沼泽型水牛ALK3基因克隆分析与表达模式研究

Cloning and Expression Pattern Investigation of Swamp Buffalo ALK3 Gene

试验旨在对沼泽型水牛ALK3基因进行克隆、生物信息学分析,并对其在水牛组织中的表达规律进行系统研究。根据GenBank中已公布的牛ALK3基因序列设计特异性引物,应用RT-PCR方法扩增、克隆获得目的基因片段;应用生物信息学方法分析和预测了水牛ALK3的遗传进化及蛋白质的理化性质、二级和三级结构;并应用QRT-PCR技术对ALK3基因在水牛组织中的表达进行了差异分析。结果表明,水牛ALK3基因编码区全长1 599bp,共编码532个氨基酸。多重序列比较分析显示,水牛ALK3核苷酸序列与牛、绵羊、猪、马、人和小鼠相应序列的同源性分别为98%、96%、95%、93%、94%和91%。系统进化树分析显示,ALK3基因在不同物种以及进化的过程中具有高度保守性。对ALK3蛋白质的分析表明该蛋白呈弱碱性,有信号肽,细胞亚定位于胞膜上,存在丝氨酸/苏氨酸激酶和GS等结构。定量分析结果显示,ALK3在水牛生殖脊、心脏、肝脏、颗粒细胞、肺脏、卵丘细胞、肾脏、下丘脑、垂体、大脑等15种组织或细胞中有不同程度的表达,其中卵巢中表达量最高,垂体、肺脏和睾丸次之,卵丘细胞表达量最低。本研究成功克隆了沼泽型水牛ALK3基因,并研究了其在不同水牛组织细胞中的表达规律,为阐明其在水牛繁殖过程中的功能及转基因载体构建中的应用研究奠定了理论基础。

Clone,sequence bioinformatics analysis and expression pattern determination were performed of ALK3 gene in swamp buffalo.A pair of special primers was designed according to the released sequence of bovine ALK3 in GenBank.ALK3 gene was amplified by RT-PCR,its gene sequence character and protein structure was systemically analyzed by bio-informatics techniques.The expression pattern of buffalo ALK3 in different tissues was also assayed with QRT-PCR.The results showed that buffalo ALK3 gene fragment including a 1 599 bp whole length CDS（coded532amino acids）was cloned and sequenced.The sequence multi-aligned results showed that buffalo ALK3 gene shared 98%,96%,95%,93%,94% and 91% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa,Equus caballus,Homo sapiens and mice,respectively.ALK3 protein sequence analysis showed that it was weakly alkaline,had signal peptide,positionedon the membrane,and with the presence of S_TKc and GS（GS motif）structure.In addition,the expression pattern analysis results showed that buffalo ALK3 expressed in all 15 tested samples,with the most abundant expression in ovary,followed by hypophysis,lung and testis,the minimal expression in cumulus cells was observed.The cloning and analysis of ALK3 gene provided an important foundation for further study regulation mechanism of ALK3 gene in buffalo in the future.