dsRNA介导的抗ToMV和CMV植物表达载体的构建

目的]构建通过表达dsRNA,诱导植物基因沉默机制可抗2种病毒的表达载体。方法]根据已报道番茄花叶病毒(ToMV)的运动蛋白基因(MP)和黄瓜花叶病毒(CMV)的沉默抑制子基因(2b)的核苷酸序列设计特异性引物,扩增到部分△MP和2b基因;然后通过重组PCR技术将2个病毒基因进行融合,获得长度为676 bp的融合基因△MP-2b。再将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。结果]构建了含两种不同病毒来源基因的植物表达载体pBIN438△MP-2b(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。结论]为利用RNA沉默原理进行植物广谱抗病研究奠定了基础。

Construction of Binary Vector Expressing dsRNA Resistant to ToMV and CMV

Objective]The purpose of this research was to obtain plant expressing vector containing fusion gene from ToMV△MP and CMV 26 to induce RNA silencing by expressing virus - derived dsRNA in plants.Method]ToMV△MP or CMV 2b was amplified by PCR with primers designed based on sequences of ToMV or CMV,respectively.Fusion gene(△MP -2b) of 676 bp was constructed by recombinant of PCR technique.Two copies of△MP -2b fusion gene were ligated with soybean intron in inverted repeat manner, and then the recombinant fragments were inserted into binary vector pBIN438 under the control of 35S promoter.Result]Recombinant plasmid pBIN438 -△MP -2b(i/r) which contained two different virus genes was constructed successfully which was tested by restriction endonuclease enzymes digestion and PCR analysis.Conclusion]This approach provides a basis for broad spectrum plant virus resistance - mediated by RNA silencing.