Cloning Of Conidiation Associated Genes Of Coniothyrium Minitans And Preliminary Study On Cmada

In this study, the efficient Agrobacterium tumefacies mediated transformation system was established to transform Coniothyrium minitans. Conidia from 14-18 dpi colony developed on PDA slant of ZS-1 were collected after centrifuge, and then the conidial concentration was adjusted and resuspended to bacterial liquid. The results showed that fifty transformants per plate were obtained. A T-DNA insertional library with 5000 transformants of ZS-1 was constructed using the method described above.From this library,23 mutants were screened out, and among them 8 mutants could not produce any conidia on PDA plate,10 mutants could produce a few conidia,6 mutants produced abnormal pigment as compared with ZS-1,3 mutants grew faster on PDA than ZS-1,3 mutants grew slower than ZS-1, and hyphae of two mutant collapsed and then degraded. These mutants were significantly different in hyphal growth rate, antibiotics production, parasite of sclerotia, colony morphology and so on. Genomic DNA fragment flanking the insertional T-DNA were cloned from six mutants using Inverse-PCR technique, including ZS-1 TN29,ZS-1TN28,ZS-1TN5012,ZS-1TN5498 and ZS-1TN6222. Putative protein were CMARGJ, CMGAL,CMHMGR,CMPurD,CMMAPKKK respectively by conserved domain analysising.Mutant ZS-1TN250 was studied in detail. The growth rate of mutant ZS-1TN250 was significantly faster than ZS-1, but the biomass in PDB was less. Sporulation was declined significantly and could produce a few conidia on PDA plate. The pycnidia could not break to release conidia, and most of the spores could not mature. This mutant could parasitize sclerotia of Sclerotinia sclerotiorum, but could not produce conidia on sclertia and rotting capacity was decreased significantly. Just like ZS-1, ZS-1TN250 could secrete antibiotic substances. Genomic DNA fragment flanking the insertional T-DNA of ZS-1TN250 was obtained by Inverse-PCR. Based on the flanking DNA, the sequence of a gene was abtained. The full length gene included 1522bp with 4 exons and 3 introns, and cDNA length was 996 bp, encoding a protein with 332 aa. The T-DNA inserted into the second exon, insertion point was at the 191nt position. The deduced amino acid sequence shared 61% identity with adenosine/AMP deaminase family protein of Pyrenophora tritici-repentis. Therefore, we named the gene as CMADA. Further primers were designed, and 3'end was amplified from the cDNA library of C. minitans. Southern blot analysis confirmed that single T-DNA was inserted in the ZS-1TN250. RT-PCR showed that CMADA gene was expressed in the wild strain ZS-lbut not in ZS-1TN250. This indicates that T-DNA insertion induced the mutant phenotype. The protein activity was detected with ADA activity determination kit. The enzyme activity could be detected in the wild strain, whereas could not in mutant ZS-1TN250. The results suggested that CMADA gene is related to mycelial growth and conodia formation of Coniothyrium minitans.