Gene Cloning And Functional Analysis Of Protein Kinases In Fusarium Verticillioide And Olpitrichum Tenellum

Fusarium verticillioide is an important plant pathogenic fungus all over the world, and can cause maize seedling blight , maize ear rot,etc. Olpitrichum tenellum is an important biotrophic mycoparasite of Fusarium verticillioide,In order to probing into the mechanism of biological recognition, interaction and parasitization between Fusarium verticillioide with Olpitrichum tenellum and maize ,This research try to clone the protein kinase genes from the two fungi ,and study their function in the process of interaction,and look for the factors by establishing muntants throμgh DNA insertion. As a resμLt ,4 protein kinase genes were cloned , two protein kinase's function were understood by gene disrupting . Also the genetic transformation system of Fusarium verticillioide were established by using the system of REMI and ATMT, and abtained many mutation .1.Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation systems of F. verticillioide and Olpitrichum tenellum, conditions for the protoplasts isolation and regeneration of the mycelia of F. verticillioide and Olpitrichum tenellum were examined, including the effects of the hyphal age, enzymes and their concentration, time and temperature of digestion, osmotic stabilizers on the protoplast preparation and the effect of osmotic stabilizers on the protoplast regeneration. The resμLts indicated that optimum conditions for preparing protoplasts of F. verticillioid were: the condia cμLtured in liquid PDB medium for 14 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 20mg/mL Lywallzyme used to digest the hyphae for 4 hours at 30℃. And 0.7mol/L sucrose was the osmotic stabilizer for the regeneration of protoplasts. Optimum conditions for preparing protoplasts of Olpitrichum tenellum were: the condia cμLtured in liquid PDB medium for 36~48 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 30mg/mL Lywallzyme used to digest the hyphae for 5 hours at 30℃.2.To study the pathogenicity mechanism, the restriction enzyme-mediated integration approach were used to transform the protoplasts of F. verticillioide with vector pUCATPH which has hygromycin B-resistant gene, over 300 transformants were obtained.It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification. Most of the transformants were fμLly stable after five rounds successive cμLture,many phenotypic mutants and 2 weak pathogenicity mutants were gained by REMI. But no transformant was got after transforming Olpitrichum tenellum.3.The plant expression vector pROKII was ligated to hygromycin B resistance gene cassette (PtrpC-hph-TtrpC) with the fungus promoter from plasmid pUCATPH. Thus, the vector pROKII-PtrpC-hph-TtrpC was constructed, and then transferred into Agrobacterium LBA4404. By using the system of Agrobacterium tumefacines-mediated transformation, we successfμLly transformed F. verticillioide and obtained T-DNA insertion mutants. But no transformant was got after transforming Olpitrichum tenellum.Under the optimal conditions (F. verticillioide spores:106 /mL;A.tumefaciens OD600:0.15~0.20; acetosyringone:200μg/ mL ; co-cμLtivation:36h), transformation efficiency reached 60~120 transformants per 106spores. More than 1000 transformants were obtained. Most of them were quite stable after five rounds of successive cμLtures. PCR amplification showed that the T-DNA was integrated into the genome. The transformation system is the basis for studying on pathogenicity mechanism and functional gene of the fungi.4.By using degenerate PCR,TAIL-PCR and RACE technique,four protein kinase genes were cloned in the two fungi.They were named of fpk1,fmk1,opk1 and omk1 .All their DNA and cDNA fμLl-length sequences were acquired ,and their GenBank accession were EF405958,EU417814,EU417815 and EU479712.fpk1Included 1854bp DNA sequence from ATG to TAA,with 1680bp coding region , three intron (their length :66bp,54bp and 54bp),the predicated protein of fpk1 gene had 559aa.Sequence analyse indicated that fpk1 belong to PKA.opk1Included 2223bp DNA sequence from ATG to TAA,with 2031bp coding regin , two introns (their length :108bp,84bp),the predicated protein of opk1 gene had 676aa.fmk1Included 1242bp DNA sequence from ATG to TAA,with 1068bp coding regin , three introns (their length :58bp,56bp,60bp),the predicated protein of fmk1 gene had 355aa.omk1Included 1198bp DNA sequence from ATG to TAA,with 1680bp coding regin , two introns (their length :57bp,73bp),the predicated protein of omk1 gene had 355aa.5.The fpk1 and fmk1 gene-disruption vetor were constructed based on the gene homologous combination theory. The gene-disruption vetor were PBS-kinase-hph and pUCATPH-KS-KX, and hygromycin B resistance gene as the screening label. By transforming protoplasts with the gene-disruption vector, a fpk1 gene-disruption isolate and fmk1 gene-disruption isolate were successfμLly screened from more than 200 transformants. homologous combination was testified by PCR and southern blotting in the mutation .It was clear that the two gene's function were related with hyphal development, conidiophore producing,virμLence and cell wall biosynthesis.