Gene Analysis And Function Preliminary Study Of V-ATPase H Subunit Of Penicillium Digitatum

Penicillium digitatum is the main pathogen causing citrus postharvest diseases,causing huge losses in citrus production.Nowadays,fungicide treatment was still the main method for controlling the postharvest diseases of citrus fruits.However,most of the chemical fungicides belong to the single-target drug,which are prone to forming resistant strains and short the life time.The research and development of fungicides with new drug targets and action mode are of great significance for the safe production of postharvest citrus.For most of the pathogenic fungi,the homeostasis of intracellular pH is essential for cell growth,development and virulence.Yeast is a single-celled eukaryotic model organism with simple structure and less protein.It is a good model organism for studying the physiological processes of eukaryotes.The V-ATPaseH subunit,a gene being essential for fungal growth,extracellular acid production and azole drugs,was screened from a yeast mutant library in which proton pump-related genes were deleted.The role of VmaH in P.digitatum was investigated.The main results are as follows:(1)The key gene VMA13,that is the V-ATPaseH subunit,which has a role in fungal growth,extracellular acid production and azole drugs,was screened from the yeast mutant library.(2)VmaH of P.digitatum had two very conserved domains,namely N-terminal domain and C-terminal domain,similar to other fungi.The amino acid similarity of VmaH in fungal species was 79%.Moreover,VmaH of P.digitatum had a close relationshipwith that of Penicillium and Aspergillus.VmaH maintained a high gene expression level throughout the growth and infection of P.digitatum.Besides,it exhibited different expression patterns under different external environmental stress conditions.(3)In this experiment,the pCAMBIA3300 plasmid was used as the backbone to successfully construct the VmaH gene overexpression vector p-5HPV3-3300 and two sets of RNA interference vectors p-PS-A-Hyg B-3300 and p-pgpd A-VmaH-PtrpC-Hyg B-3300.Using Agrobacterium tumefaciens mediated transformation of P.digitatum,one VmaH overexpression mutant and one VmaH silent mutant were obtained,respectively.(4)VmaH had little effect on the mycelium morphology of P.digitatum.Silence ofthe VmaH gene leaded to the delay of germination time of P.digitatum,slow down the growth rate of mycelium and decrease of spore production.The VmaH gene had a positive regulation effect on infecting citrus.The VmaH gene had a positive regulation on the pathogenicity of P.digitatum on citrus fruits.The lesions diameter of the fruits inoculated with the overexpressed strain was significantly larger than that of wild-type strain;And the lesions diameter of the fruits inoculated with the silent strain was smaller than that of wild-type strain.Besides,there was no visible hyphae growth and green spores spreading.The pH of the culture solution of the overexpressed strain of VmaH and the wild-type strain were not significantly different.However,the pH of the VmaH silent strain culture solution was significantly lower than that of the control strain and overexpressed strain.