Rodríguez-Martínez Raquel, Labrenz Matthias, del Campo Javier, Forn Irene, Jürgens Klaus, Massana Ramon
Institut de Ciències del Mar, CMIMA (CSIC), Passeig Marítim de la Barceloneta, Barcelona, Spain.
Environ Microbiol. 2009 Feb;11(2):397-408. doi: 10.1111/j.1462-2920.2008.01779.x.
Molecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2-3 microm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal-offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml(-1)) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importance.
对海洋微微型真核生物的分子调查揭示了大量与培养生物无关的序列，比如那些构成海洋不等鞭毛藻（MAST）-4进化枝的序列。最近的荧光原位杂交（FISH）数据表明，MAST-4细胞是大小为2-3微米的未培养异养鞭毛虫，在非极地海水中全球分布。然而，FISH耗时且难以应用于海洋科考期间产生的众多样本，因此我们开发了一种实时定量聚合酶链反应（Q-PCR）方案，以利用环境DNA快速测定该类群的丰度。我们设计了一套靶向MAST-4的18S核糖体RNA基因（rDNA）的引物，并使用以目标序列为插入片段的质粒对Q-PCR方案进行了优化和校准。然后将Q-PCR应用于沿三条海洋断面定量MAST-4 rDNA分子，这三条断面分别是印度洋的纵向断面、德雷克海峡的纬度断面以及地中海的海岸-近海断面，还应用于地中海一个海岸站点的时间序列研究。在所有处理的样本中均检测到了MAST-4（平均丰度在500至1000个rDNA分子每毫升之间），除了在中层和南极样本中几乎未检测到。总体而言，它在海岸比近海更丰富，在深层叶绿素最大值处比表层更丰富。在严格控制的微生物培养中对Q-PCR和FISH信号的比较表明，MAST-4细胞具有约30个rDNA操纵子拷贝。这种Q-PCR检测方法快速产生了未培养的MAST-4细胞的定量数据，并证实了它们的广泛分布和假定的生态重要性。
