Molecular cloning and characterization of mating type and fruiting related genes in Ganoderma lucidum

• Main Authors: Hung-Yi Wu, 吳竑毅
• Other Authors: 曾顯雄
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/90454338060630910444

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 93 === 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.