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

Abstract

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

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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