Zonula occludens (ZO) family proteins were found as members of membrane-associated guanylate kinase (MAGUK) family proteins of tight junctions (TJs), among them, ZO-1 was the first studied gene.When ZO-1 interacts with other cellular junction proteins through its PDZ region, it not only participated in the regulation of cell material transport and maintenance of epithelial polarity, but also associated with information transmission, regulation of cell proliferation and differentiation, tumor cell metastasis, gene transcription and other processes.To further explore the function of ZO-1 gene in development of pig early embryos and proliferation of pig granulosa cells by get and loss way, in this study, we cloned the pig ZO-1 gene and its effective shRNA fragments were screened.(1) A 3033bp CDS of pig ZO-1 mRNA was cloned from total RNA of Duroc Landrace Yarkshire (DLY) three-breed crossbred pigs.It had 99% similarity compared with that of published pig ZO-1 gene on NCBI genebank.The fusion expression vector of pig ZO-1 gene was constructrd by inserting the CDS fragment into pDsRed-N1 vector.The expression level of ZO-1 gene in transfected 293 cells was detected using QRT-PCR method.(2) Two shRNA fragmnets targeting pig ZO-1 were designed, they were inserted into the pSicoR-GFP vector to construct the shRNA recombinant expression vector,_respectively.pSicoR-GFP-1864 vector was used as negative control(NC).After confirmed by restriction enzyme digestion and sequencing methods, the fusion expression vector of pig zo-1 (pDsRed-N1-ZO-1) and the pSicoR-GFP vector were co-transfected into HEK293 cells using Lipofectamine(R) 3000, respectively.The inhibition efficiency of the shRNA fragment was detected by flow cytometry analysis and QRT-PCR method.