【作者】 邹礼平；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2005， 博士

【摘要】 抗坏血酸即维生素C，在生物体中具有重要的代谢功能和抗氧化作用。抗坏血酸是保证人类健康所必需的营养物质，人类自身已经丧失了合成能力，必须从食物中摄取，而植物为人类提供了主要的抗坏血酸来源。抗坏血酸还与植物细胞的分裂、伸长和植株生长有关。抗坏血酸及其代谢相关酶类在清除活性氧方面的作用使得抗坏血酸在植物抵抗环境胁迫中也起着重要作用。通过调控植物抗坏血酸生物合成与代谢相关酶基因的表达，可以提高抗坏血酸的含量，改变某些代谢酶类的活性，从而增加植物产品的营养价值，增强植物对逆境的抗性。 番茄是一种世界上广为栽培的高营养、高价值的蔬菜作物，然而，目前番茄抗坏血酸生物合成与代谢途径中大部分基因都没有克隆到，对其进行转基因调控的报道几乎没有。因此，本研究以番茄这一重要的蔬菜作物为材料，鉴定和克隆抗坏血酸生物合成和代谢相关酶基因，通过基因工程调控番茄抗坏血酸的代谢，分析转基因番茄的植株生长、生理生化及抗氧化逆境的特性等，以期获得具有应用价值的抗坏血酸含量增加的番茄材料。本研究获得的主要结果如下： 1．克隆了番茄抗坏血酸生物合成途径中上游的GDP-D-甘露糖焦磷酸化酶(GMP)基因的全长cDNA，其GenBank登录号为AY605668，测序结果表明该cDNA全长1535bp，包含一个长为1086bp的完整开放读码框，编码361个氨基酸，氨基酸序列比对结果显示番茄GMP与其他植物GMP的同源性较高。根据GenBank中已公布的番茄抗坏血酸生物合成途径中最后一步合成酶L-半乳糖酸-1，4-内酯脱氢酶(GLDH)的全长cDNA序列，克隆了番茄GLDH基因，该基因编码区长1767bp，编码588个氨基酸。根据已报道的序列克隆了番茄抗坏血酸代谢途径中促进抗坏血酸再生的单脱氢抗坏血酸还原酶(MDHAR1)基因的全长cDNA，该基因编码433个氨基酸。利用RACE技术克隆了2个促进抗坏血酸再生的另一关键酶脱氢抗坏血酸还原酶(DHAR)基因的全长cDNA，其GenBank登录号分别为AY971873和AY971874，DHAR1的cDNA全长991bp，编码210个氨基酸，DHAR2的cDNA全长1024bp，编码268个氨基酸，分析表明DHAR1属于胞质类型，而DHAR2属于质体类型。利用RACE技术克隆了一个新的番茄胞质抗坏血酸过氧化物酶(APX)基因，其GenBank登录号为AY974805，该基因的cDNA全长1169更多还原

【Abstract】 L-ascorbic acid (AsA), also named vitamin C, has a multitude of functions in organisms. AsA is an essential component of human nutrition, but humans lack the ability to synthesize AsA as a result of a mutation in the last enzyme required for AsA biosynthesis. AsA, therefore, must be acquired regularly from dietary sources, primarily from plants rich in AsA. AsA is involved in the process of cell division, cell expansion and plant growth. AsA also plays an important role as an antioxidant and protects the plant during oxidative damage by scavenging free radicals and reactive oxygen species that are generated during photosynthesis, oxidative metabolism and various stresses. By regulating the expression of genes involved in plant ascorbic acid biosynthesis and metabolism, it is possible to increase the AsA contents of plants, thus improve the nutritive values of plant foods and the resistance to environmental stresses.Tomato is a worldwide vegetable with high nutritive values. However, most genes involved in tomato ascorbic acid biosynthesis and metabolism have not been cloned and there are nearly no reports about the gene regulation. Therefore, this study aims to clone and regulate the expression of genes involved in tomato AsA biosynthesis and metabolism and expects to obtain tomato materials with high content of AsA by transgenic techniques. The main results were as follows:1. The full-length cDNA of GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22), which catalyzes the formation of GDP-D-mannose, was cloned by RACE-PCR and the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AY605668. The full-length of GMP cDNA is 1535 bp and contains a complete open reading frame (ORF) of 1086 bp, which encoding 361 amino acids (aa). Homology analysis of tomato GMP showed high homology with GMP from other plants. The full-length cDNA of L-galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3), which is the last key enzyme of plant ascorbic acid biosynthesis, was cloned by RT-PCR based on the published sequence (AB080690). The complete ORF of GLDH更多还原