草原红牛Bcl2L13的CDS克隆及其在不同组织的表达规律研究  

Cloning of Bcl2L13 Gene CDS Sequence and Its Expression in Different Tissues of Red Cattle

作　　者：刘理想 高一 秦立红 吕阳 薛佳佳 吴健[1,3,4] 胡忠昌 张国梁 LIU Lixiang;GAO Yi;Qin Lihong;LYV Yang;XUE Jiajia;WU jian;HU Zhongchang;ZHANG Guoliang(Animal Husbandry Division,Jilin Academy of Agricultural Sciences,Jilin Gongzhuling 136100,China;College of Animal Science and Technology,Jilin Agricultural University,Jilin Changchun 130000,China;Jilin Kuncheng Animal Husbandry Technology Development Co.,Ltd.,Jilin Gongzhuling 136100,China;Jilin Beef cattle breeding and breeding Technology Innovation Center,Jilin Gongzhuling 136100,China)

机构地区：[1]吉林省农业科学院畜牧分院,吉林公主岭136100 [2]吉林农业大学动物科学技术学院,吉林长春130000 [3]吉林坤成牧业科技发展有限公司,吉林公主岭136100 [4]吉林省肉牛繁育及养殖技术科技创新中心,吉林公主岭136100

出　　处：《中国畜牧杂志》2020年第7期69-75,共7页Chinese Journal of Animal Science

基　　金：国家肉牛牦牛产业技术体系(CARA-37);国家重点研发计划(2018YFD0501802);吉林省科学技术厅重点科技研发项目(20180201039NY);肉牛肉质性状优异基因挖掘与鉴定(20160204006NY);吉林省畜禽良种工程项目(肉用草原红牛培育);吉林省农业科技创新工程(高品质肉牛培育);家养动物种质资源平台;吉林省农业科学院创新工程(CXGC2019DC005)。

摘　　要：本研究旨在初步探究草原红牛Bcl2L13的基因功能,并对其进行生物信息学分析,检测Bcl2L13在草原红牛不同组织中的表达差异。本实验以草原红牛为研究对象,根据GenBank公布的牛Bcl2L13基因序列(GenBank登录号:NM001078082.2)设计引物,PCR扩增获得Bcl2L13的完整CDS并进行测序,利用分析软件进行序列同源性比对并构建系统进化树;获得对应的氨基酸序列并分析蛋白理化特性及蛋白亚细胞结构、蛋白亲疏水性和磷酸化位点;预测蛋白二级结构并进行蛋白质三级结构模型构建;采用实时荧光定量PCR方法检测Bcl2L13基因在草原红牛各组织中的表达规律。结果显示:草原红牛Bcl2L13基因核苷酸序列与普通牛和牦牛的同源性高(100%和99.3%),与瘤牛和亚洲水牛的同源性较高(91.8%、90.5%);Bcl2L13基因CDS大小为839bp,编码279个氨基酸,该蛋白质分子式为C1348H2108N348O439S5,分子量为30.374 ku,理论等电点为4.51,蛋白质不稳定指数为77.71,总平均亲水性为-0.250。亚细胞定位分析表明,Bcl2L13蛋白分布在内质网(4.3%)、线粒体(4.3%)、过氧化物酶体(8.7%)、细胞质(65.2%)、细胞骨架(4.3%)、细胞核(13.0%);磷酸化位点分析发现Bcl2L13蛋白存在33个磷酸化位点;二级结构主要形式有α-螺旋(22.4%)、β-转角(12.1%)、β折叠(31.0%)、无规则卷曲(34.5%),与三级结构预测相同;Bcl2L13在背最长肌中表达量最高,在肺组织中表达量最少。综上,Bcl2L13基因在生物进化过程中具有低保守性,其编码的氨基酸组成的蛋白质结构不稳定,属于水溶性蛋白,主要在细胞质中发挥作用,在草原红牛不同组织的表达量有显著差异。This experiment was aimed to study the gene function of Bcl2L13 in the red cattle using bioinformatics,and investigate the mRNA expression of Bcl2L13 gene in different tissues of red cattle.Primers were designed According the predicted sequence of Bcl2L13 gene(accession No.:NM_001078082.2)in GenBank.The complete CDS region of Bcl2L13 gene was cloned by RT-PCR and sequencing.Sequence homology alignment and phylogenetic tree of Bcl2L13 gene constructed by software.The corresponding amino acid sequence was obtained.The physical and chemical property and subcellular structure was analysed by online prediction software.Protein hydrophilicity and hydrophobicity was analysed.Phosphorylation site was analysed.Protein secondary and tertiary structure was analysed by DNAStar software and SWISSMODEL.Real-time PCR was used to detect the mRNA expression of Bcl2L13 gene.The results showed that the nucleotide sequence of Bcl2L13 gene of grassland red cattle had high homology with Bos taurus and Bos mutus(100%and 99.3%),and with Bos indicus and Bos indicus(91.8%and 90.5%).The CDS of Bcl2L13 gene was 839 bp,encoding 279 amino acids.The molecular formula of the protein:C1348H2108N348O439S5,molecular weight:30.374 ku,theoretical isoelectric point:4.51.The protein instability index was 77.71,which belonged to unstable protein,and the total average hydrophilicity was-0.250.The results of subcellular localization showed that Bcl2L13 protein was located in the endoplasmic(4.3%),mitochondria(4.3%),peroxisomal(8.7%),cytoplasmic(65.2%),cytoskeletal(4.3%),nuclear(13.0%).There were 33 phosphorylation sites.The main forms of the secondary structure wereα-helix(22.4%),β-turn(12.1%),β-fold(31.0%),and irregularcurl(34.5%),which was the same as the prediction of the tertiary structure.Bcl2L13 gene was expressed most in Longissimus dorsi.It was rarely expressed in lung tissue.The Bcl2L13 gene was lowly conserved in the process of biological evolution.Bcl2L13 protein was structurally unstable,and it belonged to water soluble protein.The protein

关 键 词：草原红牛 Bcl2L13基因 克隆 生物信息学分析 表达差异 

分 类 号：S823.2[农业科学—畜牧学]