甘蓝花叶病病毒种类鉴定及BrYV遗传变异分析

为了明确引起甘蓝花叶病的病毒种类，利用小干扰RNA（small interfering RNA，siRNA）高通量测序技术，并结合分子生物学方法查找甘蓝样品中的病毒序列，发现存在芜菁花叶病毒（turnip mosaic virus，TuMV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）、蚕豆萎蔫病毒2（broad bean wilt virus2，BBWV2）、烟草花叶病毒（tobacco mosaic virus，TMV）和芸薹黄化病毒（brassica yellows virus，BrYV）等5种病毒。采用RT-PCR技术进行验证，结果显示样品检测到BrYV、TuMV和CMV共3种病毒。对获得的BrYV外壳蛋白基因（coat protein，cp）进行测序和系统进化分析，获得1条576 bp的BrYV cp基因全长序列，与GenBank中BrYV分离物cp基因核苷酸序列同源率为88.0%~96.4%，氨基酸序列同源率为90.1%~95.8%。基于cp基因序列构建系统发育树，BrYV-GL为BrYV-C基因型，与中国内蒙古油菜BrYV（GenBank登录号EF079907）分离物cp基因序列聚集在一起，亲缘关系较近。BrYV cp基因共检测到4个潜在重组事件，可为后续甘蓝花叶病的研究和防治提供参考。

Identification of Viruses Causing Cabbage Mosaic Disease and Genetic Variation in BrYV

To identify viral pathogens associated with cabbage mosaic viruses,we conducted a high-throughput sequencing of the small interfering RNA (siRNA) of the leaf samples. Based on the siRNA sequences,potential viral pathogens were detected,including the turnip mosaic virus (TuMV),cucumber mosaic virus (CMV),broad bean wilt virus2 (BBWV2),tobacco mosaic virus (TMV) and brassica yellows virus (BrYV). To confirm the existence of the viruses by RT-PCR,BrYV,TuMV and CMV were detected. Moreover,cloning and phylogenetic analysis were carried out on the coat protein (cp) of the BrYV. The cp fragments of the BrYV was 576 bp. Pairwise comparison analysis indicated that the nucleotide and amino acid sequence identities of the BrYV cp genes with other published BrYV BrYV isolates in NCBI were from 88.0%-96.4% and 90.1%-95.8%,respectively. A phylogenetic tree was reconstructed for BrYV using the cp gene sequence,indicating a close relationship between BrYV-GL (classified as BrYV-C genotype) and an isolate from Inner Mongolia (GenBank accession no. EF079907).Four potential evidence of recombination was found among the 15 complete cp sequences. The results of this study may provide a theoretical reference for future research and control of the disease.