【作者】 蔡晓锋；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2014， 博士

【摘要】 L-抗坏血酸(AsA, vitamin C),是一种重要的高丰度小分子的水溶性化合物,是植物和动物中重要生物活性的抗氧化剂,在植物和动物的生长发育方面起重要的调控作用。由于人类及其他灵长类中合成AsA的最后一步酶L-古洛糖-1,4-内酯氧化酶突变导致功能丧失不能自主合成AsA,只能从膳食中获得,而新鲜的水果和蔬菜由于富含AsA是人类及其他哺乳动物AsA的主要来源。番茄(Solanum lycopersicum)属于茄科茄属植物在世界各地广泛栽培,是重要的一年生商业化栽培蔬菜。由于番茄中含有丰富的维生素C和A、纤维素以及不含胆固醇,是人类极其重要的营养来源。目前番茄基因组和转录组测序数据的发表为研究番茄基因家族成员提供了很好的基础和技术平台；而番茄完善的遗传转化体系则是研究番茄基因功能的重要手段。Dof (DNA-binding with one finger)蛋白是植物特有的一类转录因子,在植物生长发育过程中起着重要的作用。南瓜一个Dof转录因子可以与南瓜抗坏血酸氧化酶启动子结合调控其在生长素诱导下的表达,但是否影响抗坏血酸的含量积累则没有详细研究。有关番茄抗坏血酸的合成D-甘露糖/L-半乳糖途径中的相关基因的功能及对抗坏血酸含量的调控效果已比较清楚,但D-半乳糖醛酸途径与再生代谢途径中的相关基因在抗坏血酸含量积累中的功能作用还没有鉴定。本研究首次在全基因组水平筛选鉴定了番茄中的Dof家族成员,并利用生物信息学的方法对该家族成员基因进行了系统的研究；同时鉴定了一个与番茄SlAO基因启动子结合的Dof转录因子,转基因功能验证结合基因芯片技术阐述了Dof转录因子在番茄抗坏血酸含量积累和盐胁迫中的作用机制。另一方面,研究了番茄抗坏血酸再生途径基因SIDHAR1和草莓中抗坏血酸合成重要途径中D-半乳糖醛酸途径中的关键基因FaGalUR在番茄抗坏血酸含量积累及对非生物逆境胁迫抗性中的作用。主要研究结果如下：1、番茄Dof基因家族全基因组分析。在番茄中共有34个Dof基因,除在第7和第12上没有分布外,在其他染色体上均有分布,且在第7和第6条染色体上分布最多,分别含有9和7个Dof基因；其中15个Dof基因只含有1个内含子,4个Dof基因含有2个内含子,其他的不具有内含子；系统发生学结果表明番茄中的34个Dof基因可以分为四个亚家族,与双子叶植物拟南芥存在更近的亲缘关系；保守基序分析表明,除了含有Dof保守域外,不同的亚家族中含有其他的保守结构；基因表达谱结果说明Dof基因在转录水平和表达模式上均有各自的表达特性,表明Dof基因存在功能特异性。2、番茄SlDof22在AsA含量积累及盐胁迫抗性中的机理解析。通过与已报道的Dof蛋白进行氨基酸序列比对筛选出了一个与南瓜CmAOBP同源性最高的Dof蛋白,SlDof22,同源性为49.47%。利用酵母单杂交方法证明SlDof22蛋白可以和SIAO基因启动子中的CTTT串联重复元件特异性结合,说明SlDof22蛋白编码一个抗坏血酸氧化酶启动子结合蛋白。SlDof22基因为组成型表达,在番茄各个组织中的表达量均较高,但在成熟叶片中表达量最高,幼叶、花和未成熟绿熟期果实中次之；SlDof22基因表达受激素、MV和盐胁迫诱导。干涉SlDof22基因显著下调转基因植株叶片和果实中SlDof22基因的表达量,提高番茄叶片和果实AsA含量。在叶片和果实中AsA含量比对照分别提高33%和64%左右,分别达到72.2mg/100g FW和43.3mg/100g FW。另外转基因植株对200mM NaCl胁迫表现敏感,具体表现为：盐胁迫处理后转基因植株叶绿素含量只有对照的70%,而地上部鲜重和地下部鲜重分别是对照的80%和60%。抗氧化和SOS途径相关基因表达水平分析表明SlDof22可能参与调控SISOS1基因的表达；酵母单杂交结果证明S1Dof22可以和SlSOS1基因启动子结合调控其表达。基因芯片分析结果表明,干涉SlDof22基因转基因植株破色期果实中显著影响与光合有关和糖类代谢有关的代谢途径,同时也影响了与次级代谢、胁迫和激素相关基因的表达。3、番茄SlDHAR1在番茄AsA含量积累中的功能分析。利用实验室前期构建的SlDHAR1转化材料研究番茄SlDHAR1基因在AsA含量积累及非生物逆境胁迫抗性中的作用,为番茄品质和抗性育种提供新的育种材料。超量表达SlDHAR1基因显著提高叶片和果实中AsA含量,在叶片和果实中AsA含量最高分别增加了1.5倍和1.3倍左右。并且超量表达SlDHAR1转基因幼苗在盐胁迫处理后维持较高的叶绿素含量和地上部和地下部鲜重；此外,离体叶盘实验也表明超量表达SlDHAR1转基因叶片可以增强对NaCl和MV诱导的盐胁迫和氧化胁迫抗性。4、草莓FaGalUR在番茄AsA含量积累中的功能分析。番茄叶片和果实中GalUR酶活性和AsA含量检测分析发现两者之间存在相关性,因此并将草莓FaGalUR基因转入番茄品系AC,转基因番茄叶片和果实中AsA含量均提高了1.5-2.0倍。同时FaGalUR基因通过上调果胶降解途径PE和PG基因的表达增加其合成前体物质,促进AsA合成能力,并增强AsA再生能力抑制AsA的代谢进而增加转基因番茄中AsA的积累。同时,超量表达FaGalUR基因转基因植株也增强了对氧化、盐和冷害等非生物逆境胁迫的抗性。更多还原

【Abstract】 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.更多还原