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Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1)

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Abstract

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5′-/3′-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 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 belonging to Monocot. Recombinant GST–CeCPI fusion protein was overexpressed in Escherichia 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. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 μg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150–200 μg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.

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Abbreviations

CeCPI:

Taro cysteine protease inhibitors

GST:

Glutathione-S-Transferase

IPTG:

Isopropyl -β-D-thiogalactoside

BANA:

N-benzoyl-DL-arginine-2-naphthylamide

E64:

trans-epoxysuccinyl-L-leucylamino (4-guanidino)-butane

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Acknowledgements

This work was financially supported by National Science Council grants (NSC-89-231-B-002-018, NSC-89-231-B-002-055, and NSC-90-2311-B-002-009). We are also grateful to Dr. Shun-Fa Tai (Kaoshiung District Agricultural Improvement Station) for kindly supplying all taro cultivars employed in this study. The authors thank Dr. Ashok Kumar for critically reading the manuscript.

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Correspondence to K. W. Yeh.

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Yang, A.H., Yeh, K.W. Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1). Planta 221, 493–501 (2005). https://doi.org/10.1007/s00425-004-1462-8

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