由中央研究院植物所 蕭介夫老師實驗室 楊智元博士在研究青花菜老化的扣除雜交實驗中發現到一個抗真菌蛋白基因片段(Bo-AFP3)，大小約為240 bp並含有一個signal peptide (30 amino acid)，預測之分子量為8.6 kDa、pI值為8.23，屬於antifungal protein中的defensins，具有高度的保留區，含有8個cysteine，預測可形成4個雙硫鍵。其序列與木瓜中的AFP3 (Cp-AFP3)相似度達到95.92 % 。 將此基因轉殖到Pichia pastoris及Escherichia coli中大量表現，在Pichia系統中，可以觀察到所產生的抗真菌蛋白質對Rhizoctonia solani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、完全展開葉與黃葉，有明顯的表現，並且會受到不同的植物荷爾蒙、黑暗處理以及切傷處理，而有所改變；經由RT-PCR的分析ABA、GA、JA、IAA、乙烯、切傷、黑暗處理等，皆會誘導Bo-AFP3基因的表現，而BA與SA則會延緩Bo-AFP3基因的表現。 An antifungal protein gene (Bo-AFP3) was isolated from broccoli by subtractive hybridization method. Bo-AFP3 encoded a 79-amino-acid protein containing a 30 amino-acid signal peptide in the N-terminal end. The predicted molecular weight was 8.6 kDa and its pI value was 8.23. Bo-AFP3 structure belongs to plant defensins AFP3 family with eight highly conserved cysteine residues linked by 4 disulfide bridges. The deduced amino acid sequences of Bo-AFP3 and Cp-AFP3 shared 95.92 % similarity Bo-AFP3 protein was overexpressed in Pichia pastoris and Escherichia coli system. The recombinant Bo-AFP3 expressed in P. pastoris was shown to possess antifungal activity against Rhizoctonia solain. RT-PCR analysis showed that Bo-AFP3 gene was expressed in root, mature green and yellow leaves. It was affected by different plant hormone, wounding and dark treatment. RT-PCR analysis showed ABA, GA, JA, IAA and ethylene induced Bo-AFP3 gene expression, while BA and SA delay the expression.
