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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of motif occurrence in introns At-iEF1α (1), At-iANT1 (2), and At-iAct7 (3). Each shaded box with the indication “+1” represents one motif. Base numbers of the intron sequence are indicated on the bottom of the graph.

FIG. 2 is a schematic representation of modification of the intron At-iEF1α. A is the native intron with one copy of the motif SEQ ID NO:17 (indicated by a blank box with an arrow showing the direction of the motif sequence); B is a modified At-iEF1α with an extra copy of the motif inserted approx. 100 bp downstream from the original motif; C is a modified At-iEF1α la with two extra motifs inserted approximately 100 bp and 200 bp, respectively, downstream from the original motif, wherein all motifs are in the forward direction; D is a modified At-iEF1α with two extra motifs inserted approx. 100 bp and 200 bp, respectively, downstream from the original motif, wherein one inserted motif is in the reverse direction.

Claims

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; andiv) 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, andiv) 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, andiii) 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.