Preliminary Studies On The Function Of PDEL And PDEH Genes Coded Phosphodiesterases In The Tangerine Pathotype Of Alternaria Alternata

Alternaria alternata causing Citrus Alternaria brown spot,is one of important phytopathogenic fungi.A.alternata is specific to tangerines and its hybrid.It attacks twigs,young leaves and fruits.severely infected leaves and fruits and always results in significant yield losses.c AMP signal transduction pathway mediated by G-proteincoupled receptors is one of the major signal transduction pathways in pathogenic fungi,and it has important regulatory effects on the growth and development,morphogenesis and pathogenicity of fungi.c AMP phosphodiesterase effectively regulate the intracellular basal c AMP level,so the mechanism of action of phosphodiesterase in c AMP signal transduction pathway has become a hot topic in recent years.In this research,one high-affinity phosphodiesterase gene Aa PDEH and one low-affinity phosphodiesterase gene Aa PDEL were cloned and explored by creating the gene-knockout mutants.Main results in this research were as follows:1.Homologous genes were obtained,based on BLAST alignment in the complete sequence database of Alternaria alternata according to the pde1 and pde2 protein sequences of S.cerevisiae,named Aa PDEL and Aa PDEH respectively.Aa PDEL and the low-affinity c AMP phosphodiesterase from S.cerevisiae,M.oryzae,A.fumigatus,A.flavys and C.albicans sequence similarity was from 18.5% to 52.59% on amino acid level.PDEase_Ⅱ conserved sites was found in Aa PDEL.Metal dependent phosphodiesterase HD_domain and PDEase_Ⅰ3′5′cyclic nucleotide phosphodiesterase conserved site was found in Aa PDEH,whereas high-affinity c AMP phosphodiesterase was 30.26% ~69.59% sequence similarity to he highaffinity c AMP phosphodiesterase from S.cerevisiae,M.oryzae,A.fumigatus,A. flavys and C.albicans.Through bioinformatics analysis and comparison,it was found that pde in Alternaria alternata and S.cerevisiae has many identical properties,suggesting the conservation between different fungi.2.Aa PDEL and Aa PDEH gene-disruption vectors were constructed based on the gene doublecross homologous combination theory and PEG mediated protoplast transformation system.Transformants were picked out and verified by hygromycin B,PCR and Southern blot.The complementary strains PDEL-CP and PDEH-CP were got after PEG mediated protoplast transformation system,the complemented strains PDEL-CP and PDEH-CP were obtained by G418 and PCR with specific primer.3.The function of mutants were analyzed the function of phosphodiesterase genes.Results indicate that Aa PDEL gene is involved in cell wall integrity,production of sporulation,laccase activity and pathogenicity in A.alternata.Respectively,Aa PDEL might have an important role in laccase activity but no regulatory effect on others.