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Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

文献类型： 外文期刊

作者： Tuo, Decai 1 ; Shen, Wentao 1 ; Yan, Pu 1 ; Li, Xiaoying 1 ; Zhou, Peng 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Minist Agr, Key Lab Biol & Genet Resources Trop Crops, Haikou 571101, Peoples R China

关键词： PLDMV;infectious cDNA clone;In-Fusion;intron;papaya

期刊名称：VIRUSES-BASEL （ 影响因子：5.048； 五年影响因子：5.127 ）

ISSN： 1999-4915

年卷期： 2015 年 7 卷 12 期

页码：

收录情况： SCI

摘要： Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion((R)) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli.In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

  • 相关文献

[1]Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses. Tuo, Decai,Shen, Wentao,Yang, Yong,Yan, Pu,Li, Xiaoying,Zhou, Peng,Tuo, Decai. 2014

[2]混合感染番木瓜PRSV/PLDMV株系基因组全长cDNA的克隆和实时荧光定量PCR分析. 刘芳,庹德财,沈文涛,言普,黎小瑛,周鹏. 2016

[3]海南地区番木瓜花叶病病原的分子鉴定与多样性分析. 张雨良,黄启星,朱丽娣,孔华,曾会才. 2014

[4]海南地区番木瓜畸形花叶病毒的发现与鉴定. 杨勇,庹德财,沈文涛,言普,黎小瑛,周鹏. 2013

[5]海南番木瓜PRSV和PLDMV病毒发生情况及分子鉴定. 张雨良,黄启星,郭安平,曾会才,赵辉,孔华,刘志昕. 2013

[6]Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation. Tuo, Decal,Fu, Lanlan,Shen, Wentao,Li, Xiaoying,Zhou, Peng,Yan, Pu. 2017

[7]Antioxidant Activity of Papaya Seed Extracts. Zhou, Kaibing,Wang, Hui,Mei, Wenli,Li, Xiaona,Luo, Ying,Dai, Haofu,Zhou, Kaibing. 2011

[8]Cloning and expression analysis of phytoene desaturase and zeta-carotene desaturase genes in Carica papaya. Yan, P.,Gao, X. Z.,Shen, W. T.,Zhou, P.,Gao, X. Z.,Zhou, P.. 2011

[9]Diversity and Application of Lactic Acid Bacteria from Papaya. Zhang, Lijuan,Yang, Jinsong,Ma, Liwei,Tan, Haisheng,Liu, Xiangqian,Zhou, Hanlin. 2014

[10]Complete genomic sequence of a Papaya ringspot virus isolate from Hainan Island, China. Lu, Y. W.,Shen, W. T.,Zhou, P.,Tang, Q. J.,Niu, Y. M.,Peng, M.,Lu, Y. W.,Xiong, Z..

[11]NIa-Pro of Papaya ringspot virus interacts with Carica papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G). Gao, Le,Tuo, Decai,Shen, Wentao,Yan, Pu,Li, Xiaoying,Zhou, Peng,Gao, Le,Tuo, Decai,Shen, Wentao,Yan, Pu,Li, Xiaoying,Zhou, Peng,Gao, Le.

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