侵染蚕豆ClYVV的鉴定及其衍生的小干扰RNA的深度测序鉴定研究

对安徽省合肥市采集到的具有明显典型花叶症状的蚕豆叶片进行深度测序,鉴定到样品中存在三叶草黄脉病毒(Clover yellow vein virus, ClYVV),并获得了ClYVV的完整基因组序列,随后通过直接测序扩增基因组区域获得完整的基因组ClYVV-HF。获得的ClYVV-HF基因组由9 585个核苷酸组成,不包括poly(A)尾巴。ClYVV-HF的全部核苷酸序列与之前报道的来自日本的分离株(AB011819、NC_003536)和来自韩国的分离株(KF975894)分别具有95.45%和95.10%的核苷酸同源性。进一步分析了ClYVV-HF衍生的小干扰RNA的表征。vsiRNAs的大小为21~22 nt,vsiRNAs的5' 末端碱基偏向于A / U。ClYVV-HF来源的小干扰RNA大多来源于其基因组链中的7个热点,其中5个热点以21 nt vsiRNAs为主。生物学实验表明,ClYVV可通过汁液摩擦接种多种豆科作物。这是有关中国ClYVV分离株全部核苷酸序列的首次报道。

Identification of Clover yellow vein virus infecting broad bean in China by deep sequencing and characterization of virus-derived small interfering RNAs

The broad bean samples causing prominent mosaic on leaves were collected in Hefei City, Anhui Province in China, and viruses in samples were detected by deep sequencing. The results showed that Clover yellow vein virus(ClYVV) existed in the broad bean samples and the complete genome ClYVV-HF was obtained by direct sequencing of the amplified genomic region, subsequently. The ClYVV genome of China consists of 9 585 nucleotides excluding the poly(A) tail, the full nucleotide sequence of ClYVV-HF shared a nucleotide identity of 95.45% and 95.10% with that of isolates from Japan(AB011819, NC_003536) and an isolate from Korea (KF975894) reported previously. Characterization of ClYVV-derived small interfering RNAs was further analyzed. The prevalent vsiRNAs size was 21-22 nt, and the 5'-terminal base of vsiRNAs was biased towards A/U. The ClYVV-derived small interfering RNAs were most derived from its genomic-strand RNAs, among the 7 hot spots across the genome, 5 hot spots were predominated by 21 nt vsiRNAs. Biological experiments showed that ClYVV could be used to frictionally inoculate many leguminous crops through juice. This is the first report about the full nucleotide sequence of ClYVV isolate from China.