马铃薯卷叶病毒中国株(PLRV-Ch)复制酶基因结构研究  

Study on the Replicase Gene Structure of Potato Leafroll Virus Chinese Isolate

作　　者：张鹤龄[1] 梁成罡[1] 张彤[1] 哈斯阿古拉[1] 赵国芬[1] 

机构地区：[1]内蒙古大学生物系,呼和浩特010021

出　　处：《中国病毒学》2000年第3期255-263,共9页Virologica Sinica

基　　金：内蒙自然科学基金资助项目!(96 10E2 8)

摘　　要：用设计合成的两对特异性引物 ,以马铃薯卷叶病毒中国分离株PLRV ChRNA为模板 ,经反转录PCR扩增 ,将复制酶基因 3′端 0 .6kb和 5′端 1.2kb ,克隆于 pUC19中 ,分别构建了重组质粒pLR3和 pLR5,并分五段进行了序列分析。将获得的核苷酸序列及氨基酸序列与国外报导的四个PLRV分离株的相应区段的序列同源性进行了比较。结果表明具有高度同源性。文中对核苷酸序列中存在的可能移码序列和其下游的茎环结构或假节结构、以及特征性三次重复区及氨基酸序列中复制酶蛋白N端的碱性氨基酸序列以及C端区域中包括GDD在内的 8个特征序列进行了讨论。作者发现移码序列上游的三次重复的核苷酸序列可以形成连续折叠的互补双链区和发夹结构 ,这一结构可能和转译移码有关。此外PLRV复制酶蛋白N端部分氨基酸序列易变 ,而C端氨基酸序列十分保守 ,可能和复制酶功能有更重要关系。According to the genomic sequence of PLRV S, two pairs of specific primers were designed and synthesized. The cDNA of the replicase gene of PLRV Chinese isolate (PLRV Ch) was synthesized and amplified by RT PCR using the viral RNA as a template. The synthesized 3′ and 5′ cDNA fragments of PLRV Ch replicase gene were inserted into pUC 19 and cloned in E.coli JM109 and sequenced respectively. The homology of nucleotide sequences between PLRV Ch and PLRV S (Scotland, UK), PLRV N (Netherlands), PLRV C (Canada), PLRV A (Australia) are 98.8%, 97.7%, 97.8% and 96.2%, and the homology of putative amino acid sequences are 98.9%, 97.4%, 97.6% and 95.8% respectively. In 5′ region of replicase gene a slippery sequence (heptanucleotide motif) for 1 frameshift and its down stream “stem—loop” or “pseudoknot”, and upstream nucleotide sequence repeats were found. Authors suggested that the nucleotide repeat sequences characteristic for PLRV could form a tight successively folded complementary double stranded regions and hairpins. This structure possibly has something to do with 1 frameshift. The amino acid sequence of C terminus region was very conservative unlike the N terminus region in which the amino acid sequence is more diversified. The amino acid domains: motifs Ⅰ Ⅷ in the C terminus and the basic amino acid sequence region in the N terminus of RNA dependent RNA polymerase of PLRV were discussed.

关 键 词：马铃薯卷叶病毒 基因克隆 序列分析 复制酶 

分 类 号：Q939.4[生物学—微生物学]