Functional Analysis Of The Environmental PH Signal Transduction Factor PalF Gene Of Penicillium Digitatum In Ctrius

Green mold is a disease with the most serious consequences of citrus after harvest.Penicillium digitatum is the pathogen causing green mold.When it infects citrus,it can reduce the pH around the fruit wound,and its own pH metabolism will also change due to changes in the environmental pH.This experiment hopes to find out the key genes capable of regulating the pH of Penicillium digitatum,and to clarify the environmental pH signal transduction by studying the pH signal transduction pathway of Penicillium digitatum,receiving and transmitting pH signal and activating the key factor PalF of the transcription factor PacC.The relationship between the genes in the pathway and the physiological and biochemical characteristics of Penicillium digitatum.The PalF gene was knocked out by Agrobacterium-mediated homologous recombination,and the physiological and biochemical and PalF functions of the knockout mutant were studied.The results are as follows:1.Analysis of genes expression and copy number of the system: 70 optimized transformants were obtained by optimization.After screening,the positive knockout transformant,?PalF,was successfully obtained,and by Quantitative real-time PCR assays(qRT-PCR).It was found that with the deletion of PalF gene,The expression levels of PalI ? PalA and PalB were up-regulated,and PalB was significantly up-regulated.The expression of PacC gene was inhibited and the expression level decreased,PalH and PalC are not significantly different from WT.It was confirmed by resequencing that ?PalF was a single copy,and the T-DNA insertion site coincided with the PalF site.2.Physiological analysis of ?PalF and WT:(1)The growth rate of PalF was significantly slower than that of WT;The difference in colony diameter was significant after four days of growth.The sporulation of PalF was significantly higher than that of wild WT,which was 2.2 times of wild.(2)By designing different pH gradients of pH3,pH5,pH7 and pH9,the colony diameter of ?PalF and WT was slightly smaller than that of WT under acidic and neutral pH conditions.In thealkaline condition pH 9 environment,the growth rates of ?PalF and WT were inhibited,but ?PalF hardly grew,the colony diameter was only 20.92 mm,and WT was 2.6 times that of ?PalF.It indicates that PalF is a key factor regulating growth in an alkaline environment.The sporulation of ?PalF was still higher than that of WT at different pHs,which is consistent with the results of electron microscopy.The conidiophores of ?PalF are coarser than the conidiophores of WT.3.Regulation of environmental pH and pathogenicity:(1)Under the condition of in vitro culture,the pH of ?PalF was stable at about 4.9,and the pH of WT was stable at about 4.7;Under the living conditions,the pH of the fruit inoculated with?PalF decreased slightly slower than that of wild type WT.(2)For the pathogenicity analysis of citrus,the fourth day of inoculation with ?PalF and WT,the coverage of wild-type WT mycelium reached 90% of inoculation,while the rot rate and mycelial coverage of the fruit inoculated with ?PalF were relatively low,indicating that after the PalF gene was knocked out The knockout of the mutant ?PalF was slightly less pathogenic to the wild type WT,but had no significant effect on the pathogenicity of the fruit.