Metabolic Engineering Of Ascorbic Acid Synthesis Pathway In Arabidopsis By A Dual-gene Vector System

Vitamin C (ascorbic acid) is a main part of the plant antioxidant system and it plays an important role in photoprotection, flowering, fruit ripening, cell senescence, division, expansion, and cell wall metabolism. However, humans have lost the ability to synthesize AsA, thus have to obtain their AsA regularly from plants. Engineering plant AsA metabolism has now become feasible because of the increased understanding of its biosynthetic pathway and key enzymes. All of the six genes which involved in AsA biosynthetic pathway were cloned by PCR from Arabidopsis thaliana cDNA and evaluated. Four dual-gene plant expression vectors were constructed and transformed into Arabidopsis thaliana for the comparison between single-gene transformation and dual-gene transformation, and also for the selection of the best gene combination.In most of the biothyntic gene transgenic lines, the expression of the target gene was increased on RNA level(real-time PCR). The AsA content was detected using HPLC. The GGT-GLDH transgenic lines increased the AsA content to 4.09-fold compared with the blank transformation(CK); the GGT, GLDH, GMP-GME, GGT-GPP, GDH-GLDH transgenic lines increased the AsA content to 3.63-fold,1.77-fold,1.29-fold,4.09-fold,1.21-fold compared with CK respectively. This result shows that GGT catalyze the only rate-limiting step in L-galactose pathway. The other five genes contribute little to AsA accumulation. This is the reason why dual-gene transformation does not perform markedly better to single-gene transformation. The result also indicates that GGT has the pull-up effect to the other genes.It is the first time to compare the single-gene transformation and dual-gene transformation for their merits and faults. The regulation of multi-gene transformation will help us understand the mechanisms that control plant AsA accumulation and select the best combination, thus providing the opportunity to enhance nutritional quality and stress tolerance of crop plants.