Purification and characterization of basic proteins with in vitro antifungal activity from seeds of cotton, Gossypium hirsutum.

摘要

Basic proteins from seeds of cotton (Gossypium hirsutum) were purified by a procedure involving cation exchange chromatography on carboxymethyl cellulose (CM52) and reversed phase high performance liquid chromatography (HPLC). The basic protein fraction from these seeds contains a multiplicity of vicilin-derived 9-11 kDa proteins, a 16.3 kDa protein that has an N-terminal sequence nearly identical to that of a cotton cysteine-rich small vicilin protein and a 46.3 kDa protein having an N-terminal sequence with 61% identity and 75% similarity to the cDNA-based sequence of a gamma-conglutin from Lupinus angustifolius. Edman N-terminal sequencing, electrospray ionization mass spectrometry (ESMS) and literature cDNA-based sequencing data were used to define the sequences of the vicilin-derived proteins. The inferred N- and C-terminal sequences of the 9-11 kDa proteins correspond to N-terminally truncated forms of cysteine-rich small vicilin proteins (such as the 16.3 kDa protein) suggesting that bothcould be processed from the vicilin (alpha-globulin) preproprotein. The various cotton basic proteins were found to have selective growth inhibitory activity in vitro against the filamentous fungi Botrytis cinerea, Alternaria brassicicola, Chalara elegans [Thielaviopsis basicola] and Fusarium oxysporum. These proteins differ, however, from numerous other seed antifungal proteins in being neither substrates nor inhibitors of signal transduction elements such as wheat germ Ca2+-dependent protein kinase (CDPK), rat liver cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK), rat brain Ca2+- and phospholipid-dependent protein kinase (PKC) and chicken gizzard calmodulin-dependent myosin light chain kinase (MLCK).