愛玉子瘦果第一類病程相關蛋白之選殖、表達與功能特性分析

愛玉子(Ficus awkeotasang Makino)屬台灣特有種植物之一。用水搓洗愛玉種子後所形成之愛玉凍可作為炎炎夏日在台灣的傳統消暑飲料。愛玉凍內含有不少蛋白質，其中已鑑定出的含量較多的蛋白質包括果膠甲基酯酶(pectin methylesterase,PME)、幾丁質水解酶(chitinase)和似索馬甜蛋白(thaumatin-like protein, TLP)。由MALDI-TOF分析結果顯示，愛玉凍中可能含有微量的病程相關之蛋白，如第一類病程相關之蛋白(PR-1)等。PR-1可能在宿主植物防禦系統中發揮其作用。根據pI值將PR-1分為酸性及鹼性兩大類。於此論文，我們利用PCR技術選殖出愛玉PR-1的三個全長(PR1-1、PR1-2、PR1-4)以及一轉譯後含蓋部分成熟蛋白(PR1-3)的cDNA片段。預測這些成熟蛋白(PR1-1至PR1-4)的pI值依次是6.44、7.12、8.25和8.92，其成熟蛋白分子量各為14.8、21.3、16.0和16.0 kDa。重組PR1-1、PR1-2及PR1-3在大腸桿菌中以包涵體形式表達，並完成其抗體之製備。進一步以西方墨點法分析偵測到愛玉凍蛋白中含有天然PR-1蛋白。在酵母菌Pichia系統成功地分泌表達重組PR1-1、PR1-3及PR1-4。且純化後的PR1-1蛋白對Glomerella cingulata具有抑制真菌活性。我們進一步的研究發現，純化後的 PR1-3及PR1-4之結構不僅以單聚體(monomer)形式也可能以二聚體(dimer)之形式存在。活性測試結果推測 PR1-3可能具有極微量serine蛋白酶活性。

關鍵字

愛玉；第一類病程相關之蛋白；抗真菌；抗細菌

並列摘要

Jelly fig (Ficus awkeotasang Makino) is one of the endemic plants in Taiwan. Water extract from jelly fig achenes is utilized to produce jelly curd for making a popular drink in local markets. MALDI-TOF analysis suggested that there is pathogenesis related-1 protein (PR-1) in jelly curd. Plant PR-1s may play a role in host defense system, and are classified into acidic and basic types based on pI value. Here, four cDNA fragments encoding three full-length (PR1-1, PR1-2 and PR1-4) and one partial-length of jelly fig PR-1 isoforms (PR1-3) were obtained by PCR cloning. The pI values of these predicted mature proteins are 6.44, 7.12, 8.25 and 8.92, and their predicted mature molecular mass were 14.8, 21.3, 16.0 and 16.0 kDa, respectively. PR1-1, PR1-2 and PR1-3 over-expressed as insoluble proteins in Escherichia coli were used to raise antibodies. Western blot analyses suggested that PR-1 isoforms may presence as minor pericardial proteins extracted from jelly fig achenes. PR1-1, PR1-3 and PR1-4 were successfully expressed in yeast Pichia. The purified PR1-1 exhibited antifungal activity toward Glomerella cingulata. In addition, we found that PR1-3 and PR1-4 may form dimers structure. Moreover, PR1-3 probably exhibits weak serine protease activity using azocasein as substrate.

並列關鍵字

Jelly fig ； PR-1 ； antifungal ； anti-microbial

參考文獻

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愛玉子瘦果第一類病程相關蛋白之選殖、表達與功能特性分析

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