从江香猪SP1基因组织表达特征及其超表达对间质细胞自噬和凋亡的影响

Tissue expression characteristics of SP1 gene and effects of its overexpression on autophagy and apoptosis in Leydig cells from Congjiang Xiang pigs

  • 摘要: 【目的】分析SP1基因在从江香猪不同组织及不同发育阶段睾丸中的表达情况及其对睾丸间质细胞自噬、凋亡的转录调控作用,为探究SP1基因调控间质细胞自噬的分子机制及提高从江香猪雄性繁殖性能提供理论基础。【方法】选取性早熟的从江香猪作为研究对象,PCR扩增得到其SP1基因编码区(CDS)序列,应用相关在线软件对CDS序列进行生物信息学分析,荧光定量PCR检测性成熟期从江香猪不同组织中SP1表达量,利用实时荧光定量PCR和Western blotting检测不同日龄从江香猪睾丸中的SP1基因表达量。【结果】生物信息学分析结果显示,SP1基因CDS序列全长为2361 bp,编码786个氨基酸残基,蛋白二级及三级结构以无规则卷曲和延伸链为主,无跨膜结构域和信号肽剪切位点,且为不稳定蛋白。SP1氨基酸序列有174个磷酸化位点,通过氨基酸同源性分析发现猪SP1与绵羊和牛的亲缘关系最近。对SP1在各组织的表达量进行检测,结果表明SP1基因相对表达量在脾脏中最高;此外,SP1的蛋白水平在180 d的香猪睾丸中有最高表达量,基因水平在30和180 d的香猪睾丸中有较高表达量。进一步构建SP1基因超表达载体,转染至从江香猪睾丸间质细胞,结果显示pEGFP-C1-SP1组的SP1相对表达量显著高于pEGFP-C1组(P<0.05,下同),而自噬通路相关因子基因mTOR、LC3B、Beclin-1和凋亡相关因子基因Caspase-3、Bcl-2的相对表达量在超表达pEGFP-C1-SP1组显著低于pEGFP-C1组,但自噬凋亡信号因子ERK1/2和凋亡基因Bax的相对表达量无显著变化(P>0.05)。推测SP1基因可通过降低LC3B、Beclin-1、Caspase-3的表达来抑制睾丸间质细胞自噬凋亡的发生,或通过降低mTOR及抗凋亡基因Bcl-2的表达来促进凋亡发生。【结论】 SP1基因在从江香猪不同组织及睾丸发育不同阶段均有表达,且通过影响睾丸间质细胞自噬、凋亡相关基因表达,而在从江香猪初情期和性成熟期发挥重要的生理学作用。

     

    Abstract: 【Objective】 To analyze SP1 gene expressionin tissues of Congjiang Xiang pigs of different age and its transcriptional regulation of Leydig cell autophagy and apoptosis, and to provide a theoretical basis for investigating molecular mechanism of SP1 gene regulation of Leydig cell autophagy and improving male reproductive performance of Congjiang Xiang pigs.【Method】 Congjiang Xiang pigs of precocious puberty were selected, the sequence of coding region (CDS) of SP1 gene was obtained by PCR amplification, and the sequence of CDS region was bioinformatically analyzed by applying online software. Fluorescence quantitative PCR(qRT-PCR) was used to detect SP1 expression of in different tissues of Congjiang Xiang pigs at the stage of sexual maturity, and qRT-PCR and Western blotting were used to detect SP1 expression in testes of Congjiang Xiang pigs at different age.【Result】 Bioinformatics analysis showed that the CDS sequence of SP1 gene was 2361 bp, encoding 786 amino acids residues, and the secondary and tertiary structures of the protein were mainly randomly coiled and extended chains, without transmembrane structural domains or signal peptide shear sites, and it was an unstable protein. 174 phosphorylation sites were found in the SP1 amino acid sequence, and amino acid homology analysis revealed that pig SP1 had the closest genetic relationship with sheep and cattle.According to SP1 gene expression test in Congjiang Xiang pig tissues, SP1 gene relative expression was the highest in the spleen; and the highest SP1 protein expression was found in testes of 180 day-old pig and high SP1 gene expression was found in 30 day-old and 180 day-old pigs. SP1 gene overexpression vector was further constructed and transfected into the Leydig cells. The qRT-PCR results showed that SP1 gene relative expression was significantly higher in pEGFP-C1-SP1 group than in pEGFP-C1 group(P<0.05, the same below), while the relative expression of autophagy pathway-related factors genes mTOR, LC3B, Beclin-1 and apoptosis-related factors genes Caspase-3 and Bcl-2 was expressed significantly lower in the overexpression pEGFP-C1-SP1 group than in the pEGFP-C1 group, but no significant difference of autophagy and apoptosis factor ERK1/2 relative expression and apoptosis gene Bax was detected(P>0.05). It was hypothesized that SP1 gene could inhibit the occurrence of apoptosis in Leydig cells by decreasing the expression of LC3B, Beclin-1, and Caspase-3, or promote apoptosis by decreasing the expression of mTOR and anti-apoptotic gene Bcl-2.【Conclusion】 SP1 gene is expressed in tissues of Congjiang Xiang pigs in different testis growth periods and by influencing expression of genes related to autophagy and apoptosis of the pigs, SP1 gene plays an important role physiologically during puberty and sexual maturity of Congjiang Xiang pigs.

     

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