Gene Expression Profiling Of Ascorbic Acid Biosynthesis Pathway In Non-Heading Chinese Cabbage And Functional Analysis Of BCPMI2and BCGME

L-Ascorbic acid (AsA) is also called Vitamin C. It is a hexose lactone compound essential for both plants and animals to maintain life activities. AsA is not only a main antioxidant but also a coenzyme factor regulating plant growth and development. AsA biosynthesis is considered to be the main source of AsA accumulation in plant cells. L-galactose pathway is the main pathway for the biosynthesis of AsA in plant, in which eight enzymes are involved. To find the method regulating AsA biosynthesis and accumulation to obtain non-heading Chinese cabbage with high AsA content, the relationship of gene expression and AsA accumulation were researched.The main results are as follows:1. We investigated the AsA content and transcription level of the L-galactose pathway genes during diurnal changes and after MeJA treatment in leaves of non-heading Chinese cabbage. Light significantly increased the levels of ascorbate. Expression patterns of seven genes (PMI2, PMM, GMP, GME, GGP, GalDH and GalLDH) during the whole day showed a roughly similar expression profile except GPP. The transcripts increased rapidly in light at the beginning few hours and then decreased and showed lower in the dark. Among which, notably, the gene expression level of GGP peaked very earlier than that of the others. High concentration treatment of MeJA decreased AsA content of the leaves accompanying with decrease of transcriptional expression in GME, GGP, GPP and GalDH. While treated with low concentration of MeJA, AsA level of ’Suzhouqing’ increased evidently. All these suggested the accumulation of AsA influenced by exogenous MeJA, at least in part, by affected transcription of MeJA-responsive genes in L-galactose pathway of non-heading Chinese cabbage.2. Mannose-6-phosphate isomerase (PMI) is an enzyme which catalyses the first step of L-galactose pathway for ascorbic acid biosynthesis in plants. The content of AsA was lower in transgenic tobaccos than that in the wild type in normal condition. However, the AsA level in BcPMI2overexpression plants was significantly increased under stress. The germination rate of T1seeds of transgenic tobacco plants exhibited significantly higher level than that of wild type under NaCl or H2O2treatments. Meanwhile, higher tolerance was found in transgenic lines than in wild lines. It implied that overexpression of BcPMI2improved the AsA biosynthetic ability as well as AsA accumulation and obviously enhanced tolerance to oxidative and salt stresses, although the AsA level was lower than wild-type tobacco in normal condition.3. GDP-D-mannose-3’,5’-epimerase (GME) is an enzyme which catalyses a key step in the biosynthetic pathway of AsA. It can catalyse GDP-D-mannose not only to be GDP-L-galactose but also to be GDP-L-gulose. In this experiment, a whole fragment encoding GDP-D-mannose-3’,5’-epimerase was cloned from non-heading Chinese cabbage used to construct plant overexpression vector. A total of12non-heading Chinese cabbage and3Arabidopsis seedlings resistant to Km were obtained using the planta Agrobacterium-mediated gene transformation with infiltration. The result showed that plants at beginning-flower stage and3min+5min discontinuous vacuum infiltration made higher transformation frequency.