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研究生:王玄芳
研究生(外文):Hsuan-Fan Wang
論文名稱:利用hetR基因序列探討龜山島海域束毛藻(Trichodesmiumspp.)的種類組成與相對豐度
論文名稱(外文):Species composition based on het R gene sequences and relative abundance of Trichodesmium spp. in the vicinity of Gueishan Island
指導教授:張正張正引用關係
指導教授(外文):Jeng Chang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:68
中文關鍵詞:龜山島束毛藻種類組成相對豐度het R
外文關鍵詞:Gueishan IslandTrichodesmiumSpecies compositionrelative abundancehet R
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束毛藻(Trichodesmium spp.)為絲狀的固氮藍綠細菌,是開放性大洋中主要的氮鹽來源。本論文的目的在利用光學顯微鏡觀察與基因序列分析來探討龜山島附近海域束毛藻的種類組成與相對豐度。經由顯微鏡觀察發現束毛藻主要分佈在50公尺以淺之水層,且以全體藻絲的豐富度而言,2004和2005年的春季(四、五月)及秋季(九月)的豐度高,可達到224.6 trichomes L-1,而夏季(七月)及冬季(十一、十二月)的豐度低。藻絲長度依季節而略有不同,在四月、五月及九月風浪較小的時候,藻絲長度皆較其他月份為長。藉由16S rRNA、het R及nif H三種退化引子來分析單一束毛藻群體的基因序列,結果顯示龜山島海域束毛藻的het R序列可以清楚的分為四群(cluster):第一群(Cluster I)為T. thiebautii、第二群(Cluster IIa)是和來自赤道太平洋的束毛藻樣本B49及B51-3相似的一群、第三群(Cluster IIb-IIc)為龜山島附近海域特有的新分類群、第四群(Cluster III)則是T. erythraeum,胺基酸序列也支持這項結果。由混合樣本所取得的核酸序列顯示屬於T. erythraeum的序列佔有率為33∼80%,但顯微鏡觀察則顯示T. thiebautii與T. erythraeum大約各佔一半。對於束毛藻這一屬來說,由於群體形態多變且藻絲細胞變異大,所以不能單以群體或藻絲作為分類的依據,必須輔以基因序列分析才能更精準的描述種間的親源關係。
Trichodesmium spp. is a nitrogen-fixing filamentous cyanobacterium that contributes a major fraction of new nitrogen input to the open ocean. In this study, microscope observation and sequences analysis were used to determine the species composition and relative abundance of Trichodesmium spp. in the vicinity of Gueishan Island. The results indicated that Trichodesmium always existed in the upper water column with depths shallower than 50 m. In 2004 and 2005, high abundance (224.6 trichomes L-1 ) was observed in spring (April and May) and fall (September), while low abundance was observed in summer (June) and winter (December). The length of trichomes varied with the seasons. Trichomes became longer when the sea was calm (April, May, and September). Based on sequence analysis of het R, 16S rRNA, and nif H individual colonies of Trichodesmium spp. were grouped into 4 main clusters. The first group (Cluster I) included species related to T. thiebautii. The second group (Cluster IIa) included specimens closely related to B49 and B51-3, two specimen collected in Equatorial Pacific Ocean. The third group (Cluster IIb-IIc) is a novel Trichodesmium lineage. The fourth group(Cluster III) is T. erythraeum. Amino acid sequences suggested a similar trend. The sequences of mixed samples showed that T. erythraeum was the dominated species, contributing 33~80% to total Trichodesmium sequences. However, the microscope observation revealed that T. thiebautii and T. erythraeum contributed approximately equally. Because colony morphology is changeable and cell dimensions makes a varies greatly, identification of species is difficult using morphological characteristics alone. Sequence analysis of genes is a good supplementary method to describe the phylogenetic relationship in Trichodesmium genus.
目錄

中文摘要………………………………………………………………I
英文摘要……………………………………………………………....III
目錄……………………………………………………………………V
表目錄…………………………………………………………………VI
圖目錄……………………………………………….……….......…...VII
前言…………………………………………………………………… 1
材料與方法………………………………………………………….... 6
結果…………………………………………………………………... 13
討論……………………………………………………………………22
參考文獻………………………………………………………………28
圖表……………………………………………………………………34
附錄……………………………………………………………………59












表目錄

表一、根據文獻描述與顯微鏡觀察所定出的各種束毛藻的形態特徵。
……………………………………………………………… 34
表二、2004年至2005年龜山島附近海域表層(5 m)束毛藻數量百
分比的季節變化。…………………………………………… 35
表三、利用het R基因分析龜山島附近海域單一束毛藻群體各Cluster的基因序列數。……………………………………………… 36
表四、利用het R基因分析比較五個單一束毛藻群體間內部相似度。
…………………………………………………………………37
表五、利用het R基因序列分析龜山島附近海域混合樣本中束毛藻的藻種組成。…………………………………………………… 38
表六、龜山島附近海域四群束毛藻分類群之核酸序列比對。……… 39
表七、利用光學顯微鏡觀察2005年三個航次拖網樣本中束毛藻之藻種豐度及藻絲相關常數。………………………………………40
附錄一、2004及2005年各測站束毛藻藻絲長度及細胞長度之平均值。
……………………………………………………………..…59
附錄二、實驗藥品。……………………………………………………62








圖目錄

圖一、龜山島附近海域的測站位置圖。……………………………… 41
圖二、龜山島附近海域T. thiebautii群體與藻絲的形態特徵。………42
圖三、龜山島附近海域T. erythraeum群體與藻絲的形態特徵。…… 43
圖四、2004至2005年龜山島附近海域表層(5 m)兩種束毛藻豐度的季節性變化。………………………………………………..44
圖五、2004年龜山島附近海域束毛藻沿中間測線(St. 6a –10b)水平
與垂直分佈。…………………………………………………..45
圖六、2004年至2005年龜山島附近海域三種束毛藻藻絲的平均長度。
..…………………………………………………………………47
圖七、2004年至2005年龜山島附近海域三種束毛藻細胞的平均長度。
………………………………………………………………….48
圖八、2004年至2005年龜山島附近海域三種束毛藻細胞長度的頻度分佈圖。……………………………………………………….49
圖九、增殖基因片段後以電泳分離 PCR產物。……..…………… 50
圖十、龜山島附近海域單一束毛藻群體het R核酸序列之親緣關係樹
狀圖。………………………………………………………….51
圖十一、龜山島附近海域單一束毛藻群體het R胺基酸序列之親緣關係樹狀圖。………………………………………………….52
圖十二、龜山島附近海域單一束毛藻群體16S rRNA核酸序列之親緣關係樹狀圖。……………………………………………….53
圖十三、龜山島附近海域單一束毛藻群體nif H核酸序列之親緣關係樹狀圖。……………………………………………………54

圖十四、龜山島附近海域2005年五月第1278航次het R核酸序列之親緣關係樹狀圖。………………………………………….55
圖十五、龜山島附近海域2005年七月第1297航次het R核酸序列之親緣關係樹狀圖。………………………………………….56
圖十六、龜山島附近海域2005年九月第1306航次het R核酸序列之親緣關係樹狀圖。………………………………………….57
圖十七、龜山島附近海域2005年十二月第1320航次het R核酸序列之親緣關係樹狀圖。……………………………………….58
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