曾顯雄臺灣大學：植物病理與微生物學研究所陳良坤Chen, Liang-KunLiang-KunChen2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/58079牛樟芝（Antrodia cinnamomea），為台灣特有藥用真菌，含有許多具生理活性的成分，其萜類化合物被認為在其生理活性上扮演相當重要的角色，因此擬經由萜類生合成途徑在基因層面的研究，以究明其生理功能。首先應用先前本系所應用真菌研究室所建構之cDNA基因資料庫，設計專一性引子對利用聚合酶連鎖反應（PCR，polymerase chain reaction）增幅出牛樟芝基因體DNA之farnesyl diphosphate synthase（FDS）（長約300 bp）、geranylgeranyltransferase（GT）（長約700 bp）、sesquiterpene cyclase（SC）（長約1 kb）及squalene monooxygenase（SM）（長約700 bp）等基因之核酸片段，再以DIG標識作為探針，篩選所建構之牛樟芝基因體之Fosmid library，挑選出呈正反應的Fosmid clone進行解序並註解，其中bdf01008-B24選殖株對FDS探針呈正反應，定序後得到長為37,783 bp之DNA序列，其上涵括七個基因：兩個hypothetical protein，一個heat shock protein，一個expressed protein，一個有關monooxygenase的蛋白質，兩個farnesyl diphosphate synthase；而bdf01020-E06選殖株對GT探針呈正反應，定序後得到長為34,826 bp之DNA序列，涵蓋有六個基因：兩個hypothetical protein，一個glutamate dehydrogenase/leucine dehydrogenase，一個phosphoethanolamine N-methyltransferase，一個ppg3，一個geranylgeranyltransferase；而bdf01010-N11選殖株對SC探針呈正反應，定序後得到長為37,395 bp之DNA序列，包括七個基因：兩個hypothetical protein，一個Pol-like protein Pol-2，一個pol protein，一個H25N7.04，一個nuclear mRNA splicing protein, via spliceosmoe-related protein，一個trichodiene synthase；而bdf01002-P14選殖株對SM探針呈正反應，定序後得到長為26,567 bp之DNA序列，承載六個基因：四個hypothetical protein，一個vicilin storage protein，一個squalene monooxygenase。從已知cDNA序列設計專一性引子對，利用rapid amplification of cDNA ends（RACE）也已獲得FDS、GT、SC及SM等四個萜類生合成基因之全長度cDNA序列，更近一步分析open reading frame（ORF）以及所轉譯的胺基酸序列之功能性分析。FDS cDNA全長度為1359 bp， ORF長為1092 bp，可轉譯出364個胺基酸；GT cDNA全長度為1181 bp，ORF長為1026 bp，轉譯342個胺基酸；SC cDNA全長度為1402 bp，ORF長為831 bp，轉譯277個胺基酸；SM cDNA全長度為1546 bp，ORF長為1443 bp，轉譯481個胺基酸。利用四個解序出的Fosmid clones之序列，設計專一性引子對以增幅FDS、GT、SC及SM四個基因兩端同源性序列，並以pAN7-1之抗hygromycin B之hph cassette作為selection marker，建構四個置換載體（FDS、GT、SC及SM replacement vector），應用基因槍將置換載體送入單核菌株AC T1 MT14以進行基因破壞，挑選出突變菌株，再利用hph cassette專一性引子對進行PCR以初步檢測，但目前尚未成功獲得突變菌株來進行後續的功能性分析，因此，仍需再進行基因破壞的工作，並期望各別將此四個基因的基因體DNA（包含exon及intron）轉殖於酵母菌中，以確認這四個基因的功能。Antrodia cinnamomea (Neu-Chang-Tsu), a potent medicinal resupinate mushroom, was documented with the capacity to produce numerous metabolites with biological activity, notably the terpenoids, and merit for study further. Attempt at molecular level to reveal the activity of these secondary metabolites of A. cinnamomea, we conducted cloning and characterization of the terpenoid biosynthesis genes. The genomic DNA gene fragments of farnesyl diphosphatesynthase (FDS) (ca. 300 bp ), geranylgeranyltransferase (GT) (ca. 700 bp), sesquiterpene cyclase (SC) (ca. 1 kb), and squalene monooxygenase (SM) (ca. 700 bp) were obtained by using specific primers derived from previously constructed cDNA EST library by polymerase chain reaction (PCR). The unique genomic DNA fragments were labeled to probe the constructed Fosmid library. Four Fosmid clones, bdf01008-B24 (37,783 bp), bdf01020-E06 (34,826 bp), bdf01010-N11 (37,395 bp), and bdf01002-P14 (26,567 bp) which exhibited positive signal towards FDS, GT, SC, and SM specific probes, respectively, were accessed by Fosmid colony hybridization. Of the four Fosmid clones selected and sequenced, in addition to FDS, GT, SC, and SM, 22 putative genes, including monooxygenase, heat shock protein, expressed protein, glutamate dehydrogenase/leucine dehydrogenas, phosphoethanolamine N-methyltransferase, ppg3, Pol-like protein Pol-2, pol protein, H25N7.04, nuclear mRNA splicing protein, vicilin storage protein, and 10 hypothetical proteins, were annotated after BlastX with the NCBI database. Furthermore, the full-length cDNA of FDS, GT, SC, and SM were achieved by rapid amplification of cDNA ends (RACE), of them, FDS cDNA was 1359 bp , with 1092 bp open reading frame (ORF), encoding translated 364 amino acids; GT 1181 bp, ORF 1026 bp, translated 342 amino acids; SC 1402 bp, ORF 831 bp, translated 277 amino acids; SM 1546 bp, ORF 1443 bp, translated 481 amino acids, respectively. In order to verify the function of the four FDS, GT, SC, and SM genes by gene disruption, four specific replacement vectors were constructed by insertion the pAN7-1 vector with the hygromycin resistance cassette (hph cassette) flanked with the terminal specific homologus sequences of FDS, GT, SC, and SM genes. Although the four constructed replacement vectors have been tried to transform the monokaryotic strain AC T1 MT14 of A. cinnamomea by biolistic gun, no positive transformants have been secured yet, and not available for functional analysis. To overcome the obstacles, refine the transformation protocol and expression of these genes in yeast host will be continued later on.中文摘要………………………………………………………………………………1 英文摘要………………………………………………………………………………3 前言……………………………………………………………………………………5 壹、前人研究…………………………………………………………………………7 一、牛樟芝……………………………………………………………………………7 （一）分類地位………………………………………………………………………7 （二）型態特性………………………………………………………………………8 （三）生物活性成分…………………………………………………………………8 1. 一般成分分析………………………………………………………………...8 2. 萜類化合物………………………………………………………………… ..9 3. 類固醇……………………………………………………………………….10 4. 其他………………………………………………………………………….10 （四）功能研究………………………………………………………………………11 1. 抗氧化作用………………………………………………………………….11 2. 免疫調變的作用…………………………………………………………….12 3. 抗發炎作用………………………………………………………………….12 4. 細胞凋亡作用……………………………………………………………….13 5. 抗腫瘤作用………………………………………………………………….14 6. 保肝作用…………………………………………………………………….15 7. 抗病毒作用………………………………………………………………….15 8. 血管舒張…………………………………………………………………….16 9. 抗血管新生………………………………………………………………….16 10. 急性毒性研究………………………………………………………...……16 11. 其他………………………………………………………………………...17 二、三萜類之功能特性……………………………………………………………..17 三、萜類之生合成…………………………………………………………………..18 （一）萜類生合成途徑……………………………………………………………..18 I. Isoprene 基本單元生合成途徑……………………………………………...18 1. Mevalonate pathway………………………………………………………..18 2. Non-mevalonate pathway…………………………………………………..18 3. Mevalonate pathway 與non- mevalonate pathway 的探討……………...20 II. Isoprenoid symthesis pathway…………………………………………….21 1. Isoprenyl pyrophosphate synthase………………………………………...21 2. Prenyltransferase…………………………………………………………...21 3. Terpenoid cyclase…………………………………………………………...22 III. 牛樟芝之萜類生合成可能途徑…………………………………………..22 （二）萜類生合成相關基因之探討………………………………………………..22 1. Farnesyl diphosphate synthase（FPPS）……………………………………22 2. Geranylgeranyltransferase（GGTase）……………………………………23 3. Sesquiterpene cyclase（SC）……………………………………………….24 4. Squalene monooxygenase（SM）………………………………………….24 貳、材料與方法………………………………………………………………………26 ㄧ、菌株與其培養……………………………………………………………………26 （一）菌株……………………………………………………………………………26 （二）菌體培養………………………………………………………………………26 二、基因體DNA 製備………………………………………………………………26 （一）基因體DNA 萃取……………………………………………………………26 （二）DNA 電泳分析………………………………………………………………28 三、萜類生合成相關基因之選殖……………………………………………………29 （一）聚合酶連鎖反應Polymerase chain reaction（PCR）…………………….29 （二）純化PCR 產物………………………………………………………………30 （三）TA cloning…………………………………………………………………….31 （四）質體DNA 萃取………………………………………………………………32 四、牛樟芝Fosmid library 中萜類生合成相關基因之clone 篩選及定序……….35 （一）DIG（Digoxigenin）探針標定之合成………………………………………35 （二）Fosmid colony hybridization………………………………………………...36 （三）Colony hybridization…………………………………………………………38 （四）挑選正反應訊號之Fosmid clone 建構Shotgun library…………………….41 五、RNA 製備……………………………………………………………………….43 （一）Total RNA 萃取………………………………………………………….……43 （二）RNA formaldehyde 變性電泳膠分析…………………………………….….44 （三）mRNA 製備…………………………………………………………………..45 六、萜類生合成相關基因之RACE（Rapid Amplification of cDNA Ends）............46 （一）生合成First-strand cDNA…………………………………………………...46 （二）快速增幅cDNA Ends…………………………………………………..........47 七、Antrodia cinnamomea AC T1-MT14 單核菌株………………………………..49 （一）單核菌株的取得………………………………………………………………49 （二）單核菌株的交配型分析………………………………………………………50 （三）AC T1-MT14 對Hygromycin B 抗藥性測試………………………………50 八、基因破壞（Gene knockout）………………………………………………….51 （一）基因置換所需之載體的建構…………………………………………………51 （二）基因槍轉型（Biolistic transformation）…………………………………...53 九、序列分析…………………………………………………………………………57 參、結果………………………………………………………………………………58 一、萜類生合成相關基因之選殖……………………………………………………58 二、牛樟芝Fosmid library 中萜類生合成相關基因之選殖（cloning）及定序…….59 （一）Fosmid clone 篩選……………………………………………………………59 （二）Fosmid clone 定序……………………………………………………………59 三、萜類生合成相關基因之RACE（Rapid Amplification of cDNA Ends）……62 四、萜類生合成相關基因genomic DNA 的特性………………………………….66 五、Antrodia cinnamomea AC T1-MT14 單核菌株……………………………….67 六、基因破壞…………………………………………………………………………68 （一）基因置換載體建構……………………………………………………………68 （二）Biolistic transformation………………….…………………………………..69 肆、討論………………………………………………………………………………70 伍、圖表………………………………………………………………………………77 陸、參考文獻………………………………………………………………………117 附錄一 實驗試劑配方……………………………………………………..............123 附錄二 培養基成分……………………………………………………………… .126 附錄三………………………………………………………………………………128 附錄四………………………………………………………………………………1376159213 bytesapplication/pdfen-US牛樟芝萜類生合成farnesyl diphosphate synthasegeranylgeranyltransferasesesquiterpene cyclasesqualene monooxygenaseAntrodia cinnamomeaterpenoid biosynthesisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58079/1/ntu-95-R92633001-1.pdf