以農桿菌媒介轉形法作為鮑魚菇異源表達之工具

Agrobacterium tumefaciens mediated transformation as a tool for heterologous expression in Pleurotus ostreatus

蔡媛禎(Yuan-Chen Tsai)

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(2009年)

https://doi.org/10.6342/NTU.2009.00625

摘要

鮑魚菇在食用菇中的產量僅次於洋菇，其栽培容易且可利用多種農業廢棄材做為基質，受到各國食用菇農業的青睞；建立穩定的鮑魚菇轉形方法可做為發展
鮑魚菇作為異源表達系統之工具，進而應用至食用疫苗的開發。
農桿腫瘤菌被譽為「自然的基因工程師」，農桿腫瘤菌媒介之轉形法相較其他轉形方法有操作便利、耗費較低等優點。轉入的異源基因多以單一嵌入位嵌入宿主染色體內，因此能穩定表現。在本研究中，我們成功建立了鮑魚菇以農桿菌媒介之轉形系統，將質體帶有不同啟動子啟動報導基因綠色螢光蛋白質(Green fluorescent protein, GFP)與篩選標記潮黴素磷酸轉移酶(Hygromycin phosphotransferase, hph)之農桿菌和鮑魚菇共培養之測試結果，鮑魚菇以蕈褶組織做為轉形材料與農桿菌株(AGL-1、LBA4404)共培養能夠得到較多轉形株，約80~90%的轉形效率，而均質菌絲液為轉形材料得到較少轉形株，以菌絲塊則無法得到轉形株。螢光顯微鏡結果顯示，花椰菜鑲嵌病毒35S啟動子(CaMV 35S)與洋菇的甘油醛-3-磷酸脫氫酶 (glyceraldehyde-3-phosphate dehydrogenase, GPD) 啟動子啟動之綠色螢光蛋白質傾向呈現點狀螢光，類似聚集體(aggresome)的情形；當鮑魚菇GPD啟動子啟動C端融合內質網停留訊息胜肽的GFP時，亦有點狀綠色螢光分佈菌絲內。而鮑魚菇、金針菇GPD啟動子啟動之 GFP 則在菌絲內呈現分散的螢光。推測融合訊息胜肽可能有助於鮑魚菇內GFP的累積而導致GFP聚集。
以三明治法酵素連結免疫吸附分析轉形株內可溶性GFP，每克總可溶性蛋白質含量最高為43.83 ng。此外，異源基因在轉形株內於有絲分裂與減數分裂後皆能穩定表現。

關鍵字

鮑魚菇 ； 農桿菌媒介轉形 ； 甘油醛-3-磷酸脫氫酶啟動子 ； 綠色螢光蛋白質

並列摘要

Pleurotus ostreatus is the second largest edible mushrooms next to Agaricus bisporus. P. ostreatus is popular worldwide due to ease of and use of agricultural wastes as substrates. Establishing a stable transformation system of P. ostreatus may provide a tool for its heterologous expression and even oral vaccine production in the future. Agrobacterium tumefaciens has been known as “natural engineer”. There are many advantages of Agrobacterium tumefaciens mediated transformation (ATMT) over other transformation methods such as convenience and less expenssive. The transformed heterologous gene can be integrated into host genome by mostly single insertion, and consequently more stable.
In this study, we successfully developed the A.tumefaciens mediated transformation of Pleurotus ostreatus. Agrobacterium harboring plasmid with different promoters driving green fluorescent protein (GFP) and hygromycin phosphotransferase gene (hph) was co-cultivated with Pleurotus ostreatus. P. ostreatus gill tissues used as explants co-cultivating with A.tumefaciens AGL-1 and LBA4404 could get more transformants, with transformation efficiency around 80 ~ 90%. However, only a few of transformants were obtained when homogenized mycelia was used as starting material, and no transfomant was found using mycelia modified pellets (MMP). Fluorescent microscopic observation revealed that GFP can be driven by the califlower mosaic virus (CaMV) 35S promoter and Agaricus bisporus glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter, in which GFP tended to appear as aggresome-like fluorescent spot. When GFP gene with C terminal endoplasmic reticulum retention signal was driven by P. ostreatus homologous GPD promoters, the GFP also formed green fluorescent spots. In the other hand, the Flammulina velutipes GPD and P. ostreatus homologous GPD promoters without retention signal showed scattered green fluorescence. These results suggested that fused signal peptide led to GFP accumulation in P. ostreatu as aggregates. The highest amount of soluble GFP extracted from P. ostreatus detected by ELISA is 43.83 ng per gram total soluble protein. Besides, heterologous gene could be stably expressed after mitosis and meiosis.

並列關鍵字

Pleurotus ostreatus ； Agrobacterium tumefaciens mediated transformation (ATMT) ； glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter ； green fluorescent protein (GFP)

參考文獻

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