摘 要：試驗旨在對廣靈驢的激素敏感脂酶(hormone sensitive lipase，HSL)基因進行克隆和序列分析，並對HSL基因在廣靈驢不同組織中的差異表達水平進行分析。使用RT-PCR法擴增並克隆廣靈驢HSL基因CDS區部分序列，將序列拼接後得到HSL基因完整的CDS區全長序列，並對序列進行一系列生物信息學分析，通過實時螢光定量PCR檢測HSL基因mRNA在廣靈驢的心臟、肝臟、脾臟、肺臟、腎臟、背最長肌和皮下脂肪7個組織中的表達情況。結果顯示，廣靈驢HSL基因完整的CDS區全長為2 286 bp，共編碼761個胺基酸，序列已提交到NCBI，登錄號：MN231003。廣靈驢HSL基因的核苷酸序列與馬、羊駝、駱駝、豬、牛、山羊、小鼠、綿羊相應序列的同源性分別為99.6%、88.9%、88.6%、88.1%、86.9%、85.6%、80.8%、79.1%。系統進化樹預測表明，廣靈驢HSL基因與馬的親緣關係最近，與小鼠的親緣關係最遠。生物信息學分析發現，HSL蛋白的理論等電點為6.51，不穩定指數為56.83，親水性的總平均值為-0.048，說明HSL是酸性不穩定的水溶性蛋白。蛋白保守域中存在N-末端結構域、α/β水解酶摺疊結構域以及調節結構域。蛋白序列中共存在88個磷酸化修飾位點、25個糖基化修飾位點。蛋白中存在較強的疏水性區域，沒有信號肽及跨膜區域。二級結構顯示此蛋白是由α-螺旋、延伸鏈、β-轉角和無規則捲曲構成的，分別佔45.33%、11.70%、5.65%、37.32%。實時螢光定量PCR檢測結果顯示，HSL基因mRNA在廣靈驢的7種組織中均有表達但存在差異，在皮下脂肪中表達量最高，在心臟中表達量最低，說明廣靈驢HSL基因可能在體內脂肪沉積中發揮著非常重要的作用。該試驗為進一步研究HSL蛋白功能及其在廣靈驢脂肪沉積中代謝調控機制提供了理論基礎。
關鍵詞：廣靈驢；HSL基因；克隆；生物信息學分析；差異表達
Abstract: 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.
Key words: Guangling donkey; HSL gene; cloning; bioinformatics analysis; differential expression
李武峰, 孫瑜彤, 趙婧微，等. 廣靈驢HSL基因克隆、序列分析與差異表達[J]. 中國畜牧獸醫, 2020, 47(8): 2348-2358.
doi: 10.16431/j.cnki.1671-7236.2020.08.002