甘蔗黄叶病毒CP蛋白基因ihpRNA表达载体构建及烟草转化

Construction of ihpRNA Expression Vector of Sugarcane Yellow Leaf Virus CP Protein Gene and Genetic Transformation in Tabacco

为提高甘蔗抗病性,本研究根据甘蔗黄叶病毒海南分离物ScYLV-CHN-HN1全基因组序列(GenBank no.HQ342888),利用病毒CP蛋白介导的RNAi技术,针对病毒外壳蛋白CP,设计两对含有酶切位点的特异性引物,CPsf1/CPsr1和CPasf1/CPasr1,以构建好的pMD19-T/CP质粒为模板,pRNAi1017为中间载体,分别合成构建干扰载体的正反向片段pRNAi-CP-F-R,将CP正反向片段分别插入表达载体pCAMBIA2300的相应位置,构建含有发卡结构的RNAi载体p2300-CP-F-R,经过PstⅠ酶切鉴定,证明载体构建成功。通过农杆菌介导的方法,以干扰表达载体p2300-CP-F-R转化烟草,经过PCR检测,得到12株阳性转基因植株,Southern blot杂交和半定量RT-PCR对其检测,证明干扰片段已经整合烟草基因组中并进行了转录,该结果为RNAi介导抗病毒甘蔗育种研究奠定基础。

To improve the sugarcane resistance against disease,a suagrcane yellow leaf virus CP protein mediated RNA interference technology was set up.In the paper,a RNAi expression vector harboring CP protein gene fragment of sugarcane yellow leaf virus was constructed.On the basis of the complete genome sequence of sugarcane yellow leaf virus Hainan isolate（GenBank accession no.HQ342888）,two pairs of specific primers,CPsf1/CPsr1 and CPasf1/CPasr1,containing different enzyme sites were designed.With the template of pMD19-T/CP plasmid constructed and middle vector of pRNAi1017,positive sense strand and antisense strand were obtained,which were separately inserted into the plant expressional vector pCAMBIA2300.The RNAi vector p2300-CP-F-R containing a hairpin structure was confirmed by the digestion of restriction enzyme PstⅠ.The p2300-CP-F-R was transformed into tobacco by Agrobacterium-mediated transformation system.The results revealed that 12 transgenic tobacco plants were obtained by PCR,Southern blot and semiquantitative RT-PCR.The CP gene was succeeded in being integrated into genome of tobacco and transcripted.This work laid the foundation for breeding of plant mediated RNAi technology in sugarcane against disease.