Brittle stalk 2 gene family and related methods and uses

Abstract

This invention relates to isolated polynucleotides encoding BRITTLE STALK 2-like (Bk2L) family polypeptides. The invention also relates to the construction of a chimeric gene encoding all or a portion of a Bk2L polypeptide, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the Bk2L polypeptide in a transformed host cell.

1 . An isolated polynucleotide comprising:
 (a) a nucleotide sequence encoding a polypeptide associated with stalk mechanical strength, wherein said polypeptide has an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, or any other integer in between 80% and 100%, based on the Clustal V method of alignment, when compared to SEQ ID NOs:16 or 18; or   (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         2 . A recombinant DNA construct comprising the polynucleotide of  claim 1  operably linked to a promoter that is functional in a plant. 
     
     
         3 . A method for altering stalk mechanical strength of a plant, comprising:
 (a) introducing into a regenerable plant cell the recombinant DNA construct of  claim 2  to produce a transformed plant cell; and   (b) regenerating a transgenic plant from said transformed plant cell, wherein said transgenic plant comprises in its genome said recombinant DNA construct and wherein said transgenic plant exhibits an alteration in stalk mechanical strength, when compared to a control plant not comprising said recombinant DNA construct.   
     
     
         4 . The method of  claim 3 , further comprising (c) obtaining a progeny plant derived from said transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct. 
     
     
         5 . The method of  claim 3 , wherein the transgenic plant exhibits an increase in stalk mechanical strength. 
     
     
         6 . A plant comprising in its genome the recombinant DNA construct of  claim 2 . 
     
     
         7 . The isolated polynucleotide of  claim 1 , wherein said stalk mechanical strength is measured by the three-point bend test. 
     
     
         8 . A method of evaluating stalk mechanical strength in a plant, comprising:
 (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i);   (b) regenerating a transgenic plant from said transformed plant cell; and   (c) evaluating said transgenic plant for stalk mechanical strength.   
     
     
         9 . The method of  claim 8 , further comprising:
 (d) obtaining a progeny plant derived from said transgenic plant; and   (e) evaluating said progeny plant for stalk mechanical strength.   
     
     
         10 . A method of evaluating stalk mechanical strength in a plant, comprising:
 (a) introducing into a regenerable plant cell a recombinant DNA construct to produce transformed plant cells, said recombinant DNA construct comprising a promoter that is functional in a plant operably linked to (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, and 18, or (b) a full-length complement of said polynucleotide of (a)(i);   (b) regenerating a transgenic plant from said transformed plant cell;   (c) obtaining a progeny plant derived from said transgenic plant; and   (d) evaluating said progeny plant for stalk mechanical strength.   
     
     
         11 . A plant comprising in its genome:
 (a) a first recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a first isolated polynucleotide selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and 
 (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and 
   (b) a second recombinant DNA construct comprising at least one promoter that is functional in a plant operably linked to at least one of a second isolated polynucleotide selected from the group consisting of:
 (iv) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; 
 (v) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and 
 (vi) a full-length complement of the polynucleotide of (b)(iv) or (b)(v) 
   and wherein said plant exhibits increased cell wall cellulose content or enhanced growth rate when compared to a control plant not comprising said first recombinant DNA construct and said second recombinant DNA construct.   
     
     
         12 . A plant comprising in its genome at least one regulatory sequence operably linked to:
 (a) at least one isolated polynucleotide selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and 
 (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and 
   (b) at least one isolated polynucleotide selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41; and 
 (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii), 
   and wherein said plant exhibits increased cell wall cellulose content or enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).   
     
     
         13 . The plant of  claim 12 , wherein:
 (a) said at least one polynucleotide is selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:2; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:1; and 
 (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and 
   (b) said at least one isolated polynucleotide is selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:38, 40, and 42; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:37, 39, and 41; and 
 (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii), 
   and wherein said plant exhibits increased cell wall cellulose content when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).   
     
     
         14 . The plant of  claim 12 , wherein:
 (a) said at least one polynucleotide is selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:5; and 
 (iii) a full-length complement of the polynucleotide of (a)(i) or (a)(ii); and 
   (b) said at least one isolated polynucleotide is selected from the group consisting of:
 (i) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:20, 32, and 34; 
 (ii) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:19, 31, and 33; and 
 (iii) a full-length complement of the polynucleotide of (b)(i) or (b)(ii), 
   and wherein said plant exhibits enhanced growth rate when compared to a control plant not comprising said at least one regulatory sequence operably linked to said (a) and (b).   
     
     
         15 . A plant comprising in its genome at least one regulatory sequence operably linked to at least two isolated polynucleotides selected from the group consisting of:
 (a) a polynucleotide encoding a polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, and 18;   (b) a polynucleotide having a nucleic acid sequence of at least 60% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17; and   (c) a full-length complement of the polynucleotide of (a) or (b).   
     
     
         16 . A plant comprising in its genome:
 a suppression DNA construct comprising a promoter functional in a plant operably linked to:
 (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or 
   (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a polypeptide selected from the group consisting of Bk2, Bk2L1, Bk2L3, Bk2L4, Bk2L5, Bk2L6, Bk2L7, Bk2L8 and Bk2L9,   and wherein said plant exhibits reduced stalk mechanical strength when compared to a control plant not comprising said suppression DNA construct.   
     
     
         17 . The plant of  claim 16 , wherein said suppression DNA construct comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct. 
     
     
         18 . A plant comprising in its genome:
 a suppression DNA construct comprising a promoter functional in a plant operably linked to:
 (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or 
 (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L3 polypeptide, 
   and wherein said plant exhibits reduced plant height and/or reduced organ size when compared to a control plant not comprising said suppression DNA construct.   
     
     
         19 . A plant comprising in its genome:
 a suppression DNA construct comprising a promoter functional in a plant operably linked to:
 (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:10, or (ii) a full-length complement of the nucleic acid sequence of (a)(i); or 
 (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a Bk2L5 polypeptide, 
   and wherein said plant exhibits male sterility when compared to a control plant not comprising said suppression DNA construct.