Abstract: 【Objective】 The objective of this study is to construct the knockout vector of gene Vmgtp1 encoding GTP cyclohydrolase II using the strategy of reverse genetics, and analyze the gene preliminary function by homologous recombination, so as to lay a foundation for the comprehensive analysis of pathogenic molecular mechanisms in Valsa mali and provide a theoretical basis for the technology and pharmaceutical research of apple rot disease. 【Method】 Based on the analysis of the transcriptome data of V. mali, the gene Vmgtp1 (tentatively named) was obtained. After inoculating for 5 days, the expression in the infected tissue was increased. So it is predicted that Vmgtp1 may be related with pathogenicity in V. mali. Using Double-joint PCR method, the up and downstream fragment of Vmgtp1 and the selectable marker hygromycin-phosphotransferase gene (hph) to one fragment were connected. The PCR product and plasmid pHIG2RHPH2-GFP-GUS were double-digested, connected with T4 ligase and transformed into JM109. Then the positive plasmid was transformed into V. mali through PEG-mediated genetic transformation. Next, the hgromycin-resistant mutant strains which can grow normally on PDA medium containing hygromycin were selected. The selected homologous recombinant mutants were then confirmed by PCR with four pairs of primers and Southern Blot. At last, the phenotypes of wild-type strain 03-8 and mutant were analyzed through observation colony color, colony size and propagulum production, the pathogenicities were tested by observing the lesion sizes on the apple limbs which inoculated with wild-type strain 03-8 and mutant. The significance of differences was analyzed by the software of SPSS.【Result】The knockout vector of gene Vmgtp1 encoded GTP cyclohydrolase II using the above-mentioned method was constructed successfully and 101 mutant strains with PEG-mediated transformation were obtained. Only one homologous recombination mutant ΔVmgtp1-90 was obtained after PCR and Southern Blot testing the 101 mutant strains. Compared with the wild type 03-8, the mutant ΔVmgtp1-90 showed slower colony growth rate on PDA medium, the average growth rate of ΔVmgtp1-90 was 11.33 mm?d-1, and the average growth rate of strain 03-8 was 24.67 mm?d-1. At the same time, ΔVmgtp1-90 had lighter colony color and sparser aerial mycelium, had not produced any propagulum on the PDA medium 40 days later under light conditions, but strain 03-8 could produce propagulum after 15 days and overflow conidium after 30 days cultivation on the PDA medium under light conditions. The result of pathogenicity testing also showed that the pathogenicity of ΔVmgtp1-90 was significantly weakened. At 7 days after inoculation, the average lesion diameter on the branch of Fuji apple tree was 24.8 mm after inoculated with strain 03-8, but the average lesion diameter only was 9.3 mm after inoculated with ΔVmgtp1-90.【Conclusion】A mutant of the gene Vmgtp1 encoding GTP cyclohydrolase II through gene knockout and genetic transformation was obtained. Compared the difference of phenotype and pathogenesis of Vmgtp1 mutant ΔVmgtp1-90 and the wild-type strain 03-8, it could conclude that the gene Vmgtp1 may affect mycelial growth and regulate of sporulation of V. mali, and may play a role in the process of V. mali infection. But further research should be done to know whether it plays a major role or not.

Key words: Valsa mali , homologous recombination , PEG , pathogenicity