The Key Laboratory of Mariculture, Education Ministry of China, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
Fish Shellfish Immunol. 2011 Jan;30(1):280-6. doi: 10.1016/j.fsi.2010.10.019. Epub 2010 Oct 29.
In the present study, the cDNA of heat shock protein 90 from Pacific abalone Haliotis discus hannai Ino (HdhHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of HdhHSP90 was of 2660 bp, including an open reading frame (ORF) of 2187 bp encoding a polypeptide of 728 amino acids with predicted molecular weight of 84.134 kDa and theoretical isoelectric point of 4.619. BLAST analysis revealed that HdhHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HdhHSP90, which indicated that HdhHSP90 should be a cytosolic member of the HSP90 family. The expression levels of HdhHSP90 in haemocytes and hepatopancreas were measured by real-time PCR after Pacific abalone were fed with semi-purified diets containing graded levels of selenium (0.15, 1.32 and 48.70 mg Kg(-1)) for 20 weeks, respectively. The results showed that the expression levels of HdhHSP90 transcript were significantly up-regulated and reached the maximum (0.47-fold) in hepatopancreas of Pacific abalone fed with optimal dietary Se (1.32 mg Kg(-1)) (p < 0.05). However, these levels significantly decreased in hepatopancreas at the excessive dietary Se (48.70 mg Kg(-1)) (p < 0.01). In haemocytes, the expression of HdhHSP90 mRNA increased and reached the maximum (0.96-fold) at the excessive dietary Se (48.70 mg Kg(-1)) (p < 0.01). It is implied that the expression levels of HdhHSP90 could be affected by dietary Se in hepatopancreas and haemocytes, and HdhHSP90 was potentially involved in the anti-oxidation responses in Pacific abalone H. discus hannai.
在本研究中，通过同源克隆和快速扩增 cDNA 末端（RACE）方法克隆了太平洋鲍 Haliotis discus hannai Ino（HdhHSP90）的热休克蛋白 90 cDNA。HdhHSP90 的全长 cDNA 为 2660 bp，包含一个 2187 bp 的开放阅读框（ORF），编码一个 728 个氨基酸的多肽，预测分子量为 84.134 kDa，理论等电点为 4.619。BLAST 分析表明，HdhHSP90 与其他已知的 HSP90 具有高度相似性，并且在 HdhHSP90 中也鉴定出了 HSP90 蛋白家族特征定义的五个保守氨基酸块，这表明 HdhHSP90 应该是 HSP90 家族的细胞质成员。用含有不同水平硒（0.15、1.32 和 48.70 mg Kg(-1)) 的半纯化饲料分别饲养太平洋鲍 20 周后，通过实时 PCR 测量了血细胞和肝胰腺中 HdhHSP90 的表达水平。结果表明，在最优膳食 Se（1.32 mg Kg(-1)) （p < 0.05）条件下，肝胰腺中 HdhHSP90 转录本的表达水平显著上调并达到最大值（0.47 倍）。然而，在过量膳食 Se（48.70 mg Kg(-1)) 时，这些水平在肝胰腺中显著降低（p < 0.01）。在血细胞中，HdhHSP90 mRNA 的表达增加，并在过量膳食 Se（48.70 mg Kg(-1)) 时达到最大值（0.96 倍）（p < 0.01）。这表明，膳食 Se 可以影响肝胰腺和血细胞中 HdhHSP90 的表达水平，HdhHSP90 可能参与了太平洋鲍 H. discus hannai 的抗氧化反应。
