大麦条纹花叶病毒新疆株(BSMV-XJ)RNA_2的cDNA克隆及5′端序列分析

CLONING, MAPPING AND SEQUENCING OF RNA2 FROM BARLEY STRIPE MOSAIC VIRUS XINGJIANG STRAIN

分离了大麦条纹花叶病毒(BSMV)新疆株基因组的3个RNA组份。以RNA2为模板,3′端互补寡核苷酸为引物,合成了第一条cDNA链和第二条cDNA链,将ds-cDNA重组在pUC9质粒中,转化大肠杆菌细胞,获得含RNA3′端的克隆,并证明所选克隆的cDNA含有新疆株几近全长的RNA2组份。对于插入片段为3.3kbp的112号克隆进行了酶谱分析,得到了与国外典型株类似的结果;用双脱氧终止法分析了相当于RNA 2 5′端250bp的cDNA酶切片段,表明与国外典型株有十分相似的一级结构。

A set of ds-cDNA clones were synthesized using fractionated RNA2 of BSMV-XJ strain as the template and a 20nt synthetic primer complementary to the extreme 3' -end of BSMV-RNAs, and the modified Gubler and Hoffman method for the 2nd strand synthesis and annealing of the ( dC ) -tailed ds-cDNA with ( dG )-tailed pUC9 at Pst I site.The longest inserts Covering the 3'-end of RNA were selected by in situ hybridization with α-32P-labelled synthetic primer as probe,their size measured by electrophoresis after Pst I digestion and the clone was finally identified by Northern blot.The restriction map of the selected clone, No.112, was compared with that of Type strain. The result showed that the clone was long enough to cover both 3'-and 5'-end of RNA2. Sequencing data showed that the 1st nt at the 5'-end of No. 112, when compared with the complete sequence of Type strain published by G. Gustafson, corresponds to the 4th one of the Type strain. There are high homology of the 5'-leading sequence extending to the first part of the coat protein cistron.