CN113528568B - Jute yellow vein virus infectious clone and construction method thereof - Google Patents
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Abstract
The invention provides a jute yellow vein virus invasive clone and a construction method thereof, belonging to the technical field of plant genetic engineering. The invention constructs the invasive clones pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B of jute vein virus for the first time, and the invasive clones can lead the inoculated jute to generate symptoms such as vein, flower leaf, mottle, leaf curl and the like, which lays a foundation for researching the structure and function of Huang Mahuang vein virus genome and the pathogenic mechanism thereof.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a jute vein virus invasive clone and a construction method thereof.
Background
Huang Mahuang the vein virus (Corchorus yellow vein Vietnam virus, coYVV) is a single-stranded, circular plant DNA gemini virus belonging to the genus Phaseolus golden mosaic virus whose genome is two-component (i.e., DNA-A and DNA-B). CoYVV was first found in 2006 on Jute in Vietnam and was also identified in 2015 in Fujian area of China. Although a virus found in the old world, coYVV has the characteristic of a typical new world gemini virus, i.e., does not encode the AV2 protein. Jute (Corchorus capsularis), also known as green hemp, etc., is an important fiber crop of the genus jute (Corchord) of the family Tiliaceae, and the breadth of planting and use is inferior to cotton, which is widely cultivated in the south of the Yangtze river in China. CoYVV can infect jute and cause symptoms such as leaf vein and plant dwarf, and the like, influence the yield and quality of the jute and bring potential threat to the jute industry in China. Therefore, the research on the genome structure, the function and the pathogenic mechanism of CoYVV has important significance for jute production. The construction of virus infectious clones is the primary choice for studying the pathogenic mechanism of viruses.
Disclosure of Invention
The invention aims to provide a jute vein virus infectious clone and a construction method thereof. The invention constructs the infectious clone of the jute yellow vein virus for the first time, and lays a foundation for researching the structure and the function of the virus genome and the pathogenic mechanism thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the construction method of the jute yellow vein virus infectious clone mainly comprises the following steps:
(1) Extracting total DNA of jute leaves, taking the total DNA as a template, designing specific primers CoYVV-DNA-A-F1/CoYVV-DNA-A-R1 and CoYVV-DNA-A-F2/CoYVV-DNA-A-R2, and carrying out PCR amplification to obtain a fragment A with the length of 2.7kb and a fragment B with the length of 0.5 kb.
(2) And (3) connecting the fragment A and the fragment B after gel purification with an enzyme digestion product pBINPLUS-SB obtained by double enzyme digestion of a pBINPLUS vector by BamH I and Sma I by utilizing a multi-fragment seamless cloning technology, extracting plasmids after converting competent cells of escherichia coli by a connection product heat shock method, verifying recombinant plasmids by utilizing BamH I and Sma I double enzyme digestion, and screening the obtained positive clone to obtain DNA-A invasive clone, wherein the DNA-A invasive clone is marked as pBINPLUS-CoYVV-1.2A.
(3) Taking the total DNA of jute leaves as a template, performing Rolling Circle Amplification (RCA) by using phi 29DNA polymerase, and performing enzyme digestion on an amplified product by using BamH I to obtain a fragment of 2.7kb, and connecting the fragment with a pTOPO vector to obtain the pTOPO-DNA-B.
(4) Double digestion of pTOPO-DNA-B with BamH I and Sma I was performed, 2.6. 2.6kb fragment C was recovered after agarose gel electrophoresis, fragment C was ligated to the digested product pBINPLUS-SB obtained by double digestion of pBINPLUS vector with BamH I and Sma I, plasmid was extracted after transformation of E.coli competent cells by ligation product heat shock, and positive clones were selected by double digestion with BamH I and Sma I and labeled pBINPLUS-DNA-B.
(5) The pBINPLUS-DNA-B plasmid was digested singly with BamH I, recovered by agarose gel electrophoresis, and subjected to dephosphorylation.
(6) And (3) carrying out single enzyme digestion on pTOPO-DNA-B by utilizing BamH I, recovering 2.7kb fragment D after agarose gel electrophoresis, connecting the fragment D with the dephosphorylated vector in the step (5), extracting plasmids after converting escherichia coli competent cells by a connection product heat shock method, detecting the inserted fragments and the directions thereof by utilizing a primer B-F570/B-R570, and screening positive clones to obtain DNA-B invasive clones, wherein the DNA-B invasive clones are marked as pBINPLUS-CoYVV-2.0B.
(7) The invasive clones pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B containing DNA-A and DNA-B were transferred into competent cells of Agrobacterium GV3101, respectively, by electric shock; treating agrobacterium bacterial liquid containing virus DNA-A and DNA-B infectious clone by using an inoculation buffer solution, and inoculating jute leaves with 2-3 true leaves just growing, so that the inoculated jute produces yellow vein, flower leaves, mottle and leaf rolling symptoms; and viral components were detected using PCR and Southern blot.
The specific primer sequences in the step (1) are as follows:
CoYVV-DNA-A-F1：5’-CAGGTCGACTCTAGAGGATCCACCGTGCAGCAGCCCCGC-3’；
CoYVV-DNA-A-R1：5’-GGCTGCTGCACGGTAATATTATAGGCGTGCAGCAGCG-3’；
CoYVV-DNA-A-F2：5’-AATATTACCGTGCAGCAGCCCCGC-3’；
CoYVV-DNA-A-R2：5’-AATTCGAGCTCGGTACCCGGGTGACCTTCCCTGTATGAGCA-3’。
the primer sequence in the step (6) is as follows:
B-F570：5’-GACGAACCGTCACGTGCATCCACG-3’；
B-R570：5’-TGTCGACAGCAAAGCACTCTGTTG-3’。
the PCR detection primer sequence in the step (7) is as follows:
A-F534：5’-AGGATCTATCGGACCTATAGGTCC-3’；
A-R534：5’-AGGATTAGAGGCATGAGTACATGC-3’；
B-F673：5’-ACGAATATCGTCTGACTCATGACC-3’；
B-R673：5’-TCCAGCATACTTGTGCTGAGTCTG-3’。
the obtained jute yellow vein virus infectious clone is constructed by the method.
Further, the method constructs the obtained jute yellow vein virus invasive clone pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B.
The jute vein virus infectious clone is applied to the research on the structure and the function and the pathogenic mechanism of the jute vein virus genome.
Compared with the prior art, the invention has the beneficial effects that:
the invention constructs the invasive clones pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B of jute vein virus for the first time, and the invasive clones can lead the inoculated jute to generate symptoms such as vein, flower leaf, mottle, leaf curl and the like, which lays a foundation for researching the structure and function of Huang Mahuang vein virus genome and the pathogenic mechanism thereof.
Description of the drawings:
FIG. 1 CoYVV DNA-A and DNA-B infectious clone construction schematic.
FIG. 2 shows the result M of the restriction enzyme digestion (DNA-A) and PCR (DNA-B) identification of the invasive cloning vector, which represents the molecular weight standard; lane 1 represents DNA-A cleavage assay; lane 2 represents DNA-B PCR detection.
FIG. 3 infectious clone induces the host plant to produce symptoms CK representing healthy jute; DNA-A+DNA-B represents virus-infectious clones inoculated with Corchorus olitorius.
FIG. 4 PCR detection M of invasive clone inoculated jute represents molecular weight standard; lanes 1-3 represent DNA-A; lanes 4-6 represent DNA-B; CK stands for healthy jute.
FIG. 5 Southern blot detection of viral DNA-A components after inoculation of the invasive clone with jute. Lane 1 represents virus inoculated jute; CK stands for healthy jute.
Detailed Description
The invention is further described below with reference to examples, which are provided to illustrate the invention and not to limit the invention.
Example 1
The construction method of the jute yellow vein virus infectious clone mainly comprises the following steps:
(1) Extraction of total DNA from plants (CTAB method)
Taking fresh leaf tissue of a disease plant, namely 0.1 g, loading the fresh leaf tissue into a centrifuge tube, adding steel balls, soaking the fresh leaf tissue in liquid nitrogen for 2 minutes, fixing the centrifuge tube by using a centrifuge tube rack precooled in advance, and shaking the centrifuge tube forcefully to fully crush a sample; 1 mL of a 2% (w/v) CTAB extract (containing 2% (v/v) beta-mercaptoethanol, 1% (w/v) PVP-360) preheated at 65℃was added, incubated at 65℃for 30 min, gently inverted several times every 15 min; taking out the steel balls, centrifuging at 12,000 rpm for 10 min, transferring the supernatant to a new 1.5 mL centrifuge tube, adding chloroform and isoamyl alcohol (volume ratio is 24:1) which are equal to the supernatant in volume, and uniformly mixing by vortex vibration for 30 s until the water is milky; centrifuging at 10,000 rpm for 10 min, transferring the upper aqueous phase to a new centrifuge tube; adding 2/3 volume of isopropanol into the water phase, gently inverting for several times, separating out floccules, centrifuging at 10,000 rpm for 10 min, sucking out the supernatant, and retaining the following precipitate; adding 1 mL of 75% ethanol into the precipitate, centrifuging at 10,000 rpm for 5min, sucking out the supernatant, reserving the precipitate, and naturally drying at room temperature for 10-15 min; adding 30-50 mu L of the solution Buffer, and carrying out vortex oscillation for 2 min until the solution is fully and uniformly mixed, thus obtaining the total DNA of the plant.
(2) Construction of DNA-A invasive clones
1) The total plant DNA is used as a template, and primers CoYVV-DNA-A-F1 (5'-CAGGTCGACTCTAGAGGATCCACCGTGCAGCAGCCCCGC-3')/CoYVV-DNA-A-R1 (5'-GGCTGCTGCACGGTAATATTATAGGCGTGCAGCAGCG-3') and CoYVV-DNa-F2 (5'-AATATTACCGTGCAGCAGCCCCGC-3')/CoYVV-DNA-A-R2 (5'-AATTCGAGCTCGGTACCCGGGTGACCTTCCCTGTATGAGCA-3') are used for amplifying DNA-A respectively to obtain a fragment A with the length of 2.7kb and a fragment B with the length of 0.5kb respectively, wherein the amplification system is as follows: 1. Mu.L of total DNA, 1. Mu.L of forward primer (10. Mu.M), 1. Mu.L of reverse primer (10. Mu.M), 0.5. Mu.L of dNTP Mix, 2X Phanta Max Buffer 12.5.5. Mu.L, phanta Max Super-Fidelity DNA Polymerase, ddH 2 O8. Mu.L. The reaction procedure is: pre-denaturation at 95℃for 3 min, denaturation at 95℃for 15 sec, annealing at 52℃for 15 sec, extension at 72℃for 1 min40 sec,35 cycles, and a final extension of 10 min at 72 ℃. And (5) recovering agarose gel.
2) The pBINPLUS vector was double digested with FastDigest BamH I and FastDiget Sma I, and the cleavage reaction system was: 1. Mu.g of plasmid DNA, bamH I1. Mu.L, sma I1. Mu.L, 10X FastDigest Green buffer. Mu.L, ddH 2 O was added to 20. Mu.L, and the reaction was carried out at 37℃for 30 min. The vector cleavage product was labeled pBINPLUS-SB and recovered by agarose gel.
3) The gel-purified fragments A and B and the cleavage product pBINPLUS-SB were mixed in a defined ratio according to the instructions of the multi-fragment seamless cloning kit (MultiS One Step Cloning Kit). The reaction system is as follows: 5 XCE MultiS Buffer 4. Mu.L, exnase 2. Mu.L, fragment A54 ng, fragment B10 ng, cleavage product pBINPLUS-SB 240 ng, ddH 2 O was added with water to 20. Mu.L, and the reaction was carried out at 37℃for 30 min. After transformation of E.coli DH 5. Alpha. Competent cells by ligation, plasmids were extracted and verified by double restriction enzyme digestion with FastDigest BamH I and FastDiget Sma I to obtain 12 kb vector fragment and 3.3 kb fragment (FIG. 2), indicating successful construction of DNA-A invasive clone, labeled pBINPLUS-CoYVV-1.2A.
(3) Construction of DNA-B invasive clones
1) The total DNA of plants is taken as a template, rolling Circle Amplification (RCA) is carried out by utilizing phi 29DNA polymerase (GE Healthcare) according to the instruction of a kit, the amplified product is subjected to enzyme digestion by utilizing FastDigest BamH I to obtain a fragment of 2.7kb, and the fragment is connected with a pTOPO vector, namely pTOPO-DNA-B.
2) The pTOPO-DNA-B was double digested with FastDigest BamH I and FastDiget Sma I, digested with the same reagents as above, and after agarose gel electrophoresis, the smaller fragment C, 2.6kb, was recovered and ligated into the digested product pBINPLUS-SB, the ligation system was as follows: fragment C6. Mu.L, pBINPLUS-SB 2. Mu.L, T4 Ligase 1. Mu.L, 10 XT 4 Ligase Buffer 1. Mu.L, total volume 10. Mu.L, and ligation product heat shock method were used to transform competent cells of E.coli, plasmids were extracted, positive clones were screened using FastDigest BamH I and FastDiget SmaI double restriction verification, and labeled pBINPLUS-DNA-B.
3) The pBINPLUS-DNA-B plasmid was subjected to single cleavage by FastDigest BamH I, and after recovery by agarose gel electrophoresis, it was subjected to dephosphorylation treatment in the following manner: 17.5 mu L of single enzyme cutting carrier fragment, 2 mu L of 10 XCIAP buffer solution, 0.5 mu L of CIAP (TaKaRa), 20 mu L of total volume, reacting for 30 min at 37 ℃, and purifying by column.
4) The pTOPO-DNA-B was subjected to single cleavage using FastDigest BamH I in the following manner: 1. Mu.g of plasmid DNA, 1. Mu.L of BamHI, 10X FastDigest Green buffer. Mu.L, ddH 2 O was added to 20. Mu.L, and the reaction was carried out at 37℃for 30 min. The smaller fragment D, 2.7kb, was recovered after agarose gel electrophoresis. The fragment D was ligated with the pBINPLUS-DNA-B vector from the previous step, and after transformation of E.coli competent cells by ligation product heat shock, the plasmid was extracted, and the insert and its orientation were detected using primers B-F570 (5'-GACGAACCGTCACGTGCATCCACG-3')/B-R570 (5'-TGTCGACAGCAAAGCACTCTGTTG-3'), resulting in a fragment of 0.5kb (FIG. 2), indicating successful construction of DNA-B invasive clone, labeled pBINPLUS-CoYVV-2.0B.
(4) Transformation of invasive clones
The two invasive clones pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B obtained above were transferred into competent cells of Agrobacterium GV3101, respectively, using electric shock. The method comprises the following specific steps: 1. Mu.L of infectious clone recombinant plasmid is added into 50. Mu.L of agrobacteria competence melted on ice; drying the electric shock cup in a baking oven at 25 ℃ for 10 min, and pre-cooling on ice for 5 min; adding the plasmid-containing competence into a electric shock cup, drying the cup body, and carrying out electric shock; adding 600 mu L of antibiotic-free LB culture medium to a electric shock cup, uniformly mixing, and sucking out to a 1.5 mL centrifuge tube; shake culturing at 28deg.C to recover thallus 1 h; mu.L of LB medium plates containing Kan (50. Mu.g/mL) and Rif (50. Mu.g/mL) were plated by pipetting; and (5) culturing the strain at 28 ℃ for 36-48 hours until colonies are visible.
(5) Agrobacterium inoculation and detection
1) Inoculating agrobacteria solution containing virus DNA-A and DNA-B infectious clone into 5 mL LB liquid medium containing Kan (50 μg/mL) and Rif (50 μg/mL), respectively, and shaking culturing at 28deg.C at 220 rpm overnight to OD 600 And approximately 1.0. Centrifuging the bacterial liquid at 5,000 rpm for 5min, discarding supernatant, and retaining precipitate. Adding a certain volume of inoculation buffer solution to suspend and precipitate to make OD 600 And (3) standing at room temperature for 2-5 h until the value reaches 1.0. The inoculation buffer solution comprises the following components: 10 mM MES, 200. Mu.M Acetostinone and 10 mM MgCl 2 . During inoculation, a 1 mL disposable injector with a medical needle removed is adopted, agrobacterium tumefaciens bacteria liquid of DNA-A and DNA-B which are mixed in equal proportion is absorbed, leaf infiltration method is adopted to inoculate jute leaves with 2-3 true leaves just grown, the inoculated jute is placed in an insect-proof greenhouse for cultivation, meanwhile, non-inoculated control is arranged, and symptoms are observed regularly. 30 days after inoculation, the inoculated jute produced yellow veins, flowers and leaves, mottle, volume She Zhengzhuang (fig. 3).
2) 60 days after inoculation, the jute leaves were collected to extract their total DNA, and PCR detection of the two viral components was performed using primers A-F534 (5'-AGGATCTATCGGACCTATAGGTCC-3')/A-R534 (5'-AGGATTAGAGGCATGAGTACATGC-3') and B-F673 (5'-ACGAATATCGTCTGACTCATGACC-3') B-R673 (5'-TCCAGCATACTTGTGCTGAGTCTG-3'). The results showed that the specific bands for DNA-A and DNA-B were detected for both inoculated jute, whereas healthy plants did not have any bands (FIG. 4), indicating that the constructed invasive clones had successfully infected jute.
3) Meanwhile, after 60 days of inoculation, jute leaves 0.5. 0.5 g are collected, and total genomic DNA is extracted for Southern blot (Roche DIG High Prime Lab/Detection Kit) Detection. The results showed that the accumulation of viral DNA-A components was successfully detected by inoculating the jute plants, whereas healthy jute plants were unable to detect any specific bands (FIG. 5), indicating that the constructed invasive clones had been largely replicated in jute bodies.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Fujian university of agriculture and forestry
<120> a jute yellow vein virus infectious clone and its construction method
<130> 10
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> DNA
<213> CoYVV-DNA-A-F1
<400> 1
caggtcgact ctagaggatc caccgtgcag cagccccgc 39
<210> 2
<211> 37
<212> DNA
<213> CoYVV-DNA-A-R1
<400> 2
ggctgctgca cggtaatatt ataggcgtgc agcagcg 37
<210> 3
<211> 24
<212> DNA
<213> CoYVV-DNA-A-F2
<400> 3
aatattaccg tgcagcagcc ccgc 24
<210> 4
<211> 41
<212> DNA
<213> CoYVV-DNA-A-R2
<400> 4
aattcgagct cggtacccgg gtgaccttcc ctgtatgagc a 41
<210> 5
<211> 24
<212> DNA
<213> B-F570
<400> 5
gacgaaccgt cacgtgcatc cacg 24
<210> 6
<211> 24
<212> DNA
<213> B-R570
<400> 6
tgtcgacagc aaagcactct gttg 24
<210> 7
<211> 24
<212> DNA
<213> A-F534
<400> 7
aggatctatc ggacctatag gtcc 24
<210> 8
<211> 24
<212> DNA
<213> A-R534
<400> 8
aggattagag gcatgagtac atgc 24
<210> 9
<211> 24
<212> DNA
<213> B-F673
<400> 9
acgaatatcg tctgactcat gacc 24
<210> 10
<211> 24
<212> DNA
<213> B-R673
<400> 10
tccagcatac ttgtgctgag tctg 24
Claims (5)
1. The construction method of the jute yellow vein virus invasive clone is characterized by mainly comprising the following steps:
(1) Extracting total DNA of jute leaves, taking the total DNA as a template, designing specific primers CoYVV-DNA-A-F1/CoYVV-DNA-A-R1 and CoYVV-DNA-A-F2/CoYVV-DNA-A-R2, and carrying out PCR amplification to obtain a fragment A with the length of 2.7kb and a fragment B with the length of 0.5kb respectively;
(2) Connecting the fragment A and the fragment B after gel purification with an enzyme digestion product pBINPLUS-SB obtained by double enzyme digestion of a pBINPLUS vector by BamH I and Sma I by utilizing a multi-fragment seamless cloning technology, extracting plasmids after converting competent cells of escherichia coli by a connection product heat shock method, verifying recombinant plasmids by utilizing BamH I and Sma I double enzyme digestion, and screening the obtained positive clone to obtain DNA-A invasive clone, wherein the DNA-A invasive clone is marked as pBINPLUS-CoYVV-1.2A;
(3) Performing rolling circle amplification by taking the total DNA of jute leaves as a template and utilizing phi 29DNA polymerase, and performing enzyme digestion on an amplification product by utilizing BamH I to obtain a 2.7kb fragment which is connected with a pTOPO vector to obtain pTOPO-DNA-B;
(4) Double digestion of pTOPO-DNA-B with BamH I and Sma I, recovery of 2.6kb fragment C after agarose gel electrophoresis, ligation of fragment C to the digested product pBINPLUS-SB from double digestion of pBINPLUS vector with BamH I and Sma I, transformation of E.coli competent cells by ligation product heat shock, extraction of plasmid, double digestion with BamH I and Sma I, screening of positive clones, labeling of pBINPLUS-DNA-B;
(5) Single enzyme cutting is carried out on the pBINPLUS-DNA-B plasmid by utilizing BamH I, and after agarose gel electrophoresis recovery, dephosphorylation treatment is carried out;
(6) Single enzyme cutting pTOPO-DNA-B by BamH I, recovering 2.7kb fragment D after agarose gel electrophoresis, connecting the fragment D with the dephosphorylated vector in the step (5), extracting plasmid after converting escherichia coli competent cells by a connection product heat shock method, detecting the inserted fragment and the direction thereof by using a primer B-F570/B-R570, screening positive clone, namely DNA-B invasive clone, and marking as pBINPLUS-CoYVV-2.0B;
(7) The invasive clones pBINPLUS-CoYVV-1.2A and pBINPLUS-CoYVV-2.0B containing DNA-A and DNA-B were transferred into competent cells of Agrobacterium GV3101, respectively, by electric shock; the agrobacterium solution containing virus DNA-A and DNA-B infectious clone is treated by an inoculation buffer solution, and then is inoculated with jute leaves with 2-3 true leaves just growing, so that the inoculated jute produces yellow vein, flower leaves, mottle and leaf rolling symptoms; and detecting viral components using PCR and Southern blot;
the specific primer sequence in the step (1) is as follows:
CoYVV-DNA-A-F1：5’-CAGGTCGACTCTAGAGGATCCACCGTGCAGCAGCCCCGC-3’；CoYVV-DNA-A-R1：5’-GGCTGCTGCACGGTAATATTATAGGCGTGCAGCAGCG-3’；
CoYVV-DNA-A-F2：5’-AATATTACCGTGCAGCAGCCCCGC-3’；
CoYVV-DNA-A-R2：5’-AATTCGAGCTCGGTACCCGGGTGACCTTCCCTGTATGAGCA-3’。
2. the method for constructing a jute vein virus infectious clone according to claim 1, wherein the method comprises the following steps: the primer sequences in step (6) are as follows:
B-F570：5’-GACGAACCGTCACGTGCATCCACG-3’；
B-R570：5’-TGTCGACAGCAAAGCACTCTGTTG-3’。
3. the method for constructing a jute vein virus infectious clone according to claim 1, wherein the method comprises the following steps: the PCR detection primer sequence in the step (7) is as follows:
A-F534：5’-AGGATCTATCGGACCTATAGGTCC-3’；
A-R534：5’-AGGATTAGAGGCATGAGTACATGC-3’；
B-F673：5’-ACGAATATCGTCTGACTCATGACC-3’；
B-R673：5’-TCCAGCATACTTGTGCTGAGTCTG-3’。
4. a jute yellow vein virus infectious clone constructed by the method of claim 1.
5. The use of the invasive clone of jute vein virus according to claim 4 in research of structure and function of jute vein virus genome and its pathogenic mechanism.
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Citations (2)
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| WO2017136895A1 (en) * | 2016-02-10 | 2017-08-17 | Queensland University Of Technology | Constructs and methods for conferring virus resistance |
| CN110669776A (en) * | 2019-10-16 | 2020-01-10 | 河南省农业科学院植物保护研究所 | Sweet potato Hubei leaf curl virus infectious clone and construction method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017136895A1 (en) * | 2016-02-10 | 2017-08-17 | Queensland University Of Technology | Constructs and methods for conferring virus resistance |
| CN110669776A (en) * | 2019-10-16 | 2020-01-10 | 河南省农业科学院植物保护研究所 | Sweet potato Hubei leaf curl virus infectious clone and construction method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Infectious clone construction and pathogenicity confirmation of Cotton leaf curl Multan virus (CLCuMuV), Ramie mosaic virus (RamV) and Corchorus yellow vein Vietnam virus (CoYVV) by southern blot analysis;Muhammad Arif等;PLoS One;第16卷(第5期);1-10 * |
| 黄麻黄脉病毒侵染性克隆的构建及应用;林文忠等;福建农林大学学报(自然科学版);第51卷(第3期);317-321 * |
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