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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48798

Title: 與三角褐指藻矽殼合成相關之矽親合蛋白的序列鑑定與功能探究
Identification and Characterization of Cell Wall Associated Silica Affinity Protein of Phaeodactylum tricornutum
Authors: Chien, Ya-Yun
錢亞筠
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 矽藻;三角褐指藻;矽殼;矽親和蛋白
Phaeodactylum tricornutum;silica;silaffin;cingulin
Date: 2017
Issue Date: 2018-08-22T06:31:25Z
Abstract: 矽藻是真核單細胞藻類的一個主要類群,同時也是海洋中最常見的一種浮游植物。矽藻最特殊的地方就是由玻璃組成的奈米花紋細胞壁,這層擁有物種特異性花紋的細胞壁被稱為矽殼。科學家也藉由矽殼的外型將矽藻分為中心矽藻 (Coscinodiscophyceae) 及羽紋矽藻 (Pennales)。目前對於矽藻矽殼的相關研究大多集中於一種中心矽藻-假微型海鏈藻 (Thalassiosira pseudonana)。這種中心矽藻矽殼的合成主要是由矽親和蛋白 (silica affinity protein) 將海水中的液態矽酸鹽沉澱成固態二氧化矽的奈米粒子或矽板,進而形成完整的矽殼。從矽殼中分離出來且具有沉澱二氧化矽的矽親和蛋白可以簡單分為兩大類:可溶性矽親和蛋白 (silaffin) 及不可溶性矽親和蛋白 (cingulin)。 從目前被鑑定出的矽親和蛋白來看,彼此之並沒有明顯的序列保留性。但是這些蛋白仍然具有一些特徵,例如含有高比例的離胺酸以及絲氨酸,並且可能具備一些重複序列。本計畫擬以三角褐指藻 (Phaeodactylum tricornutum) 作為研究對象,找尋其可能參與矽殼形成的矽親和蛋白。 首先我透過生物資訊的方法,將基因組資料庫中具有矽親合蛋白特性的蛋白篩選出來。將這些預測蛋白制備為一個可在三角褐指藻表現且帶有螢光蛋白的重組蛋白質體,利用基因槍進行基因轉殖。轉殖成功的矽藻透過顯微鏡初步判定蛋白在細胞表現位置是否與矽殼相關,並且萃取該矽藻之矽殼,進一步確認螢光蛋白是否表現在矽殼上。最後透過測定預測蛋白於缺矽復矽培養下的基因變化量,更進一步確認蛋白與矽藻矽營養鹽生理調控的關係。 期望得以透過比較兩種矽藻的矽親合蛋白對於矽藻矽殼的演化有初步的了解,也可以藉由三角褐指藻細胞外型的多變型態結合矽親和蛋白發展出更多元的應用。
Diatoms, a kind of single-celled algae, are the dominant phytoplanktons in the ocean. Each diatom has a cell wall, also known as frustule, which is made of biosilica (SiO2) and bearing the species-specific pattern. Based on the pattern of fructule, diatoms may be denoted as Coscinodiscophyceae and Pennales. Currently, the study of fructule formation was focused on Coscinodiscophyceae, especially in Thalassiosira pseudonana. Some proteins isolated from fructule are identified as silica affinity proteins, which can convert and deposit silicate(aq) into silica(s), including soluble silaffin and insoluble cingulin. However, sequence homologous proteins from was not identified in Pennales, such as Phaeodactylum tricornutum. In this study, we screened the genome database of P. tricornutum and found several candidate proteins with unusual high percentage of lysine and serine residues that is one of the characteristic of silica affinity protein. Next, these candidates were cloned and recombinant with a green fluorescent protein (EGFP) gene in a transgenic vector, respectively. After transfection and screening, transgenic P. tricornutum were observed under a fluorescence microscope to identify the cellular location of these candidate genes. We also isolated frustule of trangenic P. tricornutum to make sure which fusion protein was located in frustule. In addition, the gene expression patterns were also examed when P. tricornutum cultured in different silicate condition. Though our investigation, one silaffin-like protein in P. tricornutum was identified. We are going to further characterize this protein and use it to study the frustule formation mechanism in Pennales.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B017.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48798
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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