The Application Of Site Specific Recombination System For Selection Marker Gene Excision In Poplar

Poplar is one of the widely cultivated species in China. It plays an irreplaceable huge role in the ecological construction, the city greening and timber production. Using conventional breeding method often can not effectively directional cultivate new varieties, therefore transgenic technology has become an effective means in poplar genetic improvement. However with the transgenic technology in the application of breeding, its biological safety problems caused people more and more attention.The new site-specific recombination system as the basis, the loxFRT fusion recognition sites instead of traditional single recognition sites lox or FRT, using physical inducible promoter and chemical inducible promoter accurately control the expression of recombination enzyme FLP, builting a new safety and efficient poplar transgenic technology, effectively removing genetically modified poplar’s marker genes which may bring the biological safety problems, radically preventing genetically modified through the pollen escape, for genetically modified poplar accumulating promotion lay the foundation. Our detailed results are as following:(1) Transforming into Zoysia japoncia Steud callus by particle bombardment, testing the transient expression of gus gene, we verify the entire genetic transformation system feasibility of the pLFGNHFLP and pLFGNXVE.(2) We transformed pLFGNHFLP and pLFGNXVE into poplar by agrobacterium mediated genetic transformation, and there were23and48positive transgenic plants detected by PCR. Heating shock the13pLFGNHFLP transgenic plants, we obtained5non-selective marker gene transgenic poplars. We made4different concentration17-(3-estrodial into4pLFGNXVE positive transgenic plants and obtained4non-selective marker gene transgenic poplars. We obtained the most suitable17-β-estrodial concentration to induce the recombinase.(3) We constructed maker gene delete vector PYL3cry3A which contained the insect-resistant. By agrobacterium mediated genetic transformation, we obtained24positive transgenic plants detected by PCR. The mBt-Cry3A ImmunoStrip detected the positive transgenic poplar, only1plant expressed protein. Heating shock the24transgenic plants, we obtained8non-selective marker gene transgenic insect-resistant poplars.