Functional Characterization Of Both ThDREB And TheIF1A Genes From Tamarix Hispid

In plant genetic engineering, compared with importting a functional genes, the effectiveness of importting a key control gene improved is better. Regulation of gene expression levels is a complex event attended in the multi-stage, in which gene expression of transcription initiation is the most effective aspects of the regulation, and translation and post-translational processing of gene expression is the basic control points. In this paper, two cDNA librarys which encoding DRE-binding transcription factor (ThDREB) and eukaryotic translation initiation factor(TheIF1A) were cloned by 5'RACE method from Tamarix hispid, and which expression and function of the two gene were researched further.(1) The time course expression pattern both of ThDREB and TheIF1A genes were investigated under abiotic stress conditions by using real-time RT-PCR. The results showed that both ThDREB and ThelFIA were response to treatments of NaCl,PEG,NaHCO3 and CdCl2 had tissue specific expression pattern.(2) Using particle bombardment mediated transient transformation, the ThDREB and TheIF1A fused GFP protein were transiently expressed in onion epidermal cell, and the green fluorescence were detected in the nucleus and cytoplasm of onion cell. The results revealed that both the ThDREB and TheIF1A protein were targeted to nucleus.(3) Both the ThDREB and TheIF1A genes were inserted into the yeast expression vector pYES2 and recombinant plasmid (pYES2-ThDREB and pYES2-TheIF1A) and pYES2 were transformed into Saccharomyces cerevisiae INVSc1 to characterize the tolerance both of ThDREB and ThelFIA to different abiotic stresses. The results demonstrated that ThDREB and TheIF1A increased the stress tolerance of yeast, including salt, drought, alkali, oxidative, cold, heat and heavy metal stress.(4) Transgenic tobacco over-expressing ThDREB and TheIF1A were generated. Three independent transgenic tobacco plants lines (line D-4, D-10 and D-15 or line 1A-1,1A-3 and 1A-9) and wild-type plants (WT) were grown on 1/2MS agar supplemented with different concentrations of NaCl (0,100 and 150 mmol/L)and Mannitol(350 mmol/L), and their fresh weight and seed germination were further measured and compared. The results showed that both the fresh weight and seed germination of all transgenic plants were better than WT plants. In particular, the transgenic lines D-15 and 1A-3 exhibited increased height and root growth relative to other transgenic lines and WT plants. Therefore, the line lines D-15 and 1A-3 were tested under 250 mmol/L NaCl and Mannitol (150,250 and 350 mmol/L) stresses. The results showed that transgenic tobacco accumulated higher fresh weight under long-term osmotic stress than non-transgenic, and increased the stress tolerance to high salt and drought tolerance. The enhanced tolerance of transgenic lines to abiotic stresses suggests that ThDREB and TheIF1A were candidate genes for genetic engineering to improve plant tolerance to stresses.(5) To investigate the expression pattern of ThelFIA, the promoter of ThelF1A (PTheIF1A) was isolated using TAIL-PCR approach. Sequence analysis showed that the promoter is 1352bp in length and contains some stress-response cis-acting elements such as HSE, MBS, TGACG-motif, TC-rich repeats, W-box and so on. The promoter PTheIF1A was inserted into a plant expression vector pCAMBIA1301 to replace CaMV 35S promoter. Transient transformation was performed on tobacco leaves. The results showed that GUS activity was detected in leave of tobacco, suggesting that PTheIF1A has transcriptional activity. The Arabidopsis seedlings tansformed with PTheIF1A:GUS were obtained. The histochemical GUS staining analysis showed that the PTheIF1A mainly expressed in roots and leaves, and also expreesed in shoot.