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

摘要/Abstract

摘要：

试验旨在克隆猪SMYD3（SET and MYND domain-containing protein 3）基因并对其进行序列分析，研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因，根据其他物种SMYD3基因siRNA和shRNA序列，经同源性比对分析，获得两条猪SMYD3基因shRNA序列，分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体，转染HEK293T细胞，利用实时荧光定量PCR分析干扰效率，筛选出抑制效率较好的shRNA，并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体，同时分析SMYD3基因对猪成纤维细胞的增殖作用，检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示，试验克隆得到1 404 bp的猪SMYD3基因编码区序列，生物信息学分析发现，德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达（P<0.05），抑制效果分别是34%和54%，选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞，均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示，与空白对照组及阴性对照组相比，过表达SMYD3基因促进猪成纤维细胞增殖，Nanog和DNMT1基因表达显著升高（P<0.05）；抑制SMYD3基因表达，细胞增殖受到抑制，Nanog、DNMT1、DNMT3a基因表达显著降低（（P<0.05），说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。

关键词: SMYD3基因; shRNA; 猪成纤维细胞; 细胞增殖

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

中图分类号:

Q785

佘春, 张艳, 张彩玉, 尹桂军, 石德顺, 李湘萍. 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[J]. 《中国畜牧兽医》, 2019, 46(3): 782-791.

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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