广灵驴CAPN1基因克隆、生物信息学分析及表达研究

本研究旨在对钙蛋白酶1（CAPN1）基因CDS区进行克隆及生物信息学分析,并鉴定其在广灵驴各组织中的表达量。应用生物信息学方法对广灵驴CAPN1基因CDS区进行序列分析,并对其编码蛋白的理化性质、亚细胞定位、翻译后修饰结构和蛋白结构进行预测;利用实时荧光定量PCR技术对CAPN1基因在心脏、肝脏、脾脏、肺脏、肾脏和背最长肌6种组织中的表达水平进行分析。结果显示,广灵驴CAPN1基因CDS区全长2 148 bp,可编码715个氨基酸,已提交至NCBI,登录号为：MN158194,其核酸序列与马、绵羊、牛、人、山羊、小鼠、猪的同源性分别为99.7%、92.3%、92.5%、92.0%、92.5%、85.9%和92.9%;CAPN1蛋白的分子质量为82.01 ku,理论等电点为5.59,平均疏水性为-0.374,不稳定系数为36.42,不存在跨膜区及信号肽;其编码蛋白的二级结构由无规则卷曲、α-螺旋、β-转角和延伸链组成;CAPN1基因在广灵驴的6种组织中均表达,其中在背最长肌中的表达量最丰富,其次是肝脏,在心脏中的表达最低。本研究成功克隆了广灵驴CAPN1基因CDS,并对其在不同组织中的表达量进行了分析,为进一步研究CAPN1基因在肌肉嫩度方面的表达调控功能及发展地方品种广灵驴肉制品产业提供了理论依据。

Cloning,Bioinformatics and Expression Analysis of CAPN1 Gene in Guangling Donkey

This study was aimed to clone the calcium protease 1 (CAPN1) gene CDS and analyze its bioinformatics,and identify its expression in different tissues of Guangling donkey.The CAPN1 gene was amplified,cloned and sequenced,the physicochemical,subcellular localization,modified structure and protein structure of CAPN1 protein were predicted using bioinformatic methods.The expression level of CAPN1 gene in heart,liver,spleen,lung,kidney and longissimus dorsi muscle of Guangling donkey were analyzed by Real-time PCR.The results showed that the length of CAPN1 gene CDS was 2 148 bp,and encoded 715 amino acids,which had been submitted to GenBank,the accession number was MN158194.The homology of nucleic acid sequence of CAPN1 gene in Guangling donkey was 99.7%,92.3%,92.5%,92.0%,92.5%,85.9% and 92.9% with Equus caballus,Ovis arise,Bos taurus,Homo sapiens,Capra hircus,Mus musculus and Sus scrofa,respectively.The molecular weight,isoelectric point,instability index of CAPN1 protein were 82.01 ku,5.59 and 36.42,respectively,and the average hydrophobic index was -0.374.CAPN1 protein had no signal peptide and no transmembrane region.The secondary structure of CAPN1 protein was consisted of random coil,alpha helix,beta turn and extended chain.The CAPN1 gene was expressed in all six tissues,the expression in longissimus dorsi muscle was the highest,followed by liver,and the expression in heart was the lowest.This experiment successfully cloned CAPN1 gene of Guangling donkey and analyzed its expression in different tissues,it provided a theoretical basis for further study of the expression and regulation function of CAPN1 gene in the muscle tenderness and developing the meat products industry of local species Guangling donkey.