原核表达的PRSV HC-Pro基因同源dsRNA诱导番木瓜抗性的研究  

Studies of Bacterially Expressed dsRNAs Targeting PRSV HC-Pro Gene Inducing Resistance to PRSV

作　　者：杨国峰[1,3] 沈文涛[2] 言普[2] 黎小瑛[2] 周鹏[1,2] 

机构地区：[1]海南大学农学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,分析测试中心,海口571101 [3]海南师范大学生命科学学院,海口571158

出　　处：《园艺学报》2013年第7期1269-1277,共9页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31171822,31000844);海南省重点科技计划项目(ZDXM20120027)

摘　　要：利用番木瓜环斑病毒(PRSV)HC-Pro基因3′端824bp区段,通过OZ-LIC法构建了含有PDK内含子的发夹RNA编码结构,并选用pSP73和M-Jm109LacY分别作为宿主载体和宿主菌,构建了高效的同源dsRNA原核表达工程菌M-Jm109LacY/pSP73-RNAi-H824,经IPTG诱导表达的dsRNA不被DNaseI和RNaseA降解,稳定性较好。采用喷洒dsRNA粗制品的方式对番木瓜植株进行保护性处理和治疗性处理,症状观察及ELISA、Real-time RT-PCR分析结果表明,保护性处理能有效诱发对番木瓜环斑病毒的抗性(发病率低,发病时间晚);治疗性处理(植株已接种PRSV 25d)能在处理初期引起番木瓜环斑病毒积累量发生短暂降低。A Hairpin RNA coding structure of the 824 bp region（from 2 274 to 3 097）of PRSV HC-Pro gene was constructed by the OZ-LIC method. After inserted into the plasmid pSP73,the structure was transformed into M-Jm109LacY of the RNaseⅢ-deficient strain and induced with IPTG. The recombinant E. coli strain could express H824-dsRNA that remained stable in the presence of DNase I enzyme and RNase A enzyme. We carry out a protective resistance assay and a therapeutic resistance assay. In the protective resistance assay,dsRNA was used to spray onto the plant surface before inoculation of papaya leaves with PRSV. Protective treatment experimental results showed dsRNA derived from the functional gene of PRSV could protect papaya plants from virus infection. ELISA analysis and Real-time RT-PCR results confirmed that the virus accumulation could be inhibited by dsRNA. In the therapeutic resistance assay,dsRNA was used to spray onto the plant surfaces 25 days after inoculation of papaya leaves with PRSV. ELISA analysis and Real-time RT-PCR results showed that the virus accumulation declined slightly after spraying dsRNA for 3 days.

关 键 词：番木瓜环斑病毒 HC-PRO DSRNA 原核表达 RNA沉默 

分 类 号：S667.9[农业科学—果树学]