利用聚合酶鏈鎖反應－限制片段長度多型性（PCR-RFLP）技術探討該核醣體DNA及內轉錄間隔區（ITS 1-5.8 S-ITS 2）用於區分紅麴菌種之可行性

The Study on the Use of Ribosomal DNA and Internal Transcribed Spacer (ITS1-5.8S-ITS2) in Identifying Monascus spp. with the PCR-RFLP Technique

林世斌(Shih-Bin Lin) ； 張嘉珮(Jia-Pei Chang)

《宜蘭大學生物資源學刊》 2卷1期 (2005/12) Pp. 9-15

https://doi.org/10.6175/job.2005.21.12

摘要

本研考利用聚合酶鏈鎖反應－限制片段長度多型性（PCR-RFLP）技術，探討紅麴菌核醣體DNA，用於區別紅麴菌屬內種間、種內群體的可行性。針對紅麴菌屬（Monascus）九株菌體之DNA，以真菌rDNA的PCR擴散通用引子，有效的擴增5.85 rDNA與其兩端之內轉錄間隔區（internal transcribed spacer；ITS），區域長度為540-630 bp。利用七種核酸內切酵素HaeⅢ、DraⅠ、HindⅢ、EcoRⅠ、BamHⅠ、CfoⅠ、HinfⅠ進行ITS1-5.8S rDNA-ITS2區域之剪切，結果顯示BamHⅠ、DraⅠ及HindⅢ均無切位，故無任何鑑別效果。然而除了M. floridanus明顯不同外，其他酵素在M. purpureus、M. sanguineus、M. albus、M. ruber、M. pilosus及M. kaoliang所產生的片段形式皆相似，是以無法有效鑑別紅麴菌的屬內種間。除此之外，M. floridanus在馬鈴薯葡萄糖培養基（PDA）上的菌落顏色（橄欖綠）亦與其他紅麴菌種（紅色或白色）極不相同，因此推論該菌種似乎不適分類於Monascus中。

關鍵字

紅麴菌屬 ； 5.8S核榶體DNA ； 聚合酶鏈鎖反應－限制片段長度多型性 ； 核醣體內轉錄間隔區

並列摘要

This study is to realize the possibility by using ribosomal RNA gene to distinguish between Monascus spp. and between Monascus purpureus strains through the polymerase chain reaction-restriction fragment length polymorphism (RFLP) technique. The 5.8S rDNA and laterally flanked internal transcribed spacer (ITS) were successfully amplified by PCR from nine Monascus spp. with the universal primer and were between 540-630 bp in length. The PCR products were then cut by each of the seven restriction enzymes: HaeⅢ, DraⅠ, HindⅢ, EcoRⅠ, BamHⅠ, CfoⅠ, HinfⅠ, to obtained their RFLP profiles. The results demonstrated that HaeⅢ, DraⅠ and HindⅢ did not provide any cut and have no differential power. The RFLP profile obtained from M. prupureus, M. sanguineus, M albus, M. uber, M. pilosus and M. kaolian were similar except M. floridanus and so that these enzymes did not effectively provide differential power among Monascus spp. Besides, the colony color of M. floridanus grown on potato dextrose agar (PDA) was so different from the color, red or white, Obtained from the other Monascus spp. Used in this study. It seemed not probable to classify the M. floridanus into Monascus genus.

並列關鍵字

Monascus ； 5.8S rDNA ； PCR-RFLP ； ITS ； fungal identifcation