太平洋真宽水蚤(Eurytemora pacifica)热休克蛋白70(HSP70)基因克隆及在金属胁迫下的表达分析

摘要

Heat shock protein 70 (HSP70) is an important protective protein in organism and cells.The full length sequences cDNA of HSP70 in Eurytemora pacifica (short named Ep.HSP70) was cloned for the first time using the RT-PCR and RACE techniques.The sequence length is 2252bp,the GenBank accession number is KY807149;the open reading frame (ORF) is 1947bp encoding 649 amino acids,the 5'terrninal 99bp and the 3'terminal 206bp.The predicted protein molecular weight is 70.81kDa,isoelectric point is 5.16,and it is a hydrophilic protein with no signal peptide and transmembrane domain.In addition,it is rich in alpha helical structure (37.60％) and beta fold (18.80％).Multiple homologous amino acid sequence alignment showed high conservatism between Ep.HSP70 and others HSP70 of crustaceans;especially,the conserved signature sequences of HSP70 family are highly conserved in crustaceans.Phylogenetic analysis showed that the evolutionary relationships between E.pacifica and Pseudodiaptomus annandalei is the nearest than others,and the intraspecific homology of copepods is higher than shrimp and crab.Moreover,the homology with shrimps is higher than crabs.Fluorescent quantitative PCR showed that the expression level of Ep.HSP70 had significant time effect and concentration effect under different concentrations stresses of Cu,Cd and Zn.The order of inhibition effect of the three metals on Ep.HSP70 were Cu＞Cd＞Zn.The successful cloning of Ep.HSP70 gene and expression analysis under heavy metal stresses are of great significance for the further study on the biological function of HSP70 protein.%热休克蛋白70(HSP70)是生物体内一种重要的机体与细胞保护性蛋白.本文利用RT-PCR以及RACE技术首次克隆获得太平洋真宽水蚤(Eurytemora pacifica)HSP70简称Ep.HSP70 cDNA的全长序列,序列全长为2252bp (KY807149),开放阅读框(ORF)长1947bp,编码649个氨基酸,5'端99bp,3'端206bp;预测蛋白分子量为70.81kDa,等电点为5.16,为一种亲水性蛋白,不存在信号肽及跨膜区,含有丰富的α螺旋结构(37.60％),β折叠(18.80％).同源氨基酸序列比对发现,与其他甲壳动物的同源基因保守性较高,尤其是HSP70家族典型的结构位点序列在甲壳类动物中具有高度保守性.系统进化分析表明,太平洋真宽水蚤和安氏伪镖水蚤(Pseudodiaptomus annandalei)进化关系最近;桡足类种内同源性要高于虾蟹类,与虾类同源性高于蟹类.荧光定量数据分析表明,不同浓度铜、镉、锌胁迫下太平洋真宽水蚤HSP70基因表达水平具有显著的时间效应与浓度效应的特征,三种金属对Ep.HSP70抑制效应呈现Cu＞Cd＞Zn的趋势.Ep.HSP70基因的成功克隆及金属胁迫下的表达分析为深入研究HSP70蛋白生物学功能具有重要意义.