鲢鱼骨骼肌轻酶解肌球蛋白基因的cDNA克隆

cDNA Cloning of Silver Carp Fast Skeletal Light Meromyosin Genes

DOI：

中文关键词: 鲢鱼 轻酶解肌球蛋白 同工型基因 cDNA克隆

英文关键词: silver carp light meromyosin Isoform gene cDNA cloning

基金项目:上海市浦江人才基金项目(06PJ14081)

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中文摘要:

通过RT-PCR法和3′-RACE法从冬季和夏季鲢鱼的骨骼肌中分离到两种肌球蛋白重链同工型基因,分别命名为低温型(sc-w)和高温型(sc-s)。两种同工型基因的cDNA序列包含了部分3′-翻译区和全部3′-非翻译区,编码了鲢鱼轻酶解肌球蛋白(light meromyosin,LMM)羧基端的一部分氨基酸序列。在终止密码子的上游和下游区域,两种同工型基因之间在核苷酸序列上显示了明显的差异,其序列同源性为79.5%,推断的氨基酸序列的同源性为88.1%。根据与草鱼和鲤鱼相关同工型氨基酸序列的比对结果,发现在15个氨基酸残基位点上鲢鱼低温型(sc-w)与草鱼10℃型(gc10)一致,但两者显示了与其它同工型不同的变异,并且其中的11个位点属高度保守性残基。这些结果似乎表明了基于低温驯化的淡水鱼表达的肌球蛋白重链同工型必须在它们的初级结构上发生某些氨基酸残基的变异才能有助于其适应低温的栖息环境,在蛋白质水平上则展现出完全不同于高温栖息鱼类的肌球蛋白功能和性质。另一方面,根据分子系统树的分析结果,表明了来自相似栖息环境温度的鱼的肌球蛋白重链之间具有更近的基因距离,进一步证明了环境温度对广温性淡水鱼肌球蛋白重链的基因歧化和功能进化的影响。

英文摘要:

In this study,two types of myosin heavy chain isoform gene were isolated from fast skeletal muscles of silver carp(Hypophthalmichthys molitrix) acclimatized in winter and summer by RT-PCR and 3’-RACE,and named low temperature-type(sc-w) and high temperature-type(sc-s),respectively.These two isoform genes encompassed parts of 3′-translated and entire 3′-untranslated regions and encoded amino acid sequences of the C-terminal for light meromyosin(LMM).The marked differences between sc-w and sc-s were observed in the upstream and downstream regions of the stop codon.The nucleotide and deduced amino acid sequences of these two isoforms showed 79.5% and 88.1% identities,respectively.Isoform-specific differences were clearly observed between sc-w and sc-s in 15 amino acid residues.Furthermore,among these amino acid mutations,11 mutations occurred at the highly conserved residue sites of LMM heavy chain from other freshwater fish belonging to Cyprinidae except for the grass carp 10 C type(gc10).These results seem to suggest that myosin heavy chain isoforms expressed in low temperature-acclimated freshwater fish require some amino acid substitutions to adapt its assembly characteristics to different environmental temperature,and subsequently reveal functional properties different from those expressed in high temperature-acclimated freshwater fish at protein level.On the other hand,phylogenetic analysis further indicated that the myosin heavy chain isoforms from fish inhabiting similar environmental temperatures have close gene distances,demonstrating the effects of temperature on genomic divergence and functional evolution of myosin heavy chains from eurythermal freshwater fish.

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