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

doi:10.11707/j.1001-7488.20170805

摘要/Abstract

摘要： [目的]研究BaMV福州分离物BaMV-TMS1及其携带的卫星RNA（satBaMV）分离物satBaMV-TMS1的全基因组特征，明确其系统发育关系；对BaMV进行cDNA侵染性克隆构建方法的改进，为其反向遗传学体系的快速建立提供简便的方法。[方法]根据已报道的BaMV和satBaMV全长序列保守区分别设计2对和1对扩增引物，从感染BaMV的竹叶中扩增、克隆获得BaMV-TMS1和satBaMV-TMS1的全长序列，并进行序列特征分析和系统发育树构建。采用多片段无缝克隆方法将扩增得到的2个病毒DNA片段和载体进行连接并转化农杆菌，接种本氏烟后检验其侵染性。[结果]测序获得的BaMV-TMS1和satBaMV-TMS1分离物核苷酸序列全长分别为6 366和834 nt（不含3'端的多聚腺苷酸尾），具有典型的BaMV和satBaMV基因组结构特征。BaMV-TMS1分离物与已报道的其他分离物序列同源性为82%~83%，而satBaMV-TMS1分离物与其他分离物的序列同源性为92%~93%。系统进化树分析表明，BaMV-TMS1分离物可单独形成一簇，satBaMV-TMS1分离物也单独形成一个新的分支。侵染性克隆通过农杆菌注射接种本氏烟10天以后，RT-PCR检测以及透射电镜负染观察结果都验证病毒成功侵染。[结论]BaMV-TMS1和satBaMV-TMS1分离物与已知序列有着较高的变异度且生成新的系统发育树分支，体现了该病毒与卫星RNA较高的遗传多样性。本研究成功构建具有生物活性的BaMV侵染性克隆且构建方法相对简单快速。

关键词: 竹花叶病毒, 卫星RNA, 全基因组, 系统发育, 侵染性克隆

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.

Key words: bamboo mosaic virus, satellite RNA, complete genome, phylogeny, cDNA infectious clone

中图分类号:

S763.11

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.

Yan Wenkai, Lin Wenwu, Yang Wenting, Du Yaxin, Wu Zujian, Yang Liang. Complete Genome Sequence Analysis and Infectious Clone Construction of Bamboo Mosaic Virus Isolated from Fuzhou[J]. Scientia Silvae Sinicae, 2017, 53(8): 35-42.

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