摘要
为探索细菌表达目标基因dsRNA介导的RNAi技术是否在家蚕Bombyx mori可行,本研究引入了在其他物种中广泛应用的细菌表达dsRNA的RNAi系统:HT115细菌株和L4440质粒。利用L4440载体两端含有T7启动子的特点,设计并构建了针对家蚕核受体FTZ-F1基因的RNA干扰(RNA interference)载体,将构建好的质粒转入大肠杆菌Escherichia coliHT115,在IPTG诱导下成功获得目标基因对应双链RNA(dsRNA)。结果显示:通过对5龄第7天家蚕幼虫注射IPTG诱导后提取的FTZ-F1基因对应的dsRNA25μg,85%的蛹变态发育过程明显延迟,不能实现幼虫到蛹的形态完全转变。荧光定量PCR分析显示目标基因的表达得到了特异的抑制。实验结果初步表明,通过细菌表达目标基因dsRNA介导的RNAi策略,以其经济、高效的特点,具有广泛应用于家蚕基因功能研究中的潜力。
We developed a method of RNA interference based on bacterially expressed dsRNA in the silkworm,Bombyx mori.By inserting the target,FTZ-F1 gene fragment,between the two convergent T7 polymerase promoters in opposite orientation in L4440 dsRNA expression vector,the recombinant plasmid was formed.Then the recombinant plasmid was transformed into Escherichia coli HT115,an RNase-III deficient strain.dsRNA was extracted from the E.coli HT115 after being treated with isopropyl-β-D-thio-galactopyranoside（IPTG）.RNAi treatment was performed by injecting the extracted dsRNA（25 μg） into body cavity of silkworm at the 7th day of 5th instar.The RNAi of FTZ-F1 gene resulted in 85% of the insects with delay of pupal metamorphosis and disablement in pupa formation.Real-time quantitative PCR analysis revealed that the expression of FTZ-F1 gene was specially inhibited after the insects were treated with dsRNA of FTZ-F1 gene.The results suggest that the bacterially expressed dsRNA has potential to be used in silkworm functional genome analysis in an economical and efficient way.
出处
《昆虫学报》
CAS
CSCD
北大核心
2011年第5期596-601,共6页
Acta Entomologica Sinica
基金
国家自然科学基金项目(30901054
31001034
30671591)
长江学者和创新团队发展计划(IRT0750)
