中国农业科学 ›› 2015, Vol. 48 ›› Issue (2): 270-279.doi: 10.3864/j.issn.0578-1752.2015.02.07

• 植物保护 • 上一篇    下一篇

PVYNTN-NW榆林分离物的全基因组序列测定与分析

高芳銮1,常飞1,沈建国2,谢联辉1,詹家绥1   

  1. 1福建农林大学植物病毒研究所/福建省植物病毒学重点实验室,福州 350002
    2福建出入境检验检疫局检验检疫技术中心/福建省检验检疫技术   研究重点实验室,福州 350001
  • 收稿日期:2014-07-07 出版日期:2015-01-16 发布日期:2015-01-16
  • 通讯作者: 谢联辉,Tel:0591-83769704;詹家绥,Tel:0591-83856973
  • 作者简介:高芳銮,E-mail:raindy@fafu.edu.cn。常飞,E-mail:changfei2010@yeah.net。高芳銮与常飞为同等贡献作者。
  • 基金资助:
    国家现代农业马铃薯产业技术体系(CARS-10)、福建省自然科学基金(2013J01088)、国家质检总局科技计划(2013IK094)、质检公益性行业科研专项(201410076)

Complete Genome Analysis of a PVYNTN-NW Recombinant Isolate from Yulin of China

GAO Fang-luan1, CHANG Fei1, SHEN Jian-guo 2, XIE Lian-hui 1, ZHAN Jia-sui1   

  1. 1Institute of Plant Virology, Fujian Agriculture and Forestry University/Key Laboratory of Plant Virology of Fujian Province, Fuzhou 350002
    2Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection and Quarantine Bureau/Fujian Key Laboratory for Technology Research of Inspection and Quarantine, Fuzhou 350001
  • Received:2014-07-07 Online:2015-01-16 Published:2015-01-16

摘要: 【目的】马铃薯Y病毒(Potato virus Y,PVY)是马铃薯病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)代表成员,是马铃薯和烟草生产中分布最为广泛并造成严重经济损失的病毒之一。研究旨在揭示PVY榆林分离物ShX14的全基因组特征,并准确判断其株系分子类型。【方法】根据已经报道的PVY不同基因保守区设计11对简并引物,采用片段重叠法从感染 PVY的马铃薯病叶中扩增、克隆获得榆林分离物ShX14的全长序列,并对其序列特征、重组位点、系统发育关系等进行分析。同时,应用系统发育与性状关联分析(phylogeny-trait association analysis)评估PVY分离物与株系的关联性。【结果】测序获得的ShX14分离物核苷酸序列全长为9 724 nt(不含3′端的多聚腺苷酸尾)。该病毒基因组含有一个9 186 nt的开放阅读框,编码3 061个氨基酸的多聚蛋白(polyprotein)。在P3顺反子中+2相位上也发现由移码(reading frame shift)产生的PIPO蛋白。全基因组序列比较分析显示,ShX14分离物与HN2、SYR-NB-16分离物(PVYNTN-NW株系SYR-I型)的核苷酸、氨基酸序列的一致性分别为98%—99%和98%—100%。该分离物的P1、HC-Pro/P3和CP基因的5′端均检测到显著的重组信号,重组位点分别位于2 318、5 674 和8 385 nt,与PVYNTN-NW株系(SYR-I型)的重组位点相似。系统发育分析结果显示该分离物与HN2、SYR-NB-16分离物相聚成簇,表明其在系统发育关系上,与PVYNTN-NW株系(SYR-I 型)的亲缘关系最近。系统发育分析与性状关联分析结果显示该分离物与PVYNTN-NW株系(SYR-I 型)的关联系数(association index, AI)、简约分值(parsimony score, PS)和最大单系分支(maximum monophyletic clade, MC)3个统计检验均显著,表明其与PVYNTN-NW株系(SYR-I型)存在显著的关联性。同时,应用多重RT-PCR成功扩增出约为1 000、600和400 bp的3个特异性片段,与PVYNTN-NW株系(SYR-I型)的特异条带大小相一致。这些结果也进一步确定该分离物属于 PVYNTN-NW株系(SYR-I型)。【结论】ShX14 分离物为N×O重组分离物,属于PVYNTN-NW株系(SYR-I型),为后续深入开展该分离物的生物学等研究奠定了基础。

关键词: 马铃薯Y病毒, 全基因组, 重组, 系统发育, 系统发育与性状关联分析

Abstract: 【Objective】 Potato virus Y (PVY) is a typical member of the genus Potyvirus in the family Potyviridae. It is one of the most destructive pathogens affecting potato and tobacco and causes significant economic losses worldwide. The objective of this study is to determine the genomic structure of an isolate of PVY from Yulin of China and its phylogenetic relationship with reported PVY strains.【Method】The complete genome of the ShX14 was amplified and sequenced from overlapping fragments using 11 pairs of primers designed from the conserved regions of the known PVY isolates. Genomic structure and recombination events of the isolate were evaluated by various bioinformatics approaches. Phylogenetic tree was reconstructed by maximum likelihood (ML) method using nucleotide sequences of coding regions. In addition, phylogeny-trait association analysis was used to evaluate the relationship between PVY isolate and the reported stains. 【Result】The complete sequence of ShX14 had 9 724 nucleotides, excluding the 3′- terminal poly (A) tail. It contains a single open reading frame of 9 186 nucleotides and encodes a polyprotein of   3 061 amino acids. An additional protein, termed ‘PIPO’, is also translated by +2 nucleotide frame shifting within the P3 cistron. The isolate shares 98%-99% nucleotide identity and 98%-100% amino acid identity with HN2 and SYR-NB-16 (PVYNTN-NW strain, SYR-I genotype), respectively. Similar to the PVYNTN-NW (SYR-I) genomic structure, three recombination breakpoints were identified at nucleotide positions 2 318, 5 674 and 8 385 in the P1, HC-Pro/P3 and the 5′-terminus of CP gene respectively with high confidence. Phylogenetic analysis indicated that ShX14 was clustered together with HN2 and SYR-NB-16, suggesting that it shared high sequence homology with PVYNTN-NW (SYR-I) strain. Association index (AI), parsimony score (PS) and maximum monophyletic clade (MC) all indicated that ShX14 was strongly associated with the PVYNTN-NW (SYR-I) group. Three fragments of about 1 000, 600 and 400 bp in size were also amplified from the isolate by a multiplex RT-PCR, consisting with the expected band of the PVYNTN-NW (SYR-I) strain. Overall, these analyses strongly indicate that ShX14 is likely to be a PVYNTN-NW strain(SYR-I). 【Conclusion】 ShX14 is a N×O recombinant isolate, classified to PVYNTN-NW strain (SYR-I). It will provide useful information in the further study of the biology of this pathogen.

Key words: Potato virus Y, complete genome, recombination, phylogeny, phylogeny-trait association analysis