臺灣博碩士論文加值系統

靈芝（Ganoderma lucidum）為一種高價值之藥用真菌，且向來被認為對人體健康有益，本實驗室以靈芝為模式物種進行交配型基因座的研究，在之前的研究中已經界定出四個A交配型基因以及三十九個B交配型基因，並確認其分別在不同交配型的靈芝菌株基因體中存在（A1B1、A1B2、A2B1、A2B2）。為對A交配型基因座之基因進行進一步之探討，本論文首先利用四個A交配型基因a1、a2、b1、b2的探針進行靈芝基因組DNA的南方氏雜合，確認a1、a2存在於A1交配型基因座而b1、b2存在於A2交配型基因座之中，且均以單一copy的方式存在。在進行fosmid library clone的篩選之後，挑出各帶有A1及A2 locus基因序列的fosmid進行shotgun定序，每個fosmid再分別和兩個fosmid組合成兩個分別帶有靈芝A1及A2交配型基因的片段（contig），長度分別為93 kb以及99 kb，在A1交配型基因座上註解出18個保守性基因、6個hypothetical基因，而在A2交配型基因座則為18個保守性基因及8個hypothetical基因。註解完成之A1及A2交配型基因座經與其他五種擔子菌的A交配型基因座做synteny的基因排列比較後，可將其依保守性基因的排列方式區分為兩群，其中分別包含多孔菌目及傘菌目下的菌種。
分別以RACE （Rapid Amplification of cDNA Ends）進行b1及b2 cDNA的5’及3’增幅後，可設計引子對增幅出基因的全長，並發現靈芝A交配型基因座之基因會有alternative splicing的現象發生，因而產生許多不同的isoforms，目前已選殖到的基因全長包括一個a1 isoform、兩個a2 isoforms、四個b1 isoforms以及兩個b2 isoforms。於a1、a2、b1、b2蛋白質的N端可以預測出保守的helix區域，此區域在其他擔子菌研究中為A交配型基因進行heterodimerization之區域。a1及b1蛋白質和其它擔子菌的HD1蛋白質相似度較低，且缺乏homeodomain的區域。a2及b2蛋白質有很保守的homeodomain區域，而在N-端則被預測出帶有mTP（mitochondrial targeting peptide），因此可能會被送入粒線體中，由於在A交配型基因座中發現許多存在於粒線體的基因，故於本論文中也將討論A交配型基因是否具有其他不同於交配型決定與辨識的能力。

Ganoderma lucidum is a potent medicinal fungi and convinced to be beneficial to health. We use Ganoderma lucidum as model organism to study mating loci gene. In our previous studies, 4 A mating type genes and 39 B mating type genes were characterized in four different Ganoderma lucidum mating types (A1B1, A2B2, A1B2, A2B2, respectively). In order to further characterize the A mating type locus in Ganoderma lucidum, genomic DNA was digested with HindIII and hybridized with 4 probes of A mating type genes, a1, a2, b1, and b2. The results indicate that a1 and a2 are single copy and localized in only A1 mating type locus, whereas b1 and b2 in the A2 mating type locus. Fosmid library clones are hybridized with the same probes, and the clones harboring the A1 and A2 mating type genes were shotgun sequenced. The contigs of genomic region containg A1 and A2 loci genes were further connected and extended with two other overlap fosmids to work out the full length of 93 kb A1 locus and 99 kb A2 locus, each locus except the mating type genes contains 18 conserved genes and 6 or 8 hypothetical genes, respectively. Synteny comparison between the two loci in G. lucidum, and also A loci in other five Agaricomycetes were performed. According to the synteny map, six species can be separated into two groups, the Aphyllophorales and the Agaricales. RACE (Rapid Amplification of cDNA Ends) was performed to characterize the full length of both b1 and b2 cDNA. We found that alternative splicing events take place in A mating type genes, which results in different isoforms. One a1 isoform, two a2 isoforms, four b1 isoforms, and two b2 isoforms were cloned. The helical regions which participate in heterodimerization between different A locus genes were predicted at the N-terminal of a1, a2, b1, and b2. a1 and b1 proteins are extremely variable compared to HD1 genes from other basidiomycetes, including the homeodomain region. Neverthless, a2 and b2 proteins are very conserved in homeodomain, and a N-terminal mTP (mitochondrial targeting peptide) was predicted in both protein, which may target a2 and b2 protein to mitochondria. Since many protein in A locus were targeted to mitichondria, their possible functions other than mating type determination and recognition were also discussed.

目錄
誌謝………………………………………………………………………i
中文摘要………………………………………………………………ii
英文摘要…………………………………………………………… iv
前言……………………………………………………………………1
壹、前人研究……………………………………………………………2
一、靈芝簡介…………………………………………………………2
二、真菌的交配（mating）……………………………………………3
三、真菌的交配型基因座（mating type locus）………………………4
四、Homeodomain locus與Pheromone locus………………………5
五、擔子菌的生活史………………………………………………6
六、交配型基因座在不同擔子菌中之研究………………………7
七、擔子菌中的HD1/HD2之功能分析相關研究………………10
八、靈芝交配型之現今研究………………………………………14
貳、材料與方法………………………………………………………16
一、靈芝菌株之來源、培養與保存……………………………16
二、靈芝之細胞核染色……………………………………………17
三、靈芝基因體DNA之抽取與分析……………………………17
四、聚合酶鏈鎖反應以及產物的分析與純化……………………19
五、南方氏雜合分析………………………………………………24
六、Fosmid library screening………………………………………26
七、靈芝交配型基因座A1及A2的組合與定序…………………27
八、靈芝RNA的抽取及分析……………………………………27
九、Rapid Amplification of cDNA Ends（RACE）………………30
十、靈芝交配型基因全長之選殖…………………………………31
十一、靈芝轉型系統之建立………………………………………31
十二、Yeast-two-hybrid assay………………………………………35
十三、序列分析……………………………………………………36
參、結果………………………………………………………………38
一、靈芝細胞核行為之研究………………………………………38
二、靈芝轉型系統之建立………………………………………38
三、靈芝A交配型基因座之界定及註解………………………39
四、靈芝A交配型基因座之synteny分析……………………40
五、靈芝基因體DNA南方氏雜合………………………………41
六、靈芝A2 locus基因之RACE…………………………………41
七、靈芝A交配型基因之全長選殖………………………………42
八、靈芝HD1基因之功能性區域分析…………………………42
九、靈芝HD2基因之功能性區域分析…………………………43
十、靈芝HD1及HD2基因產物之交互作用分析………………43
肆、討論…………………………………………………………………44
一、靈芝細胞核學之研究…………………………………………44
二、靈芝轉型系統的建立…………………………………………44
三、靈芝A交配型基因座序列之分析…………………………47
四、靈芝與其他擔子菌之A交配型基因座的synteny分析………47
五、靈芝A交配型基因之功能分析………………………………48
六、未來展望………………………………………………………51
伍、圖表…………………………………………………………………53
陸、參考文獻……………………………………………………………90
附錄……………………………………………………………………95








圖目錄
圖一、pCGl-a2的建構流程圖…………………………………………53
圖二、pGem-gpd-Hyg的建構流程圖…………………………………54
圖三、建構完成之pGADT7-a1 FL-24及pGBKT7-a1 FL-5’…………55
圖四、建構完成之pGADT7-b2 FL-36及pGBKT7-b2 FL-36………56
圖五、靈芝菌株A1B1及A2B2配對後以giemsa進行染色之結果…57
圖六、靈芝菌株配對後以giemsa進行染色之結果…………………58
圖七、靈芝菌株配對後以giemsa進行染色之結果…………………59
圖八、以a1的探針和靈芝菌株A1B1的fosmid菌落膜進行雜合…60
圖九、以a2的探針和靈芝菌株A1B1的fosmid菌落膜進行雜合…61
圖十、以b1的探針和靈芝菌株A2B2的fosmid菌落膜進行雜合…62
圖十一、以b2的探針和靈芝菌株A2B2的fosmid菌落膜進行雜合…63
圖十二、A1及A2 locus的組合………………………………………64
圖十三、註解完成的A1 locus…………………………………………65
圖十四、註解完成的A2 locus…………………………………………66
圖十五、A1及A2 loci的序列並比結果……………………………67
圖十六、靈芝的A1及A2 loci synteny和Coprinus cinerea、Coprinellus disseminatus、Laccaria bicolor、Phanerochaete chrysosporium、Pleurotus djamor的A locus比較之結果……………………………68
圖十七、靈芝A1及A2 loci的synteny和Phanerochaete chrysosporium之比較……………………………69
圖十八、靈芝A1及A2 loci的synteny和Coprinus cinerea之比較70
圖十九、靈芝A1及A2 loci的synteny和Pleurotus djamor之比較71
圖二十、Coprinus cinerea、Coprinellus disseminatus和Laccaria bicolor的A locus synteny之比較72
圖二十一、Phanerochaete chrysosporium、Pleurotus djamor和Coprinus cinerea的A locus synteny之比較73
圖二十二、以a1及a2為探針雜合被HindIII酵解之靈芝基因體DNA的結果74
圖二十三、以b1及b2為探針雜合被HindIII酵解之靈芝基因體DNA的結果74
圖二十四、以RACE增幅b1及b2 cDNA的5’及3’端序列76
圖二十五、a2基因中alternative splicing發生的情形77
圖二十六、b1基因中alternative splicing發生的情形78
圖二十七、b2基因中alternative splicing發生的情形79
圖二十八、於a1 isoform-1的N- terminal可以預測出兩個Helices區域以及一個NLS（Nuclear Localization Signal）80
圖二十九、於a2 isoform-1及a2 isoform-2的N- terminal皆可以預測出兩個Helices區域，以及一個homeodomain和一個NLS81
圖三十、於四個b1 isoforms的N- terminal皆可以預測出兩個Helices區域，以及一個NLS（Nuclear Localization Signal）82
圖三十一、於三個b2 isoforms的N- terminal皆可以預測出兩個Helices區域，而除了b2 isoform-3之外，b2 isoform-1及b2 isoform-2中皆可預測出有homeodomain及NLS83
圖三十二、將a1 isoform-1、b1 isoform-1與其他擔子菌Coprinus cinerea、Laccaria bicolor、Schizophyllum commune、Pleurotus djamor、Phanerochaete chrysosporium的HD1 N-terminal進行並比分析84
圖三十三、將a1 isoform-1、b1 isoform-1與其他擔子菌Coprinus cinerea、Laccaria bicolor、Schizophyllum commune、Pleurotus djamor、Phanerochaete chrysosporium的HD1 N-terminal進行並比分析及二級結構預測85
圖三十四、將a2 isoform-1、b2 isoform-2與其他擔子菌Coprinus cinerea、Schizophyllum commune、Pleurotus djamor、Phanerochaete chrysosporium的HD2 N-terminal進行並比分析及二級結構預測86
圖三十五、Yeast-two-hybrid assay87



表目錄
表一、靈芝A locus上所註解出之保守性基因其Gene Ontology之相關資訊…………………………………………………………………88
表二、以TargetP預測A locus蛋白質的localization…………………89

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