摘要
RNA诱导的基因沉默复合体(RNA-induced silencing complex,RISC)核心蛋白为Argonaute(AGO)家族蛋白,作为miRNA及RNAi功能途径中的关键蛋白,能够结合miRNA和siRNA,降解靶标基因mRNA或抑制其翻译。因此,利用AGO蛋白抗体通过免疫沉淀分离其结合RNA是目前规模化分离鉴定miRNA及相关小RNA靶基因的重要方法。首先对家蚕AGO1基因进行克隆表达和抗体制备,用自制的兔多克隆抗体免疫沉淀家蚕组织内源BmAGO1蛋白,成功分离BmAGO1蛋白结合的RNA;同时在BmN细胞中采用6×His标签融合表达BmAGO1蛋白,用His单抗免疫沉淀His-BmAGO1蛋白,同样成功分离获得其结合RNA。qRT-PCR检测发现,BmAGO1蛋白结合的RNA中含有家蚕miRNA靶基因BmEm4的mRNA。研究结果为批量鉴定家蚕miRNA靶基因奠定了基础,同时也为深入研究BmAGO1蛋白在家蚕中的功能提供了依据。
Core proteins in small RNA-induced silencing complex(RISC)belong to argonaute(AGO)family proteins.As the key proteins in miRNA and RNAi functional pathways,AGO proteins can bind miRNA and siRNA to degrade target mRNA or supress its translation. Therefore,isolation of AGO bound RNAs by Co-Immunoprecipitation is an important method for large scale identification of miRNA and related small RNA target genes. In this study,AGO1 gene of Bombyx mori(BmAGO1)was cloned and expressed to prepare antibody. The endogenous BmAGO1 from Bombyx mori was immunoprecipitated using rabbit polyclonal antibody to isolate BmAGO1 bound RNAs successfully. Meanwhile,His-tagged BmAGO1 protein was expressed in BmN cells,and then the fusion protein was immunoprecipitated using His monoclonal antibody to isolate bound RNAs successfully. qRT-PCR results suggested that BmAGO1 bound RNAs contain the mRNA of BmEm4 which is the miRNA target gene in Bombyx mori. This study can not only lay a foundation for large-scale identification of miRNA target genes,but also facilitate further research on the function of BmAGO1 in silkworm,Bombyx mori.
作者
杨帆
高珍
马亚飞
蒋彩英
吕正兵
盛清
聂作明
Yang Fan;Gao Zhen;Ma Yafei;Jiang Caiying;Lyu Zhengbing;Sheng Qing;Nie Zuoming(College of Life Sciences and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China)
出处
《蚕业科学》
CAS
CSCD
北大核心
2019年第3期346-352,共7页
ACTA SERICOLOGICA SINICA
基金
国家自然科学基金项目（No.31772677）
浙江省自然科学基金项目（No.LGF18H250004）
