家蚕microRNA 7靶基因Bmhairy的鉴定和转录表达模式

doi:10.3864/j.issn.0578-1752.2016.01.018

摘要/Abstract

摘要： 【目的】microRNA是一类长约22个碱基的非编码RNA，通过与其靶基因的特异性结合来调控新陈代谢和生长发育等多种生命活动。鉴定家蚕(Bombyx mori)Bmhairy，比较Bmhairy和bmo-miR-7的表达模式，验证Bmhairy是否为bmo-miR-7的靶基因，为研究家蚕的变态发育机制提供线索。【方法】用家蚕胚胎反转期的总RNA反转录合成的cDNA模板，克隆Bmhairy编码区（coding DNA sequence，CDS）和3′端非翻译区（3′ untranslated region，3′UTR）并进行序列分析。基于荧光定量PCR和芯片数据比较bmo-miR-7和Bmhairy的表达模式。利用RNAhybrid和MiRTif，预测和分析Bmhairy的mRNA 3′UTR上bmo-miR-7的靶位点。构建荧光素酶报告基因载体，用家蚕胚胎细胞系（BmE）进行共转染试验，验证bmo-miR-7对Bmhairy的3′UTR结合位点。【结果】在家蚕中克隆了Bmhairy，含2个内含子和3个外显子，CDS长654 bp，编码217个氨基酸。Bmhairy的mRNA 3′UTR长366 nt。Bmhairy高度保守，含有bHLH、ORANGE和WRPW结构域。Bmhairy与鳞翅目（Lepidoptera）蛱蝶科（Nymphalidae）中的黑脉金斑蝶（Danaus plexippus）相似度最高。Bmo-miR-7在家蚕胚胎期高量表达，在5龄第3天的精巢中相对较高，而Bmhairy在成虫期的表达量最高，在5龄第3天的头部相对较高。Bmhairy mRNA 3′UTR上有bmo-miR-7一个靶位点。共转染试验表明，bmo-miR-7下调Bmhairy。【结论】Bmhairy的家蚕时期表达和5龄第3天组织表达均与bmo-miR-7的表达呈相反的模式。靶基因预测和双荧光素酶转染试验表明Bmhairy是bmo-miR-7的靶基因，为进一步研究bmo-miR-7和Bmhairy在家蚕体内的生物学功能及调控关系提供了依据。

关键词: 家蚕, 微小RNA 7, 靶基因, Bmhairy, 表达模式

Abstract: 【Objective】microRNAs are a class of non-coding RNAs with about 22 nucleotides and post-transcriptionally regulate the target genes responsible for the metabolism, growth, development and some other important life activities of organisms. The objective of this study is to clone Bmhairy, characterize the expression profiles of Bmhairy and bmo-miR-7 in the silkworm (Bombyx mori) and investigate whether Bmhairy is the target gene of bmo-miR-7. 【Method】The total RNA isolated from the embryo of B. mori was used to synthesize the first strand of cDNA of Bmhairy, which has served as the PCR template for producing CDS (coding DNA sequence) and 3′UTR. The expression patterns of bmo-miR-7 and Bmhairy were compared based on the results from fluorescence quantitative PCR and the previous microarray data. The binding sites of bmo-miR-7 within the 3′UTR of Bmhairy were predicted with the online tools, RNAhybrid and MiRTif, followed by experimental confirmation through the luciferase reporter gene assay in the BmE cells in vitro.【Result】The homologue of Drosophila hairy in B. mori was cloned, and named as Bmhairy, which consists of two introns and three exons; its CDS is 654 bp, encoding 217 amino acids, and its 3′UTR is 366 nt. The predicted protein comprises three domains, namely bHLH, ORANGE and WRPW, which are highly conserved in vertebrates and invertebrates. Bmhairy is most similar to the homologue in the Lepidoptera insect, Danaus plexippus. Bmo-miR-7 was highly expressed in the embryo and testis of the day 3 of 5th instar larva of B. mori. However, Bmhairy was highly expressed in the adult moth and in the head of the day 3 of 5th instar larva. Combined use of RNAhybrid and MiRTif revealed a target site of bmo-miR-7 within the 3′UTR of Bmhairy, which was supported by luciferase report gene assay in the BmE cell lines cotransfected with bmo-miR-7 mimics and Bmhairy 3′UTR luciferase reporter vector. 【Conclusion】The expression profile of Bmhairy showed opposite changes to that of bmo-miR-7 in the embryo and adult stages of B. mori as well as in the head and testis of the day 3 of 5th instar larva. Bmhairy is one target of bmo-miR-7 based on the results from the prediction and luciferase report gene assay and hopefully might lay a base for further study of the roles of bmo-miR-7 and Bmhairy as well as their relationship.

Key words: silkworm (Bombyx mori), microRNA 7, target gene; Bmhairy; expression profile

刘仕平，吴小燕，张丹宇，黄亚玺，王伟，赵萍，夏庆友. 家蚕microRNA 7靶基因Bmhairy的鉴定和转录表达模式[J]. 中国农业科学, 2016, 49(1): 195-204.

LIU Shi-ping, WU Xiao-yan, ZHANG Dan-yu, HUANG Ya-xi, WANG Wei, ZHAO Ping, XIA Qing-you. Identification of Bmhairy as the Target of bmo-miR-7 and Its Transcriptional Expression Profiles in the Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2016, 49(1): 195-204.

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