不同嫩度牦牛背最长肌中SERPINE1基因克隆、生物信息学及表达分析

Cloning,Bioinformatic and Expression Analysis of SERPINE 1 Genein Longissimus Dorsi Muscle of Yaks with Different Tenderness

【目的】克隆牦牛丝氨酸蛋白酶抑制剂家族E成员1(serpin family E member 1,SERPINE1)基因序列,同时探究不同嫩度牦牛背最长肌组织中SERPINE1基因核苷酸序列差异,为进一步研究其对牦牛肉嫩度的影响提供试验数据。【方法】根据GenBank中牦牛SERPINE1基因序列设计特异性引物,通过PCR扩增克隆获得高、低嫩度牦牛背最长肌中的SERPINE1基因序列全长,并对其结构及编码蛋白进行生物信息学分析,通过实时荧光定量PCR检测不同嫩度牦牛背最长肌中SERPINE1基因表达量。【结果】高、低嫩度牦牛背最长肌组织中SERPINE1基因全长分别为8 315和8 318 bp,均编码402个氨基酸且氨基酸序列完全相同。高、低嫩度背最长肌组织中SERPINE1基因非编码序列中存在3个碱基缺失及22个碱基突变(4个碱基颠换、18个碱基转换)。牦牛SERPINE1基因核苷酸序列与普通牛、瘤牛、美洲野牛、绵羊、山羊、家猪、人、小鼠的相似性分别为99.26%、99.26%、99.59%、96.94%、97.02%、90.49%、86.52%和80.10%。SERPINE1蛋白是一个含有23个氨基酸信号肽的亲水性稳定膜外蛋白,位于细胞外,具有34个潜在磷酸化位点,包含1个反应中心环(reactive center loop, RCL)。SERPINE1蛋白二级结构主要由α-螺旋(44.78%)和无规则卷曲(35.32%)构成。实时荧光定量PCR结果显示,SERPINE1基因在高嫩度牦牛背最长肌中表达量极显著高于低嫩度牦牛背最长肌(P<0.01)。【结论】本研究成功从高、低嫩度牦牛背最长肌组织中克隆SERPINE1基因全长并进行了生物信息学特征分析,为后续研究SERPINE1基因参与调控牦牛肉嫩度机制提供理论参考。

【Objective】This study was aimed to clone the serpin family E member 1(SERPINE1)gene sequence in yak,and explore the nucleotide sequence difference of SERPINE1 gene in longissimus dorsi muscle of yak with different tenderness,so as to provide experimental data for further study on its effect on the tenderness of yak meat.【Method】Based on the yak SERPINE 1 gene sequence in GenBank,specific primers were designed to amplify the full length of SERPINE 1 gene sequence in longissimus dorsi muscle of yak with high and low tenderness by PCR,and their structure and encoded protein were analyzed by bioinformatics.Real-time quantitative PCR was used to detect SERPINE 1 gene expression in longissimus dorsi muscle of yak with different tenderness.【Result】The total length of SERPINE 1 gene in longissimus dorsi muscle of yak with high and low tenderness was 8315 and 8318 bp,respectively,encoding 402 amino acids with identical amino acid sequence.There were 3 base deletions and 22 base mutations(4 base translocations and 18 base transitions)of SERPINE 1 gene in high and low tenderness longissimus dorsi muscle.The nucleotide sequence of SERPINE 1 gene in yak showed 99.26%,99.26%,99.59%,96.94%,97.02%,90.49%,86.52%and 80.10%similarity to Bos taurus,Bos indicus,Bison bison bison,Ovis aries,Capra hircus,Sus scrofa,Homo sapicns and Mus musculus,respectively.SERPINE1 protein was a hydrophilic stable extracellular protein containing 23 amino acid signaling peptides,and located in the extracellular with 34 potential phosphorylation sites,including 1 reactive center loop(RCL).The secondary structure of SERPINE1 protein was mainly composed of alpha helix(44.78%)and random coil(35.32%).Real-time quantitative PCR results showed that the expression of SERPINE 1 gene in longissimus dorsi muscle of yak with high tenderness was extremely significantly higher than that in longissimus dorsi muscle of yak with low tenderness(P<0.01).【Conclusion】The full length of SERPINE 1 gene was successfully cloned from longissimus dorsi muscle of yak with high and low tenderness and the bioinformatics characteristics were analyzed,which provided theoretical reference for the subsequent research on the mechanism of SERPINE 1 gene involved in the regulation of yak meat tenderness.