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

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: 2016-09-07
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 its interference vector and application
  • Verticillium dahliae adenylate kinase target gene fragment and its interference vector and application
  • Verticillium dahliae adenylate kinase target gene fragment and its interference vector and application

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, SEQ ID No.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 leaves of Nic...

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Abstract

The invention discloses adenylate kinase target gene fragment of Verticillium dahliae and its interference carrier and application, and belongs to the field of cloning and application of key genes of Verticillium dahliae disease course. The present invention adopts the host-induced gene silencing technology to screen and obtain the target gene fragment in the adenylate kinase gene that can significantly reduce the plant disease index; further constructs a Gateway interference carrier to obtain stable genetically modified tobacco with significantly improved resistance to pathogenic bacteria. Through disease index, fungal biomass analysis and target gene transcription level detection, the adenylate kinase target gene interference segment with the best anti-V. dahliae effect was finally screened. The invention applies the Verticillium dahlia adenylate kinase target gene fragment and the RNA interference carrier to improve the disease resistance ability of plants to the Verticillium dahliae and to cultivate new varieties of transgenic plants resistant to the Verticillium dahliae.

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 the epidemic in successive years. China Cotton. 2003,...

Claims

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Application Information

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Patent Type & Authority Patents(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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