表达dsRNA的细菌提取液可抑制黄瓜花叶病毒对烟草的侵染  

Crude extracts of bacterially expressed dsRNA can be used to protect tobaccos against CMV infections

作　　者：张德咏 朱春晖 成飞雪 何明远 张战泓[2] 刘勇 

机构地区：[1]湖南省植物保护研究所,长沙410125 [2]湖南省蔬菜研究所,长沙410125

出　　处：《植物病理学报》2008年第3期304-311,共8页Acta Phytopathologica Sinica

基　　金：国家“十一五”科技支撑计划项目(2006BAD17B08;2006BAD08A08)

摘　　要：利用RT-PCR分别克隆了CMV P3613株系的RNA2片段、MP(movement protein)基因片段及CMV AN株系的CP(coat protein)基因片段。以CP基因为中间间隔序列,分别构建了含有RNA2片段和MP基因反向重复片段的原核表达载体。体外转录试验表明:两个载体转录后都能形成预期大小的dsRNA。经过IPTG诱导,在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase和RNaseA消化处理,证实为dsRNA。将表达病毒基因dsRNA的细菌超声破碎后处理烟草,进行保护和治疗试验,结果表明:表达CMV MP基因和RNA2片段dsRNA的细菌破碎液能够诱导烟草对CMV产生抗性。接种病毒60d后,保护效果试验病株率分别为45%和60%,治疗效果试验病株率分别为75%和85%,而其他对照发病率均为100%。本研究结果证明了利用RNA沉默的原理,构建具有反向重复序列的原核表达载体,用细菌表达dsRNA的粗提取物可防治CMV对烟草的侵染。RNA2 fragment, movement protain （MP） gene fragment of CMV isolate P3613 and coat protein （CP） gene fragment of CMV isolate AN were amplified by RT-PCR, respectively. Three fragments were cloned into vector pBluescript SK（ - ）. Two inverted-repeat expression vectors, using CP gene as a space, of CMV RNA2 and MP, were built up under the control of T7 promotor. The expression of expected dsRNAs, MP dsRNA or RNA2 dsRNA of CMV were confirmed in vitro. Furthermore, the two plasmids were transformed into E. coli HTll5, an RNase-Ⅲ deficient strain, respectively. The two distinct expression crudes of large amount dsRNA were used for spraying plants to compare the curative or protective efficiency. 45% and 60% treated plants were infected in 60 days in MP dsRNA and RNA2 dsRNA protective assay whereas 75% and 85% plants were infected in curative assay, respectively. The results showed that the MP dsRNA bacterial crude was more efficient than RNA2 dsRNA for inducing plant resistance to the virus. The results lead a way for plant virus control using dsRNA through post transcriptional gene silencing （PTGS） approach. Now we are treating the virus infected plants in greenhouse to find the best condition for using this technique in field in near future.

关 键 词：黄瓜花叶病毒 RNA沉默 双链RNA表达 细菌 保护 病毒侵染 

分 类 号：S432.1[农业科学—植物病理学]