Characterization Of APX,AOBP And AO Genes Related With Ascorbic Acid Metabolism

L-ascorbic acid (AsA), also named vitamin C, a hexose lactone, plays an extensive role in plant and most animals. It has a multitude of functions in organisms, and is an essential component of human nutrition; moreover, is involved in the process of cell division, cell expansion and plant growth. But some animals including 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 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. The technique of RNAi has already been extensively applied in gene function research. This study intends to identify the function of genes associated with AsA metabolism preliminarily by regulating the expression of genes involved in plant metabolism using the RNAi technique and expects to obtain tomato materials with highcontent of vitamin C and the resistance to environmental stresses.Tomato is a worldwide vegetable with high nutritive values. However, most genes involved in tomato ascorbic acid metabolism have not been cloned and there are nearly no reports about the gene regulation. Therefore, this study aims to regulate the expression of genes involved in tomato AsA metabolism including ascorbate oxidase(AO), ascorbate oxidase promoter-binding protein (AOBP) and two ascorbate peroxidases (APX). Then, they were inserted into RNAi vectors for pHGRV and transgenic plants were obtained via Agrobacterium-mediated transformation method. We analyzed plant growth , the physiological and biochemical characteristics and the resistance, and expects to obtain tomato materials with high content of AsA by transgenic techniques. The main results were as follows:1. RNAi plant expression vectors were constructed with cDNA fragments by RT-PCR and transgenic tomato plants were obtained from genotype of ZS5# using the Agrobacterium-mediated transformation method.2. A total of 11 putative transgenic tomato plants were obtained. The PCR and Southern blot analysis of the putative transgenic plants showed that the T-DNA was integrated into the tomato genome, with various copy numbers.3. Measurements of the content of AsA, the activities of AO (AY971876) and tAPX(AF413573) of T₁ transgenic plants were performed and the results indicated that the fruit AsA contents of most RNAi transgenic lines were higher than the control and the highest transgenic line increased 0.59-fold, 1.16-fold respectively. The activities of most RNAi transgenic plants were lower than the control and the lowest transgenic line decreased up to 53.59% , 55.23% respectively. And there were significant...