抗水稻黑条矮缩病毒和南方水稻黑条矮缩病毒的双价RNA沉默载体的构建

为利用RNAi技术获取抗水稻黑条矮缩病毒(RBSDV)和南方水稻黑条矮缩病毒(SRBSDV)植株,分别针对两种病毒的S6和S10基因构建RNA沉默载体。采用重组PCR方法将RBSDV S6基因片段(R6)和SRBSDVS6基因片段(SR6)进行融合,获得600 bp的R6-SR6融合基因;将RBSDV S10基因片段(R10)和SRBSDV S10基因片段(SR10)进行融合,获得600 bp的R10-SR10融合基因。融合基因以反向重复的方式连入pBS载体,并定向插入到pCAMBIA1301载体上,获取了含有发夹结构的植物表达载体pCAMBIA1301-hp(R6-SR6)和pCAMBIA1301-hp(R10-SR10)。抗RBSDV和SRBSDV RNA沉默载体的构建为利用RNA沉默进行植物抗病毒研究奠定了基础。

Construction of RNA silencing expression vectors resistant to RBSDV ( rice black-streaked dwarf virus )and SRBSDV (southern rice black-streaked dwarf virus)

In order to obtain rice plants resistant to RBSDV(rice black-streaked dwarf virus) and SRBSDV(southern rice black-streaked dwarf virus) using RNAi technology,RNA silencing expression vectors targeting S6 or S10 gene from the two viruses were constructed.A 600-bp gene R6-SR6 was produced through the fusion of S6 gene segment(R6) of RBSDV and S6 gene segment(SR6) of SRBSDV by recombinant PCR.A 600-bp gene R10-SR10 was produced as well through the fusion of S10 gene segment(R10) of RBSDV and S10 gene segment(SR10) of SRBSDV.Fusion genes R6-SR6 and R10-SR10 were ligated to pBS vector in a inverted repeat manner,then were inserted into pCAMBIA1301,respectively.The binary expression vectors pCAMBIA1301-hp(R6-SR6)and pCAMBIA1301-hp(R10-SR10)containing hairpin structures were constructed,respectively.This work provided a basis of application of RNA silencing for the researches on transgenic plant resistant to RBSDV and SRBSDV.