臺灣博碩士論文加值系統

樟芝為台灣本土特有之藥用真菌，其所具有的生物活性已被研究並加以證實，但關於樟芝成分的生合成機制仍少有報告。控制羊毛脂醇去甲基酶表現的CYP51基因，在細胞色素P450的超基因家族中是最廣泛分布的一個家族，其跨越了原核生物和真核生物。對真菌而言，羊毛脂醇去甲基酶是麥角固醇生合成路徑中的必要酵素，而麥角固醇則是構成真菌細胞膜主要成分之一。另一方面，許多三萜類的生成都是以羊毛脂醇作為骨架，而這些三萜類化合物可能具有特殊的生物活性或重要的生理功能。在本論文中，我們以樟芝的表現序列基因資料庫中，一段與CYP51同源的cDNA序列片段AcCYP51，利用5''RACE和Genome Walking的方式確定這段序列的cDNA全長，並繼續以Genome walking在AcCYP51五端找到了三個基因。續以PCR的方式找出AcCYP51的基因體全長。其包含了8個exon 和7個intron，共2,045個核苷酸，編碼區的全長為1,635個核苷酸。其所表現出的蛋白質AcCyp51p經過比對可能在N端的前40個胺基酸，具有將此蛋白質送入細胞膜表現的訊號序列。將AcCYP51胺基酸序列和目前已知的CYP51胺基酸序列比對，顯示其和模式木材腐朽菌Phanerochaete chrysosporium具有最高的相似程度。利用半定量RT-PCR實驗結果顯示，相較於實驗室培養的各種菌株生活世代，AcCYP51在野生的子實體有較高的表現量。在培養基中添加羊毛脂醇以及生成羊毛脂醇的前驅物鯊烯，此基因的表現量皆會上升。經過預測啟動子片段，顯示此基因有三個主要可能的修飾片段。分別以熱休克，改變液態培養碳源和氮源，以及外加itroconazole處理液態培養的樟芝，皆會影響此基因的表現量，顯示這些因子和啟動子片段的關係。將AcCYP51建構至蛋白質表現載體 pET 和GST系統顯示，GST系統可以表現相對較多且穩定的可溶性蛋白，供後續的活性分析。利用AcCYP51的重組蛋白進行酵素反應，顯示此蛋白質亦具有催化羊毛脂醇結構上第14個碳的去甲基化的功能。本論文所得之成果，可提供後續之研究，如利用專一抗體針對蛋白質表現來進一步證實其cDNA表現係受啟動子影響；同時，麥角固醇生合成途徑的活化和子實體形成的相互關係亦是未來值得探討的題目。此外，亦可以此蛋白質對樟芝其他特有固醇類進行活性分析，探討其是否具有類似的去甲基反應，產生特殊的萜類化合物。

Antrodia cinnamomea is a well known edible medicine fungus with various proved benefits of bioactivity compounds for humans in Taiwan. However, reports of biosynthesis of these active compounds are few. Sterol 14α-demethylase (CYP51) is the most widely distributed in all members of the cytochrome P450 superfamily and is one of the key enzymes for sterol biosynthesis in fungi. It is the only CYP family which has been found in both prokaryotes and eukaryotes. In this study, a cytochrome P450 cDNA, AcCyp51, possibly encoding lanosterol 14α-demethylase was obtained and sequenced by using 5'' RACE and genome walking with primers designed from the sequences of EST libraries of A. cinnamomea. Full-length AcCyp51 consists of 2,045 bp and contains 7 introns. The coding region of AcCYP51 was 1,635 bp. By genome walking, there were 3 genes discovered from 5'' region of AcCyp51. AcCyp51p may have N-terminal signal peptide, which would be transferred to plasma membrane with possibly cleavage site at the 40th amino acid. Comparison of the protein sequence with a number of major Cyp51 homologs revealed that the AcCyp51p was highly conserved, and most closely related to the Cyp51p homologs from Phanerochaete chrysosporium, the model white rot basidiomycetous fungus. The expression of CYP51 in A. cinnamomea is higher in wild type basidiome stages than in artificial culture according to the semiquantative RT-PCR analysis. Three predicted promoter motifs of the AcCYP51 were tested by semiquantative RT-PCR for the regulation of expression by heat shock, carbon and nitrogen sources changing and azole stress. Protein expression in Escherichia coli BL21 showed lower soluble form in pET expression system and higher in GST system. GST constructed protein revealed the capacity for catalyzing the demethylation of lanosterol. Analysis of AcCYP51p protein expression from treated A. cinnamomea strains by using an anti-CYP51p antibody could be the next step to confirm the RT-PCR data. The role that ergosterol biosynthesis played in the formation of fruiting body could be an interesting topic. In addition, whether AcCYP51p demethylates any other lanosterol analogues on 14α-carbon specific in A. cinnamomea is a direction to study.

Abstract in Chinese………………………………………………………….............. I
Abstract...……………………………………………………………........................ III
1. Introduction…..………………………….………………..….…............................. 1
1.1 Antrodia Cinnamomea…………..…….………………...…………………. 1
1.2 Ergosterol biosynthesis………………..…………………………………… 6
1.3 lanosterol 14α-demethylase (CYP51)………………………........................ 9
2. Materials and Methods...…..……………………………………………............... 14
2.1 Experimental fungus strains and media………………..…………………... 14
2.2 RNA and Genomic DNA extraction…….……………………………...….. 14
2.3 Cloning and sequencing....………………….………..…………………….. 15
2.3.1 Cloning full-length cDNA……………………...…………………….. 15
2.3.2 Sequence analyses and alignments……………………...……………. 16
2.4 Promoter assay and semiquantative RT-PCR……………………………… 16
2.5 Subcloning of cDNA, expression in E. coli and enzyme purification…....... 17
2.5.1 Expression in vector pET 20b(+)…………………………………...... 17
2.5.2 Expression in vector pET100/D-TOPO®…………………………….. 18
2.5.3 Expression in vector pGEX4T-1…………………………………........ 18
2.6 Reconstituted protein purification and analysis……………………………. 19
2.6.1 Protein expression and purification…………………………………… 19
2.6.2 Protein assay with SDS-PAGE and Western…………………………... 19
2.7 Reconstituted protein catalytic activity assay……………………………… 20
3. Results and discussions…………………….…………………………………….. 21
3.1 Gene Cloning and Sequence Analysis……………………………………... 21

3.2 RT-PCR of different culture stages………………….……………………. 24
3.3. Genome Walking…………………….…………………………………… 25
3.4 Prediction of promoters…………………….…………………………....... 26
3.5 Protein expression in BL21 and activity assay…………………….………. 28
4. Conclusions…………………….……………………………………………….... 29
5. References…………………….………………………………………………….. 31
6. Tables & Figures………………………………………………………………….. 39

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