黑鲷×真鲷杂交子代与真鲷的Calmodulin基因克隆与表达分析

Cloning and expression analysis of Calmodulin from the hybrid F_1 of Acanthopagrus schlegelii male × Pagrus major female and P. major

采用cDNA末端快速扩增技术(RACE)获得的黑鲷(Acanthopagrus schlegelii,♂)×真鲷(Pagrus major,♀)的杂交子代F_1(AP)与真鲷(Pm)的两组Calmodulin(CaM)基因序列。通过生物软件分析了两者的差异及所表达的蛋白质特性;同时,应用实时荧光定量PCR技术检测了APCaM与PmCaM在仔鱼及2龄鱼的6种不同组织中的表达特征。结果表明:1)克隆得到APCaM与PmCaM的cDNA全长分别为1 230 bp、1 210bp,两基因序列均具有一个450 bp的开放阅读框(Open reading frame,ORF),编码了一个由149个氨基酸组成的多肽,该多肽包含有高度保守的4个EF-hand功能结构域。杂交F_1与真鲷的CaM基因非翻译区差异位点主要在3'端,ORF区域有5个氨基酸差异。2)两基因与其它鱼类的核苷酸序列相似性最高为95%,与氨基酸序列相似性最高为100%;从基因序列结构及蛋白质属性表明APCaM与PmCaM属保守的CaM基因家族。3)定量分析表明CaM在检测组织中均有表达,其中APCaM在脑中表达量最高,PmCaM在性腺中表达最高;APCaM与PmCaM在脑、肌肉、性腺及鳃中的表达存在显著差异(P<0.01),在肝、肾组织及仔鱼中的表达无显著差异(P>0.05);结合杂交F_1与双亲本在生长及性腺发育上的性状,推测CaM可能与生长及性腺发育调控有关。这些结果为探讨CaM基因在杂交F_1及真鲷亲本中的作用及鲷科育种研究提供基础资料。

In order to understand the biological function of the Calmodulin（ CaM） gene in the hybrid F1 of black porgy（ Acanthopagrus schlegelii,♂） × red porgy（ Pagrus major,♀） and the P. major parent（ Pm）,the full length cDNA sequences of Calmodulin（ CaM） genes（ named as APCaM and PmCaM） were cloned and identified from the hybrid F1 and P. major for the first time using cDNA RACE amplification kit.The expression profiles of CaM genes in different tissues of 2-year-old individuals and 30-day-old larvae were evaluated by real-time quantitative PCR. The full length cDNA of APCaM and PmCaM gene are 1 230 bp and1 210 bp,respectively,each of which includes an open reading frame（ ORF） of 450 bp encoding a 149 amino acids polypeptide. The deduced amino acid sequence contains 4 highly conserved EF-hand domains known to be critical for the function of CaM. The sites for sequence differences of non-coding region were mainly in the 3＇,and 5 differences for amino acid were found in the ORF region between the two genes. The alignments of the predicted proteins from PmCaM and APCaM exhibited 99% identity to each other and more than 97% identity with calmodulins of other fishes. Sequence alignment, structure comparison and bioinformatics analyses reveal APCaM and PmCaM are members of the conservative CaM family. The CaM protein appeared to be constitutively expressed in early larval development phase and in 6 tissues from their adults,and APCaM mRNA expression was highly significantly different from PmCaM in brain,gonad,muscles and gill（ P 0. 01）,but had no difference for PmCaM among liver,kidney and the larva（ P 0. 05）. The stage-specific enrichment in brain and muscles,and the inverse situation in gonad of CaM transcripts may signify an essential role of the CaM in the growth and the development of gonad for the hybrid F1. These results provide an essential material for the hybrid breeding of Sparidae and further research of CaM function gene.