CAJ | 학술논문

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.
극씨원오하시아국담수하류양식적중요품충,구유흔강적저어각충배경협박화각충자격적능력.본문이해하위대상,통과기인극륭이급종기인수평탐토HSP70s여배경응격지간적관계,위심입연구수생무척추동물HSP70s공능제공기출.채용RT-PCR화RACE방법종극씨원오하(Procambarus clarkii)심장조직중극륭득도일충HSP70 cDNA(scHSP70),기전장위2271bp,포괄1902bp적완정편마서렬、142bp적5'급221bp적3'단비번역구,GenBank등륙호DQ301506.기인조DNA확증표명해기인부유일개외현자조성.근거cDNA서렬추도출scHSP70유635개안기산조성,분자량위69.6kD,이론등전점위5.34.해서렬존재진핵세포HSP70가족적삼개특정표첨.SWISS-MODEL단백삼유결구예측현시scHSP70재N단형성ATP매결구역.재근C단형성저물태결합결구역.극씨원오하재계통발생수상적진화지위여전통분류학상일치.반정량RT-PCR실험표명,scHSP70유엄범적조직분포,재심장중표체량최고,재혈액중최소.열격후해기인대량표체,설명해기인시일충유도형HSP70.저위종단백수평연구극씨원오하HSP70여배경응격지간적관계제공기출.
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.