Establishment A Transformation System For Thermomyces Lanuginosus And Subcellular Location And Construction Gene Knockout Vector 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. Through genetic transformation, exterior plasmid insertion into genome randomly to establish the mutant of this fungus is meaningful to research the thermophilic mechanism. In addition, it may provide wide application in gene over expression in this fungus and separating functional gene.Agrobacterium tumefaciens-mediated transformation (ATMT) as a simple and efficient method for transforming and insertional mutagenesis has been widely used in plants and fungi, but has not been used in any thermophilic fungus so far. In this study, a stable transformation system of the thermophilic fungus T. lanuginosus was established and the transformation procedure was optimized. Pre-germinating spores of T.lanuginosus used at co-cultivated period was prerequisite.5×106germinating spores per plate co-cultivated with Agrobacterium tumefaciens stain EHA105at28℃for48h achieved the highest transformation efficiency. The addition of Acetosyringone (AS) during the pre-culture of A. tumefaciens and the co-cultivation was an absolute requirement and the concentration up to500μM in co-cultivation medium was more appropriate. Southern blot analysis suggested that83.3%of transformants contained a single insert of T-DNA. Unique hybridization patterns along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation was a tool for insertional mutagenesis of T.lanuginosus. Besides, transformation systerm was also constructed with plasmid pUCATPH transformed into protoplast of T.lanuginosus by electroporation.Protein kinase gene pspk is a new gene cloned from T.lanuginosus. Through the bioinformatics analysis, speculate it may have the function of mRNA cut out, but the exact function is unknown. Prediction of protein subcellular location is one of the key functional characters to understand its biological function. To analysis the function of the T.lanuginosus protein kinase gene pspk, we constructed the plasmid pROK Ⅱ-GFP-pspk used for subcellular location. A. tumefaciens strain LBA4404contained the pROK Ⅱ-GFP-pspk vector was used to infect onion epidermal cells. In fluorescence microscope, we found the green fluorescence appeared in the nucleus and predicted the subcellular location of this protein kinase was nucleus. For further analysis of the gene function, the pspk gene-disruption vetor were constructed based on the gene homologous combination theory. pUCATPH/PK and pROK Ⅱ-hygro/PK vector are used for electroporation and ATMT to disrupt the gene, respectively. The pspk gene knockout vectors were constructed by using of the homologous fragment was739bp and737bp. The constrction of the targeting vector is an importent material for the reaserch of the gene’s function.