机构地区: 中国水产科学研究院南海水产研究所
出 处: 《中国水产科学》 2007年第5期726-732,共7页
摘 要: 采用同源克隆和RT-PCR技术对合浦珠母贝(Pinctada fucata)热休克蛋白hsp70基因进行了克隆和表达分析。获得cDNA全长序列2 365 bp,其中3’非编码区域(UTR)为318 bp,5’UTR为88 bp,开放阅读框(ORF)为1 959 bp,编码652个氨基酸,分子量约为71.39 kD,理论等电点为5.22,并含有3个HSP70家族的签名序列IDLGTTYS、DLGGGTFD和EEVD。同源性分析表明,合浦珠母贝HSP70的氨基酸序列与太平洋牡蛎(Crassostrea gigas)等双壳贝类的相似性高达86%以上,基于氨基酸序列的聚类分析表明,合浦珠母贝与牡蛎属种类亲缘关系最近。高温、高盐刺激后,半定量RT-PCR检测发现hsp70基因的表达明显增加,高温刺激的表达量高于高盐刺激,高温刺激组不同组织的表达量由大到小依次为鳃、消化腺、外套膜、肌肉、性腺,高盐刺激组不同组织的表达量由大到小依次为鳃、外套膜、肌肉、消化腺、性腺,表明HSP70参与了机体对刺激的应答过程。该基因的克隆为进一步深入研究合浦珠母贝的抗逆机理及其遗传改良奠定了重要基础。 Heat shock protein 70 gene was cloned from pearl oyster Pinctada fucata by homology clone and RT-PCR. The full length cDNA of hsp70 obtained is 2 365 bp,containing a 3' UTR of 318 bp,a 5' UTR of 88 bp, and an ORF of 1 959 bp, encoding 652 amino acid residues with estimated molecular weight of 71.39 kD and theoretical isoelectric point of 5.22. Three signature sequences of HSP70 family: IDLGTTYS,DLGCTFD and EEVD were detected in the predicted amino acid sequenc. High identity of HSP70 in amino acid sequence (more than 86% ) was observed between P. fucata and the other bivalve organisms including Crassostrea gigas through blast analysis, indicating that the cDNA cloned in this study belonged to the family of heat shock protein 70. Phylogenetic tree showed that P. fucata was closely related to Crassostrea oysters based on amino acid sequences of HSP70. After pearl oysters were stimulated by heat shock or highly salty seawater, transient expressions of hsp70 gene were measured by semi-quantitative RT-PCR. The expression was up-regulated by the stimulations and higher expression occurred under heat shock than under high salinity. The order of tissue expression amounts from large to small was gill, digest gland, mantle ,muscle,sex gland under heat shock treatment, while gill, mantle, muscle, digest gland, sex gland under high salinity treatment. This indicated that HSP70 in P. fucata responded actively to environmental stimulations. Thus the cloning of hsp70 gene in the present study provided a basis for further investigation on anti-stress mechanism and genetic improvement of stress-tolerance in P. fucata. [Journal of Fishery Sciences of China, 2007,14 (5) : 726 - 732 ]
领 域: [生物学]