| Nelumbo nucifera is one of the ancient species in plant and has enormous economic value as aquatic vegetable in China. Research of Nelumbo nucifera has been mainly focused on its morphology, taxology and physiology that is related to clinic medicine. With rapid development in functional genomics, it is important to breed new species of high production, high quality and stress tolerance, however few studies of molecular cloning and transgenic engineering were reported in N. nucifera so far. In this thesis work, we have constructed cDNA libraryin Nelumbo nucifera and cloned stress-related genes and analyzed the fuctions of these genes in Nelumbo nucifera. Our results, for the first time, provide molecular evidence for transgenenic research and application in Nelumbo nucifera.1Construction of cDNA library in Nelumbo nuciferaTo discover functional gene that is related to stress tolerance, quality and transcriptional regulation, we successfully constructed the cDNA library of apical bud in Nelumbo nucifera. The titer of primary cDNA library is4.2×106pfu/mL and the ratio of combinant is100%. The inserted fragment over0.5kb occupied97%of the whole library and the titer of amplified cDNA library is8.5×10pfu/mL, which guarantee the acquisition of full length cDNA. We screened the library through the known NnUBC3gene and successfully obtained the586bp full length cDNA sequence with459bp of coding sequence and127bp of3’UTR. These data suggest that the cDNA library is effective and suitable for sequencing and functional analysis in the future. 2Cloning of NnGPXl and its functional analysis in response to abiotic stressGPX is an antioxidant enzyme that is able to catalyze the reduction of H2O2or organic hydroperoxides to water or corresponding alcohols and protect organism from oxidant. We cloned full length cDNA of GPX in Nelumbo nucifera named NnGPX1and found that NnGPX1, unlike GPX protein in other species, is not a selenium-dependent glutathione peroxidase. Through expression analysis in various tissues at different developmental stages, we found that NnGPX1is up-regulated in leaf of seedling and apical bud at flowring while it is highly expressed after treatment of chilling, hot, mechanical wounding, salty, ABA and PEG. This is not consistent with previous data in Arabidopsis that different GPX functions in different stress condition. All of data indicate that NnGPX1play a critical role in development, biotic and abiotic stress. Moreover, we purified the NnGPX1protein and predicted its structure for further functional analysis.3Cloning of NnUBC and its functional analysisUBC is ubquitin conjugating enzyme catalyzing ubiquitination with E1and E3in eukaryote. Histone H2B monoubiquitination regulate plant flowering time and ubiquitination has also been found important in defense of biotic and abiotic stress. We cloned full length cDNA of four NnUBC genes and found that they show different expression pattern, especially NnUBC3expressed highly in all tested organs through expression analysis in various tissues. Consequently, we cloned the full length genomic DNA sequence of NnUBC3and discovered that it is able to restore the phenotype of mutant by introducing full CDS of NnUBC3into ubcl,2mutant of Arabidopsis. The subcellular localization of NnUBC3resembles its location in Arabidopsis. All the results suggest that NnUBC3is a E2. In conclusion, NnUBC3may play important role in response to NaCl, ABA and PEG.4Functional analysis of VvPYLl in ABA signalling pathwayABA is an important regulator in response to biotic and abiotic stress, and control plant growth through regulating ABA responsive gene, especially in drought and salty condition. We cloned three VvPYL genes from Vitis Vinifera and discovered they are homolog of PYL by alignment of amino acid sequence and analysis of phylogenetic tree. Expression analysis in various tissues showed that VvPYLl is highly expressed in all tested tissues and upregulated in response to ABA treatment. ITC assays indicate that VvPYL1can interact with ABA at78uM of Kd value. Phosphatase activity assay demonstrates that VvPYL1is able to completely inhibit the phosphatase activity of ABI1in an ABA-dependent manner. VvPYL1is present both in cytosol and nucleus by Agroinfiltration in tobacco(Nicotiana benthamiana), which resembles the subcellular location of PYL in Arabidopsis. All these data, for the first time, suggest that VvPYL1is an ABA receptor in Vitis Vinifera. |
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