Coloning And Expression Analysis Of Pectate Lyase Genes And Preliminary Research Pathogenicity Function Of Cgpel3 From Colletotrichum Gloeosporioides,the Pathogen Of Mango Anthracnose Disease

Mango(Mangifera indica L.),known as the "tropical fruit king",is a valuable tropical and subtropical fruit with high economic value.Anthracnose caused by Colletotrichum gloeosporioides Penz.&Sacc.is one of the most serious fungal diseases in mango producing areas around the world,causing serious economic losses to the mango industry.In this paper,the pectate lyase activity of C.gloeosporioides was analyzed.The full-length DNA and cDNA sequences of the pectin lyase genes Cgpell,Cgpel2 and Cgpel3 were obtained by PCR.The bioinformatics of pectate lyase genes family and the evolutionary relationship with other fungal pectate lyase genes were systematically analyzed,and the functions of the three pectate lyase genes were preliminarily inferred.The expression of Cgpell,Cgpel2 and Cgpel3 in the process of C.gloeosporioides infecting mango were quantitatively analyzed using quantitative Real-time PCR method,and the effect of Cgpel3 was the most obvious.The relationship was determined between the three pectate lyase genes and pathogenicity of C.gloeosporioides.Using the In-Fusion HD(?)Cloning Kit,the Cgpel3 gene knockout vector was constructed and used to transform C.gloeosporioides protoplast by PEG method.Knockout mutants of Cgpel3 gene were obtained by PCR verification,target strip sequencing and gene expression analysis.Phenotypes were measured and pathogenic functions were analyzed.The main results were as follows:1.With the prolongation of culture time in PD solution,the activity of extracellular pectate lyase of C.gloeosporioides increased firstly and then decreased.It enzyme activities reached the maximum on the 6th day,with 61.65 U/mL.That indicated C.gloeosporioides could produce pectate lyase and had strong ability to produce enzymes.C.gloeosporioides was highly sensitive to fungicide Fluazinam,with an EC50 0.1536 and a slope of 3.4890,which has great potential for controlling mango anthracnose.Enzyme activity analysis showed that Fluazinam could effectively inhibit the secretion of pectate lyase from C.gloeosporioides.2.Three pectin lyase genes Cgpel1(MH395929.1),Cgpel2(MH410608.1)and Cgpel3(MH423503.1)were cloned from C gloeosporioides causing mango anthracnose.The complete DNA sequence of Cgpell,Cgpel2 and Cgpel3 gene were 1037bp,1498bp and 1089bp,respectively.The complete cDNA sequence were 975bp,1380bp and 978bp,encoding 324,459 and 325 amino acids,with a specific conserved domain of pectate lyase.Cgpel1,Cgpel2 and Cgpel3 protein contained a typical signal peptide and no transmembrane structure.In their secondary structure,there were 16.05%,20.26%and 16.92%a-helix,28.09%,21.79%and 29.54%extension strand,5.86%,8.93%and 7.38%β-turn and 50.00%,49.02%and 46.15%random coil.Phylogenetic analysis showed that amino acid sequence of Cgpell,Cgpel2 and Cgpel3 were respectively homologous to C.tofieldiae pectate lyase(KZL77240.1),C.gloeosporioides pectate lyase(XP-007274932.1)and C.incanum pectate lyase(KZL84476.1)with a similarity over 88%.The Quantitative Real-time PCR results showed that three pectate lyase genes were highly expressed continuously throughout the infection process and Cgpel3 was expressed higher than Cgpell and Cgpel2.Preliminary speculation they may mainly regulate pathogenicity.3.The phenotypes of Cgpel3 gene knockout mutant △Cgpel3 were analyzed.Compare with the blackish-brown wild type,the colony of mutant △Cgpel3 became light in color and was always white,and hyphae growth rate decreased by aboutl8%-29.5%,with sparse hyphae.The diameter of mycelium of mutant △Cgpel3 was not changed,while branch and septa of hyphae reduced.Conidial production of mutant △Cgpel3 decreased obviously and could not form appressorium on the tip of germ tube.There was little difference in the optimum temperature range for mycilial growth,but the lethal temperature of mycelia decreased.The utilization ability for part carbon and nitrogen sources was different from the wild type.The sensitivity of mutant △Cgpel3 to osmotic stress and the resistance to H2O2 were declined.The activity of pectate lyase decreased by aboutl4%-58%.Mutant △Cgpel3 could infect the wounded mango leaves and fruits,but the virulence decreased by about 30%～34%and 50%～60.1%.The unwounded mango leaves and fruits could not be infected.Quantitative Real-time PCR analysis showed that the pectin lyase gene Cgpel3 was almost not expressed in the mutant △Cgpel3,and the expression quantity of seven genes related to appressorium formation and melanin synthesis were less than 1,and the expression quantity of pectin lyase gene Cgpell and glucanase gene CgCBH1 were respectively was 2.01 and 1.25 times that of the wild type.The results revealed that the Cgpel3 plays an important role in the regulation of mycelial growth,development,differentiation,conidia production,appressorium formation,adaptability,sensitivity to fungicide Fluazinam,pathogenicity to mango,and expression of other pathogenicity related genes in C.gloeosporioides.In this paper,the ability to produce pectin lyase,the characteristics of pectin lyase gene family,and the function of pectin lyase gene Cgpel3 of C.gloeosporioides were systematically analyzed from the bioinformatics,molecular biology,physiology and biochemistry,etc.A new fungicide also has been found,which has the potential to control mango anthracnose.