Cloning, expression and function analyses of eukaryotic translation initiation factor 4E(BmeIF4E) gene from Silkworm pupa (Bombyx mori) | 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

Chino. 本研究从自建的家蚕(Bombyx mori)蛹期cDNA文库中筛选到一条cDNA序列，发现其在序列和结构上与其他物种的真核细胞翻译起始因子4E基因(BmeIF4E)具有较高的相似性，推测可能是家蚕eIF4E基因，将该序列命名为BmeIF4E，GenBank登录号为DN443192。为研究BmeIF4E的生物学功能，将该基因插入原核表达载体pET-28a(+)中，构建重组质粒pET-28a(+)-BmeIF4E，转化感受态细胞BL21(DE3)，IPTG诱导表达并纯化该重组蛋白，制备多克隆抗体。利用家蚕Bm5细胞进行亚细胞定位研究结果表明，BmeIF4E既存在于细胞质中也存在于细胞核中。荧光定量PCR和Western blot分析结果表明，在不同组织及发育时期该基因的表达有差异，在各组织中BmeIF4E表达量从高到底依次是马氏管、表皮、脂肪体、气管、卵巢、中肠、头和丝腺；在卵、幼虫、蛹和蛾4个发育时期中，蛾中的表达量最高，其次是蛹，再次是五龄幼虫，而在卵中表达量较低。以该基因的ORF为模板体外转录dsRNA，脂质体法转染Bm5细胞，72 h后提取细胞总蛋白作Western blot检测RNA干扰情况，发现BmeIF4E基因被干扰后，总蛋白中目的蛋白含量明显低于阴性对照组。MTT法检测结果表明，干扰后细胞活力也明显下降。研究结果提示，BmeIF4E作为一种重要的管家基因，在家蚕的整个生命周期中均有表达，起着十分重要的作用。

Inglés. 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. Immunostaining 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 of Bombyx mori as a valuable house-keeping gene.