Production of ascorbic acid in plants

Abstract

The present invention relates to a polynucleotide in isolated form, which polynucleotide codes for a protein with the activity of the enzyme L-galactono-γ-lactone dehydrogenase, which polynucleotide comprises at least the L-galactono-γ-lactone dehydrogenase activity-determining parts of the coding part of the sequence or a sequence derived therefrom on the basis of the degeneration of the genetic code. The invention further relates to the use of the polynucleotide in the production of transgenic plants, plant cells, or other eukaryotic cells.

1 . Polynucleotide in isolated form, which polynucleotide codes for a protein with the activity of the enzyme L-galactono-γ-lactone dehydrogenase, which polynucleotide comprises at least the L-galactono-γ-lactone dehydrogenase activity-determining parts of the coding part of the nucleotide sequence, which is shown in FIG. 3, or a sequence derived therefrom on the basis of the degeneration of the genetic code.  
     
     
         2 . Polynucleotide as claimed in  claim 1 , which polynucleotide is a cDNA which codes for the enzyme L-galactono-γ-lactone dehydrogenase and at least substantially comprises the coding part of the nucleotide sequence which is shown in FIG. 3.  
     
     
         3 . Polynucleotide as claimed in  claim 1  or  2  for use in the production of transgenic plant cells, plant tissues or plants with an increased content of the enzyme L-galactono-γ-lactone dehydrogenase relative to non-transgenic plant cells, plant tissues or plants.  
     
     
         4 . Polynucleotide as claimed in  claim 1  or  2  for use in the production of transgenic plant cells, plant tissues or plants with an increased content of ascorbic acid relative to non-transgenic plant cells, plant tissues or plants.  
     
     
         5 . Polynucleotide as claimed in  claim 1  or  2  for use in the transformation and/or transfection of eukaryotic cells in order to bring about expression of the polynucleotide therein.  
     
     
         6 . Transgenic plant cells which carry in their genome a polynucleotide as claimed in  claim 1  or  2  not naturally present therein.  
     
     
         7 . Transgenic plant cells as claimed in  claim 6  which form part of a transgenic plant tissue and/or a tranagenic plant.  
     
     
         8 . Transgenic plant tissue consisting at least partially of transgenic plant cells as claimed in  claim 6 .  
     
     
         9 . Transgenic plant tissue as claimed in  claim 8  which forms part of a transgenic plant.  
     
     
         10 . Transgenic plant which consists at least partially of plant cells as claimed in  claim 6 .  
     
     
         11 . Transgenic plant as claimed in  claim 10 , obtainable by transforming a plant cell with a polynucleotide as claimed in  claim 1  or  2  and by regenerating a plant from the transformed plant cell.  
     
     
         12 . Transgenic plant as claimed in  claim 10  or  11 , characterized in that the plant is thale cress ( Arabidopsis thaliana ), tobacco ( Nicotiana tabacum ), tomato, potato or corn.  
     
     
         13 . Transformed and/or transfected eukaryotic cell, comprising in its genome a polynucleotide as claimed in  claim 1  or  2 .  
     
     
         14 . Recombinant L-galactono-γ-lactone dehydrogenase, obtainable by expression of a polynucleotide as claimed in  claim 1  or  2  in a suitable host.  
     
     
         15 . Recombinant L-galactono-γ-lactone dehydrogenase as claimed in  claim 14  which is isolated from a transgenic plant tissue as claimed in  claim 7  or  8 , a transgenic plant as claimed in  claim 9  or  10 , or a eukaryotic cell as claimed in  claim 13 .  
     
     
         16 . Transformation system, comprising a transformation vector or set of vectors, at least one of which includes a nucleotide sequence which codes for the enzyme L-galactono-γ-lactone dehydrogenase.  
     
     
         17 . Transformation system as claimed in  claim 15 , comprising Agrobacterium and a binary vector comprising a polynucleotide as claimed in  claim 1  or  2 .  
     
     
         18 . Use of polynucleotide as claimed in  claim 1  or  2  and/or the transformation system as claimed in  claim 16  or  17  for producing a transgenic plant or plant tissue with an increased content of L-galactono-γ-lactone dehydrogenase compared with a non-transgenic plant or plant tissue.  
     
     
         19 . Use of the polynucleotide as claimed in  claim 1  or  2  and/or the transformation system as claimed in  claim 16  or  17  for producing a transgenic plant or plant tissue with an increased ascorbic acid content compared to a non-transgenic plant or plant tissue.  
     
     
         20 . Use as claimed in  claim 17  or  18 , wherein the plant is thale cress ( Arabidopsis thaliana ), tobacco ( Nicotiana tabacum ), tomato, potato or corn.  
     
     
         21 . Use of a polynucleotide as claimed in  claim 1  or  2  for transfecting and/or transforming a eukaryotic cell.  
     
     
         22 . Method for producing plants or plant tissues with an increased ascorbic acid content, comprising of transformation of a plant cell with a gene construct which comprises at least the polynucleotide as claimed in  claim 1  or  2 , optionally in the presence of suitable transcription and/or translation regulation factors, and regeneration of a transgenic plant or plant tissue from the plant cell.  
     
     
         23 . Method for producing the enzyme L-galactono-γ-lactone dehydrogenase, comprising of transfecting and/or transforming a eukaryotic cell with a gene construct which comprises at least the polynucleotide as claimed in  claim 1  or  2 , optionally in the presence of suitable transcription and/or translation regulation factors, expressing the enzyme in the transfected and/or transformed cell and optionally isolating the enzyme from the cell and/or its culture medium.  
     
     
         24 . Gene construct comprising a polynucleotide as claimed in  claim 1  or  2 , optionally in the presence of transcription and/or translation regulation factors.  
     
     
         25 . Gene construct as claimed in  claim 24 , further comprising targeting sequence for targeting the encoded enzyme to various parts of the plant cell.  
     
     
         26 . Gene construct as claimed in  claim 25 , wherein the parts of the plant cell are the cytoplasm, vacuoles, chloroplasts, mitochondria, lysosomes, endoplasmatic reticulum, Golgi apparatus.  
     
     
         27 . Method for purifying the enzyme L-galactono-γ-lactone dehydrogenase, comprising of: 
 a) passing a protein extract of cauliflower florets through an ion-exchange column:  
 b) collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions;  
 c) combining the fractions with GLDase activity and passing them through a Phenyl Sepharose CL 4B column;  
 d) collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions;  
 e) combining the fractions with GLDase activity and passing them through a gel filtration column;  
 f) collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions;  
 g) combining the fractions with GLDase activity and passing them through an FPLC Resource Q-column;  
 h) collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions;  
 i) combining the fractions with GLDase activity and passing them through a FPLC Poros 20 SP-column;  
 j) collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions.  
 
     
     
         28 . Method for increasing the L-ascorbic acid levels in plants, comprising: 
 a) provision of plants that have been transformed with the sense version of the GLDase gene, and    b) providing the said plants with the precursor L-galactono-γ-lactone in order to induce increased L-ascorbic acid synthesis.    
     
     
         29 . Transgenic plants having in their genome an antisense version of the GLDase gene resulting in a decreased amount of ascorbic acid as compared to non-transgenic plants for use a model system or biosensor for oxidative stress.