Cloning And Function Analysis Of Protein Kinase From Alternaria Longipes

Signal transduction plays an important role in the processes of environment-cell or cell-cell interaction. A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction.Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade.The best methods and conditions were studied for protoplast preparation and regeneration of Alternaria longipes. Protoplast with high concentration and strong ability can be obtained when the hypha were digested with 10 mg / mL Lywallzyme at 30℃for 3 h.The hypha used in this process were cultivate for 24h in PDB,and the osmotic stabilizers used in digestion and regeneration were NaCl and sucrose.The protoplast of Alternaria longipes were successfully transformed with restriction enzyme-mediated integration and the transformation system has been examined, including the enzymes and their concentration, Alternaria longipes of protoplast, concentration of plasmid, concentration of PEG. Over 600 transformants were obtained when protoplasts were transformed with 2μg linear pUCATPH (digested by HindⅢ),20 U HindⅢ, 107 / mL protoplast and 60% of PEG3350。It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification.Most of the transformants were fully stable after five rounds successive culture.By using the system of Agrobacterium tumefacines-mediated transformation,we successfully transformed Alternaria longipes and obtained T-DNA insertion mutants.Under an optimum condition(Alternaria longipes concentration:106/mL;A.tumefacinesOD600=0.25; Acetosyringone concentration: 300μg/mL, co-cultivationtime: 48h), 85 transformants have been obtained and most of them were quite stable after five rounds successive culture.PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division.Degenerate primers designed on the conserved domain of other reported serine proteases, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The full length of aapk1 cDNA gene is 1905bp, which contained an ORF of 1476bp encoding 491amino acids. The cDNA and DNA sequence of gene aapk1 has been registered in Genbank with accession number EU381116 and EU381117 respectively. The catalytic domain of aapk1 was compared with the common protein kinase,and the result showed that aapk1 has the common features,Such as GTGSFGRV in subdomainⅠ,K72 in subdomainⅡ,E91 in subdomainⅢ,YRDLKPEN in subdomainVIB,DFG in subdomainⅦ,GTPDYLAPE in subdomainⅧ,D220 in subdomainⅨ,R280 in subdomain XI。The aapk1 gene targeting vector was constructed by double-joint PCR (DJ-PCR), and the homologous fragment was 652 bp and 620 bp, respectively. Protoplasts of A. longipes were transformed, and PCR was used to screen aapk1 gene deletion mutants(△aapk1). Southern blot and RT-PCR method were used to verify△aapk1 mutant. The wild type of A. longipes and△aapk1 mutants were compared and contrasted. the resulted showed that the differences between the conidia sporulation and the shape, size, color of conidia were not obvious, but△aapk1 transformat showed slow growth rate on PDA .Also the dry weight of△aapk1 mutants was lower than that of wild type of A. longipes.From these result we concluded that the aapk1 gene plays an important role in the mycelia growth of A. longipes.The pathogenicity test showed that the lesion which caused by inoculation with spores suspension of△aapk1 mutant occurred later than that of wild type of A. longipes,but the lesion size showed no significant difference.