黑鲷钙调磷酸酶B同源蛋白2(CHP2)基因克隆及表达差异  

Molecular Cloning and mRNA Expression Analysis of Calcineurin B Homologous Protein 2 Gene in Black Porgy Acanthopagrus schlegelii

作　　者：陈淑吟[1] 张志勇[1] 孙瑞健[1] 贾超峰[1] 孟乾 祝斐[1] 张志伟[1] 吴国均[1] CHEN Shuyin;ZHANG Zhiyong;SUN Ruijian;JIA Chaofeng;MENG Qian;ZHU Fei;ZHANG Zhiwei;WU Guojun(Marine Fisheries Research Institute of Jiangsu Province,Nantong 226007,China)

机构地区：[1]江苏省海洋水产研究所,江苏南通226007

出　　处：《水产科学》2021年第5期661-669,共9页Fisheries Science

基　　金：江苏省农业重大新品种创制项目(PZC201744);江苏省农业科技自主创新资金资助项目(CX(17)2021);江苏省科技项目(BK20181202).

摘　　要：钙调磷酸酶B同源蛋白2(CHP2)为参与细胞内pH及细胞增殖调控的关键因子。用RACE末端PCR方法得到黑鲷CHP2(AsCHP2)基因全长cDNA,并利用生物信息学方法分析该基因的序列特征与同源性;同时利用实时荧光定量PCR方法检测AsCHP2的mRNA在黑鲷整体仔鱼,90d、180d黑鲷,杂交F_(2)(真鲷♂×黑鲷♀)及回交F_(1)(黑鲷♂×杂交F_(1)♀)的脑、心脏、肌肉、肝脏和性腺等组织中的表达。研究结果表明:(1)AsCHP2cDNA序列全长1722bp,5'-非编码区(UTR)长度69bp,3'-UTR长度1069bp,开放阅读框长度为585bp,编码194个氨基酸;其结构域包含2个EF-hand结构及1个核输出信号;其蛋白分子质量为21.87ku,理论等电点为4.89;同源建模获得了CHP2的二级与三维结构。AsCHP2基因的核苷酸序列与其他鱼类的相似性为86%~81%,编码的氨基酸序列与其他鱼类的相似性为80%~72%。邻接系统树显示了黑鲷CHP2氨基酸序列与其他鱼类的进化关系。序列比对与结构分析表明,AsCHP2基因具有典型的CHP2基因结构。(2)AsCHP2的mRNA在黑鲷的仔鱼期(5~35d)及大规格个体的脑、肝脏及肌肉组织中均有表达,在90d个体各组织中的表达量均高于在180d个体各组织中的表达量;在180d黑鲷、杂交F_(2)及回交F_(1)的个体中,基因在脑、肝脏、肌肉及性腺等不同组织中均有表达,且均在心脏中高表达、在性腺中低表达。这些结果显示,CHP2基因在海水鱼类的生长、发育过程中发挥功能。Full length cDNA sequence of calcineurin B homologous protein 2(CHP2)gene as a key molecule involved in regulation of intracellular pH and cell proliferation was amplified in black porgy Acanthopagrus schlegelii(AsCHP2)by RACE PCR,and the sequence characteristics and homology of the AsCHP2 were analyzed by bioinformatics.The expression of AsCHP2 mRNA was detected in whole black porgy larvae,and brain,heart,muscle,liver and gonads of 90 d or 180 d old black porgy hybrid F_(2)(red sea bream Pagrus major♂×black porgy♀)and,backcross F_(1)(black porgy♂×hybrid F_(1)♀)by real-time fluorescent quantitative PCR.It was found that the AsCHP2 cDNA was 1722 bp in length,including a 69 bp 5′-untranslated region(UTR),a 1069 bp 3′-UTR and a 585 bp open reading frame encoding a peptide of 194 amino acids.The protein contained two conserved EF-hand domains and a nuclear export sequence,the molecular weight of about 21.87 ku,pI of about 4.89.Secondary and three-dimensional structure of AsCHP2 was developed by homology modeling.Nuclear sequence alignment revealed that AsCHP2 shared higher identities(86%—81%)with other fishes,and the alignment of the predicted proteins sequence of AsCHP2 had 80%—72%of similarity with other fishes.The evolutionary relationships of AsCHP2 amino acid sequences with other kinds were analyzed using the methods of NJ tree and ML tree.Sequence alignment and structure comparison revealed that AsCHP2 were members of the conservative CHP2.The AsCHP2 appeared to be constitutively expressed in early larval development(5—35 d)and the 90-day and 180-day old individuals,and had high expression level in all tissues from the individuals of 90-day old.CHP2 mRNAs was ubiquitously expressed in all tested tissues,and far more abundant in the heart,as about several times as that in gonad in black porgy,the hybrid F_(2) and the backcross F_(1).These findings suggest that the CHP2 gene might be involved in the growth and development of sea fishes.

关 键 词：黑鲷 钙调磷酸酶B同源蛋白2基因 基因克隆 MRNA表达 

分 类 号：Q959.4[生物学—动物学]