臺灣博碩士論文加值系統

牛樟芝(Antrodia cinnamomea)為台灣特有種真菌，對宿主具有專一性，僅生長在台灣特有植物老齡牛樟樹(Cinnamomum kanehirae)中空腐朽心材內。早期被原住民作為解酒之用，目前許多研究指出牛樟芝菌絲體及子實體的萃取物對於抗癌、抗發炎、降血壓、抗氧化、糖尿病具有功效。牛樟芝由於數量稀少、價錢昂貴，取得不易，遂變成山老鼠盜採之對象。本實驗利用18S rRNA基因進行牛樟芝菌株的分子鑑定，接著以不同菌種的牛樟芝，接種在置換營養源和成份遞減的人工固態培養基中央，黑暗下、室溫倒置培養八周觀察牛樟芝的生長差異。另外，分別抽取不同菌株，菌絲內、中和外部(或子實體)的cDNA，進行即時定量PCR，分析四條跟老化有關的基因在菌絲體內部，中間和外部的(或子實體)相對表現量。此四條基因分別是cytochrome P450，glutathione-S-transferase，peroxiredoxin和manganese superoxide dismutase。實驗結果顯示，以18S rRNA基因進行牛樟芝菌株的分子鑑定，確認本實驗所用的三株菌株都與食品工業研究所的菌株BCRC35398序列相同，確認為牛樟芝。在人工培養基的營養源和成份遞減實驗中，發現各菌株在適合的培養條件下，都有菌絲形態、顏色改變及原基的發生，但無子實體的多孔性狀，顯示牛樟芝菌株對於子實體的形成，具有菌株特異性。而在即時定量PCR中，分析的四條基因中，cytochrome P450及glutathione-S-transferase在子實體發生時，表現量也隨之增高，推測這兩條基因在子實體形成上，可能扮演重要角色。

Antrodia cinnamomea is a endemic fungal species in Taiwan which only parasitizes inside the decayed heart wood of Cinnamonum kanehirae. Recent researches indicated that A. cinnamomea extractions had the medicinal effects in anti-cancer, anti-inflammation, anti-hypertension, anti-oxidation and diabetes mellitus. A. cinnamomea has, therefore, become the target of illegal harvesting due to its low production and high price. This study characterized four strains of A. cinnamomea based on 18S rRNA gene sequencing. These four different strains of A. cinnamomea were cultured on solid-state cultivate media with adjusted major ingredients and reduced nutrients in a dark environment at room temperature for 8 weeks. Moreover, cDNA’s were isolated from inner mycelia, middle mycelia and outer mycelia (or fruiting body) of A. cinnamomea. Those cDNA’s were later used in real-time PCR to analyze the relative abundance of aging genes, cytochrome P450, glutathione-S-transferase, peroxiredoxin and manganese superoxide dismutase. The results showed that the four strains of A. cinnamomea carried the same 18S rRNA gene sequencing as the strain BCRC35398 from the Food Industry Research and Development Institute, inticating that they were A. cinnamomea. In the experiments of adjusting major ingredients and reducing nutrients of various solid-state cultivate media, we found that when strains were cultivated in suitable conditions, they grew into mycelia form, changing color and forming primordia. However, they did not carry the porous morphological characteristics. This result reveals the capacity of forming the fruiting body of A. cinnamomea varies differently among different strains. In the experiment of real time PCR, two of the four genes analyzed, cytochrome P450 and glutathione-S-transferase show increased expression levels during fruitification. Suggesting that this two genes play an important role in the formation of fruiting body.

中文摘要--------------------------------------------------------------------------Ι
英文摘要--------------------------------------------------------------------------III
研究目的--------------------------------------------------------------------------V
目錄------------------------------------------------------------------------------VI
圖、表目錄-----------------------------------------------------------------------XII
第一章 文獻回顧-----------------------------------------------------------------1
一、牛樟樹簡介--------------------------------------------------------------1
二、牛樟芝的介紹------------------------------------------------------------1
三、牛樟芝的命名經過------------------------------------------------------2
四、牛樟芝在分類學上的地位---------------------------------------------3
五、牛樟芝的型態與生長環境---------------------------------------------3
六、牛樟芝的化學成份------------------------------------------------------4
1.一次代謝物-----------------------------------------------------------5
2.二次代謝物-----------------------------------------------------------5
3.多醣體-----------------------------------------------------------------5
4.三萜類-----------------------------------------------------------------6
七、牛樟芝的功效與價值---------------------------------------------------8
1.保護肝臟--------------------------------------------------------------8
2.抗氧化能力----------------------------------------------------------10
3.增強免疫力及抗癌-------------------------------------------------12
4.抗B型肝炎病毒----------------------------------------------------13
5.抗發炎----------------------------------------------------------------14
6.降血糖----------------------------------------------------------------15
7.抗高血壓-------------------------------------------------------------16
8.安全性----------------------------------------------------------------16
第二章 利用18S rRNA基因序列進行牛樟芝的分子鑑定--------------19
一、前言---------------------------------------------------------------------19
二、材料方法---------------------------------------------------------------20
1.牛樟芝的菌株與來源--------------------------------------------20
2.去氧核醣核酸萃取-----------------------------------------------20
3.設計引子-----------------------------------------------------------21
4.聚合酵素鏈鎖反應-----------------------------------------------21
5. DNA clean up-----------------------------------------------------22
6.去氧核醣核酸定序-----------------------------------------------23
三、結果與討論------------------------------------------------------------23
第三章 牛樟芝的栽培及人工固態培養基---------------------------------25
一、前言---------------------------------------------------------------------25
1.牛樟芝的培養條件-----------------------------------------------25
2.牛樟芝的培養方式-----------------------------------------------25
3.人工固態培養基的子實體生成--------------------------------26
4.人工培養基養分遞減對牛樟芝生長之影響-----------------27
二、材料方法--------------------------------------------------------------28
1.牛樟芝的人工固態培養基--------------------------------------28
2.牛樟芝的繼代培養-----------------------------------------------29
3.人工培養基的營養源置換--------------------------------------29
4.人工培養基的成份遞減-----------------------------------------30
三、結果與討論-----------------------------------------------------------31
第四章 即時定量PCR---------------------------------------------------------37
一、前言--------------------------------------------------------------------37
二、材料方法--------------------------------------------------------------38
1. Total RNA的抽取------------------------------------------------38
2.逆轉錄PCR--------------------------------------------------------39
3.設計引子-----------------------------------------------------------39
4.即時定量PCR-----------------------------------------------------39
三、結果與討論-----------------------------------------------------------40
第五章 總結---------------------------------------------------------------------45
第六章 參考文獻---------------------------------------------------------------47



圖、表目錄
圖1-1、牛樟芝子實體的外觀--------------------------------------------------57
圖2-1、核醣體基因圖譜和引子的相對位置--------------------------------58
圖2-2、牛樟芝18S 核醣體基因序列中，NS1，NS3，NS4至NS8的片段。------------------------------------------------------------------------------59
圖3-1、原基示意圖-------------------------------------------------------------60
圖3-2、牛樟芝四個菌種在50% MEA上培養八周的觀察----------------61
圖3-3、牛樟芝Strain A在薏仁培養基(JTA Medium)的觀察------------62
圖3-4、牛樟芝Strain A在綠豆培養基(MGA Medium)的觀察----------63
圖3-5、牛樟芝Strain A在馬鈴薯培養基(PDA Medium)的觀察--------64
圖3-6、牛樟芝Strain A在台梗九號培養基(RICE Medium)的觀察----65
圖3-7、牛樟芝Strain B在薏仁培養基(JTA Medium)的觀察------------66
圖3-8、牛樟芝Strain B在綠豆培養基(MGA Medium)的觀察----------67
圖3-9、牛樟芝Strain B在馬鈴薯培養基(PDA Medium)的觀察---------68
圖3-10、牛樟芝Strain B在台梗九號培養基(RICE Medium)的觀察--69
圖3-11、牛樟芝Strain ACI3在薏仁培養基(JTA Medium)的觀察------70
圖3-12、牛樟芝Strain ACI3在綠豆培養基(MGA Medium)的觀察----71
圖3-13、牛樟芝Strain ACI3在馬鈴薯培養基(PDA Medium)的觀察--72
圖3-14、牛樟芝Strain ACI3在台梗九號培養基(RICE Medium)的觀察--------------------------------------------------------------------------------------73
圖3-15、牛樟芝Strain O在薏仁培養基(JTA Medium)的觀察-----------74
圖3-16、牛樟芝Strain O在綠豆培養基(MGA Medium)的觀察---------75
圖3-17、牛樟芝Strain O在馬鈴薯培養基(PDA Medium)的觀察-------76
圖3-18、牛樟芝Strain O在台梗九號培養基(RICE Medium)的觀察---77
圖4-1、peroxiredoxin分別在牛樟芝Strain A，Strain B及Strain O的表現量差異--------------------------------------------------------------------78
圖4-2、glutathione-S-transferase分別在牛樟芝Strain A，Strain B及Strain O的表現量差--------------------------------------------------------------------79
圖4-3、manganese superoxide dismutase分別在牛樟芝Strain A，Strain B及Strain O 的表現量差異------------------------------------------------------80
圖4-4、cytochrome P450分別在牛樟芝Strain A，Strain B及Strain O的表現量差異--------------------------------------------------------------------------81
表2-1、本次實驗所使用的菌株編號及來源--------------------------------82
表2-2、18S核醣體基因序列分析所使用的引子---------------------------83
表3-1、目前牛樟芝培養的方式----------------------------------------------84
表3-2、牛樟芝各菌種培養八周後，具有原基出現的培養基------------85
表4-1、即時定量PCR所使用的引子列表----------------------------------86
表4-2、牛樟芝Strain A菌絲體之基因Ct值--------------------------------87
表4-3、牛樟芝Strain B菌絲體及子實體之基因Ct值--------------------88
表4-4、牛樟芝Strain O菌絲體之基因Ct值--------------------------------89
表4-5、牛樟芝Strain A菌絲體基因的delta Ct值(ΔCt)------------------90
表4-6、牛樟芝Strain B菌絲體及子實體基因的delta Ct值(ΔCt)------91
表4-7、牛樟芝Strain O菌絲體基因的delta Ct值(ΔCt)------------------92
表4-8、牛樟芝Strain A菌絲體基因的deltadelta Ct值(ΔΔCt)及2-delta deltaCt值-----------------------------------------------------------------------------93
表4-9、牛樟芝Strain B菌絲體基因的deltadelta Ct值(ΔΔCt)及2-delta deltaCt值-----------------------------------------------------------------------------94
表4-10、牛樟芝Strain O菌絲體基因的deltadelta Ct值(ΔΔCt)及2-delta deltaCt值-----------------------------------------------------------------------------95

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