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Tomato yellow leaf curl virus isolate TYLCV-BJ, infectious clone construction method and application

A TYLCV-BJ, TYLCV-BJ-FW-F technology, applied in the field of tomato yellow leaf curl virus isolate and its invasive clone construction, can solve unfavorable experiment development, TYLCV-SH2 infectivity is not strong, etc. question

Inactive Publication Date: 2020-05-15
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the known tomato yellow leaf curl virus TYLCV-SH2 is not very infective, which is not conducive to the development of experiments.

Method used

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  • Tomato yellow leaf curl virus isolate TYLCV-BJ, infectious clone construction method and application
  • Tomato yellow leaf curl virus isolate TYLCV-BJ, infectious clone construction method and application
  • Tomato yellow leaf curl virus isolate TYLCV-BJ, infectious clone construction method and application

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Experimental program
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Effect test

Embodiment Construction

[0029] Tomato Yellow Leaf Curl Virus Isolate TYLCV-BJ and Construction Method of Infectious Clones:

[0030] (1) Take tomato samples suspected of geminivirus infection from the greenhouse;

[0031] (2) Utilize CTAB to extract the DNA of the tomato sample;

[0032] (3) Take 100ng of tomato sample DNA, use the geminivirus merger primer PA / PB to amplify a DNA fragment of about 500bp, connect it to the Quan's gold pEASY-Blunt Simple Cloning T vector (referred to as the T vector), and transform the recombinant vector into In Escherichia coli DH5a, use universal primers M13-F and M13-R (as shown in SEQ ID: No.12-13) to screen positive clones; select positive clones and culture them; extract and cultivate the plasmids of the target clones; through DNA sequencing Verify; obtain the sequence SEQ ID: as shown in No.1;

[0033] M13-F: TGTAAAACGACGGCCAGT

[0034] M13-R: CAGGAAACAGCTATGACC

[0035] PA: TAATATTACCKGWKGVCCSC

[0036] PB: TGGACYTTRCAWGGBCCTTCACA

[0037] *B=C, T or G, K...

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Abstract

The invention discloses a tomato yellow leaf curl virus isolate TYLCV-BJ, an infectious clone construction method and an application. The tomato yellow leaf curl virus isolate TYLCV-BJ has a whole genome sequence as shown in SEQ ID NO: 2 in a sequence table, and has a preservation number of CGMCC NO. 19119. The infectious clone construction method comprises the following steps: constructing 1.4 TYLCV-BJ genome sequences which are repeated in series onto a vector pCambia2300 so as to obtain a pCambia2300-TYLCV-BJ-1.4 DNA recombinant plasmid; and introducing the pCambia2300-TYLCV-BJ-1.4 DNA recombinant plasmid into agrobacterium tumefaciens EHA105 so as to obtain agrobacterium tumefaciens capable of successfully infecting tobacco and tomato plants. The recombinant vector pCambia2300-TYLCV-BJ-1.4 DNA has the following characteristics: 1) the recombinant vector pCambia2300-TYLCV-BJ-1.4 DNA has infectivity and can successfully infect tobacco, tomatoes and arabidopsis thaliana plants; 2) therecombinant vector pCambia2300-TYLCV-BJ-1.4 DNA can induce obvious virus symptoms on tobaccos, tomatoes and arabidopsis plants and accumulate DNA of virus; 3) the recombinant vector pCambia2300-TYLCV-BJ-1.4 DNA can be used for researching the functions of virus genes by mutating virus genes; and 4) the recombinant vector pCambia2300-TYLCV-BJ-1.4 DNA can be used as a virus source for screening anti-virus tomato varieties and anti-virus arabidopsis mutants.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a tomato yellow leaf curl virus isolate and a construction method and application of an infective clone thereof. Background technique [0002] Tomato yellow curl virus (Tomato yellow curl virus, TYLCV) is a single-stranded, circular plant DNA virus, which belongs to the family Geminiviridae and belongs to the genus Begomovirus. TYLCV can infect a variety of vegetable crops and economic crops of Solanaceae, and is an important pathogen in agricultural production in my country and the world. In general, to study the pathogenicity and function of plant viruses, it is necessary to first obtain an infectious clone of the virus. [0003] Infectious cloning of viruses is the construction of infectious viruses in vitro by means of molecular biology and genetic engineering techniques. However, the known tomato yellow leaf curl virus TYLCV-SH2 has weak infectivity, which is not conduci...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/34C12N15/82C12N15/66C12N1/21C12R1/01
CPCC12N7/00C12N15/66C12N15/8205C12N2750/12021
Inventor 李方方龚攀李浩周雪平
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI