Abstract:

Banana bunchy top virus Chinese Zhangzhou isolate (BBTV-ZZ) DNA 4 was amplified by PCR and cloned. Sequence analysis showed that BBTV-ZZ DNA 4 is 1039 nucleotides (nts) in length and this virus could be one member of BBTV Asian group. Transcriptional initiation site A, which is at the 269 nucleotide, was preliminarily determined by using 5′ RACE method. BBTV-ZZ DNA 4 non-coding region was sub cloned by PCR and inserted into upstream of gfp∶∶gus plant expression vector pCAMBIA 1304 to construct recombinant plasmid pTA2.Agrobacterium tumefaciens harboring pTA2 was injected into leaves of the tobacco (Nicotiana tabacum L. cv. Xanthi NC) via Agrobacterium infiltration procedure. Transient expressions of GUS and GFP were determined in injected leaves 3-5 d later. GUS activities of pTA2,pCAMBIA 1304 injected and noninjected tobacco leaves respectively were 1.0070 pmol MU·μg-1·min-1, 2.0690 pmol MU·μg-1·min-1 and 0.0214 pmol MU·μg-1·min-1. Indirect ELISA for GFP in 1 mg total protein from pTA2, pCAMBIA 1304 injected and non injected leaves showed an A 490 nm value of 89.577, 100.440 and 3.287, respectively. These results showed that the non coding region of BBTV-ZZ DNA 4 has a promoter activity not only in the virus replication in monocot, but also in driving the expression of a foreign gene in dicot plants.

香蕉束顶病毒中国漳州分离物DNA 4的克隆及非编码区的启动子活性初探
孙德俊 魏红艳 蔡文启* 田颖川
（中国科学院微生物研究所，北京100080）

摘要： 应用PCR方法克隆了香蕉束顶病毒中国漳州分离物(Banana bunchy top virus Chinese Zhangzhou isolate, BBTV-ZZ) DNA 4.序列分析表明其序列全长为1 039 nt,归属于亚洲组.5′ RACE分析确定其转录起始位点是269 nt处的A.利用PCR方法亚克隆了BBTV-ZZ DNA 4非编码区序列并将其插入到植物表达载体pCAMBIA 1304中的 gfp∶∶gus 基因上游得到重组质粒pTA2.将含pTA2和pCAMBIA 1304的根癌土壤杆菌(Agrobacterium tumefaciens)注射进烟草(Nicotiana tabacum L. cv. Xanthi NC)叶片,3～5 d后剪下注射部位的叶片进行GUS和GFP的表达分析.pTA2(含BBTV-ZZ DNA 4非编码区)、pCAMBIA 1304(含CaMV 35S启动子)和未注射的烟草叶片的GUS活性分别为1.007 0 pmol MU*μg-1*min-1 , 2.069 0 pmol MU*μg-1*min-1和0.021 4 pmol MU*μg-1*min-1.注射含pTA2和pCAMBIA 1304植物表达载体根癌土壤杆菌以及未注射的烟草叶片的每毫克总蛋白的GFP间接ELISA在490 nm的吸光值分别为89.577、100.440和3.287.

关键词： 香蕉束顶病毒漳州分离物DNA 4；5′RACE；启动子；GFP；GUS

通讯作者。E-mail: caowq @ sun.im.ac.cn

Key words: Banana bunchy top virus Chinese Zhangzhou isolate DNA 4, 5′RACE, promoter, GFP, GUS