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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/44192

Title: 以單一細胞聚合酶連鎖反應探討纖毛蟲種類組成調查之可行性
Feasibility study on ciliate community by single-cell PCR
Authors: Liu, Wen-Hsin
劉溫馨
Contributors: 國立臺灣海洋大學:海洋環境與生態研究所
Keywords: 種類組成;舞毛目纖毛蟲;寡毛目纖毛蟲;小次單元去氧核醣核酸(18S rDNA)
community structure;Choreotrichida;Oligotrichida;18S rDNA
Date: 2016
Issue Date: 2017-11-15T08:45:44Z
Abstract: 寡毛綱纖毛蟲是一群具有高歧異度的原生生物,且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,寡毛綱纖毛蟲的群聚結構是非常重要的一個基礎調查工作。生態研究一般以Lugol’s iodine solution固定海水樣本後,於倒立式顯微鏡下將寡毛綱纖毛蟲區分為Strombidium、Strobilidium、Mesodinium、Tontonia和Tintinnid五大類,分別進行計數,以探討寡毛綱纖毛蟲種類組成及數量變化。本研究結合傳統生態研究法,即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,在同一玻片中挑取單一寡毛綱纖毛蟲細胞並置 於一微小離心管裡,最後取得之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。 本研究於台灣東北沿岸採集寡毛綱纖毛蟲樣本,並進行拍照及紀錄大小,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。研究期間共獲得330個單一細胞樣本,其中僅102個樣本於PCR過程中成功取得目標序列產物,但最後只有32條可對應到寡毛綱纖毛蟲序列,成功率僅約10%。由於本研究所用引子(primer) 與NCBI上已知相關寡毛綱纖毛蟲18S rDNA序列比較,發現125條相關纖毛蟲序列中,僅有6條序列同時包含本研究所使用引子的F端及R端,推測此引子對寡毛綱纖毛蟲專一性不佳。 在種類鑑定比較上,本研究發現這兩個方法所得結果有不同程度的差異。五大類中,除了Mesodinium屬與Tintinnid的形態辨種結果和18S rDNA序列結果無差異外,其餘三大類皆有明顯的差異存在。其中以形態辨識為Strombidium屬之樣本差異最大,序列結果顯示其中僅約54%為Strombidium屬,亦即將近46%的形態辨識種發生錯誤,當中包括約31%為Tontonia誤認為Strombidium屬、約15%之誤認為Tintinnid,推測為尾部受固定刺激脫落或角度問題,使得無法觀察到Tontonia蟲體特徵(尾部)。以形態分類為Strobilidium屬樣本,均未發現任何Strobilidium屬的序列,其序列顯示為Mesodinium及Tintinnid,因此誤判率非常高, Mesodinium屬之纖毛環繞蟲體中間,俯面觀察與Strobilidium屬之俯面極為相似;而Tintinnid則與Strobilidium屬同樣具有封閉之口部,因此當無殼時易造成誤判。
Owing to the highly diverse biodiversity and variable trophic strategies in oligotrich ciliates, it is important and imperative to survey oligotrich community structures before investigating the carbon cycles and/or environmental changes. The seawater samples were preserved with non-acid Lugol’s iodine solution. The oligotrichs were divided into five groups, i.e. Strombidium, Strobilidium, Mesodinium, Tontonia, and tintinnids, and enumerated under the inverted microscope according to the morphological characteristics. A new method was conducted in the present study to combine 1) the enumeration and identification of the oligotrichs by inverted microscopy and 2) the single-cell polymerase chain reaction (single-cell PCR) for 18S rDNA sequences of each cell. The feasibility of the new method and the diverse results in species identification based on morphological data and 18S rDNA sequences were discussed. Oligotrich ciliate samples were collected from the coastal waters of northeastern Taiwan. For each oligotrich cell, before transferring each cell to an Eppendorf for PCR to obtain 18S rDNA sequences, morphological characters, cell dimensions and photos were recorded. 330 single cell samples were collected during the study period and 102 DNA extracts were obtained, however only 32 sequences were identified as oligotrich ciliates, a success rate of 10%. Comparing the primers we used with those shown in NCBI, only 6 sequences matched the forward and reverse primers in 125 oligotrich sequences, suggesting the poor specificity of the primers for oligotrich ciliates. In the comparison of species (groups) identified with morphological classification and with 18S rDNA sequences, we found the presence of differences. Only Mesodinium spp. and tintitnids had identical results by morphological and molecular identifications, all other groups showed significant identification differences by these two methods. The discrepancy was largest in morphological and molecular data for Strombidium spp.. Among the morphologically identified Strombidium spp., as indicated by molecular identification, only 54% of them belonged to Strombidium, the remaining 46% were Tontonia spp. (31%) and tintinnids (15%). The former may be misidentified by the loss of the tail (the specific character of Tontonia spp.) while the latter by the disappearance of the lorica (the specific character of tintinnids), caused by the fixation and the disturbance of sample collection. Two morphologically-identified Strobilidium spp. were found, to be Mesodinium spp. and tintinnid, respectively, according to molecular identification, indicating high misidentification rate in morphological identification. We may have assumed the cilia distributed on the middle of cell in Mesodinium spp. as the adoral membranelles in Strobilidium spp. on the top view. It is also easy for tintinnids to be misidentified as Strobilidium spp. with the same closed-shape adoral zone of membranelles, when we missed their lorica.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010483002.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/44192
Appears in Collections:[海洋環境與生態研究所] 博碩士論文

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