侵染白术的大豆花叶病毒的全基因序列测定及分析

Identification and Analysis of Complete Genomic Sequences of Soybean Mosaic Virus Isolates from Atractylodes macrocephala Koidz

大豆花叶病毒(soybean mosaic virus,SMV)是影响大豆产量与质量的重要病原体,且自然寄主范围窄。本研究对疑似感病白术叶片进行非序列依赖性扩增(sequence-independent amplification,SIA)检测,结果表明,感病白术为SMV侵染,并命名为SMV-Am。为明确其基因组结构特征及系统进化关系,本研究利用双链RNA(double-stranded RNA,dsRNA)提取、RT-PCR及RACE技术对其进行全基因组扩增,并进行生物信息学分析。结果表明,SMV-Am基因组全长9587 nt,具有显著的马铃薯Y病毒属(Potyvirus)基因组结构特征;核苷酸与氨基酸序列相似性分析表明,SMV-Am与来自中国的SMV-Liaoning分离物相似性最高,核苷酸与氨基酸序列相似性分别为96.57%和98.86%;系统进化分析也显示与SMV-Liaoning分离物亲缘关系最近;对SMV-Am蛋白序列进一步分析发现,存在与功能位点相关的氨基酸突变,经I-TASSER和PyMOL软件进行蛋白建模分析,发现SMV-Am的P1、HC-Pro、P3、6K2、NIa-Pro、NIb蛋白结构均发生不同程度的变化,且P1蛋白最明显;重组分析结果显示,在SMV-Am基因组的6560~8950 nt位置存在重组事件,其主要亲本和次要亲本分别为SMV-XFQ012(GenBank登录号:KP710875.1)和SMV-pCB301-SC15(GenBank登录号:MH919386.1)。这是SMV侵染白术的首次报道,研究结果有望为防范SMV病害在白术上爆发流行以及防治提供科学依据。

Soybean mosaic virus(SMV)is a critical pathogen that reduces the soybean yield and seed quality worldwide,and SMV has a restricted natural host range.In this study,we used sequence-independent amplification(SIA)methods to identify the viruses that may cause the Atractylodes macrocephala Koidz disease.Results revealed that there is SMV in diseased Atractylodes macrocephala leaves,and we named this isolate as SMV-Am.To further characterize the genomic structural and phylogenetic relationships of SMV-Am,genomic dsRNA was extracted,and the genomic sequence was amplified by RT-PCR and RACE.The results of bioinformatics analysis showed that the genomic RNA of SMV-Am is 9587 nucleotides in length,which conforms to the typical characteristics of Potyvirus.According to the sequence alignment of complete nucleotide sequences,SMV-Am showed the highest level of nucleotide homology and amino acid sequences to SMV-Liaoning,96.57%and 98.86%,respectively.In addition,phylogenetic analysis based on the nucleotide sequences of other SMV isolates of SMV-Am revealed that SMV-Am was most closely related to SMV-Liaoning.Then,the SMV-Am protein was further analyzed by I-TASSER and PyMOL software,revealing that the key amino acid mutations led to the structural changes of P1,HC-Pro,P3,6 K2,NIa-pro and NIb proteins,with P1 the most obvious.Finally,recombination has been detected at the position of 6560-8950 nucleotides,the main parent is the isolate SMV-XFQ012(accession number KP710875.1),and the secondary parent is isolate SMV-pCB301-SC15(accession number MH919386.1).This study is the first report of SMV in Atractylodes macrocephala Koidz,and these results are expected to provide a scientific basis for the prevention and control of SMV infection on Atractylodes macrocephala Koidz.