农业生物技术学报2011，Vol.19Issue(2)：206-213,8.DOI:10.3969/j.issn.1674-7968.2011.02.002
家蚕真核细胞翻译起始因子(BmeIF4E)基因的克隆、表达及功能分析
Cloning, Expression and Function Analyses of Eukaryotic Translation Initiation Factor 4E(BmeIF4E) Gene from Silkworm Pupa (Bombyx mori)
摘要
Abstract
The full open reading frame (ORF) of eukaryotic translation initiation factor 4E (eIF4E) was obtained from silkworm(Bombyx mori pupal cDNA library and was named BmeIF4E (GenBank accession No.DN443192).In order to study the biological function of BmeIF4E, the ORF of this gene was cloned and the procaryotic expression recombinant plasmid pET-28a(+)-BmeIF4E was constructed to express His-BmeIF4E fusion protein in Escherichia coli BL21 (DE3).Because the rBmeIF4E in E.coli was soluble, the protein was purified with metal-chelating affinity chromatography directly, rBmeIF4E was used to generate anti-BmeIF4E polyclonal antibodies, to determine the subcellular localization of BmeIF4E.Immnunostaining indicated that BmeIF4E protein could be found in both the cytoplasm and nucleus.The gene expression features in different organizations and different developmental stages were analyzed using Real-time RT-PCR and Western blotting analyses.The results showed that BmeIF4E were expressed in all tissues and the expression levels were highest in malpighian tubes,followed by epidermis, fat body, spiracle, ovary, gut and head, and were lowest in the silk glad.Additionally,BmeIF4E expression began during the egg stage, increased during the larvae stage and pupa stage, reached a peak during the moth stage.Furthermore, RNA interference technology was applied to silence the target mRNA and the cell viability was assessed by Western blot and MTT assay.After 72 h of RNAi, the MTT assay indicated that when BmeIF4E was knocked down in Bm5 cells, the cell viability was decreased significantly.BmeIF4E may play an important role in the whole life cycle ofBombyx mori as a valuable house-keeping gene.关键词
家蚕/BmeIF4E/原核表达/亚细胞定位/RNAiKey words
Bombyx mori, BmeIF4E, Procaryotic expression, Subcellular localization, RNAi引用本文复制引用
于威,韩剑秋,张耀洲..家蚕真核细胞翻译起始因子(BmeIF4E)基因的克隆、表达及功能分析[J].农业生物技术学报,2011,19(2):206-213,8.基金项目
本课题由国家高技术研究发展计划(863)课题(No.2007AA100504)、浙江省自然科学基金(No.Y3090339)和浙江省钱江人才计划项H(No.QJD0702014)共同资助 （863）
