Functional Analysis Of Growth And Conidiation Associated Gene CmTERT In Coniothyrium Minitans

Coniothyrium minitans is a very important biocontrol agent of rapeseed stem rot caused by Sclerotinia sclerotiorum. In previous study, a T-DNA insertional library was construted with the wild type strain ZS-1 by Agrobacterium tumefa-mediated transformation technique（ATMT） and a conidial deficient mutant ZS-1TN4685 was screened from the library. By cloning the flanking sequences of T-DNA insertion site and comparing it to the genome sequences of C.minitans, a gene Cm₀3805 was found to be disrupted. Here in this research, mutant ZS-1TN4685 and the function of the gene Cm₀3805 were preliminarily studied. The results were as follows:1. Compared with wild type ZS-1, the mutant ZS-1TN4685 showed delayed growth,irregular colony morphology, increased branching, bending hyphal tip, decreased sporulation and it stopped growing on PDA plate in the end, but the parasitic capacity of conidia to sclerotia did not be influenced.2. Gene Cm₀3805, containing the RNA-binding domain and telomerase reverse transcriptase specific domain by alignment in NCBI, was named Cm TERT. By cloning and analyzing this gene, it revealed that it included 10 exons, nine introns and encoded1168 amino acids.3. Cm TERT was a single copy in genome of C.minitans by local blast analysis.Phylogenetic analysis indicated that the deduced amino acid sequences of Cm TERT had high homology with Stemphylium lycopersici. Dynamic analysis of Cm TERT expression revealed that the gene was expressed in the initial stage of pycnidium development.4. Four gene knockout transformants of Cm TERT were obtained by the principle of homologous recombination. All the transformants showed same phenotypes as the mutant ZS-1TN4685, namely delayed growth, irregular colony morphology, increased branching,bending hyphal tip, inceased ROS, decreased sporulation and the same parasitic capacity of conidia to sclerotia.All the results above showed that Cm TERT played critical roles in regulating the growth and conidia production of C.minitans, but it had no influce in parasitic capacity of conidia to sclerotia. This research will be of great significance for understanding the growth and development of C.minitans.