陆川猪热休克蛋白90基因的克隆与序列分析

Gene cloning and sequence analysis of heat shock protein 90 in Luchuan pig

【目的】对陆川猪热休克蛋白90(HSP90)基因进行克隆与序列分析,为研究HSP90与陆川猪抗热应激的相关性及寻找猪耐热性分子标记奠定基础。【方法】根据Gen Bank已发表的猪HSP90基因序列(登录号NM_213973.1)设计引物,RT-PCR扩增HSP90基因编码区序列,并应用Laser Gene、Meg Align等生物软件进行序列分析及蛋白质结构预测。【结果】克隆获得陆川猪HSP90基因编码区序列长2202 bp,编码733个氨基酸,分子质量84774.8 U,理论等电点4.93;与野猪的亲缘关系最近,其氨基酸同源性为100.0%,与牛、绵羊、人类、大猩猩的氨基酸同源性也在96.2%以上。陆川猪HSP90蛋白N端有1个HATPase_c superfamily保守结构域,C端最末端有一个MEEVD保守序列,氨基酸序列中存在5个HSP90家族特有保守信号区,其成熟肽包含有α螺旋、β转角、延长线和无规卷曲4种二级结构元件。【结论】猪HSP90基因高度保守,可作为研究陆川猪抗热应激的候选基因之一。

【Objective】In order to lay the foundation for further researching HSP90 gene and anti-heat stress ability,and screening heat-resistant molecular marker of pig, the heat shock protein gene（HSP90） of Luchuan pig was cloned,and its gene sequence was analyzed. 【Method】Based on pig HSP90 gene sequence published in Gen Bank（Accession No.NM_213973.1）,three pairs of primers were designed to amplify HSP90 gene from Luchuan pig using RT-PCR technique.Then the Laser Gene, Meg Align and other biological softwares were used to analyze sequence of HSP90 gene and predict structure of coded protein. 【Result 】The results showed that, the coding region of cloned HSP90 gene was 2202 bp in length, coding a total of 733 amino acids. The molecular weight of HSP90 gene was 84774.8 U, and its theoretical isoelectric point（p I） was 4.93. In addition, Luchuan pig had the closest genetic relationship with wild boar（Sus scrofa）, and the HSP90 protein of Luchuan pig shared 100.0% homology of amino acid sequence with that of wild boar, which shared above 96.2% of amino acids sequence with those of Bos taurus, Ovis aries, Homo sapiens and Pan troglodytes. Furthermore, the HSP90 protein of Luchuan pig contained HATPase_c superfamily conservative domain on N-terminal, MEEVD conservative sequence on C-terminal and five specific conservative signal domains in amino acid sequence. Beside, the HSP90 mature peptide contained four secondary structural elements viz., α-helix,β-corner, extension cords and random coil. 【Conclusion】HSP90 gene is highly conservative, thus which can be used as one of candidate genes for researching anti-heat stress of Luchuan pig.