Analysis Of The Function Of MAPK Genes And The Relationship Between MAPK Genes And Toxin,Melanin-related Genes In Curvularia Lunata

Curvularia leaf spot,caused by Curvularia lunata(Wakker)Boedijn,is one of the most serious leaf fungal diseases in corn.At present,there is a preliminary study in the pathogenesis of C.lunata.It has proved that MAPK signal pathway has an important role in the aspect of pathogenic in C.lunata,and the main way to damage the plants is by production and accumulation of toxin and melanin.But it is still no research on whether there is a correlation between the MAPK signal pathway and the toxin,melanin pathway.Meanwhile,it shows that acetyl-CoA and malonyl-CoA have important roles in accumulation of toxin and melanin that are not clear in C.lunata when C.lunata produces toxin and melanin during secondary metabolism.Therefore,on the basis of the previous research,the function of Clh1 gene in C.lunata that is homologous to Hog1 in Saccharomyces cerevisiae is studied,at the same time,an acetyl-CoA gene ClMfp was obtained in C.lunata.Then,researched the possibly regulating relationship among MAPK,virulence,melanin and acetyl-CoA,malonyl-CoA in C.lunata.1.Definitized how Clh1 gene affects pathogenic factorsObtained the gene sequences which are the highest homologous to Hog1 in S.cerevisiae from the C.lunata whole genome sequence by Blast.The gene length is 2.782 kb,cDNA length is 1.134 kb.In order to analyze the function of Clh1,homologous recombination is used to construct the vector and agrobacterium mediated transformation(ATMT)is used to screen the transformations.Compared Clh1 mutant to the wild type strain CX-3,the growth rate becomes more slowly but Clh1 mutant stops growing.The concentration of glycerinum in the mutant is distinctly decreased and the growth rate of germ tube slows down.The pathogenicity of mutant is decreased and that is more serious when inoculated the mutant on the wound leaf.The activities of cellulose and cutinase are both decreased.Therefore,Clh1 gene is involved in the regulation of osmotic pressure that is one of the most important mechanisms to help fungus to invade into the host by mechanical stress.2.MAPK genes accelerate the production of toxin and melanin and inhibit the expression of resistant genesReal-time quantitative pcr is used to test the expression of toxin,melanin,pathogenicity and defense genes(Clt1,PKS18,Brn1,Scd,VelB,eEF3,Sod,Ace1,Hsp104,Tsa1)in MAPK mutants(?Clk1,?Clm1,?Clh1).It shows that the expressions of toxin,melanin genes(Clt1,PKS18,Brn1,Scd)are all decreased.The expression of PR protein is significantly increased during the wild-type strain CX-3 infects the leaves,on the contrary,during the MAPK mutants infect the leaves,the expression of PR protein is decreased(P<0.01)that proves that MAPK genes accelerate the host defense response.In addition,the expression of Rp1-dP2 gene is increased after MAPK mutants infect leaves so the MAPK genes can instantly reduce the expression of NB-LRR gene in order to promote its infection.The concentrations of Methyl-hydroxy methyl(5')furan-2-carboxylic acid and DHN decreased in the mutants by using HPLC.3.ClMfp gene mainly affected the production of toxin and melaninHomologous recombination and ATMT are also used to research ClMfp gene that is related to acetyl-coA.The gene length is 3.853 kb,cDNA length is 1.15 kb.The color of the colony is lighter in the mutant than that is in the CX-3.The number of conidium and the ability of infection are significantly decreased,and it can't damage the host when it is inoculated on the unwounded leaf.The concentration of Methyl-hydroxy methyl(5')furan-2-carboxylic acid is decreased in the mutant by using HPLC and the expression of toxin,melanin and disease-related genes are all decreased distinctly.4.Analyzed the relationship between MAPK genes and acetyl-coA,malonyl-coAIt found that the concentrations of acetyl-coA and malonyl-coA are distinctly decreased in MAPK mutants and the same as the acetyl-coA genes.The expressions of MAPK genes are also decreased in ClMfp mutant.In addition,it shows that the pathogenicities of the MAPK mutants are all increased after adding acetyl-coA into the spore suspension and the content of furan is also increased.All in all,it proves that MAPK gene participated the production of toxin,melanin and pathogenicity during the secondary metabolism in fungus.And MAPK pathway can affect the production of acetyl-coA and malonyl-coA that can also influence the expression of MAPK genes.