ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2018, Vol. 49 ›› Issue (7): 1366-1376.doi: 10.11843/j.issn.0366-6964.2018.07.005

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Construction of Yak Smad 4 Gene 3'UTR Dual-luciferase Reporter Vector and Its Targeting Validation to bta-miR-146a

NIU Jia-qiang1,2, WANG Yu-heng1,2, SUOLANG Si-zhu1,2, CHANGBA Yang-zong1, XU Ye-fen1,2*, GUO Min3, CHENG Ling-hua3, YANG Shi-cheng1,2   

  1. 1. Department of Animal Science, Tibet Agricultural and Animal Husbandry College, Linzhi 860000, China;
    2. Provincial Key Laboratory of Tibet Plateau Animal Epidemic Disease Research, Tibet Agricultural and Animal Husbandry College, Linzhi 860000, China;
    3. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
  • Received:2017-11-27 Online:2018-07-23 Published:2018-07-19

Abstract:

The aim of this study was to identify the targeting relationship between Smad 4 gene and bta-miR-146a, which would provide a theoretical basis for their regulating mechanism in follicular development of yak ovary. The conservation of miR-146a in different species was analyzed by miRBase database and MEGA 6.0 software. At the same time, TargetScan software was used to predict the potential target genes of miR-146a, and the targeting validation was performed by construction of psiCHECK2 dual-luciferase reporter vector. The results of bioinformatics analysis showed that the miR-146a mature sequence was highly conserved in vertebrates, and a total of 178 potential target genes related to ovarian follicular development were predicted, then, among these genes, Smad 4 gene was selected to be further analyzed, showing that its 3'UTR had the binding site to bta-miR-146a. The wild and mutant type psiCHECK2 dual-luciferase reporter vectors of 3'UTR of yak Smad 4 gene were successfully constructed, and the dual-luciferase assay result showed that the luciferase activity of wild vector of 3'UTR of yak Smad 4 gene was distinctly down-regulated by the bta-miR-146a mimics, and was significantly lower than that of mutant vector and NC group(P<0.05), respectively. In conclusion, this research preliminarily confirmed that yak Smad 4 gene was the target gene of bta-miR-146a, suggesting that bta-miR-146a might play roles in the development of yak follicles by regulating Smad 4 gene.

Key words: yak, bta-miR-146a, Smad 4, luciferase activity

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