Study On Chimeric Gene Of Spider Insecticidal And Bt Transformation For P.nigra×P.deltoids "108"

Poplar is important quick-growing tree widespread cultivated on the mid-latitude area of the world. But poplar's pest types are so many, and the harm done by the pests is extremely serious, especially, on the large area of the planted poplar forest. Poplar anti-insect genetic engineering provides an effective way for solving this difficult problem. Populus nigra×Populus deltoids "108" is Populus nigra and Populus deltoids artificial hybrid clone. The research took Populus nigra ×Populus deltoids "108" as explant, using agrobacterium-mediated transforming anti-insect gene (the chimeric genes of spider insecticidal and Bt) into the acceptor successfully. Through the experiments ,we have drawn conclusions below:(1) Through the bacteriophages screening experiment, we could draw a conclusion that the cefazolin-sodium's (CEZ) sterilizing effect was the best including 7 kind of bacteriophages. Therefore, we used the CEZ when sterilized bacterium during the experiment;(2) Through the sensitivity experiment of the leaves of Populus nigra ×Populus deltoids "108" to the CEZ, we drew the conclusion that the concentration of the CEZ was 1000mg/L, when sterilize bacterium during the experiment;(3) Through sensitivity experiment of leaves to Kanamycin, the concentration of Kanamycin was 30 mg/L when screening transformed hybrid buds;(4) Through sensitivity experiment of rooting, finally we determined to select that the concentration of Kanamycin was 20 mg/L in root medium;(5) Successfully established the Populus nigra xPopulus deltoids "108" transformation procedure: stimulating bacterium → the leaves preparing invaded 2 3d→as bacterium liquid OD₆₀₀=0.05 -0.1, invade 5 10min→ 37 ℃, cultivating together 2³d→CEZ 1000 mg/L to sterilize bacterium, Kanamycin 30mg/L to screen transformed hybrid buds → sterilizing bacterium and screening several times→obtain transformed hybrid buds resist Kanamycin →after a month , take the transformed hybrid buds into the rooting medium (CEZ 1000 mg/L to sterilize bacterium, Kanamycin 20mg/L to screen) ,rooting culture→ obtain seedling resisting Kanamycin;(6) Had transformed 95 trays, 1,713 leaves, had obtained 8 ones which resisting Kanamycin, the trans-formation rate was 4.67 ‰;(7) Through PCR analysis, the agarose gel electrophoresis result demonstrated that 6 ones had target genes of the 8 plants resist Kanamycin;(8) In the above six plants which had resistance bud differentiation of target gene, chose three plants in each sampling on average and selected their leaves for GUS detection. The result shaw that all the leaves were positive and the positive rate was 100%;(9) Through PCR-Southern blots analysis, the result further indicated that the target genes (the chi- meric genes of spider insecticidal and Bt) had been inserted into the genomes of Populus nigraxPopulus deltoids "108" pants successfully;The success of the experiment, will provide a basal theoretics for the further research of the transformation of the Populus nigraxPopulus deltoids "108".