Study on Oligotrich Ciliate Communities by Single-Cell Pcr

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Project title

Code/計畫編號

MOST106-2611-M019-023

Translated Name/計畫中文名

以單一細胞聚合酶連鎖反應探討寡毛類纖毛蟲種類組成調查

Project Coordinator/計畫主持人

Sheng-Fang Tsai

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Center of Excellence for the Oceans

Website

https://www.grb.gov.tw/search/planDetail?id=12232296

Year

2017

Start date/計畫起

01-08-2017

Expected Completion/計畫迄

31-07-2018

Bugetid/研究經費

1262千元

ResearchField/研究領域

海洋科學

寡毛亞綱纖毛蟲是一群歧異度極高的原生生物，同時其營養模式非常多樣，因此在研究海洋生態碳傳遞路徑或環境變遷，寡毛亞綱纖毛蟲的群聚結構是非常重要的一個基礎調查工作。以往生態研究一般僅以Lugol’s iodine solution 固定海水樣本後，於倒立式顯微鏡下將寡毛亞綱纖毛蟲區分為 Strombidium、Strobilidium、Mesodinium、Tontonia 和Tintinnid，並計數個別的數量，以探討研究海域寡毛亞綱纖毛蟲種類組成及數量變化。然而此方法僅靠蟲體固定後的粗略外形作為類群鑑定的依據，勢必存在數據可靠性的問題。本研究設計一新的生態研究法，即不僅於倒立式顯微鏡下觀察計數寡毛綱纖毛蟲，同時搭配單一細胞PCR 法，在同一玻片中挑取所有單一寡毛綱纖毛蟲細胞，並置於一微小離心管裡，最後將取得之18S rDNA 序列作為種類判定的結果。本研究主要想探討此一新實驗方法的可行性，同時藉此方法亦可瞭解傳統生態研究下五大類分類之準確性。寡毛亞綱纖毛蟲樣本預計於台灣東北沿岸進行採集，並拍照及紀錄大小，作為外部形態鑑種依據。隨後將蟲體進行single-cell PCR，所得18S rDNA 序列作為分子序列辨種依據。在種類鑑定比較上，預期此兩個方法所得結果有不同程度的差異。"Because of the highly diverse biodiversity and variable trophic strategies in oligotrich ciliates, it is imperative to survey oligotrich community structures before investigating the carbon cycles and/or environmental changes. Normally, seawater samples were preserved with Lugol’s iodine solution for identifying oligotrichs in five groups, i.e. Strombidium, Strobilidium, Mesodinium, Tontonia, and tintinnids, and enumerated under the inverted microscope. However, merely depending on the rough morphological characteristics for identifying five oligotrich groups should exist problems of reliability. A new method was conducted to combine 1) the enumeration and identification of the oligotrichs by inverted microscopy and 2) the single-cell polymerase chain reaction for 18S rDNA sequences of each cell. The feasibility of the new method and the diverse results in species identification based on morphological and molecular data are going to be discussed. Oligotrich ciliates were collected from the coastal waters of northeastern Taiwan. For oligotrich cells, before transferring each cell to an Eppendorf for PCR to obtain 18S rDNA sequences, their morphological characters, cell dimensions and photos were recorded. In the comparison of species (groups) identified with morphological classification and with 18S rDNA sequences, we anticipate the existence of discrepancy between morphological and molecular data."

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Study on Oligotrich Ciliate Communities by Single-Cell Pcr

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