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

Lanting Zhou1,2 Ting Zhou1 Junling Gao1 Wei Wang1 Xiaoyan Wu1 Yaxi Huang1 Qingyou Xia1 Shiping Liu1

(1.State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China 400716)
(2.Medical College, Hubei University of Arts and Science, Xiangyang, Hubei, China 441053)

【Abstract】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 SilkDB. 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 larvae, 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 of 1 434 bp in length was cloned by rapid-amplification of cDNA 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.

【Keywords】 silkworm (Bombyx mori); heterochronic gene; bmo-let-7 miRNA; Bmlin-41; expression profile; target gene;

【DOI】

【Funds】 National Basic Research Program of China (973 Program) (No. 2012CB114602) National Basic Research Program of China(973 Program)(No.2012CB114602) National Natural Science Foundation of China (Nos. 31071136, 1571334) National Natural Science Foundation of China(Nos.31071136,1571334) Fundamental and Advanced Research Projects of Chongqing (No. cstc2014jcyjA00025) Fundamental and Advanced Research Projects of Chongqing(No.cstc2014jcyjA00025)

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(Translated by 白小东)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 635-647

May 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Discussion
  • 4 Conclusions
  • References