猪ASB2基因CDS区克隆、生物信息学分析及表达规律研究

本研究旨在克隆猪锚蛋白重复序列和细胞信号抑制因子盒蛋白2（ankyrin repeat and suppressor of cytokine signalling box containing protein 2，ASB2）基因完整CDS区序列，通过生物信息学方法分析CDS序列和蛋白质基本特性，探讨其在晋汾白猪不同组织及卫星细胞诱导成肌过程中的表达规律。选取1日龄晋汾白猪为研究对象，依据GenBank中猪ASB2基因预测核苷酸序列设计引物，以背最长肌组织cDNA为模板，采用分段扩增法进行猪ASB2基因的克隆。利用生物信息学软件分析ASB2氨基酸序列及编码蛋白质的结构和功能，利用实时荧光定量PCR技术检测ASB2基因在猪不同组织及卫星细胞诱导成肌过程中的表达水平。结果显示，猪ASB2基因完整CDS区序列长1 824 bp，共编码607个氨基酸。猪ASB2基因核苷酸序列与山羊和牛的相似性最高。生物信息学分析发现，ASB2蛋白为亲水性蛋白，共有54个磷酸化位点，11个O-糖基化位点，1个N-糖基化位点，没有信号肽。保守结构域分析结果表明存在11个ANK基序和1个SOCS基序。猪ASB2蛋白二级结构中无规则卷曲、α-螺旋、β-转角和延伸链分别占43.99%、40.36%、10.05%和5.60%。实时荧光定量PCR结果显示，ASB2基因在猪腰大肌组织中表达量最高，其次为背最长肌和心脏，且与其他组织中表达量具有极显著差异（P<0.01）；在诱导卫星细胞成肌过程中发现该基因表达量呈先升高后降低趋势，提示其可能参与调控肌肉生长过程。本研究结果可为进一步探讨猪ASB2基因功能及作用机制提供参考依据。

Study on Cloning,Bioinformatics Analysis and Expression Profile of ASB2 Gene CDS in Pig

The objective of this study was to clone the complete CDS region of porcine ankyrin repeat and suppressor of cytokine signalling box containing protein 2 (ASB2) gene,analyze the CDS sequence and the basic characteristics of proteins by bioinformatics methods,and explore its expression profiles in different tissues of one-day-old Jinfen White pig and satellite cell myoblast induction.Based on the predicted nucleotide sequence of pig ASB2 gene in GenBank.Primers were designed to amplify the CDS region of ASB2 gene by RT-PCR using cDNA of longissimus dorsi muscle as template.The amino acid sequence and the structure and function of ASB2 were analyzed using bioinformatics software.The expression profiles in different tissues and during the procession of myogenic differentiation of satellite cells were investigated by Real-time quantitative PCR.The results showed that the complete CDS region of porcine ASB2 gene was 1 824 bp in length,which encoded a total of 607 amino acids.The nucleotide sequence of porcine ASB2 gene had the highest similarity with those of goat and cow.Bioinformatics analysis results showed that ASB2 was a hydrophilic protein,including 11 O-glycosylation sites,1 N-glycosylation site,and no signal peptide.There were 11 ANK motifs and 1 SOCS motif.The secondary structure of the protein was predicted to be 43.99%,40.36%,10.05% and 5.60% with random coil,alpha helix,beta turn and extended strand.Real-time quantitative PCR results showed that the highest expression level was found in porcine psoas muscle,and followed by longissimus dorsal muscle and heart,which were extremely significantly different from those in other tissues (P<0.01).During the procession of myogenic differentiation of satellite cells,the expression of ASB2 gene was increased at the beginning,reached the peak after 2 d induction,then decreased,which suggested that ASB2 gene might be involved in regulating muscle growth.The results of this experiment provided a reference for further exploring the function and mechanism of porcine ASB2 gene.