Abstract: This paper was aimed to clone 3’UTR of Stra8 gene of Rugao yellow chicken, and predicted the microRNAs (miRNA) targeting the gene to provide theoretical basis for further function study of Stra8 in the process of embryonic stem cells differentiation into the male germ cell mediated by miRNAs. 3'UTR of Stra8 was cloned by 3'RACE based on primers designed from cDNA of Gallus gallus in NCBI, then the vectors of Luciferase expression and mutation was constructed. Targetscan bioinformatics database was used to predict the prospective miRNAs targeting to Stra8 and synthesize miRNA mimics. The candidate miRNA mimics and the vectors of Stra8-3’UTR were transiently co-transfected with into DF-1 cells, and pRL-TK vector was used as an internal control reporter. The dual-luciferase reporter assay system was used to evaluate the activity of luciferase. The results showed that 3'UTR of Stra8 was cloned. Four miRNAs has been predicted that targeting to Stra8 by using the online bioinformatics database and their lentiviral vectors were constructed successfully. Dual luciferase activity test results showed that gga-miR-1a,gga-miR-31,gga-miR-218 can inhibit gene expression of Stra8, but inhibitor of gga-miR-31 was the best. The gga-miR-31 could significantly inhibit expression of Stra8, the results will provide references for the investigation on Stra8 gene regulatory networks in male germ cell differentiation.

Key words: Gallus gallus, miRNA, Stra8, gga-miR-31, dual luciferase activity detection