Virus induced gene silencing system and use thereof

A gene silencing and virus technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as unpredictable results, inability to study multi-gene families, heavy workload, and time-consuming

Inactive Publication Date: 2009-04-29
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

T-DNA and transposon tagging technologies are very effective in generating mutants, but cannot study the function of existing multigene families, it is difficult to achieve genome saturation, and common loss of multiple genes is often caused by insertional diversity. live
Antisense RNA (asRNA) and cosuppression using tissue-specific promoters can prevent early developmental repression, but are labor-intensive, time-consuming, and have unpredictable outcomes

Method used

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  • Virus induced gene silencing system and use thereof
  • Virus induced gene silencing system and use thereof
  • Virus induced gene silencing system and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of TMV silencing vector pTMV-rCPc61-VIGS

[0022] 1) Introduction of multiple cloning sites

[0023] The construction of TMV cDNA cloning vector pTMV20 refers to Wu, L.G., Jiang, L.B., Zhou, Z.A., Fan, J.H., Zhang, Q.Q., Zhu, H.H., Han, Q.and Xu, Z.K. (2003) Expression offfoot-and-mouthdisease Virus epitopes in tobacco by a tobacco mosaic virus-based vector. Vaccine 21, 4390-4398. The KpnI / XhoI / EcoRV / NotI sequence of the multiple cloning site region was synthesized into primers KXEN5(+) / KXEN3(-), and the KpnI / XhoI / EcoRV / NotI sequence of the multiple cloning site region was introduced into the pTMV20 vector by the method of secondary PCR After the middle CP stop codon, the plasmid pTMV-VIGS was obtained.

[0024] It should be understood that the use of the multiple cloning sites of the present invention in the entire VIGS system is only for introducing inserts, and corresponding sites can be introduced according to the needs of one's own experim...

Embodiment 2

[0032] Example 2: Efficiency test of gene silencing vector

[0033] Taking the phytoene desaturase (PDS) of Nicotiana benthamiana as an example, the effect of the silencing vector is illustrated. PDS is a key enzyme in the carotenoid synthesis pathway. When the gene is silenced, the plant will produce albinism, and the leaves of the plant will turn white, which is caused by a significant decrease in the level of PDS mRNA.

[0034] 1. Clone in vitro transcription: Linearize the recombinant TMV cDNA clone in Example 1 by Pst I digestion; add T4 DNA polymerase to a final concentration of 0.1U / μl, dNTP to a final concentration of 0.1mM / ul, and react at 16oC for 30min. Used to fill in the 3' end after enzyme digestion; phenol: chloroform extraction, 2.5 times the volume of ice ethanol precipitation plasmid, 75% ethanol washed twice; 2 μg linearized plasmid as template, catalyzed by T7RNA polymerase for capping in vitro For transcription, see Xu, Z.K., Anzola, J.V., and Nuss, D.L. ...

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Abstract

The invention relates to a virus-induced gene silencing system in particular using tobacco mosaic virus TMV as a vector. In the system, a DNA segment of a target gene is inserted between a termination codon and a 3' untranslated region of coat protein CP of the tobacco mosaic virus TMV; a polyclonal restricted endo enzyme cutting point is introduced between the termination codon and the 3' untranslated region of the coat protein CP of the tobacco mosaic virus TMV and is used for the insertion of the DNA segment of the target gene; and a section of a basic group sequence on the bottom end of CP3' is introduced in the front of the 3' untranslated region so as to strengthen the stability of the silencing vector. The DNA segment of the target gene is not more than 1 kb. The gene silencing system is utilized to carry out silencing on the target gene and is economic, feasible, convenient and effective.

Description

technical field [0001] The invention relates to a virus-induced gene silencing system and its application. In particular, a virus-induced gene silencing system using tobacco mosaic virus TMV as a vector. technical background [0002] Plant molecular biology essentially relies on forward genetics; that is, by mutating and cloning mutant genes to find the wild-type gene sequence that controls the phenotype. In the past few years, with the completion of the genome sequencing of the model plants Arabidopsis and rice and the successive production of sequence information databases for other species, it means that a technology for studying gene function that is superior to traditional forward genetics is emerging . One of them is reverse genetics, which is the functional study of mutant phenotypes by changing the expression of a gene or sequence. T-DNA and transposon tagging technologies are very effective in generating mutants, but cannot study the function of existing multi-ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/83
Inventor 许政暟李茫茫贺燕云金菲李平宋任涛
Owner SHANGHAI UNIV
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