Preliminary Study On Adenylate Cyclase Gene In Coniothyrium Minitans

Coniothyriumminitans is a sclerotial mycoparasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, it is an important biocontrol agent against Scleroinia sclerotiorum. Inprevious studies, we suspected that C.minitans could obtain active substance like hypoxanthineor that was related with hypoxanthine from the host in the process of parasitizing sclerotia to which cAMP belonged. Intracellular cAMP can be formed by the catalysis of adenylate cyclase(AC), an dadenylate cyclase is an important component of the cAMP signaling system. In eukaryotes, many signaling pathways are involved in the process of other genes, transferring external signals to the nucleus through the cell membrane, and cAMP signaling pathway is an important one. As a second messenger, cAMP plays an important role in vegetative growth, formation of infection structure and pathogenicity. Thus, the Study of cAMP signaling system in C.minitansis meaningful in both theory and practice aspect.A gene named as CMAC, which encoded adenylate cyclase in C. minitans, was obtained through BLASTp analysis of the amino acid sequence from the yeast (Saccharomyces cerevisiae) with wild type ZS-1strain genome. The length of the gene was6908bp, and it encoded a protein of2114amino acids. It contained four exons and three introns. Sequence alignment result in NCBI showed that the gene had high analogy to adenylate cyclase in many fungi. Furthermore, phylogenetic analysis showed that similarity degree of the gene with Pyrenophora tritici-repentis and Sclerotinia sclerotiorum were80%and67%seqaratly.In order to study the function of the gene in C.minitans, CMAC was knocked-out in wild type ZS-1with Agrobactirium tumfaciens mediated-transformation(ATMT) and two knocked-out transformants called KO86and KO245were got. The colony morphology of transformants were significantly different from the wild type ZS-1on potato dextrose agar (PDA), including slow growth speed, less aerial mycelium, dense mycelium, and growing into the medium of the hyphae, changed hyphae tip branching pattern. The colonies had irregular edges, lack of melanin, significantly decreased sporulation; the pycnidium were relatively hard, rarely secretes mucus in the late of the growth. Sclerotium decomposed level with the parasitism of transformants had significant difference with the wild type ZS-1, transformants basically loss the capabilities of parasite. The results showed that the gene CMAC of C. minitans has an important regulatory role in mycelia growth, sporulation, spore germination and sclerotial parasitizing. The results show that the gene CMAC of C. minitans has an important regulatory role in mycelia growth, sporulation, spore germination and sclerotial parasitizing.Contacting with the hyphae of Sclerotinia mycelial with a period of time, Knocked-out transformants had some changes, growing more mycelial, forming acircle of Sclerotium around the transformants contacting with Sclerotinia, and Sclerotiniain activated EP-1A367confrontation. It will not happen contacting with the dead hyphae of Sclerotinia mycelial. Meanwhile, the vegetative growth of the transformants mycelium were promoted after parasite sclerotial, thick mycelium was generated on the surface of Sclerotia, but spore formation had not been restored. Results show that gene CMAC knocked-out transformants can capture cAMP to restore their vegetative growth in the process of contacting with Sclerotinia promote sclerotium generated.In this research, apreliminary analysis of cAMP signaling system in Coniothyrium provid a basis for the study of the role of the interaction between Coniothyrium and Sclerotinia.