Preliminary Study On Biological Function Of DHS In Plant

eIF-5A (Eukaryotic translation initiation factor 5A) is the only known protein which contains the special amino acid-hypusine so far. The protein is involved in many cellular biological functions, especially correlates closely with both cell proliferation and cell senescence. DHS(deoxyhypusine synthase)catalyses the first step of hypusination. eIF-5A is the only known substrate of DHS. Lots of researches about DHS and eIF-5A in human being and yeast indicate that they involved in many cellular activities, but their exact functions remained unknown so far. In the present study we concentrate on, firstly, testing the expressional characteristic of DHS gene in plants by using semiquantitive RT-PCR. And then, the expression of DHS gene was depressed in Nicotiana benthamiana and Nicotiana tabacum by the techniques of VIGS(virus induced gene silence) and Antisense RNA, respectively, to investigate the biofunction of DHS and eIF-5A in plants. We also explored whether it can be a new way of genetically modifying plants by downregulating DHS gene expressional level. We have obtained several progress:1. Test expressional characteristic of DHS in N. tabacum, Arabidopsis thaliana and poplar by semiquantitive RT-PCR, and then analyse the expression pattern of DHS in plants.2. Extract cDNA sequence of DHS gene form Nicotiana benthamiana by RT-PCR, construct the transgenic virus expression carrier, and then restrain the expression of target gene-DHS in the plant by the VIGS system, then we obtained the function-loss phenotype of DHS in N.benthamiana. Comparing to the wild type plants, the DHS restricted ones grow faster, with delayed in leaves senescing and flowering and tolerance against drought.3. Construct the antisense-DHS expression carrier, and transform it into the N. tabacum mediated by Agrobacterium. We have obtained several transgenic plants. Studies of phenotypes on the T1 generation of transgenic tabacum, the results reveal that the transgenic plants exhibit obvious tolerance to drought, cold and salt stresses. So it is inferred that down-regulating DHS expression may be a new way to modify genetic trait in plants.4. In order to explore the actual application of down-regulating DHS expression in plant genetical modification, and concerning that DHS restraining may influence the yield of seeds, we choose poplar, which vegetative organ(stem) was used as our economic target. Extract cDNA sequence of poplar DHS gene, construct the sense/antisense DHS expressioncarriers, transform poplar mediated by Agrobacterium. And we have already obtained 20 transgenic seedlings which were confirmed by using PCR. During our study, we also make an initial exploration to improve the method of transforming poplar mediated by Agrobacterium.5. Isolate the promoter sequence of DHS from poplar and Arabidopsis and that of 3 eIF-5A in Arabidopsis, construct the expression carrier, whose the reporter gene GUS was inserted downstreaming these promoters. And transforming poplar and Arabidopsis are underway. These works pave the foundation for studying characteristics of these promoters.