Study on generation of ascorbic acid-enriched transgenic rice

L-抗坏血酸（阿萨），又称维生素C m，在植物组织中积累到毫摩尔浓度。L-半乳糖酸-1，4-内酯脱氢酶（GalLDH; EC 1.3.2.3）是植物L-抗坏血酸（阿萨）生物合成途径中的最终酶，催化L-半乳糖酸-1，4-内酯氧化为阿萨。通过在花椰菜花叶病毒35 S启动子的控制下过量表达GalLDH cDNA，获得了过量生产AsA的转基因烟草BY-2细胞系。过量表达GalLDH mRNA可使GalLDH活性和蛋白质含量增加。检测到该转基因烟草的阿萨水平约为野生型（W）细胞的6-18倍。有趣的是，在这些富含AsA的转基因细胞培养物中，细胞布朗宁明显受到抑制，表明较高水平的阿萨抑制了这些转基因细胞的衰老。此外，这些过量产生阿萨的转基因细胞可以获得对产生某些活性氧的百草枯的抗性。因此，过量产生AsA的转基因烟草细胞具有更高的抑制活性氧的能力。水稻GalLDH cDNA也被克隆。GalLDH的表达量与水稻抗坏血酸含量呈正相关。通过9种抗坏血酸前体物质的补充试验，研究了水稻抗坏血酸的生物合成途径。在水稻根中，抗坏血酸含量的增加促进根的伸长。此外，抗坏血酸的增加抑制了紫外线引起的叶绿素降解。

L-ascorbic acid (AsA), Called vitamin Cm is accumulated up to millimolar concentrations in plant tissues. L-Galactono-1, 4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the final enzyme in L-ascorbic acid (AsA) biosynthesis pathway of plant, and it catalyzes the oxidation of L-galactono-1, 4-lactone to AsA. AsA-overproducing transgenic tobacco BY-2 cell-lines were generated by over-expression of the GalLDH cDNA under the control of cauliflower mosaic virus 35S promoter. Over-expression of GalLDH mRNA let to increases in the GalLDH activity and fine protein. AsA levels of this transgenic tobacco were detected about 6-18 fold as much as wild type (W) cells. Interestingly in these AsA-enriched transgenic cell cultures, cell browning markedly restrained, suggesting that a higher level of AsA suppressed senescence of these transgenic cells. Furthermore, these AsA over-producing transgenic cells could acquire resistance against paraquat that produces some active oxygen species. Thus, AsA-overproducing transgenic tobacco cells were suggested to have a higher ability to scavenge active oxygen species. Rice GalLDH cDNA was also clones. The expression of GalLDH was positively correlated with ascorbic acid content of rice. In rice, the biosynthetic pathway of ascorbic acid was investigated by supplement experiment of 9 precursors of ascorbic acid. In rice root, an increase in ascorbic acid content stimulated root elongation. Furthermore, the increase in ascorbic acid inhibited degradation of leaf chlorophyll caused by UV.