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研究生:王威閔
研究生(外文):Wei-Min Wang
論文名稱:利用細胞核傳送方法進行牛樟芝的擬有性雜交
論文名稱(外文):Parasexual Crosses of Antrodia Camphorata By nuclear transfer method
指導教授:吳瑞鈺吳瑞鈺引用關係
指導教授(外文):Rey-Yuh Wu
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:129
中文關鍵詞:牛樟芝擬性生殖細胞核傳送
外文關鍵詞:Antrodia CamphorataParasexualnuclear transfer
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牛樟芝Antrodia camphorata(Zang & Su)被喻為台灣最珍貴真菌,有台灣紅寶石之稱,為台灣特有的真菌之一。在傳統療法上牛樟芝常被利用為解毒劑(antidote),用以治療癌症(cancer)、高血壓(hypertension)及肝癌 (liver cancer), 目前已有報告指出牛樟芝子實體可被分離出固醇酸(steroid acid) 等成分。牛樟芝目前仍無法以人工栽培的方式生產子實體,但隨著生物科技的發展牛樟芝目前正發展菌絲體培養法進行發酵,大量生產菌絲體及有效成分,因此企望能經由選種或育種,找出具有生長快速、有效成分含量高的優良菌種,以進行工業化生產。
傳統真菌育種大多是以原生質體融合的方式來進行育種,本實驗開發傳送 核 入 原 生 質 體的方法,以二株不同的牛樟菌株CCRC35396(C96)與CCRC353989 (C98)當親本,分別將C96的核傳送入C98原生質體(68傳送組)及 C98的核傳送入 C96原生質體(86傳送組)來進行雜交育種。首先以酵素Novozyme 234 分別製備原生質體然後純化出細胞核,再分別進行傳送核入原生質體,而所獲得的68傳送組與86傳送組的雜交體共有10群,這些雜交體經過多次繼代培養與單株化後共獲得13株穩定的雜交株系這些雜交株系包括S68A、S68B、S68C、S8D S68E、S68F、S68G、S68H、S68I、S68J、與S86A、S86B、S86C等。雜交株系的外觀表現經分析後發現13個雜交株系在19.5、27.5、35.5℃三種溫度與 BM、MEA、 PDA三種培養基培養,其菌落型態與顏色皆呈現不同的表現型。利用AFLP DNA指紋分析技術,由64組引子對中篩選出可以擴增標誌片段並區別親本的E1M3、E2M4、E4M6、E7M3四組引子,再利用這四組引子對進行牛樟菌親本與雜交株系的 AFLP DNA圖譜分析,用以鑑別其基因型,經由4組引子的DNA標誌片段的比對分析,S68D的基因型與 C98相同,而其他12株系皆呈現不同基因型。比對牛樟菌二親本 (CCRC35396、CCRC35398)與13個雜交株系的外表型(phenotype)與基因型(genotype),發現13個雜交株系的外表型態與基因型有一致性,而且可以加以區別,顯示13個雜交株系為不同於C98與C96牛樟菌的新菌株。
本實驗建立牛樟芝以傳送核入原生質體的擬有性生殖育種方法、輔以菌種外表型鑑別法與AFLP DNA指紋分析技術鑑別牛樟菌菌種之雜交株系,藉由這些方法建立,更育出13種不同的牛樟芝新株系。
Antrodia camphorata , is considered the most precious and unique fungus in Taiwan. It was also nicknamed as “Ruby of Taiwan”. It has been utilized as an antidote in the treatment for cancer, hypertension, and hepatocellular carcinoma in Taiwan folk medicine. Studies indicated that steroid acid and other active components could be isolated from the fruiting bodies of A. comphorata. But it was still unsuccessful to reproduce the fruiting body through mycelium cultivation. However, the rapid progress in biotechnology has made the mass production of these active components possible, by scale-up fermentation of the mycelium of A. comphorata.
The conventional methods for fungi breeding mainly depends on the successful protoplast fusion technology. In our studies, a nuclei transfer method was developed and used for A. comphorata breeding. Two parental stocks of A. comphorata ( C96 and C98 ) were purchased from Culture Collection and Research Center(CCRC). The nuclei were isolated and purified from protoplast of mycelium treated by Novozyme 234. The isolated nuclei from C96 and C98 were transferred into C98 and C96 protoplasts, respectively, to regenerate new A. comphorata hybrids. In the beginning, 10 groups of hybrids with a differential characteristic in colony were obtained after the first subculture. Finally, 13 stable hybrid strains (designated as S68A S68B S68C S68D S68E S68F S68G S69H S68I S68J and S86A S86B S86C) were generated through a serial of subculture and clonization process.
Different culture conditions, including 3 temperature of 19.5 27.5 35.5 C and 3 media of BM MEA PDA, had been tested to differentiate these 13 hybrid strains. The morphology and pigment production of fungal colonies were compared and distinct from each other under some culture conditions. The AFLP DNA fingerprinting assay with 64 pairs of primers was further used to study the genotype of hybrid strains. Results showed that E1M3, E2M4, E4M6, and E7M3, 4 out of the 64 primer pairs, were able to distinguish the PCR-amplification products of C96 from those of C98. Using these 4 primer pairs, we had completed the genotype identification of 13 hybrid strains and compared these data with those of their parental stocks, C96 and C98. The results demonstrated that these 13 hybrid strains were distinct in both genotype and phenotype and they should be identified as new strains of A. comphorata.
In our studies, we demonstrated the success of parasexual reproduction of A. comphorata by transferring nuclei into protoplast. In addition to the morphological classification method, the AFLP DNA fingerprinting method could be utilized for identification of hybrid strains and their parental stocks. In this report, 13 new strains of A. comphorata were obtained and identified.
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目錄..................................................Ι
一:摘要
(一)中文摘要..........................................Ⅲ
(二)英文摘要..........................................Ⅴ
圖目錄................................................Ⅶ
表目錄................................................Ⅹ
二:緒論..............................................1
(一)牛樟芝之介紹:.....................................1
1.牛樟芝的發現。......................................1
2.牛樟芝的鑑定與命名。................................3
3.牛樟芝的型態特徵與分類地位。........................4
4.牛樟芝的成分與功能。................................5
5.牛樟芝的重要性。...................................12
(二)研究背景:........................................15
1.擔子菌的生活史。...................................15
2.擔子菌的育種方法。.................................18
2(1)單孢系間的雜交。.................................18
2(2)雜交育種。.......................................18
2(3)原生質體融合。...................................19
2(4)電融合。.........................................20
2(5)分子育種。.......................................20
2(6)傳送核進原生質體。...............................21
3.擬有性生質之介紹。.................................26
4.擔子菌菌種鑑定。...................................28
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三:材料與方法.......................................30
(一)牛樟菌菌種之取得與培養準備。.....................30
(二)菌種之培養、比較。...............................30
(三)牛樟菌細胞核傳送之雜交。.........................31
(四)雜交融合體之單株化。.............................37
(五)牛樟菌雜交體之分析。.............................38
四:結果.............................................46
(一)菌種之培養、比較。...............................46
(二)牛樟菌細胞核傳送之雜交。.........................51
(三)雜交融合體之單株化。.............................63
(四)牛樟菌雜交體之分析。.............................76
1.外表型(phenotype)分析。 76
2.基因型(genotype)分析。.............................86
五:討論.............................................96
六:參考文獻 .......................................104
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圖 目 錄
圖1-1:牛樟樹在台灣的分布情形。...............................1
圖1-2:牛樟芝子實體。.........................................2
圖1-3:牛樟芝的型態。.........................................8
圖1-4:Cherng et al., 1995.由牛樟芝分離之triterpenoids。......9
圖1-5:Chen et al., 1995.由牛樟芝分離出二種steroid acid。.....9
圖1-6:Cherng et al., 1996.由牛樟芝分離到的三種triterpenoids。9
圖1-7:Yang et al., 1996.由牛樟芝分離到steroid acid三種。....10
圖1-8:Shen et al., 1997.由牛樟芝分離到的steroidal acid。....11
圖1-9:典型的擔子菌之生活史。................................24
圖4-1:牛樟菌株C96分別培養於PDA,MEA,BM三種固體培養基
21天菌落生長曲線之比較。...............................47
圖4-2:牛樟菌株C98分別培養於PDA,MEA,BM三種固體培養基
21天菌落生長曲線之比較。...............................47
圖4-3:C96及C98在BM固體培養基培養21天菌落
生長曲線之比較。.......................................48
圖4-4:CCRC35396與CCRC35398分別培養於MEA與PDA
21天後菌落生長的比較。.................................49
圖4-5:CCRC 35396及CCRC 35398培養於BM
21天後菌落生長的比較。...............................50
圖4-6:CCRC35396菌絲於處理酵素1.5小時後
原生質體釋出的情形。...................................55
圖4-7:CCRC35398菌絲於處理酵素1.5小時後
原生質體釋出的情形。...................................56
圖4-8:CCRC35396細胞核經DAPI染色多成圓形。...................57
圖4-9:CCRC35398細胞核經DAPI染色有破損情形。.................57
圖4-10:原生質體利用Rhodamine 123染色可見到螢光反應,
表示含有粒腺體及高倍下觀察粒腺體染色的情形。...........58
圖4-11:CCRC35396經核純化過程所得懸浮液,利用Rhodamine 123染色
未見螢光反應。.........................................58
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圖4-12:86傳送組與C96原生質體的再生菌落之確認與比較。........59
圖4-13:68傳送組與C98原生質體的再生菌落之確認與比較。........60
圖4-14:86傳送組與C96原生質體的再生菌落之確認與比較。........61
圖4-15:68傳送組與C98原生質體的再生菌落之確認與比較。........62
圖4-16:68A群。..............................................67
圖4-17:68B群。..............................................68
圖4-18:68C群。..............................................69
圖4-19:68D群。..............................................70
圖4-20:68E群。..............................................71
圖4-21:68F群。..............................................71
圖4-22:68G群。..............................................72
圖4-23:68H群。..............................................72
圖4-24:86A群。..............................................73
圖4-25:86B群。..............................................74
圖4-26:86C群。..............................................74
圖4-27:牛樟菌經核傳送獲得之雜交菌株經5次以上的
單株繼代培養獲得的13株生長穩定的雜交株係。.............75
圖4-28:牛樟菌C96、C98與13個雜交株係在BM培養基中以
19.5℃培養28天後之菌落生長之比較。.....................81
圖4-29:牛樟菌C96、C98與13個雜交株係在MEA培養基中以
19.5℃培養28天後之菌落生長之比較。.....................81
圖4-30:牛樟菌C96、C98與13個雜交株係在PDA培養基中以
19.5℃培養28天後之菌落生長之比較。.....................81
圖4-31:牛樟菌C96、C98與13個雜交株係在BM培養基中以
27.5℃培養28天後之菌落生長之比較。.....................82
圖4-32:牛樟菌C96、C98與13個雜交株係在MEA培養基中以
27.5℃培養28天後之菌落生長之比較。.....................82
圖4-33:牛樟菌C96、C98與13個雜交株係在PDA培養基中以
27.5℃培養28天後之菌落生長之比較。.....................82
圖4-34:牛樟菌C96、C98及13個雜交株係在BM培養基中以
35.5℃培養28天後之菌落生長之比較。.....................83
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圖4-35:牛樟菌C96、C98與13個雜交株係在MEA培養基中以
35.5℃培養28天後之菌落生長之比較。.....................83
圖4-36:二親本與13個雜交株系以BM培養基培養於三種
不同的溫度16天後菌落生長半徑之比較。...................84
圖4-37:二親本與13個雜交株系以MEA培養基培養於三種
不同的溫度16天後菌落生長半徑之比較。...................84
圖4-38:二親本與13個雜交株系以PDA培養基培養於三種
不同的溫度16天後菌落生長半徑之比較。 ..................84
圖4-39:二親本(C96與C98)與13個雜交株係基因組
以E1M3當引子所擴增進行AFLP基因圖譜分析。...............87
圖4-40:二親本(C96與C98)以E1M3引子擴增所產生之
獨特DNA片段位置區與13個雜交株系之比較。................88
圖4-41:二親本(C96與C98)與13個雜交株係基因組
以E2M4當引子所擴增進行AFLP基因圖譜分析。...............89
圖4-42:二親本(C96與C98)以E2M4引子擴增所產生之
獨特DNA片段位置區與13個雜交株系之比較。................90
圖4-43:二親本(C96與C98)與13個雜交株係基因組
以E4M6當引子所擴增進行AFLP基因圖譜分析。...............91
圖4-44:二親本(C96與C98)以E4M6引子擴增所產生之獨特
DNA片段位置區與13個雜交株系之比較。....................92
圖4-45:二親本(C96與C98)與13個雜交株係基因組
以E7M3當引子所擴增進行AFLP基因圖譜分析。...............93
圖4-46:二親本(C96與C98)以E7M3引子擴增所產生之獨特
DNA片段位置區與13個雜交株系之比較。....................94
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表 目 錄
表1-1:薄孔菌屬、似薄孔菌屬靈芝在生殖菌絲、骨架菌絲、
擔子及擔子,型態上之比較情形。..........................13
表1-2:文獻上有關牛樟芝所分離出的成分,採集地點
及牛樟樹學名。 .........................................14
表1-3:常見的幾種擔子菌在製備原生質體時所使用的
酵素及等張溶液濃度。 ...................................25
表1-4:常見的單倍體化的藥劑。................................27
表4-1:牛樟菌C96與C98於BM,MEA,PDA三種固體
培養基培養21天菌落顏色之比較。..........................48
表4-2:牛樟菌株C96與C98於三次原生質體製備試驗中
所獲原生質體數及活性指標之比較。 .......................53
表4-3:C96與C98二菌株細胞核與原生質體於核傳送
前後菌落再生數的比較。..................................53
表4-4:經核傳送獲得的雜交體子代與其親本於菌落顏色之比對。 ...54
表4-5:68傳送組與86傳送組再生菌落繼代後的分群與生長
型態變化比較。..........................................66
表4-6:牛樟菌13個雜交株系於三種不同的溫度與三種不同的
培養基的菌落生長型態與顏色之比較分析與分群。............85
表4-7:牛樟菌13個雜交株系利的AFLP基因圖譜合親本的
DNA標誌片段之比較分析。.................................95
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