Mutant Screening For Thermomyces Lanuginosus And Over Expression Of Protein Kinase

Thermomyces lanuginosus is a thermophilic fungus with higher growth temperature. The fungus can produce thermostable enzynes, and its some thermostable enzymes genes have been cloned and expressed. However, it is unclear about the mechanism how thermophilic fungi recept and transfer signal.Protein kinase gene pspk is a new gene cloned from T.lanuginosus,but its exact function is unknown.Through genetic transformation, exterior plasmid insertion into genome randomly to establish the mutant of this fungus is meaningful to research the thermophilic mechanism. Our laboratory first successfully established its genetic transformation system using Agrobacterium tumefaciens-mediated transformation method.It may provide an important tool for study the function of genes, separate functional genes and an effective way for exogenous gene expression in T.lanuginosus.In addition,using the method of subcellular localization,transform the gene expression vector fusion with GFP gene into onion epidermal cells and express it,then in fluorescence microscope, we observed the green fluorescence appeared in the nucleus,and predicted the onion epidermal cells subcellular location of this protein kinase was nucleus.Genetic transformation system used in the experiment has been successfully constructed Thermomyces lanuginosus, screening of large numbers of mutants by Agrobacterium-mediated method,access to a variety of different mutant phenotype.These mutants and wild strain in colony morphology, color,sporulation and so has obvious difference.This is for the study of gene function, gene isolation function provides materials, provide materials for exploring the mechanism of the resistance of thermophilic bacteria.In this experiment,we studied the T.lanuginosus protein kinase,constructed its expression vector pROK Ⅱ-hph-pspk,and optimized its genetic transformation system established by our laboratory.It eventually determined that when the agrobacterium strains LBA4404in spore germination4.5h,spore concentration at5×106cells/dish,AS concentration at250μmol/L in co-cultivation medium, the expression vector has the highest conversion efficiency.Using fluorescence quantitative techniques determined the expression amount of protein kinase gene pspk in the transformed strains is1.729times in the wild strains,proving that we obtained the over expression strain of the protein kinase gene. By the biological phenotype analysis of over expressing strain, we found they are in a consistent biological phenotype and their thermostability than wild strains is decreased obviously.Through the above study speculated that protein kinase gene pspk may be related to its heat resistant function, help to understand the molecular and physiological characteristics of Thermomyces lanuginosus, provide the basis and the reference for studying the gene function.