惠蓀林場關刀溪流域鵝膏屬真菌分子之系統研究

Abstract

惠蓀實驗林場關刀溪流域是全球變遷計劃，長期生態調查（LTER） 中的一站，主要作為森林生態系研究，其林相豐富，水量充沛，孕育各種不同型態的野生真菌，其中包含許多鵝膏屬真菌（Amanita）。本實驗之目的係利用分子鑑定的方式，建立蕙蓀林場中鵝膏屬真菌之ITS（Internal Transcribed Spacer）分子標誌，並藉由分子資料的比對，探討蕙蓀林場中可能分佈之鵝膏菌種類及屬內種間之親緣關係。 本研究係利用實驗室現有鵝膏屬真菌子實體標本為材料，抽取其總體DNA，以此些總體DNA為模板，分別以ITS1-F、ITS4-B；ITS1-F、ITS-4；二組真菌共通引子，對鵝膏菌之染色體DNA進行多聚合酶聯鎖反應（PCR），以增幅18S-ITS1-5.8S-ITS2-28S之片段，其大小約為700-900bp。將實驗結果來自不同菌株之rDNA-ITS序列，送至NCBI中，以BLAST程式進行相似性序列比對，搜尋物種或屬間之特定保留序列，另將此序列以CLUSTAL W的程式進行多序列排對，由繪製親緣樹來瞭解鵝膏菌群間特有之關係，並藉由標準菌株之比對證實實驗之準確性。實驗結果發現這群鵝膏菌其5.8 S- rDNA序列完全相同，而ITS1及ITS2序列片段其差異性很大。

Long-term ecological research（LTER）located in Guantauchi area of Huisun Experimental Forest, which belongs to global climatic change research project, is conducted primarily in the forest ecosystem. In this study, we try to establish the method of molecular identified for Amanita ITS molecular marker. We also try to use the data of ITS sequence analysis to survey about the species and distribution of Amanita in Huisun Experimental Forest. The genomic DNA was extracted for the template of PCR. Universal primers ITS1F / ITS4B and ITS1F / ITS4 of amplified 700-900 base pair of 18S-ITS1-5.8S-ITS2-28S fragments highly conserved regions of Basidiomycotina ribosomal DNA. The results of the alignments of the ITS sequences of Amanita spp. using the BLAST program in NCBI show the similarities and dissimilarities of molecular identified and traditional classification systems. We also had a result of multiple sequence alignments by CLUSTWAL program which shows that the 18S、5.8S and 28S coding regions have highly conserved. However within Amanita species there are great variations in the ITS1 and ITS2 region. Keywords：Huisun Experimental Forest, Amanita spp. , ITS , PCR.