茄子斑驳皱缩病毒病病原物的鉴定及分子进化分析

Identification and molecular evolutionary analysis of viruses causing eggplant mottle crinkle disease

为了鉴定引起茄子斑驳皱缩病毒病的病原物,利用siRNA高通量测序技术(Small interfering RNA,siRNA),结合生物信息学的方法发现2个表现斑驳和皱缩的茄子样品中存在蚕豆萎蔫病毒2号(Broad bean wilt virus,BBWV2)、葡萄A病毒(Grapevine virus A,GVA)和烟草花叶病毒(Tobacco mosaic virus,TMV)。采用RT-PCR技术对该结果进行验证,2个样品中均检测到BBWV2和TMV,未检测到GVA。通过对BBWV2外壳蛋白(Large coat protein,LCP)大亚基基因进行测序和分子进化分析,获得了2条1126 bp的BBWV2 LCP基因片段,2条BBWV2 LCP基因的核苷酸序列及其编码的氨基酸序列一致性分别为99.6%和99.3%;与NCBI中已发表的23个BBWV2 LCP基因核苷酸序列及其编码的氨基酸序列一致性分别为79.9%~95.2%和80.5%~97.2%。基于LCP基因核苷酸序列进行种群结构和遗传多样性分析,结果表明在系统发育进化树中,BBWV2可分为两大支。不同寄主BBWV2 LCP基因的遗传距离在0.049~0.332。

To identify the viruses causing eggplant mottle crinkle disease,the high-throughput sequencing of small interfering RNA(siRNA)and the bioinformatics methods were used to detect the potential viral pathogens,including the broad bean wilt virus 2(BBWV2),grapevine virus A(GVA)and tobacco mosaic virus(TMV).The results were validated by RT-PCR.BBWV2 and TWV were detected in the samples,and GVA was not detected.Through sequencing and molecular evolutionary analysis of the large subunit gene of BBWV2 coat protein(LCP),two 1126 bp BBWV2 LCP gene fragments were obtained.The nucleotide and coding amino acid sequences identities of the two BBWV2 LCP genes were 99.6%and 99.3%,respectively.The nucleotide and amino acid sequences identities of the 23 BBWV2 LCP genes published in NCBI were 79.9%-95.2%and 80.5%-97.2%,respectively.The analysis of population structure and genetic diversity based on LCP gene nucleotide sequence showed that BBWV2 could be divided into two branches in the phylogenetic tree.The genetic distance of BBWV2 LCP gene between different hosts ranged from 0.049 to 0.332.