Studies On Genetic Mechanism For Heat Tolerance And Its Relationship With Eukaryotic Iniation Factor 5A Gene In Chinese Rose (Rosa Chinenses)

eIF-5A (Eukaryotic translation initiation factor 5A) is the only found protein that contains the special hypusine lyside. Hypusine ([Ne-(4-amino-2-hydroxybutyl)-lysine] is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The first reaction leading tothe formation of hypusine is catalyzed by deoxyhypusine synthase, an enzyme that adds butylamine to the conserved lysine of eIF-5A to form deoxyhypusine. A subsequentreaction in which deoxyhypusine is converted to hypusine is catalyzed by deoxyhypusine hydroxylase. Hypusine modification apparently ensues immediately following translation of eIF-5A inasmuch as there is no accumulation of either eIF-5A precursor (lysine form) or eIF-5A intermediate (deoxyhypusine form) unless cells are treated with inhibitors of either deoxyhypusine synthase or deoxyhypusine hydroxylase.Full-length cDNA clones encoding DHS and eIF-5A have been isolated from several plant species. Moreover, recombinant plant DHS (like its mammalian counterpart) catalyses the formation of deoxyhypusine on inactive eIF-5A, and recombinant plant eIF-5A (like its mammalian counterpart) is capable of being deoxyhypusine-modified. Plant eIF-5A sequences are highly conserved (80–90% identity at the amino acid level) but have only 50–60% amino acid identity with human, yeast and fungal eIF-5A.This study uses Chinese Rose (Rosa chinensis) as research material. As leading role of urban greening, Chinese Rose is famous for its continuous anthesis, polychrome flower colors, sweet fragrance and insensitive to temperature. There is no relating research on the flowering inhibition of high temperature in China, if we can make breakthrough in this research about Chinese Rose, and carry on researches on physiological and genetic mechanisms of heat-resistant Rose, providing theoretical basis for breeding heat-resistant flowers in other plants.This research retrieved the cDNA encoding eIF-5A of Chinese Rose through RT-PCR, and validated its function by over-expression in E. coli. At the same time, we transformed anti-sense eIF-5A of Chinese Rose into yeast to inhibit the expression of endogenous eIF-5A to validate its function. The copy number of eIF-5A in Chinese Rose and its expression pattern in different plant organs were also examined, indicating that different eIF-5A exist in Chinese Rose to control its development and senescence.The results of this research are listed below:1. Obtained the cDNA encoding eIF-5A from heat-shocked Chinese Rose leaves by RT-PCR. By Southern blot, we analyzed the copy number of this gene in Chinese Rose genome, finding low-copy in Chinese Rose genome.2. By RT-PCR, we examined the different expression pattern of eIF-5A in different organs of Chinese Rose, and high expression level was found in young leaves and old leaves.3. By constructing prokaryotic expression vector with the cDNA of RceIF-5A, it was expressed in E. coli. Transgenic lines of E. coli showed significant resistance to high and low temperature than the control.4. The cDNA of RceIF-5A was expressed in yeast by an eukaryotic expression vector. The transgenic lines also showed better resistance to high temperature than the control.