沉默抑制子CMV 2b基因的克隆及其反向重复植物表达载体的构建

Cloning of Silencing Suppressor CMV 2b Gene and Its Construction of Plant Expression Vector Containing Inverted Repeat Fragments

从安徽亳州采集表现明显CMV症状的烟草样本,取ELISA检测阳性反应最强的样本提取总RNA。设计特异性引物扩增CMV RNA2部分片段,克隆并测序。其中包含的2b基因全长336个核苷酸,编码111个氨基酸。将来源于安徽烟草的CMV 2b基因与其它CMV 2b基因进行序列比对,结果表明,来源于安徽烟草的CMV2b基因与来源于韩国的2b基因AF033667的核苷酸序列相似性最高,达98.8%。构建2b基因系统关系树,也表明来源于安徽烟草的CMV 2b基因与AF033667亲缘关系最近。以安徽CMV 2b为模板,分别扩增大小约为300 bp的正向片段CMV 2b(r)和反向片段CMV 2b(i)。先后将反向片段CMV 2b(i)和正向片段CMV 2b(r)分别插入载体pSK-In所含intron两侧,获得重组质粒pSK-2b(r)-In-2b(i)。再将含intron的正反向片段插入pBIN438,最终得到含CMV 2b部分序列反向重复的植物表达载体pBIN-2b(r)-In-2b(i)。

The diseased tobacco samples showing visible CMV symptoms were collected from Bozhou, Anhui Province. The total RNA was extracted from the sample showing the strongest positive reaction by ELISA. CMV RNA2 partial fragment was amplified by designing specific primer pair, and then it was cloned and sequenced. 2b gene included in it is 336 nucleotides, encoding 111 amino acids. Comparing CMV 2b gene derived from Anhui with the other CMV 2b genes, the result showed that nuclear sequence of CMV 2b gene derived from Anhui shared the highest sequence similarity （98.8%） with that of 2b gene from Korea （AF033667）. A phylogenetic tree based on alignment of nucleotide sequences of 2b genes was constructed. The results also showed that CMV 2b gene from Anhui had the closest relationship with AF033667. Approximately 300 bp length sense fragment CMV 2b （r） and antisence fragment CMV 2b （i） were amplified by using 2b gene from Anhui as template. Antisence fragment CMV 2b （i） and sense fragment CMV 2b（r） were inserted into the two sides of intron of vector pSK-In in priority order, and the recombinant plasmid pSK-2b（r）-In-2b（i） was constructed. Sense and antisence fragment containing intron was further inserted into plant expression vector pBIN438, and the recombinant vector pBIN-2b （r）-In-2b（i） containing inverted repeats of partial sequence of CMV 2b gene was acquired ultimately.