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Title: 紅藻細翼枝菜上的真菌群落多樣性
Total and active community of fungi associated with the red alga Pterocladiella capillacea
Authors: He,Chih-Chiao
何止喬
Contributors: NTOU:Institute of Marine Biology
國立臺灣海洋大學:海洋生物研究所
Keywords: 紅藻;海洋真菌;真菌群落多樣性;培養法;高通量定序
Red alga;Marine fungi;Diversity;Culture-dependent technique;Next-generation sequencing
Date: 2019
Issue Date: 2020-07-03T08:34:03Z
Abstract: 關於生長在藻類上的真菌研究缺乏,特別是海洋大型藻類,而前人只針對小部份褐藻、綠藻和紅藻進行初部研究。因此本研究選取臺灣北部的優勢紅藻細翼枝菜 (Pterocladiella capillacea) 作為研究對象,以培養法分離真菌及非培養法 (metabarcoding) 結果比較細翼枝菜上的真菌多樣性。於培養法中,將洗淨的健康及死亡藻體置於葡萄糖-酵母萃取物-蛋白腖瓊脂海水培養基 (GYPS) 及人工海水瓊脂培養基 (SWA) 上,待菌絲從藻體生長出來後便繼代於玉米粉瓊脂培養基 (CMAS) 上,依其菌落形態分群。利用聚合酶連鎖反應 (polymerase chain reaction, PCR) 擴增其核醣體基因 (rDNA) 內轉錄間隔區 (internal transcribed spacer, ITS) 及核醣體大亞基 (large subunit, LSU) 並進行測序,最後與NCBI的GenBank資料庫序列進行比較以鑑種。於高通量定序中,從冷凍乾燥後的細翼枝菜藻體萃取總DNA和RNA。先將RNA反轉錄成cDNA後,並使用巢式PCR擴增DNA及cDNA的ITS1區域 (230−422 bp),所得到的擴增產物利用Illumina Miseq進行測序。結果顯示基於相對豐富度 (每個物種數量佔全部數量的百分比) 的計算,從培養法中只獲得子囊菌門 (88.75%) 和擔子菌門 (11.25%)。於高通量定序中,rDNA樣品大部份序列屬於未被分類之真菌群 (75.04%),擔子菌門 (16.23%) 和子囊菌門 (8.72%) 為優勢群。擔子菌門 (52.31%) 在rRNA樣品中為優勢群,其次是子囊菌門 (40.56%),有少量的壺菌門序列 (0.004%),而未被分類之真菌群佔總序列的7.13%。基於相對豐富度的計算,優勢真菌於分離法中為Hypoxylon monticulosum (11.25%)、Byssochlamys spectabilis (7.5%) 和Emericella sp. (7.5%);於rDNA樣品中為Mycosphaerella sp. (2.49%)、Malassezia globosa (1.73%) 和 Auricularia polytricha (1.23%);於rRNA樣品中為Nigrospora sphaerica (12.52%)、Peniophora sp.1 (9.23%) 和Auricularia polytricha (8.09%)。香農-維納多樣性指數 (H’) 的結果顯示rRNA樣品具有最高的多樣性 (3.644),其次為可培養的真菌 (3.414) 和最低為rDNA 樣品 (2.077)。這些結果皆表明紅藻細翼枝菜上有高真菌之多樣性,利用培養技術及分子技術所獲得的藻類真菌多樣性並不一致,且擔子菌可能與紅藻有共生關係。
Information on the diversity of fungi associated with macroalgae is lacking. Previous studies only focused on fungi of selected species of brown, green and red algae. This study, therefore, investigates the diversity of fungi associated with Pterocladiella capillacea, a dominant red alga in northern Taiwan, using a culture-dependent technique and a metabarcoding approach. For isolation, mycelia growing out from washed healthy and dead thalli on GYPS (glucose-yeast extract-peptone seawater agar) and SWA (seawater agar) media were subcutlured onto CMAS (cornmeal seawater agar) medium and grouped into colony morphotypes. ITS (internal transcribed spacers of the rDNA) and LSU (large subunit of the rDNA) of the fungi were sequenced and compared with reference sequences in NCBI for identification. For high-throughput sequencing, total RNA and DNA were extracted from freeze-dried Pterocladiella capillacea thalli. RNA was reverse-transcribed into cDNA, and together with the extracted total DNA, a region of the ITS1 (230−422 bp) of DNA and cDNA using a nested PCR approach was amplified for Illumina Miseq sequencing. For relative abundance (no. of a particular species / no. of total species  100 %), Ascomycota (88.75%) and Basidiomycota (11.25%) were the only phyla obtained from the isolation approach. For the metabarcoding analyses, unidentified fungi (75.04%) were dominant in the rDNA sequences, followed by Basidiomycota (16.23%) and Ascomycota (8.72%). Basidiomycota (52.31%) was dominant in the rRNA samples, followed by Ascomycota (40.56%) and Chytridiomycota (0.004%) while unidentified fungi constituted 7.13% of the total sequences. The dominant fungi from isolation based on relative abundance were Hypoxylon monticulosum (11.25%), Byssochlamys spectabilis (7.50%) and Emericella sp. (7.50%); Mycosphaerella sp. (2.49%), Malassezia globosa (1.73%) and Auricularia polytricha (1.23%) in the rDNA samples; Nigrospora sphaerica (12.52%), Peniophora sp.1 (9.23%) and Auricularia polytricha (8.09%) in the rRNA samples. Shannon’s diversity index (H’) suggests that the rRNA samples had the highest diversity (3.644), followed by the culturable collection of fungi (3.414) and the rDNA samples (2.077). These results collectively suggest that different methods uncovered different fungal diversity on the alga, and the Basidiomycota might play a symbiotic role with the red alga.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010634003.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53570
Appears in Collections:[海洋生物研究所] 博碩士論文

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