剑尾鱼热应激蛋白HSP70 cDNA片段的克隆与序列分析

Isolation and characterization of a cDNA clone encoding a 70-kDa heat shock protein in green swordtail(Xiphophorus helleri)

为了进一步开展剑尾鱼Xiphophorus helleri——标准化实验动物在抗逆方面的相关研究,以及热应激蛋白(HSP70)在抗逆方面的作用,作者用RT-PCR方法首次克隆得到了剑尾鱼的HSP70的cDNA片段,该片段包含HSP70的特征性序列(GAT CTC GGT GGT GGC ACT TTT GAT)。通过将克隆片段与已发表的其它鱼类HSP70的核苷酸序列和其编码的氨基酸序列进行同源性比较,发现剑尾鱼核苷酸序列同源性较高,与牙鲆Paralichthys olivaceus HSP70核苷酸序列同源性最高为89%,与川鲽Platichthys flesus、虹鳟Oncorhyn-chus mykiss和黄锡鲷Rhabdosargus sarba的同源性皆在85%以上;与国外学者发表的HSP70的规律类似,所克隆的片段与其它鱼类HSP70基因编码的氨基酸序列同源性较相应核苷酸序列同源性更高,与牙鲆和川鲽的HSP70氨基酸序列同源性最高达到99%,与斑马鱼Danio rerio、虹鳟和黄锡鲷的氨基酸序列同源性均高于97%。不同鱼的HSP70 cDNA核苷酸序列存在一定差异,但氨基酸序列并未受到影响,经对照,这种不同相当一部分存在于密码子的第3个碱基,因为密码子的简并性,编码氨基酸没有发生改变。

To continue researching on stress tolerance of laboratory animal green swordtail（Xiphophorus helleri） and HSP70, the cDNA partial clones encoding HSP70 protein has been first isolated in green swordtail. The isolated genes contains a consensus HSP70 motif sequence（ GAT CTC GGT GGT GGC ACT TIT GAT）. Comparison of the nucleotide and deduced amino acid sequences to entries in the GenBank revealed that the cDNA clone encodes a heat shock 70 kDa protein. The isolated cDNA clone nucleotide sequence exhibits a high degree of homology to the DNA sequences of HSP70 proteins from other organisms. The highest percentage of homology at the nucleotide level found to that of Japanese flounder Paralichthys olivaceus at 89%. Other fish whose homology is greater than 85% include Platichthysflesus, rainbow trout Oncorhynchus mykiss and Rhabdosargus sarba. Surprisingly, the degree of homology was even higher for the amino acid sequence of HSP70 counterpart （greater than 97% ）. A significantly high degree of homology was also found with the Japanese flounder and Platichthys flesus HSP70 at 99%. The degeneracy of the genetic code allows for changes in the nucleotide sequence that do not affect the amino acid sequence of protein due to silent substitutions.