Cloning And Functional Analysis Of L-galactono-1,4-lactone Dehydrogenase Gene In Potato

L-Ascorbic acid (AsA), also known as vitamin C, is one of the most abundant antioxidants and cofactor for several enzymes. It has been reported that AsA is necessary for plant growth and development, and it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Moreover, Plant-derived ascorbate also provides the major source of vitamin C for the human, because humans are incapable of synthesizing AsA and must secure it by means of dietary uptake to be healthy. Increasing the plant vitamin C content is important for peoples'health.It had been well confirmed that L-galactose or Smirnoff-Wheeler pathway was the major biosynthetic pathway of ascorbate in plants, and GLDH was the key enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In the previous study,we isolated a full-length cDNA clone encoding L-galactono-1,4-lactone dehydrogenase (GLDH) from the leaf of potato (Solanum tuberosum L.) cv. Favorita and subcloned into the binary vector pBI121 to construct sense and antisense recombinant plant expression vectors respectively. The recombinant were introduced into potato, and 4 sense transgenic potato lines and 2 anti-sense ones were obtained. Both the StGLDH expression level, StGLDH activity and AsA content in leaf and tuber of potato were investigated. Because of the postulated role of AsA as a stress-related Antioxidants, the expression of NaCl stress-responsive were examined in transformed and nontransformed potato plantlets grown in vitro. From which we get some information about the founction of GLDH in AsA metabolism system in Potato, as well as the overexpression of StGLDH. The main results were as follows:1. A full-length cDNA clone encoding L-galactono-1,4-lactone dehydrogenase (GLDH), named as StGLDH (GenBank:FJ755844), was isolated from the leaf of potato (Solanum tuberosum L.) cv. Favorita StGLDH transcript is 2563 nt long with an open reading frame of 1773 bp and encodes a polypeptide of 590 amino acids. The StGLDH amino acid sequence predicted a molecular weight of 67 119 Da and a pI of 8.5, which contained a putative mitochondrial-targeting domain in its first 83 amino acids and a cleavage site (FR/YA) similar to other known GLDHs. Three possible transmembrane regions were predicted between residues 50 and 68,247 and 269,479 and 503. Analysis of StGLDH amino acid sequence identified a putative FAD binding domain between residues 105 and 240, wherein was located a region (134VGSGLSP140) common to all GLDHs characterized to date. Sequence analysis showed that deduced StGLDH protein was highly homologous to other GLDH proteins from different species, especially with Solanum lycopersicum, Capsicum annuum, Nicotiana tabacum, about 90.6%-95.9% identity in amino acid sequence.2. The full-length cDNA, as well as partial cDNA of GLDH, were subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form a sense construct. Then the constructs were introduced into potato via Agrobacterium fumeracens-mediated procedure. PCR and Real time PCR results indicated that the StGLDH had been recombined into potato genome,4 sense transgenic potato lines and 2 anti-sense ones were obtained.3. Among the six transgenic potato lines, anti-GLDH 13 lines exhibited an obviously phenotypic difference from others. Its aerial parts was reduced and displayed very severe growth defects such as stunted plants with deformed leaves. Detailed characterization revealed that the plant growth rate, plant height, node height, leaf size, stem diameter and fruit weight were all reduced, and the epidermal hair density increased compared to the wild type lines, and the To tuber germinate more earlier than the others. Accordingly, the ability of anti-GLDH 13 line against stress was also decreased.The sense transgenic lines showed a slightly phenotypic difference from wild type lines, the node height and leaf thickness were increased compared to the wild type lines. Sense transgenic lines turn slightly round in leaf shape and To Tuber. Furthermore, the leaf color of sense transgenic lines was greener than that of the wild type and the anti-sense ones. But there was no obviously differerce in plant growth rate, except GLDH 155 lines showed a rapid growth in earlier period.4. Two sense transgenic lines GLDH 516, GLDH 518 overexpress StGLDH. Overexpression of StGLDH resulted in enhanced GLDH activity. AsA content, DHA content, AsA+DHA content increased in both leaves and tubers correspondly. AsA content in leaves of GLDH 516 and GLDH 518 lines increased 47.16% and 13.1% respectively compared to wild type line, and AsA content in tubers increased 10.3% and 6.8% respectively. An adverse result occurred to the anti-sense transgenic lines'anti-GLDH 13', which had a decrease in AsA content in both leaves (28.82%) and tubers (10.3%). AsA content in leaves showed large variations in amplitude than that of in tubers.5. Feeding potato leaves with AsA precursor was very effective at increasing AsA. Fed with L-GalL, the GLDH activity, AsA content, DHA content and AsA+DHA content in either transgenic or wild potato leaves were all significantly higher than those of treated with distilled water. L-GalL feeding leaves were approximately 2-fold DHA content and AsA+DHA content more than their corresponding controls.The difference in AsA content among 7 potato lines decreased after L-GalL feeding. AsA content in GLDH 516 and GLDH 518 lines were still higher than that of others, while anti-GLDH 13 lines had the lowlest AsA content in both leaves and tubers as yet.6. NaCl-stress could regulate GLDH mRNA expression in vitro potato plantlets. Under 0～250 mmol·L-1NaCl concentration and within 5 days, NaCl stress could significantly up regulated the expression of GLDH mRNA in the planetlets grown in vitro, then down regulate it continuously with the prolongation of time. The AsA level was positively correlated with the mRNA transcript expression and also GLDH activity. But there existed great difference of variation amplitude between GLDH mRNA expression level and AsA content, as well as GLDH activity.7. Sense expression of StGLDH in GLDH 516, GLDH 518 lines conferred better tolerance to NaCl stresses, while anti expression of StGLDH in GLDH 13 had observed adverse effects. The NaCl tolerance of potato plantlets was associated with AsA level before stress. After 5 days treatment, APX activity, MDHAR activity, DHAR activity and MDA content increased while the AO activity decreased. The increase of AsA level in better tolerance potato planets, such as GLDH 516, GLDH 518 lines, was resulted from the cooperation of rapid AsA synthesis and regeneration. The decrease of AsA level in the salt sensitive lines, such as anti-GLDH 13 lines, was resulted from the cooperation of decreased AsA synthesise and rapid oxidation, not for the reason of AsA regeneration. Anti-GLDH 13 line showed evident injured symptom due to high level of membrane lipid peroxidation initiated by NaCl stress.Consequently, the level of AsA accumulation in potato plantlets was directly correlated with their ability to withstand NaCl stresses. These results further demonstrated that the expression of GLDH and GLDH activity had played an important role on regulating AsA level as well as tolerance of potato plantlets to NaCl stresses.