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標題: 彩虹草腺毛形態與外泌蛋白分析
Analysis of secretory trichome and protein in carnivorous plant Byblis guehoi
作者: 陳冠于
Alvin K.Y. Chen
關鍵字: 彩虹草
Alvin K.Y. Chen
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摘要: 食肉植物大多生長在土壤貧瘠的地區,並且各自演化出不同的捕食器官以便從其他生物身上獵取其所缺乏的氮源及磷源。現今對食肉植物的研究主要集中在他們的演化、形態以及少數物種的蛋白質體分析,鮮少有人在基因與轉殖上多作著墨。谷霍腺毛草 (Byblis guehoi) 又稱為彩虹草,有著生長快速、構造簡單且佈滿大量分泌型腺毛的特性,所以我們選擇用它做為研究對象。經由共軛交顯微鏡分析發現彩虹草的葉子上共有三種腺毛,長型、短型與基底型。以組織貼盤測試可知基底型腺毛可分泌蛋白酶與磷酸酶,然而長型與短型腺毛則無明顯上述活性。經嘗試多種萃取與純化方式後,我們成功從極具黏性的葉子表面收集到蛋白,膠體電泳分析得知蛋白主要集中於 72 kDa 的位置,將之回收進行蛋白質質譜身分鑑定後,發現其中至少有五種可能的蛋白質,包括蛋白酶與磷酸酶等,進一步使用陰離子交換樹脂 (Q-column)分離蛋白質,發現蛋白酶與磷酸酶這兩種主要蛋白的電性差異極大。為了找出主要蛋白的基因序列,將質譜比對到的物種蛋白進行比對分析後,設計退化引子 (degenerate primer),並以彩虹草的 cDNA 為模板 進行PCR 放大後,已得到部分的磷酸酶序列片段,且並非NCBI網站已有的序列片段。未來將進行 RACE 找出目標蛋白的基因序列全長,再向上游搜尋相應的啟動子序列 (secretion signal peptide)。而在轉殖系統的建構上,初步發現彩虹草可以透過農桿菌的感染暫時性表現 GUS 活性,暗示未來可使用農桿菌進行彩虹草的基因轉殖。由組織培養測試可知彩虹草對 kanamycin 有很高的抗性,然而對 hygromycin 卻非常的敏感。後續的實驗將使用分泌型綠色螢光蛋白表現載體,對彩虹草的外泌系統進行評估與測試。本實驗的最終目標是使用彩虹草生產外源蛋白,尤其是需要被外泌的蛋白,並達到只需洗過就能回收的效果。
Carnivorous plants usually grow in nutrient-poor wetlands. They have evolved specialized organs to capture and digest small organisms for nutrition. Researches today for carnivorous plants are mostly focused on their evolution, morphology, classification, with some proteomic studies but rarely at the gene levels. Here we used Byblis for further research due to its rapid growth, simple structure and very large amount of dew drop secretion. Three kinds of the trichomes, long, short and basal forms, were observed under confocal microscope. Tissue printing on substrate plate revealed activities of protease and phosphatase, likely secreted from the basal forms but not long or short forms of the trichome. To recover proteins from the viscous dew drop, several extraction methods were tested. One major banding at about 72 kDa was detected on SDS-PAGE. LC-MS/MS analysis reveals five candidate proteins including one subtilisin-like protease and one purple acid phosphatase. These two proteins showed very different affinities in the ion-exchanger (Q column) chromatography, suggested their differences in isoelectric points of proteins. We performed degenerate PCR on Byblis cDNA and obtained one DNA fragment that putatively encodes purple acid phosphatase. Further RACE and genomic PCR will be used to obtain the full-length cDNA and promoter fragments. Moreover, transient Agrobacterium infiltration to Byblis stem caused a prominent GUS signal, suggested that the Agrobacterium-mediated transformation may be feasible. Antibiotic resistance test showed that Byblis had a high tolerance to kanamycin but were very sensitive to hygromycin. Tissue culture and plant regeneration protocols will be tested for the purpose of Byblis transformation. Our final goal is to build up a Byblis bioreactor in which the engineered proteins can be secreted and harvested simply by wash.
文章公開時間: 2020-08-24
Appears in Collections:生物科技學研究所



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