Novel polypeptides and polynucleotides relating to the alpha- and beta-subunits of glutamate dehydrogenases and methods of use

Abstract

Amino acid and nucleotide sequences relating to the glutamate dehydrogenase (GDH) enzyme are described. The GDH enzymes described herein were discovered in the alga Chlorella sorokiniana in the form of seven different inducible isoenzymes. These isoenzymes are found in the algae as chloroplast-localized hexamers composed of alpha- and beta-subunits. Plants transformed with nucleotide sequences encoding the alpha- or beta-subunits of the enzyme show improved properties, for example, increased growth and improved stress tolerance. A heterohexamer having both α- and β-subunits can have higher aminating:deaminating activity ratio than α-homohexamers or β-homohexamers.

Claims:  
     
         1 . A recombinant DNA molecule comprising a constitutive promoter operably linked to a sequence encoding a polypeptide, said polypeptide comprising a chloroplast transit peptide operably linked to a NADP-GDH polypeptide, said sequence encoding a polypeptide being operably linked to a transcriptional termination region.  
     
     
         2 . The recombinant DNA of    claim 1   , wherein said NADP-GDH polypeptide is a bacterial polypeptide.  
     
     
         3 . A transgenic plant comprising an expression cassette having a transcription initiation region functional in a plant cell of said transgenic plant, and a genetically engineered DNA sequence that encodes a NADP-GDH enzyme in said plant cell; whereby said transgenic plant evidences detectable increases in said NADP-GDH enzyme activity when compared to non-transgenic plants, which increases the transgenic plants yield relative to that of non-transgenic plants.  
     
     
         4 . The transgenic plant of    claim 3   , wherein said NADP-GDH enzyme is a bacterial enzyme.  
     
     
         5 . A transgenic plant according to    claim 4   , wherein said plant is a dicot.  
     
     
         6 . A transgenic plant according to    claim 4   , wherein said plant is a monocot.  
     
     
         7 . A transgenic plant according to    claim 6   , wherein said plant is  Zea mays.    
     
     
         8 . Transgenic plant cells comprising an expression cassette having a transcription initiation region functional in said transformed plant cells; a DNA sequence that encodes an NADP-GDH enzyme in said transformed plant cells; and a transcription termination region functional in said transformed plant cells, wherein said expression cassette imparts increased yield to a transformed plant resulting from the transformed plant cells relative to wild-type plants resulting from wild-type plant cells.  
     
     
         9 . Cells according to    claim 8   , wherein at least one of said transcription region and said termination region is not naturally associated with said sequence.  
     
     
         10 . Cells according to    claim 8   , wherein said NADP-GDH enzyme is a bacterial enzyme.  
     
     
         11 . Cells according to    claim 10   , wherein said DNA sequence is modified to enhance expression in plant cells.  
     
     
         12 . Cells according to    claim 10   , further comprising a chloroplast transit peptide adapted to target the chloroplasts.  
     
     
         13 . Cells according to    claim 10   , wherein said transcription initiation region is constitutive in action.  
     
     
         13 . Cells according to    claim 10   , wherein said transcription initiation region is tissue specific.  
     
     
         14 . Cells according to    claim 13   , wherein said tissue specific initiation region is seed specific.