- 學位論文
靈芝交配型與出菇相關基因之選殖與分析
Molecular cloning and characterization of mating type and fruiting related genes in Ganoderma lucidum
摘要
靈芝為一種極具價值的藥用真菌，曾被報導具多種抗癌與增進免疫的功能；過去在靈芝交配型與出菇有過基礎生物學之研究，但在分子生物學層次之深入探討則尚付闕如。為瞭解靈芝調控交配型與出菇之基因，首先利用已建構之靈芝基因體與表現序列標記(Expressed Sequence Tag)資料庫，藉由比對與分析，找出可能位於靈芝A mating type locus 之4個交配型基因，以及可能位於靈芝B mating type locus的19個pheromone receptor與20個putative pheromone precursor相關基因，並組合出長約15 kbp的靈芝A mating type locus contig，再藉由RACE (Rapid amplification of cDNA ends)選殖出位於A locus之a1與a2 mating type基因，其中a1 cDNA全長1428 bp，與Pleurotus djamor 之HD1 homeodomain mating type protein具有48%之相似度，並具有與mating type protein 結合與辨識之domain，以及被預測出可能的NLS (Nuclear Localization signal)： RKRRR；a2 cDNA全長1680 bp，與P. djamor 之HD2 homeodomain mating type protein具有39%之相似度，並具有保守之homeobox domain，以及可能的NLS：RRSRCRKE。並藉由基因專一性引子對，偵測出交配型A1具有a1與a2基因，A2具有b1與b2基因，B1具有PR3、PR5、PR15與PR19基因，B2具有PR3、PR7與PR8基因。並根據不同單核體交配型之交配試驗，經由細胞學與發生學之鏡檢與觀察，證實並探討不同交配基因調控交配型所獲之結論。為證實a1與a2的功能，已完成建構以靈芝glyceraldehyde - 3 - phosphate dehydrogenase (gpd)基因之promoter驅動下游抗抗生素基因與a1或a2基因表現之binary vector：pCGl-a1與pCGl-a2，並將藉由農桿菌轉型法(Agrobacterium - mediated - transformation)於不同之交配型內表現；並且為了組合出更完整的A與B locus，分別以b1與PR7基因片段做為探針，篩檢已建構完成之Fosmid library，已獲得呈正反應訊號，於A mating type locus有8個選殖株，B mating type locus有5個選殖株，將再進行雜合加以驗證，並做Fosmid兩端定序，與靈芝基因體相關資料庫比對，以建構完整之A、B基因座之基因圖譜(gene map)。另一方面，建構靈芝成熟子實體與菌絲體之雙向扣除式cDNA資料庫(subtractive cDNA library)，並配合於靈芝基因體與EST資料庫比對分析，初步結果發現一些可能與靈芝子實體型態分化相關之基因，未來並擬利用基因剔除(gene knockout)之方法來分析個別基因之功能。
並列摘要
Ganoderma lucidum is a well-known and important medicinal mushroom demonstrated with anti-cancer and immunomodulatory activities. Although classic study on the mating and fruiting in G. lucidum have been carried out in recent decades and accumulated tremendous valuable knowledge, in-depth exploration of the biologically interesting phenomenon via molecular biology approaches was unavailable and deserved further study. In order to understand the genes in G. lucidum which regulated the mating and fruiting, first we BLAST the Expressed Sequence Tag (EST) from G. lucidum cDNA library, and accessed 4 mating type genes in A mating type locus, and 20 pheromone receptor genes and 19 pheromone precursor genes which controlled mating in B mating locus. A 15 kbp A mating type locus was assembled by linking contigs in whole genomic DNA library of G. lucidum. Full-length open reading frames of genes, a1, a2, encoded homeodomain protein HD1 and HD2, respectively, were cloned by Rapid Amplification of cDNA Ends (RACE). a1 gene is 1428 bp in full-length, having 48% similarity to HD1 of Pleurotus djamor, and also with Nuclear Localization Signal (NLS)：RKRRR, while a2 gene is 1680 bp in full length, having 39% similarity to conserved HD2 of P. djamor, and also with NLS：RRSRCRKE. By polymerase chain reaction using specific primers derived from mating genes a1, a2, b1, b2, PR3, PR5, PR7, PR8, PR15 and PR19, A1 mating type was demonstrated possessing a1, a2 genes; A2 mating type b1, b2 genes; B1 PR3, PR5, PR15 and PR19 genes; B2 PR3, PR7 and PR8 genes, respectively. The mating genes in A1B1, A1B2, A2B1, A2B2 of G. lucidum were further verified by dual mating in vitro on YMSA plates based on cytology, and ontology of clamp connection and pseudoclamp connection formation. And also, two binary vectors pCGl-a1 and pCGl-a2, having the a1 or a2 gene insertions, driven by glyceraldehyde - 3 - phosphate dehydrogenase (gpd) promoter and the downstream hygromycin resistance gene, were constructed, and will be transformed into compatible mating type to prove the function of these genes in mating. In addition, a Fosmid library constructed from monokaryotic strain BCRC 37180 (A2B2) of G. lucidum screened by labeled b1 and PR7 gene probes indicated 8 clones showing positive signals against A locus genes, and 5 clones to B locus genes. These specific Fosmid clones will be both terminal ends sequenced and assembled with the EST and genome databases to constructed a complete A, B, mating type locus genetic map. We also constructed a subtracted cDNA library of G. lucidum, genes which differentially expressed during fruiting body development will be cloned and their function proved by gene disruption.