美仁牦牛MYL 9基因克隆、生物信息学分析及组织表达研究

Cloning,Bioinformatics Analysis and Tissue Expression of MYL 9 Gene in Meiren Yak

【目的】克隆美仁牦牛肌球蛋白轻链9(myosin light chain 9,MYL9)基因CDS区并对其进行生物信息学分析,检测MYL 9基因的组织表达特征,为探究该基因功能提供一定理论依据。【方法】以美仁牦牛肌肉组织cDNA为模板,利用PCR扩增并克隆美仁牦牛CDS区序列,与其他物种进行相似性比对及系统进化树构建;通过在线软件对MYL9蛋白进行生物信息学分析,利用实时荧光定量PCR检测MYL 9基因在美仁牦牛心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脂肪和睾丸组织中表达情况。【结果】美仁牦牛MYL 9基因CDS区长516 bp,共编码171个氨基酸。系统进化树结果显示,美仁牦牛与野牦牛、普通牛亲缘关系最近,与高山倭蛙亲缘关系最远。MYL9蛋白分子式为C 858 H 1324 N 234 O 274 S 12,原子数为2702,理论等电点为4.85,不稳定系数和总平均亲水性分别为37.22和―0.772,属于稳定亲水性蛋白。MYL9蛋白无信号肽和跨膜螺旋结构,属于非分泌性蛋白;主要定位于线粒体、细胞核、细胞质中,具有17个特异性磷酸化位点。MYL9蛋白二级结构主要由α-螺旋、无规则卷曲、β-折叠和延伸链构成,三级结构预测结果与二级结构一致。实时荧光定量PCR结果显示,MYL 9基因在美仁牦牛心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脂肪和睾丸组织中均表达,且肝脏和肌肉组织中表达量显著高于其他组织(P<0.05)。【结论】本研究成功克隆美仁牦牛MYL 9基因CDS区,探究了MYL 9基因的生物学功能及组织表达特征,研究结果为进一步探究牦牛MYL 9基因功能特征提供了参考。

【Objective】The objective of this experiment was to clone the CDS region of myosin light chain 9(MYL9)gene in Meiren yak and perform bioinformatics analysis,and detect the tissue expression characteristics of MYL 9 gene,so as to provide a theoretical basis for exploring the function of this gene.【Method】Using the cDNA of Meiren yak muscle tissue as template,PCR was used to amplify and clone the CDS region sequence of Meiren yak,similarity comparison with other species and phylogenetic tree construction were carried out.Bioinformatic analysis of MYL9 protein was performed by online software.The expression of MYL 9 gene in heart,liver,spleen,lung,kidney,muscle,fat and testis of Meilen yak was detected by Real-time quantitative PCR.【Result】The CDS region of MYL 9 gene in Meiren yak was 516 bp,encoding 171 amino acids.Phylogenetic tree results showed that Meiren yak had the closest genetic relationship with Bos mutus and Bos taurus,and the farthest genetic relationship with Nanorana parkeri.The molecular formula of the protein encoded by the gene was C 858 H 1324 N 234 O 274 S 12,the number of atoms was 2702,the theoretical isoelectric point was 4.85,and the instability coefficient and the total average hydrophilicity were 37.22 and―0.772,respectively,which belonged to stable hydrophilic protein.MYL9 protein was a non-secreted protein without signal peptide and transmembrane helix structure.It was mainly localized in mitochondria,nucleus and cytoplasm,and had 17 specific phosphorylation sites.The secondary structure of MYL9 protein was mainly composed of alpha helix,random coil,beta turn and extended chain,and the predicted tertiary structure was consistent with the secondary structure.Real-time quantitative PCR results showed that MYL 9 gene was expressed in heart,liver,spleen,lung,kidney,muscle,fat and testis,and the expression level in liver and muscle was significantly higher than that in other tissues(P<0.05).【Conclusion】The CDS region of MYL 9 gene in Meiren yak was successfully cloned,and the biological function and tissue expression characteristics of MYL 9 gene were explored.The results provided a reference for further research on the functional characteristics of MY L9 gene in yak.