Nucleic acids and proteins with thioredoxin reductase activity

Abstract

The present invention relates to the use of a variety of methods for generating functional thioredoxin reductase variants in which at least one physical, chemical or biological property of the variant is altered in a specific and desired manner when compared to the wild-type protein.

1 - 79 . (canceled)  
     
     
         80 . A plant or portion thereof comprising a nucleic acid sequence encoding a variant thioredoxin reductase protein, said variant thioredoxin reductase protein having the formula:  
         S1-A1-A2-S2-A3-A4-A5-S3-A6-S4  
       wherein 
 a) S1 comprises SEQ ID NO:6;  
 b) S2 comprises SEQ ID NO:13;  
 c) S3 comprises SEQ ID NO:20;  
 d) S4 comprises SEQ ID NO:27;  
 e) A1 is the amino acid serine;  
 f) A2 is the amino acid is the amino acid alanine;  
 g) A3 is the amino acid is the amino acid histidine;  
 h) A4 is an amino acid moiety selected from the group consisting of arginine and tryptophan;  
 i) A5 is an amino acid moiety selected from the group consisting of arginine, valine, leucine, isoleucine, methionine, phenylalanine, and tyrosine; and  
 j) A6 is an amino acid moiety selected from the group consisting of arginine, glycine, asparagine, glutamine, serine, and threonine, wherein only zero, one, or two of A4, A5 or A6 are arginine.  
 
     
     
         81 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid valine; and    j) A6 is the amino acid arginine.    
     
     
         82 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid methionine; and    j) A6 is the amino acid glycine.    
     
     
         83 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid isoleucine; and    j) A6 is the amino acid serine.    
     
     
         84 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid methionine; and    j) A6 is the amino acid serine.    
     
     
         85 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid leucine; and    j) A6 is the amino acid serine.    
     
     
         86 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid arginine; and    j) A6 is the amino acid threonine.    
     
     
         87 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid tryptophan;    i) A5 is the amino acid valine; and    j) A6 is the amino acid glycine.    
     
     
         88 . The plant or portion thereof of  claim 80 , wherein 
 a) S1 comprises SEQ ID NO:6;    b) S2 comprises SEQ ID NO:13;    c) S3 comprises SEQ ID NO:20;    d) S4 comprises SEQ ID NO:27;    e) A1 is the amino acid serine;    f) A2 is the amino acid alanine;    g) A3 is the amino acid histidine;    h) A4 is the amino acid arginine;    i) A5 is the amino acid tyrosine; and    j) A6 is the amino acid asparagine.    
     
     
         89 . The plant or portion thereof of  claim 80 , wherein said variant thioredoxin protein has altered cofactor specificity as compared to a naturally occurring thioredoxin reductase protein.  
     
     
         90 . The plant or portion thereof of  claim 89 , wherein said variant thioredoxin protein has altered cofactor specificity for a cofactor selected from the group consisting of NADPH and NADH.  
     
     
         91 . The plant or portion thereof of  claim 90 , wherein said variant thioredoxin protein has altered cofactor specificity for NADH.  
     
     
         92 . The plant or portion thereof of  claim 89 , wherein said variant thioredoxin protein preferentially binds NADPH compared to NADH.  
     
     
         93 . The plant or portion thereof of  claim 89 , wherein said variant thioredoxin protein exhibits improved catalytic efficiency for NADPH as compared to a naturally-occurring protein in an untransformed plant.  
     
     
         94 . The plant or portion thereof of  claim 80 , wherein the said portion of said plant comprises a seed.  
     
     
         95 . The plant or portion thereof of  claim 80 , wherein the plant or portion thereof consists of a seed.  
     
     
         96 . The plant or portion thereof of  claim 80 , wherein said nucleic acid sequence is a recombinant nucleic acid sequence.  
     
     
         97 . The plant or portion thereof of  claim 96 , wherein said nucleic acid sequence is part of a vector.  
     
     
         98 . The plant or portion thereof of  claim 96 , wherein said nucleic acid sequence is incorporated into the genome.  
     
     
         99 . The plant or portion thereof of  claim 80 , wherein the plant is a hybrid.  
     
     
         100 . A method of making the plant or portion thereof of  claim 80 , comprising: 
 a) introducing an expression cassette comprising a promoter functional in a plant cell operably liked to a DNA molecule encoding said variant thioredoxin reductase protein according to claim  1 , to produce one or more transformed plant cells; and    b) regenerating said transformed plan cells to provide a differentiated transformed plant, wherein expression of said DNA molecule encoding said variant thioredoxin reductase protein in said plant alters the co-factor specificity as compared to the untransformed plant.