Cloning And Characterization Of Ambient PH Signal Regulatory Gene PalF From Mango Anthracnose Causal Agent

Mango is an important fruit trees in tropical of China.Anthracnose, is one of the main disease of mango.The disease infect mango tender tip caused the symptom such as leaf spot and branch dieback, as well as infect the the dropping of flowers and fruits of Mango, affect the output of the fruit enormously.Futhermore,Mango anthracnose have obvious characteristics of latent infection, which caused a lot of fruit rot during storage transport, and lead to serious economic loss.So far the mango anthracnose prevention and control is given priority to with chemical methods, not only a limited impact, but also lead to aspirin resistant and the problem of pesticide residue.Therefore, the study of pathogen and host interaction mechanism, can provide new ideas for prevention and control technology measures of new technology measures and theoretical basis.Ambient pH sensing signal transduetion pathway involved in regulation of anthracnose in pathogenicity of mango. Based on the separation from mango anthracnose spore wild strain A2as materials, clone and characterization of ambient pH signal regulatory gene PalF;build a knockout vector of PalF, obtainPalF knockout mutant and PalF:Pacl double knockout mutant; Through the analysis of wild strain A2, PalF knockout mutant, Pacl knockout mutant and PalF:Pacl double gene knockout mutant phenotype and pathogenicity, initial identification the function of the PalF gene, and Furthmore to explore whether PalF has other functions besides ambiton pH signal transduction pathways. The results of the study are as follows:(1) Using the traditional homogene cloning method, PalF gene was cloned from Colletotrichum gloeosporioides of Mango, and the objective gene fragment with the size of2369bp were acquired. The gene sequence analysis showed that it included intact open reading frame, and it have100%sequence homology with PalF gene of Colletotrichum gloeosporioides from Rubber. The cDNA sequence of PalF gene obtained by RT-PCR has a length of2310bp with the gene sequence analysis; it includes one59bp intron encoding a protein of769amino acid residues. The analysis of conserved regions of amino acid sequence of PalF gene indicated that PalF protein contains a conservative domain of Arrestin-C, and presumed that PalF gene participated in the regulation of Ambient pH-sensing signal transduetion pathway.(2) Application In-Fusion HD Cloning Kit built PalF knockout vector based on Bar selection markers, used Fungal protoplast reforming process which mediated by polyethylene ethanol,As to transformation for wild strain A2and Pacl knockout mutant protoplast get transformant.Though PCR test, determine the conversion for PalF knockout (3) mutant and PalF:Pacl double knockout mutant genes.(4) Analysis on A2and palF、Pac1knockout mutant、palF:pacl knockout mutant,the results show that colony morphology and spore production of palF knockout mutan are less than A2and other two mutants;compared with other three mutants,palF:Pac1knockout mutan is serious decline; Four strains in different pH of culture medium and in mango leaves and fruits on the determination results show that under the condition of alkaline, PalF knockout mutant, PalF:Pacl knockout mutant of irulence is smaller than the pathogenicity of acidic conditions.(5) The above experimental results show that ambition pH related gene PalF involved in signal transduction pathways regulating in pathogenicity of mango, PalF and Pacl gene function have superposition effect.