牛Trf2基因的cDNA克隆和生物信息学分析

Analysis of cDNA Cloning and Biological Information of Cattle Trf 2 Gene

[目的]探讨牛Trf2基因的生物学功能,为进一步研究该基因对牛精液品质的影响奠定基础。[方法]以牛睾丸组织为材料,根据GenBank中发表的人Trf2基因序列,结合生物信息学技术,设计并合成3对引物,采用RT-PCR方法克隆牛Trf2cDNA,并运用DNA-MAN软件及在线工具对所得到的序列进行生物信息学分析。采用牛肾脏、肝脏、睾丸、肺、瘤胃、子宫、小肠、心脏、脾脏、卵巢和脂肪组织共11个组织,以牛-βactin基因为内参基因,对牛Trf2基因的组织表达谱进行分析。[结果]牛Trf2基因cDNA长度为1 701 bp(Gen-Bank登录号EU140625),包含由561个碱基组成的开放读码框(ORF),该ORF包含1个UGA终止密码子,编码186个氨基酸;在进化关系上,牛首先和人、大鼠、狗聚成一类,然后与袋鼠聚成第1群,再与小鼠聚成第2群。Trf 2蛋白含有2个结构域TLF和SPT15。牛Trf2基因在各个组织中都有表达,在睾丸组织中表达最高。[结论]获得了牛Trf2基因的cDNA序列(GenBank登录号EU140625);牛Trf 2蛋白可能对牛精液品质有影响。

[Objective] The study aimed to discuss the biological function of Cattle Trf 2 gene, and laid the foundation for the further study on the effects of this gene on cattle sperm quality.[Method] Cattle testis tissue was used to material, according to Trf2 gene sequence published by GenBank and combining with biological information technology, 3 pair of primers was designed . Cloning Trf 2 eDNA used by RT-PCR method and biological information analyzing used by DNAMAN software and online tool. 11 tissues including bovine kidney, liver, testicle, lung, spleen, rumen, uterus, small intestine, heart, ovary and fat tissues were used to experimental material. Cattle β-actin gene was reference gene, the tissues expression profile of cattle Trf2 gene were analyzed. [Result] The result showed that the sequence of eDNA of cattle Trf2 gene was 1701bp （GenBank Accession No. EU140625 ） containing an open reading frame （ORF） of 1 UGA stop codon which encoded 186 amino acids. Phylogenetic tree of amino acid analysis indicated that cattle, rat, human and dog were located in first embranchment, this embranchment was near with kangaroo. Mouse was located in the other embranchment. Only two TLF and SPT15 domain was contained in Trf 2 protein through the bioinformatics analysis. Trf2 gene expression was studied on all kinds of tissues , and highest in testis tissue.[Conclusion] The cDNA of Trf2 gene was cloned （GenBank accession No. EU140625 ; earle Trf 2 protein probably had effects on sperm quality.