Tom M, Douek J, Yankelevich I, Bosch T C, Rinkevich B
National Institute of Oceanography, Israel Oceanographic and Limnological Research, Haifa, 31080, Israel.
Biochem Biophys Res Commun. 1999 Aug 19;262(1):103-8. doi: 10.1006/bbrc.1999.1165.
The branching coral Stylophora pistillata, one of the most abundant hermatypic corals along the coasts of the Red Sea, has been used for many years as a model species for coral biological studies. Here we characterize the first coral heat shock protein 70 gene (SP-HSP70), cloned from S. pistillata, to be used as a tool for studying coral stress response. The cloning was carried out by a combination of PCR methods using heterologous, degenerate HSP70-based primers, followed by plaque-lift screening of a genomic library. The sequenced clone (5212 bp), contains a complete 1953 bp, intronless open reading frame, and 5' and 3' flanking regions of 1,935 and 1,324 bp, respectively. TATA, CAAT, and ATF boxes as well as 11 putative heat shock elements were identified in the SP-HSP70 5' flanking region. A polyadenylation site was identified in the 3' flanking region. SP-HSP70 protein sequence resembles the cytosolic/nuclear HSP70 cluster. RT-PCR studies confirmed SP-HSP70 mRNA expression in corals grown within their normal physiological conditions. Furthermore, SP-HSP70 has been shown to belong to the coral genome and not to its symbiotic algae one, as revealed by SP-HSP70 PCR amplification, using purified algal and coral DNA templates.
分支珊瑚细指鹿角珊瑚是红海沿岸最为常见的造礁珊瑚之一，多年来一直被用作珊瑚生物学研究的模式物种。在此，我们对首个从细指鹿角珊瑚中克隆出的珊瑚热休克蛋白70基因（SP-HSP70）进行了表征，将其作为研究珊瑚应激反应的工具。克隆过程采用了多种PCR方法，使用基于异源简并HSP70的引物，随后对基因组文库进行菌斑原位杂交筛选。测序后的克隆片段（5212 bp）包含一个完整的1953 bp无内含子开放阅读框，以及分别为1935 bp和1324 bp的5'和3'侧翼区域。在SP-HSP70的5'侧翼区域鉴定出了TATA、CAAT和ATF框以及11个假定的热休克元件。在3'侧翼区域鉴定出了一个聚腺苷酸化位点。SP-HSP70蛋白序列类似于胞质/核HSP70簇。RT-PCR研究证实了在正常生理条件下生长的珊瑚中SP-HSP70 mRNA的表达。此外，使用纯化的藻类和珊瑚DNA模板进行的SP-HSP70 PCR扩增表明，SP-HSP70属于珊瑚基因组，而非其共生藻类基因组。
