木瓜幾丁質?之選殖與分析

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Title:	木瓜幾丁質?之選殖與分析
Other Titles:	Cloning and analysis of papaya chitinase.
Authors:	黃怡萍 Huang, I-Ping
Contributors:	蔡正宗;蕭介夫 Tsai, Tsun-Chuang;Shaw, Jei-Fu 東海大學食品科學系
Keywords:	木瓜;幾丁質? papaya;chitinase
Date:	2004
Issue Date:	2011-07-26T03:56:36Z (UTC)
Abstract:	由中央研究院植物所蕭介夫老師實驗室陳玉婷博士在研究木瓜老化的扣除雜交實驗中發現到一個幾丁質?基因片段，大小約為288 bp，即由此片段設計primer而夾出幾丁質?全長基因(CpCHI)，預測之分子量為29.1 kDa、pI值為6.59，此全長基因含有一個訊號??約28個胺基酸，而其成熟蛋白 (無訊號??)預測之分子量為26.2 kDa、pI值為6.32。其胺基酸序列與植物中第四類的幾丁質?(例如：歐洲種金香葡萄的VvCHI及阿拉伯芥的AtCHI )在演化上最相近，因而推測CpCHI屬於第四類的植物幾丁質?。將此基因轉殖到大腸菌中大量表現，可以觀察到所產生的幾丁質?具有活性，以乙二醇幾丁質為基質進行水解，其最適反應pH 值為6，最適反應溫度為30 ℃；進行抗菌實驗可觀察到CpCHI對Fusarium oxysporum有抗真菌活性表現。另外此蛋白質的基因表現在老化的組織中，如根及老葉，有明顯增加表現，並且會受到不同的植物荷爾蒙、乙烯處理以及切傷處理，而有所改變；經由RT-PCR及南方點墨法的分析後發現ABA、IAA、乙烯、切傷處理等，皆會誘導CpCHI基因的表現。 A chitinase cDNA clone (CpCHI, 288 bp) was isolated from papaya fruit CpCHI encoded a 96-amino-acid protein containing a 28 amino-acid signal peptide in the N-terminal end. The predicted molecular weight was 29.1 kDa and its pI value was 6.59. The predicted molecular weight of mature protein was 26.2 kDa and its pI value was 6.32. Based on its amino acid sequence homology with other plant chitinases, it was classified as class 4 chitinase which includes VvCHI and AtCHI. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed optimum activity at pH 6 and the optimum reaction temperature at 30 ℃.The recombinant CpCHI expressed in E. coli was shown to possess antifungal activity against Fusarium oxysporum. RT-PCR analysis revealed that CpCHI was upregulated in senescent organ such as root and old leaf. RT-PCR analysis also showed CpCHI gene was upregulated by the ABA, IAA, ethylene and wounding treatment.
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