梅病毒A新疆蟠桃分离物的全基因组序列分析与侵染性克隆构建

Complete Genome Sequence Analysis and Infectious Clone Construction of Mume Virus A Peach Isolate pp in Xinjiang

【目的】梅病毒A(mume virus A,MuVA)是新发现的感染桃树的病毒,其全基因组序列分析研究甚少,国内尚无报道。本研究旨在探明MuVA在我国新疆蟠桃中的感染情况,分析MuVA pp分离物的基因组、系统进化及致病性,为MuVA的防治提供科学依据。【方法】使用RT-PCR方法对田间蟠桃样品进行MuVA检测,采用5′/3′cDNA末端快速扩增(RACE)技术确定MuVA pp分离物的全基因组序列,并运用生物信息学软件分析MuVA pp分离物的基因组序列特征和系统进化关系。利用Gibson组装策略构建该分离物的基因组全长cDNA克隆,并通过农杆菌介导的方法对其侵染性进行接种测试。【结果】RT-PCR检测结果表明,30份疑似病毒病样本中有10份感染了MuVA,不同蟠桃品种感染率由高到低分别为‘七月蟠’(4/9)、‘八一蟠桃’(5/13)、‘中熟八一’(1/8);MuVA pp分离物基因组全长为7 647 nt,基因组由5′非翻译区(5′UTR)、3′UTR和两个重叠的开放阅读框(ORF)组成,编码甲基转移酶(Met)、RNA解旋酶(Hel)、RNA依赖性RNA聚合酶(RdRp)、外壳蛋白(CP)和运动蛋白(MP);序列分析结果显示,MuVA pp分离物与已报道的MuVA pm14分离物基因组的核苷酸和多聚蛋白氨基酸序列一致性分别为80.8%和82.7%,两者的多聚蛋白存在406个氨基酸残基的差异,分布在Met(21个)、Hel(29个)、RdRp(19个)、CP(18个)和其他(319个)。与MuVA pm14分离物显著不同的是5′UTR,核苷酸序列一致性仅为74.6%,编码的蛋白质中,MP变异最大,氨基酸一致性为80.9%,RdRp最保守,氨基酸一致性最高,为94.0%;系统发育分析结果显示,MuVA pp分离物与MuVA pm14分离物的亲缘关系最近,且MuVA有宿主和地理特异性的趋势,李子分离物与梅分离物聚集成簇,而桃分离物单独成支;构建的pCB301-MuVA侵染性克隆可以系统侵染苋色藜,并未引起症状,导致三生烟接种叶产生过敏性坏死,但不能引起系统侵染,也不能侵染昆诺藜、心叶烟、西方烟、本氏烟、番茄、黄瓜和南瓜。【结论】成功获得了蟠桃MuVA pp分离物的全基因组序列,其基因组为单链正义RNA,大小为7 647 nt,不含3′端poly(A)尾,编码两个ORF;通过摩擦接种证实MuVA不能侵染草本植物;构建了具有侵染活性的MuVA pp分离物的全长c DNA侵染性克隆载体pCB301-MuVA,可系统侵染苋色藜,且无明显症状,导致三生烟接种叶产生过敏性坏死,未引起系统侵染,这为进一步研究MuVA的致病分子机理提供了参考依据。

【Objective】Mume virus A peach isolate(MuVA pp)is a newly discovered virus infecting peach(Prunus persica)trees,and its complete genome sequence study has not been reported in China.Therefore,the purpose of this study is to analyze the genome,phylogenetic evolution and pathogenicity of MuVA pp isolate,and to investigate its prevalence in peach in Xinjiang,China,so as to provide scientific basis for the prevention and control of MuVA.【Method】RT-PCR was used to detect MuVA in field samples of peach,and 5′/3′rapid amplification of cDNA ends(RACE)technology was used to determine the complete genome sequence of MuVA pp isolate.The genome organization and phylogenetic relationships were analyzed by bioinformatics methods.A full-length infectious cDNA clone was constructed using Gibson assembly,and its infectivity was tested by inoculation with Agrobacterium tumefacieas.【Result】The results of RT-PCR showed that 10 of the 30 suspected viral disease samples were infected with MuVA,and the infection rate of different peach varieties was from high to low,namely‘July flat peach’(4/9),‘flat peach on August 1’(5/13)and‘medium mature August 1’(1/8),respectively.The genome of MuVA pp is 7647 nt in length and consists of 5′UTR,3′UTR and two overlapping open reading frames(ORFs),encoding methyltransferase(Met),RNA helicase(Hel),RNA-dependent RNA polymerase(RdRp),coat protein(CP)and movement protein(MP).The sequence analysis showed that MuVA pp isolate has 80.8%and 82.7%identities with previously reported MuVA isolate pm14 at the nucleotide sequence and amino acid sequence levels,respectively.The polyproteins encoded by these two isolates differ by 406 amino acid residues,which are distributed in Met(21),Hel(29),RdRp(19),CP(18),and other(319).In addition,the 5′UTR of the genome was more different,and the identity of the nucleotide sequence was only 74.6%.Of these encoded proteins,MP has the greatest variability(80.9%identity),while RdRp is the most conserved(94.0%identity).Phylogenetic analysis showed that MuVA pp isolate was closely related to MuVA pm14 isolate,and MuVA showed a tendency of host and geographic specificity.Plum isolates and mume isolates clustered into clusters,while peach isolates formed clays alone.The Chenopodium amaranticolor plants could be systemically infected by MuVA infectious clone,but symptomless.Tobacco(Nicotiana tabacum var.Samsun NN)agroinfiltrated showed allergic necrosis in inoculated leaves,but no systemic infection occurred.Other indicator plants,including C.quinoa,N.glutinosa,N.occidentalis,N.benthamiana,or Solanum lycopersicum,Cucumis sativus and Cucurbita moschata,could not be infected by MuVA infectious clones.【Conclusion】The complete genome sequence of the peach isolate pp of MuVA was sequenced successfully.The genome is a single-stranded positive-sense RNA with a length of 7647 nt,lacking a 3′terminal poly(A)tail,and encoding two ORFs.Mechanical inoculation proved that MuVA could not infect herbaceous plants.pCB301-MuVA,a MuVA pp full-length cDNA infectious cloning vector,was constructed.It could infect C.amaranticolor systematically and N.tabacum var.Samsun NN locally.The results provide a reference for further study of the pathogenic molecular mechanism of MuVA.