Developing a DNA Microarray for the Detection of Phytoplankton Physiological States in Natural Environment

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Project title

Code/計畫編號

NSC92-2313-B019-050

Translated Name/計畫中文名

發展核酸微矩陣來偵測浮游藻類在自然界的生理狀態

Project Coordinator/計畫主持人

Jeng Chang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Institute of Marine Biology

Website

https://www.grb.gov.tw/search/planDetail?id=849759

Year

2003

Start date/計畫起

01-08-2003

Expected Completion/計畫迄

01-07-2004

Bugetid/研究經費

700千元

ResearchField/研究領域

生物技術(農)
自然生態保育

本申請案的目標為設計一個核酸微矩陣並以此作為一個簡單的方法來同時偵測自然水體中浮游藻類的種類組成、生長的快慢、以及受環境因子限制的程度。我們計劃針對五個主要的海洋浮游藻類的分類群分別偵測五種生理指標基因的表現，總數為 25 條。初步選定的藻株分別屬於藍綠藻、矽藻、渦鞭藻、綠鞭藻 (prasinophytes)、和三毛黃鞭藻 (haptophytes)。而在生理指標基因方面，則以 rubisco 的 rbcL 做為光合作用的指標、以 PCNA 基因做為細胞複製的指標、以 Nrt, Amt, 與 GSII 做為氮限制的指標、以 PHO 做為磷限制的的指標、而以 flavodoxin 基因做為鐵限制的指標。在計畫執行期間已使用聚合脢鎖鏈反應 (PCR) 選殖得到 20 條相關的基因片段，並由相似度比對確認他們的身份。另外又使用少數片段在培養藻種中進行雜合反應上的靈敏度與專一性測試，結果顯示雜合反應可以順利偵測到單個基因的表現量。本計畫至此已達成當初預設的目標，並有後續工作在國科會支助下繼續進行中。 The goal of this research project is to design a DNA microarray, and use it as a simple means to simultaneously detect phytoplankton species composition, growth rate, and the degree of light and nutrient limitation in marine environments. A DNA microarray of 25 genes will be fabricated to contain 5 physiological indicator genes from each of the 5 major taxonomical groups in marine phytoplankton, including cyanobacteria, diatoms, dinoflagellates, prasiophytes, and haptophytes. In each group, the rbcL gene of rubisco will be used as the indicator for photosynthetic rate. In addition, PCNA gene, Nrt gene, Amt gene, GSII gene, TcPHO gene, and flavodoxin gene will be used respectively as the indicators for cell proliferation, nitrogen limitation, phosphorus limitation, and iron limitation. During the execution period of this project, we have cloned 20 gene fragments using the technique of polymerase chain reaction. The identity of each gene was confirmed by similarity analysis based on known sequences in the GenBank. In addition, a limited amount of gene fragments were used in a dot-blot hybridization test to evaluate the sensitivity and specificity of this technique. Our results indicated that the expression levels of individual genes were readily detectable by this approach. We feel that the goals set for the first year have been reached, and continuing works are currently in progress under the support from National Science Foundation.

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Developing a DNA Microarray for the Detection of Phytoplankton Physiological States in Natural Environment

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