Rearch On Induction Expression Of Alien DREB1B And CBF1 Transcriptional Factor In Transgenic Wheat

Drought is one of environmental stresses that effects on the growth of plants and the productivity of crops. The products of these genes induced by drought stress can be classified into two groups: those that directly protect against environmental stresses, and those that regulate gene expression and signal transduction in the stress response. The first group includes proteins and enzymes that likely function by protecting cells from dehydration, such as late-embryogenesis-abundant (LEA) proteins, enzymes required for biosynthesis of proline, and aquaporins(AQP), et al. The second group of gene products includes transcription factors, protein kinases, and enzymes involved in phosphoinositide metabolism, such as bZIP, MYC, DREB. Recently scientist mostly concerns the second group of gene. DREB (Dehydration Responsive Element Binding) and CBF1(CRT/DRE binding factor 1) are a cis-acting element identified in Arabidopsis. Expression of the DREB and CBF1 under control of promoter in transgenic plants gave rise to strong constitutive expression of the 12 stress-inducible genes, of which the dehydration-responsive element (DRE) is essential in the promoter regions. , such as RD29,COR15,RD17 et al. and increased tolerance to drought, salt loading, and freezing stresses. In this study, DREB1B and CBF1 gene were isolated from Arabidopsis, transferred into wheat, and in order to improve wheat drought-tolerance.Plasmid pBAC128F(7.0kb) and pBAC127F(6.9kb), which contain RD29B (induced by drought stress) promoter driven DREB1B gene and CBF1 gene, and CaMV 35S promoter plus maize (Zeamays L.) Adh1 first intron driven bar gene were injected immature calluses as explants of common wheat cultivars, 8901, 5-98, 99-92, Baofenl04 and Jinghua No.1 by biolistic microprojectile bombardment (PDS-1000/He). And 79 plants were regenerated and the leaves of T₁ transgenic lines were applied Basta(100mg/L). Some transgenic lines took on obvious resistance.PCR, DNA and RNA dot blotting analysis of transgenic lines proved that DREB1B gene has been integrated into genome of some lines, and was well expressed in offspring of different line.Relative water content (RWC), contents of chlorophyll and proline, the rate of photosynthesis in leaves of T₂ lines were determined and analysised. Among these transgenic lines, 10 transgenic lines exhibited increaseing. RWC in leaves of transgenic lines is all above15%. It is higher than CK(7.19⁹. 13%). Contents of chlorophyll a are all 0. 07 y g/ml, more than CK (0. 02—0. 05 y g/ml) . Contents of chlorophyll b are all 0. 14y g/ml, more than CK(0. 5—0. 9 y g/ml). The content of proline in leaves of transgenic lines are more than 1000 y g/g, increased 2-3 folds. The rate of photosynthesis in leaves of transgenic lines are above 8. 0 y mol C02/m2/s, whereas CK are2. 27². 47 y mol C02/m2/s. Under drought condition, transgenic lines and wild type control stopped to water for 15 days. Leaves of transgenic lines kept green, while control showed fading. Then both transgenic lines and control were treated with water for 10 days, leaves of transgenic lines recovered green, while wild type control dead.Dot blotting analysis of transgenic lines with the probes of CBF1 gene and bar proved that CBFlgene has been integrated into genome of some transgenic lines. And transgenic plant lines identified by PCR with primers 5'-bar and 3-NOS showed that 2 transgenic plant lines had the 800bp DNA band. The result was the same as DNA dot blotting analysis.Our research suggests that enhancement expression of alien DREB1B and CBF1 transcriptional factors into wheat is an effective way to improve its drought tolerance ability.