广灵驴HSL基因克隆、序列分析与差异表达

试验旨在对广灵驴的激素敏感脂酶（hormone sensitive lipase，HSL）基因进行克隆和序列分析，并对HSL基因在广灵驴不同组织中的差异表达水平进行分析。使用RT-PCR法扩增并克隆广灵驴HSL基因CDS区部分序列，将序列拼接后得到HSL基因完整的CDS区全长序列，并对序列进行一系列生物信息学分析，通过实时荧光定量PCR检测HSL基因mRNA在广灵驴的心脏、肝脏、脾脏、肺脏、肾脏、背最长肌和皮下脂肪7个组织中的表达情况。结果显示，广灵驴HSL基因完整的CDS区全长为2 286 bp，共编码761个氨基酸，序列已提交到NCBI，登录号：MN231003。广灵驴HSL基因的核苷酸序列与马、羊驼、骆驼、猪、牛、山羊、小鼠、绵羊相应序列的同源性分别为99.6%、88.9%、88.6%、88.1%、86.9%、85.6%、80.8%、79.1%。系统进化树预测表明，广灵驴HSL基因与马的亲缘关系最近，与小鼠的亲缘关系最远。生物信息学分析发现，HSL蛋白的理论等电点为6.51，不稳定指数为56.83，亲水性的总平均值为-0.048，说明HSL是酸性不稳定的水溶性蛋白。蛋白保守域中存在N-末端结构域、α/β水解酶折叠结构域以及调节结构域。蛋白序列中共存在88个磷酸化修饰位点、25个糖基化修饰位点。蛋白中存在较强的疏水性区域，没有信号肽及跨膜区域。二级结构显示此蛋白是由α-螺旋、延伸链、β-转角和无规则卷曲构成的，分别占45.33%、11.70%、5.65%、37.32%。实时荧光定量PCR检测结果显示，HSL基因mRNA在广灵驴的7种组织中均有表达但存在差异，在皮下脂肪中表达量最高，在心脏中表达量最低，说明广灵驴HSL基因可能在体内脂肪沉积中发挥着非常重要的作用。该试验为进一步研究HSL蛋白功能及其在广灵驴脂肪沉积中代谢调控机制提供了理论基础。

Cloning,Sequence Analysis and Differential Expression of HSL Gene in Guangling Donkey

The aim of the experiment was to clone the hormone sensitive lipase (HSL) gene of Guangling donkey and analyze its sequence,and further analyze the differential expression level of HSL gene in different tissues of Guangling donkey.In this experiment,RT-PCR method was used to amplify and clone the partial sequence of CDS region of HSL gene of Guangling donkey.After splicing the sequence,the full length sequence of CDS region of HSL gene could be obtained,and a series of bioinformatics analysis was performed on the sequence.Real-time quantitative PCR was used to detect the expression of HSL mRNA in heart,liver,spleen,lung,kidney,longissimus dorsi muscle and subcutaneous fat in Guangling donkey.The results showed that the complete CDS region of HSL gene in Guangling donkey was 2 286 bp in length,encoding a total of 761 amino acids,and the sequence was successfully submitted to NCBI,accession No.MN231003.The homology of the nucleotide sequence of HSL gene in Guangling donkey with the corresponding sequences of Equus caballus,Vicugna pacos,Camelus ferus,Sus scrofa,Bos taurus,Capra hircus,Mus muqulus and Ovis aries were 99.6%,88.9%,88.6%,88.1%,86.9%,85.6%,80.8% and 79.1%,respectively.Phylogenetic tree prediction showed that HSL gene of Guangling donkey was closely related to the horse and the furthest to the mouse.Bioinformatics analysis found that the theoretical isoelectric point of HSL protein was 6.51,and the instability index was 56.83,and the total average hydrophilicity was -0.048,indicating that HSL was an acid-labile water-soluble protein.There were N-terminal domains and α/β hydrolase folding domains as well as regulatory domains in the conserved domains of proteins.There were 88 phosphorylation modification sites and 25 glycosylation modification sites in the protein sequence.There were strong hydrophobic regions in the protein,without signal peptides and transmembrane regions.The secondary structure showed that this protein was composed of alpha-helix,extended chain,beta-turn and random coil,which account for 45.33%,11.70%,5.65%,37.32%,respectively.Real-time quantitative PCR detection results showed that HSL gene mRNA was expressed in 7 kinds of tissues in Guangling donkeys,but there were differences.The highest expression was in subcutaneous fat and the lowest expression in heart,indicating that Guangling donkey HSL gene might be play a very important part in fat deposition in vivo.It provided a theoretical basis for further studying the function of HSL protein and its metabolic regulation mechanism of fat deposition in Guangling donkey.