biologia plantarum

Biologia plantarum 46:175-179, 2003 | DOI: 10.1023/A:1022877905774

Brownish Acidic Protein Induced in Pumpkin Callus by a High Concentration of 2,4-Dichlorophenoxyacetic Acid

M. Fujita¹, M.Z. Hossain¹

¹ Department of Plant Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan

An unknown brownish protein was purified by ammonium sulfate precipitation and DEAE-cellulose column and hydroxyapatite column chromatographies from pumpkin callus treated with a high concentration of 2,4-D. The apparent molecular mass and isoelectric point of the purified protein were estimated to be 38 kD and 4.6, respectively. The absorption spectra of the protein showed a shoulder at around 280 nm and a sharp peak at 405 nm. In order to determine what the purified protein is, a cDNA library of the callus treated with a high concentration of 2,4-D was immunoscreened with antiserum raised against the purified protein. The obtained positive cDNA clone encoded a thioredoxin h having a predicted molecular mass of 13 123 D and a predicted isoelectric point of 5.24, suggesting that the purified protein might be a trimer that was formed by oxidative polymerization of the thioredoxin h.

Keywords: antiserum; cDNA cloning; Cucurbita maxima; 2,4-D; immunoscreening; purification; thioredoxin h; Western blotting

Subjects: cDNA cloning; Cucurbita maxima; 2,4-dichlorophenoxyacetic acid; immunoscreening; proteins, unknown, induced by 2,4-D; pumpkin, brownish acidic protein; thioredoxin h; Western blotting

Prepublished online: March 1, 2003; Published: September 1, 2003  Show citation