Cloning And Functional Characterization Of VdCPMO Gene In Verticillium Dahliae

Verticillium dahliae,an ubiquitous soilborne fungus,can infect over 660 plant species and causes huge economic loss.Microsclerotia can survive for 14 years in soil as dormancy structures and serve as the primary inoculum on many hosts.Germination of microsclerotia perceived as dormancy structures is the prerequisite condition to infect host successfully.However,up to now,only few genes related to microsclerotium germination have been reported.According to the whole genome wide expression profiles on germination of microsclerotia,VDAG₀3943,coding cyclopentanone 1,2-monooxygenase,was the most highly expressed during the germination process.In present study,we cloned the sequence and characterized the function of VDAG₀3943.The main results were exhibited as follows.1.Based on the genome database of V.dahliae strain Vd Ls.17,we cloned and analyzed the sequence of VDAG₀3943.This gene,named Vd CPMO,has a full length of 1929 bp.The c DNA coding region of Vd CPMO contains 1668 bp nucleotides that encode a protein consisting of 555 amino acids.Knockout plasmid of Vd CPMO,called p OSCAR-CPMO,was achieved successfully by one step method for construction of vector.2.Vd CPMO was knocked out from wild type strain JY using Agrobacterium tumefaciens-mediated transformation method and 26 deletion transformant strains were obtained through resistance screening of hygromycin.Confirmation of two deletion mutant strains,ΔCPMO-1 and ΔCPMO-2,was carried out by PCR detection with the hygromycin primers（Hyg-F/R）and target gene primers（CPMO-F/R）.Subsequently,mutant complementation was conducted to get 14 complementary transformant strains via polyethylene glycol（PEG）mediated protoplast transformation approach.The results of G418 resistance screening,phenotypic identification,and PCR amplification detection revealed that we got 3 complementary mutant strains.A strain named ΔCPMO-C was chosen for our research.3.The measurement of colony growth,germination rate of microsclerotia,average length of germ tube,and pathogenicity was performed on two deletion mutant strains ΔCPMO-1 andΔCPMO-2,complementary mutant strain ΔCPMO-C,and wild type strain JY.Resultssignified that deletion mutant strains,ΔCPMO-1 and ΔCPMO-2,exhibited some developmental defects containing slower growth of colony,decreased germination rate of microsclerotia,and decreased average length of germ tube when compared to the wild type strain JY.There was no significant difference between complementary mutant strainΔCPMO-C and wild type strain JY.Furthermore,the microsclerotia of deletion mutant strains,ΔCPMO-1 and ΔCPMO-2 lost the pathogenicity to cotton and showed reduced pathogenicity to tobacco.The pathogenicity of complementary mutant strain ΔCPMO-C displayed no discrepancies with wild type strain JY.