Agrobacterium-mediated Transformation (AMT) And The Expression Of Green Fluorescent Protein In Trichoderma Reesei

Saprophytic fungus Trichoderma reesei can produce a complete set of extracellular cellulases for the degradation of cellulose and hemicellulose.It is a biotechnically important filamentous fungus.As a lower eukaryote,T.reesei has simple structure and fast growth rate,so it's a kind of significant biomaterial for the study of fungi heredity and metabolism.The genome sequence of T.reesei has been completed,and it's useful for the development of functional genomics.In order to research the molecular mechanism of heredity and metabolism,it is important to establish the Agrobacterium-mediated transformation(AMT)system,form mutant library and analyse the different mutants to work out the biological function of these genes.This is important to the molecular biology research and genetic modification of T.reesei.Green fluorescent protein(GFP)is widely used in the study of prokaryote and eukaryote as a new reporter gene.The green fluorescence is excitated by UV or blue light.It is stable,high sensitive and it doesn't need substrates or helper factors.Since successfully expressed in Escherichia coli and Caenorhabditis elegans,GFP and other fluorescent proteins have been widely used for many studies.In this study,filamentous fungus T.reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1.T-DNA binary vector pBI-hph was constructed by introducing the hygromycin B phosphotransferase gene(hph)isolated from pAN7-1 into pBIN121.Co-cultivating of A.tumefaciens containing pBI-hph with T.reesei conidia or protoplasts,hygromycin B-resistant clones were selected and the integration of the hph gene into T.reesei genome was determined by PCR and Dot blot analysis.T-DNA borders and the flanking genome sequences were successfully rescued from the putative transformants by TAIL-PCR.Now,T-DNA tagged T.reesei mutants had been obtained from the AMT transformation.It suggests that Agrobacterium-mediated transformation is a potentially powerful tool for the gene transfer technology of T.reesei.Plasmid pIG1783 is transformed into two sac fungi,and egfp gene in this vector can be expressed successfully.In this study,the egfp gene cassette was obtained,and T. reesei M23 was co-transformated with this cassette and plasmid pAB4-1.Two transformants with stable heredity of the egfp gene were selected by PCR analysis. The RT-PCR,SDS-PAGE results indicated that egfp gene was transcripted and translated successfully.Detecting with fluorescence microscope,green fluorescence can be found in hypha and spores of T.reesei transformants.To improve the screening efficiency,egfp gene and orotidine-5'-phosphate decarboxylase(pyrG)gene were used as marker genes.Two binary vectors were constructed with a pCAMBIA0390 backbone.The binary vector pCMBIA-P containing pyrG gene,was transformed into A.tumefaciens EHA105.After co-cultivation between A.tumefaciens and T.reesei,resistant transformants were selected.PCR analysis established that pyrG gene was incorporated into the genome. Another double-selective-marker binary vector is pCMBIA-OEP,containing the pyrG and egfp genes.T.reesei M23 was co-cultivated with A.tumefaciens containing pCMBIA-OEP.Resistant transformants were selected and detected with fluorescence microscope.Green fluorescence can be found in hypha of T.reesei transformants.The analysis of transformants is undergoing experimental procedures.Agrobacterium-mediated transformation(AMT)system was constructed and egfp gene was expressed successfully in T.ressei.An efficient and high throughput screening system is hoped to build up and we try to make deep analysis to T.reesei transformants.