Cloning And Identification Of Two Chitinase Genes From Limonium Bicolor

Chitinase is a kind of glycosidase catalyzing the degredation of chintin,an important constituent of fungal cell wall.Chitinase have roles in suppressing the growth of fungi, enhancing the resistant ability to fungal pathogens in plants.Though chintin is not found in plant,the chintinase is existed widely in plant.Recently,accompanying of further study of the catalytical mechanisms,biochemical function and expression regulation,control plant pathogens by transformed the plants with chitinase is becaming an effective method.In this study,two chitinase genes,chi31 and chi32,were isolated from Limonium bicolor, and they were deposited in GenBank with the accession number DQ431248 and DQ431249, respectively.The DNA GeneBank accession number EU744178 and EU744179,respectively.Real-time RT-PCR was employed to investigate the differential expression of chi31 and chi32 in roots and leaves of Limonium bicolor when inoculated with Fusarium oxysporum.The results showed that,when roots were infected with Fusarium oxysporum,chi31 was highly expressed in root and the chi32 transcripts were very low in root;however,both of them expressed slightly in leaves.In case of a fungi isolation of leaf infected leaves,the chi31 was continuously up-regulated,whereas the chi31 presented an oscilation pattern.Thus,the chi31 respond to roots pathogens invasion,and the chi32 was stimulated by leaves pathogens infection.Thees two chitinase genes respose synergistically in different tissues and periods of infection,each plays indispensible roles in the pathogen resistant system,constitute an integrated defence system to outer stimuli.Two recombinant secretion expression vectors,pPIC19-chi31 and pPIC-chi32,harboring the chitinase genes chi31 and chi32 gene,were construted and transfomed into yeast Pichia pastoris.The yeast strains,which can secrete recombinant chitinases were identified by SDS-PAGE examination of the fermentation supernatant.Enzymatic activities in the fermentation supernatant was determined by the modified Schales method,and the results demonstrated that the CH131 and CHI32 can be expressed and secreted into medium with full biological activities in two Pichia pastoris strains GS115 and KM71.The most suitable reaction condition for the activity of CHI31 is 5mmol/L of Mn²⁺,40℃,and the pH of 5.0,using the Fusarium oxysporum cell wall as substrate.Under this condition,the enzymatic activity of CHI31 was 0.79U in the crude fermentation media.Correspondingly,the recombinant CHI32 most optimal condition is 5mmol/L of Ba²⁺,40℃,and the pH of 5.0,with the substrate of Valsa sordida cell wall,the activity of CHI32 is 1.21U in the crude fermentation media under this condition.Moreover,the recombinant CHI31 demonstrated better hydrolytic ability to cell walls of different fungal than synthetic chitins,suggesting a high potentiality utilization of CHI31 on biocontrol.However,the recombinant CHI32 showed a high capacity of decomposing hydrolytic synthetic chitins than fungal cell wall,.These results suggested that the two chintinase genes may play roles in defence the invasion of fungal pathogens.The recombinant chitinase expressed by Pichia pastoris have much values in controlling plant pathogen in agriculture and forestry.Production of recombination enzyme by using Pichia pastoris can decrease the cost of production and the expense on control plant pathogen. This study also provides solid foundation for the further application of chitinase genes in pathogen-resistant plant molecular breeding.