家蚕Bmyan基因的克隆表达和作为microRNA 7靶基因的验证

Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7

摘要:

microRNAs (miRNAs) 是一类长约22 nt的非编码RNA，通过与其靶基因3′端非翻译区 (3′-UTR) 的结合来调节各项生命活动。克隆表达家蚕Bmyan基因，验证其是否是bmo-miR-7的靶基因对于深入研究家蚕变态发育机制有重要意义。基于同源性检索和PCR扩增，克隆了家蚕Bmyan基因CDS全长，编码476个氨基酸。序列分析表明，家蚕YAN蛋白的氨基酸序列保守，含SAM-PNT和ETs结构域。芯片数据、RT-PCR和定量PCR的检测结果表明，Bmyan在五龄3 d的家蚕头部、体壁、卵巢中高量表达，在其余组织中低量表达或不表达。在幼虫期，Bmyan表达水平相对较低，但在上蔟期和蛹期前4 d高量表达。通过3′ RACE克隆了Bmyan基因的3′-UTR。RNAhybrid在线软件预测了其3?-UTR上bmo-miR-7的两个靶位点。构建了含有Bmyan基因3′-UTR和荧光素酶报告基因的转染载体，将该载体与bmo-miR-7的mimics序列共转染到家蚕胚胎细胞系BmE中，通过测定荧光素酶的活性，证明了Bmyan基因是bmo-miR-7的靶基因。本研究为进一步揭示bmo-miR-7和Bmyan在家蚕体内的生物学功能奠定了基础。

Abstract:

microRNAs (miRNAs) are an extensive class of ~22-nucleotide (nt) endogenous noncoding RNAs regulating life activities of metazoans through binding to 3′-untranslated regions (3′-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3′-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3′RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3′-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.