首页|PVYNTN-NW榆林分离物的全基因组序列测定与分析

PVYNTN-NW榆林分离物的全基因组序列测定与分析

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【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9724 nt(不含3′端的多聚腺苷酸尾)。该病毒基因组含有一个9186 nt 的开放阅读框,编码3061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2318、5674和8385 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1000、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。
Complete Genome Analysis of a PVYNTN-NW Recombinant Isolate from Yulin of China
[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.

Potato virus Ycomplete genomerecombinationphylogenyphylogeny-trait association analysis

高芳銮、常飞、沈建国、谢联辉、詹家绥

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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

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

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

国家现代农业马铃薯产业技术体系福建省自然科学基金国家质检总局科技计划质检公益性行业科研专项

CARS-102013J010882013IK094201410076

2015

中国农业科学
中国农业科学院

中国农业科学

CSTPCDCSCD北大核心
影响因子:1.899
ISSN:0578-1752
年,卷(期):2015.(2)
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