Establishment Of A Target Gene Knockout System And Research On Polyketide Synthase KzPKS18 In Kabatiella Zeaegene

Maize eyespot is a corn leaf disease caused by Kabatiella zeae Narita et Hiratsuka.In recent years,due to changes in variety and cultivation measures,the occurrence of diseases has shown an increasing trend resulting in a reduction of 5-10% per year.At present,the research on this disease in China and abroad mainly focuses on theisolation,identification,biological characteristics of pathogens,the occurrence of diseases and the prevention and control techniques.Although some scholars have used the Agrobacterium tumefaciens-mediated technique(ATMT)to establish a mutant library of K.zeae.However,the method for studying the pathogenic mechanism of the pathogen is not clear,and studies on the function of pathogenic related genes have not been reported.Polyketide synthase gene PKS18 is a key gene for melanin synthesis.It has been proved that the pathogenicity of PKS18 with pathogens caused by various corn leaf diseases,such as Cochliobolus heterostrophus and Curvularia lunata.In this paper,the Kz PKS18 gene obtained by cloning was used as the research object,and bioinformatics software was used to predict and analyze the physical and chemical properties and functions of the protein encoded by the gene.The target gene knockout system of K.zeae was established and optimized.On the basis of this,the biological characteristics,anti-stress ability and pathogenicity of Kz PKS18 were studied,which laid a foundation for further study of the function of Kz PKS18 gene.The main findings results were as follows:1.The full sequence of the PKS18 gene of K.zeae was cloned and the physicochemical properties of Kz PKS18 gene were obtained by bioinformatics analysisAccording to the PKS18 gene and protein sequence of Aureobasidium pullulans,A.melanogenum,A.subglaciale and A.namibiae,the Clustal W software was used for the alignment and the specific primers were designed.After 5 rounds of PCR amplification,the full sequence of the Kz PKS18 gene sequence(gene sequence number MK416207)was obtained.Bioinformatics software was used to predict the characteristics of Kz PKS18 gene and the physicochemical properties,secondary structure and subcellular localization of the protein.The results showed that the PKS18 gene of the k.zeae was 6689 bp,the open reading frame was 6649 bp,encoding 2162 amino acids,and the theoretical isoelectric point was 5.87.It was a stable,hydrophilic protein,no glycosylation site;subcellular localization results indicate that it was located in the cytoplasm;The protein had four secondary structures,the mainly secondary structures are random coils and ?-helix;Kz PKS18 had Pks D belonging to PKS-KS super famliy and PT_fungal_PKSdomain belonging to hot-dog super family and so on.The phylogenetic tree showed that the PKS18 of K.zeae and the polyketide synthase gene of Aureobasidium were clustered into one group.2.The knockout vector of K.zeae was constructed,and the target gene knockout system of K.zeae was constructed and optimized,and the Kz PKS18 knockout transformant was obtained.In this study,the Kz PKS18 knockout vector p PZP100HGPKS18 containing hygromycin and green fluorescent marker gene was constructed,and the Kz PKS18 knockout vector was introduced into Agrobacterium AGL-1 by electroporation.The concentration of conidia,Agrobacterium and conidia mixing ratio,the concentration of Agrobacterium,induction time and co-culture time of genetic transformation conditions are optimized,the ATMT knockout system was constructed.The optimal genetic transformation conditions were as follows: after culturing for 7 days in PDA medium at 25 ° C in the dark,the concentration of conidia of K.zeae was 106·m L-1,germination for 3 days under dark conditions at 25 °C,the concentration of Agrobacterium was OD600=0.6.The Agrobacterium and conidia were mixed in a ratio of 1:1 for 1 h,and co-cultured for 48 d at 25 °C in the dark.The average efficiency of transformation was 90.67/106 m L-1.3.The effects of Kz PKS18 on growth,stress resistance and pathogenicity of K.zeae were studied.The Kz PKS18 gene has an effect on melanin synthesis,growth and development,stress tolerance and pathogenicity of anthracnose bacteria in K.zeae.Compared with the wild-type strain,the ?kzpks18 colony ruled,the pleats were less,the colony color was lighter,and the colony edge was light brown,indicating that the gene was involved in the melanin synthesis of the pathogen;The formation rate of ?kzpks18conidial adherent cells decreased slightly;the mycelial growth rate and sporulation of ?kzpks18 increased significantly,indicating that Kz PKS18 negatively regulates the mycelial growth and sporulation of the pathogen;spore germination rate and attachment cell formation rate of ?kzpks18 decreased significantly,indicating that the Kz PKS18 gene positively regulates conidial germination and attachment cell formation.The sensitivity of ?kzpks18 to SDS,Na Cl and H2O2 was significantly lower than that of wild type,indicating that Kz PKS18 gene is related to the cell wall integrity,osmotic stress and oxidative stress of the pathogen.The virulence test showed that the Kz PKS18 gene affects the pathogenicity of the pathogen.