摘要
利用基因沉默的原理构建了能转录产生dsRNA,诱导植物发生RNA沉默的沉默载体。根据已报道的番茄花叶病毒(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(Rep)的核苷酸序列设计特异性引物,扩增到△MP基因片段和Rep基因片段;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711bp的融合基因△MP-Rep。将融合基因以反向重复的方式与大豆内含子相连,得到含有融合基因反向重复序列的重组质粒pBlue SK-Intron△MP-Rep(i/r)。再用限制性内切酶BamHⅠ切下包含Intron在内的反向重复的融合基因,并定向插入到植物表达载体pBIN438上,构建了含2种病毒基因的植物表达载体pBIN438△MP-Rep(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。该研究结果为利用RNA沉默原理进行植物广谱抗病研究奠定了基础。
Plant expressing vector containing fusion gene from ToMV ΔMP and CMV ΔRep was obtained to induce RNA silencing by expressing virus-derived dsRNA in plants.ToMV ΔMP and CMV ΔRep were amplified by PCR with primers designed on the basis of sequences of ToMV and CMV,respectively.Fusion gene（△MP-Rep） of 711bp was constructed by recombinant PCR technique.Two copies of △MP-Rep fusion gene were ligated with soybean intron in inverted repeat manner,and then recombinant plasmid pBlucSK-Intron-△MP-Rep（i/r） was constructed.The recombinant fragments containing intron and two copies of △MP-Rep were inserted into binary vector pBIN438 under the control of 35S promoter.Recombinant plasmid pBIN438-△MP-Rep（i/r） containing two different virus genes was constructed successfully which was tested by restriction endonuclease enzymes digestion and PCR analysis.This approach provides a basis for the research into broad spectrum plant virus-resistance by using RNA silence theory.
出处
《石河子大学学报（自然科学版）》
CAS
2012年第4期429-433,共5页
Journal of Shihezi University(Natural Science)
基金
国家自然科学基金项目(30860162)
