Bacterially expressed dsRNA protects maize against SCMV infection
Bacterially expressed dsRNA protects maize against SCMV infection
复制标题
DOI：
10.1007/s00299-010-0911-z
复制
发表时间：
2010-11-01
影响因子：
6.2
通讯作者：
Cheng, Beijiu
中科院分区：
文献类型：
--
作者：
Gan, Defang;Zhang, Jiao;Cheng, Beijiu
RNA干扰（RNA interference，RNAi）是一种序列特异性的转录后基因沉默（posttranscriptional gene silencing，PTGS）过程。在这项研究中，我们报告了一种有效的方法来生产dsRNA使用细菌表达系统。利用RT-PCR技术扩增了甘蔗花叶病毒（Sugarcane Mosaic Virus，SCMV）外壳蛋白（Coat protein，CP）基因的2个片段，并将其克隆到反向重复克隆载体pUCCRNAi中。将两个重组质粒分别转化到E. coliHT 115，一株RNase-III缺陷型菌株，用异丙基-β-d-硫代半乳糖苷（IPTG）诱导表达。E.将含有大量dsRNA的大肠杆菌HT 115作为喷雾剂施用到植物上，实验证实了预防功效。我们的研究结果表明，喷洒粗dsRNA含有提取物抑制SCMV感染，和来自上游区域（CP 1）的dsRNA比来自下游区域（CP 2）的SCMV CP基因的dsRNA更有效。这些结果为使用dsRNA和PTGS方法控制植物病毒提供了有价值的工具。
RNA interference (RNAi) is a sequence-specific, posttranscriptional gene silencing (PTGS) process in plants that is mediated by dsRNA homologous to the silenced gene(s). In this study, we report an efficient method to produce dsRNA using a bacterial expression system. Two fragments of the Sugarcane Mosaic Virus (SCMV) CP (coat protein) gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi. The two recombinant plasmids were transformed individually into E. coli HT115, an RNase-III deficient strain, and dsRNA was induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). The crude extracts of E. coli HT115 containing large amounts of dsRNA were applied to plants as a spray and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (CP1) was more effective than was dsRNA derived from a downstream region (CP2) of the SCMV CP gene. The results provide a valuable tool for plant viral control using dsRNA and the PTGS approach.