Cloning Of High Efficient Promoter Fragments And Construction Of Transgenic Vectors Of Fungi

Bursaphelenchus xylophilus is one of the most serious plant pathogens in the forest of China. It canfeed on blue stain fungus which is one of endophytic fungi on pine trees after it infects and invadsplants. Fungi play an important role in the population reproduction and disease epidemic of nematode inthe forest. And currently Botrytis cinerea is also the source of food in cultivating B. xylophilus. Thestudy of fungi-nematodes interaction, construction of tool vector for fungal transgenes could providetheoretical foundation for the study on epidemicology and prevention and cure of pine wilt diseasecaused by B. xylophilus, lay the foundation for fungal transformation system as well.This study used pCAMBIA1300vector as basic vector，modifying to make it suitable for fungitransgenic strategy. First，Sclerotinia sclerotiorum genome DNA was used as template to clonepromoter segment of acp and gpd；Hygromycin resistant gene—hph and green fluorescein gene—gfpwere isolated from pCAMBIA1300plasmid and pEGFP-N1plasmid, repectively. These four DNAfragments were then ligated by using overlap extension PCR technology and linked the two longfragments in vitro. At the end, the linked large fragments were inserted into pCAMBIA1300, producingtransgenic vector pCAGG-N1.Agrobacterium mediated transformation method were used for B. cinerea transgentic construction.A GFP expressed B. cinerea was constructed.In this dissertation, we construct a vector which could be widely used in fungal transgeneticconstruction by cloning the high efficient promoter fragment, and it make heterologous expression workin B. cinerea. It clarifies the study of fungi-nematode interaction, nematode-controled with geneticallymodified fungus and other researches-related.