Functional Analysis Of GLDH And AO 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.Effects of factors including explants,seedling age,hormone combinations,agar concentration,AgNO₃ and AsA on differentiation of adventitious bud in non-heading Chinese cabbage were evaluated.The results showed:the explants from 4～7 days old seedling are more efficient to induce adventitious buds;the combination of 4 mg/L 6-BA and 0.5 mg/L NAA can improve adventitious buds induction;the culture medium with 9 g/L Agar,5～7.5 mg/L AgNO₃ and 0.1～0.5 mmol/L AsA can increase the frequence of differentiation and the quality of buds.A procedure for induction of adventitious buds and regeneration system for non-heading Chinese cabbage is established.Using this system, high regeneration frequence can be obtained.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 over-expression vector was constructed,which consists of the sence of GLDH cDNA sequence from non-heading Chinese cabbage.A total of 46 regenerate plants were obtained using the Agro bacterium-mediated transformation method,9 of which were putative transgenic plants according to PCR detection,GLDH gene have been introduced into Non-heading Chinese cabbage(Wutacai)genome.Measurements of AsA contents and real-time PCR of transgenic plants were performed,and the results indicated that GLDH transcript level in transgenic plants were higher than that of the control and the highest one increased up to 28.5 fold.The leaf AsA contents in transgenic plants were higher than the control and the highest one increased up to 1.8 fold.3.According to the principle of hpRNA(hairpin RNA) in the plant,a 484 bp cDNA fragment encoding L-galactono-1,4-laetone dehydrogenase(GLDH) was cloned from non-heading Chinese cabbage by the method of RT-PCR on the basis of the specific primers which with double enzyme sites.Ligate GLDH fragment with YYT interval region at forward and reverse orientation to construct the GLDH-RNAi expression cassette.The binary vector pRNAi-GLDH harboring GLDH-RNAi expression cassette was constructed through three sub-cloning via mid-clone vector and then transformed into Agrobaeterium LBA4404.A total of 5 transgenic plants were obtained using the Agrobacterium-mediated transformation method,which provided an effective tool for the further study of GLDH gene function.4.Using RT-PCR and RACE technique,the full-length cDNA of ascorbate oxidase gene was cloned from non-heading Chinese cabbage cultivar 'Suzhouqing' Sequence analysis indicated that BcAAO gene consisted of 1854 nucleotides encoding a 64.28 kD peptide containing 577 amino acids with pI of 9.07.Further,BcAAO shows high similarity with AO from other plants.Phylogenetie analysis revealed the evolutionary conservation of these proteins among diverse species.The sequence has been submitted to the GenBank database and the accession number is AB369266.Southern blotting analysis indicated that there was more than one copy of AO gene in non-heading Chinese cabbage genome. Real-time PCR analysis revealed the gene was constitutively expressed with lower level, and the corresponding mRNA was accumulated most abundantly 72 h after infected by Alternaria brassicicola.