鲢肌球蛋白重链球状结构域的cDNA克隆及结构解析

cDNA cloning and structural analysis of the globular head for myosin heavy chain from silver carp (Hypophthalmichthys molitrix)

根据已经获得的两种鲢肌球蛋白重链同工型基因(低温型sc-w和高温型sc-s)在3′端展现的明显差异,设计了2个特异性的反向引物,以鲤科鱼类肌球蛋白重链5′端的保守序列为正向引物,通过Long-PCR对编码鲢两种肌球蛋白重链同工型的球状结构域(Subfragment-1,S1)的全长基因进行了克隆和测序,并推断出它们一级结构的氨基酸序列。研究结果表明,sc-w与sc-s在S1的初级结构上显示80.5%的同源性、与已经报道的草鱼低温型(gc10)有97.2%的高同源性;sc-s则与草鱼中间型(gcI)和高温型(gc30)显示了分别为98.4%和97.1%的高同源性。低温型的sc-w和gc10在S1初级结构上展现的特有变异主要发生在43个氨基酸残基位点,其中15个属保守性残基。对S1区域中两个功能性的表面环loop1(与ATP结合位点有关)和loop2(与肌动蛋白结合位点有关)的结构解析发现,sc-w和gc10在两个表面环的长度、残基电荷分布和氨基酸组成等方面与其它同工型之间存在明显差异,揭示了这两个表面环的结构差异可能影响了栖息于不同环境温度下的淡水鱼的肌球蛋白"分子马达"功能。分子系统树的分析结果进一步证明,鱼类栖息环境温度对其肌球蛋白同工型的基因和功能歧化的影响。

In the previous study, we reported that two types of myosin heavy chain isoform genes were isolated from fast skeletal muscles of silver carp （Hypophthalmichthys molitrix ） acclimatized in winter and summer, and named the low temperature-type（sc-w） and the high temperature-type （sc-s）, respectively. In this study, two gene-specific reverse primers were designed according to the striking differences in the nucleotide sequences of 3 ＇-terminals between the two types of silver carp myosin heavy chain isoform, and a forward primer was designed with reference to conserved nucleotide sequences in 5＇-terminal of cyprinidae. Long-PCR was performed to amplify the complete cDNAs encoding myosin subfragment-1 （S1） heavy chains for the two types of isoform. DNA sequencing of both strands was carded out, and the amino acid sequences for the two types of myosin S1 heavy chain were deduced. The results showed that the primary structure of myosin S1 heavy chain produced 80.5% identity between sc-w and sc-s. Whereas a high sequence identity of 97.2% was found between sc-w and gc10, which was reported to be isolated from 10 ℃-acclimated grass carp. In contrast, the amino acid sequence of S1 heavy chain for sc-s revealed much higher identity to those of gcI （98.4%） and gc30 （97. 1% ）, which were isolated from 30℃-acclimated grass carp. Compared with the other myosin S1 heavy chain isoforms, isoform-specific differences for sc-w and gc10 were clearly observed in 43 amino acid residues. Furthermore, among these amino acid mutations, 15 mutations occurred at the conserved residue sites. Additionally, sc-w and gc10 showed striking differences compared with the other S1 heavy chain isoforms in the two surface loops, loop 1 located near the ATP-binding domain and loop 2, which is one of the actin binding domains, suggesting that the changes in the flexibility of two surface loops were caused by modulations in length, amino acid composition and charge can play an important role in adaptation of motor function to low environmental temperature. Phylogenetic analysis further demonstrated the effects of environmental temperature on genomic divergence and functional evolution of fish myosin isoforms.