Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd, Qingdao 266071, China.
Fish Shellfish Immunol. 2011 Jan;30(1):154-62. doi: 10.1016/j.fsi.2010.09.020. Epub 2010 Oct 8.
Dopamine beta hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines, and also plays an important role in complex neuroendocrine-immune regulatory network. In the present study, the cDNA encoding dopamine beta hydroxylase (designated CfDBH) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The full-length cDNA of CfDBH was of 2302 bp, containing a 5' untranslated region (UTR) of 32 bp, a 3' UTR of 461 bp with a poly (A) tail, and an open reading frame (ORF) of 1809 bp encoding a polypeptide of 603 amino acids. The deduced amino acid sequence of CfDBH contained a signal peptide, a DOMON domain and a Cu2_monooxygen domain, and it shared 39.4%-42.9% similarity with other reported DBHs. The conserved domains in CfDBH and the amino acid sequence similarity with other DBHs strongly suggested that it was a homologue of DBH in C. farreri. The mRNA expression of CfDBH in various tissues and its temporal expression in haemocytes of scallops stimulated with LPS were ascertained by Quantitative real-time RT-PCR. The mRNA transcripts of CfDBH were detected in all the examined tissues with the highest expression level in hepatopancreas. The expression level of CfDBH in haemocytes was up-regulated after LPS stimulation and increased to hundreds fold higher than that of the control group at 12 h, and then decrease significantly to 0.36-fold and 0.31-fold at 24 h and 48 h respectively. The results suggested pathogen infections significantly induced the expression level of CfDBH, and the activation of DBH could influence the immune response of scallop C. farreri through changing the concentration of catecholamines.
多巴胺β羟化酶（DBH）是儿茶酚胺生物合成中的关键酶，在复杂的神经内分泌-免疫调节网络中也起着重要作用。本研究采用快速扩增 cDNA 末端（RACE）和表达序列标签（EST）分析方法，从栉孔扇贝中克隆得到多巴胺β羟化酶（命名为 CfDBH）的 cDNA。CfDBH 的全长 cDNA 为 2302bp，包含 32bp 的 5'非翻译区（UTR）、461bp 的 3'UTR 带有 poly（A）尾，以及编码 603 个氨基酸的开放阅读框（ORF）。CfDBH 的推导氨基酸序列包含一个信号肽、一个 DOMON 结构域和一个 Cu2_单加氧酶结构域，与其他报道的 DBHs 具有 39.4%-42.9%的相似性。CfDBH 中的保守结构域和与其他 DBHs 的氨基酸序列相似性强烈表明它是栉孔扇贝 DBH 的同源物。通过定量实时 RT-PCR 确定 CfDBH 在各种组织中的 mRNA 表达及其在 LPS 刺激的扇贝血细胞中的时间表达。在所检测的组织中均检测到 CfDBH 的 mRNA 转录本，在肝胰腺中的表达水平最高。在 LPS 刺激后，血细胞中 CfDBH 的表达水平上调，并在 12h 时比对照组增加数百倍，然后在 24h 和 48h 时分别显著下降至 0.36 倍和 0.31 倍。结果表明，病原体感染显著诱导 CfDBH 的表达水平，DBH 的激活可以通过改变儿茶酚胺的浓度来影响栉孔扇贝 C. farreri 的免疫反应。
