Molecular Cloning, Recombinant Gene Expression and Antifungal Activity of Cystatin from Taro (Colocasia esculenta L. Schott )

• Main Authors: Ai-hua Yang, 楊藹華
• Other Authors: Kai-Wen Yeh
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/46938736587555092229

博士 === 國立臺灣大學 === 植物科學研究所 === 93 === Taro (Colocasia esculenta L. Schott) is one of the oldest cultivated crops grown for its edible corms and leaves. Taro corm is a well-differentiated organ and contains approximately 1.4 ~ 3.0% storage proteins on a fresh weight basis. In order to investigate the proteinase inhibitors of storage protein in corm of four local taro cultivars, the affinity analysis of the cystatin/papain was performed by using the mild denaturing gelatin /PAGE. Results from substrate polyacrylamide gel electrophoresis revealed that the cultivar-Kaohsiung No. 1 contains cystatin in corm. Subsequently, a cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from corm of taro using both degenerated primers/RT-PCR amplification and 5’- / 3’- RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 205 amino acid residues, with deduced MW. 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to eudicots rather than to monocots, despite taro belongs to monocot. Recombinant GST-CeCPI fusion protein was overexpressed in E.coli and its inhibitory activity against papain was identified on gelatin/ SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. The investigation of its antifungal activity clearly revealed the toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at the concentration of 80 μg of recombinant CeCPI per ml. Moreover, the mycelium growth was completely inhibited and the sclerotia lysed at the concentration of 150 μg – 200 μg per ml. Further studies have demonstrated that the recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium. The predicted tertiary structure of CeCPI was modeled based on the known crystal structure of the rice OC-I, which shares 50.5% (48/95) sequence identity with the taro cystatin. The data indicate that tarocystatin consists of six?helix and seven-strand antiparallel β-sheet. Using polymerase chain reaction (PCR) approaches (Table 1), six conserved amino acids, Gln48, Gln49, Trp80, Trp114, Ser134 and Ile135 were chosen for site-directed mutagensis. The mutation at Q49→P lead to a significant loss in the papain-inhibitor activity of tarocystatin. The W80 →G and W114 →G mutants showed a higher resistance to Sclerotium rolfsii than the wild type, although they also lost most inhibitory activity toward papain.