南瓜蚜传黄化病毒P0蛋白基因ihpRNA载体构建及烟草转化

P0蛋白是南瓜蚜传黄化病毒基因组编码的基因沉默抑制子。根据已克隆的CABYV P0蛋白基因序列,设计带酶切位点的特异性引物,PCR扩增用于构建RNAi表达载体的P0蛋白基因正反义片段。将正反义片段分别插入到含有内含子的中间载体pBSint上,得到重组的中间载体pBSint-P0-F-R。用Sal I和Sac I双酶切pBSint-P0-F-R,将切下的包括内含子和正反义P0蛋白基因在内的片段定向克隆到植物表达载体pCambia1300上,得到含有发卡结构的RNAi表达载体pCambia-P0-F-R。通过农杆菌介导的方法将该表达载体转化本生烟,经PCR检测,得到4株阳性转基因植株。为设计出更广谱、特异有效的抗病毒策略及研究病毒蛋白的功能特性及其侵染机制提供实验材料。

Construction of ihpRNA expression vector of Cucurbit aphid-borne yellows virusp0 protein gene and genetic transformation in tobacco

CABYV infects a wide range of hosts,including plants of families of Cucurbitaceae,causes significant losses of horticultural produce.There is no effective chemical controlling measures.Transgenic technology could expand ways for prevention and controlling of plant viral disease.P0 is a gene silencing suppressor encoded by Cucurbit aphid-borne yellows virus(CABYV).The specific primers were designed based on the cloned P0 protein gene of Cucurbit aphid-borne yellows virus(CABYV)to amplify positive-sense and antisense strands of P0 protein gene,respectively.The obtained positive sense and antisense strands were inserted separately into the middle vector pBSint which contains an intron.The obtained recombinant middle vector pBSint-P0-F-R was digested with Sal I and Sac I,and the restriction products were cloned into the expression vector pCambia1300 to construct the ihpRNA vector pCambia-P0-F-R.The pCambia-P0-F-R was transformed into Nicotiana benthamiana mediated by Agrobacterium tumefaciens.PCR testing showed that four transgenic plants were obtained.This provided experimental materials for designing a more extensive,specific and effective strategies to control virus and studying the function of protein and infection mechanism of virus.