CN115927757A - A method for efficient screening of antiviral germplasm resources based on PEMV-1 and PEMV-2 invasive clones - Google Patents

Abstract

The invention relates to a method for efficiently screening antiviral germplasm resources based on PEMV-1 and PEMV-2 invasive clones; comprising: a root soaking step of Agrobacterium. The present invention has the following advantages: 1. The method of the present invention is easy to operate, and has the advantages of saving time and more convenience than manual friction inoculation; 2. The method of the present invention can control the inoculum amount of the virus by adjusting the concentration of invasive clones, and the same The error of inoculum amount between different plants of batches is small; 3, an infective clone in the method of the present invention only has the infective activity of a kind of virus, has solved the problem of toxin source impurity, has satisfied the specific pathogen inoculation requirement Experimental conditions; 4, the inventive method has a good inoculation effect, and the infection rate of the plant is high; 5, the inventive method makes the plant acquire the virus early, and the time for the virus to accumulate in the plant is long; 6, the inventive method cost is relatively low , The inoculation operation time is short.

Description

Method for efficient screening of antiviral germplasm resources based on PEMV-1 and PEMV-2 infectious clones

Technical Field

The invention belongs to the technical field of molecular biology, and in particular to the technical field of methods for efficiently screening antiviral germplasm resources based on PEMV-1 and PEMV-2 infectious clones.

Background Art

Pea mosaic virus 1 (PEMV-1) is a virus of the genus Enamovirus in the family Solemoviridae, and pea mosaic virus 2 (PEMV-2) is a virus of the genus Umbravirus in the family Tombusviridae. The combined infection of the two causes pea mosaic disease (PEMD). PEMD is widely distributed and has been reported in the United States, Germany, the United Kingdom, France, the Czech Republic and other countries. It has caused a pea yield loss of up to 50% in England. PEMV-1 and PEMV-2 mainly infect leguminous plants in the field, including 23 leguminous plants such as peas, broad beans, chickpeas, soybeans, and kidney beans. At the same time, PEMV-1 and PEMV-2 can also infect non-leguminous plants such as tomatoes, peppers, chicory ^[13] , globe amaranth, and Nicotiana benthamiana. PEMV-1 and PEMV-2 infection can cause plant growth distortion, stunted development, mosaic, yellowing of leaves with necrotic spots, chlorotic leaf veins, ear-like protrusions on the back of leaves and pods, pods are often deformed, and the fruiting rate is significantly reduced, resulting in serious losses. Both PEMV-1 and PEMV-2 can replicate themselves independently. PEMV-1 can be transmitted by aphids in a cyclical non-proliferative manner, PEMV-2 requires the CP of PEMV-1 to be encapsidated for aphid transmission, and PEMV-2 can assist PEMV-1 in systemic infection and mechanical transmission. Many aphids are vectors of PEMV (PEMV-1 and PEMV-2), such as pea aphids, cowpea aphids, peach aphids, and eggplant aphids. These aphids are widely distributed in China and around the world, and have large populations, which makes PEMD have a high risk of transmission and epidemic.

There are no effective methods reported to control PEMD. According to relevant literature, the use of insecticides to control PEMD vectors, aphids, has little effect. Therefore, the best control method is to use disease-resistant varieties. Artificial inoculation is the basis for screening disease-resistant germplasm resources and breeding for disease resistance. It is also a common method for studying the pathogenicity and transmission characteristics of viruses and the interaction between viruses and vector insects.

Traditional methods of artificially rubbing peas with virus sources have many limitations, while virus-infectious cloning refers to the use of gene recombination technology to insert the full-length genome gene sequence of the virus into a specific vector to obtain a recombinant vector with the function of infecting the host. Viruses can easily infect plants by injection inoculation, but injection inoculation has problems such as high cost and cumbersome operation. Developing a more efficient and convenient inoculation method is one direction of infectious cloning, which is of great significance for the study of the pathogenic mechanism of the virus and the screening of disease-resistant germplasm resources.

The traditional friction inoculation technology has the following problems: it is time-consuming and labor-intensive, and requires rubbing leaves one by one, usually more than two leaves per plant, and the inoculated plants must be cleaned after rubbing;

1. It is difficult to control the strength of artificial friction, which is technically difficult. Excessive friction may cause cell damage or even plant death.

2. The virus source is impure. The diseased juice obtained by directly grinding the diseased leaves may contain two or more viruses, and the purpose of single pathogen inoculation cannot be achieved;

3. The inoculation cannot be quantified. There is a large error in the amount of virus inoculation between different plants in the same batch, and the amount of diseased sap used for each inoculation cannot be fixed.

Summary of the invention

The present invention solves the above problems and defects and provides a method for efficiently screening antiviral germplasm resources based on PEMV-1 and PEMV-2 infectious clones. The present invention lays a foundation for studying the pathogenicity, transmission characteristics and interaction between viruses and insect vectors of the virus and solves the technical problems in the screening of antiviral pea germplasm resources.

The present invention is implemented by adopting the following technical solutions.

A primer for detecting PEMV-1, the primer comprising:

PEMV-1-801-F: 5’-AGTTCGTCCTGGTGTCCTGG-3’;

PEMV-1-801-R: 5’-CATACCACTTCCCATCCCGC-3’.

A primer for detecting PEMV-2, the primer comprising:

PEMV-2-483-F: 5’-TTCAGGAGCACCCGAAACAC-3’;

PEMV-2-483-R: 5’-CGTAGTGAGAGGCATGGCAT-3’.

The reaction system of the primers of the present invention is: 5 μL Tap mix, 3.6 μL dd H ₂ O, 0.2 μL Primer (For) and 0.2 μL Primer (Rev), 1 μL cDNA; the reaction program is: 94°C 3min; 94°C 30s, 56°C 30s, 72°C 50s, 35 cycles; 72°C 5min.

A primer for amplifying the 5' and 3' end sequences of the PEMV-1 genome, the primer being: PEMV-1 5'RACE-GSP: 5'-GCGGTAGTTGAGGCTGCTCAATTCC-3';

PEMV-1 3'RACE-GSP: 5'-AGGCCAGGAGTTCTCTGCCTGTGAG-3'.

A primer for amplifying the 5' and 3' end sequences of the PEMV-2 genome, the primer being: PEMV-2 5'RACE-GSP: 5'-TACTGTCAGCATCGCGGCGCGCTCG-3';

PEMV-2 3'RACE-GSP: 5'-ACACCCTGCCACGAGGTGCGTGGA-3'.

A method for amplifying PEMV-1, the invention divides PEMV-1 into three fragments: PEMV-1-1, PEMV-1-2 and PEMV-1-3; uses primer sets pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R and PEMV-1-3F/pCB301-PEMV-1-3R to amplify the fragments PEMV-1-1, PEMV-1-2 and PEMV-1-3 of PEMV-1 respectively;

The PCR amplification system of the method is 20 μL: 5× PrimeSTAR GXL Buffer 4 μL, dNTP mixture (2.5 mM each) 1.6 μL, upstream primer 0.6 μL, downstream primer 0.6 μL, PrimeSTAR GXL DNA Polymerase 0.4 μL, dd H ₂ O 10.8 μL, cDNA 2 μL; primer pCB301-PEMV-1-1F/PEMV-1-1R annealing temperature 54.5°C, extension 3min; PEMV-1-2F/PEMV-1-2R annealing temperature 60°C, extension 3min; PEMV-1-3F/pCB301-PEMV-1-3R annealing temperature 60°C, extension 2min; reaction procedures are: 98°C 30s; 98°C 10s, Y°C 15s, 68°C Zmin; 68°C 10min; 4°C storage.

A method for amplifying PEMV-2. The invention divides PEMV-2 into three fragments: PEMV-2-1, PEMV-2-2, and PEMV-2-3; uses primer sets pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R, and PEMV-2-3F/pCB301-PEMV-2-3R to amplify the fragments PEMV-2-1, PEMV-2-2, and PEMV-2-3 of PEMV-2 respectively;

The PCR amplification system of the method is 20 μL: 5× PrimeSTAR GXL Buffer 4 μL, dNTP mixture (2.5 mM each) 1.6 μL, upstream primer 0.6 μL, downstream primer 0.6 μL, PrimeSTAR GXL DNA Polymerase 0.4 μL, dd H ₂ O 10.8 μL, cDNA 2 μL.

(Primers pCB301-PEMV-2-1F/PEMV-2-1R annealing temperature 60℃, extension 2min30s; PEMV-2-2F/PEMV-2-2R annealing temperature 60℃, extension 2mins; PEMV-2-3F/pCB301-PEMV-2-3R annealing temperature 58℃, extension 1min), the reaction procedure is 98℃30s; 98℃10s, Y℃15s, 68℃Zmin, 68℃10min; stored at 4℃.

A primer set for determining the infectivity of PEMV-1 and PEMV-2, the primer set comprising:

PEMV-1-CP-F: 5'-ATGCCGACTAGATCGAAATC-3';

PEMV-1-CP-R: 5'-TCAGAGGGAGGCATTCATTA-3';

PEMV2-ORF3-F: 5'-ATGACGATAATCATTAATG-3';

PEMV2-ORF3-R: 5'-TCACCCGTAGTGAGAGGCA-3';

A method for constructing an infectious cloning vector of ear mosaic virus comprises the following steps: Step 1):

Total RNA was extracted from pea plants infected with PEMV-1 and PEMV-2 collected in Dali, Yunnan Province;

Step 2):

PEMV-1 and PEMV-2 were divided into three fragments and amplified to obtain the full-length nucleotide sequences of PEMV-1 and PEMV-2;

Step 3):

Primers for amplification of the whole genome sequence were designed based on the genome sequences of PEMV-1 and PEMV-2 and the binary vector pCB301-2X35S-MCS-HDVRZ-NOS-1, and PEMV-1 and PEMV-2 were amplified by dividing them into three fragments with 21-33 bp of nucleotide overlap at the ends of the fragments;

Step 4):

Primer sets pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R, and PEMV-1-3F/pCB301-PEMV-1-3R were used to amplify fragments PEMV-1-1, PEMV-1-2, and PEMV-1-3 of PEMV-1;

Using cDNA as a template, three fragments of PEMV-1 were amplified and connected to the linearized vector pCB301; the correctly sequenced recombinant plasmid pCB301-PEMV-1 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method.

Step 5):

The other primer pairs pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R, and PEMV-2-3F/pCB301-PEMV-2-3R amplified the PEMV-2 fragments PEMV-2-1, PEMV-2-2, and PEMV-2-3, respectively;

Using cDNA as template, three fragments of PEMV-2 were amplified and connected to the linearized vector pCB301; the correctly sequenced recombinant plasmid pCB301-PEMV-2 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method;

Step 6):

Agrobacterium root dipping: pea seeds with roots were placed in Agrobacterium LB culture medium containing PEMV-1 and PEMV-2 infectious cloning vectors with _OD600 = (0.5-1.0) and immersed in a 28°C incubator for 2-3 hours. Then, the bacterial solution was not completely poured out and co-culture was continued under the same conditions for 6-7 hours before sowing.

Furthermore, step 6) of the present invention is root soaking with Agrobacterium: the pea seeds with roots are placed in Agrobacterium LB culture medium containing PEMV-1 and PEMV-2 infectious cloning vectors with _OD600 = (0.5-1.0) and immersed in a 28°C incubator for 3 hours. After 3 hours, the bacterial solution is not completely poured out, and co-culture is continued under the same conditions for 6 hours, and then the seeds are sown.

The beneficial effects of the present invention are:

1. The method of the present invention is easy to operate and has the advantages of being more time-saving and more convenient than manual friction inoculation;

2. The method of the present invention can control the virus inoculation amount by adjusting the concentration of the infectious clone, and the error of the inoculation amount between different plants in the same batch is small;

3. In the method of the present invention, one infectious clone has only one virus's infectious activity, which solves the problem of impure virus source and meets the experimental conditions requiring specific pathogen inoculation;

4. The inoculation method of the present invention has good effect and high infection rate on plants;

5. The method of the present invention enables the plant to acquire the virus earlier, and the virus accumulates in the plant for a long time;

6. The method of the present invention has relatively low cost and short inoculation operation time.

The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of the root length of pea seeds of the present invention;

Fig. 2 is a schematic diagram of pea seed root soaking according to the present invention;

3 is an electrophoretic diagram of PEMV-1 and PEMV-2 detection of the present invention; lanes 1-14 are PEMV-1 detection results, with a band size of 570 bp; lanes 15-28 are PEMV-2 detection results, with a band size of 693 bp; +CK represents a positive control, and -CK represents a negative control;

Fig. 4 is an electrophoresis diagram of PEMV-1 detection of different pea varieties treated differently according to the present invention; lanes 1-9, lanes 10-20, and lanes 21-24 are respectively the PEMV-1 detection results of the pink flower pod bean king without root puncture, the pink flower pod bean king with root puncture, and the Taiwan longevity pea kernel root without root puncture, and the band size is 570bp;

FIG5 is an electrophoresis diagram of PEMV-1 detection of Taiwan longevity pea kernel roots without puncture and puncture treatment of the present invention; lanes 1-8 and lanes 9-15 are respectively the PEMV-1 detection results of Taiwan longevity pea kernel roots without puncture and Taiwan longevity pea kernel roots punctured, and the band size is 570bp;

Fig. 6 is an electrophoresis diagram of PEMV-2 detection of different pea varieties treated differently according to the present invention; lanes 1-9, lanes 10-20, and lanes 21-23 are respectively the PEMV-2 detection results of the pink flower pod bean king without root puncture, the pink flower pod bean king with root puncture, and the Taiwan longevity pea kernel root without root puncture, and the band size is 693bp;

FIG7 is an electrophoresis diagram of PEMV-2 detection of Taiwan longevity pea kernel roots without puncture and puncture treatment of the present invention; lanes 1-9 and lanes 10-16 are respectively the PEMN-1 detection results of Taiwan longevity pea kernel roots without puncture and Taiwan longevity pea kernel roots punctured, and the band size is 693 bp;

FIG8 is an electrophoretic diagram of PEMV-1 detection of roots treated with puncture and without puncture according to the present invention; lanes 1-11 and lanes 12-17 are the PEMV-1 detection results of roots punctured and roots not punctured, respectively, and the band size is 801 bp;

Fig. 9 is an electrophoresis diagram of PEMV-1 and PEMV-2 detection of roots treated with no puncture and puncture according to the present invention; lanes 1-6 are the detection results of PEMV-1 without puncture, with a band size of 801 bp; lanes 9-17 are the detection results of PEMV-2 with puncture, with a band size of 483 bp; +CK positive control is invalid;

FIG10 is an electrophoretic diagram of PEMV-2 detection of roots treated with puncture and without puncture according to the present invention; Lanes 1-2 are the detection results of PEMV-2 with punctured roots, with a band size of 483 bp; Lanes 3-14 are the detection results of PEMV-2 with unpunctured roots; +CK positive failure;

Fig. 11 is an electrophoretic diagram of PEMV-1 detection of roots inoculated with PEMV-1 and PEMV-2 respectively after different treatments of the present invention; lanes 1-6 and lanes 7-12 are the PEMV-1 detection results of roots punctured and roots not punctured and only inoculated with pCB301-PEMV-1-YDL, respectively, with a band size of 801 bp; lanes 13-17 are the PEMV-1 detection results of roots punctured and only inoculated with pCB301-PEMV-2;

Fig. 12 is an electrophoretic diagram of PEMV-1 and PEMV-2 detection in roots inoculated with PEMV-2 in different treatments according to the present invention; lanes 1 and 2-3 are the detection results of PEMV-1 inoculated with pCB301-PEMV-2 only with and without root puncture, respectively; +CK positive control is invalid; lanes 6-15 are the detection results of PEMV-2 inoculated with pCB301-PEMV-2 only with root puncture, with a band size of 483 bp; +CK positive control is invalid;

Fig. 13 is an electrophoretic diagram of the present invention for detecting a mixed sample of PEMV-1 and PEMV-2 inoculated peas by friction with a virus source that is infected with PEMV-1 or PEMV-2 separately or simultaneously; lanes 1-7 are the detection results of PEMV-1 of the mixed sample 1-7, with a band size of 801 bp; lanes 10-16 are the detection results of PEMV-2 of the mixed sample 1-7, with a band size of 483 bp;

Fig. 14 is a single sample electrophoresis diagram of PEMV-1 and PEMV-2 inoculated peas with a virus source that infects PEMV-1 and PEMV-2 separately or simultaneously according to the present invention; lanes 1-4 and 5-7 are the single sample PEMV-1 detection results of mixed samples 1 and 3 in the previous step, respectively, with a band size of 801 bp; lanes 10-13 and 14-16 are the single sample PEMV-2 detection results of mixed samples 1 and 3 in the previous step, with a band size of 483 bp;

FIG15 is a diagram showing symptoms of peas inoculated with PEMV-1, PEMV-2, and PEMV-1+PEMV-2 according to the present invention.

DETAILED DESCRIPTION

A method for constructing an infectious cloning vector of ear mosaic virus comprises the following steps:

Extract plant total RNA;

(a) Clean the work surface and tools with 75% ethanol;

(b) Add 1 ml of Trizol reagent to a 1.5 ml centrifuge tube;

(c) taking a small amount of the above sample, grinding it thoroughly in liquid nitrogen, grinding the sample into powder, adding it to a centrifuge tube, and shaking to mix;

(d) Centrifuge at 12000 rpm for 10 min at 4°C, transfer the supernatant to a new 1.5 ml centrifuge tube and place at room temperature for 5 min;

(e) Add 300 μL of chloroform;

(f) After mixing, place at room temperature for 2-3 minutes;

(g) Centrifugation at 12000 rpm for 15 min at 4°C;

(h) Carefully pipette the aqueous phase (supernatant) of the sample into a new centrifuge tube, taking care not to pipette the organic phase below;

(i) Add an equal volume of 100% isopropanol and leave at room temperature for 10 min;

(j) Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant;

(k) Resuspend the precipitate in 1 ml of 75% ethanol and let it stand for several minutes;

(l) Repeat the above steps 2 times;

(m) standing at room temperature for 5 min to remove excess anhydrous ethanol;

(n) Dissolve RNA in RNA-free sterile water and set aside.

cDNA synthesis and virus detection were performed using plant total RNA (primers PEMV-1-801-F/R predicted to amplify an 801 bp product and primers PEMV-2-483-F/R predicted to amplify a 483 bp fragment were used for molecular detection of PEMV-1 and PEMV-2, respectively);

(a) Using downstream Primer (Rev) as a primer or using random primers, the total plant RNA in the above step was reverse transcribed into cDNA using Reverse Transcriptase M-MLV (RNase H ^- ) (TaKaRa), as follows: first add 3 μL dd H ₂ O, 2 μL RNA template and 1 μL random primer, mix well, place the system at 70°C for 10 min, quickly take out and cool on ice for 2 min, centrifuge for a few seconds, add the following reagents: 2 μL 5×M-MLV Buffer, 1 μL dd H ₂ O, 0.5 μL dNTP mix (10 mM) and RTase M-MLV (200u/μL), after adding the reagents, mix well, place the system at 42°C for 1 h and 75°C for 15 min, and obtain the cDNA template;

(b) The primer pair PEMV-1-801-F/R predicted to amplify an 801 bp product and the primer pair PEMV-2-483-F/R expected to amplify a 483 bp fragment were used for the molecular detection of PEMV-1 and PEMV-2, respectively.

The primer sequences are as follows:

PEMV-1-801-F: 5’-AGTTCGTCCTGGTGTCCTGG-3’;

PEMV-1-801-R: 5’-CATACCACTTCCCATCCCGC-3’;

PEMV-2-483-F: 5’-TTCAGGAGCACCCGAAACAC-3’;

PEMV-2-483-R: 5’-CGTAGTGAGAGGCATGGCAT-3’;

Using cDNA as template, PEMV-1-801-F/R and PEMV-2-483-F/R were used to amplify the genomic sequences of PEMV-1 and PEMV-2, as follows: 5 μL Tap mix, 3.2 μL dd H ₂ O, 0.4 μL Primer (For) and 0.4 μL Primer (Rev), 1 μL cDNA were added. The reaction program was: 94°C for 3 min; 94°C for 30 s, 56°C for 30 s, 72°C for 50 s, 35 cycles; 72°C for 5 min.

The amplified PCR products were purified using the SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, Shanghai, China), cloned into the pMD19-T vector (TaKaRa Biotechnology, Dalian, China) using the TA cloning strategy, and sequenced by Sanger sequencing. The sequences were assembled and analyzed using EditSeq software (Lasergene 7.0, USA DNASTAR Inc.).

Subsequently, 5′ and 3′ amplification primers were designed according to the obtained sequences of PEMV-1 and PEMV-2, namely PEMV-1 5′RACE, PEMV-1 3′RACE, PEMV-2 5′RACE and PEMV-2 3′RACE.

RACE 5/3(TaKaRa Biotechnology，中国大连)扩增了PEMV-1、PEMV-2基因组的5′和3′端序列。克隆到pMD19-T载体中并进行sanger测序。PEMV-1和PEMV-2的完整核苷酸序列通过软件Lasergene 7.0组装和分析。use

The 5′ and 3′ end sequences of the PEMV-1 and PEMV-2 genomes were amplified by RACE 5/3 (TaKaRa Biotechnology, Dalian, China). The sequences were cloned into the pMD19-T vector and subjected to Sanger sequencing. The complete nucleotide sequences of PEMV-1 and PEMV-2 were assembled and analyzed by the software Lasergene 7.0.

In order to obtain the full-length infectious clones of PEMV-1 and PEMV-2, primers for amplification of the whole genome sequence were designed based on the genome sequences of PEMV-1 and PEMV-2 and the binary vector pCB301-2X35S-MCS-HDVRZ-NOS-1. PEMV-1 and PEMV-2 were divided into three fragments for amplification, and the ends of the fragments had 21-33 bp of nucleotide overlap.

Primer set pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R and

PEMV-1-3F/pCB301-PEMV-1-3R was used to amplify the PEMV-1 fragments PEMV-1-1, PEMV-1-2 and PEMV-1-3.

In addition, other primer pairs pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R, and PEMV-2-3F/pCB301-PEMV-2-3R were used to amplify the fragments PEMV-2-1, PEMV-2-2, and PEMV-2-3 of PEMV-2, respectively;

Amplification of PEMV-1 fragments and construction of infectious cloning vectors:

Using the aforementioned cDNA as a template, PEMV-1 was amplified using primers pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R and PEMV-1-3F/pCB301-PEMV-1-3R to obtain three fragments with lengths of 3002bp (PEMV-1-1), 1479bp (PEMV-1-2) and 1317bp (PEMV-1-3).

The PCR amplification system was 20 μL: 5× PrimeSTAR GXL Buffer 4 μL, dNTP mixture (2.5 mM each) 1.6 μL, upstream primer 0.6 μL, downstream primer 0.6 μL, PrimeSTAR GXL DNA Polymerase 0.4 μL, ddH ₂ O 10.8 μL, cDNA 2 μL. (Primers pCB301-PEMV-1-1F/PEMV-1-1R annealing temperature 54.5°C, extension 3 min; PEMV-1-2F/PEMV-1-2R annealing temperature 60°C, extension 3 min; PEMV-1-3F/pCB301-PEMV-1-3R annealing temperature 60°C, extension 2 min) The reaction program was: 98°C 30 s; 98°C 10 s, Y°C 15 s, 68°C Z min; 68°C 10 min; stored at 4°C. Then, the target fragment was recovered using the SanPrep Column DNA Gel Extraction Kit.

TaKaRa restriction endonucleases SmaI and Stu I were used to digest the pCB301 binary vector to obtain the pCB301 linear vector. The reaction system was as follows: 20 μL (0.8 μg) of the expression vector pCB301 plasmid, 5 μL of 1×QuickCut Green Buffer, 1.0 μL of restriction endonuclease Sma I, and 23 μL of ddH ₂ O. Reaction conditions: incubate at 30°C for 2 h. Then 1 μL of Stu I restriction endonuclease was added and incubated at 37°C for 2 h. After electrophoresis verification, the product was recovered using the SanPrep Column DNAGel Extraction Kit.

Ultra One Step Cloning Kit试剂盒将PEMV-1三个片段的PCR产物连接到pCB301线性载体。使用TaKaRa限制性内切酶SmaI和Stu I酶切pCB301双元载体，获得pCB301线性载体，反应体系：表达载体pCB301质粒20μL(0.8μg)，1×QuickCut GreenBuffer 5μL，限制性内切酶Sma I 1.0μL，ddH₂O 23μL。反应条件：30℃温育2h。再加入1μLStu I限制性内切酶，37℃温育2h。电泳验证后利用SanPrep Column DNAGel ExtractionKit试剂盒进行产物回收。use

The Ultra One Step Cloning Kit was used to connect the PCR products of the three fragments of PEMV-1 to the pCB301 linear vector. The pCB301 binary vector was digested with TaKaRa restriction endonucleases SmaI and Stu I to obtain the pCB301 linear vector. The reaction system included 20 μL (0.8 μg) of the expression vector pCB301 plasmid, 5 μL of 1×QuickCut GreenBuffer, 1.0 μL of restriction endonuclease Sma I, and 23 μL of ddH ₂ O. Reaction conditions: incubate at 30°C for 2 hours. Then 1 μL of Stu I restriction endonuclease was added and incubated at 37°C for 2 hours. After electrophoresis verification, the product was recovered using the SanPrep Column DNAGel ExtractionKit.

Ultra One Step Cloning Kit试剂盒将PEMV-1三个片段的PCR产物连接到pCB301线性载体。通过冻融法将测序正确的重组质粒pCB301-PEMV-1转化到根癌农杆菌EHA105中。其中，同源重组体系为：2×ClonExpress Mix 10μL，片段PEMV-1-1 1μL，片段PEMV-1-2 1μL，片段PEMV-1-3 1μL，线性化pCB301 7μL。反应条件：50℃温育30min。use

The Ultra One Step Cloning Kit was used to connect the PCR products of the three fragments of PEMV-1 to the pCB301 linear vector. The correctly sequenced recombinant plasmid pCB301-PEMV-1 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method. Among them, the homologous recombination system was: 2×ClonExpress Mix 10μL, fragment PEMV-1-1 1μL, fragment PEMV-1-2 1μL, fragment PEMV-1-3 1μL, linearized pCB301 7μL. Reaction conditions: incubate at 50℃ for 30min.

Amplification of PEMV-2 fragment and acquisition of its infectious cloning vector

Using the aforementioned cDNA as a template, primers pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R and PEMV-2-3F/pCB301-PEMV-2-3R were used to amplify PEMV-2, and three fragments of approximately 2264 bp (PEMV-2-1), 1344 bp (PEMV-2-2) and 740 bp (PEMV-2-3) in length were obtained.

The PCR amplification system was 20 μL: 5× PrimeSTAR GXL Buffer 4 μL, dNTP mixture (2.5 mM each) 1.6 μL, upstream primer 0.6 μL, downstream primer 0.6 μL, PrimeSTAR GXL DNA Polymerase 0.4 μL, ddH ₂ O 10.8 μL, cDNA 2 μL. (Primers pCB301-PEMV-2-1F/PEMV-2-1R annealing temperature 60°C, extension 2 min 30 s; PEMV-2-2F/PEMV-2-2R annealing temperature 60°C, extension 2 mins; PEMV-2-3F/pCB301-PEMV-2-3R annealing temperature 58°C, extension 1 min), the reaction program was 98°C 30 s; 98°C 10 s, Y°C 15 s, 68°C Z min, 68°C 10 min; stored at 4°C. Then, the target fragment was recovered using the SanPrep Column DNA Gel Extraction Kit.

Ultra One Step Cloning Kit试剂盒将PEMV-2-YDL三个片段的PCR产物连接到pCB301线性载体。TaKaRa restriction endonucleases Sma I and Stu I were used to digest the pCB301 binary vector to obtain the pCB301 linear vector. The reaction system included 20 μL (0.8 μg) of the expression vector pCB301 plasmid, 5 μL of 1×QuickCut Green Buffer, 1.0 μL of restriction endonuclease Sma I, and 23 μL of ddH2O. Reaction conditions: incubate at 30°C for 2 h. Then 1 μL of Stu I restriction endonuclease was added and incubated at 37°C for 2 h. After electrophoresis verification, the product was recovered using the SanPrep Column DNA Gel Extraction Kit.

The PCR products of the three fragments of PEMV-2-YDL were ligated to the pCB301 linear vector using the Ultra One Step Cloning Kit.

TaKaRa restriction endonucleases Sma I and Stu I were used to digest the pCB301 binary vector to obtain the pCB301 linear vector. The reaction system was as follows: 20 μL (0.8 μg) of the expression vector pCB301 plasmid, 5 μL of 1×QuickCut Green Buffer, 1.0 μL of restriction endonuclease Sma I, and 23 μL of ddH2O. Reaction conditions: incubate at 30°C for 2 h. Then 1 μL of Stu I restriction endonuclease was added and incubated at 37°C for 2 h. After electrophoresis verification, the product was recovered using the SanPrep Column DNA Gel Extraction Kit.

Ultra One Step Cloning Kit试剂盒将PEMV-2-YDL三个片段的PCR产物连接到pCB301线性载体。通过冻融法将测序正确的重组质粒pCB301-PEMV-2转化到根癌农杆菌EHA105中。其中，同源重组体系为：：2×ClonExpress Mix 10μL，片段PEMV-2-1 1μL，片段PEMV-2-2 1μL，片段PEMV-2-3 1μL，线性化pCB301载体7μL。反应条件：50℃温育30min。use

The Ultra One Step Cloning Kit was used to connect the PCR products of the three fragments of PEMV-2-YDL to the pCB301 linear vector. The correctly sequenced recombinant plasmid pCB301-PEMV-2 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method. Among them, the homologous recombination system was: 2×ClonExpress Mix 10μL, fragment PEMV-2-1 1μL, fragment PEMV-2-2 1μL, fragment PEMV-2-3 1μL, linearized pCB301 vector 7μL. Reaction conditions: incubate at 50℃ for 30min.

Transformation with infectious cloning vectors:

(a) Add 1-10 μL of pCB301-PEMV-1/pCB301-PEMV-2 plasmid to EHA105 competent cells and place on ice for 30 min;

(b) Place in liquid nitrogen for 5 min;

(c) 37°C water bath for 5 min;

(d) Place on ice for 2 min;

(e) Add 890 μL of LB liquid medium without antibiotics into the clean bench and shake the culture at 28°C, 220 rpm for 3-4 h;

(f) 5000 rpm, 5 min, remove the supernatant, leaving 100 μL of resuspended precipitate, spread the liquid on LB solid medium containing 100 μg/ml Kan+Rif, and culture inverted at 28°C for 36-48 h;

(g) Pick a single colony and add it to 3 ml of LB liquid medium containing 100 μg/ml Kan+Rif and shake at 28°C for 36-48 h.

Propagation of infectious cloning vectors:

(a) Prepare a certain number of sterile test tubes (enough to hold LB medium for root immersion) and add LB medium to 2/3 of the test tubes;

(b) Add 0.1-1 ml of transformed bacterial solution to each test tube and shake in a shaker for 8-10 hours until the bacterial solution reaches an appropriate concentration.

Infectivity-based cloning by Agrobacterium root dip inoculation:

(a) Soak pea seeds in water. After about 2 days, pea seeds will have roots of 1-2 cm in length (Figure 1);

(b) Place the rooted pea seeds in a container, add LB medium containing the infectious cloning vector of PEMV ( _OD600 between 0.5-1.0) until the roots of the pea seeds are just covered (Figure 2) (Before soaking the roots, pierce the roots of the pea seeds for better results. The specific operation is: use a fine needle (embroidery needle) to gently pierce 3 to 4 holes from the base to the end of the root, and be careful not to cause too much damage to the root) (Figure 1);

(c) After culturing in an incubator set at 28°C and 100% illumination for 3 hours, pour out the LB medium (be careful not to pour out completely to prevent the cultured seeds from drying out too much), cover the container with a transparent bag, and continue culturing in the incubator for 6 hours;

(d) Rinse the cultured seeds with clean water (to remove the LB medium on the surface of the seeds) and then sow them.

Determination of infectivity:

Through preliminary experiments (the _OD600 value of the LB culture medium containing the infectious cloning vectors of PEMV-1 and PEMV-2 was about 0.5, cultured at 18°C for 2 hours, and then cultured for another 24 hours), it was found that by treating the roots of the pea variety "Pink Flower Pod King" whose roots had broken through the seed coat with Agrobacterium, RT-PCR detection was performed using primers PEMV1-CP-F/PEMV1-CP-R and PEMV2-ORF3-F/PEMV2-ORF3-R (Figure 3), the detection rates of PEMV-1 and PEMV-2 were 83.3% and 50%, respectively.

Then the second batch of root dipping experiments (LB medium containing PEMV-1 and PEMV-2 infectious cloning vectors with an OD ₆₀₀ value of about 0.5, cultured at 28°C for 2 hours, and then placed for 12 hours) was tested (Table 1): On the 10th day after sowing the root-dipped seeds, primers PEMV1-CP-F/PEMV1-CP-R and PEMV2-ORF3-F/PEMV2-ORF3-R were used to perform PCR tests on two pea varieties that were simultaneously root-dipped and inoculated with pCB301-PEMV-1 and pCB301-PEMV-2 (Figures 4-7). The test results are shown in the following table:

Table 1 Systemic infection rates of PEMV-1 and PEMV-2 in different pea varieties under different treatments

The second batch of test results showed that the pea ear mosaic virus could successfully infect pea plants in this experiment, and the effect was good, indicating that the method of inoculating pea plants by Agrobacterium root dipping based on infectious clones is feasible. Based on the test results of PEMV-1 and PEMV-2, the detection rate of root puncture treatment was higher than that of non-puncture treatment, indicating that root puncture treatment is conducive to PEMV infection.

After that, the third batch of root immersion experiments was conducted (the OD ₆₀₀ value of the LB culture medium containing the infectious cloning vectors of PEMV-1 and PEMV-2 was about 0.8, cultured at 28°C for 3 hours, and then placed for 6 hours). In addition to the improvement in the treatment conditions (Figures 8-10), separate root immersion inoculation of pCB301-PEMV-1 and pCB301-PEMV-2 was set up (tested on the 14th day after sowing) (Figures 11-12) (Table 2) and composite friction inoculation and separate friction inoculation of pCB301-PEMV-1 and pCB301-PEMV-2 (tested on the 14th day after friction inoculation; mixed samples 1-4 were inoculated with pCB301-PEMV-1 and pCB301-PEMV-2 at the same time, mixed sample 5 was inoculated with only pCB301-PEMV-1, and mixed samples 6-7 were inoculated with only pCB301-PEMV-2) (Figures 13-14) (Table 3). The test results are shown in the following table:

Table 2 Systemic infection rates of PEMV-1 and PEMV-2 in pea plants under different treatments

Table 3 Systemic infection rate of PEMV-1 and PEMV-2 on pea plants after friction inoculation

The test results of the third batch of root immersion inoculation showed that PEMV had a higher infection rate, among which the PEMV infection rate of the root puncture treatment was greater than that of the non-puncture treatment, and the infection rate of PEMV-2 was greater than the detection rate of PEMV-1, while the test results of the second batch showed that the detection rate of PEMV-1 was greater than that of PEMV-2. It is speculated that the difference may be caused by the different OD ₆₀₀ values of the bacterial solution. In addition, PEMV-1 or PEMV-2 can also successfully infect peas when inoculated by root immersion alone, and the infection rate is relatively high. From the results of friction inoculation, the infection rates of PEMV-1 and PEMV-2 are significantly low. After 20 days of simultaneous root immersion inoculation of PEMV-1+PEMV-2, pea plants began to show symptoms, but pea plants inoculated with PEMV-1 or PEMV-2 alone did not show symptoms. (Figure 15). The above results show that compared with the friction inoculation method, the inoculation method of the present invention can enable PEMV to effectively infect pea plants, which is convenient and time-saving.

PEMV-1 full-length sequence: (Gene accession number: OP113929);

PEMV-2 full-length sequence: (Gene accession number: OP113928).

The above are only some specific embodiments of the present invention. The known specific contents or common sense in the scheme are not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims (10)

1. A primer for detecting PEMV-1, wherein the primer is:

PEMV-1-801-F：5’-AGTTCGTCCTGGTGTCCTGG-3’；

PEMV-1-801-R：5’-CATACCACTTCCCATCCCGC-3’。

2. a primer for detecting PEMV-2, wherein the primer is:

PEMV-2-483-F：5’-TTCAGGAGCACCCGAAACAC-3’；

PEMV-2-483-R：5’-CGTAGTGAGAGGCATGGCAT-3’。

3. the primer according to claim 1 or 2, wherein the reaction system is: 5 μ L of Tap mix, 3.6 μ L of dd H ₂ O, 0.2. Mu.L Primer (For) and 0.2. Mu.L Primer (Rev), 1. Mu.L cDNA;

the reaction procedure is as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 50s at 72 ℃,35cycles; 5min at 72 ℃.

4. A primer for amplifying 5 'and 3' terminal sequences of the genome of PEMV-1, wherein the primer is:

PEMV-1 5'RACE-GSP：5'-GCGGTAGTTGAGGCTGCTCAATTCC-3'；

PEMV-1 3'RACE-GSP：5'-AGGCCAGGAGTTCTCTGCCTGTGAG-3'。

5. a primer for amplifying 5 'and 3' terminal sequences of the PEMV-2 genome, wherein the primer is:

PEMV-2 5'RACE-GSP：5'-TACTGTCAGCATCGCGGCGCGCTCG-3'；

PEMV-2 3'RACE-GSP：5'-ACACCCTGCCACGAGGTGCGTGGA-3'。

6. a method for amplifying PEMV-1, wherein the PEMV-1 is divided into three fragments: PEMV-1-1, PEMV-1-2 and PEMV-1-3; the fragments PEMV-1-1, PEMV-1-2 and PEMV-1-3 of PEMV-1 are respectively amplified by using primer sets pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R and PEMV-1-3F/pCB 301-PEMV-1-3R;

the primer sequence of PEMV-1 is as follows:

pCB301-PEMV-1-1F：

5’-AGTTCATTTCATTTGGAGAGGGTGAAATAATTGTAAGAAAGCTCTAG-3’；

PEMV-1-1R：

5’-TTGTTACGAATCTTCTCCATCTTATGTGGTTCCAGC-3’；

PEMV-1-2F：5’-GGAACCACATAAGATGGAGAAGATTCGTAACAAGCGC-3’；

PEMV-1-2R：

5’-CGACCGAAAGTGGCAGACCCATTGGAACGA-3’；

PEMV-1-3F：

5’-ATGGGTCTGCCACTTTCGGTCGTGAAATTC-3’；

pCB301-PEMV-1-3R：

5 'GAGATGCCATGCCGACCTGCGATAATCCCATGAAAACGCTG-3'; the PCR amplification system of the method is 20 mu L:5 XPrimeSTAR GXL Buffer 4. Mu.L, dNTP mix (2.5 mM each) 1.6. Mu.L, upstream primer 0.6. Mu.L, downstream primer 0.6. Mu.L, primeSTAR GXL DNA polymerase 0.4. Mu.L, dd H ₂ O10.8. Mu.L, cDNA 2. Mu.L; the annealing temperature of the primer pCB301-PEMV-1-1F/PEMV-1-1R is 54.5 ℃, and the extension is 3min; the annealing temperature of the PEMV-1-2F/PEMV-1-2R is 60 ℃, and the extension time is 3min; the annealing temperature of the PEMV-1-3F/pCB301-PEMV-1-3R is 60 ℃, and the extension is 2min; the reaction procedure is as follows: 30s at 98 ℃; 10s at 98 ℃, 15s at Y ℃, and Zmin at 68 ℃;10 min at 68 ℃; storing at 4 ℃.

7. A method for amplifying PEMV-2, wherein the PEMV-2 is divided into three fragments: PEMV-2-1, PEMV-2-2 and PEMV-2-3; respectively amplifying fragments PEMV-2-1, PEMV-2-2 and PEMV-2-3 of PEMV-2 by using primer sets pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R and PEMV-2-3F/pCB 301-PEMV-2-3R;

the primer sequences of PEMV-2 are as follows:

pCB301-PEMV-2-1F：

5’-AGTTCATTTCATTTGGAGAGGGGGTATTTATAGAGATCGGTATGAAC-3’；

PEMV-2-1R：

5’-CGTTCTTCCATAAAGGGTGTGACTGGCACTTAGTCC-3’；

PEMV-2-2F：

5’-CTAAGTGCCAGTCACACCCYYTATGGAAGAACGARGGG-3’；

PEMV-2-2R：

5’-GCGAAGCCTCACTTAGAAGCCTGGGTACACA-3’；

PEMV-2-3F：

5’-ACCCAGGCTTCTAAGTGAGGCTTMGCTTCC-3’；

pCB301-PEMV-2-3R：

5’-GAGATGCCATGCCGACCCGGGCGCCAGGGAGGTAACCACCTGGC-3’；

the PCR amplification system of the method is 20 mu L:5 XPrimeSTAR GXL Buffer 4. Mu.L, dNTP mix (2.5 mM each) 1.6. Mu.L, forward primer 0.6. Mu.L, downstream primer 0.6. Mu.L, primeSTAR GXL DNA Polymerase 0.4. Mu.L, dd H ₂ O10.8. Mu.L, cDNA 2. Mu.L; annealing the primer pCB301-PEMV-2-1F/PEMV-2-1R at 60 ℃, and extending for 2min and 30s; the annealing temperature of the PEMV-2-2F/PEMV-2-2R is 60 ℃, and the extension is 2mins; the annealing temperature of the PEMV-2-3F/pCB301-PEMV-2-3R is 58 ℃, the extension is 1min, and the reaction program is 98 ℃ for 30s; 10s at 98 ℃, 15s at Y ℃, zmin at 68 ℃ and 10min at 68 ℃; storing at 4 ℃.

8. A primer set for determining the infectivity of PEMV-1 and PEMV-2, wherein the primer set comprises:

PEMV-1-CP-F：5'-ATGCCGACTAGATCGAAATC-3'；

PEMV-1-CP-R：5'-TCAGAGGGAGGCATTCATTA-3'；

PEMV2-ORF3-F：5'-ATGACGATAATCATTAATG-3'；

PEMV2-ORF3-R：5'-TCACCCGTAGTGAGAGGCA-3'。

9. a construction method of an auris mosaic virus infectious cloning vector is characterized by comprising the following steps: step 1): extracting total RNA of pea plants infected with PEMV-1 and PEMV-2 simultaneously;

step 2): dividing the PEMV-1 and the PEMV-2 into three fragments for amplification to obtain full-length nucleotide sequences of the PEMV-1 and the PEMV-2;

and step 3): designing a whole genome sequence amplification primer based on genome sequences of the PEMV-1 and the PEMV-2 and a binary vector pCB301-2X35S-MCS-HDVRZ-NOS-1, dividing the PEMV-1 and the PEMV-2 into 3 fragments for amplification, wherein the tail ends of the fragments have nucleotide overlap of 21-33 bp;

and step 4): the primer group pCB301-PEMV-1-1F/PEMV-1-1R, PEMV-1-2F/PEMV-1-2R and PEMV-1-3F/pCB301-PEMV-1-3R is used for amplifying PEMV-1-1, PEMV-1-2 and PEMV-1-3 fragments of PEMV-1;

the PCR reaction was performed using cDNA of the PEMV-1 isolate as a template, and the amplified PCR product was ligated into the binary vector pCB301; transforming the recombinant plasmid pCB301-PEMV-1 with correct sequencing into agrobacterium tumefaciens EHA105 by a freeze-thawing method;

and step 5): the other primer pairs pCB301-PEMV-2-1F/PEMV-2-1R, PEMV-2-2F/PEMV-2-2R and PEMV-2-3F/pCB301-PEMV-2-3R respectively amplify the fragments PEMV-2-1, PEMV-2-2 and PEMV-2-3 of the PEMV-2;

the PCR reaction was performed using cDNA of the PEMV-2 isolate as a template, and the amplified PCR product was ligated into the binary vector pCB301; transforming the recombinant plasmid pCB301-PEMV-2 with correct sequencing into agrobacterium tumefaciens EHA105 by a freeze-thawing method;

step 6): root soaking of agrobacterium: placing the rooted pea seeds on OD ₆₀₀ Soaking in LB culture medium containing PEMV-1 and PEMV-2 infectious cloning vectors for 2-3 hr at 28 deg.C, completely pouring out the strain, co-culturing under the same conditions for 6-7 hr, and sowing.

10. The method according to claim 9, wherein the step 6) is agrobacterium rhizolysis: placing the rooted pea seeds on OD ₆₀₀ The agrobacterium of = (0.5-1.0) containing the PEMV-1 and PEMV-2 infectious cloning vectors was immersed in LB medium in an incubator at 28 ℃ for 3 hours, and after 3 hours, the suspension was not completely poured out, and the mixture was cultured under the same conditions for 6 hours, and then seeded.

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