Functional Characterization Of An EIF5A Gene From Tamarix Androssowii

Drought and salt stresses are the main factors that seriously affect on plant growth and development. The genetic engineering is an effective way to improve plant stress resistance. One of the basis for plant genetic engineering is study on the function of stress-related gene. The eukaryotic translation initiation factor 5A (eIF5A) is the only protein with special amino acid (hypusine) residues among all the eukaryotic organisms. To date, the cellular function of eIF5A in plants is not well understood. Previous studies showed that eIF5A is involved in many bioprocesses and participate in stress responses in plant.To determine the function of eIF5A gene, an eIF5A gene (TaeIF5Al) from Tamarix androssowii was investigated in the present study.The time-course expression pattern of TaeIF5A gene was investigated under abiotic stress conditions by real-time RT-PCR. The results showed that TaeIF5A was response to treatments of NaCl,PEG,NaHC3,CdCl2 and ABA, and had tissue specific expression pattern. Using particle bombardment mediated transient transformation, the TaeIF5A fused GFP protein was transiently expressed in onion epidermal cell, and the green fluorescence was detected in the nucleus and cytoplasm of onion cell. The results revealed that the TaeIF5A protein was targeted to both nucleus and cytoplasm nucleus.To investigate the expression pattern of TaeIF5A, the promoter of TaeIF5A was isolated using TAIL-PCR approach. Sequence analysis showed that the promoter is 1550bp in length and contains some stress-response cis-acting elements such as MYB, MYC, GT1, WRKY and so on. The promoter of TaeIF5A was inserted into a plant expression vector pCAMBIA1301 to replace CaMV 35S promoter (TaeIF5A promoter:GUS). Transient transformation was performed on tobacco leaves. The results showed that GUS activity was detected in leave of tobacco, suggesting that TaeIF5A promoter has transcriptional activity. The Arabidopsis seedlings tansformed with TaeIF5A promoter:GUS were obtained. The histochemical GUS staining analysis showed that the TaeIF5A mainly expressed in roots, and also expreesed in leaves.The TaeIF5A gene was inserted into the yeast expression vector pYES2 and recombinant plasmid (pYES2-TaeIF5A) and empty pYES2 (served as negative control) were transformed into Saccharomyces cerevisiae INVScl to characterize the tolerance of TaeIF5A to different abiotic stresses. The results demonstrated that TaeIF5A exhibits a wide range of abiotic stresses, including salt, drought, alkali, oxidative, and heavy metal stress.To further characterize the function of TaeIF5A under abiotic stresses in plant, the transgenic populus overexpressing TaeIF5A were generated. Ten transgenic lines and WT plants were grown on a medium supplemented with 0.6% NaCl for 20 d, and their height and root growth were further measured and compared. The results showed that the height growth of all transgenic plants (except line 7) were better than WT plants. The transgenic lines 2 and 3 exhibited increased height and root growth compar to other transgenic lines and WT plants. Therefore, the line 2 and 3 were selected for stress-study under ZnCl2, CuSO4, CdCl2, sorbitol and NaHCO3. The results showed that non-transgenic and transgenic populus can not grow and died in the medium with 5mM NaHCO3, while overexpression of TaeIF5A increased the stress tolerance to heavy metals and drought tolerance. To further confirm the mechanism of salt tolerance of TaeIF5A, WT and 10 transgenic lines were grown in the greenhouse until their heigth reaching about 60～70cm, and were treated with 0.8% NaCl to measure and compare the soluble protein content, POD activity, SOD activity, relative electrical conductivity and relative chlorophyll content. The results showed that the soluble protein content, POD activity, SOD activity and relative chlorophyll content in most transformed plants were higher than that in WT populus under salt stress. The relative electrical conductivity in most transgenic lines was lower than that in WT populus under NaCl conditions. In addition, growth in relative height and basal diameter were subsequently compared between transgenic and WT plants. Results showed that the most transgenic lines exhibited higher growth than WT plants. The increase in height and basal diameter under NaCl conditions suggested that the TaeIF5Al gene conferred salt tolerance to the transgenic plants.The enhanced tolerance of transgenic lines to abiotic stresses suggests that TaeIF5A is a candidate gene for genetic engineering to improve plant tolerance to stresses.To determine the proteins which could interact with TaeIF5A, a cDNA library of Tamarix androssowii was constructed using yeast strain as host, and the TaeIF5A gene was constructed to the Bait vector. The proteins interacted with eIF5A were screened using yeast two-hybrid system. Six proteins that potentially interacted with TaeIF5A were detected. Three of them were further verified that they interacted with TaeIF5A strongly. One is an unknown-function protein, the other is a Xyloglucan endotransglucosylase/hydrolase (XTHs) protein precursor and ARF GTPase activator. The interaction of TaeIF5A with XTHs precursor protein and ARF GTPase activator indicated that TaeIF5A may play a role in cell growth, cell wall structural changes and abiotic stress responses; however, the detailed process and the functional significance of their interaction need to be further studied.The cis-acting elements from TaeIF5A promoter associated with salt stress response, including GT-1, MYC/E-box, and W-box, were selected for yeast one-hybrid analysis, to identify the upstream regulatory genes. The tandem repeat sequence of the cis-element was constructed into the reporter vector pHIS2, respectively,then the yeast one-hybrid screening was performed to explore upstream regulatory genes of TaeIF5A. GT-1 cis-acting element screening cDNA library revealed six proteins potentially interacted with cis-acting element. Among them, four proteins are associated with the chloroplast protein, one was ubiquitin conjugating enzyme involved in DNA repair, and the other is glucose-6-phosphate dehydrogenase which playing important roles in growth and development in plants. Seven proteins potentially interacted with MYC/E-box cis-acting element were obtained, four of them were associated with photosynthesis; one of them was an unknown function protein, the other two were hydroxymethylglutaryl-CoA reductase that is the rate-limiting enzyme in metabolic pathway of isoprene; and RNA polymeraseβ'subunit involved in DNA recognition and binding in the process of transcription activation.Five proteins that potentially interacted with W-box cis-acting element were found, one of them is AP2 transcription factor involved in plant drought, high salt and low temperature stress-responsive; one is mini-chromosome maintenance protein that is a member of the MCMs family closely related to DNA replication; one was the fructose-bisphosphate aldolase, which is one of the key enzymes of photosynthesis and respiration in plants; one was ubiquitin-conjugating enzyme E2 that playing a key role in the specificity, exact time and space of the ubiquitin modification. The other is the RNA polymeraseβ'subunit. Whether these proteins can regulate the expression of TaeIF5A through interaction with the cis-acting elements need further investigation.