China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (9): 3109-3117.doi: 10.16431/j.cnki.1671-7236.2021.09.001

• Biotechnology • Previous Articles     Next Articles

Construction of Ovine INHBB,SMAD4 and FGF18 Genes Dual-luciferase Reporter and Validation of Their Targeting Relationship with miR-370-3p

LI Zhifeng1,2, CHU Mingxing2, SUN Wei1,3   

  1. 1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
    2. Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    3. Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou 225009, China
  • Received:2021-03-31 Online:2021-09-20 Published:2021-09-17

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Abstract: To investigate the targeting relationship between miR-370-3p and inhibin subunit beta B (INHBB), SMAD family member 4 (SMAD4) and fibroblast growth factor 18 (FGF18) genes that were associated with oviductal function in sheep, in this study, the sequences of miR-370-3p in multiple species (Ovis aries, Homo sapiens, Mus musculus, Rattus norvegicus, Macaca mulatta, Cavia porcellus and Oryctolagus cuniculus) were compared and RNAhybrid was used to predict the potential binding sites of ovine miR-370-3p to the 3'UTR of INHBB, SMAD4 and FGF18 genes. Then wild type and mutant type dual-luciferase reporter vectors for INHBB, SMAD4 and FGF18 gene 3'UTR were constructed and co-transfected with miR-370-3p mimics and mimics NC into HEK293T cells to detect dual-luciferase activity, respectively. The results showed that the ovine miR-370-3p sequence was different from the other six species but was somewhat conserved, and was predicted by RNAhybrid to have binding sites to the 3'UTRs of the INHBB, SMAD4 and FGF18 genes. The electrophoresis results and sequencing results indicated that the wild type and mutant type vectors of INHBB, SMAD4 and FGF18 gene 3'UTR were successfully constructed. The dual-luciferase activities of the co-transfected INHBB, SMAD4 and FGF18 wild type vectors and miR-370-3p mimics were extremely significantly or significantly lower than the corresponding controls (P<0.01;P<0.05). While the dual-luciferase activities of the three mutant type vectors and miR-370-3p mimics co-transfected were not significantly different from the corresponding controls (P>0.05). It was shown that the 3'UTR region of INHBB, SMAD4 and FGF18 genes could all bind to miR-370-3p and inhibit dual-luciferase activity, which verified that INHBB, SMAD4 and FGF18 genes were both target genes of miR-370-3p and provided a basis for further study on the molecular mechanism of oar-miR-370-3p affecting oviductal function and sheep fecundity.

Key words: INHBB; SMAD4; FGF18; miR-370-3p; dual-luciferase

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