猪SMYD3基因的克隆、序列分析及其对猪成纤维细胞增殖的影响

doi:10.16431/j.cnki.1671-7236.2019.03.017

Abstract

Abstract:

This experiment was aimed to clone and analyze the sequence of porcine SET and MYND domain-containing protein 3 (SMYD3) gene,and study its effect on the proliferation of porcine fibroblasts.Firstly,SMYD3 gene was cloned,according to the siRNA and shRNA sequences of other species SMYD3 gene,two shRNA sequences of SMYD3 gene were obtained by homologous alignment analysis,pSicoR-GFP-SMYD3 shRNA1/shRNA2 expression vector were constructed and transfected into HEK293T cells.The inhibition efficiency of shRNA was analyzed by Real-time PCR,and screened out shRNA with better inhibition efficiency.Then the pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,the proliferation of porcine fibroblasts was analyzed,and the expression levels of Nanog,DNMT1 and DNMT3a genes were detected by Real-time quantitative PCR.The results showed that the cloned CDS length of porcine SMYD3 gene was 1 404 bp.Bioinformatics analysis results showed that the homology of the SMYD3 gene was 99.5% between pig and Sus scrofa,which shared 93.8% and 92.9% identity with Capra hircus and Bos mutus,respectively.The expression of SMYD3 gene was significantly inhibited by shRNA1/shRNA2 (P<0.05),and the inhibitory rates were 34% and 54%,respectively.pSicoR-GFP-SMYD3 shRNA2 was selected for follow-up studies.The pLVX-IRES-ZsGreen1-SMYD3 and pSicoR-GFP-SMYD3 shRNA eukaryotic expression vectors were constructed,and clear green fluorescent signal was observed when the plasmids were transfected into HEK293T cells by liposome method.The results of lentiviral infection and Real-time quantitative PCR showed that compared with the negative and blank control groups,overexpression of SMYD3 gene promoted the porcine fibroblast proliferation,and the expression of Nanog and DNMT1 genes were significantly increased (P<0.05);And inhibition of SMYD3 gene inhibited porcine fibroblasts proliferation while the expression of Nanog,DNMT1 and DNMT3a genes were significantly decreased (P<0.05).This results indicated that the expression of SMYD3 gene was significantly associated with the proliferation of porcine fibroblasts.

Key words: SMYD3 gene; shRNA; porcine fibroblasts; cell proliferation

CLC Number:

Q785

SHE Chun, ZHANG Yan, ZHANG Caiyu, YIN Guijun, SHI Deshun, LI Xiangping. Cloning and Sequence Analysis of Porcine SMYD3 Gene and Its Effects on Proliferation of Porcine Fibroblasts[J]. , 2019, 46(3): 782-791.

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Cloning and Sequence Analysis of Porcine SMYD3 Gene and Its Effects on Proliferation of Porcine Fibroblasts

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