Gene Cloning And Expression Profiling Of Ascorbic Acid Biosynthesis In Non-Heading Chinese Cabbage And Functional Analysis Of BcPMI2

Ascorbic acid (AsA), also called Vitamin C, is essential for the maintenance of life in plant and animal. AsA content in plant is the result of the balance of biosynthesis and consumption of AsA. The main pathway of the biosynthesis of AsA in plant is the L-galactose pathway, in which L-galactose is a key precursor and eight enzymes are involved. Enzyme activities can be regulated by controlling the gene expression and the biosynthesis rate and accumulation of ascorbic acid is then controlled. Artificial changes of some enzyme activities could affect the accumulation of AsA in plant. 1. Using alkaline treatment as a background, under 245 nm, ascorbic acid can be determined directly by UV spectrophotometry. A method for the determination of AsA content in plant was developed as that it is determined by UV spectrophotometry after determination applied alkaline treatment. The method was applied in the determination of the content of AsA in non-heading Chinese cabbage, and the result was similar with that of the bathophenanthroline method. The spectrophotometry determination method is very applicable for the determination of AsA content of non-heading Chinese cabbage. The coefficient of recovery is over 90%. The standard deviation of the same variety is between 2%-12%.2. Carbon dioxide (CO2) treatment can retard senescence, and reduce the loss of AsA, soluble protein and chlorophyll. The activities of hydrogen peroxide scavenging enzyme system including catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) were also inhibited by CO2 treatment. The descent of the AsA content could be delayed by treatment with both 50% CO2 and 100% CO2, and the effect of 50% CO2 was more significant than that of 100% CO2. At 25℃, the effect of CO2 treatment was better than that of control, but the distinction between CO2 and control was not significant at 4℃. Besides, under the same CO2 concentration treatment, temperature has little effect on the AsA content.3. Accumulation of AsA partially depends on the expression of genes in L-galactose pathway. In this study, we have cloned some of genes involved in the L-galactose pathway from non-heading Chinese cabbage, and examined the expression patterns of these genes in non-heading Chinese cabbage with real-time PCR. The analysis of gene expressions in AsA biosynthesis pathway between'Wutacai'and'Erqing', two non-heading Chinese cabbage varieties with differential content of AsA, suggest that the difference of AsA content in different cultivars is not due to the differentail expression of some one gene of the biosynthesis pathway of AsA. The AsA content of leaves shows a temporary increasing after treatment with 5mM methyl-jasmonate. The expressions of the seven genes in L-galactose pathway, BcPMI2, BcPMM, BcVTC1, BcGME, BcVTC2, BcGDH and BcGLDH, can be induced by 5 mM methyl-jasmonate in the leaves of non-heading Chinese cabbage. And BcVTC1, BcGME, BcVTC2 and BcGLDH, the four genes may play more important roles in the regulation of AsA accumulation after methyl-jasmonate treatment, since the expressions of the four genes was induced rapidly after methyl-jasmonate treatment.4. Mannose-6-phosphate isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate to mannose-6-phosphate, the first step in the L-galactose pathway for the biosynthesis of L-ascorbic acid (AsA). In this study, through the GUS test and PCR detection, we get 8 positive transgenic tobacco plants with over-expression of BcPMI2, the gene cloned from non-heading Chinese cabbage and encoded PMI. Two transgenic plants, P1 and P2, were selected for the further study because the RT-PCR detection indicated higher expression ofBcPMI2 in their leaves. Instead of the AsA content increasing, over-expression of BcPMI2 lead to a decreasing of the AsA content in tobacco leaves and follow a decreasing of the saccharine content. Over-expression of BcPMI2 lead to a high tolerance against H2O2 comparing to wild-type in tobacco leaves. A higher content of AsA in leaf-plates of transgenic tobacco than that of wild-type detected after 3 d treated with low concentration H2O2. It suggests that over-expression of BcPMI2 in tobacco increases the potential of the AsA accumulation under oxidative stress.