Transformation Of Drought And Salt Resistant Gene In Populus×euramericana Cv.'Nanlin895'

Poplar has been recognized as a model tree species by Forest Genetists due to its fast growth, easy propagation, wide adaptation, and its small genome size. DREB1C transcription factor (Dehydration Responsive Element Binding) is correlated with stress and it can distinguish DRE (dehydration responsive element) cis-acting element in the promoter region of stress response genes about drought, high salt, low temperature and so on. Moreover, it can regulate downstream resistance gene's expression in combination with DRE cis-acting element, sequentially enhance stress resistance of plant. In order to broaden cultivation of poplar, especially in drought and salt area, it is very important to develop transgenic polpar resistant to drought and salt.In this study, leaf disc of Polulus×euramericana cv.'Nanlin89'was used as experiment material, adapting to Agrobacterium-mediated to introduce DREB1C gene. Meantime, stress-induced promoter rd29A was homologous cloned from Arabidopsis, and new expression vector was constructed and transferred into Nanlin895 in the same way. The main conclusions of this study were as following:1. Primers were designed according to sequence reported in NCBI, and then stress-induced promoter rd29A was cloned from Arobdopsis. Plant expression vector pBrd-GUS was constructed and transferred into tobacco in order to test the activity of rd29A promoter. PCR and GUS histochemical stain of transgenic tobacco showed that rd29A promoter was induced by low temperature, dehydration and high salt stress to increase expression of gene GUS. Therefore, rd29A promoter could be used in plant gene engineering for stress resistance improvement.2. The expression vector pDR2-rd29A was constructed by fusing DREB1C gene with the stress-induced promoter rd29A. The expression vectors were then introduced into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method and proved by enzyme digestion and PCR methods.3. Antibiotic sensitivity experiment of leaf regeneration and radication were made separatly. Result showed Hyg 3mg/L was critical concentration of leaf regeneration, and Hyg 1.5mg/L was critical concentration of radication.4. Leaf disc of Nanlin895 was used as experiment material. The expression vectors pDR2 and pDR2-rd29A containing the DREB1C gene under the control of the constitutive promoter CaMV 35S and the stress-induced promoter rd29A respectively were transferred into Nanlin895 by Agrobacterium-mediated method. 80 Hyg resistant plants with 35S::DREB1C gene and 40 resistant plants with rd29A::DREB1C gene were gained via strictly selection by Hyg. PCR and real-time PCR analysis of resistant plants proved 30 plants positive to 35S::DREB1C gene and 15 plants positive to rd29A::DREB1C gene. It could be concluded that DREB1C gene was probably integrated into genome of Nanlin895.5. Morphological observation of the transgenic plants showed that the growth characteristics of them were normal and Dwarf growth was not observed in this study. Two kinds of transgenic plant growed normally under drought and salt stress, and its growth was much better than the control plant, indicating that the tolerance of transgenic plants to drought and salt was better than that of the control plant.