CN115927757A

CN115927757A - Method for efficiently screening antiviral germplasm resources based on PEMV-1 and PEMV-2 infectious clones

Info

Publication number: CN115927757A
Application number: CN202211567418.1A
Authority: CN
Inventors: 陈小姣; 陈泓霖; 谢敏; 兰平秀; 谭冠林; 李凡
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-04-07

Abstract

The invention relates to a method for efficiently screening antiviral germplasm resources based on the infectious clone of PEMV-1 and PEMV-2; the method comprises the following steps: and (5) carrying out root soaking on agrobacterium. The invention has the following advantages: 1. the method is simple and convenient to operate, and has the advantages of being more time-saving and more convenient than manual friction inoculation; 2. the method can control the inoculation amount of the virus by adjusting the concentration of the infectious clone, and the error of the inoculation amount among different plants in the same batch is small; 3. in the method, one infectious clone only has the infection activity of one virus, so that the problem of impure virus source is solved, and the experimental condition of requiring specific pathogen inoculation is met; 4. the method has good inoculation effect and high infection rate to plants; 5. the method of the invention ensures that the virus acquisition time of the plant is early and the accumulation time of the virus in the plant is long; 6. the method has relatively low cost and short inoculation operation time.

Description

Method for efficiently screening 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 particularly relates to the technical field of a method for efficiently screening antiviral germplasm resources based on the infectious clone of PEMV-1 and PEMV-2.

Background

Pea ear mosaic virus No. 1 (PEMV-1) is a virus of the genus Eumosaic virus (Enamovirus) of the family Solemoviridae, and Pea ear mosaic virus No. 2 (PEMV-2) is a virus of the genus Empovirus (Umbravirus) of the family Solanaceae, and the compound infection of the two causes Pea ear mosaic virus (PEMD). PEMDs are widely distributed and reported in the united states, germany, uk, france, czech, etc., which have caused yield loss of peas in england as high as 50% of PEMV-1 and PEMV-2 to infect mainly leguminous plants in the field, including 23 leguminous plants including peas, broad beans, chickpeas, soybeans, and kidney beans. At the same time, PEMV-1 and PEMV-2 also infect non-leguminous plants, such as tomato, pepper, sonchus oleraceus ^[13] Qianliang, benshi tobacco, etc. The infection of the PEMV-1 and the PEMV-2 causes plant growth distortion, growth retardation, leaf yellowing, necrotic spots, chlorosis of leaf veins, ear-shaped protrusions appear on leaf backs and bean pods, the bean pods are often deformed, the seed setting rate is obviously reduced, and serious loss is caused. Both PEMV-1 and PEMV-2 are capable of autonomous self-replication. While PEMV-1 can be transmitted by aphids in a recurrent, non-proliferative manner, PEMV-2 requires PEMV-1 CP for encapsidation for aphid transmission, and PEMV-2 can assist PEMV-1 in systemic and mechanical transmission. The transmission of PEMV (PEMV-1 and PEMV-2) by a variety of aphidsMediators such as pea aphids, cowpea aphids, green peach aphids, eggplant aphids and the like are widely distributed in China and the world, and have large population quantity, thereby causing high epidemic risk of PEMD.

At present, no effective method for preventing and treating PEMD is reported. According to the relevant literature, the use of insecticides to control PEMD has seen little effect on vector aphids, and therefore the best control method is to use disease resistant varieties. Artificial inoculation is the basis of disease-resistant germplasm resource screening and disease-resistant breeding, and is also a common method for researching virus pathogenicity, transmission characteristics and interaction with viruses and vector insects.

The traditional manual friction inoculation of peas by using a virus source has a plurality of limitations, and virus infectious cloning refers to the fact that a gene sequence containing a virus full-length genome is inserted into a specific vector by using a gene recombination technology to obtain a recombinant vector with a host infection function. The virus is easy to infect plants by injection inoculation, but injection inoculation has the problems of high cost, complex operation and the like. The development of a more efficient and more convenient inoculation method is a direction of infectious cloning, and has important significance on the research of virus pathogenic mechanism, disease-resistant germplasm resource screening and the like.

The traditional friction inoculation technology has the following problems: the method is time-consuming and labor-consuming, the leaves need to be rubbed one by one, generally, each plant needs to be rubbed for more than two leaves, and the plants need to be cleaned after the rubbing;

1. the manual friction force is difficult to control, and certain technical difficulty is caused, for example, the excessive force can cause cell damage and even plant death;

2. the source of the virus is impure, and the disease juice obtained by directly grinding the diseased leaves possibly has two or more than two viruses, so that the aim of single pathogen inoculation cannot be achieved;

3. inoculation can not be quantified, the error of virus inoculation amount among different plants in the same batch is large, and the amount of disease juice dipped by each inoculation can not be fixed.

Disclosure of Invention

The invention provides a method for efficiently screening antiviral germplasm resources based on the infectious clone of PEMV-1 and PEMV-2, aiming at solving the defects of the problems. The invention lays a foundation for researching the pathogenicity and the propagation characteristics of the virus and the interaction between the virus and vector insects, and solves the technical problem in screening of the virus-resistant pea germplasm resources.

The invention is realized by adopting the following technical scheme.

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

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

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

a primer for detecting PEMV-2, the primer being:

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

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

the reaction system of the primer of the invention is as follows: 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 ℃.

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

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

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

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

the invention discloses a method for amplifying PEMV-1, which divides the PEMV-1 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 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 GXLDNA Polymerase0.4μL，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 ℃.

The invention discloses a method for amplifying PEMV-2, which divides the PEMV-2 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 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 O 10.8μL，cDNA 2μL。

(the annealing temperature of the primers pCB301-PEMV-2-1F/PEMV-2-1R is 60 ℃, the annealing temperature of the primers PEMV-2-2F/PEMV-2-2R is extended for 2mins, the annealing temperature of the primers PEMV-2-3F/pCB301-PEMV-2-3R is 58 ℃, and the extension time is 1 min), wherein the reaction program is 98 ℃ for 30s; 10s at 98 ℃, 15s at Y ℃, zmin at 68 ℃ and 10min at 68 ℃; storing at 4 deg.C.

A primer set for determining the infectivity of PEMV-1, PEMV-2, said 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'；

the invention relates to a construction method of an auris mosaic virus infectious clone vector, which comprises the following steps: step 1):

extracting total RNA infected with PEMV-1 and PEMV-2 pea plants collected from the university city of Yunnan province;

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;

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;

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;

amplifying three fragments of the PEMV-1 by taking the cDNA as a template and connecting the three fragments to a linearization vector pCB301; the correctly sequenced recombinant plasmid pCB301-PEMV-1 was transformed into Agrobacterium tumefaciens EHA105 by a 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 respectively amplify the fragments PEMV-2-1, PEMV-2-2 and PEMV-2-3 of the PEMV-2;

amplifying three fragments of the PEMV-2 by taking the cDNA as a template and connecting the three fragments to a linearization 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.

Further, the step 6) of the invention is that the agrobacterium rhizogenes is soaked: 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.

The beneficial effects of the invention are as follows:

1. the method is simple and convenient to operate, and has the advantages of saving time and being more convenient than manual friction inoculation;

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

3. in the method, one infectious clone only has the infection activity of one virus, so that the problem of impure virus source is solved, and the experimental condition of requiring specific pathogen inoculation is met;

4. the method has good inoculation effect and high infection rate to plants;

5. the method of the invention ensures that the time for obtaining the virus of the plant is early and the time for accumulating the virus in the plant is long;

6. the method has relatively low cost and short inoculation operation time.

The invention is further explained below with reference to the drawings and the detailed description.

Drawings

FIG. 1 is a schematic view of the root length of pea seeds according to the invention;

FIG. 2 is a schematic view of the root soaking of pea seeds according to the present invention;

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

FIG. 4 is a detection electrophoretogram of PEMV-1 treated differently for different pea varieties according to the present invention; the detection results of PEMV-1 of not puncturing pink podded bean roots, not puncturing pink podded bean roots and not puncturing Taiwan longevity pea roots in lanes 1-9, lanes 10-20 and lanes 21-24 respectively show that the strip size is 570bp;

FIG. 5 is a PEMV-1 detection electrophoretogram of Taiwan longevity pea kernel root treated by non-puncture and puncture; lanes 1-8 and lanes 9-15 are PEMV-1 detection results of no puncture of Royle of longevity peas in Taiwan and puncture of Royle of longevity peas in Taiwan, and the size of the strip is 570bp;

FIG. 6 is a PEMV-2 detection electrophoretogram of different pea varieties subjected to different treatments according to the invention; lanes 1-9, lanes 10-20, and lanes 21-23 show the detection results of PEMV-2 with no puncture of Rohdea roseoflavalia, rohdea roseoflavalia and Rohdea taiwanensis, respectively, and the size of the bands is 693bp;

FIG. 7 is a PEMV-2 detection electrophoretogram of Taiwan longevity pea kernel root treated by non-puncture and puncture; lanes 1-9 and lanes 10-16 are PEMN-1 detection results of no puncture of Taiwan long-life pea kernel root and puncture of Taiwan long-life pea kernel root respectively, and the size of the strip is 693bp;

FIG. 8 is a PEMV-1 detection electropherogram of the root with and without puncture treatment according to the present invention; the detection results of the PEMV-1 with and without root puncture are respectively shown in lanes 1-11 and lanes 12-17, and the size of the strip is 801bp;

FIG. 9 is the electrophoretogram of PEMV-1 and PEMV-2 detection of non-punctured and punctured roots according to the invention; lanes 1-6 are the detection results of PEMV-1 with non-punctured roots, and the size of the band is 801bp;9-17 lanes are the detection results of root-penetrating PEMV-2, and the size of the band is 483bp; + CK positive control failure;

FIG. 10 is a PEMV-2 detection electropherogram of the root with and without puncture treatment according to the present invention; 1-2 lanes show the detection result of root-penetrating PEMV-2, and the size of the band is 483bp; lanes 3-14 are the root-prick-free PEMV-2 detection results; + CK positive failure;

FIG. 11 is a PEMV-1 detection electrophoretogram of PEMV-1 and PEMV-2 inoculated to roots of the invention respectively after different treatments; 1-6 lanes and 7-12 lanes are the detection results of the PEMV-1 which is only inoculated with pCB301-PEMV-1-YDL and has a root punctured and a root not punctured respectively, and the size of a strip is 801bp; lanes 13-17 are the result of PEMV-1 detection in which only pCB301-PEMV-2 was inoculated for root puncture;

FIG. 12 is a graph showing the electrophoresis of the detection of PEMV-1 and PEMV-2 by different treatments of roots according to the present invention, in which only PEMV-2 is inoculated; lanes 1 and 2-3 are the detection results of PEMV-1 inoculated with pCB301-PEMV-2 alone with and without root puncture, respectively; + CK positive control failed; the 6-15 Lane is the detection result of the PEMV-2 inoculated with pCB301-PEMV-2 only and the size of the band is 483bp; + CK positive control failed;

FIG. 13 is the detection electrophoresis chart of the PEMV-1 and PEMV-2 mixed sample of peas frictionally inoculated with the poison sources separately or simultaneously infected with PEMV-1 and PEMV-2 according to the invention; lanes 1-7 are the detection results of PEMV-1 of the mixed sample 1-7, and the size of the band is 801bp; lanes 10-16 are the detection results of PEMV-2 in the mixed sample 1-7, and the size of the band is 483bp;

FIG. 14 is a single sample detection electrophoretogram of PEMV-1 and PEMV-2 obtained by frictionally inoculating peas with a virus source infected with PEMV-1 and PEMV-2 respectively or simultaneously according to the present invention; lanes 1-4 and 5-7 are single sample PEMV-1 detection results of the

mixed samples

1 and 3 in the previous step, respectively, and the band size is 801bp; lanes 10-13 and 14-16 show the single-sample PEMV-2 detection results of the

mixed samples

1 and 3 of the previous step, and the band size is 483bp;

FIG. 15 is a graph of the symptoms of peas inoculated with PEMV-1, PEMV-2 and PEMV-1+ PEMV-2 in accordance with the present invention.

Detailed Description

A construction method of an auris mosaic virus infectious cloning vector comprises the following steps:

extracting total RNA of plants;

(a) Cleaning the surface of the worktable and tools by using 75% ethanol;

(b) 1ml of Trizol reagent is added into a 1.5ml centrifuge tube;

(c) Taking a small amount of the sample, fully grinding the sample in liquid nitrogen, adding the sample into a centrifuge tube after grinding the sample into powder, and shaking and uniformly mixing the sample and the centrifuge tube;

(d) Centrifuging at 12000rpm for 10min at 4 deg.C, transferring the supernatant into a new 1.5ml centrifuge tube, and standing at room temperature for 5min;

(e) Adding 300 mu L of chloroform;

(f) Mixing, standing at room temperature for 2-3min;

(g) Centrifuging at 12000rpm for 15min at 4 deg.C;

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

(i) Adding 100% isopropanol with the same volume, and standing at room temperature for 10min;

(j) Centrifuging at 12000rpm for 10min at 4 deg.C, and removing supernatant;

(k) Suspending the precipitate with 1ml of 75% ethanol, and standing for several minutes;

(l) Repeating the above steps for 2 times;

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

(n) dissolving the RNA in sterile water of RNA-free for use.

cDNA synthesis and virus detection are carried out by using plant total RNA (a primer PEMV-1-801-F/R for predicting the amplified 801bp product and a primer PEMV-2-483-F/R for predicting the amplified 483bp fragment are respectively used for molecular detection of PEMV-1 and PEMV-2);

(a) Using Reverse Transcriptase M-MLV (RNase H) with downstream Primer (Rev) as a Primer or using a random Primer ^- ) (TaKaRa) reverse transcribing the total plant RNA in the above step into cDNA as follows: add 3. Mu.L of dd H first ₂ O, 2 mu LRNA template and 1 mu L random primer, mixing uniformly, placing the system at 70 ℃ for reaction for 10min, quickly taking out of ice for cooling for 2min, centrifuging for several seconds, and adding the following reagents: 2 μ L of 5 XM-MLV Buffer, 1 μ L of dd H ₂ O, 0.5 mu L dNTP mix (10 mM) and RTase M-MLV (200 u/mu L), mixing uniformly after the reagents are added, and placing the system at 42 ℃ for 1h and 75 ℃ for 15min to obtain a cDNA template;

(b) The primer pair PEMV-1-801-F/R for predicting the amplification of 801bp products and the primer pair PEMV-2-483-F/R for expecting the amplification of 483bp fragments are respectively used for the molecular detection of the PEMV-1 and the PEMV-2.

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 a template, and using PEMV-1-801-F/R and PEMV-2-483-F/R to amplify the genomic sequences of the PEMV-1 and the PEMV-2, wherein the specific steps are as follows: first, 5. Mu.L of Tap mix and 3.2. Mu.L of dd H were added ₂ O, 0.4. Mu.L Primer (For) and 0.4. Mu.L LPrimer (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 ℃.

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, mass., china) using the TA cloning strategy, and sequenced by Sanger sequencing. Sequences were assembled and analyzed using EditSeq software (Lasergene 7.0, USA DNASTAR Inc.).

5 'and 3' amplification primers, i.e., PEMV-15'RACE, PEMV-1 3' RACE, PEMV-2 'RACE and PEMV-2' RACE were then designed based on the sequences of the obtained PEMV-1 and PEMV-2, respectively.

Use of

RACE 5/3 (TaKaRa Biotechnology, chinese Dalian) amplified the 5 'and 3' end sequences of the PEMV-1 and PEMV-2 genomes. Cloned into pMD19-T vector and sanger sequenced. The complete nucleotide sequences of PEMV-1 and PEMV-2 were assembled and analyzed by the software Lasergene 7.0.

In order to obtain full-length infectious clones of PEMV-1 and PEMV-2, full-genome sequence amplification primers are designed based on the genome sequences of the PEMV-1 and the PEMV-2 and a binary vector pCB301-2X35S-MCS-HDVRZ-NOS-1, and the PEMV-1 and the PEMV-2 are divided into 3 fragments for amplification, wherein the tail ends of the fragments have nucleotide overlap of 21-33 bp.

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

the PEMV-1-3F/pCB301-PEMV-1-3R is used for amplifying the fragments PEMV-1-1, PEMV-1-2 and PEMV-1-3 of PEMV-1.

In addition, 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 are used for respectively amplifying the fragments PEMV-2-1, PEMV-2-2 and PEMV-2-3 of PEMV-2;

amplification of each fragment of the PEMV-1 and construction of an infectious cloning vector thereof:

using the cDNA as a template, the primers 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 PEMV-1 to obtain three fragments with lengths of 3002bp (PEMV-1-1), 1479bp (PEMV-1-2) and 1317bp (PEMV-1-3).

Wherein, the PCR amplification system 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 Polymerase0.4μL，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 ℃, the annealing temperature of the primer is extended for 3min, the annealing temperature of the primer PEMV-1-2F/PEMV-1-2R is 60 ℃, the annealing temperature of the primer PEMV-1-3F/pCB301-PEMV-1-3R is 60 ℃, and the extension is 2 min) comprises the following reaction procedures: 30s at 98 ℃; 10s at 98 ℃, 15s at Y ℃, and Zmin at 68 ℃;10 min at 68 ℃; storing at 4 deg.C. Then, the desired fragment was recovered using SanPrep Column DNAsel Extraction Kit.

The pCB301 binary vector is cut by TaKaRa restriction enzymes SmaI and Stu I to obtain a pCB301 linear vector, and the reaction system comprises the following steps: expression vector pCB301 plasmid 20. Mu.L (0.8. Mu.g), 1 XQuickcut Green Buffer 5. Mu.L, restriction enzyme Sma I1.0. Mu.L, ddH ₂ O23. Mu.L. The reaction conditions are as follows: incubate at 30 ℃ for 2h. mu.L of Stu I restriction enzyme was added and incubated at 37 ℃ for 2h. And recovering the product by using a SanPrep Column DNASELL Extraction Kit after electrophoretic verification.

Use of

The Ultra One Step Cloning Kit ligated PCR products of three fragments of PEMV-1 into pCB301 linear vector. The pCB301 binary vector is cut by TaKaRa restriction enzymes SmaI and Stu I to obtain a pCB301 linear vector, and the reaction system comprises the following steps: expression vector pCB301 plasmid 20. Mu.L (0.8. Mu.g), 1 XQuickcut Green Buffer 5. Mu.L, restriction enzyme Sma I1.0. Mu.L, ddH ₂ O23. Mu.L. Reaction conditions are as follows: incubate at 30 ℃ for 2h. Additional 1. Mu.L of LStu I restriction enzyme was added and incubated at 37 ℃ for 2h. And recovering the product by using a SanPrep Column DNASELL Extraction Kit after electrophoretic verification.

Use of

The Ultra One Step Cloning Kit ligated PCR products of three fragments of PEMV-1 into pCB301 linear vector. The correctly sequenced recombinant plasmid pCB301-PEMV-1 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method. Wherein, the homologous recombinants are: 2 × Clonexpress Mix 10 μ L, fragment PEMV-1-1 μ L, fragment PEMV-1-2 μ L, fragment PEMV-1-3 μ L, linearizationpCB 301. Mu.L. Reaction conditions are as follows: incubate at 50 ℃ for 30min.

Amplification of PEMV-2 fragment and obtaining of infectious cloning vector thereof

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, obtaining three fragments of about 2264bp (PEMV-2-1), 1344bp (PEMV-2-2) and 740bp (PEMV-2-3) in length.

Wherein, the PCR amplification system is 20 μ 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. (the annealing temperature of the primers pCB301-PEMV-2-1F/PEMV-2-1R is 60 ℃, the annealing temperature of PEMV-2-2F/PEMV-2-2R is 60 ℃, the annealing temperature of the extended primers is 2mins, the annealing temperature of the PEMV-2-3F/pCB301-PEMV-2-3R is 58 ℃, the extension time is 1 min), and the reaction program is 98 ℃ for 30s; 10s at 98 ℃, 15s at Y ℃, zmin at 68 ℃ and 10min at 68 ℃; storing at 4 ℃. Then, the desired fragment was recovered using SanPrep Column DNAsel Extraction Kit.

Using TaKaRa restriction endonucleases Sma I and Stu I to cut the pCB301 binary vector to obtain a pCB301 linear vector, wherein the reaction system comprises the following steps: the expression vector pCB301 plasmid 20. Mu.L (0.8. Mu.g), 1 XQuickcut Green Buffer 5. Mu.L, restriction enzyme Sma I1.0. Mu.L, ddH2O 23. Mu.L. Reaction conditions are as follows: incubate at 30 ℃ for 2h. mu.L of Stu I restriction enzyme was added and incubated at 37 ℃ for 2h. And after electrophoretic verification, the product is recovered by using a SanPrep Column DNA Gel Extraction Kit. Use of

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

Using TaKaRa restriction endonucleases Sma I and Stu I to cut the pCB301 binary vector to obtain a pCB301 linear vector, wherein the reaction system comprises the following steps: the expression vector pCB301 plasmid 20. Mu.L (0.8. Mu.g), 1 XQuickcut Green Buffer 5. Mu.L, restriction enzyme Sma I1.0. Mu.L, ddH2O 23. Mu.L. The reaction conditions are as follows: incubate at 30 ℃ for 2h. mu.L of Stu I restriction enzyme was added and incubated at 37 ℃ for 2h. And after electrophoretic verification, the product is recovered by using a SanPrep Column DNA Gel Extraction Kit.

Use of

The Ultra One Step Cloning Kit connects PCR products of the three fragments of PEMV-2-YDL to pCB301 linear vector. The correctly sequenced recombinant plasmid pCB301-PEMV-2 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method. Wherein, the homologous recombinants are: :2 × Clonexpress Mix 10 μ L, fragment PEMV-2-1 μ L, fragment PEMV-2-2 μ L, fragment PEMV-2-3 μ L, linearized pCB301 vector 7 μ L. Reaction conditions are as follows: incubate at 50 ℃ for 30min.

Transformation of infectious cloning vectors:

(a) Adding 1-10 μ L pCB301-PEMV-1/pCB301-PEMV-2 plasmid into EHA105 competent cell, and standing on ice for 30min;

(b) Standing in liquid nitrogen for 5min;

(c) Water bath at 37 deg.C for 5min;

(d) Standing on ice for 2min;

(e) Adding 890 microliter of LB liquid culture medium without antibiotic into the super clean bench, at 28 deg.C and 220rpm, and shaking for 3-4h;

(f) 5000rpm,5min, removing supernatant, leaving 100 μ L heavy suspension precipitate, spreading the liquid on LB solid culture medium containing 100 μ g/ml Kan + Rif, and performing inverted culture at 28 ℃ for 36-48h;

(g) A single colony was picked and added to 3ml LB liquid medium containing 100. Mu.g/ml Kan + Rif for 36-48h at 28 ℃.

Amplification of infectious cloning vectors:

(a) Preparing a certain number of sterilized test tubes (the amount of LB culture medium enough to soak roots can be filled in the test tubes), and adding the LB culture medium to 2/3 of the test tubes;

(b) Adding 0.1-1ml of transformed bacteria liquid into each test tube, and expanding and shaking for 8-10 hours in a shaking table until the bacteria liquid concentration is proper.

Root-dipping inoculation by agrobacterium based on infectious clones:

(a) Soaking semen Pisi Sativi in water for about 2 days, and allowing the semen Pisi Sativi to emerge with 1-2cm root (figure 1);

(b) Putting the rooted pea seeds into a container, adding a PEMV-containing infectious cloning vector (OD) ₆₀₀ Between 0.5 and 1.0) to just under the root of the pea seed (fig. 2) (before root soaking, the root of the pea seed is punctured, the effect is better, and the specific operation is as follows: gently puncture 3 to 4 holes from the root base to the tip using a fine needle (just an embroidery needle) taking care not to cause too much damage to the root (fig. 1);

(c) Culturing in an incubator set at 28 deg.C and 100% illumination for 3 hr, pouring out LB medium (not completely pouring out to avoid over-drying of cultured seeds), covering a transparent bag at the container mouth, and culturing in the incubator for 6 hr;

(d) And (4) rinsing the cultured seeds once with clear water (removing LB culture medium on the surfaces of the seeds) and then sowing.

Determination of infectivity:

by means of preliminary experiments (OD of LB culture medium containing the infectious clone vectors PEMV-1 and PEMV-2) ₆₀₀ The value is about 0.5, the pea variety 'pink pod king' with the root breaking through the seed coat is cultured for 2 hours at 18 ℃, and the pea variety is continuously placed and cultured for 24 hours), the agrobacterium is subjected to root soaking treatment, and RT-PCR detection is carried out by using primers PEMV1-CP-F/PEMV1-CP-R and PEMV2-ORF3-F/PEMV2-ORF3-R (figure 3), and the detection rates of the PEMV-1 and the PEMV-2 are 83.3 percent and 50 percent respectively.

Followed by a second root-soaking experiment (OD of LB medium containing the infectious clone vectors PEMV-1 and PEMV-2) ₆₀₀ Value of about 0.5, incubation at 28 ℃ for 2h, and further standing for 12 h) (table 1): on the 10 th day after sowing of the root-soaked seeds, PCR detection was performed on two pea varieties inoculated with pCB301-PEMV-1 and pCB301-PEMV-2 by the primers PEMV1-CP-F/PEMV1-CP-R and PEMV2-ORF3-F/PEMV2-ORF3-R, both of which were root-soaked (FIG. 4-FIG. 7). The results are shown in the following table:

TABLE 1 systematic infestation of PEMV-1 and PEMV-2 on different pea varieties treated differently

The second batch of detection results show that the pea auris mosaic virus in the experiment can successfully infect pea plants, and the effect is good, which shows that the inoculation of pea plants by an agrobacterium rhizophoresis method based on infectious clone of the invention has feasibility. The detection rate of the root puncture treatment is higher than that of the non-puncture treatment by combining the detection results of the PEMV-1 and the PEMV-2, which shows that the root puncture treatment is favorable for the infection of the PEMV.

Thereafter, a third root-soaking experiment (OD of LB medium containing the infectious clone vectors PEMV-1 and PEMV-2) was performed ₆₀₀ A value of about 0.8, 3 hours at 28 ℃ and 6 hours for further standing cultivation), in addition to the improvement in the treatment conditions (fig. 8-10), separate root-soaking inoculation (14 days after sowing) (fig. 11-12) of pCB301-PEMV-1 and pCB301-PEMV-2 and combined rubbing inoculation and separate rubbing inoculation (14 days after rubbing inoculation) of pCB301-PEMV-1 and pCB301-PEMV-2 were set (table 2); mix 1-4 was inoculated with pCB301-PEMV-1 and pCB301-PEMV-2 simultaneously, mix 5 was inoculated with pCB301-PEMV-1 only, and mix 6-7 was inoculated with pCB301-PEMV-2 only (FIGS. 13-14) (Table 3). The results are shown in the following table:

TABLE 2 systematic infestation of PEMV-1 and PEMV-2 on differently treated pea plants

TABLE 3 systematic infestation on pea plants after tribo-inoculation of PEMV-1 and PEMV-2

The test result of the third batch of root soaking inoculation shows that the PEMV has higher infection rate, wherein the PEMV infection rate of the root puncture treatment is greater than that of the non-puncture treatmentAnd the infection rate of the PEMV-2 is greater than the detection rate of the PEMV-1, and the detection result of the second batch is that the detection rate of the PEMV-1 is greater than that of the PEMV-2, and the presumption possibly is that the OD of the bacterial liquid ₆₀₀ The difference caused by the difference in value. In addition, pea can be successfully infected when the PEMV-1 or the PEMV-2 is singly used for root soaking and inoculation, and the infection rate is high. From the results of the tribological inoculation, the infection rates of PEMV-1 and PEMV-2 are significantly low. Pea plants started to have symptoms 20 days after PEMV-1+ PEMV-2 is simultaneously inoculated in root soaking, but pea plants inoculated with PEMV-1 or PEMV-2 alone have no symptoms. (FIG. 15). The above results show that compared with the friction inoculation method, the inoculation method of the present invention enables PEMV to effectively infect pea plants, which is convenient and time-saving.

Full-length sequence of PEMV-1: (Gene accession No. OP 113929);

full-length sequence of PEMV-2: (Gene accession No.: OP 113928).

The foregoing is only a part of the specific embodiments of the present invention, and the specific contents or common general knowledge in the schemes are not described herein too much (including but not limited to the shorthand, abbreviation, units commonly used in the art). It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

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.