Cloning And Functional Analysis Of STK Genes Regulating The Growth, Development And Pathogenicity Of Setosphaeria Turcica

Northern Com Leaf Blight, caused by Setosphaeria turcica, is one of the most important diseases in corn planting area. MAPK, the universal signal tranduction pathway in fungi, is one of the key protein kinase in regulating the growth, development and pathogenicity of fungal pathogens. Three MAPK genes, STKI (Setosphaeria turcica mitogen activated protein kinase 1), STK2 (Setosphaeria turcica mitogen activated protein kinase 2), STK3 (Setosphaeria turcica mitogen activated protein kinase 3), were cloned. The phylogenetic analyses revealed that STK1 possibly regulated the osmosis regulation and spore production, STK2 was similar to known MAPK genes in regulating pathogenicity of fungal pathogen, and STK3 was related to cell wall integrity MAP kinase genes. The mutated STK1 gene recombination vectors were constructed to study the gene function, and the results confirmed the above phylogenetic analyses of STKI gene. This work will facilitate the studies on similar functional analyses of the other two MAPK genes in S. turcica, the composition of MAPK signal pathway, and the relation between different MAPK pathways. The antisense inhibition of STK1 gene was done to make the basis of gene-targeted disease control strategy, and the positive gene expression induction vector was constructed for the transformation of the gene and studies the relationship between different genes. The ATMT mutants with similar characteristics as STK1 mutants were screened and the STK1 gene expression in these mutants were analyzed. The normal expression of STK1 gene in these mutants has shown the complex regulation mechanisms1.Three MAPK genes, STK1, STK2, STK3, were cloned with the candidate gene cloning strategy. The cDNA and DNA full-length sequences of STK1 and STK2, a cDNA and DNA fragment of STK3 were acquired. STK1 gene was updated in GenBank(GenBank accession AY849317).2.The structure of STK1, STK2 and STK3 were revealed. STK1 included a 1506bp DNA sequence with a 1071 bp coding region, nine exons and eight introns. The predicated protein of STK1 gene had 356 aa. STK2 gene includes 1277bp with a 1059bp cDNA sequence, five exons, and four introns. The predicated protein of STK2 gene had 352 aa. The STK3 gene fragment was 591bp, with three possible introns (172bp) and four possible exons (419bp). All introns were accordance with GT-AG rules.3.Phylogenetic analysis had shown the three genes possibly belonged to three signal pathways: STK1, related to osmosis regulation, stress reaction and spore development; STK2, related to pathogenicity; STK3, related to cell wall integrity.Aβ-microtublin gene in S. turcica were cloned, sequenced and used as the controls in studying the expression of STK1 gene in high osmosis. The STK1 gene expression was stable within the first 8 hours aider the high osmosis treatment, but rapidly descended after 8 hours treatment.4.Under the high osmosis stress, the wild type isolate exhibited high changes in colony morphology, color, growth speed, but changes in the STK1 mutants were not obvious.5.A prokaryote gene expression vector of STK1 was constructed, pET28a(＋) vector, E. coli BL21, and the STK1 gene expression were confirmed by SDS-PAGE and Western blotting.Southern hybridization results have shown the STK1 gene had a single copy in the genome of S. turcica.6.The STK1 gene-disruption vector was constructed based on the gene homologous combination theory and PEG gene transformation system. A STK1 gene-disruption isolate was successfully screened from 246 transformants and named as STM-35 in the thesis. The STM-35 mutant exhibited grey colony, few aerobic mycelia, transparent hypha, cell lysis in the center colony, significantly reduced HT-toxin activity and pathogenicity, without conidial production.7.The sense and antisense STK1 gene expression induction vectors were constructed based on the plasmid pSOI, and 5 antisense STK1 gene mutants were screened from 30 transformants. The characteristics ofSTK1 antisense inhibition mutants were similar to the gene-disruption mutant STM-35.STAM-26, a mutant acquired by ATMT method, and three melanin-loss mutants induced by UV radiation, exhibit similar traits as STM-35. STK1 gene expression was undisturbed in the two mutants had predicated there were complex network regulating the growth and development in S. turcica.The above results can summarize the function of STK1: insignificant effect on mycelium development and growth but related to the pigment accumulation in hypha; key factor regulating the conidial development; related to the HT-toxin activity and pathogenicity.