Lin Xin, Zhang Huan, Huang Bangqin, Lin Senjie
State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China Department of Marine Sciences, University of Connecticut, Groton 06340, Connecticut, USADepartment of Marine Sciences, University of Connecticut, Groton 06340, Connecticut, USAState Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China Department of Marine Sciences, University of Connecticut, Groton 06340, Connecticut, USA State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China.
J Phycol. 2011 Oct;47(5):1110-20. doi: 10.1111/j.1529-8817.2011.01038.x. Epub 2011 Aug 22.
Alkaline phosphatase (AP) in phytoplankton facilitates the utilization of dissolved organic phosphorus (DOP) when the dissolved inorganic phosphorus (DIP) is limited in the environment. The AP gene sequence and its expression under DIP limitation has not been studied in dinoflagellates. In this study, we isolated the full-length cDNA of AP from the toxic dinoflagellate Amphidinium carterae Hulburt (2,112 bp, named as acaap). The deduced amino acid sequence of acaap (ACAAP, 704 amino acid residues) was identified as a membrane-associated protein, in agreement with the dominantly cell surface localization of the AP activity shown with enzyme-labeled fluorescence (ELF) labeling. ACAAP shares sequence similarity in the key domains with APs from diatoms, proteobacteria, and cyanobacteria. In accordance, phylogenetic reconstruction showed clustering of ACAAP with counterparts in those organisms, although branches were long as a result of the generally high variability of the gene sequence. The expression levels of acaap were studied for A. carterae cultured in media with different phosphate concentrations using quantitative reverse-transcription PCR (RT-qPCR) method. The result showed that the transcription level of acaap was elevated in the DIP-depleted cultures relative to the DIP-replete cultures and repressed upon resupply of DIP. The transcription level of acaap exhibited a positive correlation with AP enzyme activity. Taken together, these results demonstrate that AP activity and gene expression are regulated by the availability of DIP in A. carterae, suggesting that AP expression is a promising indicator of DIP stress in this and possibly other species of dinoflagellates.
当环境中溶解无机磷（DIP）受限的时候，浮游植物中的碱性磷酸酶（AP）有助于溶解有机磷（DOP）的利用。在甲藻中，尚未对DIP受限情况下的AP基因序列及其表达进行研究。在本研究中，我们从有毒甲藻卡特亚力山大藻（Amphidinium carterae Hulburt）中分离出了AP的全长cDNA（2,112 bp，命名为acaap）。acaap推导的氨基酸序列（ACAAP，704个氨基酸残基）被鉴定为一种膜相关蛋白，这与酶标记荧光（ELF）标记显示的AP活性主要定位于细胞表面一致。ACAAP在关键结构域与硅藻、变形菌和蓝细菌的AP具有序列相似性。相应地，系统发育重建显示ACAAP与这些生物中的对应物聚类，尽管由于基因序列普遍高度可变，分支较长。使用定量逆转录PCR（RT-qPCR）方法研究了在不同磷酸盐浓度培养基中培养的卡特亚力山大藻中acaap的表达水平。结果表明，相对于DIP充足的培养物，acaap的转录水平在DIP耗尽的培养物中升高，而在重新供应DIP后受到抑制。acaap的转录水平与AP酶活性呈正相关。综上所述，这些结果表明，卡特亚力山大藻中AP活性和基因表达受DIP可用性的调节，这表明AP表达可能是该甲藻及其他可能的甲藻物种中DIP胁迫的一个有前景的指标。
