Ilikchyan Irina N, McKay R Michael L, Zehr Jonathan P, Dyhrman Sonya T, Bullerjahn George S
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Environ Microbiol. 2009 May;11(5):1314-24. doi: 10.1111/j.1462-2920.2009.01869.x. Epub 2009 Feb 10.
We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.
我们描述了一种基于聚合酶链式反应（PCR）的检测方法，旨在检测来自蓝细菌的膦酸盐同化基因phnD的表达。phnD基因编码ABC型膦酸盐转运蛋白的膦酸盐结合蛋白，存在于许多蓝细菌基因组序列中。检测phnD的表达可以表明浮游植物利用膦酸盐的能力，膦酸盐是一种难降解的磷形式，在海洋系统中可占高分子量溶解有机磷库的25%。设计引物组以特异性扩增来自海洋和淡水聚球藻属、原绿球藻属以及来自海洋和劳伦大湖的环境样本中的phnD序列。对培养的海洋聚球藻菌株WH8102和淡水聚球藻ARC-21进行定量逆转录PCR（RT-PCR），结果表明在缺磷培养基中phnD表达被诱导，这表明phn基因与受phoRB控制的基因协同调控。最后, 对马尾藻海和太平洋环境RNA样本进行RT-PCR，检测到了当地蓝细菌种群中phnD的表达。聚球藻属phnD的表达随着磷生物可利用性梯度呈现出深度依赖性模式。相比之下，原绿球藻属的引物显示，在所有测试样本中，phnD的表达是组成型的。本文所述的方法将有助于未来旨在了解海洋中天然存在的膦酸盐利用情况以及监测淡水蓝细菌对合成膦酸盐除草剂（如草甘膦）摄取情况的研究。
