Molecular Cloning, Recombinant Gene Expression and Antifungal Activity of Cystatin from Taro (Colocasia esculenta L. Schott )
Date Issued
2005
Date
2005
Author(s)
Yang, Ai-hua
DOI
zh-TW
Abstract
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
Subjects
半胱胺酸蛋白酶
對微生物抗性
芋頭
抑制劑
taro
cysteine proteinase inhibitor(cystatin)
cysteine proteinase
antifungal
Type
other
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