基因干扰载体的构建及其鉴定

葡萄病毒A MP基因RNA干扰载体的构建及其鉴定

CONSTUCTION AND IDENTIFICATION OF THE RNA INTERFERENCE VECTOR OF GVA MP GENE

作者：丁艳杰　　导师：周宗山

中国农业科学院　　植物病理学2011届硕士

摘　要

RNA干扰（RNA interference,RNAi）:当一些小分子量的外源双链RNA（dsRNA）进入宿主细胞后，特异性降解相应序列的mRNA，从而导致转录后水平的基因沉默（PTGS）。RNA介导的抗病毒最有效的途径是构建病毒特定基因片段的反向重复序列载体，之后转入植物中，转录后生成RNA发夹结构（hpRNA）,发夹结构的柄可诱导转录后基因沉默，从而达到抗病毒的目的。

利用根癌农杆菌介导的瞬时表达系统来研究植物基因功能的报道很多，它不仅可以快速地导入外源基因，而且使转入基因在短时间内表达，从而在功能基因组学上面发挥巨大的作用。

葡萄病毒A（GVA）既是危害葡萄最为严重的病毒之一，分布广泛，又是少数能摩擦接种到草本寄主的葡萄病毒之一，易于开展研究工作，可以带动其他病毒的相关理论问题和应用时实践的研究。因此本文以GVA运动蛋白（MP）基因为模扩增出了418bp的基因片段，根据RNA干扰的基本原理，构建了RNA干扰载体，并成功转化到根癌农杆菌中，通过瞬时表达来检测干扰载体的抗性效果。主要的试验结果如下：

1.　基因片段的克隆与序列分析：实际引物克隆质粒pDriver-MP中的一个基因片段，通过引物配以不同的酶切位点，分别命名为GVAF和GVAR，长度都为418bp，经序列分析，证明这两个片段为目的基因片段，可用于构建RNA干扰载体。

2.　RNA干扰载体的构建：通过酶切、连接、PCR等方法将目的片段正反向分别插入载体pFGC5941-GVAFR，经菌液PCR和酶切鉴定证明干扰载体构建成功。

3.　表达载体向农杆菌中的转化：通过冻融法将pFGC5941-GVAFR向农杆菌中进行转化，菌液PCR检测结果表明获得了含有表达载体的农杆菌EHA105。

4.　瞬时表达分析：向本实验中注射农杆菌后接种GVA，通过表型观察和PCR检测得出：

（1）瞬时干扰作用具有时间依赖性：农杆菌渗入3d、4d、5d后接种GVA对病毒的干扰效果最好，没有病症出现，而农杆菌渗入2d后接种GVA，则没有表现出任何抗性，全部观察到病症。

（2）干扰载体pFGC5941-GVAFR对GVA的抗性达到了70%，且抗性植株上没有表现出较强的抗性，可进一步在木本植物葡萄上进行研究。

关键词　葡萄病毒A　运动蛋白基因MP　RNA干扰　载体构建　瞬时表达

Abstract

RNA interference (RNA i): When some exogenous low molecular weight double-stranded(dsRNA)get into host cells,specifically degrade the sequence-specific mRNA,leading to post-transcriptional gene silencing(PTGS).The most effective antiviral way mediated bu RNA is to construct virus-specific reverse repeat gene fragments,then transferred to plants ,and form RNA hairpin formation (hairpin-RNA,hpRNA)by transcription,the handle of hairpin structure induce gene silencing ,thus achieve the antiviral purpose.

A large number of reports about the gene function study through agrobacterium-mediated transient expression system were published ,this system can not only import the exogenous gene quickly,but also express the transferred genes in a short time ,so it played a huge role on functional genomics study.

Grapevine virus A (GVA) is both one of the most serious viruses against grape,wide distribution ,and one of the few filamentous viruses which can be inoculated onto herbaceous hosts mechanically,so it is easy to conduct research work,and benefit to the theoretical and practical applications research of other viruses. In this study,according to the basic principles of RNA interference ,two fragments(418bp) of GVA movement protein (MP) gene were amplified and RNA interference vector was constructed ,then successfully transformed into agrobacterium tumefaciens,finally the resistance effect of interference vector to GVA infection was studied by transient expression. The main results were as follows:

1.　Gene clone and sequence analysis:418bp fragment of GVA MP gene was cloned from plasmid pDriver-MP with designed primers,the fragments with different restriction sites were named as GVAF and GVAR,sequence analysis showed that the two fragment for the target gene could be used to construct RNA interference vector.

2.　RNA inherence vector construction：fragments were inserted into both sides of intron of pFGC5941-GVAFR was identified by PCR and restriction enzyme analysis.

3.　Transfomation: the RNAi vector pFGC5941-GVAFR was transformed into the agrobacterium strain EHA105 through freeze-thaw method,it is identified by mini-PCR.

4.　Transient expression analysis: N.benthamiana was first infiltrated with EHA105 (include pFGC5941-GVAFR),then mechanically inoculate GVA on the leaves,the RNAi resistance to GVA was detected by phenotype observation and PCR:

(1) Transient interference action is time-dependent: inoculate GVA on N.benthamiana after 3d,4d,5d of agrobacterium infiltration,the interference effect was the best,no symptoms appear,while inoculate GVA on N.benthamiana after 2d of agrobacterium infiltration,the plants did not show any resistance,all showed observed symptoms.

(2) The resistance of vector pFGC5941-GVAFR against GVA reached 70%,and on the resistant plants did not show any symptoms ,while the control group all showed symptoms. The interference vector pFGC5941-GVAFR in herbs N.benthamiana showed strong resistance,it can be further studied in woody grape plants.

Key words　Grapevine virus A　Movement protein gene MP　RNA interference vector　Vector construction　Transient expression