萝卜病毒RasV1基因组多片段共表达载体构建及拟南芥转化.docVIP

 萝卜病毒 RasV1 基因组多片段共表达载体 构建及拟南芥转化# 潘晟昊1，竺锡武1，曹跃芬1，王春兰1，陈集双1,2** 5 10 15 20 25 30 35 40 （1. 浙江理工大学生物工程研究所，杭州 310018； 2. 南京工业大学生物资源工程研究所，南京 210009） 摘要：dsRNA 病毒侵染性克隆主要采用原生质体法，但由于原生质体法程序复杂、不够稳定， 因而 dsRNA 病毒侵染性克隆成功的例子不多。为探索萝卜潜隐性 dsRNA 病毒 Raphanus sativus virus 1（RasV1）的基因组构成并为 dsRNA 病毒侵染性克隆的深入研究提供基础， 本研究利用多位点 Gateway 克隆技术构建 RasV1 病毒三条基因组的多片段共表达载体，通过 农杆菌花序侵染法将该多片段共表达载体导入野生型拟南芥，经卡那霉素、绿色荧光筛选并 结合基因组 DNA-PCR、基因组 DNA 斑点杂交等分子鉴定，证实萝卜病毒 RasV1 的 3 条基因序 列已成功共转化入拟南芥基因组中。但在转基因拟南芥植株中未能提取到病毒的 dsRNA，原 因有待进一步研究分析。 关键词：Raphanus sativus virus 1；多位点 Gateway 克隆；dsRNA；拟南芥 中图分类号：Q945.8 Construction of multi-fragment coexpression vector of Raphanus sativus virus 1 genome and transformation into Arabidopsis thaliana PAN Shenghao1, ZHU Xiwu1, CAO Yuefen1, WANG Chunlan1, CHEN Jishuang1,2 (1. Institute of Bioengineering, Zhejiang Sci-Tech University, HangZhou 310018; 2. Institute of Bioresource Engineering, Nanjing University of Technology, NanJing 210009) Abstract: The technique of protoplast fusion is the main method of infectious cloning of dsRNA virus, but due to its complex procedures and not high stability, there are few examples of successful infectious cloning of dsRNA. In order to study the gene element of Raphanus sativus cryptic dsRNA virus Raphanus sativus virus 1（RasV1）and provide the basis for the further research of infectious cloning of dsRNA virus, the technology of multi-site Gateway cloning was used to construct multi-fragment coexpression vector of RasV1 genome. The coexpression vector was transferred into wild Arabidopsis thaliana mediated by Agrobacterium tumefaciens. The transformations were identified by screening of Kanamyci gene, green fluorescence observation of GFP gene with microscope, gennome DNA-PCR and genome dot blot southern hybridization. However, the dsRNA of RasV1 were not extracted from the transgenic Arabidopsis thaliana, reasons should be studied for further. Keywords: Raphanus sativus virus 1; multi-site Gateway cloning; dsRNA; Arabidopsis thaliana 0 引言