Cloning And Functional Analysis Of Melanins Associated Gene CmPKS1 In Coniothyrium Minitans

Coniothyrium minitans, an important sclerotial mycoparasite of Sclerotinia sclerotiorum, has potential for biological control of plant diseases caused by S. sclerotiorum and other Sclerotinia spp. In this research, we reported the cloning and functional analysis of a gene involving in melanins of C. minitans based on T-DNA insertional melanins deficient mutant ZS-1TN25878.Unlike original strain ZS-1, ZS-1TN25878 mutant hyphae appeared bleaching. Mycelial growth rate of strain ZS-1 hsa no significant difference in the biological yield no significant differences in the ability of parasitic cause rot sclerotia was little difference, the loss of melanin produced capacity. In the PDA medium, the mutant ZS-1TN25878 strains with the original strain ZS-1 also has sporulation capacity, sporulation and no significant difference.Previously, From Coniothyrium minitans strain ZS-1 T-DNA tag insertion mutant library was screened to the production of melanin-deficient mutant ZS-1TN25878, by reverse PCR, a 500bp of DNA sequence expansion in the genome of C. minitans genes found total length of 6708bp. Sequencing analysis showed that there included a complete ORF (nucleotide positions 1834-8541) with three introns and encoded 2161aa and Located 1834nt ATG start codon for the start.BLASTp result showed that this putative protein containing a conserved domains of KS、AT、ACP、TE, and the deduced amino acid sequence shared 100% identity and 68% similarity with Peroxisomal protein KS of Bipolaris oryzae and 99% identity and 65% similarity with Peroxisomal protein KS of Botryotinia fuckeliana. Therefore, we named the gene as CmPKSl. T-DNA was inserted at the middle of CmPKS1.RT-PCR showed that CmPKS12 gene could not express in mutant ZS-1TN25878, but could express in original strain ZS-1. The CmPKS1 in original strain ZS-1 was disrupted with Agrobactirium tumfacience mediated-transformation. One CmPKSl gene disruption transformants namely PKS2-12 was obtained. Comparing to the original strain ZS-1, PKS gene knockout transformants and the original strain ZS-1 compared to bleaching appear, and the mutant showed the same traits ZS-1TN25878. Our work suggested that the CmPKSl is involving in melanins of C. minitans.