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A kind of method for cultivating anti-cucumber mosaic virus plants

A cucumber mosaic virus and plant technology, applied in the field of genetic engineering, can solve problems such as reducing the severity of virus disease, achieve broad market application prospects, improve management levels, and reduce environmental pollution.

Inactive Publication Date: 2011-12-07
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these traditional methods can reduce the severity of the virus to varying degrees, they cannot fundamentally solve the problem.

Method used

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  • A kind of method for cultivating anti-cucumber mosaic virus plants
  • A kind of method for cultivating anti-cucumber mosaic virus plants
  • A kind of method for cultivating anti-cucumber mosaic virus plants

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Experimental program
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Effect test

Embodiment 1

[0025] 1. CMV 2a Amplification and sequence analysis of gene fragments

[0026] The lily leaves with CMV symptoms were ground into powder with liquid nitrogen, transferred to a 1.5ml centrifuge tube, and the total RNA was extracted by the modified guanidine isothiocyanate method. Use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: take 8 μg Total RNA, add 50 ng oligo(dT), 4 μL ddH in sequence 2 O (RNase-free) to a reaction volume of 12.5 μL; after mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 2 μL dNTP (2.5mM each), 4 μL M-MLV 5×buffer , 0.5 μL nuclease inhibitor RNasin (200U), 1 μL reverse transcriptase M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5h, take it out and heat at 95°C for 5min to terminate the reaction. After the first strand of cDNA was synthesized, it was stored at -20...

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Abstract

The invention discloses a method for cultivating plants resistant to cucumber mosaic virus. The present invention clones the cDNA fragment of the replicase gene of cucumber mosaic virus, which is 308bp long and is located between 1207bp-1515bp of the full-length gene sequence of CMV2a, and then constructs the gene fragment on the RNAi high-throughput expression vector pHellsgate2, and then passes it through Agrobacterium tumefaciens The CMV2aRNAi plant expression vector was introduced into tobacco by the mediated genetic transformation method and stably expressed, and the virus resistance of the transgenic plants was analyzed through experiments, and finally transgenic tobacco plants with enhanced resistance to CMV were obtained; the expression of viral gene fragments in plants was confirmed by experiments The double-stranded RNA can induce the occurrence of RNA interference, and then produce the small interfering RNA of the corresponding gene, and specifically inhibit the infection of the virus.

Description

technical field [0001] The invention relates to a method for cultivating plants resistant to cucumber mosaic virus, belonging to the field of genetic engineering. Background technique [0002] Plant virus diseases are known as plant "cancer", and the economic losses caused by virus diseases to crops are about tens of billions of yuan every year. Plant viruses must live obligately in host cells. Although some viruses can only infect one or certain plants, there are also a few viruses that are very harmful, such as cucumber mosaic virus (CMV). CMV is widespread in the world and has the widest range of hosts, infecting about 885 species of plants in 65 families. Because of its importance in causing diseases, CMV has become the most important plant RNA virus worldwide, and it is also one of the most studied plant RNA viruses. [0003] CMV is a member of the genus Cucumovirus in the Bromoviridae family. Its genome consists of three positive-sense single-stranded RNAs, which are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 刘迪秋田荣欢贺欣饶健葛锋陈朝银丁为群周阿涛
Owner KUNMING UNIV OF SCI & TECH
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