靈芝A交配型基因座之選殖及功能界定

Cloning and characterization of mating type A locus in Ganoderma lucidum

袁如陵(Ju-Ling Yuan)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.03248

摘要

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。

關鍵字

靈芝 ； 交配型 ； 交配型基因座 ； 交配型基因 ； 基因排列

並列摘要

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.

並列關鍵字

Ganoderma lucidum ； mating type ； mating type locus ； mating type gene ； synteny

參考文獻

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