林业科学2017，Vol.53Issue(8)：35-42,8.DOI:10.11707/j.1001-7488.20170805
竹花叶病毒福州分离物基因组序列分析及其侵染性克隆的构建
Complete Genome Sequence Analysis and Infectious Clone Construction of Bamboo Mosaic Virus Isolated from Fuzhou
摘要
Abstract
[Objective]Bamboo mosaic virus(BaMV),a typical member of the genus Potexvirus,is widespread in bamboo cultivation region and can severely affect regular growth of bamboos. Theobjective of this study is to determine the genomic structure of a new isolate of BaMV (BaMV-TMS1) and its associated satellite RNA(satBaMV-TMS1) from Fuzhou of China and their phylogenetic relationship with reported BaMV and satBaMV strains. Moreover,it is necessary to optimize an efficientmethod of constructing BaMV cDNA infectious clone for rapid establishment of reverse genetic system.[Method]The complete genomes of BaMV-TMS1 and satBaMV-TMS1 were amplified and sequenced from bamboo leaves with mosaic symptom by respectively using 2 and 1 primer pairs designed according to the conserved regions of known sequences. Genomic structure was analyzed and phylogenetic tree was reconstructed by maximum likelihood (ML)method. Meanwhile,seamless cloningmethod of multiple segments was applied to ligate two segments of BaMV-TMS1 and an amplified vector segment. An agrobacteria-mediatedmethod was used to verify the infectivity of BaMV cDNA infectious clone.[Result] The complete sequences of BaMV-TMS1 and satBaMV-TMS1 have 6365 and 836 nucleotides, respectively,excluding the 3'-terminal poly(A) tail and contain the typical characteristic of genomic structure of BaMV and satBaMV. The isolate BaMV-TMS1 shared the highest nucleotide sequence identity of 82%-83% and 92%-93%, respectively,with all the BaMV and satBaMV isolates available in GenBank. Phylogenetic analysis indicated that BaMV-TMS1 was clustered into a new phylogenetic sub-lineage. Similarresult can be seen that satBaMV-TMS1 was grouped into a new sub-cluster. Theresult of RT-PCR detection and negative staining of plant crude extraction showed the successful and highly-efficient infection of BaMV after 10 days post-inoculation.[Conclusion] The isolates BaMV-TMS1 and satBaMV-TMS1 have a higher degree of variation and were clustered into a new phylogenetic sub-lineage considering all isolates analyzed in this study,suggesting a relatively higher genetic diversity of the virus and its associated satellite RNA. On the other hand,the study provides a rapid and efficientmethod of constructing BaMV cDNA infectious clone with biological activity.关键词
竹花叶病毒/卫星RNA/全基因组/系统发育/侵染性克隆Key words
bamboo mosaic virus/satellite RNA/complete genome/phylogeny/cDNA infectious clone分类
农业科技引用本文复制引用
闫文凯,林文武,杨文婷,杜雅馨,吴祖建,杨靓..竹花叶病毒福州分离物基因组序列分析及其侵染性克隆的构建[J].林业科学,2017,53(8):35-42,8.基金项目
教育部博士点基金项目(20113515110001) （20113515110001）
福建省自然科学基金项目(2014J06011) （2014J06011）
福建省高校杰出青年科研人才培育计划(JA13092) （JA13092）
"福建农林大学杰出青年科研人才"培养专项基金项目(xjq201402). （xjq201402）
