银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。

Cloning and expressional characterization of creatine kinase M3-CK in the gibel carp(Carassius auratus gibelio)

The full length cDNA of creatine kinase M3 CK gene was cloned from gynogenetic gibel carp( Carassius auratus gibelio ) by screening the suppression subtractive hybridization(SSH) plasmid library between early stage embryos with heart beat and tail bud stage embryos using SMART cDNA synthesis and RACE PCR.The resultant segment was 1 551 bp in length and had an open reading frame for encoding 380 amino acids.Its deduced amino acid sequence has about 95% identity with that of the common carp( Cyprinus carpio ) M3 CK.Phylogenetic analysis indicates that gibel carp creatine kinase M3 CK and other vertebrate M CK belong to a subgroup,and that M3 CKs of gibel carp and common carp( Cyprinus carpio ) are clustered together.The divergence between gibel carp M3 CK and B CKs or Mt CKs is greater than that between gibel carp M3 CK and M CKs of other species.Virtual Northern blot showed that the gibel carp M3 CK gene is differentially expressed during embryogenesis.RT PCR analysis showed that the maternal mRNAs of the gibel carp M3 CK gene was detected in mature eggs and early stage embryos.It began to transcribe from the muscle contraction stage and expression continued after that.RT PCR analysis of adult tissues indicated that it was expressed only in heart and muscle .