Characterization of chitinase activities, and cloning, analysis, and expression of genes encoding pathogenesis-related proteins in strawberry.

摘要

The goal of this dissertation research is to investigate the defense systems of strawberry plant by characterizing the genes and their products involved in plant disease-resistance. Pathogenesis-related proteins, including hydrolytic enzymes chitinases and β-1,3-glucanases, have been known to be induced in plants upon infection with various pathogens. Highest total chitinase activity was found in strawberry crown, whereas root, petiole, fruit, leaf, and runner showed successively lower activities. Chitinase isoform analysis showed that up to six acidic and two basic isoforms were present in various organs. The total chitinase activity was stable at 50°C. The pH optimum for chitinase activity was 5. Total chitinase activity was inducible in leaves when plants were treated with fungal spores, salicylic acid, ethephon, or injury.; Genes encoding a class III chitinase and two class II chitinases (designated as FaChi2-1 and FaChi2-2) were cloned and their complete nucleotide sequences were obtained. Of the two class II chitinase genes, FaChi2-1 contains one intron whereas FaChi2-2 contains two introns. cDNA clones, containing the complete protein coding regions, for the two class II chitinase genes were obtained to establish the exact intron splice junctions. All cloned genes were found to be expressed constitutively in the strawberry leaves. Southern blot analyses for the two class II chitinase genes showed that these genes belong to small multi-gene families with no more than two members per haploid genome. Transcription start site for FaChi2-1 was mapped to −87 and −102 positions from the putative translation start site by primer extension analysis. FaChi2-2's transcription start site was mapped to −52 position upstream of the putative translation start site.; Induction of total chitinase activity, pattern of acidic and basic isoforms, and expression of two class II chitinase genes were analyzed at 2, 6, 12, 24, and 48 h after fungal inoculation of plants with Colletotrichum fragariae or C. acutatum. The chitinase activity was inducible up to five fold. The expression of FaChi2-1 and FaChi2-2 was quantified by real-time PCR. FaChi2-1 was induced early within 2 h whereas FaChi2-2 was induced only at 24–48 h post-infection.