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Verticillium dahliae oligosaccharyl transferase target gene fragment and interference vector and application thereof

A technology of Verticillium dahliae and base transfer, which can be used in application, genetic engineering, plant genetic improvement and other directions, and can solve the problems of lack of very effective control measures and chemicals.

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

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Problems solved by technology

Once the plants get sick, there are no very effective control measures and pesticides in the field at present

Method used

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  • Verticillium dahliae oligosaccharyl transferase target gene fragment and interference vector and application thereof
  • Verticillium dahliae oligosaccharyl transferase target gene fragment and interference vector and application thereof
  • Verticillium dahliae oligosaccharyl transferase target gene fragment and interference vector and application thereof

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Experimental program
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Embodiment 1

[0051] Example 1 Oligosaccharyl transferase complex STT3 subunit target gene screening of Verticillium dahliae

[0052] 1. Experimental method

[0053] 1.1 Construction of VIGS interference vector

[0054] In order to screen the target gene segment with the best interference effect, five pairs of specific primers were designed according to the coding sequence of Verticillium dahliae oligosaccharyltransferase STT3 subunit (VDAG_03232.1) (Table 1) , The two ends of the primers contain EcoRI and BamHI restriction sites, and PCR amplification is performed on the target fragment respectively. 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.

[0055] Table 1 Primer information fo...

Embodiment 2

[0076] Construction and plant transformation of embodiment 2Gateway interference vector

[0077] 1. Experimental method

[0078] 1.1 Construction of stable genetic vector

[0079] 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, three DNA segments (nucleotide sequences respectively SEQ ID No. .1, shown in SEQIDNo.4, SEQIDNo.5), 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.

[0080] Table 3. Stable genetic interference primer information

[0081]

[0082] 1.2 Transformation of Nicotiana benthamiana

[0083...

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Abstract

The invention discloses a verticillium dahliae oligosaccharyl transferase complex STT3 subunit 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 oligosaccharyl transferase complex STT3 subunit 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 oligosaccharyl transferase complex STT3 subunit target gene interference fragment with the optimal verticillium dahliae resisting effect is finally screened. The verticillium dahliae oligosaccharyl transferase complex STT3 subunit 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 oligosaccharyltransferase complex STT3 subunit and the RNA transcribed by the target gene fragment, and also relates to the target gene fragment containing the STT3 subunit of the oligosaccharyltransferase complex The interference carrier, the present invention further relates to the application of the STT3 subunit target gene fragment of the Verticillium dahliae oligosaccharyltransferase complex or the interference carrier in improving the disease resistance of plants to Verticillium dahliae, which belongs to Verticillium dahliae Cloning and application fields of key genes in the course of mycobacteria. Background technique [0002] Verticillium dahliae (Verticilliumdahliae), is a soil-borne pathogenic fungus, can harm a variety of economic crops, fruit trees and ornamental flowers, causing huge economic losses (FradinEF, ThommaBP.Physiology and molecular aspects of Vert...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/11C12N15/63A01H5/00
Inventor 苏晓峰齐希梁程红梅郭惠明
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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