Functional Complementation Of A MAPK Homologous Gene(CDK1)-deficient Mutants In Corynespora Cassiicola

Hevea brasiliensis was an important economic crop in tropical area, and its product was known as one of the four industrial raw materials of modern industrial societyone. The Corynespora Leaf Fall (CLF) caused by Corynespora cassiicola (Bert.&Curt.) Wei was one of the major disease in Hevea brasiliensis. The studies on the CLF had made progress in pathogen biology, disease epidemiology and the control measures. However, there was few reports about the functions of the pathogen-related genes. However, research on pathogen-related genes would be of great scientific value so that revealing the pathogeneisis mechanisms and knowing the interaction relationship between C.corynespora and Hevea brasiliensis. The researches also would be useful for finding new control targets for this disease.In our privious studies, a MAPK homologous gene (CCK1) of C. cassiicola was disrupted and the mutant ?CCK1was acquired. To illuminate more accurately the fuction of CCK1gene in this study, as gene homologous cloning strategy, the fragment of upstream (containing the complete open reading frame of CCK1) and downstream of CCK1gene, a MAPK homologous gene, were amplified from C. cassiicola(B.&C.)Wei. The bar gene as the resistance marker was be inserted in reverse between the upstream and downstream of CCK1gene by using the In-FusionR HD Cloning Kit. Thus, the complementary vector p ?CCK1-C of the ?CCK1mutant was successfully constructed. A complementary fragment from the p?CCK1-C vector containing the5end fragment of CCKl1::bar::the3end fragment of CCK1was amplified, and be used to transform the protoplasts of ? CCK1mutant by the PEG method. After Basta plate screening, PCR verification, a complementary strains of ? CCK1mutant was acquired, named as strains CBB3. The results of phenotype were as follows. By the complementation of CCK1gene, the ?CCK1complementary strains CCB3was restored to some characteristics of the wild-type in mycelium lethal temperature, the sensitivity to different high osmosis stress, as well as toxins, laccase and cellulase production. Some characteristics were closed to the wild-type in the CCK1expression,the colony color, mycelium growth, conidium formation, and pathogenicity, but less the wild-type. The above results indicated that the protein kinase CCK1might be play a important regulatory role in mycelial growth, development, and toxins, laccase and cellulase production, and adaptability to hight temperature and high osmosis stress, as well as pathogenicity of C. gloeosporioides in Hevea brasiliensis. In addition, the CCK1could weaken the promote growth effect of exogenous Ca2+on mycelia, and enhance the inhibitory growth effect of exogenous cAMP on mycelia.