The Research On Two Insect-resistant Gene Transforming Into Populus×euramericana Cv."74/76"

Poplars is widely distributed in the global and important afforestation and ecological shelterbelt species. Due to its straight trunk and beautiful tree shape Poplar is a good landscape trees and widely used in the garden, which usually be took as plant landscaping trees and landscape background tree. Because of its feature of rapid growth and high ornamental value, Poplar has been more widely used in landscaping and economic forest. However, in recent years, the violations of pests and diseases have caused a significant loss. The obtain of transgenic hybrid poplar 107 pest can not only effectively solve the issue of pest but also take poplar as preferred specie in landscaping application due to its rapid growth nature. From the respect of social needs or economic benefits, the culture and research of transgenic hybrid poplar 107 were necessary.At present, in many species of poplar, only Leuce Duby species is relatively mature in tissue culture and genetic transformation. This exeperiment mainly used agricultural rodmediated method to do Genetic transformation research of Europe and the United States Yang 107 Populus by using bi-insect-resistant genes built in a vector (BtCry1Ac gene +API arrowhead protease inhibitor gene), for transgenic plants, molecular biology detect and preliminary feeding insects trial had been took, and had obtained transgenic hybrid poplar 107 lines.(1) According to different factors influencing genetic transformation of Europe and America 107 poplar, efficient tissue culture regeneration and genetic transformation system of 107 Populus varieties were initially established. By means of taking Material stems and leaves as explants, through selecting the best medium of their various growth stages, stable and efficient regeneration system was established. The results show that: optimal medium for adventitious buds induced differentiation was MS+6-BA 0.5 mg/L+IAA 0.15 mg/L+sucrose 25 g/L+ agar 7 g/L, for rooting medium was 1/2 MS+IBA 0.5 mg/L+ sucrose 15 g+ agar 7 g/L. Simultaneously the results showed that: main veins, germination situation in leaf petioles and stems wound were similar. Therefore, selecting a leaf or stem segments as the test materials had little effect on the experiment.(2) Agrobacterium-mediated method was used for genetic transformation, Optimization research of the various factors affecting transformation had been took. The results show that: were being baptist bacteria 15min and culturing 3 dayas altogether, resistant shoots were obtained with induction rate 16.7%. After kanamycin screening, rooting culture, hardening and transplanting, obtain resistant plants were acquiring.(3) According to PCR analysis and ELISA experimental results, the Bt gene and protease inhibitor genes had been integrated into Europe and America 107 poplar genomic DNA was proved. Through this research, we obtained transgenic hybrid Europe and America 107 poplar 10 clones. Among these7 clones was positive in the PCR assay. In ELISA detection of 10 departments, the highest expression levels of toxic protein content occupying in the total protein was 0.0336%。(4) Insects test of feeding the Hyphantria instar larvae on leaves of Europe and America 107 poplar show that deratization rate of Transgenic hybrid Europe and America 107 poplar was significantly higher than the control not genetically modified. 6 numbered leaves being feed fall webworm, among these 4 numbers mortality can reach 100%. Above of these show it is a fact that these two genes in plant cells were able to effectively express and its expression products had a strong toxicity for the fall webworm larvae.(5) Through molecular biology detection and insect test results, the target gene had been integrated into the genome of Europe and America 107 poplar was proved, and having normal growth and development and the ability of insect-resistant fine strains had been screened out from the transgenic lines .