香蕉束顶病毒中国漳州分离物DNA4非编码区启动子活性的研究  

Promoter activity of banana bunchy top virus Zhangzhou isolate DNA 4 non-coding region.

作　　者：魏红艳[1] 孙德俊[2] 李云[1] 孙卉[2] 蔡文启[2] 

机构地区：[1]北京林业大学生物科学与技术学院 [2]中国科学院微生物研究所

出　　处：《北京林业大学学报》2003年第5期34-37,共4页Journal of Beijing Forestry University

基　　金：中国科学院重大项目子课题 (KY95 1 A1 30 2 12 10 )资助

摘　　要：应用PCR方法亚克隆了香蕉束顶病毒中国漳州分离物 (BananabunchytopvirusChineseZhangzhouisolate ,BBTV ZZ)DNA 4非编码区 (Po1)、主要共同区缺失片段 (Po2 )和主要共同区及茎环共同区都缺失片段 (Po3) ,分别插入到植物表达载体pCAMBIA 130 4的gfp::gus基因上游 ,替代椰菜花叶病毒启动子 (CaMV 35S) ,得到重组质粒pTA2、pC2 6和pC45 .通过Agroinfiltration法 ,将含pTA2、pC2 6、pC45和pCAMBIA 130 4的根癌土壤杆菌 (Agrobacteriumtu mefaciens)注射进烟草 (NicotianatabacumL .cv .XanthiNC)叶片 ,进行 β 葡糖苷酸酶 (GUS)和绿荧光蛋白 (GFP)含量的瞬间表达分析 .含有pTA2、pC2 6、pC45、pCAMBIA 130 4和未注射的烟草叶片其GUS活性分别为 1 0 0 7、0 85 2、0 939、2 0 6 9和 0 0 2 1pmol·MU (μg·min) ;间接酶连免疫试验 (ELISA)结果表明每毫克总蛋白中的GFP于 490处的吸收值 (A490 )分别为 89 5 77、6 5 184、72 0 96、10 0 4 40和 3 2 87.以上表明Po1、Po2和Po3具有较强的启动子活性 .此外 ,转基因烟草的GUS组织化学定位检测试验进一步表明Po1、Po2和Po3具有维管组织特异性 .Banana bunchy top virus Chinese Zhangzhou isolate (BBTV--ZZ) DNA 4 non-coding region (Po1), and its 5′ end deletion of CR--M (Po2),and deletion of CR--M and CR--SL (Po3) were subcloned by PCR and inserted into upstream of gfp::gus plant expression vector pCAMBIA 1304 to construct the recombinant plasmid pTA2, pC26 and pC45, respectively. Agrobacterium tumefaciens harboring pTA2, pC26 and pC45 were respectively injected into leaves of the tobacco (Nicotiana tabacum L. cv. Xanthi NC) via Agroinfiltration. Transient expressions of GUS and GFP were determined in injected leaves 3～5 d later. GUS activities of pTA2, pC26, pC45, pCAMBIA 1304 injected and non-injected leaves were 1.007, 0.852, 0.939, 2.069 and 0.021 pmol·MU/(μg·min), respectively. Values of absorbence of GFP in 1 mg total protein from pTA2,pC26,pC45, pCAMBIA 1304 injected leaves and non-injected at 490 nm by indirect ELISA were 89.577,65.184,72.096,100.440 and 3.287, respectively. The results suggest that Po1, Po2 and Po3 all have strong promoter activity. And in transgenic tobacco plants, activities of Po1, Po2 and Po3 were restricted to the vascular-associated tissue by detection of GUS.

关 键 词：香蕉束顶病毒中国漳州分离物 DNA 4 启动子活性 GUS GFP 

分 类 号：S668.108[农业科学—果树学]