한국 들잔디로부터 chitinase 유전자들의 클로닝 및 특성분석

Abstract

Zoysiagrass (Zoysia japonica Steud.) is the most significant cultivar used in golf courses, athletic fields, commercially. But it is very suffering damage by large patch disease causing Rhizoctonia solani AG-2-2 (IV), and having physical and economical loss for biocontrol of this disease. In this study, two full-length chitinase genes that widely known as PR-protein were isolated from zoysiagrass. Further, structural property and expression analysis of these genes was carried out. Consequently, isolated two chitinases classified into class I (Zjchi1) and class II (Zjchi2), in a result of expression analysis showed higher expression in roots than in blade. For functional analysis, two chitinases were overexpressed in E. coli and purified using GST-affinity column chromatography. In chitinolytic activity test using 4MU-(GlcNAc), two purified recombinant chitinases showed chitin hydrolysis activity for 4-Methylumbelliferyl N,N'- diacetylchitobioside hydrate, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, and 4-Methylumbelliferyl β-D-N,N',N''-triacetylchitotriose, respectively. Also, antifungal activity against various fungi causing diseases of zoysiagrass and most important crops was investigated in the purified recombinant chitinases. Zjchi2 of the purified recombinant chitinases indicated growth inhibition of mycelia from Rhizoctonia cerealis, Fusarium culmorum, Fusarium graminearum, Trichoderma reesei, whereas not showed antifungal activity from Rhizoctonia solani AG-2-2 (IV), Rhizoctonia solani AG-1 (IA), Sclerotinia minor, Sclerotinia sclerotiorum, Neurospora crassa, Phytophthora capsici, respectively. Although both of the purified recombinant chitinases not showed antifungal activity against Rhizoctonia solani AG-2-2 (IV) causing large patch disease of zoysiagrass, this is the first study for characterization of chitinase genes isolated from zoysiagrass.