天祝白牦牛转铁蛋白(Tf)基因克隆及生物信息学分析

Molecular cloning of transferrin gene and its bioinformatics in Tianzhu White Yak

目的:克隆天祝白牦牛转铁蛋白(Transferrin,Tf)基因,并对其序列及蛋白进行遗传特性分析,为研究Tf生物学作用和生产应用提供理论依据。方法 :以成年天祝白牦牛肝脏组织为实验材料,根据牦牛Tf基因CDS区序列设计引物,利用PCR和逆转录PCR(reverse transcription,RT-PCR)技术克隆获得天祝白牦牛Tf基因编码区序列,并结合生物信息学方法分析其遗传特性。结果:PCR扩增获得了大小为2 115 bp的基因片段,为Tf基因的完整编码区,编码704个氨基酸,其N端有一个19个氨基酸组成的信号肽,含2个高度保守的同源结构域,N端结构域组成为第25到363位氨基酸(339aa),C端结构域组成为第364-693位氨基酸(330aa),两端结构域的同源性为42%;Tf基因编码的蛋白分子量为75.78 k Da,理论等电点为6.63。序列分析显示,克隆获得的Tf基因序列存在4个碱基的变异,第57位发生碱基转换(A←→G),第750位发生碱基转换(T←→C),为同义突变;第482位发生碱基转换(A←→T),第511位发生碱基转换(T←→C),为错义突变,引起第161、171位氨基酸发生改变(L→G、L→F)。DNAman软件进行氨基酸同源性分析表明,天祝白牦牛Tf基因氨基酸序列与野牦牛、牛、水牛、藏羚羊、绵羊、野猪、人、鼠、鸡和鱼Tf氨基酸序列间的同源性分别为100%、99%、96%、94%、93%、75%、69%、64%、51%、39%,说明天祝白牦牛Tf蛋白与其他物种同源,与牛类进化水平较为接近,与鸡和鱼类进化水平较远。同源建模预测Tf三级结构模型显示,天祝白牦牛Tf三级结构是在二级结构基础上经肽链折叠形成2个邻近相似的N端、C端结构域,N端结构域含N1、N2两个亚基,C端结构域含C1、C2两个亚基。结论:从天祝白牦牛肝脏组织中提取总RNA,克隆获得Tf基因编码区全长序列,并揭示了其分子遗传特性,为牦牛Tf蛋白基因工程和蛋白质功能的研究奠定了基础。

Objectives: To clone transferrin(Tf) gene in Tianzhu White Yak and analyze its characteristics of nucleotide sequence and encoding protein to provide theoretical basis for further study the function and application. Methods: Total RNA was extracted from liver of adult Tianzhu White Yak. Coding sequence(CDS) of Tf was amplified by RT-PCR using specific primers designed according to the nucleotide sequence of known Tf of Bos grunniens published in Gen Bank. The bioinformatics of Tf was also analyzed. Results: Complete CDS of Tf with 2 115 bp was cloned by RT-PCR(Gen Bank accession number:KP720624), which encode 704 amino acids(AA). Tf protein contained a signal peptide with 19 AA long in the N terminal and two highly conserved homologous domains. The N terminal domain consisted of 339 AA from 25 thto 263rdand C terminal domain consisted of 330 AA from 364 thto 693rd, which was 42% homology in AA sequence between them. The molecular weight and PI of Tf protein was 75.78 k Da and 6.63, respectively. Four base variations were found in the nucleotide sequence of Tf, including two transitions of non-synonymous mutation in A/G in 57 thand in T/C in 750 th, two transitions of mis-sense mutation in A/T in482 ndand in T/C in 511 st, leading L in 161 stand L in 171 stto G and F, respectively. The homology of AA sequence in Tf protein between Tianzhu White Yak and wild yak, bovine, buffalo, Tibetan antelope, sheep, wild boar, human mouse chicken and fish was 100%, 99%, 96%, 94%, 93%, 75%, 69%, 64%, 51% and 39%, showing Tf in Tianzhu White Yak was more close to the species of Bos. The predicted tertiary structure indicated that the secondary peptides folded up to two adjacent similar N-terminal and C-terminal structures. The N-terminal structure composed for N1 and N2 subunits, while C1 and C2 for C-terminal structure. Conclusions: The CDS of Tf in Tianzhu White Yak was successfully cloned and its bioinformatics were analyzed.