克氏原螯虾一种诱导型HSP70基因克隆及分析

克氏原螯虾是我国淡水虾类养殖的重要品种，具有很强的抵御各种环境胁迫和各种刺激的能力。本文以该虾为对象，通过基因克隆以及从基因水平探讨HSP70s与环境应激之间的关系，为深入研究水生无脊椎动物HSP70s功能提供基础。采用RT-PCR和RACE方法从克氏原螯虾（Procambarus clarkii）心脏组织中克隆得到一种HSP70 cDNA（scHSP70），其全长为2271bp，包括1902bp的完整编码序列、142bp的5′及221bp的3′端非翻译区, GenBank登陆号DQ301506。基因组DNA扩增表明该基因仅由一个外显子组成。根据cDNA序列推导出scHSP70由635个氨基酸组成，分子量为69.6kD，理论等电点为5.34。该序列存在真核细胞HSP70家族的三个特征标签。SWISS-MODEL蛋白三维结构预测显示scHSP70在N端形成ATP酶结构域，在近C端形成底物肽结合结构域。克氏原螯虾在系统发生树上的进化地位与传统分类学相一致。半定量RT-PCR实验表明，scHSP70有广泛的组织分布，在心脏中表达量最高，在血液中最少。热激后该基因大量表达，说明该基因是一种诱导型HSP70。这为从蛋白水平研究克氏原螯虾HSP70与环境应激之间的关系提供基础。

Cloning and expression analysis of an inducible heat shock protein 70 gene from red swamp crayfish, Prccambarus clarkii

Red swamp crayfish, Procambarus clarkia, with advantages of easy cultivation and availability, can live in various tough environments, so intrigued researchers pay attention to its ecology, toxicology, physiology and immunology. We studied an inducible cDNA encoding HSP70 in P. clarkia which would contribute to extensive researches of HSP70s and environmental stresses. An inducible heat shock protein 70 (HSP70) cDNA was cloned from red swamp crayfish by RTPCR and RACE, which named scHSP70. The full-length cDNA of the scHSP70 was 2271 bp, consisting of a partial 5'-terminal untranslated region (UTR) of 142bp, a 3'-terminal UTR of221bp, an open reading frame of 1902bp (ORF) and a poly (A) tail and GenBank No. DQ301506. The gene contained only one exon according to amplification of scHSPT0 from genomic DNA. The scHSP70 cDNA encoded a polypeptide of 635 amino acids. Based on phylogenetic analysis, the gene was clustered with inducible HSP70 family members from other species. The evolution relationship was consisted with traditional classification. Semi-quantitative PCR was employed to assess the temporal expression of scHSP70 mRNA levels from heat-shock treated and unstressed crayfish. Challenge of the red swamp crayfish with 2h heat shock resulted in dramatic increases in the expression of HSP70 mRNA levels in all tissues, heart, muscle, hemocytes, digestive gland, antenhal gland, testis and intestine, among which, the highest expression was found in heart. However, under normal conditions, the expressions of HSP70 mRNA level were much lower in all tissues compared to treated ones, especially in haemocytes. The upregulated mRNA expression of the HSP70 in the crayfish following heat shock indicates that scHSP70 gene is inducible. These stress proteins provide invaluable information in stress response in the crayfish.