Establishing The T-DNA Insertion Mutant Population Of Magnaporthe Grisea And Analyzing The Mutants

Plant Protection College, Environment and Plant Protection Research InstituteCATAS & SCUTA Baodao Xincun, Danzhou, Hainan, P.R. China, 571737Magnaporthe grisea (MG)is the pathogenic fungus of rice many species of grass family. On rice, it causes rice blast, which is of economic important and world-wide distribution. Functional genomic, especially pathogenic functional genomic of MG is now being widely studied because these studies are of theoretically and practically significant for the probe of the relationship between the rice and MG, breeding and selection of rice blast resistant varieties, and creating the new control strategies to this disease. In this thesis, we established a high efficient and stable transformation system of MG based on Agrobacterium tumefaciens-mediated transformation (ATMT) and obtained 2437 T-DNA insertion mutants using this system.1. The key conditions, factors and parameters of the transformation system of MG were screened and optimized. The results showed as followings: among the culture media tested, the most suitable medium for MG spore formation was oat meal, then the V8, rice chafe and dry plum medium; the spore production of MG under continuous light was higher than the one under alternate light exposure; the spore production of MG was the lowest under continuous dark incubation; hygromycin could inhibit the growth of MG, but the effect of inhabitation activities was different from culture medium to culture medium. On oat meal, the growth of MG mycelia andspores were well inhibited at 200 ug/ml of hygromycin. On V8, however, MG still grew well even at 300 ug/ml hygromycin. Therefore, 200 ug/ml of hygromycin in oat meal culture medium was used in the screening mutant in this transformation system. Among the A. tumefaciens isolates tested, the transformation efficiency of MG with isolate AGL-1 was significantly higher than the isolate LBA4404 and EH A105; the 6h incubation for A. tumefaciens in broth IM with 200um/ml of AS before co-cultivation was the best incubation duration because it resulted in highest transformation efficiency; A.tumefaciens (OD600 0.8) and MG spore (1 106 conidia per ml) solution were mixed by equal volume, and then 200ul of this mixed solution was smear on IM plate and co-cultivated at 25 for 48h, and these resulted in good transformation.2. The transformation experiment was carried out by using the transformation system of MG established with the above conditions and parameters. The transformation was also testified. The results showed as the followings: for 1 106 MG conidia, 300-400 of hygromycin B resistant colonies were obtained on average; with a pair of primers (Hf, Hr) designed by hygromycin phosphotransferase gene in plasmid pBHt2, a fragment of 819bP were separately amplified by PCR from all clones randomly selected from hygromycin B resistant colonies, confirming the hygromycin gene has really inserted in to the MG genome; the transformants still maintained the phonetype of hygromycin B resistance after 6 continuous transfers and incubated on medium that absent hygromycin B, showing the phonetype of hygromycin B resistance of the mutants obtained was stable; southern blotting analysis on the clones randomly chosen from the hygromycin B resistant clones revealed that the average insertion T-DNA was as low as 1.4 copies per line, and single copy insertion was as high as 73.5%.3.Large scale transformation on MG was carried out. The colony form and color,spore production and morphology between the MG mutants and wild type were compared. A total of 2437 T-DNA insertion mutants of MG was obtained, of which 23 were of morphology different from the wild type MG, which accounted for 0.944% of all mutants. Among the morphology mutants, spore morphology mutants, colony form, colony color and spore sterilization mutants accounted for 0.246%, 0.451%, 0.497%,0.287% of the all mutants obtained, respectively.