关键词: 梅病毒A, RT-PCR, 基因组结构, 进化分析, 侵染性鉴定

Abstract:

【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 7 647 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 7 647 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.

Key words: mume virus A (MuVA), RT-PCR, genome structure, evolutionary analysis, identification of infectivity