Specific genetic modification of the activity of trehalose-6-phosphate synthase and expression in a homologous or heterologous environment

Abstract

A method for the preparation of a eukaryotic organism, for example selected from plants, animals and fungi, showing constitutive, inducible and/or organ specific expression of a specifically modified TPS gene, which comprises the steps of providing a TPS gene; designing a suitable modification to the TPS gene by aligning the gene with the corresponding gene of yeast and establishing which part of the gene extends beyond the 5′ terminus of the yeast gene; deleting or inactivating a part of the N-terminal region of the TPS gene extending beyond the 5′ terminus of the yeast gene, in order to achieve an increased trehalose-6-phosphate synthase activity; cloning the thus modified gene into an expression vector under the control of a constitutive, inducible and/or organ-specific promoter; transforming a plant cell or tissue with the thus obtained expression vector; and regenerating a complete plant from the transformed plant cell or tissue.

What is claimed is: 
     
         1 . A method for determining the presence or quantity of trehalose-6-phosphate in a sample comprising:
 providing a sample, wherein the sample comprises trehalose-6-phosphate, glucose and trehalose;   separating the glucose and the trehalose from the trehalose-6-phosphate thereby forming a glucose-trehalose fraction;   reacting the glucose-trehalose fraction with a phosphotrehalase thereby producing a reaction product;   separating glucose from the reaction product;   determining the presence of the glucose; and   comparing the amount of the glucose to a reference standard to determine the amount of the trehalose-6-phosphate present in the sample.   
     
     
         2 . The method as claimed in  claim 1 , wherein the phosphotrehalase is from a bacterium. 
     
     
         3 . The method as claimed in  claim 2 , wherein the bacterium is  Bacillus subtilis.    
     
     
         4 . The method as claimed in  claim 2 , wherein the bacterium is  Escherichia coli.    
     
     
         5 . A chimeric DNA construct comprising a first domain and a second domain, wherein the first domain is from a TPS gene from a first species selected from the group consisting of  A. thaliana  and  S. cerevisiae , and wherein the second domain is from a TPS gene or TTP gene from a second species selected from the group consisting of  A. thaliana  and  S. cerevisiae.    
     
     
         6 . The chimeric DNA construct according to  claim 5 , wherein the first domain comprises a nucleotide fragment obtained by PCR amplification of a TPS 1 gene from  A. thaliana  using a first primer having a sequence of SEQ ID NO: 6 and a second primer having a sequence of SEQ ID NO: 15. 
     
     
         7 . The chimeric DNA construct according to  claim 5 , wherein the second domain comprises a nucleotide fragment obtained by PCR amplification of a TTPB gene from  A. thaliana  using a third primer having a sequence of SEQ ID NO: 16 and a further primer having a sequence of SEQ ID NO: 17. 
     
     
         8 . The chimeric DNA construct according to  claim 5 , wherein the second domain comprises a nucleotide fragment obtained by PCR amplification of a TPS2 gene from  S. cerevisiae  using a third primer having a sequence of SEQ ID NO: 19 and a fourth primer having a sequence of SEQ ID NO: 18. 
     
     
         9 . The chimeric DNA construct according to  claim 5 , wherein the first domain comprises a nucleotide fragment obtained by PCR amplification of a TPS 1 gene from  S. cerevisiae  using a first primer having a sequence of SEQ ID NO: 20 and a second primer having a sequence of SEQ ID NO: 21. 
     
     
         10 . A transgenic plant comprising in its genome a first domain and a second domain, wherein the first domain is from a TPS gene from a first species selected from the group consisting of  A. thaliana  and  S. cerevisiae , and wherein the second domain is from a TPS gene or TTP gene from a second species selected from the group consisting of  A. thaliana  and  S. cerevisiae.    
     
     
         11 . The transgenic plant according to  claim 10 , wherein the first domain comprises a nucleotide fragment obtained by PCR amplification of a TPS 1 gene from  A. thaliana  using a first primer having a sequence of SEQ ID NO: 6 and a second primer having a sequence of SEQ ID NO: 15. 
     
     
         12 . The transgenic plant according to  claim 10 , wherein the second domain comprises a nucleotide fragment obtained by PCR amplification of a TTPB gene from  A. thaliana  using a third primer having a sequence of SEQ ID NO: 16 and a further primer having a sequence of SEQ ID NO: 17. 
     
     
         13 . The transgenic plant according to  claim 10 , wherein the second domain comprises a nucleotide fragment obtained by PCR amplification of a TPS2 gene from  S. cerevisiae  using a third primer having a sequence of SEQ ID NO: 19 and a fourth primer having a sequence of SEQ ID NO: 18. 
     
     
         14 . The transgenic plant according to  claim 10 , wherein the first domain comprises a nucleotide fragment obtained by PCR amplification of a TPS 1 gene from  S. cerevisiae  using a first primer having a sequence of SEQ ID NO: 20 and a second primer having a sequence of SEQ ID NO: 21.