Chimeric regulatory sequences comprising introns for plant gene expression

Abstract

The present invention relates to a method of using a dicot intron or elements thereof to enhance transgene expression in plants. The present invention also provides constructs, transgenic plants and seeds containing the polynucleotide useful for expressing transgene in plants.

1 . A method of enhancing transgene expression in a dicot plant comprising:
 i) operably linking a dicot intron to a monocot promoter to form a chimeric DNA regulatory sequence;   ii) assembling an expression construct comprising said chimeric regulatory sequence in a transgene;   iii) transforming said transgene into the genome of a dicot plant cell; and   iv) regenerating said cell into a fertile plant, wherein said chimeric regulatory sequence provides enhanced transgene expression when compared to that of a corresponding transgene comprising the same monocot promoter without the linked dicot intron.   
     
     
         2 . The method of  claim 1  wherein said enhanced transgene expression comprises broadening the spatial expression pattern of a transgene in dicot plants. 
     
     
         3 . The method of  claim 2  wherein said dicot intron is selected from the group consisting of the first introns of At-EF1α, At-ANT1, and At-Act7 and said monocot promoter is the rice TPI promoter. 
     
     
         4 . The method of  claim 3  wherein said chimeric regulatory sequence comprising a dicot intron and a monocot promoter enables transgene expression in male reproductive tissue of a dicot plant. 
     
     
         5 . The method of  claim 2  wherein said transgene comprises a nucleotide sequence encoding a glyphosate tolerance protein. 
     
     
         6 . The method of  claim 5  wherein said glyphosate tolerance protein is selected from the group consisting of glyphosate oxidoreductase, glyphosate acetyltransferase, and 5-enolpyruvyl-3-phosphoshikimate synthase. 
     
     
         7 . A chimeric DNA regulatory sequence comprising a monocot promoter operably linked to a dicot intron, wherein said chimeric DNA regulatory sequence provides enhanced transgene expression in dicot plants. 
     
     
         8 . The chimeric DNA regulatory sequence of  claim 7  wherein said dicot intron is selected from the group consisting of the first introns of At-EF1α, At-ANT1, and At-Act7 and said monocot promoter is the rice TPI promoter. 
     
     
         9 . A transgene comprising the chimeric DNA regulatory sequence of  claim 8 , further comprising a sequence encoding a glyphosate tolerance protein. 
     
     
         10 . The transgene of  claim 9 , wherein said glyphosate tolerance protein is selected from the group consisting of glyphosate oxidoreductase, glyphosate acetyltransferase, and 5-enolpyruvyl-3-phosphoshikimate synthase. 
     
     
         11 . A method for identifying novel regulatory molecules for enhancing transgene expression in plants comprising:
 i) operably linking a plurality of dicot introns to the promoter of a transgene to form a plurality of transgenes comprising a dicot intron;   ii) introducing each transgene comprising a dicot intron and a corresponding transgene without a dicot intron into plants;   iii) assessing the broadening effect on the spatial expression pattern of the transgene by each dicot intron, and   iv) identifying a motif unique to the dicot introns having similar broadening effect on the spatial expression pattern of the transgene.   
     
     
         12 . A method of regulating gene expression in plants comprising:
 i) constructing a recombinant enhancer comprising the identified motif of  claim 11 ;   ii) operably linking the recombinant enhancer of  claim 11  with a promoter in a transgene, and   iii) expressing said transgene in a plant wherein a broadened spatial expression pattern is obtained when compared to that of the same transgene lacking the recombinant enhancer.   
     
     
         13 . The method of  claim 11  wherein identification of the motif is performed by subjecting DNA sequence information of a plurality of dicot introns showing a desired broadening effect to searching with a MEME program or similar position-dependent scoring matrix programs for motif searching. 
     
     
         14 . The method of  claim 12  wherein said promoter is selected from the group consisting of dicot, monocot, and viral promoters. 
     
     
         15 . The method of  claim 12  wherein said recombinant enhancer is upstream of said promoter in the transgene. 
     
     
         16 . The method of  claim 15  wherein said recombinant enhancer is placed in the transgene in a direction selected from the group consisting of forward and reverse directions. 
     
     
         17 . The method of  claim 12  wherein said recombinant enhancer is downstream of said promoter in the transgene. 
     
     
         18 . The method of  claim 17  wherein said recombinant enhancer is placed in the transgene in a direction selected from the group consisting of forward and reverse directions. 
     
     
         19 . The method of  claim 12  wherein said identified motif is SEQ ID NO: 17, and wherein said motif confers transgene expression in male reproductive tissue in dicot plants. 
     
     
         20 . The method of  claim 19  wherein said transgene comprises a nucleotide sequence encoding a glyphosate tolerance protein. 
     
     
         21 . The method of  claim 20  wherein said glyphosate tolerance protein is selected from the group consisting of glyphosate oxidoreductase, glyphosate acetyltransferase, and 5-enolpyruvyl-3-phosphoshikimate synthase. 
     
     
         22 . A recombinant enhancer comprising a sequence motif (SEQ ID NO: 17), wherein said recombinant enhancer is capable of enhancing transgene expression in male reproductive tissue of plants. 
     
     
         23 . The recombinant enhancer of  claim 22  wherein said recombinant enhancer contains two copies of said motif. 
     
     
         24 . The recombinant enhancer of  claim 22  wherein said recombinant enhancer contains three copies of said motif. 
     
     
         25 . The recombinant enhancer of  claim 22  wherein said recombinant enhancer contains four copies of said motif. 
     
     
         26 . The recombinant enhancer of  claim 22  wherein the distance between two adjacent motifs is between 0-1000 base pairs. 
     
     
         27 . The recombinant enhancer of  claim 26  wherein the distance between two adjacent motifs is between 10-250 base pairs. 
     
     
         28 . The recombinant enhancer of  claim 22  wherein said recombinant enhancer further comprises at least two elements selected from the group consisting of motifs Fac109 (SEQ ID NO:18), Fac029 (SEQ ID NO:19), and PHO2 (SEQ ID NO:20). 
     
     
         29 . A chimeric regulatory sequence comprising the recombinant enhancer of  claim 22  operably linked to a promoter selected from the group consisting of monocot, dicot, and viral promoters. 
     
     
         30 . A recombinant DNA construct comprising the chimeric regulatory sequence of  claim 29 . 
     
     
         31 . A transgenic plant stably transformed with the recombinant DNA construct of  claim 30 . 
     
     
         32 . A seed, flower, root, shoot, pollen, stem, or progeny of the transgenic plant of  claim 31 .