家蚕let-7 microRNA靶基因Bmlin-41的克隆表达与调控

Cloning and expression profile of Bmlin-41 and its regulation by the silkworm microRNA let-7

异时性基因调控细胞增殖和个体发育阶段的转换。家蚕异时性基因在家蚕变态发育过程中也很可能具有重要的调控作用,但它们的表达模式、生物学功能以及与micro RNA之间的关系却鲜有报道。本研究首先利用果蝇同源基因lin-41搜索家蚕基因组数据库中相似序列,设计引物扩增Bmlin-41的编码序列,克隆了家蚕Bmlin-41基因CDS,其长度为2 166 bp,编码721个氨基酸,含有B-box和NHL结构域;随后,利用RT-PCR、q PCR技术并结合已有的家蚕全基因组芯片数据研究了Bmlin-41在家蚕中的时空表达模式,发现Bmlin-41在从家蚕胚胎到成虫的发育过程中呈逐渐递增的表达趋势,在五龄3 d不同组织中,于卵巢里表达量最高,精巢和中肠次之,而其余组织中低量表达或不表达;最后,利用3′RACE克隆了Bmlin-41基因的3′UTR,全长1 434 bp,用在线软件RNAhybrid预测发现Bmlin-41的3′UTR上存在bmo-let-7靶位点,构建了含Bmlin-41 3′UTR的双荧光素酶报告基因载体,在S2细胞上共转染Bmlin-41 3′UTR和bmo-let-7的模拟物(Mimics)和拮抗剂(Antagomir),bmo-let-7 mimics显著下调Bmlin-41,bmo-let-7 antagomir显著上调Bmlin-41,证实了Bmlin-41是bmo-let-7的靶基因。以上研究结果为深入研究let-7 mi RNA和Bmlin-41的功能,揭示Bmlin-41和bmo-let-7在家蚕变态发育过程中的调控关系提供了新的线索。

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with micro RNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against Silk DB. The obtained full CDS（2 166 bp） of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab＇s previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3′UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of c DNA ends（3＇RACE） and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3′UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.