Construction Of Gfp Expression Vector For Fungus And Agrobacterium Tumefaciens-mediated Transformation Of Colletotrichum Gloeosporioides

Walnut(Juglans regia L.),is an important "woody oil" strategic tree species.However,with the expansion of walnut intensive cultivation area and the lack of resistant varieties,walnut disease is becoming increasingly serious.The walnut anthracnose is caused by the colletotrichum gloeosporioides,it can lead to fruit gangrene,leaf blade dry,and damage the young shoots,it is a devastating disease in walnut production.In this paper,a green fluorescent protein(gfp)expression vector suitable for fungi was constructed.Agrobacterium tumefaciens-mediated transformation(ATMT)was used to genetically transform Bacillus anthracis to obtain the green fluorescent protein Colletotrichum gloeosporioides and was used to study the mechanism of the infection of walnut and the interaction mechanism between walnut and Colletotrichum gloeosporioides.The results are as follows:1.Construction of Fungal gfp Independent Expression Vector(DL-gfp).The promoter and terminator of the fungal trp C gene promoter Ptrp C and the terminator Ttrp C were used as gfp and hph(Quanpin)genes.(Ptrp C+hph+Ttrp C)and gfp expression elements(Ptrp C+gfp+Ttrp C)were constructed by PCR recombination technique(Overlap PCR).The two expression elements were characterized by the(p Green II 62-SK)of the T-DNA region,to construct an independent expression vector DL-gfp.2.Construction of Fungal gfp Fusion Expression Vector(RH-gfp).The promoter of the trp C gene promoter Ptrp C and the terminator Ttrp C were used as promoters and terminators of gfp and Hygromycin B phosphortransferase gene(hph).The promoter Ptrp C and hph genes were recombined together by Overlap PCR,and the hph gene did not contain the stop codon.The fragment of the Overlap PCR was ligated with gfp,the terminator Ttrp C and the linearized Ti plasmid(p Green II 62-SK)by In-fusion Enzyme ligations were used to construct the fusion expression vector RH-gfp.3.Re-separation of purified virulence strong m9 coliform bacteria.The m9 strain of Bacillus subtilis,which was preserved at 4 ? in the laboratory,was cultured directly on potato dextrose agar medium(PDA),the mycelium was long,the sporulation rate was less,the sporulation capacity of the strain decreased,and the m9 strain was re-infected with walnut leaves,A large number of m9 conidia were obtained and purified on a PDA plate after purification.4.Agrobacterium tumefaciens-mediated transformation of Colletotrichum gloeosporioides.The concentration of Agrobacterium tumefaciens was 107/m L,Agrobacterium tumefaciens GV3101(containing DL-gfp vector)OD620?0.5;Acetylacetone(AS)induced concentration of 200?M,The inhibitory concentration of Bacillus thuringiensis was 100?g/m L,and the inhibitory temperature was 22°C for 2days.5.The m9 converts were obtained with fluorescent protein markers.After several subcultures,the transformants that could stably express the gfp marker gene and the hph gene.These were screened has laid the foundation for the subsequent study of the differences between the biological characteristics of transformants and wild-type strains and the observation of the interaction of the dynamic process of living between the transformants and walnut.