牦牛SIRT1基因的克隆及其在不同发育阶段睾丸中的表达

Cloning of Yak SIRT1 Gene and Its Expression Pattern in Testis at Different Development Stages

旨在克隆牦牛SIRT1基因,预测SIRT1蛋白的结构和功能,并检测其在牦牛不同组织及不同年龄睾丸中的表达和定位。9只健康雄性牦牛被划分为幼年组(0.5~1岁)、青年组(2~3岁)及成年组(4~5岁)(每组3头),其中来源于同一年龄阶段的3个组织样本视为生物学重复。采集成年期牦牛肝脏、心脏、脾脏、脑、肌肉、小肠以及3个时期牦牛睾丸组织。分别提取各组织的总RNA并利用反转录PCR(Reverse transcription PCR,RT-PCR)技术克隆获得牦牛SIRT1的基因序列,使用不同的生物信息学软件预测SIRT1基因的序列同源性,以及其编码蛋白质的氨基酸序列、结构和性质;通过实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)检测SIRT1基因的mRNA在牦牛各组织及不同发育阶段睾丸中的表达水平,利用色素原位杂交(Chromogenic in situ hybridization,CISH)技术检测SIRT1 mRNA在不同阶段睾丸中的定位,利用Western Blot技术检测SIRT1蛋白在不同阶段睾丸中的表达。结果表明,SIRT1基因的开放阅读框(Open reading frame,ORF)为1866 bp,编码621个氨基酸,其基因序列在哺乳动物中高度保守。结构预测表明,SIRT1是一个脂溶性疏水蛋白,含有一个保守的沉默信息调节子2(Silent information regulator 2,Sir2)结构域。该基因的mRNA在牦牛的各个组织中广泛表达,在睾丸、卵巢和肝脏中的表达较高。在牦牛睾丸中SIRT1的mRNA定位在除精细胞之外的各类细胞中,SIRT1的蛋白在牦牛睾丸中的表达随年龄增长呈现持续下降的趋势,其中在幼年期表达最高。研究结果为揭示SIRT1在牦牛生长发育,尤其是睾丸发育过程中的调控提供了一定的基础数据。

This research was conducted to clone the SIRT1 gene in yak,predicated the structure and function of SIRT1 protein,identified its expression pattern in different tissues and testis of different ages.Nine healthy male yaks were divided into in calve(0.5 to 1 year),juvenile(2 to 3 years)and adult(4 to 5 years)groups(three in each group).Three samples from the same age were considered as biological duplications.The samples of liver,heart,spleen,brain,muscle,small intestine from adult sage and testis from three stages were collected.Total RNAs in different testis were extracted respectively and the sequence of yak SIRT1 gene was cloned by reverse transcription PCR(RT-PCR).Different bioinformatics softwares were used to predicate the homology of SIRT1 gene sequence,amino acid sequence,structure and properties of SIRT1 protein.Real-time PCR was applied to identify the mRNA expression profile of SIRT1 in different tissues of yak and chromogenic in situ hybridization(CISH)was applied to analyse the localization of SIRT1 mRNA in yak testis.Western Blot was conducted to detect the expression pattern of SIRT1 protein in yak testis of different stages.The open reading frame(ORF)of yak SIRT1 was 1866 bp and encoded 621 amino acids.Sequence alignment analysis showed yak SIRT1 had a high sequence similarity with other mammals.Structure prediction indicated that SIRT1 was a hydrophilic liposoluble protein with a conserved silent information regulator 2(Sir2)catalytic core domain.The mRNA of SIRT1 was widely expressed in different tissues of yak and was highly expressed in testis,ovary and liver.The testicular SIRT1 protein was highly expressed in calves and the expression decreased significantly with the growing of age.SIRT1 mRNA localized in multiple kinds of cells except spermatid.This research provides a theoretical basis for further understanding the mechanism of SIRT1 in regulating yak testicular development and is of great importance in providing new insights in improving yak reproductive performance in the future.