Mutant Pathogenicity Characteristic Analysis And Related Gene Cloning In Curvularia Lunata In

Curvularia leaf spot, caused by Curvularia lunata (Wakker) Boed., is one of the most serious maize diseases. Even though controlled by application of resistant varieties in past decade, the disease still takes great risk for disease resurgence due to pathogen virulence differentiation and relatively low genetic diversity in resistant varieties. Little has been known so far about the molecular mechanisms of its pathogenicity, particularly the ways of genes how to work in vitro and in vivo. In this study, an Agrobacterium tumefaciens-mediated transformation (ATMT) system was utilized to create a wide range of insertional transformants of C. lunata. We also analyzed pathogenicity-related characteristics of transformants and cloned related gene.1. Highly virulent strain CX3 of C. lunata was used as a recipient strain for ATMT. The experimental conditions for ATMT were optimized on different germination times of conidia, the strain of Agrobacterium tumefaciens used and co-cultivation temperatures. Under our optimised transformation condition, the average transformation efficiency of C. lunata with T-DNA was 84±5 transformants per 1×106 germlings. The total number of 1454 transformatants were obtained.2. Nine transformants which exhibited obvious changes in colony morphology, color, growth speed were successfully screened from a large amount of transformants and were tested in pathogencity-related characteristics, such as sporulation, cell-wall-degration enzymes, toxin and so on. Of them, the five pathogicity-deficient transformants were obtained by pathogenicity tests on both detached leaves and the whole plants.3. PCR analysis and southern blot suggested that only one copy of T-DNA was inserted in CLM-1648. Genome walking, RT-PCR and bioinformatics were applied to clone related gene, the structure of which included a 1,175 bp DNA sequence with a 1,038 bp coding region, three exons and two introns. It shared high identity to the ATP-phosphoribosy -ltransferase of Pyrenophora tritici-repentis(78%). The predicted protein had 346 aa. Sothern blot showed that there was only one copy of the gene in C.lunata genomic DNA. RT-PCR result indicated the gene expression was significantly affected by T-DNA insertion. It is therefore inferred that the gene might be closely involved in the pathogenicity expression of isolates through histidine metabolism or other physiological processes, nonetheless, the further work should be done to give reliable verification to the spectulation.4. The isolate CLM-1648 cology exhibited relatively light color, enhanced conidial production with faster germination speed. But it failed in penetrating epidermal cells of onion ,and also decreased its pathogenicity by 70 %. The growth speed of CLM-1648 was significantly declined on MM medium and few conidia was produced. Based on above results, we summarized that CLM-1648 was obtained through the mutagenesis of wild type isolate in asexual reproduction, nourishment, which then might contribute to some extent to a sharp reduction in pathogenicity. The gene-knockout vector was also constructed according to the gene homologous combination theory for further study on gene function.