烟草反义Mlo基因表达载体的构建  

Construction of Tobacco Anti-sense Mlo Gene Expression Vector

作　　者：邬晓勇 时羽杰 李杰 罗倩 王跃华 唐媛 孙雁霞 WU Xiaoyong;SHI Yujie;LI Jie;LUO Qian;WANG Yuehua;TANG Yuan;SUN Yanxia(School of Pharmacy and Bioengineering, Chengdu University, Chengdu 610106, China)

机构地区：[1]成都大学药学与生物工程学院,四川成都610106

出　　处：《成都大学学报（自然科学版）》2017年第4期333-337,共5页Journal of Chengdu University（Natural Science Edition）

基　　金：成都大学药食同源植物资源开发四川省高校重点实验室开放基金(10Y201409)资助项目

摘　　要：获得烟草反义Mlo基因的植物表达载体pBI 121-Mlo,为进行烟草遗传转化,获得该基因表达的缺陷型植株打下基础.从烟草叶片中提取总RNA,利用RT-PCR技术扩增得到Mlo基因的c DNA,以此为模板设计反义引物,通过PCR扩增出反义Mlo基因,将此反义Mlo基因与T载体连接,测序正确后再将此反义片段与植物表达载体pBI 121连接,构建烟草反义Mlo基因的植物表达载体pBI 121-Mlo.经Kan选择筛选出反义重组菌落,碱裂解法小量提取质粒后,用Xba I和Bam HI双酶切后再进行电泳鉴定.结果表明,目的基因已与植物表达载体pBI 121连接成功.成功构建了烟草反义Mlo基因表达载体pBI 121-Mlo.Tobacco anti-sense Mlo gene plant expression vector PBI 121-Mlo was constructed not only for tobacco genetic transformation, but also to lay the foundation for the defective plant gene expression. Total RNA was extracted from tobacco leaves and the cDNA of Mlo was obtained by PT-PCR amplification. The cDNA was served as a template to amplify the anti-sense Mlo gene by the anti-sense primers.Then the an-ti-sense Mlo gene was connected with T vector and sequenced and then the anti-sense fragment was con-nected to the plant expression vector named pBI 121. The plant expression vector of the tobacco anti-sense Mo named pBI121 Mo was constructed .The recombinant bacteria were selected through Kanamycin and a few plasmids were extracted by alkaline lysis method. The plasmid was identified by electrophoresis after double digestion by Xba I and BamH I. The results illustrated that the target gene was successfully connect-ed to the plant expression vector pBI 121 .The genetic expression vector named pBI 121-Mlo of tobacco an-ti-sense Mlo was constructed.

关 键 词：MLO基因 PBI 121Mlo RT-PCR 反义片段 双酶切 

分 类 号：S572.032[农业科学—烟草工业]