猪紧密连接蛋白1基因克隆及其shRNA片段的筛选

doi:10.16431/j.cnki.1671-7236.2016.06.002

Abstract

Abstract: The aim of this study was to screen effective shRNA fragments targeting pig zonula occludens-1 (ZO-1) gene,which would lay foundation to further explore its function in pig oocytes in vitro maturation (IVM) by RNAi technology.Pig ZO-1 gene was cloned and its eukaryotic expression vector was constructed,its effective shRNA fragments were screened.The results showed that the CDS length of cloned pig ZO-1 gene was 3 036 bp.The results of sequence multialigned showed that the sequence of pig ZO-1 gene shared 88%,88%,87% and 85% homology with Taurus,Ovis aries, Equns caballus and Homo sapiens,respectively.Clear RFP red fluorescent was observed in transfected cells with the pDsRed-N1-ZO-1 eukaryotic expression plasmid transfected into HEK293T cells by X-tremeGENE HP DNA transfection reagent.Two shRNA fragments targeting pig ZO-1 gene were designed and synthesized,weaker RFP red fluorescent was observed in co-transfected cells with the target and interfered shRNA plasmid groups.The Real-time PCR results showed that the two designed shRNAs could effectively inhibit the expression of pig ZO-1 mRNA,shRNA201 fragment had significantly higher inhibition effect than that of shRNA1276 fragment (77.8% and 67.0%,P < 0.05).

Key words: pig; ZO-1 gene; clone; shRNA

CLC Number:

Q785

CAO Li-hua, WANG Ping, HAO Wen-yan, DAI Xiao-li, CHEN Pei-fang, SHI De-shun, LI Xiang-ping. Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments[J]. , 2016, 43(6): 1405-1412.

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Cloning of Pig ZO-1 Gene and Screening on its Effective shRNA Fragments

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