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

Abstract

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

CLC Number:

S763.11

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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Complete Genome Sequence Analysis and Infectious Clone Construction of Bamboo Mosaic Virus Isolated from Fuzhou

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