【摘要】 目的:根据人、小鼠HSF1cDNA保守区序列设计引物,通过PCR方法扩增海南黄牛HSF1cDNA,并进行序列分析。方法:利用RT-PCR、半巢式PCR以及3’-RACE技术分段扩增得到了海南黄牛HSF1cDNA序列,测序正确后进行拼接。用DNAMAN 生物信息学软件分析海南黄牛HSF1 cDNA与赫里福德牛、人、小鼠同源性和海南黄牛HSF1蛋白的氨基酸组成、等电点、亲/疏水区等蛋白质性质,并根据各种动物HSF1蛋白绘制进化树。结果:(1)海南黄牛的HSF1 cDNA序列全长为1 993bp,包括150bp的5’非翻译区,1 578bp的开放阅读框以及264bp(不含poly(A)尾)的3’非翻译区,编码524个氨基酸,分子量为56.42 kD,等电点(pI)为 4.79。(2)海南黄牛的HSF1 cDNA与赫里福德牛、小鼠和人HSF1 cDNA的同源性分别为98.99 %、81.78 %、87.82 %,相应编码蛋白氨基酸序列的同源性分别为98.86 %、83.84 %、89.06 %,其中N-末端和C-末端高度保守,而中间区域存在缺失或替换。(3)根据氨基酸序列构建不同动物HSF1蛋白的进化树,与采用经典遗传分类法构建的进化树基本一致。结论:首次克隆了海南黄牛 HSF1 cDNA全长,分析表明:海南黄牛HSF1蛋白是亲水性蛋白,在8种动物中,其同源性大于73 %,高度保守。海南黄牛与赫里福德牛HSF1蛋白同源性高达98.86 %,在三聚体化区域、转录调节域和激活域存在6个位点的单氨基酸突变,这些发现为进一步揭示海南黄牛抗热性状形成的分子机制提供了重要依据。更多还原

【Abstract】 Objective:HSF1 cDNA of hainan yellow cattle was cloned according to the conservative sequence of human and mouse HSF1 by PCR.Method:Three parts of HSF1 cDNA of hainan yellow cattle was cloned by RT-PCR,Semi-nested PCR and 3’-RACE separately.After sequence,the HSF1 cDNA was reconstructed and the constitution of HSF1 cDNA and amino acid of HSF1 protein in hainan yellow cattle was analyzed by DNAMAN software and a phylogenetic tree based on HSF1 protein from different animals was constructed.Result:(1)A 1993 bp length cDNA sequence containing 150 bp 5’-untranslated region,264 bp 3’-untranslated region and 1 578 bp open reading frame(ORF)encoding a 56.42 kD peptide contain 524 amino acids with pI of 4.79.(2)The homology of HSF1 gene of hainan yellow cattle are 98.99%,81.78%,87.82% compared to cattle,mouse and human,and the homology comparison of HSF1 amino acid sequence on hainan yellow cattle with human and mouse was 98.86%,83.84% and 89.06% separately,the region in the N-terminal and C-terminal was highly conservative and amino substitution or deletion existed in the middle region.(3)Phylogenetic tree based on the HSF1 protein of different animals was matched with the classical taxonomic classification of the animal.Couclusion:HSF1 protein is a hydrophilic protein and is high conservative in eight species animals.Six sits of singal amino mutation was occurred in the regulatory domain and activation domain of HSF1 protein of hainan yellow cattle.These results set a strong basis for further research of the molecular mechanism of the good thermal resistance of hainan yellow cattle.更多还原