愛玉瘦果第四類病程相關蛋白之不同區域的外源表達及其特性分析

Heterogenous Expression and Characterization of Different Domain of Jelly Fig Pathogenesis Related-4 Protein

林一弘(Yi-Hung Lin)

指導教授 ： 周文敏

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6827/NFU.2014.00069

摘要

愛玉子 (Ficus awkeotasang Makino)為台灣特有種植物，用水搓洗愛玉種子所製得的愛玉凍為夏日常見的消暑飲品；其中也含有不少蛋白質，已鑑定出的蛋白質包括果膠甲基酯酶 (pectin methylesterase, PME)、幾丁質水解酵素 (chitinase)及似索馬甜蛋白(thaumatin-like protein, TLP)，和微量的病程相關之蛋白，如:第一類病程相關蛋白 (PR-1)及第四類病程相關蛋白 (PR-4)。目前已知的PR-4可以分為N端含有額外的幾丁質結合區域的Class I，以及缺乏此區域的Class II。而愛玉PR-4基因(FaPR4) 已經被選殖出來。演譯所得之蛋白質N端含有額外的幾丁質結合區域，其C端有一小段液泡序列 (vacuolar signal)。利用引子設計及PCR技術將液泡序列去除得到FaPR4(noCt) cDNA片段，並成功地在酵母菌Pichia分泌表達出r-FaPR4(noCt)及r-FaPR4(noCt/mutant)。r-FaPR4(noCt/mutant)為r-FaPR4(noCt)中一個胺基酸發生點突變。FaPR4(NO)為去除幾丁質結合區域的愛玉PR-4。 r-FaPR4、r-FaPR4(NO)和r-FaPR4(NO/mutant)亦已在酵母菌Pichia分泌表達出來。將純化後的r-FaPR4、r-FaPR4(noCt) 、r-FaPR4(noCt/mutant)、r-FaPR4(NO)和r-FaPR4(NO/mutant)進行RNase活性分析，發現以不同鹽濃度沖提出的蛋白質具有不同程度的RNase活性。而重組蛋白之抗真菌能力分析結果發現r-FaPR4、r-FaPR4(noCt) 及r-FaPR4(noCt/mutant)對Glomerella cingulata及Fusarium oxysporum的孢子萌芽具有抑制活性，且重組蛋白經加熱後還具有一定的活性，其中以r-FaPR4的抗真菌活性最好。進一步研究結果顯示這些重組蛋白會使Rhizoctonia solani菌絲產生ROS反應 (reactive oxygen species)，其中以r-FaPR4所造成的ROS反應最明顯。

關鍵字

愛玉 ； 第四類病程相關蛋白 ； 核醣核酸酶抗真菌活性

並列摘要

Jelly fig (Ficus awkeotasang Makino) is one of the endemic plants in Taiwan. The water extract from jelly fig achenes forms jelly curd, a common summer beverage. Some pericarpial proteins extracted from the jelly curd have been identified as a pectin methylesterase (PME), chitinase, thaumatin-like protein (TLP), two PR proteins, pathogenesis related-1(PR-1) and pathogenesis related-4 (PR-4). PR-4 was classified into two groups, Class I contains extra chitin binding domain (CBD) in N-ternimus and Class II has no such domain. cDNA fragment coding for Jelly fig PR4 (FaPR4) has been cloned and its deduced protein has CBD in N-terminus and a vacuolar signal in C-terminus. Using designed primers and PCR technology obtained cDNA fragnmemt encoding FaPR4(noCt), a jelly fig PR-4 without vacuolar signal. FaPR4(noCt) and FaPR4(noCt/mutant) were successfully expressed in yeast Pichia pastoris. FaPR4(noCt/mutant) is a point-mutant of FaPR4(noCt). The purified r-FaPR4, r-FaPR4(noCt), r-FaPR4(noCt/mutant), r-FaPR4(NO) and r-FaPR4(NO/mutant) were subjected for RNase activity assay. The recombinant proteins eluted from different salt concentrate showed different RNase activity. Recombinantion r-FaPR, r-FaPR4(noCt)and r-FaPR4(noCt/mutant) exhibited antifungal activity toward Glomerella cingulata and Fusarium oxysporum. After heating at 100oC for 10 min, the recombination proteins remained some activity, and r-FaPR4 had the highest antifungal activity. In addition, recombination proteins could cause ROS response in Rhizoctonia solani hyphae, and r-FaPR4 has the most obvious effect on ROS response.

並列關鍵字

Jelly fig ； PR-4 ； RNase ； antifungal activity

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