双齿围沙蚕诱导型热休克蛋白70基因的克隆及Cu胁迫下的表达分析

Gene cloning and expression analysis of an inducible heat shock protein 70 gene from the polychaete Perinereis aibuhitensis under Cu^(2+) stress

本研究主要评估了双齿围沙蚕热休克蛋白70(HSP70)基因的分子特征,记录了其对于液态Cu^(2+)胁迫的基因表达情况,并通过测序获得的HSP70 cDNA序列与其他沙蚕及无脊椎动物HSP70同源性比对来判定蛋白特性.结果表明:该HSP70基因全长cDNA序列共2161bp,包括5'非翻译区48 bp,3'非翻译区142 bp,一个多聚腺苷酸信号序列(AATAAA)和Poly A尾巴以及开放阅读框1971 bp.阅读框共编码656个氨基酸,总分子量为71.43 kD,理论等电点为5.15.该氨基酸序列中含有HSP70家族的3个签名序列——IDLGTTYS、IFDLGGGTFDVSIL和IVLVGGSTRIPKIQK,以及细胞质特异性调控基序EEVD,C端重复序列GGMP.同源性分析表明,本研究所获双齿围沙蚕HSP70氨基酸序列与已报道的序列相似性高达94%,与其他无脊椎生物的HSP70相似性也高达79%以上.荧光实时定量PCR分析表明,Cu^(2+)(0.2~5.0mg·L^(-1))胁迫能够显著诱导沙蚕HSP70 mRNA表达,并于1d后达到峰值.本研究系统描述了双齿围沙蚕HSP70的分子特性,其可被液态Cu^(2+)诱导表达,具备作为环境污染分子生物标记物的潜力.

The objective of this study was to evaluate the molecular characteristics of heat shock protein 70 （HSP70） gene, and document changes in HSP70 gene expression upon exposure of Perinereis aibuhitensis to aqueous Cu2＋. Full length cDNA of HSP70 was sequenced and the characteristics of the translated protein were determined and compared both with other ragworms species and other invertebrates. The results showed that the cDNA was 2161 bp and consisted of a 5＇-terminal untranslated region （UTR） of 48 bp, a 3＇-termianl UTR of 142 bp with a canonical polyadeny- lation signal sequence AATAAA and a poly A tail, and an open reading frame （ORF） of 1971 bp. The HSP70 protein was 656 amino acids with a calculated molecular weight of 71.43 kD and theoretical isoelectric point of 5.15. Sequence analysis of the protein showed that HSP70 of P. aibuhitensis contained three signature sequences IDLGTTYS, IFDLGGGTFDVSIL and IVLVGGSTRIPKIQK all belonging to the HSP70 family, a cytoplasm characteristic motif of EEVD, and the C-terminal repeats of GGMP. The molecular characteristics of P. aibuhitertsis shared 94% identity with other rag-worms and 79% identity with other invertebrates. In ragworms exposed to CH2＋, expression of HSP70 mRNA increased significantly with Cu2＋ concentration （0.2-5.0 mg0 L-1）, and it reached the peak on 1-day exposure. In summary, the molecular characteristics of HSPT0 of P. aibuhitensis were described, and its gene expression was inducible by exposure to aqueous Cu2＋, suggesting HSP70 has potential to become a useful molecular biomarker of environmental pollution.