The Function Of Laccase Genes And Optimization Of Fermentation Condition For Laccase Production In Setosphaeria Turcica

Laccase is the multicopper oxidase, whic h plays an important role in life cycle, and has more and more applicatios in many industrial production fields. In this research, in order to analyze the function of laccases in the pathogenic fungi of S.turcica, the expression level of St LAC-family genes in Setosphaeria turcica was tested in the development stage and inducing conditions by q PCR. The complemented strains of St LAC1 and St LAC2 gene were obtained using the technology of PEG- mediated transformation, in order to analyze the functions of St LAC1 and St LAC2 gene. Based on single factor experiments, central composite design(CCD) combined with response surface methodology(RSM) was employed to optimize the laccase fermentation conditions of S.turcica. The main results were as follows:1. The expression pattern of laccase genes of the S. turcica was analyzed by q PCR, indicating that different laccase genes played different roles in development and inducers. The St LAC1 was highly expressed in the stage of appressorium and penetration pegs, which means that it is mainly involved in the process of infection. The expression of the St LAC2 during conidia stage was higher than the other stages. The expression level of St LAC6 and St LAC8 were at high level in all stages except for the conidia stage. The expression of St LAC2、St LAC4 and St LAC5 were at highest level when induced by resveratrol. When induced by ABTS and Cu2+, the expression level of St LAC6 and St LAC8 were higher than that of the control. These results indicated the laccase family genes played different roles in the infection process and cultivation condition.2. The complemented vectors of St LAC1 and St LAC2 were transformed into the protoplast of △St LAC1 and △St LAC2 strain using PEG- mediated transformation of protoplast, and transformants C.△St LAC1 and C.△St LAC2 were obtained.3. The phenotype analysis showed C.△St LAC1 strains grew faster, produced more melanin and performed weaker osmotic ability compared to that of △St LAC1. When the completion of St LAC1 gene in S. turcica, the infection capacity in appressorium were the same as the wild type. The laccase activity and pathogenicity were enhanced compared to the △ St LAC1. These results showed that St LAC1 gene was involved in melanin biosynthesis and the penetration and pathogenicity of appressorium.4. The phenotype analysis of wild type, △St LAC2 and C.△St LAC2 showed that the aerial hyphae of C.△St LAC2 colonies were lush, the mycelium’s cell wall were thicker, its hydrophobicity ability was stronger and C. △ St LAC2 showed weaker resistant capabilities to salt and high osmotic pressure compared with △St LAC2, which showed that the integrity of cell wall was recovered. In addition, the laccase activity were increased, the ability of melanin biosynthesis and infection was complimented. It was indicated that the St LAC2 were involved in the melanin biosynthesis, appressorium penetration and development in S. turcica.5. Building the knocked-out vector of St LAC2 using the PBS vector intergrited with the glufosinate gene, and obtained a double- gene deletion mutant △St LAC1/△St LAC2 by PEG- mediated transformation.6.The multivariate quadratic regression model which the enzymatic activity was chosen as response value was significantly different(P=0.0001), which meant the established regressing equation for activity of laccase had an excellent goodness of fit and the laccase fermented by S. turcica could be analyzed by this model. According to the model, the factors influencing the activity of laccase were in the order as followed: Cu2+>glucose>urea. The optimum conditions, which was predicted by this model, were 50.05 g/L glucose, 1 g/L KH2PO4, 1.46 g/L urea, 0.5 g/L Mg SO4, 2 g/L peptone, 0.5 g/L corn steep liquor, 0.07g/L Cu SO4 and 3m L/L Tween80, at 28℃, 150 r/min. The maximal enzyme activity was 40.00±1.20 U/m L after 7 d of cultivation. The laccase of S. turcica had a molecular mass of 80 k Da after purification, and it showed optimum p H and temperature at p H 4.2 and 50℃ respectively, using ABTS as substrate. The laccase of S. turcica exhibited high thermal stability and the stability of p H. The K m was 0.036 mmol/L, at room temperature and p H 4.2.