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1.
Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed transgenic potato plants ( Solanum tuberosum L. cv. Taedong Valley) over-expressing strawberry GalUR gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the GalUR gene in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by high-performance liquid chromatography (HPLC). The over-expression of GalUR resulted in 1.6–2-fold increase in AsA in transgenic potato and the levels of AsA were positively correlated with increased GalUR activity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen (MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of GalUR gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. 相似文献
3.
Salt-tolerance was studied in transgenic potato. It was conferred by overexpression of ascorbate pathway enzyme ( d-galacturonic acid reductase, GalUR). As genetic engineering of the GalUR gene in potato enhances its ascorbic acid content ( l-AsA), and subsequently plants suffered minimal oxidative stress-induced damage, we now report on the comprehensive aptness
of this engineering approach for enhanced salt tolerance in transgenic potato ( Solanum tuberosum L. cv. Taedong Valley). Potatoes overexpressing GalUR grew and tuberized in continuous presence of 200 mM of NaCl. The transgenic plants maintained a higher reduced to oxidized
glutathione (GSH:GSSG) ratio together with enhanced activity of glutathione dependent antioxidative and glyoxalase enzymes
under salinity stress. The transgenics resisted an increase in methylglyoxal that increased radically in untransformed control
plants under salinity stress. This is the first report of genetic engineering of ascorbate pathway gene in maintaining higher
level of GSH homeostasis along with higher glyoxalase activity inhibiting the accumulation in methylglyoxal (a potent cytotoxic
compound) under salt stress. These results suggested the engineering of ascorbate pathway enzymes as a major step towards
developing salinity tolerant crop plants. 相似文献
5.
Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has
many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining
the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated
and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two
types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of
the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results
indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic
DHAR might play main important roles in improving the AsA contents of potato tubers. 相似文献
7.
GDP-Mannose 3′,5′-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP- d-mannose to GDP- l-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members
of the GME gene family were isolated from tomato ( Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1- GFP (green fluorescent protein) and SlGME2- GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also
showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen
(paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and
root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses. 相似文献
10.
l-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco ( Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content. 相似文献
11.
l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the
AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the
non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines;
i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g −1 FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold
increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor
for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing
the AsA content in plants. 相似文献
12.
After analyzing tomato plants transformed with GalUR gene for their ascorbic acid contents, it was found that some transgenic lines contained higher levels of ascorbic acid compared to control plants. In the present study, callus induction rate was 50.2 % in the explant and shoot regeneration rate was 51.5 % from the callus with transformation efficiency of 3.0 %. Based on PCR and Southern blot analysis, three independent transformants containing the insert gene were selected. Phenotypic traits of these transgenic progeny were similar to those of control tomatoes. Tomatoes (H15) with high fruit ascorbic acid contents were selected for next generation ( GalUR T 3) analysis. Transgenic tomatoes with increased ascorbic acid contents were found to be more tolerant to abiotic stresses induced by viologen, NaCl, or mannitol than non-transformed plants. In leaf disc senescence assay, the tolerance of these transgenic plants was better than control plants because they could retain higher chlorophyll contents. Under salt stress of less than 200 mM NaCl, these transgenic plants survived. However, control plants were unable to survive such high salt stress. Ascorbic acid contents in the transgenic plants were inversely correlated with MDA contents, especially under salt stress conditions. The GalUR gene was expressed in H15 tomatoes, but not in control plants. Higher expression levels of antioxidant genes ( APX and CAT) were also found in these transgenic plants compared to that in the control plants. However, no detectable difference in SOD expression was found between transgenic plants and control plants. Results from this study suggest that the increase in ascorbic acid contents in plants could up-regulate the antioxidant system to enhance the tolerance of transgenic tomato plants to various abiotic stresses. 相似文献
13.
Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine
betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses
through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of
the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated
that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants
was between 10.8 and 11.7 U. There was a negative relationship ( y = −2.2083 x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height
increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene
glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to
tolerate drought and salt was increased when their BADH activity was increased. 相似文献
14.
Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the l-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage ( Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S:: BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T 1 seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H 2O 2 treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition. 相似文献
16.
GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp
nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot
analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal
temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to
fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments
of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1),
APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than
those of WT, while the contents of H 2O 2 and O 2
−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net
photosynthetic rate ( P
n) and the maximal photochemical efficiency of PSII ( F
v/ F
m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression
of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants. 相似文献
17.
Main conclusion
Transgenic rice expressing pigeonpea
Cc
CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in
CcCDR
-transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes.
Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants. 相似文献
19.
To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants,
a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as
the recycling was performed in the fruits of apple ( Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA
levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase ( l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase ( l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh.
By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However,
their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light
for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh.
Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by
light in the leaves. 相似文献
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