dsRNA介导的抗ToMV和CMV植物表达载体的构建

摘要

[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 26 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 -1b 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.%[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.