关键词: 苹果褪绿叶斑病毒, 基因组, 外壳蛋白, 多样性, 重组分析

Abstract: 【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 (GenBank 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.

Key words: Apple chlorotic leaf spot virus (ACLSV), genome, coat protein, diversity, recombination analysis