关键词: 牙鲆, miRNA, emx2, 定点诱变, psiCHECK-emx2-3&apos, UTR, psiCHECK-mutated-emx2-3&apos, UTR

Abstract: Taking Paralichthys olivaceus empty spiracles homeobox 2(emx2)as an example, we constructed the wild-type and mutant luciferase reporter plasmids containing emx2 3'UTR region for being utilized in miRNA target detection. The total RNA was extracted from the mixtures of testis and ovarian in adult fish with Trizol. Using previously-cloned cDNA sequences of emx2 as a reference, a pair of specific primers for emx2 3'UTR fragment were designed and synthetized, then amplified genes by RT-PCR and psiCHECK-2 vector were treated with double restriction enzyme digestion, and then ligated with T4 DNA Ligase. The ligated fragment was transformed into the competent cells of DH5α, then the wild-type recombinant plasmid were obtained by screening. In vitro site-directed mutagenesis of emx2 was carried out, and a site-directed mutant plasmid was generated by the same methods. The results indicated that the emx2 3'UTR of P. olivaceus was successfully cloned, and GACTTGA, the sequence of miRNA target sites in it was mutated to AGTCCAG. The wild-type and mutant luciferase reporter plasmids used in miRNA target detection were constructed successfully. In conclusion, with the techniques of RT-PCR, gene recombination and site-directed mutagenesis, the wild-type luciferase reporter plasmids psiCHECK-emx2-3'UTR and mutant one of psiCHECK-mutated-emx2-3'UTR used in miRNA target detection were successfully constructed, which lays the foundation for further researches on identification and function of miRNA target emx2.

Key words: Paralichthys olivaceus, miRNA, emx2, site-directed mutagenesis, psiCHECK-emx2-3&apos, UTR, psiCHECK-mutated-emx2-3&apos, UTR