Method of production of recombinant sucrose synthase, use thereof in the manufacture of kits for determination of sucrose, production of ADPglucose and production of transgenic plants whose leaves and storage organs accumulate high contents of ADPglucose and starch

Abstract

An isolated sucrose synthase peptide. Also, a method of preparing ADPglucose by incubating the isolated sucrose synthase peptide with ADP in suitable conditions and then isolating and purifying the ADPG produced. Also, an assay kit for the spectrophotometric, fluorimetric or amperometric determination of sucrose, which kit includes the isolated sucrose synthase peptide. Also, a method of producing a transgenic plant that overexpresses sucrose synthase by inserting a genetic construct containing a DNA fragment that encodes the sucrose synthase peptide into a vector and transferring to a plant genome, and a transgenic plant obtained thereby.

The invention claimed is: 
     
       1. An isolated peptide comprising the sucrose synthase of SEQ ID NO: 12. 
     
     
       2. The isolated peptide as claimed in  claim 1  that consists of SEQ ID NO: 12. 
     
     
       3. A method of preparing ADPG comprising the steps of incubating the isolated peptide of  claim 1  with ADP in suitable conditions for causing a reaction that produces ADPG followed by isolation and purification of the ADPG produced. 
     
     
       4. The method of preparing ADPG according to  claim 3 , comprising the steps of:
 a) Providing 100 ml of the following solution for the incubating step and incubating for 12 h at 37° C.: 
 
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   Sucrose 
                   1 
                   M 
                 
                     
                   HEPES, pH 7.0 
                   50 
                   mM 
                 
                     
                   EDTA 
                   1 
                   mM 
                 
                 
                 
                 
               
                     
                   Polyethylene glycol 
                   20% 
                 
                 
                 
                 
                 
               
                     
                   MgCl 2   
                   1 
                   mM 
                 
                     
                   KCl 
                   15 
                   mM 
                 
                     
                   ADP 
                   100 
                   mM 
                 
                     
                     
                 
             
                
               
               
                
                
                
               
            
             
                
               
            
             
                
                
                
                
               
            
           
         
         b) Stopping the reaction by heating, 
         c) Centrifuging at 10000 g for 10 min with formation of a supernatant, and 
         d) Chromatographing the supernatant by HPLC, and then eluting and purifying the ADPG. 
       
     
     
       5. An assay kit for the spectrophotometric, fluorimetric or amperometric determination of sucrose comprising the sucrose synthase of  claim 1 . 
     
     
       6. The assay kit as claimed in  claim 5 , comprising an incubation medium with the following components:
 a) 2 units of sucrose synthase, 
 b) 2 mM of ADP 
 c) 2 units of ADPG pyrophosphatase of plant, animal or microbial origin 
 d) 2 units of PGM 
 e) 2 units of G6PDH 
 f) 0.5 mM of NAD(P) 
 g) 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl 2 /15 mM KCl 
 h) Previously filtered test sample. 
 
     
     
       7. The assay kit as claimed in  claim 5 , comprising an incubation medium with the following components:
 a) 2 units of sucrose synthase, 
 b) 2 mM of UDP 
 c) 2 units of UDPG pyrophosphatase of plant, animal or microbial origin 
 d) 2 units of PGM 
 e) 2 units of G6PDH 
 f) 0.5 mM of NAD(P) 
 g) 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl 2 /5 mM KCl 
 h) Previously filtered test sample. 
 
     
     
       8. The assay kit as claimed in  claim 5 , comprising an incubation medium with the following components:
 a) 2 units of sucrose synthase, 
 b) 2 mM of UDP 
 c) 2 units of UDPG dehydrogenase 
 d) 0.5 mM of NAD 
 e) 100 ml of reaction buffer: 50 mM HEPES, pH 7.0/1 mM EDTA/20% polyethylene glycol/1 mM MgCl 2 /15 mM KCl 
 f) Previously filtered test sample. 
 
     
     
       9. A method of producing a transgenic plant that overexpresses sucrose synthase comprising the steps of inserting a genetic construct that contains and expresses the DNA fragment of SEQ ID NO: 11 in a suitable vector and transferring the genetic construction to the genome of a plant. 
     
     
       10. The method according to  claim 9 , wherein the vector comprises pSS5. 
     
     
       11. A transgenic plant comprising a genetic construct that overexpresses a sucrose synthase comprising SEQ ID NO: 12 such that the plant has a higher content of sucrose, G6P, ADPG and starch than a corresponding wild type plant without the genetic construct. 
     
     
       12. The transgenic plant according to  claim 11 , wherein the transgenic plant has a level of sucrose synthase enzyme activity that is 2-10 times greater than a level of sucrose synthase enzyme activity in a corresponding wild-type plant without the genetic construct. 
     
     
       13. The transgenic plant according to  claim 11 , which is selected from the group consisting of a tobacco plant, a potato plant a tomato plant and a rice plant. 
     
     
       14. The transgenic plant according to  claim 12 , which is selected from the group consisting of a tobacco plant, a potato plant a tomato plant and a rice plant. 
     
     
       15. The transgenic plant according to  claim 13 , wherein the plant has leaves with a content of sucrose, G6P, ADPG and starch and with an amylose/amylopectin ratio that is higher than those in leaves of a corresponding wild-type plant. 
     
     
       16. The transgenic plant according to  claim 14 , wherein the plant has leaves with a content of sucrose, G6P, ADPG and starch and with an amylose/amylopectin ratio that is higher than those in leaves of a corresponding wild-type plant. 
     
     
       17. The transgenic plant according to  claim 13 , wherein the plant has at least one of a root, tuber or seed with a content of sucrose, G6P, ADPG and starch and with an amylose/amylopectin ratio that is higher than those in a root, tuber or seed of a corresponding wild-type plant.