Functional expression of shuffled yeast nitrate transporter (ynt1) in maize to improve nitrate uptake under low nitrate environment

Abstract

The present invention provides methods and compositions relating to altering NT activity, nitrogen utilization efficiency and/or uptake in plants. The invention relates to a method for the production of plants with maintained or increased yield under low nitrogen fertility. The invention provides isolated nitrate transporter variant (NT variant) nucleic acids and their encoded proteins. The invention further provides recombinant expression cassettes, host cells, and transgenic plants. Plants transformed with nucleotide sequences encoding the NT variant enzyme show improved properties, for example, increased yield.

That which is claimed: 
     
         1 . An isolated or recombinant nucleic acid comprising a nitrate transporter (NT) variant polynucleotide or nitrate reductase (NR) polynucleotide;
 wherein said NT variant polynucleotide comprises a member selected from the group consisting of,   (a) an NT variant polynucleotide comprising a nucleotide that has been substituted, wherein the nucleotide substitution is one or more of the substitutions shown in Table 3, and wherein the polynucleotide encodes a polypeptide having NT activity,   (b) an NT variant polynucleotide encoding a polypeptide comprising an amino acid sequence that has been substituted, wherein the amino acid substitution is one or more of the substitutions shown in Table 2, and wherein the polypeptide has NT activity,   (c) a polynucleotide that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, 6, 8 or 10,   (d) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, 5, 7, 9 or 11,   (e) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under stringent conditions to the complement of at least one nucleotide sequence selected from the group consisting of (a), (b), (c) and (d), wherein the conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C., and wherein the polynucleotide encodes a polypeptide having NT activity,   (f) a polynucleotide having at least 80% sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9 and 11; wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters, and wherein the polynucleotide encodes a polypeptide having NT activity,   (g) a polynucleotide degenerate from any one of (a) to (f) as a result of the genetic code, and   (h) a polynucleotide complementary to a polynucleotide of any one of (a) to (g); and   wherein said NR polynucleotide comprises a member selected from the group consisting of,   (i) a polynucleotide that encodes the polypeptide of SEQ ID NO: 14 or 17,   j) a polynucleotide comprising the sequence set forth in SEQ ID NO: 12 or 16,   (k) a polynucleotide comprising at least 30 nucleotides in length which hybridizes under stringent conditions to the complement of at least one nucleotide sequence selected from the group consisting of (i) and (j), wherein the conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C., wherein the polynucleotide encodes a polypeptide having NR activity and comprising the A561G substitution, the S561 D substitution, or the A561 and S561 D substitutions,   (l) a polynucleotide having at least 70% sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 12 and 16, wherein the % sequence identity is based on the entire encoding region and is determined by BLAST 2.0 under default parameters, and wherein the polynucleotide encodes a polypeptide having NR activity and comprising the A561G substitution, the 5561D substitution, or the A561 and S561 D substitutions,   (m) an isolated polynucleotide degenerate from any one of (i) to (l) as a result of the genetic code,   (n) a polynucleotide complementary to a polynucleotide of any one of (i) to (m).   
     
     
         2 . The isolated or recombinant polynucleotide of  claim 1 , wherein said NT variant polynucleotide encodes an NT variant polypeptide that increases a plant's yield relative to the yield of a control plant that does not contain the said NT variant polynucleotide or wherein NR polynucleotide encodes an NR polypeptide that confers increased yield or nitrogen utilization efficiency under lower fertility relative to the yield or nitrogen utilization efficiency of a control plant that contain the polynucleotide encoding the NR. 
     
     
         3 . The isolated or recombinant polynucleotide of  claim 1  that encodes an NT variant polypeptide that has maintained or increased NT activity as compared to the activity of a wild type NT. 
     
     
         4 . The isolated or recombinant polynucleotide of  claim 1  that encodes an NT variant polypeptide that has NT activity that is at least 2-fold greater than the NT activity of the polypeptide of: SEQ ID NO: 2. 
     
     
         5 . The isolated or recombinant polynucleotide of  claim 4 , wherein NT activity is nitrate uptake. 
     
     
         6 . The isolated or recombinant polynucleotide of  claim 1  that encodes an NT variant polypeptide that confers to a plant increased nitrogen utilization efficiency (NUE) under lower fertility. 
     
     
         7 . A vector comprising at least one polynucleotide, or an expression cassette comprising at least one polynucleotide operably linked to a promoter; wherein the polynucleotide is the polynucleotide of  claim 1 . 
     
     
         8 . A host cell comprising at least one expression cassette of  claim 7 . 
     
     
         9 . The host cell of  claim 8 , wherein the host cell is a monocotyledonous plant cell or dicotyledonous plant cell. 
     
     
         10 . A transgenic plant comprising stably incorporated in its genome at least one expression cassette of  claim 7 . 
     
     
         11 . The transgenic plant of  claim 10 , wherein the plant is selected from the group consisting of:  Arabidopsis , maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, peanut, sugarcane and cocoa. 
     
     
         12 . A seed from the transgenic plant of  claim 10 , wherein said seed comprises said expression cassette. 
     
     
         13 . An isolated or recombinant polypeptide selected from the group consisting of:
 (a) an NT variant polypeptide comprising an amino acid sequence that has been substituted, wherein the amino acid substitution is one or more of the substitutions shown in Table 2, and wherein the NT variant polypeptide has NT activity;   (b) an NT variant polypeptide having NT activity, wherein said polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 3, 5, 7, 9 or 11, and wherein the NT variant polypeptide has NT activity;   (c) an NT variant polypeptide having NT activity and at least 80% sequence identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 4, 6, 8 and 10;   (d) a polypeptide comprising SEQ ID NO: 14 or 17;   (e) a polypeptide encoded by the polynucleotide of SEQ ID NO: 12 or 16;   (f) a polypeptide that is at least 70% identical to the amino acid sequence at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 14 and 17, wherein said polypeptide has NR activity and comprises the A561G substitution, the S561 D substitution, or the A561 and S561 D substitutions;   (g) a polypeptide that is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 70% identical to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 12 and 16, wherein said polypeptide has NR activity and comprises the A561G substitution, the 5561D substitution, or the A561 and S561 D substitutions;   (h) a polypeptide that is encoded by a nucleic acid molecule that hybridizes with a nucleic acid probe consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12 and 16, or a complement thereof following at least one wash in 0.2×SSC at 55° C. for 20 minutes, wherein said polypeptide has NR activity and comprises the A561G substitution, the 5561D substitution, or the A561 and S561 D substitutions; and   (i) a fragment comprising at least 200 consecutive amino acids of SEQ ID NO: 14 or 17, wherein said polypeptide has NR activity and wherein said fragment comprises the A561 G substitution, the S561 D substitution, or the A561 and S561 D substitutions.