RNA1-5858R respectively to obtain three fragments with the lengths of about 2617 bp, 2057 bp and 1266 bp, and carrying out homologous recombination on the three fragments and the amplified fragmenttuI and SmaConnecting the double enzyme-digested pXT1 vector, and transferring the vector into escherichia coli to obtain pSqMV-RNA1, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC number 16296;

(5) amplifying the component RNA2 by using primers HF-RNA2-1F/RNA2-939R, RNA2-921F/RNA2-2362R and RNA2-2339F/HF-RNA2-3386R respectively to obtain three fragments with the lengths of 959 bp, 1442 bp and 1068 bp, and carrying out homologous recombination on the three fragments and the amplified fragmenttuI and SmaThe double-enzyme-cut pXT1 vector is connected and transferred into escherichia coli to obtain pSqMV-RNA2, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC number 16297.

Further, a preferred embodiment of the present invention is: the recombinant vectors pSqMV-RNA1 and pSqMV-RNA2 are introduced into an agrobacterium strain GV3101 by a liquid nitrogen freeze-thawing method.

The invention has the beneficial effects that:

(1) in the construction process of the virus infectious clone vector, the fidelity of the virus sequence has great influence on the existence of the virus infectious clone vector, the infectious clone vector of the invention not only uses high fidelity enzyme in the construction process, but also respectively utilizes three pairs of primers to carry out segmented amplification on two genome components of the virus, thereby ensuring the fidelity of the virus sequence as much as possible and improving the success probability of the virus infectious clone construction;

(2) the infectious cloning vector of the invention uses 2 multiplied by 35S enhanced promoter and strong infectivity agrobacterium strain GV3101, thus greatly improving the infection capacity;

(3) the infectious cloning vector is inoculated by agrobacterium, and can be used for respectively culturing vectors containing two genome components in large quantity according to experimental requirements, so that the infectious cloning vector is low in cost and simpler to operate compared with in vitro transcription;

(4) the pumpkin mosaic virus is a seed-borne virus, and the successful acquisition of the infectious cloning vector can promote the research of a seed-borne mechanism of the pumpkin mosaic virus so as to assist the virus prevention and control work;

(5) the method used by the invention can carry out deletion, insertion, mutation and other operations on the constructed vector to obtain a series of mutants of different types for virus pathogenicity analysis.

(6) The infectious cloning vector can infect pumpkin, melon and cucumber after being inoculated by an agrobacterium-mediated method, and the method can insert a marker gene into the constructed vector, combine the two, develop interaction research between virus and host and reveal a virus pathogenic mechanism.

Drawings

FIG. 1 symptoms induced on Cucurbita pepo by the invasive clone of squash mosaic virus 10 days after Agrobacterium inoculation;

FIG. 2 shows the result of RT-PCR detection electrophoresis of Agrobacterium-inoculated plants;

FIG. 3 symptoms induced on pumpkin, melon and cucumber by the invasive clone cucurbita pepo mosaic virus 7 days after the rubbing inoculation;

FIG. 4 shows the results of RT-PCR detection electrophoresis (RNA 1) of frictionally inoculated plants;

FIG. 5 shows the results of RT-PCR detection of the tribological inoculated plants (RNA 2).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings and embodiments, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A pumpkin mosaic virus infectious clone vector comprises a clone vector with the preservation number of CGMCC No.16296 and a clone vector with the preservation number of CGMCC No. 16297, is classified and named as pumpkin mosaic virus, is preserved in the general microorganism center of China general microbiological culture Collection center in 2018, 9 and 10 days, and the detailed address is No.3 of West Lu No.1 of Xingyang district, Beijing.

A construction method of a pumpkin mosaic virus infectious cloning vector comprises the following steps:

(1) extracting plant total RNA from pumpkin leaves infected with pumpkin mosaic virus;

a) taking 0.1 g of pumpkin leaves infected with the pumpkin mosaic virus, adding 1 mL of RNAioso Plus lysate (TAKARA), uniformly mixing the mixture by vortex, and standing at room temperature until layering;

b) adding 200 μ l chloroform, shaking vigorously, mixing, standing at room temperature for 5 min, centrifuging at 4 deg.C for 15 min at 12,000 g;

c) carefully absorbing the supernatant into a new 2 mL centrifuge tube, adding isopropanol with the same volume, slightly reversing the mixture up and down, uniformly mixing, standing at room temperature for 10 min, and centrifuging at 4 ℃ for 10,000 g for 10 min;

d) discarding the supernatant, adding 1 mL of 75% ethanol, suspending the RNA precipitate, centrifuging at 4 ℃ for 5 min at 12,000 g;

e) discarding the supernatant until ethanol is completely volatilized, and adding 30 μ L of RNase free ddH₂And dissolving the RNA in O to obtain the total plant RNA.

(2) Obtaining a full-length sequence of the pumpkin mosaic virus by using the total RNA of the plant;

a) obtaining of 3' terminal sequence of pumpkin mosaic virus

The 3' -terminal sequences of the component RNAs 1 and 2 were amplified according to the instruction of the BD SMART RACE cDNA Amplification Kit (Clontech) Kit. Wherein, the primers used in the amplification process are designed according to the known genome sequence information of the cucumovirus in NCBI database, and two 3' RACE nested primers are respectively designed aiming at the components RNA1 and RNA2,

the primer sequences of component RNA1 are as follows:

3' RACE-RNA 1-5132-F: 5 '-GTGAATTTGAACAATGTGCTGA-3', as shown in SEQ ID NO. 15;

3' RACE-RNA 1-5390-F: 5 '-GTTGGATAAGGGTGTTCAGG-3', as shown in SEQ ID NO. 16;

the primer sequences of component RNA2 are as follows:

3' RACE-RNA 2-2360-F: 5 '-GAGTTCTCCTTAYATAAAGGCAAC-3', as shown in SEQ ID NO. 17;

3' RACE-RNA 2-2820-F: 5 '-GCTGATTTGATGGAATCCAC-3', as shown in SEQ ID NO. 18;

b) obtaining of 5' terminal sequence of pumpkin mosaic virus

The 5' -terminal sequences of the component RNAs 1 and 2 were amplified according to the instruction of the BD SMART RACE cDNA Amplification Kit (Clontech) Kit. Wherein, the primers used in the amplification process are designed according to the known genome sequence information of the cucumovirus in NCBI database, and two 5' RACE nested primers are respectively designed aiming at the components RNA1 and RNA2,

the primer sequences of component RNA1 are as follows:

5' RACE-RNA 1-330-R: 5 '-CATAACATCCTCAGAATGGAC-3', as shown in SEQ ID NO. 19;

5' RACE-RNA 1-660-R: 5 '-CTTGCATTTGGGCTTCCAC-3', as shown in SEQ ID NO. 20;

the primer sequences of component RNA2 are as follows:

5' RACE-RNA 2-230-R: 5 '-CCACATGAATGCAAATTAGTTG-3', as shown in SEQ ID NO. 21;

5' RACE-RNA 2-950-R: 5 '-CAACCARCAACATACCACC-3', as shown in SEQ ID NO. 22;

c) obtaining of full-length sequence of pumpkin mosaic virus

According to the obtained 3 'terminal sequence and 5' terminal sequence of the cucumovirus, seven and five primers for amplifying the full-length sequence are respectively designed aiming at component RNA1 and RNA2 by referring to the known genome sequence information of the cucumovirus in an NCBI database,

the primer sequences of component RNA1 are as follows:

RNA 1-F: 5 '-TATTAAAAATTTCTGGAGAAGAAACTTTTTAATAAC-3', as shown in SEQ ID NO. 23;

RNA 1-660-F: 5 '-GTGGAAGCCCAAATGCAAG-3', as shown in SEQ ID NO. 24;

RNA 1-2600-R: 5 '-CATAATTRTCAGGACACACAAG-3', as shown in SEQ ID NO. 25;

RNA 1-2600-F: 5 '-CTTGTGTGTCCTGAYAATTATG-3', as shown in SEQ ID NO. 26;

RNA 1-4630-R: 5 '-AGGAGATTCATTCTCATGGT-3', as shown in SEQ ID NO. 27;

RNA 1-4630-F: 5 '-ACCATGAGAATGAATCTCCT-3', as shown in SEQ ID NO. 28;

RNA 1-R: 5 '-AAAAGAAAAAGCAACATAGTAATAACG-3', as shown in SEQ ID NO. 29;

the primer sequences of component RNA2 are as follows:

RNA 2-F: 5 '-TATTAAAAATTTCTGGAAAAGAAATTATTTAATAACC-3', as shown in SEQ ID NO. 30;

RNA 2-950-F: 5 '-GGTGGTATGTTGYTGGTTG-3', as shown in SEQ ID NO. 31;

RNA 2-2360-R: 5 '-GTTGCCTTTATRTAAGGAGAACTC-3', as shown in SEQ ID NO. 32;

RNA 2-2360-F: 5 '-GAGTTCTCCTTAYATAAAGGCAAC-3', as shown in SEQ ID NO. 33;

RNA 2-R: 5 '-AAAAACAAAAGGGCAAGCATC-3', as shown in SEQ ID NO. 34;

and (2) carrying out Reverse transcription on the total plant RNA in the step (1) into cDNA by using PrimeScript II Reverse Transcriptase (TAKARA) by taking Oligo dT Primer as a Primer, wherein the Reverse transcription comprises the following specific steps: mu.L Oligo dT Primer (50. mu. mol/L), 1. mu.L dNTP (10 mmol/L each), 1. mu.L RNA and 7. mu.L RNase free dH were added₂O, mixing uniformly, placing the system at 65 ℃ for 5 min, and immediately placing on ice; then, 4. mu.L of 5 XPrimeScriptII RT Buffer, 1. mu.L of PrimeScript II Reverse Transcriptase (200U/. mu.L), 0.5. mu.L of RNase Inhibitor (40U/. mu.L) and 4.5. mu.L of RNase free dH were added to the system₂O, mixing uniformly, and placing the system at 42 ℃ for 1 h and 70 ℃ for 15 min to obtain a cDNA template;

cDNA is taken as a template, the genome sequence of the component RNA1 is amplified by respectively using primers of RNA1-F/RNA1-660-R, RNA1-660-F/RNA1-2600-R, RNA1-2600-F/RNA1-4630-R and RNA1-4630-F/RNA1-R, and the genome sequence of the component RNA2 is amplified by respectively using primers of RNA2-F/RNA2-950-R, RNA2-950-F/RNA2-2360-R and RNA2-2360-F/RNA2-RThe genomic sequence is amplified. The amplified product was recovered by Axygen gel recovery kit (Axygen), and then ligated with a cloning vector pTOPO-blunt cloning vector (Edley), and the positive clone was sent to Shanghai (Co., Ltd.) in bioengineering for sequencing analysis. Wherein, the PCR amplification system is as follows: 2 XQ 5 Hot Start Mix, 10. mu.L, 1. mu.L of forward primer (10 mM), 1. mu.L of reverse primer (10 mM), cDNA, 1. mu.L; finally using ddH₂Make up to 20. mu.l of O. The reaction procedure is as follows: 30 s at 98 ℃; 10 s at 98 ℃, 20 s at 60 ℃, 2 min at 72 ℃ and 35 cycles; 5 min at 72 ℃. The genome sequence of the component RNA1 is shown as SEQ ID NO.1, the genome sequence of the component RNA2 is shown as SEQ ID NO.2,

(3) design of primers required for construction of infectious cloning vectors

Based on the obtained sequence and combined with the plant expression vector pXT1 sequence, three pairs of primers of two components of RNA1 and RNA2 of the pumpkin mosaic virus are respectively designed,

the primer sequences of component RNA1 are as follows:

HF-RNA 1-1F: 5 '-gttcatttcatttggagaggTATTAAAAATTTCTGGAGAAGAAACTTTTTAATAAC-3', as shown in SEQ ID NO. 3;

RNA 1-2597R: 5 '-CATAATTATCAGGACACACAAG-3', as shown in SEQ ID NO. 4;

RNA 1-2576F: 5 '-cttgtgtgtcctgataattatg-3', as shown in SEQ ID NO. 5;

RNA 1-4632R: 5 '-AGGAGATTCATTCTCATGGT-3', as shown in SEQ ID NO.6,

RNA 1-4613F: 5 '-ACCATGAGAATGAATCTCCT-3', as shown in SEQ ID NO.7,

HF-RNA 1-5858R: 5 '-tggagatgccatgccgacccTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAAAGAAAAAGCAACATAGTAATAACG-3', as shown in SEQ ID NO.8,

the primer sequences of component RNA2 are as follows:

HF-RNA 2-1F: 5 '-gttcatttcatttggagaggTATTAAAAATTTCTGGAAAAGAAATTATTTAATAACC-3', as shown in SEQ ID NO.9,

RNA 2-939R: 5 '-CAACCAGCAACATACCACC-3', as shown in SEQ ID NO.10,

RNA 2-921F: 5 '-GGTGGTATGTTGCTGGTTG-3', as shown in SEQ ID NO.11,

RNA 2-2362R: 5 '-GTTGCCTTTATGTAAGGAGAACTC-3', as shown in SEQ ID NO.12,

RNA 2-2339F: 5 '-GAGTTCTCCTTACATAAAGGCAAC-3', as shown in SEQ ID NO.13,

HF-RNA 2-3386R: 5 '-tggagatgccatgccgacccTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAAAACAAAAGGGCAAGCATC-3', as shown in SEQ ID NO.14,

(4) amplification of each fragment of component RNA1 and obtaining of infectious cloning vector thereof

a) And (3) taking the cDNA in the step (2) as a template, and respectively amplifying the component RNA1 by using primers HF-RNA1-1F/RNA1-2597R, RNA1-2576F/RNA1-4632R and RNA1-4613F/HF-RNA1-5858R to obtain three fragments with the lengths of about 2617 bp, 2057 bp and 1266 bp. Wherein, the PCR amplification system is as follows: 2 XQ 5 Hot Start Mix, 10. mu.L, 1. mu.L of forward primer (10 mM), 1. mu.L of reverse primer (10 mM), cDNA, 1. mu.L; finally using ddH₂Make up to 20. mu.l of O. The reaction procedure is as follows: 30 s at 98 ℃; 10 s at 98 ℃, 20 s at 60 ℃, 2 min at 72 ℃ and 35 cycles; 5 min at 72 ℃.

b) These three fragments were ligated with each other using NEBuilder HiFi DNA Assembly Cloning Kit (NEB)StuI andSmathe I double enzyme-digested pXT1 vector is connected and transferred into escherichia coli to obtain pSqMV-RNA1, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC No. 16296. Wherein, the homologous recombinants are: NEBuilder HiFi DNA Assembly Master Mix, 10 uL, pXT1 enzyme digestion product, 5 uL; the product was recovered from RNA1-1, 2. mu.L; the product was recovered from RNA1-2, 2. mu.L; the product recovered from RNA1-3, 1. mu.L; the total volume is 20 mu L, the system is mixed evenly and incubated for 1 h at 50 ℃.

(5) Amplification of each fragment of component RNA2 and obtaining of infectious cloning vector thereof

a) And (3) taking the cDNA in the step (2) as a template, and respectively amplifying the component RNA2 by using primers HF-RNA2-1F/RNA2-939R, RNA2-921F/RNA2-2362R and RNA2-2339F/HF-RNA2-3386R to obtain three fragments with the lengths of 959 bp, 1442 bp and 1068 bp. Wherein, the PCR amplification system is: 2 XQ 5 Hot Start Mix, 10. mu.L, 1. mu.L of forward primer (10 mM), 1. mu.L of reverse primer (10 mM), cDNA, 1. mu.L; finally using ddH₂Make up to 20. mu.l of O. The reaction procedure is as follows: 30 s at 98 ℃; 10 s at 98 ℃, 20 s at 60 ℃, 2 min at 72 ℃ and 35 cycles; 5 min at 72 ℃.

b) These three fragments were ligated with each other using NEBuilder HiFi DNA Assembly Cloning Kit (NEB)StuI andSmathe double-enzyme-cut pXT1 vector is connected and transferred into escherichia coli to obtain pSqMV-RNA2, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC number 16297. Wherein, the homologous recombinants are: NEBuilder HiFi DNA Assembly Master Mix, 10 uL, pXT1 enzyme digestion product, 5 uL; the product was recovered from RNA2-1, 1. mu.L; the product was recovered from RNA2-2, 2. mu.L; the product was recovered from RNA2-3, 2. mu.L; the total volume is 20 mu L, the system is mixed evenly and incubated for 1 h at 50 ℃.

(6) Transformation of infectious cloning vectors

Separately, 5. mu.L of pSqMV-RNA1 and pSqMV-RNA1 vector plasmids are respectively added into 50. mu.L of GV3101 competent cells, kept stand on ice for 10 min, quickly frozen by liquid nitrogen for 5 min, and placed on ice for 2 min after being bathed in a water bath kettle at 37 ℃ for 5 min, 500. mu.L of LB liquid culture medium without antibiotics is added, and then placed in a shaking table at 28 ℃ and 200 rpm for recovery culture for 2 h, and then centrifuged for 1 min at 12,000g, and thalli are collected and coated on LB solid culture medium (50 ng/. mu.l Kan, 50 ng/. mu.l Rif). After the plate is cultured in an incubator at 28 ℃ for 48 hours, the unit cell grows out. Single cells were picked up and cultured overnight in 500. mu.l LB liquid medium (50 ng/. mu.l Kan, 50 ng/. mu.l Rif), and positive clones were obtained by screening respectively by PCR detection.

(7) Infectious cloning vector inoculation detection

The positive clones were transferred to LB liquid medium (50 ng/. mu.l Kan, 50 ng/. mu.l Rif) and cultured overnight, and the cells were collected by centrifugation at 6,000 g for 6 min and inoculated with an inoculation buffer (10 mM MgCl. sub.L)₂10 mM MES and 100 mu M AS), standing at room temperature for 2-3 h, mixing the two bacterial solutions at a ratio of 1:1, shaking, and injecting from the leaf back of Cucurbita pepo with 1 mL syringeAnd (4) performing injection inoculation, and after inoculation, placing the inoculated cells under the conditions of 28 ℃/25 ℃ and 16h/8h (light/dark) for continuous culture.

(8) Determination of infectivity

At one week of inoculation, new leaves of zucchini plants inoculated with both pSqMV-RNA1 and pSqMV-RNA1 began to appear with chlorosis spots. Mosaic symptoms became more and more evident on the inoculated plants as the inoculation time was prolonged, while mosaic symptoms did not appear in the uninoculated plants at all times (FIG. 1). RT-PCR detection is carried out by using primers of RNA1-4613F/RNA1-R, RNA2-921F/RNA2-2362R, each inoculated plant is found to be positive, the non-inoculated plant is found to be negative, and the detection result is further verified by sequencing and is consistent with the existence of diseases of the plant (figure 2). Grinding appropriate amount of diseased leaves with 0.01M phosphate buffer solution with pH7.0 at a ratio of 1:10, inoculating Cucurbita pepo, fructus melo and fructus Cucumidis Sativi to the juice by mechanical friction, wherein within one week after inoculation, the Cucurbita pepo, fructus melo and fructus Cucumidis Sativi are diseased successively without symptoms different from wild plants (figure 3). RT-PCR detection is carried out by using primers of RNA1-4613F/RNA1-R, RNA2-921F/RNA2-2362R, each inoculated plant is found to be positive, the non-inoculated plant is found to be negative, and the detection result is further verified by sequencing and is consistent with the existence of disease attack of the plant (figure 4 and figure 5). The result shows that the pumpkin mosaic virus can normally generate filial generation and spread, and the pumpkin mosaic virus infectious clone vector is successfully constructed.

Sequence listing

<110> Zhengzhou fruit tree institute of Chinese academy of agricultural sciences

<120> pumpkin mosaic virus infectious cloning vector and construction method thereof

<160> 34

<170> SIPOSequenceListing 1.0

<210> 2

<211> 5858

<212> DNA/RNA

<213> pumpkin mosaic Virus component RNA1(Squash mosaic virus)

<400> 2

tattaaaaat ttctggagaa gaaacttttt aataaccatc ttcaaggaat cgcgggattc 60

ccaccaaagc aatcttcaaa gtaatctcta aacattttct gttttccagc tttgcacttt 120

acggccctgt ggtagactgt gcaattgctt ttctccctcc tttcttttct tcttcaccac 180

cactttccta tcttcaagta ctctcgtttt gtttgtgcga ttaaatttat tcaaaatgaa 240

ttttactgga aaaggaagtg ttgctagtgt tcaccaagtg gtccattctg aggaggttat 300

gttttatctg aaagtatata ccaatatgac aactgacaat aagggtgcca ctttacctcg 360

tattttggcg acgctgaagg aagagcaaaa cagacacgtt ttgtatttat ccttctacgc 420

ctattgcctg gattttgatg ctgggttaat ggaatgctac tctattgacg tcgaggattt 480

tgtttttgaa caatttcatg attttgtaac tgccatgctt aaggggtgtc atagccttat 540

gcctttgaga tcttatacaa aagctgtctt tgctgagcgt ttgcagttag cagtgaattt 600

cgttccagaa atcactactg agcttggggg ctccggacct gtggaagccc aaatgcaagg 660

gctgcgaaac attgcagcca acatgctaat gtggatacct aagaaaattg gcgccctaac 720

tgtgtggaca gtagagagta ttattggaag ttttaaggaa cattttttga agatgattaa 780

tactcactgt ccaatagttt tgtccatgtt tccctggatt cttaagattt gggatcgtgt 840

cacggaatgg ttgacagcgg cagctgatga tttcgcatgg cttttagcct ctactaaaga 900

gttgatgaca tggggcatgg ccattatggc tttaaccacg gccatgagtc ttttggacaa 960

gctgctgatg gcagttggtg ccattgctga gcccatgaac ttgtccgata tttgtttgcg 1020

tactggagtt gttgccgcgt gctgttatga gctaacaaaa cagagcggta actgtggagc 1080

ccagctggtt tctcttttta gtggtgttgc taatgtcgtt gcgggggttc tgagtgcaaa 1140

atttcaaaat caaccccaga ctattttgca agattcccct ataggcttgt tggaaacact 1200

tgcagaaaag ttaacaagtc tgtgcgatgt ttccttaata aacttgggta aaacgtgtgc 1260

cgcaataaat cagattgcga catgtgccaa tacaattaag ggttttgttg caaagatttt 1320

ctgtactttg acccattatg tctgggaagc tttgggtatt aaaacctctt tcctgcgaga 1380

cgccactttt gttcttggtg aggatgttga cggttggttg cagcaaatct cccagtgtca 1440

gaatgatttt attgtacatg cttcttgttc ccaggatgaa tttcttaagc tgcaggtctt 1500

ggtggagaaa ggcaataaca tgcgaaataa gattcttcaa ggagtccgcc tttccccagg 1560

cattataagc ttggttactt caggtattgc gatgttagac aagcttcgtc gtgaggcttg 1620

ccttcagggt aatagaactg aaaggaaaat gcccttcacc attttttgtc agggaacttc 1680

tcgtgttgga aaaacacttt taacttccaa aattgttaag gatttccaag caaccctagg 1740

attggctgag gacactgtgt acagtagaaa cccggcagaa agttactgga gtggatatcg 1800

tcgtcagcct tttgttttga ttgatgactt tggtgcagtt aagactgagc cctcgtgtga 1860

ggctcaattg atccctctgg tttcttccac tccttaccct gttcctatgg cggccattga 1920

agaaaagggg atgatgtttg actcgcaatt tattgtgtgt tcaacaaact ttctggaacc 1980

cagtccagag gcaaaaatcc gtgatgatgc tgccttccgt aatcgtcgac atgtgttgat 2040

tagtgttaaa attgatagcg aaaagcagta tgattctagt gacttcacgc aaaatcaaat 2100

ttatgagata atgcgctatg agagagagac ctatgttgtg gagcaaagat tcacctctta 2160

tgctgatttg ttcgtgtttc tccaaaataa atatgaagct cacaatgttg aacaatctgc 2220

caatatagga agtgtagttc cctacaaagg caagcaaaat ttgctagtct tacgtggctt 2280

gctgaatttg gccaatgttt ctaatgctgg tttattaaaa gctcaagcca agaagcttgg 2340

tcagccagaa ggctttaggg aatatactca cttgttcact attcagcata aaaatcgttt 2400

tgcacatctt ggttttgctg acgcacatga ttctgtaata tggtatgggg aacattccga 2460

tgtcggcaaa agtgaggagg tagttaaaat gacagcaagc catgtcatga aggcttacaa 2520

gatcctcata caaggtgaaa acctgagctt actcattaaa aatcatttgc gttatcttgt 2580

gtgtcctgat aattatgatc gtgatttcaa ttttaccggg ggggttggag atacattgtt 2640

ggagcaacaa ttgctaccag atatgcaggc actccactct tgggaaagat ttgttttgtg 2700

cgcaatgggt tactatatgg aaacccagaa aatgcaacct tggtacaaga ctattactgg 2760

aaaggtcttt gagaatctta agatagccta ttcacgtgag tttagttcgt ggcccactcc 2820

tttgaaggcc atagttggca ttgttctggc agctttagtt ggtaaaggtt tttggtttgc 2880

ttataaggct cttactgaag gtggcaacgg ttctagtctc gttggagctg cttctgtcgt 2940

tttgacaagc actactaatg ctgtcgccca aagtaggaag ccaaatcgtt ttgatgtggc 3000

acaatatcgc taccgcaatg ttccattaaa gcgcaggcaa tgggccgatg cgcagatgtc 3060

tttggatcat agtagcgttg caattatgag caagtgcaag gctaactttg agtttggaaa 3120

caccaatgtg caaatcgttt tagttcctgg tcgaaggttt ttgggatatg cgcatttttt 3180

caagaccatt aagcatccca taacagttaa aatagttaaa gatggtcggc attttcttca 3240

tgtttatgat cccaaaggta tgacatattt tgatgattct gagatctgtg tttatcatag 3300

tgctagtttt gaggacatac cacacactac ttgggacgtt ttttgctggg attgggagaa 3360

aagtctttgt aagaaatttt cagctgactt tctttcctgt aaatatgaca gattaactat 3420

gtcatacgag cccacatatg ccggcattaa tgtggaaaca atttttgaga ctttggaatt 3480

gcgagccaat ggtgccgtgc gcaagctccc ttgttttttg aagtatgagg ctccgacagt 3540

tgaccgggat tgcgggagtc ttatagtcgc acaggtggaa ggacgatatc aaatcgttgg 3600

catacatatt gggggcgatg ggagaaatgg gtttgcagcc cctctgcccc acattccgca 3660

ggttgctgat gcgcagtgta ctaccaagta tttcagtttt tatccgaatg agcaggagga 3720

agaaacaggt gttgcgctag ttggtcagct taaacctgat gtgtggatac ctttgccaac 3780

aaaaacttca ttggtggaga cggaggaagc gtggcatttg gacacgaaaa gtgataaggt 3840

gcctagtatt ttgaactctg aagacccccg tatcaagcaa gggggtaatg aaggatatga 3900

cccctttagg ggaggtgtta ctaaatattc gcagccgatg gggcatttat gtggagaaac 3960

ccttggggaa gttgccaatg aaattttgga agagtggcac gattgcctcg agcctgatga 4020

ggacttcgat gatgtggatc tggaagtggc tataaatggt attgatggct tggattatat 4080

ggatcgtatt cccttggcca cttctgaagg gtttcctcac atcctgtcaa gagaaaaagg 4140

ggaaaagggc aaagggagat ttgttgagac agtgggtggt aaatgcgccc ttatagaagg 4200

cacctcagtt taccatgctt ttgaaacctt gcaacagcag tgcaagaaag aagttcctac 4260

tttgataggg attgagtgtc ccaaggacga gaaattgcct ctgcgcaagg tttttgatac 4320

cccaaagacg cgctgtttca caatactccc catggaatat aacctgttgg tgcggatgaa 4380

gttcttgaaa tttgtccgtt ttattatgag aaatcgtgag aaacttgcct gccaggttgg 4440

aattaaccct tacagcatgg agtggacaag attggccgga agccttttaa gtgttagcca 4500

gaatattttg tgctgtgact ataagtcatt tgatggcctt ctgagtaaac aggttatgac 4560

agtcattgcg accatgatta acagattgtg cggtggatct caggagagcc aaaccatgag 4620

aatgaatctc ctcatggctt gctgttcccg ttatgctatt tcgaagaatg aagtgtggcg 4680

tgttgaatgt ggtatacctt ctggattccc tttaactgtc atttgtaatt ccattttcaa 4740

tgaaatactc gtgaggtact gttatcggaa aattctggag aagaacaatg tgccacgacc 4800

tttacatgtg aattttcctc gaatggtgaa actcgtcacg tatggggacg acaatttgat 4860

ttctgttagc catgttgttg cgaatgtgtt caatggtaga actttgaagg ctgaaatggc 4920

acaatttggg gtgactataa cggatggtat tgacaagacg agccccacat tggaattccg 4980

caagttgagc aattgtgatt tcttgaagag aggatttaaa ttgaacggtc ttgtttatga 5040

ctcaccagaa gagaagagta gtctgtgggc ccagcttcac tatgttaaca cgactaatct 5100

ggataagcaa gaagcttacc ttgtgaattt gaacaatgtg ctgaaagaat tatatatgca 5160

cagtcctgag gagatgaaca tgataagaag gaaagccctg cagctaccct ggattaacaa 5220

ggatgacgtg ctaaatgggg cccagataaa agaatttttt gcctatcagc gccaacaatt 5280

gcttccagac aacgaggata gtttggatat gatgttaaag ccagatcttc taggatctct 5340

tgttcctgat gttgtgttgt tggataaggg tgttcaggtg tctggtaggc tcagaacgat 5400

aaatcttaag tacactgaac tcagtgaaaa gcgtgataat gaattctggg ttattttcaa 5460

tggacatttc cctaccaacc gtcttcctga gcactgtttg aatattaaat gggaagcagg 5520

cactggcagg gggaacttgc caacccaatc ttggataagc aataatattt ccaggcctaa 5580

ttctgagtac aacaggaaga ttaggactgc ttatgctgct ggtaaagttc tgtgtttctg 5640

tgcctggggc gatatgatac ctgttagtat tatgttatta ctttcttcag ccagaaatga 5700

ttggattcct aagggacaga cgaatgaggc tttgacctct ttcatggaat atgcaaaaag 5760

tctcaaattt ctgccacgtg aatgtgagta tgcgtttact ggcgcgaagt aaagcttcat 5820

gaatttaact acgttattac tatgttgctt tttctttt 5858

<210> 2

<211> 3386

<212> DNA/RNA

<213> pumpkin mosaic Virus component RNA2(Squash mosaic virus)

<400> 2

tattaaaaat ttctggaaaa gaaattattt aataaccatc ttcaaggaat cgcgggattc 60

ccaccaaagc aatcttcaaa gcaatcttca aacattttct gttttccggc tttgcacttt 120

acggccctgt ggtagactgt gcaattgctt ttctccctcc tttcttttct tcctctcctc 180

cattttccca tttgtatgca tttgtccaat tcaactaatt tgcattcatg tggtgttttt 240

gtgaacaagt aaacgagtgc tttgaaggtt atcataaaga ctactctgtt caaacagtcc 300

ctgtggaata tttggcttca cactatattg tcaacaagtt tagacctgac cccttagccg 360

ttctgtggct tttctgcttg gggatttggt gggagattat tcaaatactt caccatttat 420

tccagtacaa ggaaccagca ttatttatta atagctgtca ggatcttgct gcttttttag 480

aaaggaaaca ttccatggaa gtgattcaga aggaaggttt ggctgcttca gcactcaagg 540

ataaggagcg actggccgaa aaagctgtgg tcaatcagcc cctgagtaac ttgattcctc 600

attcaaataa aatgtatgag cgaagtaaga gtcttctatc tggtcttaag cgtggtttga 660

taaagcaaaa agaggtagct tttgataagc tcatgggggg ttcgacaata gacttccagc 720

atattccaac cggaactctc acacctggtg agaataaggt gctagacata ccaattgtcc 780

cgcaacattt attgaccagt acgaatataa cggattacca tcaggccaat aataagagtg 840

ctaatggtgc tactgcactt catgttggag ctatagaagt catcttagat tgctttacct 900

ctcctgatag taacatttgt ggtggtatgt tgctggttga tacagcacat ttaaacccgg 960

ataatgctat aagaagtgtt tttgtcgcgc catttattgg tggtcgccct attcgagttt 1020

tgttgtttcc agacactttg gtggaaattg ccccaaacat gaattctcga tttaaattgc 1080

tatgtactac tagcaatggc gacgttgcac cagattttaa tttagcgatg gtcaaagtca 1140

atgttgcagg ttgtgctgtt agtttgacca agacatacac tcctacagcc taccttgagc 1200

aagagctaat taaagaaaag ggggccattg ttcaatattt gaacaggcac actttttcta 1260

tgcatcggaa taatcagatg acaaaggaag agatgcaaaa gcagtgcctt tcttttagac 1320

tggaaagtgc tctcactttg caggaaaaac atcctttgca cgccaccttt tgtaagtcaa 1380

ccaattttgt ttacaagatt ggtggagatg ccaaagaagg tagtaatggt agtttgactg 1440

ttaatgagag tcagttgtcc tcacattctt cttccacaca tgttttgcat aagcataaca 1500

acagtggcga taatgaagta gaattctcgg agattggtgt ggtcgtacca ggtgctggta 1560

gaaccaaggc ttatggtcaa aatgaactag atcttgcgca actttccttg gatgatacta 1620

gttctcttcg tggatctgtg ttgcaaacca aattagccac atcccgtgtc attttaagta 1680

aaacaatggt cgggaatact atccttaggg aagatttgct cgccaccttc ttgcaggata 1740

gcaatgagag ggccgcgata gatttaatcc gcacccatgt tataagaggc aaaatacgct 1800

gtgttgcttc tatcaatgtt ccagagaata caggttgtgc tttggctatt tgcttcaata 1860

gtagcataac aggagctgct gacacggaca tatacaccac gagttctcag gacgctatag 1920

tgtggaatcc tgcttgtgag aaggctgttg agttgtcatt taaccccaat ccctgcagtg 1980

atgcctggaa ctttgtgttt ttgcagcaga caaaggcaca tttcgcagtt cagtgcgtga 2040

ctggatggac tacaacgcca cttacagact tggctctagt gcttacatgg catatcgata 2100

gaaacttgtg cgtgcctgaa actttgacga ttagttctgc acacgcttct ttcccaatca 2160

atcgctggat gggaaagtta tctttcccac aagggcctgc acgtgttcta aaaaggatgc 2220

ccttggccat cggtggtgga gccggcacca aagatgctat tctaatgaat atgccaaatg 2280

ctgttatttc actacatcgt tattttagag gggatttcgt ctttgaaata acaaagatga 2340

gttctcctta cataaaggca accattgctt tctttatagc atttggtgat atcacagagg 2400

aaatgaccaa tttggagagt tttccccaca agcttgtgca gtttgctgaa attcaggggc 2460

gcaccaccat aacatttacg caaggcgaat ttttgacggc atggtcaaca caagttttaa 2520

gcaccgttaa tcctcagaaa gatggatgtc cccatttgta tgcactcttg catgactctg 2580

ctacgtcaac cattgaagga aatttcatta ttggtgttaa actgctggac attaggaact 2640

atcgtgctta tggccataac cccggttttg agggagcccg cctgttagga atttctgggc 2700

agagtactat ggtgcagcag cttggaactt ataatccaat ttggatggtt cgtacgccct 2760

tagaaagtac agccccacaa aattttgcga gttttactgc tgatttgatg gaatccacag 2820

tgagtgggga ttctactgga aattggaata ttacagctta tccgagtcct atatccaatt 2880

tgttgaaagt ggctgcttgg aagaagggga caataagatt ccaactcatt tgccggggtg 2940

ctgccgttaa gcaatccgat tgggctgcgt cagccagaat agacttggtc aataacctct 3000

caaataaagc tttacctgcg cgctcttggt acattactaa gccacgagga ggtgatatcg 3060

agtttgactt ggaaatagcg gggccaaata atggttttga aatggccaat tccagttggg 3120

ctttccagac tacatggtat ttggaaattg ccatagacaa ccctaagcaa ttcactcttt 3180

tcgagttaaa tgcttgtctt atggaagact ttgaagtggc tggaaacacc ttaaacccac 3240

ctattttact ttcttagtct ttgtgttgct tttccctttc cttttgtttc tgggttttgt 3300

tgtggcttct ttcccagttc gctttagaag cctctctttg taaattttaa gagcttgttt 3360

tctttgatgc ttgccctttt gttttt 3386

<210> 3

<211> 56

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gttcatttca tttggagagg tattaaaaat ttctggagaa gaaacttttt aataac 56

<210> 4

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cataattatc aggacacaca ag 22

<210> 5

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cttgtgtgtc ctgataatta tg 22

<210> 6

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aggagattca ttctcatggt 20

<210> 7

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

accatgagaa tgaatctcct 20

<210> 8

<211> 77

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tggagatgcc atgccgaccc tttttttttt tttttttttt tttttttttt aaaagaaaaa 60

gcaacatagt aataacg 77

<210> 9

<211> 57

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gttcatttca tttggagagg tattaaaaat ttctggaaaa gaaattattt aataacc 57

<210> 10

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caaccagcaa cataccacc 19

<210> 11

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtggtatgt tgctggttg 19

<210> 12

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gttgccttta tgtaaggaga actc 24

<210> 13

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gagttctcct tacataaagg caac 24

<210> 14

<211> 71

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tggagatgcc atgccgaccc tttttttttt tttttttttt tttttttttt aaaaacaaaa 60

gggcaagcat c 71

<210> 15

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtgaatttga acaatgtgct ga 22

<210> 16

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gttggataag ggtgttcagg 20

<210> 17

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gagttctcct tayataaagg caac 24

<210> 18

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gctgatttga tggaatccac 20

<210> 19

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cataacatcc tcagaatgga c 21

<210> 20

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cttgcatttg ggcttccac 19

<210> 21

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccacatgaat gcaaattagt tg 22

<210> 22

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

caaccarcaa cataccacc 19

<210> 23

<211> 36

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tattaaaaat ttctggagaa gaaacttttt aataac 36

<210> 24

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gtggaagccc aaatgcaag 19

<210> 25

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cataattrtc aggacacaca ag 22

<210> 26

<211> 22

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cttgtgtgtc ctgayaatta tg 22

<210> 27

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aggagattca ttctcatggt 20

<210> 28

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

accatgagaa tgaatctcct 20

<210> 29

<211> 27

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

aaaagaaaaa gcaacatagt aataacg 27

<210> 30

<211> 37

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tattaaaaat ttctggaaaa gaaattattt aataacc 37

<210> 31

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ggtggtatgt tgytggttg 19

<210> 32

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gttgccttta trtaaggaga actc 24

<210> 33

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gagttctcct tayataaagg caac 24

<210> 34

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

aaaaacaaaa gggcaagcat c 21

Claims (3)

1. A pumpkin mosaic virus infectious cloning vector is characterized in that: comprises a cloning vector with the preservation number of CGMCC No.16296 and a cloning vector with the preservation number of CGMCC No. 16297, and is preserved in the general microbiological culture Collection center of China general microbiological culture Collection management Committee in 2018, 9 and 10.

2. A method of constructing the cucumovirus infectious cloning vector of claim 1, wherein:

(1) extracting plant total RNA from pumpkin leaves infected with pumpkin mosaic virus;

(2) obtaining a full-length sequence of the pumpkin mosaic virus by using the total RNA of the plant;

(3) based on the obtained sequence and combined with the plant expression vector pXT1 sequence, three pairs of primers of two components of RNA1 and RNA2 of the pumpkin mosaic virus are respectively designed,

the primer sequences of component RNA1 are as follows:

HF-RNA 1-1F: as shown in SEQ ID NO.3, RNA 1-2597R: as shown in SEQ ID NO. 4; RNA 1-2576F: as shown in SEQ ID NO. 5; RNA 1-4632R: as shown in SEQ ID NO. 6; RNA 1-4613F: as shown in SEQ ID NO. 7; HF-RNA 1-5858R: as shown in SEQ ID NO.8,

the primer sequences of component RNA2 are as follows: HF-RNA 2-1F: as shown in SEQ ID NO. 9; RNA 2-939R: as shown in SEQ ID NO.10, RNA 2-921F: as shown in SEQ ID NO. 11; RNA 2-2362R: as shown in SEQ ID NO. 12; RNA 2-2339F: as shown in SEQ ID NO. 13; HF-RNA2-3386R is shown as SEQ ID NO. 14;

(4) amplifying the component RNA1 by using primers HF-RNA1-1F/RNA1-2597R, RNA1-2576F/RNA1-4632R and RNA1-4613F/HF-RNA1-5858R respectively to obtain three fragments with the lengths of about 2617 bp, 2057 bp and 1266 bp, and carrying out homologous recombination on the three fragments and the amplified fragmenttuI and SmaConnecting the double enzyme-digested pXT1 vector, and transferring the vector into escherichia coli to obtain pSqMV-RNA1, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC number 16296;

(5) amplifying the component RNA2 by using primers HF-RNA2-1F/RNA2-939R, RNA2-921F/RNA2-2362R and RNA2-2339F/HF-RNA2-3386R respectively to obtain three fragments with the lengths of 959 bp, 1442 bp and 1068 bp, and carrying out homologous recombination on the three fragments and the amplified fragmenttuI and SmaThe double-enzyme-cut pXT1 vector is connected and transferred into escherichia coli to obtain pSqMV-RNA2, namely the pumpkin mosaic virus infectious clone vector with the preservation number of CGMCC number 16297.

3. The method for constructing the cucumovirus infectious cloning vector according to claim 2, wherein: the recombinant vectors pSqMV-RNA1 and pSqMV-RNA2 are introduced into an agrobacterium strain GV3101 by a liquid nitrogen freeze-thawing method.

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