Regulatory sequences for modulating transgene expression in plants

Abstract

The invention relates to gene expression regulatory sequences, specifically introns that act as enhancers of gene expression, the promoter and terminator sequences endogenously associated with these introns. Presence of these intronic enhancer sequences in proximity to promoter sequences leads to enhancement of gene expression. Methods of finding such new intronic enhancer sequences and using them to generate transgenic plants are also described.

1 . A recombinant DNA construct comprising an intron operably linked to a promoter, a heteroloqous polynucleotide and a terminator wherein the intron comprises a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118, 137 or 138. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The recombinant DNA construct of  claim 1 , wherein
 the promoter comprises a nucleotide sequence that has at least 95% identity to SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 136 or 139.   
     
     
         5 . The recombinant DNA construct of  claim 4 , wherein
 the terminator comprises a nucleotide sequence that has at least 95% identity to SEQ ID NO: 140, 141, 142 or 143.   
     
     
         6 . The recombinant DNA construct of  claim 1 , wherein the intron comprises the nucleotide sequence of SEQ ID NO: 4, 8, 13, 19, 52, 53, 56, 57, 58, 101, 102, 103, 104, 118, 137 or 138. 
     
     
         7 . The recombinant DNA construct of  claim 4 , wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 136 or 139. 
     
     
         8 . The recombinant DNA construct of  claim 5 , wherein the terminator comprises the nucleotide sequence of SEQ ID NO: 140, 141, 142 or 143. 
     
     
         9 . (canceled) 
     
     
         10 . The recombinant DNA construct of  claim 1 , wherein the intron enhances expression of the heterologous polynucleotide in plants. 
     
     
         11 . A recombinant DNA construct comprising an intron operably linked to a promoter and a heterologous polynucleotide, wherein the intron comprises a nucleotide sequence that comprises at least one copy of SEQ ID NO: 99, and wherein the intron is capable of enhancing expression of a heterologous polynucleotide in a monocotyledonous plant when compared to a corresponding recombinant DNA construct without the intron. 
     
     
         12 . The recombinant DNA construct of  claim 11 , comprising the nucleotide sequence of the intron comprises at least one copy of SEQ ID NO: 100. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . A plant comprising the recombinant DNA construct of  claim 10 . 
     
     
         17 . A seed comprising the recombinant DNA construct of  claim 10 . 
     
     
         18 . A plant comprising the recombinant DNA construct of  claim 11 . 
     
     
         19 . A seed comprising the recombinant DNA construct of  claim 11 . 
     
     
         20 . A plant comprising the recombinant DNA construct of  claim 12 . 
     
     
         21 . A seed comprising the recombinant DNA construct of  claim 12 . 
     
     
         22 . (canceled) 
     
     
         23 . A method for identifying an intron useful for enhancing transgene expression in a monocotyledenous plant comprising the steps of:
 (a) scanning a plurality of monocot introns for the presence of a sequence motif identical to SEQ ID NO: 99;   (b) selecting an intron from step (a) comprising a nucleotide sequence that contains at least one copy of a sequence motif identical to SEQ ID NO: 99; and   (c) measuring the enhancing effect of the intron of step (b) on the expression of an operably linked heterologous polynucleotide.   
     
     
         24 . (canceled) 
     
     
         25 . A method for modulating transgene expression in a plant comprising the steps of:
 (a) introducing into a regenerable plant cell the recombinant DNA construct of  claim 1 ;   (b) regenerating a transgenic plant from the regenerable plant cell after step (a) wherein the transgenic plant comprises the recombinant DNA construct; and   (c) obtaining a progeny plant derived from the transgenic plant of step (b), wherein the progeny plant comprises the recombinant DNA construct and exhibits enhanced transgene expression when compared to a plant comprising in its genome the recombinant DNA construct without the corresponding intron sequence.   
     
     
         26 . The method of  claim 25  wherein said plant is a monocot.