Functional Analysis Of Gldh And Apx Genes, Associated With Ascorbate Acid Biosynthesis And Metabolism, In Non-Heading Chinese Cabbage

Vitamin C (L-ascorbic acid, AsA) is the major soluble antioxidant found in plants, playing an extensive role in plant physiology, such as ROS detoxification, photosynthesis and photoprotection, and so on. What is more, it is also an essential component of human nutrition. By regulating the experssion 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 plant resistance to environmental stresses.This study intends to identify the function of genes associated with AsA metabolism preliminarily using the transgenic technique and expects to obtain non-heading Chinese cabbage materials with higher content of vitamin C.1. The T1generation of transgenic plants transferred over-expression vector was tested. Measurements of AsA contents、real-time PCR and photosynthetic characteristics of transgenic plants were performed, results showed that:The objective band was showed in32plants out of total160plants. The reason may be the genetic isolation of transgenic offspring or there are some genetic loss happened. GLDH transcript level in transgenic plants were higher than that of the control and the highest one increased up to1.7fold; the leaf AsA contents in transgenic plants were higher than the control and the highest one increased up to1.5fold, and the photosynthetic characteristics of transgenic plants increased compared with the controls.2. L-galactono-1,4-lactone dehydrogenase(GLDH)was a key enzyme that catalyzed the final step in the L-ascorbic acid synthetic pathway of plants. In this experiment, a plant suppress-expression vector was constructed, which consists of the sense of GLDH cDNA sequence from non-heading Chinese cabbage. A total ofl8regenerate plants were obtained using the Agro bacterium-mediated transformation method,5of which were putative trans--genic plants according to PCR detection, which showed that GLDH gene have been introduced into Non-heading Chinese cabbage (Wutacai) genome. The controls are plants regenerated from the cotyledon and hypocotyl via Agrobacterium without target gene transform. Measurements of AsA contents、real-time PCR and activity of GLDH enzymes of transgenic plants were performed, and the results indicated that relative expression of GLDH、expression of GLDH enzymes and AsA contents in transgenic plants were lower than that of the control and the lowest one decreased36.6%、27.2%and58.4%respectively.3. Using RT-PCR and RACE technique, the full-length cDNA of APXgene was cloned from non-heading Chinese cabbage cultivarPol CMS. Sequence analysis indicated that BcAPX gene consisted of1089nucleotides encoding a31.6kD peptide containing288amino acids with pI of5.52. Further, BcAPX shows high similarity with APX from other plants. BC Apxl had been cloned into PGEX4T-3and transformed into the host BL21(DE3). Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG.