| Nitraria tangutorum is a typical and native desert halophyte in northwest China with superior ability to resist salinity, alkalinity and drought. In addition, it can efficiently fix moving sands and decrease wind speed by clonal growth, which makes it an ideal plant for preventing soil desertification and alleviating the degree of soil salinity-alkalinity. However, the molecular mechanism underlying stress tolerance of this halophyte is still far from clear owing to the absence of genetic data. At the present study, NtP5CS and NtCIPK2genes were isolated from N. tangutorum to elucidate their characteristics of phylogenetic analysis, subcellular localization, promoter prediction and expression pattern, and to investigate their function in stress tolerance of Escherichia coli and Arabidopsis. Finally, NtPSCS-and NtCIPK2-overexpression Medicago sativa were obtained and preliminary validated. This study would be of great help in understanding the molecular mechanism of N. tangutorum in stress tolerance, exploring and utilizing this plant and breeding the stess-tolerant herbage. The main results are as follow:1. Two novel genes NtP5CS and NtCIPK2were isolated from N. tangutorum by rapid amplification of cDNA ends (RACE) cloning. Bioinformatics analysis demonstrated that NtP5CS and NtCIPK2shared similar function with their orthologs from other species due to the same typical characteristics, conserved domains and regulatory sites, they play the important role in Pro synthesis and Ca2+signal transduction of N. tangutorum. NtP5CS protein was limited to nuclei and at the plasmalemma, whereas NtCIPK2protein was distributed throughout the whole cells.2. To identify putative cis regulatory elements,2566bp and1600bp of the upstream sequences from the start codon of NtP5CS and NtCIPK2were isolated and analyzed. Many stress-related cis regulatory elements, such as W-box, MYB, MYC, HSE and LTR lay in the promoters. In accordance with promoter analysis, expression patterns by semi-quantative PCR were proved that the two genes showed upregulation of transcript by drought, salinity, cold and heat stress. The results suggested that NtP5CS and NtCIPK2mediated stress tolerance with regard to the adaptability of N. tangutorum to hostile environment.3. The two genes were inserted into the prokaryotic expression vector pGEX4T-1and transformed into E. coli, meanwhile; using the homologous gene AtP5CS of Arabidopsis as the representative of glycophyte, the difference in effect of NtP5CS and AtP5CS on E. coli was also assessed. The recombinant strains showed better growth than the control strains against salinity, drought, alkali, heat, cold, and high osmolyte stress, and also the NtP5CS-transformed strains grew better than the AtP5CS-transformed strains. The results indicated overexpression of NtP5CS and NtCIPK2improved the sress tolerance of E. coli and NtP5CS functioned more efficiently than AtP5CS.4. Under stressed conditions, higher seed germination rate, longer root and better growth was observed and proline synthesis, chlorophyll content, soluble sugar accumulation and MDA was significantly improved in transgenic lines compared to the control, so overexpression of NtPSCS and NtCIPK2in Arabidopsis improved the transgenic plants tolerance to drought and salt stress by osmotic adjustment, membrane oxidation alleviation and photosynthesis enhancement.5. Used the cotyledon as explants for induction, we obtained3NtP5CS-transfromed M. sativa lines and3NtCIPK2-transfromed lines, respectively. The two genes were integrated into the genome and expressed normally in the regenerated plants. |
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