杏衰退萎黄病病毒的siRNA高通量测序和RT-PCR鉴定

Identification of Viruses Causing Apricot Decline and Leaf Chlorosis Disease by siRNA High-throughput Sequencing and RT-PCR Detection

为了明确引起杏衰退萎黄病的病毒种类,利用小干扰RNA(small interfering RNA,siRNA)高通量测序技术,结合生物信息寻找杏衰退萎黄样品中的病毒序列,发现存在亚洲李属病毒1(Asian prunus virus 1,APV1)和亚洲李属病毒3(Asian prunus virus 3,APV3)。为了验证该结果,采用RT-PCR对10个待测杏样品进行APV1、APV2和APV3检测,所有样品中未检测到APV2,检出APV1和APV3的样品数分别为3和4个。对检测到病毒的外壳蛋白(coat protein,CP)进行测序和系统进化分析,分别获得了3条653 bp的APV1和4条1031 bp(或1032 bp)的APV3 CP基因片段;3条APV1 CP基因核苷酸序列一致性为99.2%~99.8%,与NCBI中已发表的26个APV1 CP基因序列一致性为44.0%~99.8%;4条APV3 CP基因序列一致性为78.5%~99.5%,与NCBI中已发表的26个APV3 CP基因序列一致性为44.7%~99.5%。

To identify viral pathogens associated with apricot tree decline and leaf chlorosis,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 Asian prunus virus(APV)1 and APV 3.To confirm the existence of the viruses in the symptomatic samples,ten leaf samples were collected and tested by reverse transcription-polymerase chain reaction.The result showed that the infected number of APV1 and APV3 was 3 and 4,respectively.Moreover,cloning and phylogenetic analyses were carried out on the coat protein(CP)of the APV1 and APV3.The CP fragments of the APV1 in the three samples were identical to each other in size of 653 bp,while the CP fragments of the APV3 in the four samples were 1031 or 1032 bp in size.Pairwise comparison analysis revealed that the nucleotide sequence identities between the three APV1 isolates were from 99.2% to 99.8%,and they shared the nucleotide sequence identities of 44.0% to 99.8% with other published APV1 isolates in NCBI.The four APV3 have nucleotide sequence identities from 78.5% to 99.5% between each other,and they shared nucleotide sequence identities of 44.7% to 99.5% to those published in NCBI.