Karyotyping and localization of functional genes on chromosomes of Ganoderma lucidum

靈芝(Ganoderma lucidum)為一種傳統的中藥材，同時靈芝也是一種會造成木材白腐的木材腐朽菌和多種樹木的病原菌。在近代的研究中證實靈芝的次級代謝產物具有抗腫瘤、降低高血壓和保護肝臟的功能。現今關於靈芝的研究很多，但大多都聚焦於靈芝的次級代謝產物上，在基礎研究上相對較為缺乏。本研究主要目的在探討靈芝的染色體特性，包含染色體核型分析以及功能性基因座落位置。在細胞學染色體核型分析，應用發芽管爆破的方式將靈芝單核株37180的染色體釋出，再以螢光染劑DAPI配合螢光顯微鏡觀察染色體的數量和型態。鏡檢顯示G. lucidum 37180 (A2B2)染色體數目為11至15條之間，平均為12.65條，長度在0.8 µm至4.1 µm之間。此外，電泳染色體核型分析則是用脈衝式電泳將染色體DNA依照分子量大小做分離，脈衝式電泳可將G. lucidum 37180的染色體DNA分離成六個訊號強度不同的條帶，大小分別為3.02、3.36、3.67、4.24、4.53和5.05 Mb。此外，也進行G. lucidum 37177(A1B1)的脈衝式電泳；37177的脈衝式電泳結果顯示具7個條帶，比37180多一個約4 Mb的條帶。將經脈衝式電泳分離的染色體DNA轉印到尼龍膜後進行南方氏雜合分析，以G. lucidum 37177 的13條染色體上的序列設計探針，可以在G. lucidum 37180脈衝式電泳的6個條帶上標示出與G. lucidum 37177同源性的13條染色體的位置，並可估計出G. lucidum 37180的基因體大小約為50.35 Mb。此外也利用專一性探針(A2之b1與B2之PR8基因)探測了G. lucidum 37180上交配型基因座的位置；A2交配型基因座位於ChI染色體上，B2交配型基因座則位於ChIII、ChV或ChVI其中一條染色體上。此外也嘗試應用掃描式電子顯微鏡、螢光原位雜交和流式細胞分析儀對G. lucidum染色體做分析。In nature, Ganoderma licidum acts as wood white-rot pathogens for many tree species; nevertheless, its fruit body has also been used for century as folk medicine. More recently, researches have shown its polysaccharides or terpenoides with anti-tumor, alleviating high blood pressure and hepatoprotective activity. Though immensive biological activity studies of G. lucidum have been focused on its secondary metabolites, the basic exploration appeared rare. Herein, we concentrated our efforts to study the chromosomal and karyotyping of G. lucidum via germ tube bursting method (GTBM), pulse field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH), flow cytometry and scanning electromicroscopy. GTBM in combination of fluorescent microscopy indicated that G. lucidum 37180 (A2B2) possessed 11-15 chromosomes, with average of 12.65, and a length of 0.8~4.1 µm, while PFGE showed the six-banded chromosomal zones of 3.02, 3.36, 3.67, 4.24, 4.53 and 5.05 Mb. One additional chromosomal band at 4.0 Mb was found in G. lucidum 37177 (A1B1). Southern blotting of the six GL 37180 chromosomes bands separated by PFGE by using specific probes derived from the thirteen deciphed GL 37177 chromosomes unraveled the 13 allelic chromosomes, respectively. In addition, by specific probe (A2: b1 gene) also can allocate GL A2 mating locus located at chromosome I, also allocating the possible location of B2 mating locus possibly at chromosome III, V or VI by specific probe (B2: PR8 gene). The same approaches undertaken in this study perhaps can be applied to study the karyotyping of other GL monokaryotic strains and other medicinal mushrooms as well