烟草花叶病毒和黄瓜花叶病毒双价RNA沉默抗病载体的快速构建*

中国生物工程杂志

2009, Vol. 29

Issue (02): 76-80

技术与方法

烟草花叶病毒和黄瓜花叶病毒双价RNA沉默抗病载体的快速构建*

牛颜冰,雷霄飞,申林炎,王德富,姚敏,郭平毅

山西农业大学

Rapid Construction of RNA Silencing Virus Resistance Vector containing TMV and CMV Virus-Derived Genes

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摘要：

利用重组PCR技术将烟草花叶病毒(TMV)部分移动蛋白基因（△MP）和黄瓜花叶病毒（CMV）部分复制酶基因（△Rep）连接起来，获得全长约1000 bp的MP-Rep融合基因，将所得融合基因以反向重复的方式与大豆内含子相连，并定向插入到植物表达载体pBIN438上35S启动子下游，构建了含两种不同病毒来源基因的植物表达载体pBIN438-MP-Rep（i/r）。酶切和PCR鉴定证明所构建的载体与预期的设计完全一致。该研究结果为利用RNA沉默原理进行植物广谱抗病研究奠定了基础。

关键词： 重组PCR;融合基因;RNA沉默;抗病性

Abstract:

MP-Rep fusion gene which size is about 1000bp was constructed by recombinant PCR technique. In order to construct fusion gene, Tobacco mosaic virus (TMV) partial movement protein gene (MP) and Cucumber mosaic virus (CMV) partial Replicase gene (Rep) were ligated. Two copies of MP-Rep fusion gene were ligated with soybean intron in inverted repeat manner, the recombinant fragments were then inserted into binary vector pBIN438 under the control of 35S promoter. Recombinant clone pBIN438-MP-Rep (i/r) which contained two different virus derived genes was constructed. Recombinant clone pBIN438-MP-Rep (i/r) in accord with expected design was certified by restriction endonuclease enzymes digestion and PCR analysis. This approach provides a basis for Broad-spectrum plant virus resistance mediated by RNA Silencing.

收稿日期: 2008-07-25 出版日期: 2009-03-31

基金资助:

山西省自然科学基金；山西农业大学科技创新基金;国家自然科学基金

通讯作者: 郭平毅

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引用本文:

牛颜冰,雷霄飞,申林炎,王德富,姚敏,郭平毅. 烟草花叶病毒和黄瓜花叶病毒双价RNA沉默抗病载体的快速构建*[J]. 中国生物工程杂志, 2009, 29(02): 76-80.

NIU Ya-Bing- Lei-Xiao-Fei- Shen-Lin-Tan- Wang-De-Fu- Tao-Min- Guo-Beng-Yi. Rapid Construction of RNA Silencing Virus Resistance Vector containing TMV and CMV Virus-Derived Genes. China Biotechnology, 2009, 29(02): 76-80.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I02/76

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