Verticillium dahliae adenylate kinase target gene fragment and interference vector and application thereof

An adenylate kinase, Verticillium dahliae technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of rapid mutation, no very effective control method, complex pathogenic mechanism, etc. disease effects

Active Publication Date: 2016-01-20
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large number of physiological races, rapid variation, and complex pathogenic mechanism, there is no very effective control method at present.

Method used

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  • Verticillium dahliae adenylate kinase target gene fragment and interference vector and application thereof
  • Verticillium dahliae adenylate kinase target gene fragment and interference vector and application thereof
  • Verticillium dahliae adenylate kinase target gene fragment and interference vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Screening of the adenylate kinase target gene of Verticillium dahliae

[0051] 1. Experimental method

[0052] 1.1 Construction of VIGS interference vector

[0053] In order to screen the target gene segment with the best interference effect, 4 pairs of specific primers (Table 1) were designed according to the coding sequence of Verticillium dahliae adenylatekinase (AK, VDAG_01040.1), with EcoRI and BamHI enzymes at both ends of the primers According to the cut site, the target fragments were amplified by PCR. Then use 1% agarose gel electrophoresis to detect PCR amplification products, and carry out fragment recovery. The target fragment and the vector were digested separately, and the T 4 Ligase constructs it into the TRV2 vector. Finally, the verified positive plasmid was transformed into Agrobacterium GV3101 by enzyme digestion and sequencing analysis.

[0054] Table 1 Primer information for different segments of AK

[0055]

[0056] Note: The sit...

Embodiment 2

[0074] Construction and plant transformation of embodiment 2Gateway interference vector

[0075] 1. Experimental method

[0076] 1.1 Construction of stable genetic vector

[0077] In order to obtain stably inherited Nicotiana benthamiana containing the target gene dsRNA, according to the change of the tobacco disease index of transient transformation in Example 1, two DNA segments (nucleotide sequences respectively SEQ ID No. .1, shown in SEQIDNo.4), redesign primers (both ends contain part of BP sites), and then use attb primers to amplify (Table 3), for the construction of stable genetic interference vectors. According to the BP reaction, the target sequence was ligated into pDONR207; then it was constructed into pK7GWIWG2(I),0 by the LR reaction. Finally, the constructed vector was transformed into Agrobacterium LBA4404.

[0078] Table 3 Stable genetic interference primer information

[0079]

[0080] 1.2 Transformation of Nicotiana benthamiana

[0081] Take the lea...

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Abstract

The invention discloses a verticillium dahliae adenylate kinase target gene fragment and an interference vector and application thereof, and belongs to the fields of clone and application of verticillium dahliae disease course key genes. A host-induced gene silencing technology is adopted to screen the target gene fragment which can obviously lower the disease index in adenylate kinase genes; the Gateway interference vector is further established, and then stably inherited transgenic tobaccos of which the resistance to pathogenic bacteria is obviously improved are obtained; through the disease index, fungal biomass analysis and target gene transcriptional level detection, the adenylate kinase target gene interference fragment with the optimal verticillium dahliae resisting effect is finally screened. The verticillium dahliae adenylate kinase target gene fragment and the RNA interference vector can be applied to improving the disease resistance of a plant to verticillium dahliae and cultivating new transgenic plant species with the verticillium dahliae resistance.

Description

technical field [0001] The present invention relates to the target gene fragment of Verticillium dahliae adenylate kinase and the RNA transcribed by the target gene fragment, and also relates to an interference vector containing the target gene fragment of adenylate kinase. The present invention further relates to the Verticillium dahliae The application of mycobacteria-related adenylate kinase target gene fragments or interference vectors in improving the disease resistance of plants to Verticillium dahliae belongs to the field of cloning and application of key genes in the course of Verticillium dahliae. Background technique [0002] Cotton Verticillium wilt (Verticillium wilt) is a typical soil-borne vascular systemic disease, which mainly affects the quality and high yield of cotton in China, and is called "cotton cancer" (Jian Guiliang, Zou Yafei, Ma Cun. Verticillium wilt of cotton The reasons and countermeasures of successive epidemics. China Cotton. 2003, 30: 13-14.)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/11C12N15/63A01H5/00
Inventor 苏晓峰齐希梁郭惠明程红梅
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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