| Apple Glomerella leaf spot is a new disease on apple(Malus pumila Mill) cvs. Gala,Qinguan, Golden delicious, and causes heavy early leaves drop and fruit necrotic spots,resulting in significant losses in China. Previous researches indicated that the pathogen of Glomerella leaf spot are Colletotrichum fructicola and C. aenigma, C. fructicola is the dominant species. In this study, the genetic transformation of C. fructicola host strain SQ06-E was developed by PEG- mediated protoplasts transformation, and two C. fructicola GFPlabeled strain was obtained. The main results are as follows:1. The conditions for the protoplast isolation and regeneration of C. fructicola are optimized: the conidia(1×106 spores per milliliter) of C. fructicola were incubated in M3 S medium at 28℃for 8 h by shocking in 200r/min, the 0.5 g mycelium were collected and hydrolyzed in 10 mL enzyme solution containing 2.5% Driselase and 1 % Lysing enzyme for3 h, 0.8 mol/L KCl was used as the osmotic stabilizer. The protoplast yield was up to 3×107cuf/mg.2. Using PEG-mediated transformation, the plasmid pCB1003 was transferred into the protoplast of SQ06-E. 76 transformants were got, and the transmation efficiency was 3-4 per g DNA. Analysis of the transformants by PCR and Southern blot showed that the selectable marker gene hph was integrated effectively into the genome of SQ06-E. After 5 subculturing on PDA, all transformants could grow on the media containing hygromycin. This stability test of transformants suggested that the foreign gene hph was stable in heredity.3. Using PEG-mediated method co-transformation, the plasmid p CB1003 and pRM7 were transferred into SQ06-E protoplast. 42 tranformants were been havested. After 5subculturing on PDA, all transformants could grow on media containing hygromycin, green fluorescence could be observed under a fluorescence microscope. PCR analysis also showed that hph and gfp genes are integrated into the genome of SQ06-E. In order to obtain strains labeled by GFP having no significant difference in biological characteristics and virulence with the wild type, 7 transformants were tested randomly, two transformants SE3 and SE36 were selected for further experiment. |
