들잔디(Zoysia japonica Steud.)Lipid Transfer Protein 유전자의 기능적 특성분석

Abstract

If disease occur on the lawn, that will damage fine view and require additional management, moreover that will make economic loss. So managers and farmers perform chemical pest control, but sprayed excess agrochemicals leads to the social problems such as pesticide residue, environmental pollution. Molecular breeding is an effective way for replace chemical control to protect grass from pathogens. In order to find available gene source for transformation, carried out characterization lipid transfer protein(LTP) genes from zoysia grass(Zoysia japonica Steud.). Plant LTP is one of Pathogenesis Related(PR) peptide due to they are induced with phytopathogens and their accumulation in the plants of related to plant resistance responses. Some of these proteins exhibit in vitro antimicrobial activities. Five putative LTP clones were found in zoysia EST library. Two(ZjLTP1 and 2) out of five had full ORF and the rest(ZjLTP3,4 and 5) had partial sequence. Through performing semi qRT-PCR, analyzed the pattern of organ specific expression, biotic, abiotic stress inducible expression of ZjLTPs. Many pathogens attack sheath or root. All ZjLTPs were expressed in sheath and stolon, but ZjLTP4 was the only detectable clone in root. ZjLTP2 expression was detectable in dormant seeds. Although, the expression pattern of ZjLTP1 was induced at earlier stage by CF of R. solani AG2-2(IV) or hyphae of R, cerealis, other LTPs were not induced significantly but just reduced steadily. In heat stress treated blade piece, expression of ZjLTP2 was greatly reduced. In subcellular localization assay, ZjLTP1 protein signal was observed in the cytosol and ZjLTP2 was observed in the peroxisome. The ZjLTP1 and 2 recombinant proteins did not show growth inhibition activity in the condition of this study. The PIP strip test result indicated that ZjLTP1 can bind Phospho-phosphatidil inositide, PA and PS which are famous for the second massage transfer molecules in plant. Arabidopsis were transformed with ZjLTP1 or ZjLTP2 harboring T-DNA by Agrobacterium mediated transformation and selected homozygote line by segregation assay about Basta resistance. When methanol extract from transgenic Arabidopsis were supplied in medium, the growth of R. solani AG2-2(IV), AG-1(IA), AG2-2(IIIB) and R. cerealis was inhibited compared with WT extract. Transgenic lines over-expressing ZjLTP2 treated with R. cerealis or F. culmorum showed a phenotype that grow up slowly and have dark green leaves. Although, ZjLTP1 and 2 proteins did not show antifungal activity, it is expected that ZjLTP1 play a role in signaling pathway which need a transfer PA and ZjLTP2 involved in lipid metabolic pathway.