ACLSV山东苹果分离物基因重组及CP序列多样性分析

Analysis of Genome Recombination and CP Sequence Diversity of ACLSV Apple Isolate from Shandong

【目的】明确苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)山东苹果分离物全基因组的分子特征、潜在重组事件及CP基因的多样性。【方法】采用RT-PCR分段扩增、克隆、拼接获得ACLSV全基因组序列,对其进行分子特征描述,并与已报道的16条全长基因组或近全长基因组序列进行比较和系统发育分析,同时扩增其CP进行多样性分析。【结果】扩增得到ACLSV山东苹果分离物QD-13全基因组序列(Gen Bank登录号KJ522693),QD-13全长7 557 nt,包含3个ORF。构建进化树分析表明,QD-13与日本苹果分离物B6聚为一簇,二者相似性最高,为85.9%。17条ACLSV分离物基因组分段比较表明,5′端和3′端差异较大;ORF3保守性相对较高,除分离物Ta Tao 5和MS外,其推导的氨基酸序列相似性均在91.2%以上。ORF1编码区527—665 aa区域保守性差,所比较的17条序列在该区域的相似性为15.4%—59.7%。基因组重组分析以P≤0.05为标准,3种以上算法同时检测到则为有意义重组事件,结果显示,QD-13存在3个重组事件,它们分别发生在6 507—6 247、6 397-6 497和3 851—4 760 nt。扩增QD-15、QD-20和YT-24分离物ACLSV的CP,共获得22条序列,可分为3种类型。对CP氨基酸序列进行比对分析表明,3种类型为不同氨基酸保守位点的组合,分别是Met60-Ser73-Ser79-Asp82-Asn97-Gly98、Leu59-Met83-Ile193和Ala40-Leu60-Ala72-Phe75-Ile86-Arg88-Ser130-Gly137-Met184。【结论】报道了中国ACLSV苹果分离物的完整基因组序列;明确了其基因组分子特点并发现了3个主要的潜在重组事件;ACLSV山东苹果分离物CP存在3种序列类型,具有丰富的多样性。

【Objective】The objective of this study is to determine the molecular characterization and potential recombination events of genome of Apple chlorotic leaf spot virus（ACLSV） isolated from apple in Shandong Province, and diversity of CP gene of these ACLSV isolates.【Method】The whole genome sequence of ACLSV was obtained by RT-PCR through segment amplification, cloning and gene splicing. The molecular characterization and phylogeny of whole genome of ACLSV were then described and analyzed by comparison with reported sixteen full-length genome or near full-length genome sequences. The CP of ACLSV isolates was amplified for phylogenetic and diversity analysis. 【Result】The whole genome sequence of the ACLSV isolate, QD-13, was cloned and sequenced. The molecular characterization showed that QD-13（Gen Bank accession number KJ522693） was 7 557 nt in length containing three ORFs, and shared the highest identity of 85.9% with Japanese apple isolate B6. The phylogenetic tree showed that the isolate QD-13 was grouped into the same cluster with isolate B6. Segmented comparisons of seventeen genome sequences of ACLSV isolates indicated that there were great differences in the sequences of 5′UTR and 3′UTR. ORF3 was relatively conserved and all of the identities of putative amino acid sequences were more than 91.2% except isolates Ta Tao 5 and MS. The ORF1 coding region between 527-665 aa was least conservative and the identities of this region were 15.4%-59.7% among all the seventeen compared sequences. Recombination event was considered as significant when it was simultaneously detected by more than three detection methods with P-Value less than 0.05. The results showed that there were three potential significant recombination events occurred in 6 507-6 247, 6 397-6 497 and 3 851-4 760 nt, respectively. The isolates QD-15, QD-20 and YT-24 were randomly selected for CP amplification. The diversity analysis showed that twenty-two sequences of CP obtained from QD-15 and YT-24 includes three types. Comparison of amino acid sequences of CP revealed that the three types are different combinations of amino acid conserved sites which were Met60-Ser73-Ser79-Asp82-Asn97-Gly98, Leu59-Met83-Ile193 and Ala40-Leu60-Ala72-Phe75-Ile86-Arg88-Ser130-Gly137-Met184, respectively. 【Conclusion】The complete genome sequence of ACLSV apple isolate in China was reported. The molecular characterization of QD-13 was confirmed and three major recombination events occurred in the genome of QD-13. Three types of CP gene were found in the isolates of ACLSV from Shandong Province, which indicated that CP gene of ACLSV has rich diversity.