| L-ascorbate (AsA, vitamin C), is a high abundant, small molecular weight and water-soluble antioxidant, and plays very important roles in a range of cellular processes in both plants and animals. However, humans and other primates are unable to synthesize AsA because the terminal enzyme in the animal pathway, L-gulono-1,4-lactone oxidase, has been mutated and non-functional. Accordingly, they depend on dietary intake to cover their requirements and mainly obtain from plant sources. Fruit and vegetables are rich in AsA as an important source of human diets, and the contents of AsA vary with different kinds of fruit and vegetables. Tomato (Solarium lycopersicum) belongs to the family Solanaceae, and is commercially cultivated as an annual crop. Tomato fruit is regarded as a most important vegetable crop due to rich in vitamins C, A and fiber and cholesterol free. To date, completed sequencing and assembly of tomato genome and transcriptomes sequenceing data provides a foundation for further identifiy and analysis of tomato gene families; besides, it is transformable (genetic transformation system) play an important role in study gene function in tomato.The Dof (DNA-binding with one finger) domain proteins are plant-specific transcription factors with a highly conserved DNA-binding domain, and play critical roles as transcriptional regulators in plant growth and development. It was report that one of Dof protein involved in regulate the expression of ascorbate oxidase during the auxin treatment in pumpkin, but the functional analysis in ascorbate accumulate was not report. The positively contribution of D-mannose/L-galactose pathway to the accumulation of AsA has beed well clarified, but the function of genes from metabolic pathways and D-galacturonic acid pathways involved in AsA accumulation of still need more evidence in tomato.In this study, we firstly screening and identified the Dof family genes in tomato (Solarium lycopersicum L.). The systematic overview of SlDof genes in tomato is presented, including the gene structures, chromosome locations, phylogeny, protein motifs, evolution pattern and gene expression analysis. In addition, one of SIDof genes binding to the promoter of SlAO gene was identified and functional anslysis in tomato. On the other side, the function of SIDHAR1and FaGalUR involved in AsA accumulation and abiotic stresses was also analysis in tomato. The main results are presented as following:1. Genome wide analysis of tomato Dof genes family. Using bioinformatical analysis,34Dof family genes were identified in tomato (Solanum lycopersicum L.), and a complete overview of SlDof genes in tomato is presented, including the gene structures, chromosome locations, phylogeny, protein motifs, evolution pattern and gene expression analysis. Tomato Dof family genes distributed on11chromosomes except for chromosome7and12, and chromosome2and6had a maximum of nine and seven Dof genes, respectively. The gene structural analyses shown that15SlDof genes contained one intron, four genes contained two introns, and the remained genes were intronless, besides, all the introns were located upstream of the Dof domain. Phylogenetic analysis of34SlDof proteins resulted in four classes constituting six clusters and shown high similarity with Arabidopsis Dof genes. In addition, conserved motif analysis indicated that S1Dof proteins consisted in same group shared similar conserved motifs except for known Dof domain. Furthermore, the SlDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth conditions.2. The identification and functional analysis of tomato SlDof22gene in AsA accumulation and salt resistence. One of Dof genes, SlDof22, shown high similar with pumpkin CmAOBP, according to the proteins sequences alignment and phylogenetic analysis of reported Dof proteins, and the SlDof22displayed the highest similarity with CmAOBP, with49.47%identical. Yest one hybrid analyss demonstrated that SlDof22protein could bind to the CTTT elements in promoter of SI A O gene in tomato, suggesting SlDof22encodes an ascorbate oxidase binding protein. The expression of SlDof22was constitutively high expressed in roots, stems, leaves, flowers and different development stages of fruits, whereas the highest expression levels were detected in leaves, followed with in young leaves, flowers and immature fruits, and the expression of SlDof22was induced by hormone, MV and salt treatment. RNA interference of SlDof22gene significant increased AsA contents in leaves and fruits, accompaning with reduced SlDof22gene expression level. The AsA contents of leaves and fruits were enhanced33%and64%in transgenic lines as compared to wild type, up to72.2mg/100g FW and43.3mg/100g FW, respectively. In addition, the transgenic plants displayd susceptibility to200mM NaCl stress. The chlorophll content of transgenic plants was70%of that in wild type, and the fresh weight of aerial part and underground part was80%and60%of that in wild type after NaCl treatment, respectively. The expression level of SISOS1gene was siginificatly down regulated, yeast one hybrid analysis shown SlDof22protein could bind to the promoter of SlSOS1gene. Furthermore, gene chip analysis shown that RNA interference of SlDof22gene significant changed the expression level of genes involved in photosynthetic and carbonhydrate metabolism, and also changed the expression of genes related to secondary metabolism, stresses and hormone.3. The functional analysis of tomato SlDHAR1in AsA accumulation and abiotic stresses resistence. The SlDHAR1overexpressing lines were isolated and generated by previously study. The transgenic plants exhibited a significant increase in ascorbic acid in functional leaves and red fruits compared with wild-type plants, positively correlated with SlDHAR1expression abundances and DHAR activity. The AsA content improved1.5and1.3fold in leaves and red ripe fruits of transgenic lines, respectively. Furthermore, the seedings of transgenic plants showed enhanced salt stress tolerance with higher chlorophyll content and fresh weight of aerial part and under-ground part than those of WT plants. In addition, the transgenic plants also exhibited considerable tolerance to salt and oxidative damage induced by NaCl and methyl viologen (MV) based on less chlorophyll content loss under methyl viologen (paraquat) and salt treatment with leaves disc.4. The functional analysis of strawberry FaGalUR gene in AsA accumulation and abiotic stresses resistence in tomato. The activity of D-galacturonic acid reducta.se was detected parallel to AsA accumulation in crude extract of tomato leaves and fruits. Subsequently, transgenic tomato lines overexpressing strawberry FaGalUR gene were generated. The results showed that introducing a single gene GalUR led to1.5-2.0fold increase in AsA level in tomato leaves and fruits, which correlated positively with GalUR transcriptional abundance and GalUR activity. The expression level of PE and PG genes, which were invovled in degradation of cell wall pectins, was up-regulated in transgenit lines. Furthermore, FaGalUR overepxressing lines showed enhanced tolerance to abiotic stresses induced by oxidization (methyl viologen), salt (NaCl) and cold as compared to the wild-type plants. |
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