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研究生:楊淳翔
研究生(外文):Chun-Hsiang Yang
論文名稱:光線對北蟲草二次代謝物及wc-1基因表現之影響
論文名稱(外文):Effects of light on the secondary metabolite and wc-1 gene expression in Cordyceps militaris
指導教授:陳淑德
指導教授(外文):Su-Der Chen
口試委員:林世斌鄭永祥孫舜國
口試委員(外文):Shih-Bin LinYeong-Hsiang ChengShun-Kuo Sun
口試日期:2014-01-07
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術與動物科學系生物技術碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:161
中文關鍵詞:北蟲草蟲草素光誘導LOV功能區塊wc1基因光形態發生
外文關鍵詞:Cordyceps militariscordycepinlight inductionLOV domainWC-1photomorphogenesis
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人工栽培的北蟲草(Cordyceps militaris)需要經過長時間且週期性的光線刺激下,才會開始由白色菌絲轉為黃色菌絲。在轉型成為黃色菌絲的同時,菌絲表面會出現很多直徑約1㎜,結實且尖凸的原基(primordium),且隨著光照的持續誘導下,原基會開始膨大並延伸,形成具有性生殖的世代,此為亮菊色且長棒狀的子實體,同時蟲草素(cordycepin)也會大量地在子實體中累積。故以各種光線(紅光、藍光、白光和黑暗)去刺激蛹蟲草,並觀察液態培養、培養皿培養及可形成子實體之固態培養狀況下,結果顯示,蛹蟲草經過紅光的誘導下,雖然菌絲不會因為紅光的刺激而產生黃色色素,但是其蟲草素含量為最高。另外也發現在藍光可誘導促進子實體形成,該菌種可能是藉由含LOV domain的蛋白質進行光線感應,並引發產生子實體的訊息調控。進一步參考各文獻中提出其他物種對光誘導具有感應能力的功能性蛋白之基因高度保留區設計引子,抽取子實體的Total RNA進行RT-PCR轉成cDNA之後,用設計的引子進行PCR。在PCR產物中,找到一條與Neurospora crassa wc-1 基因有高度相似度的序列;具有PAS domain及Zinc finger domain。以此序列透過Q-PCR等方法研究北蟲草之光誘導前菌絲、光誘導後之菌絲、原基及子實體各階段的成長下,發現除了高度4㎜的子實體之wc1 mRNA表現量明顯偏低外,隨著子實體的形成而wc1 mRNA表現量均逐漸增加。
The artificial cultivation of Cordyceps militaris needs light induction with long period of time and daily cycle to turn the color of mycelium from white to yellow. There are many solid and pin shape primordia appeared on culture surface during production of yellow mycelium. Primordia grows bigger and elongates to form a sexual generation, and then to form fruiting bodies with bright orange color and long stick rod shade containing enormous amount cordycepin with continuous light induction. Therefor various light conditions (red, blue, white, and dark) was used to induce Cordyceps militaris in liquid culture, plate culture and solid-state culture for fruiting body formation. The results showed that Cordyceps militaris with red light induction did not obtain yellow fruiting body, but it contained the highest amount of cordycepin. Moreover, only blue light induced Cordyceps militaris to form fruiting bodies, and the species could contain a light sensing protein with LOV domain (Light-Oxygen-Voltage domain), and transfer signal to form fruiting body. To investigate the light on the gene expression of Cordyceps militaris, the primers were designed for other species with a highly conserved gene region which could translate a functional protein sensing the light used for PCR. The Total RNA from fruiting bodies was extracted and transformed into cDNA by reverse-transcription PCR, then amplified by PCR with the primers. In the PCR amplification, a sequence was found with high genetic similarity to Neurospora crassa wc-1 gene which contained PAS and Zinc finger domain. Furthermore, investigating wc1 mRNA exprestion from mycelium without light inducing or with light inducing, primordia and fruiting body in Cordyceps militaris was extracted, and the difference of expression level in these different growth stages was studied.
中文摘要 I
Abstract II
總目錄 IV
表目錄 VIII
圖目錄 IX
壹、文獻回顧 1
一、前言 1
二、北蟲草 3
(一) 北蟲草簡介 3
(二) 商業用途 5
(三) 毒性研究 8
(四) 液態培養 9
(五) 固態培養 10
(六) 北蟲草代謝物 11
(七) 光照對北蟲草培養的影響 13
(八) 生理活性和功效 14
三、光誘導之研究 22
(一) 光與生物的關係 22
(二) 光誘導與真菌的關係 27
(三) 光誘導之真菌行為表現案例 33
(四) Neurospora crassa的光誘導與生物週期的關係 38
(五) LOV domain特性 43
四、北蟲草培養問題及研究動機 44
(一) 光誘導與北蟲草的關係 44
(二) 探討的問題 47
貳、研究目的 48
參、實驗架構 49
一、光線對北蟲草生長影響的實驗設計與架構圖 49
二、藍光受器及Actin基因找尋與表現量變化之實驗架構圖 50
肆、材料與方法 51
一、實驗藥品材料及設備 51
(一) 實驗藥品材料 51
(二) 實驗設備 52
二、北蟲草培養方法 54
(一) 菌種 54
(二) 平盤培養 54
(三) 液態培養 55
(四) 固態培養及子實體誘導 56
(五) 菌種保存 57
三、光誘導方法 57
(一) 光模組設計 57
(二) 液態培養光誘導模組 58
(三) 平盤及固態培養光誘導模組 58
四、分析方法 59
(一) 蟲草素及腺苷之HPLC分析方法 59
(二) 光誘導色素變化分析 61
五、基因表現實驗方法 62
(一) 樣品收集 62
(二) Total RNA抽取 62
(三) RT-PCR、純化cDNA及定量 66
(四) PCR引子設計及PCR條件 69
(五) 製作洋菜電泳膠及進行電泳 69
(六) q-PCR 69
六、統計分析 70
伍、結果 71
一、光線對平盤培養的影響 71
二、光線對北蟲草液態培養的影響 74
三、光線對北蟲草固態培養的影響 75
四、藍光受器及Actin基因的找尋 76
五、藍光受器及Actin基因在北蟲草栽培過程中的表現變化 80
陸、討論 82
柒、結論 88
捌、參考文獻 89
玖、圖表 105

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