Methods of producing nucleic acids coding for proteins have raffinose synthase activity

Abstract

Raffinose is produced by allowing a raffinose synthase having the following properties to act on sucrose and galactinol: (1) action and substrate specificity: produces raffinose from sucrose and galactinol; (2) optimum pH: about 6 to 8; (3) optimum temperature: about 35 to 40° C.; (4) molecular weight: (i) about 75 kDa to 95 kDa estimated by gel filtration chromatography; (ii) about 90 kDa to 100 kDa estimated by polyacrylamide gel electrophoresis (Native PAGE); and (iii) about 90 kDa to 100 kDa estimated by SDS-polyacrylamide gel electrophoresis under a reduced condition (SDS-PAGE); and (5) inhibition: inhibited by iodoacetamide, N-ethylmaleimide, and myo-inositol.

1 - 17 . Canceled.  
     
     
         18 . A method of producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, which method comprising screening a cDNA library prepared from a plant by hybridization using a probe whose sequence is a portion of the nucleotide sequence of SEQ ID NO: 23, to select the DNA.  
     
     
         19 . A method of producing a vector comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose an galactinol, which method comprising producing the DNA by the method of  claim 18 , and inserting the DNA to a vector.  
     
     
         20 . A method of producing a transformed plant, which method comprising producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol by the method of  claim 18 , and transforming a plant with the DNA.  
     
     
         21 . The method according to  claim 20 , wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.  
     
     
         22 . A method of producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, which method comprising producing the DNA by the method of  claim 18 , and operably linking the DNA to the transcription regulatory region expressible in plant cells.  
     
     
         23 . A method of producing a transformed plant, which method comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of  claim 22 , and transforming a plant with the chimeric gene.  
     
     
         24 . The method according to  claim 23 , wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.  
     
     
         25 . A method of modifying the content of raffinose family oligosaccharides in a plant, which method comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of  claim 22 , and transforming the plant with the chimeric gene, thereby changing the content of raffinose family oligosaccharides in the plant.  
     
     
         26 . A method of producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, which method comprising subjecting a DNA coding for a protein comprising SEQ ID NO: 24 to a mutagenesis and screening for a mutant DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol.  
     
     
         27 . A method of producing a vector comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, which method comprising producing the DNA by the method of  claim 26 , and inserting the DNA to a vector.  
     
     
         28 . A method of producing a transformed plant, which method comprising producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol by the method of  claim 26 , and transforming a plant with the DNA.  
     
     
         29 . The method according to  claim 28 , wherein the plant is sobybean, rapeseed, cotton, sugar beet, or sugar cane.  
     
     
         30 . The method of producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, which method comprising producing the DNA by the method of  claim 26 , and operably linking the DNA to the transcription regulatory expressible in plant cells.  
     
     
         31 . A method of producing a transformed plant, which method comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of  claim 30 , and transforming a plant with the chimeric gene.  
     
     
         32 . The method according to  claim 31 , wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.  
     
     
         33 . A method of modifying the content of raffinose family oligosaccharides in a plant, which method comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of  claim 30 , and transforming the plant with the chimeric gene, thereby changing the content of raffinose family oligosaccharides in the plant.