为探索利用PRSV-NIb基因同源dsRNA的原核表达产物在体外防治番木瓜环斑病毒的可行性,利用较保守的PRSV-NIb基因3’端的312、501和809 bp区段分别构建了含有PDK内含子的3个发夹RNA编码结构,并选用pSP73和M-Jml09LacY分别作为宿主载体和宿主菌构建了高效的dsRNA原核表达工程菌M-Jml09LacY/pSP73-RNAi-N312、M-Jml09LacY/pSP73-RNAi-N501、M-Jm109LacY/pSP73-RNAi-N809,经IPTG诱导成功表达了dsRNA,并证明dsRNA不被DNase I和RNase A降解,稳定性较好.还利用PRSV-NIb基因构建了GFP瞬时植物融合表达载体pNIb-GFP,对经3种dsRNA和GFP瞬时融合表达载体共转化的番木瓜叶片原生质体的共聚焦显微镜观察及通过半定量RT-PCR对其中mRNA表达量的分析,结果表明融合基因NIb-GFP的表达均发生了不同程度的下调,说明dsRNA在原生质体中引发了针对同源基因的沉默.%To explore the use of PRSV-NIb gene prokaryotic expression of homologous dsRNA product feasibility for controlling papaya ringspot virus in vitro,three hairpin RNA coding structures with PDK intron were built with 3'-end 312 bp,501 bp and 809 bp sections of a more conservative PRSV-NIb gene.After inserted into the plasmid pSP73,the structures were transformed into M-Jml09LacY of the RNaseⅢ-deficient strain and induced with IPTG,respectively.The Escherichia coli strains could express N312-dsRNA,N501-dsRNA and N809-dsRNA,respectively.The plant transient expression vector encoding the PRSV-NIb with an N-terminal GFP fusion was constructed.Protoplasts were co-transfected using the transient GFP-fusion expression vector and the corresponding bacterially expressed dsRNA.The results of GFP fluorescence intensity using a confocal microscope and semiquantitative RT-PCR analysis of the PRSV-NIb mRNA expression showed that the effectively bacterially expressed dsRNA-triggered gene silencing was detected.Our work laid a foundation for a potential green and effective approach protecting papaya from PRSV.
展开▼
