绵羊STAT5a基因的组织表达及核苷酸序列变异分析

摘要

信号传导与转录激活因子5a(signal transducer and activators of transcription 5a,STAT5a)作为产奶性状的候选基因,在调控动物乳腺发育中具有重要的作用.本研究利用实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)技术检测藏绵羊(Ovis aries)和小尾寒羊STAT5a基因在心脏、肝脏、脾脏、肺脏、肾脏、小脑和乳腺7个不同脏器组织中的表达量、研究绵羊品种和组织间差异表达;利用多聚酶链式反应-单链构象多态(polymerase chain reaction-single strand conformation polymorphsim,PCR-SSCP)技术和测序法分析藏绵羊、湖羊和多浪羊STAT5a基因在P1和P2两个扩增区域内的核苷酸序列变异及群体遗传特征.结果表明,STAT5a基因在绵羊7个组织中均有表达,并表现出明显的品种差异性和组织差异性,在藏绵羊和小尾寒羊中,STAT5a基因在乳腺组织中的表达量显著高于其他6个组织(P＜0.01),而2个品种之间,小尾寒羊乳腺组织的表达量显著高于藏绵羊(P＜0.05),但在肝脏、肺脏、肾脏和小脑的表达量显著低于藏绵羊(P＜0.05);在P1引物的扩增区域中,各绵羊群体中均检测到c.2150-154C＞T和c.2150-50T＞C 2个SNPs和对应的等位基因A、B,其中等位基因B为优势等位基因,基因型BB为优势基因型,藏绵羊多态信息含量为0.25 ＜PIC＜0.5,属中度多态,湖羊和多浪羊PIC＜0.25,属低度多态.3个绵羊群体的x2值均未达到显著水平(P＞0.05),处于Hardy-Weinberg平衡状态.在P2引物的扩增区域中,未检测到多态性.本研究结果可以丰富绵羊基因组学内容,为研究乳腺性状形成的分子机制提供基础性资料.%Signal transducer and activator of transcription 5a (STAT5a),considered as an important candidate gene for milk production traits,plays key roles in the process of development of regulating animal mammary gland.In the present study,seven tissues included heart,liver,spleen,lung,kidney,cerebellum and mammary gland of Tibetan and Small tail han sheep were detected by qRT-PCR to research the tissues distribution and the differences in expression between different sheep breeds and tissues.The P1 and P2 primer amplification regions of STAT5a in three sheep breeds (Tibetan sheep,Hu sheep and Duolang sheep) using PCR-SSCP and sequencing methods to analysis nucleotide sequence variation and population genetic characteristics.The results indicated that STAT5a gene was expressed in all seven tissues and showed significant differences in species and tissues.The expression of mammary gland was significantly higher than other six tissues in Tibetan and Small tail han sheep (P＜0.01).Between these two breeds,the expression of mammary gland in small han sheep was highly than Tibetan sheep(P＜0.05),but the expression of kidney,cerebellum,and liver were lower than Tibetan sheep (P＜0.05).Two SNPs (c.2150-154C＞T and c.2150-50T＞C) and corresponding alleles A and B were detected in P1 primer amplification regions of STAT5a in sheep.The allele B and genotype BB were the dominant allele and genotype and Tibetan population was moderately polymorphic (0.25＜PIC＜0.5),but other populations were low polymorphic (PIC＜0.25).The x2 values of the three sheep populations were non-significant (P＞0.05),so those populations lay in the Hardy-Weinberg equilibrium.However,there was no polymorphism in the P2 primer amplification region.In conclusion,these results enrich the contents of the sheep genome and provide the basic information for the molecular mechanism of the formation of mammary gland.