Study On The Function Of CgAAP Gene In Colletotrichum Gloeosporioides

Mango is an important tropical fruit.Anthracnose is one of the most serious diseases of mango in the world,Colletotrichum gloeosporioides(Penz.)Penz.&Sacc.is the main pathogen of mango anthrax.The research took colletotrichum gloeosporioides as object.In the early stage of the study,transcriptome sequencing was performed on the strains of C.gloeosporioides with less sporulation.Through bioinformatics analysis,a gene of AAP family of amino acid transporters was screened and named CgAAP,then a preliminary analysis of its function.The following results were are obtained:In this study,the DNA sequence and cDNA sequence of CgAAP were amplified by PCR and RT-PCR.Gene sequencing and analyzing results indicate that the CgAAP gene is 1858 bp with four introns and its cDNA is 1593bp.The CgAAP gene encodes a 530-amino acid polypeptide,the molecular weight of which is approximately 57.1 kD and the pI of the isoelectric point is 6.24.This polypeptide is a neutral protein with amino acid osmotic enzyme domain.The knockout vector pCB 1532-CgAAP,the overexpression vector CgniaD-ToxA-GFP-CgAAP and the complementary vector pBARGPEl-Hygro-CgAAP were constructed to explore the function of the CgAAP gene.The knockout vector and overexpression vector were transformed into wild-type protoplasts of C.gloeosporioides by PEG-mediated transformation,the complementary vector pBARGPEl-Hygro-CgAAP were transformed into knockout mutant converters of C.gloeosporioides.knockout mutant converters(ACgaap),complementary mutant converters,and overexpressed mutant converters were obtained through the screening of chlorsulfuron/hypopycin resistance and the identification of converters.The results showed that the growth rate of the wild-type strain(WT)was basically the same as that of the converters of the complementary mutant.The over-expressing mutant converters grew faster than the other three transformants.Knockout mutant converters(ΔCgaap)growth speed slightly slower than the wild type strains and complementary strains.At the same time,the spore yields of shaking bacteria in PDB medium,cultivateing in PDA medium were compared.Of the three converters,the spore yield of the over-expressing mutant was the largest,indicating that CgAAP is involved in regulating the sporulation of Anthracnose.Further study found that A Cgaap intensity of A Cgaap’s inhibition of oxidative stress tolerance was obviously higher than that of WT and over-expressing mutants.However,the complementary mutants were completely inhibited when cultured in PDA containing 20 mM concentration H2O2 for 6 days,probably due to the complements some functions and also destroys some functions of the CgAAP gene,indicating that CgAAP is involved in regulating the oxidative stress response of C.gloeosporioides.By pathogenic analysis shows that the incidence of the A Cgaap decrease is lower than the wild type stain.The expression level of CgAAP gene in A Cgaap when the mycelium was not scratched and was treated for 18h was close to 0,The expression of CgAAP gene in the overexpressing mutant converters without scratching mycelium was 80 times that of WT,and the expression of 18 hours after scratching mycelium was 12 times that of WT.The expression level of CgAAP gene was similar to that of WT when the mycelium was not scratched in the converters of the complementary mutant,and the expression of CgAAP gene was 0.01 when the mycelium was scratched for 18 hours.It may be that only part of the function of the CgAAP gene was replenished,and the stress response of the complementary mutant hyphae was damaged during the scratch treatment.The gene was not expressed in the strain after the scratch.