SGER: Development of an Immunoassay for Nitrogenase for Estimating Nitrogen Fixation Rates in Trichodesmium

毛霉菌是一种广泛分布于热带和亚热带海洋的丝状非异囊蓝藻。木霉在这些环境的初级生产力中是重要的，但也是在开放海洋中发现的主要固氮生物。最近对中央海洋环流氮收支的研究已经指出了木霉作为氮源的重要性。木霉只在白天固定氮，而且是浮力的，在整个欣快区分布不均匀。因此，估计木霉自然种群的深度综合日固氮率需要在一天中的不同时间和不同深度进行多次固氮率测量。虽然测量固氮率相对简单，但它们确实需要船上的孵育和专门的设备，并且需要在海上进行。该研究利用了最近发现的固氮速率与固氮酶（催化固氮的酶）成分丰度之间的关系，为估计海中菌毛菌的固氮作用提供了一种新的方法。这项探索性研究的小额拨款的目标是开发一种免疫测定方法来估计木霉的固氮速率。这一建议是基于初步结果，结果显示在木霉的培养中，氮酶的活性形式铁蛋白的丰度与固氮率之间存在很强的相关性。本研究是探索性的，因为铁蛋白在木霉中的修饰机制尚不清楚。然而，如果能够制造出一种抗血清来区分铁蛋白的活性形式和非活性形式，则可以开发出一种免疫测定法来估计固氮活性，而无需样品容器和孵育。这种免疫分析方法将有可能在大的空间和时间尺度上获得木霉中N2固定的估计，并且是首次使用免疫分析方法来估计海洋生物地球化学循环速率的方法之一。该项目将提供海洋科学方面的博士后培训，并为开发免疫学方法在海洋科学中的新应用提供条件。

Trichodesmium is a filamentous nonheterocystous cyanobacterium which is widely distributed throughout tropical and subtropical seas. Trichodesmium is important in the primary productivity of these environments, but also is the major nitrogen fixing organism found in the open ocean. Recent studies of the N budgets of the central oceanic gyres has pointed towards the importance of Trichodesmium as a source of nitrogen. Trichodesmium fixes nitrogen only during the day, and also is buoyant and not evenly distributed throughout the euphoric zone. Thus, estimates of depth integrated daily nitrogen fixation rates for natural populations of Trichodesmium require multiple nitrogen fixation rate measurements at different times of day and at different depths. Although nitrogen fixation rate measurements are relatively simple, they do require shipboard incubations and specialized equipment, and need to be perfomed at sea. The proposed research exploits a recently discovered relationship between nitrogen fixation rates and abundance of a component of nitrogenase, the enzyme that catalyzes nitrogen fixation, to provide a new approach for estimating nitrogen fixation by Trichodesmium in the sea. The goal of this small grant for exploratory research is to develop an immunoassay approach for estimating the rate of nitrogen fixation by Trichodesmium. The proposal is based on preliminary results showing a strong correlation between abundance of the active form of the Fe protein of nitrogenase and nitrogen fixation rates in a culture of Trichodesmium. The proposed research is exploratory in that the mechanism of modification of the Fe protein in Trichodesmium is not yet known. However, if an antiserum can be made that distinguishes between the active and inactive forms of the Fe protein, an immunoassay can be developed for estimating nitrogen fixation activities, without the need for sample containment and incubation. This immunoassay approach will make it possible to obtain estimates of N2 fixation in Trichodesmium over large spatial and temporal scales, and is one of the first uses of an immunoassay approach for estimating rates of biogeochemical cycles in the sea. This project will provide post doctoral training in ocean sciences, as well as provide for the development of a new application of immunological approaches in the marine sciences.