利用同源甘油醛-3-磷酸脫氫酶啟動子進行金針菇轉形系統之研究

Study on the transformation system of Flammulina velutipes(Curt.:Fr.)Sing.using homologous glyceraldhyde-3-phosphate dehydrogenase promoter

周書瑜(Chou, Shu-Yu)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01094

摘要

基因轉形技術能夠使特定基因在不同物種間移動。利用此技術除了進行基因體學之相關研究外，亦可將一物種之基因轉移至另一物種進行異源表達。隨著絲狀真菌於分子生物學及基因轉形之發展，食用菇類於基因轉形之研究與應用在過去十年也逐漸引起科學家的重視。
本論文以台灣地區最大宗生鮮食用菇類金針菇為試驗材料，進行其轉形系統之相關研究。本研究中成市堨艉妒鱁w菇轉形系統，是以發芽之金針菇擔孢子為材料，經酵素處理後利用電穿孔法進行轉形，並使用金針菇之甘油醛-3-磷酸脫氫酶（gpd）啟動子及大腸桿菌之潮黴素抗藥性基因（hygromycin B phosphotransferase gene, hph）為篩選標記。由電穿孔條件測試結果顯示，以濃度為4 mg/mL 之lysing enzyme處理擔孢子2小時，並以電穿孔條件為25 μF、100 Ω、12.5 kV/cm或25 μF、200 Ω、5 kV/cm進行電脈衝，能夠得到最佳之轉形效率為每μg DNA可得到51至56株的轉形株。另外，利用所建立之轉形系統與同源之gpd啟動子，能夠成左穛{異源基因hph及β-glucuronidase（GUS）基因於金針菇體中。轉形株經南方氏雜合分析得知轉入之目標基因是以隨機的方式插至金針菇的染色體DNA中，並由穩定度之實驗結果亦得知，轉形株所呈現hygromycin
B抗藥性能夠穩定存在至少三個月。除此之外，經進一步分析金針菇之gpd啟動子發現到，使用轉譯起始點上游約336 bp之啟動子核酸序列長度就足以啟動下游基因之表現。

關鍵字

電穿孔 ； 金針菇 ； 啟動子 ； 轉形系統 ； 甘油醛-3-磷酸脫氫酶

並列摘要

Transformation is a powerful technology whereby genes can be transferred within or between different species. The application of transformation is not only to aid the studies in genetic, but also can express heterologous genes in specific hosts. As the progress of molecular biology and genetic transformation of filamentous fungi were developed, the applications of transformation to edible mushrooms attracted scientists’ attention for the past decade.
The goal of this study is to establish a transformation system of Flammulina velutipes, the major fresh edible mushroom in Taiwan. A simple, reliable, and efficient transformation procedure has been developed using electroporation of germinated basidiospores, a F. velutipes glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter and a E. coli hygromycin B phosphotransferase gene (hph) as a selectable marker. The highest transformation efficiencies, 51-56 transformants per μg of DNA, were achieved with using basidiospores pretreated by 4 mg/mL lysing enzyme and electrioporation conditions as following : 25 μF capacitance, 100 Ω resistance, 12.5 kV/cm field strength and 25 μF capacitance, 200 Ω resistance, 5 kV/cm field strength. In addition, we demonstrated that heterologous genes, hph and β-glucuronidase (GUS) gene, could be successfully expressed in F. velutipes using this transformation procedure and the homologous gpd promoter. Southern analysis of transformants indicated the target genes randomly integrated into F. velutipes genome. The results of the stability test of the target gene revealed that the hygromycin B resistance of transformants was maintained stably for at least three months. Furthermore, The deletion analysis of the gpd promoter suggested that the 336 bp of the promoter region upstream from the transcription start point should be sufficient to express the downstream gene.

並列關鍵字

gpd promoter ； GUS ； Flammulina velutipes ； Transformation system ； electroporation ； hygromycin B resistance

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