小麦丛矮病毒(WRSV)的近全长cDNA基因文库的构建

Construction of a Near Full-length cDNA Library of Wheat Rosette Stunt Virus(WRSV)

用大肠杆菌Poly（A）聚合酶，在纯化的小麦丛矮病毒的单链基因组RNA3＇末端加多聚腺苷酸，以此RNA作模板，12－18寡聚脱氧胸核着酸作引物，合成cNDA后，分别用限制性内切酶PstI和EcoRI对该cDNA酶切，同时再分别用PstI单酶和SmaI与EcoRI双酶对pUC载体酶切，经连接、转化感受态细胞、筛选和酶切鉴定后，共得到10种不同大小的非定向克隆，其大小之和约14kb，同时还得到定向克隆47个。已知N蛋白基因3＇端含有SacI酶切位点，故在用限制性内切酶SacI对10种插入片段进行分析后发现，仅有约6kb的插入片段含有SacI位点，再用合成的与WRSVNN蛋白mRNA3＇端顺序相同的引物对该克隆进行顺序分析证明，它含有WRSVN蛋白、G蛋白、M蛋白和部分L蛋白基因。对47个定向克隆进行顺序测定后，用DNA顺序分析软件将它与其它几种弹状病毒基因组的3＇前导（leader）和5＇拖尾（trailer）RNA进行比较后发现，有两个定向克隆分别含有它们的3＇前导或5＇拖尾的高保守区顺序，构建了一个近全长的小麦丛矮病毒（WRSV）cDNA文库。

A near full - length cDNA library of Wheat Rosette Stunt Virus (WRSV) was constructed.Purified viral RNA was polyadenylated by E. coli poly(A) polymerase. The poly(A) - tailed RNA was used as a template complementary to oligo dT(12 - 18) primer for cDNA synthesis.The cDNA was separately digested with restriction enzymes Pst I and EcoR I/Sma I. The pUC19 plasmid was digested with the same enzymes to poduce the both cohesive - ends, or one blunt and the other cohesive - ends respectively. Ten inserts with different sizes were obtained and the total size of them reached approximately 14 kb. One insert of near 6 kb length was identified as the insert containing the N protein gene and covering the Ns, M, G, genes as well as a part of L gene.The cDNA inserts with orientated - direction were sequenced and the sequence was compared to 5' and 3' ends of the genomic RNAs of two other plant rhabdoviruses, SYNV and LNYV using computer assisted searching method with the Software Sequence Analysis Version 3. 00.