建立藥用真菌樟芝的高效率基因轉殖系統

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標題:	建立藥用真菌樟芝的高效率基因轉殖系統 Development of high efficiency gene transformation system for basidiomycetous medicinal fungus Antrodia cinnamomea
作者:	洪婉禎
貢獻者:	江建民嘉南藥理科技大學：生物科技系暨研究所
關鍵字:	香菇GPD啟動子甘油醛-3-磷酸脫氫酶靈芝電轉殖樟芝農桿菌介導轉殖 ATMT Electroporation glyceraldehydes-3-phosphate dehydrogenase Ganderma lucidum Antrodia cinnamomea
日期:	2010
上傳時間:	2010-10-12 15:11:24 (UTC+8)
摘要:	樟芝(Antrodia cinnamomea)及靈芝(Ganderma lucidum)皆屬於藥用真菌，二者產生的二次代謝物，像是三萜類、多醣體、固醇類，具有抑制癌細胞、增加免疫力及抗發炎等的作用。然而對於這些二次代謝物的產生及累積方面的相關資訊仍是很少，尤其樟芝在成長速度來說過於緩慢，尚未達到大量生產之要求。本研究構築了含有香菇中取中取得的甘油醛-3-磷酸脫氫酶(glyceraldehydes-3-phosphate dehydrogenase，GPD)的啟動子來驅動篩選標記Hygromycin抗性基因的載體，已成功的利用了農桿菌介導轉殖法及電轉殖對樟芝的節孢子、菌絲及靈芝的菌絲、原生質體進行基因轉殖。農桿菌介導轉殖的最佳條件下以樟芝節孢子108作為宿主細胞可獲得230個穩定的轉殖株 ; 靈芝菌絲以0.1g作為宿主細胞可獲得64.3個轉殖株。電轉殖的最佳條件下樟芝節孢子108可獲得37個穩定轉殖株 ; 靈芝原生質體是以209個轉殖株。所獲得的轉殖株表現出色澤改變或生長較慢的不同性狀，利用PCR檢測GFP基因和hph基因，可確定基因轉入這些轉殖株中。Western blot偵測中GFP蛋白質的表現量並不是非常的明顯，在未來必須更深入探討。本研究所開發的方式可成功轉殖靈芝及樟芝，將有助於研究與二次代謝物合成之相關基因，以及改善其生長和二次代謝的生產。 Antrodia cinnamomea and Ganoderma lucidium are medicinal fungi that attract attention because of its effective inhibition on tumor proliferation and anti-inflammation by their secondary metabolites, such as triterpenoids and polysaccharides. However, the genetic information about the production and accumulation of these metabolites is rarely known. The growth rate of Antrodia cinnamomea is too slow to fit the commercial requirement. In this study, the vectors that consist of fragment of glyceraldehyde–3 phosphate dehydrogenase promoter from Lentinus edodes and the hygromycin as a selection marker were constructed for transformation. Via the Agrobacterium tumefaciens -mediated transformation (ATMT) and electroporation, the arthrospore and mycelia of Antrodia cinnamomea as well as mycelia and protoplast of Ganoderma lucidium, were successfully transformed against hygromycin. For ATMT, 230 stable transformants were obtained for 108 arthrospore of A. cinnamomea transformed; while 64.3 stable transformants were obtained for 0.01g mycelia of G. lucidium transformed. For electroporation, 37 stable transformants were obtained for 108 arthrospore of A. cinnamomea transformed, while 209 stable transformants were obtained for 108 protoplast of G. lucidium transformed, under the optimized condition. The stable transformants exhibit differential properties such as different color or slower growth rate that can be seen on the culture plate. PCR for hph and gfp genes indicated their insertion into chromosome, although the faint fluorescence and western blot signal of GFP indicated trace expression of gfp gene under this construct. The methods developed in this study provide efficient transformation of A. cinnamomea and G. lucidium, and may facilitate the scientific research of their secondary metabolite related genes and the possible improvement of the growth rate and accumulation of specific metabolite.
關聯:	校內校外均不公開，學年度：98，110頁
顯示於類別:	[生物科技系(所)] 博碩士論文

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