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A method of 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 level, and reduce the effects of use

Inactive Publication Date: 2011-12-21
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

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Embodiment 1

[0027] 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°C for future use.

[0028] Use the synthesized first-strand cDNA as a tem...

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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 1a of cucumber mosaic virus, which is 344bp long, and is located between 1673bp-2016bp of the full-length gene sequence of CMV1a, and constructs this gene fragment on the RNAi high-throughput expression vector pHellsgate2, and then passes The genetic transformation method mediated by Agrobacterium tumefaciens introduces the RNAi plant expression vector into tobacco and stably expresses it, and finally obtains virus-resistant tobacco plants; it is confirmed by experiments that the double-stranded RNA expressing viral gene fragments in plants can induce RNA interference , and then produce the small interfering RNA of the corresponding gene, and specifically inhibit the infection of the virus, and the screened transgenic tobacco has obvious resistance to the infection of cucumber mosaic 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. A certain virus can only infect one or some plants, but there are also a few viruses that are very harmful, such as cucumber mosaic virus (CMV). CMV can invade more than 1,000 kinds of plants, including common cucurbitaceae, solanaceae, and cruciferous crops, as well as agricultural and forestry crops such as Paulownia, banana, and corn, as well as farmland such as chickweed, geranium, bamboo leaf grass, and sorrel Common weeds seriously affect the normal growth of crops. [0003] CMV is a member of the genus Cucumovirus in the Bromoviridae family. Its genome consists of three posit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113A01H5/00
Inventor 刘迪秋田荣欢贺欣饶健葛锋陈朝银周阿涛丁为群
Owner KUNMING UNIV OF SCI & TECH
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