以冬蟲夏草性別基因作為即時定量系統標的之研究

Abstract

冬蟲夏草Cordyceps sinensis (Berk.) Sacc.為我國滋肺補腎的珍貴藥材，來源稀少、採集困難又無法進行人工栽培，因此價值逐年高漲。由於成本高昂，市售的冬蟲夏草產品多以人工培養無性世代的冬蟲夏草菌絲體取代野生的子實體。然而冬蟲夏草無性世代菌種中國被毛孢 (Hirsutella sinensis) 菌絲體的生長緩慢，導致來源有限，故常有以其他真菌菌絲體摻雜或替代於冬蟲夏草產品中。因此建立真正冬蟲夏草菌體含量的檢測系統為確保品質的首要課題。 本研究中首先比對已發表蟲草屬菌種性別基因中，較保守的alpha box與HMG box序列，設計簡併性引子 (degenerate primer) 擴增出冬蟲夏草的HMG box基因，由不同冬蟲夏草菌株的HMG box基因序列分析結果，設計僅可專一性擴增出冬蟲夏草HMG box基因的引子對 (HMGsp F/R)，經確定引子對的物種專一性、靈敏度以及結果再現性後，進行冬蟲夏草菌體即時定量系統的建立。在生活史推測部份，由於在不同冬蟲夏草菌株及子實體標本中只擴增出一種HMG box的性別基因，所以尚無法推測冬蟲夏草為雌雄同體型或雌雄異體型。但是由此HMG box序列所設計的專一性引子對(HMGsp F/R)針對冬蟲夏草具有極高的專一性，可作為核糖體基因外另一種鑑定冬蟲夏草的分生指標。以此專一性引子對 (HMGsp F/R)所建立的即時定量系統，可得HMG box基因拷貝數，再將拷貝數推算回原始總核酸量，最後帶入總核酸量與菌絲乾重的回歸方程式，即可推算出分析樣本中含有多少真正的冬蟲夏草。

Cordyceps sinensis (Berk.) Sacc is a very expensive medicinal fungus of Traditional Chinese Medicine, because it is hard to get and couldn’t process artificial propagation. If we would like to process artificial propagation, the first thing we have to know is life cycle of C. sinensis. So far, C. sinensis life cycle still unclear. This study would like to figure out whether C. sinensis belong to homothallism or heterothallism by mating gene. Due to C. sinensis is very expensive, Hirsutella sinensis mycelium was usually used to replace C. sinensis to make C. sinensis product. Since H. sinensis mycelium growth slowly leading cost higher, so products often mix another species mycelium or additive. It is very important to build quantitative system to detect the quality of C. sinensis products. In the study, first is to design degenerate primer to clone alpha box and HMG box, conserve sequence in mating gene MAT1-1 and MAT1-2, and I obtained HMG box. Following, designed HMG box specific primer (HMGsp F/R). After chick the specific, sensitive and repeatability of this primer, and to build quantitative system by real-time PCR. In the part of presume C. sinensis life cycle, since I just obtained one mating gene in C. sinensis, so I couldn’t identify whether C. sinensis belong to homothallism or heterothallism, but due to the specific HMG box primer it could offer another molecular systematics of C. sinensis. Finally, using HMGsp F/R to build quantitative system could detect the quantity of H. sinensis mycelium successfully.