Isolated DNA molecule for enhancing gene expression of a coding sequence, fragment, genetic variant, cassette, vector, cell, plant and seed containing said molecule

Abstract

An isolated DNA molecule for enhancing gene expression of a coding sequence, fragment, genetic variant, cassette, vector, cell, plant and seed containing said molecule, wherein the molecule comprises introns for induce an increase in the expression level of a transgene, useful in DNA constructions for transforming plant cells, wherein the cell or plant has a construction containing one of these introns under control of a promoter and upstream of a coding sequence stably integrated in its genome displaying higher expression levels as compared to non-transformed cells or plants, or cells or plants transformed with constructions that lack the inventive intron, wherein the sequences of 5′-non-coding sequences of the genes mentioned above also comprise promoters and exonic sequences in DNA constructions displaying synergism with the effect produced by the intron, and wherein the 5′-non-coding sequences of COX5-c genes lacking said intron promote tissue-specific expression in pollen when they are comprised in DNA constructions for plant transformation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example in the following drawings wherein:

FIG. 1 shows COX5c genes schemes (A, upper panel) and (B, lower panel), which contain an intron within the 5′-non-coding region.

(A) Scheme of the six sequenced COX5c genes available in data banks: three genes from Arabidopsis thaliana (AtCOX5c) and one each from Ipomoea batatas (IbCOX5c), Oryza sativa (OsCOX5c), and Lotus corniculatus (LcCOX5c). All genes contain a single intron (white boxes) located in the 5′-non-coding region. The OsCOX5c intron is not drawn to scale due to its length (2.4 kbp). Non-coding and coding regions of exons are shown in light and dark grey, respectively.

(B) Scheme of the different constructs used to analyse COX5c regions required for expression. Different regions of the COX5c-1 and COX5c-2 genes, containing 5′non coding region (black boxes), non coding regions of exons 1 and 2 located upstream of the start codon (light grey boxes), and the leader intron (white box) were fused to the gus coding region and introduced into plants. In a similar way, COX5c-2 untranslated leader sequences were fused to the COX5b-1 promoter (striped boxes) in both orientations.

FIG. 2 shows histochemical localization of GUS activity in Arabidopsis plants transformed with constructs carrying different portions of the COX5c-2 gene fused to gus. Plants transformed with fragments containing non-transcribed upstream regions plus either the complete transcribed 5′-non-coding region (A-J), only exon 1 sequences (K-O), or the 5′-non-coding region without the intron (P-T) were analysed. Two-(A), 3-(B, K, P), 15-(D, F), and 20-d-old plants (C, E) are shown. Leaves (G, L, Q, R), flowers (H, I, M, N, S), anthers (T), and siliques (J, O) from adult plants are also shown.

FIG. 3 shows bar-graphic illustrating that removal of the COX5c intron produces a pronounced decrease in expression of the reporter gene. GUS-specific activity of extracts from plants transformed with construct pBI5c2 (5c2+I) or the respective constructs in which either the intron plus a portion of exon 2 (5c2-I/E2) or only the intron (5c2-I) were removed was measured using the fluorogenic substrate MUG and protein extracts prepared from rosettes of 20-d-old plants. GUS activity was also measured in extracts from non-transformed plants (wt) or from plants carrying the promoterless gus gene (pBI101) or the gus gene under the control of the CaMV 35S promoter (pBI121). Error bars represent SE of three independent measurements with one representative line from each construct. Similar results were obtained with different independent lines. The inset shows an enlargement of the bars corresponding to plants that display low GUS activity values.

FIG. 4 shows histochemical localization of GUS activity in Arabidopsis plants transformed with the COX5b-1 promoter fused to the COX5c-2 leader intron. (A, B) Three- and 15-d-old plants transformed with a 609 bp COX5b-1 promoter fragment. (C-F) Similar plants transformed with the same promoter fragment fused to the COX5c-2 leader intron in the sense (D, E) or antisense (C, F) orientation. (G-L) Leaves from adult plants carrying the promoter alone (G, J) or with the intron in the sense (H, K) or the antisense (I, L) orientation. (M-P) Flowers and siliques from plants transformed with the COX5b-1 promoter fragment without (M, N) or with the COX5c-2 intron (O, P).

FIG. 5 shows a graph illustrating that the COX5c-2 leader intron increases expression from the unrelated COX5b-1 promoter. Specific GUS activity in extracts from plants transformed with a 609 bp COX5b-1 promoter fragment fused to gus (5b) or with a similar construct in which the COX5c-2 leader intron was inserted between the promoter and the gus coding region either in the sense (5b+Is) or antisense (5b+Ias) orientation was measured using the fluorogenic substrate MUG. GUS activity was also measured in extracts from non-transformed plants (wt) or from plants carrying the promoterless gus gene (pBI101) or the gus gene under the control of the CaMV 35S promoter (pBI121). Error bars represent SE of three independent measurements with one representative line from each construct. Similar results were obtained with different independent lines.

FIG. 6 shows a northern blot analysis of gus steady-state transcript levels using total RNA from plants transformed with constructs containing different portions of the COX5c-2 gene fused to gus. Total RNA (20 μg) from plants transformed with fragments containing COX5c-2 non-transcribed upstream regions and either the complete transcribed 5′-non-coding region (lane 1), only exon 1 sequences (lane 2), or the 5′-non-coding region without the intron (lane 3). Lanes 4, 5, and 6 contain RNA from plants transformed with a 609 bp COX5b-1 promoter fragment or the same fragment fused to the COX5c-2 leader intron in the antisense or sense orientation, respectively. Plants transformed with the promoterless gus gene (lane 7), non-transformed plants (lane 8), and plants transformed with the gus gene under the control of the CaMV 35S promoter (lane 9) were also analysed.

FIG. 7 shows DNA sequences isolated in the present invention:

SEQ N^o 1: COX5c-1 promoter;

SEQ N^o 2: COX5c-1 exon 1;

SEQ N^o 3 COX5c-1 intron;

SEQ N^o 4: COX5c-1 exon 2

FIG. 8 shows DNA sequences isolated in the present invention:

SEQ N^o 5: COX5c-2 promoter;

SEQ N^o 6: COX5c-2 exon 1;

SEQ N^o 7: COX5c-2 intron;

SEQ N^o 8: COX5c-2 exon 2

FIG. 9 shows DNA sequences isolated in the present invention:

SEQ N^o 9: COX5c-3 promoter;

SEQ N^o 10: COX5c-3 exon 2;

SEQ N^o 11: COX5c-3 intron;

SEQ N^o 12: COX5c-3 exon 2

Claims

1. An isolated DNA molecule comprising at least one of the following introns: COX5c-1 gene intron, SEQ No 3;COX5c-2 gene intron, SEQ No 7;COX5c-3 gene intron, SEQ No 11, or a portion thereof.

2. The molecule of claim 1, wherein intron sequence SEQ ID No 3 goes from nucleotides 22 to 698 from the start codon of COX5c-1;intron sequence SEQ ID No 7 goes from nucleotides 12 to 591 from the start codon of COX5c-2;intron sequence SEQ ID No 11 goes from nucleotides 25 to 1058 from the start codon of COX5c-3;and wherein said portion comprises a fragment, genetic variant or deletion of at least one of said sequences that conserve the capability of enhancing gene expression in plant cells when it is located in a construction under control of a non-related promoter and upstream of at least one coding sequence.

3. The fragment, genetic variant or deletion of the DNA molecule of claim 2, wherein the molecule includes at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 22 and 698 when the sequence is SEQ ID No 3; at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 12 and 591 when the sequence is SEQ ID No 7; and at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 25 and 1058 when the sequence is SEQ ID No 11.

4. An expression cassette for promoting high expression levels of at least one coding sequence, wherein the cassette comprises at least one of the introns of claim 1 associated with a coding sequence and regulatory elements of gene expression.

5. A plant expression vector including the expression cassette of claim 4.

6. A plant expression vector of claim 5, wherein the effect of said expression cassette is produced in the whole plant.

7. A cell comprising the vector of claim 5 and any descendant of the cell.

8. The cell of claim 7, wherein the cell is a bacterium.

9. The cell of claim 7, wherein the cell is a plant cell.

10. The cell of claim 9 expressing a coding sequence under control of a promoter contained in the expression cassette.

11. The cell of claim 9 comprising the expression cassette, stably integrated in its genome.

12. The cell of claim 9 comprising the vector, stably integrated in its genome.

13. A transgenic plant and any seed and descendant thereof, wherein the plant is regenerated from the plant cell of claim 11.

14. The transgenic plant of claim 13 expressing in at least one of its cells a coding sequence under control of the expression cassette.

15. The plant of claim 13, wherein the plant is a monocot plant.

16. The plant of claim 13, wherein the plant is a dicot plant.

17. An isolated DNA molecule comprising at least one of the sequences: COX5C-1 promoter-exon 1-intron-exon 2, SEQ No 1-2-3-4;COX5C-2 promoter-exon 1-intron-exon 2, SEQ No 5-6-7-8;COX5C-3 promoter-exon 1-intron-exon 2, SEQ No 9-10-11-12, or a portion thereof.

18. The DNA molecule of claim 17, wherein: the COX5C-1 promoter-exon 1-intron-exon 2, SEQ No 1-2-3-4; is from nucleotides 1 to 1211 of Cox5C-1,the COX5C-2 promoter-exon 1-intron-exon 2, SEQ No 5-6-7-8 is from nucleotides 1 to 1153 from Start Codon upstream to non coding region of Cox5C-2, and the COX5C-3 promoter-exon 1-intron-exon 2, SEQ No 9-10-11-12 is from nucleotides 1 to 2205 of Cox5C-3,and wherein said portion comprises a fragment, genetic variant or deletion of at least one of said sequences that conserve the capability of enhancing gene expression of at least one coding sequence of a gene of interest.

19. The fragment, genetic, variant or deletion of the DNA molecule of claim 18, wherein the molecule includes at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 1211 when the sequence is SEQ ID No 1-2-3-4; at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 1153 when the sequence is SEQ ID No 5-6-7-8; and at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 2205 when the sequence is SEQ ID No 9-10-11-12.

20. An expression cassette comprising the DNA molecule of claim 17 associated with a coding sequence.

21. A plant expression vector comprising the cassette of claim 20.

22. The plant expression vector of claim 21, wherein the effect of said expression cassette is produced in the whole plant.

23. A cell and any descendant thereof, transformed with the vector of claim 21.

24. The cell of claim 23, wherein the cell is a bacterium.

25. The cell of claim 23, wherein the cell is a plant cell.

26. The plant cell of claim 25 expressing a coding sequence under control of a promoter comprised in the expression cassette.

27. The plant cell of claim 25, wherein the cell has the cassette stably integrated in its genome.

28. A transgenic plant and any seed and descendant thereof, wherein the plant is regenerated from the plant cell of claim 27.

29. The transgenic plant of claim 28 expressing in at least one of its cells a coding sequence under control of the expression cassette.

30. The plant of claim 29, wherein the plant is a monocot plant.

31. The plant of claim 29, wherein the plant is a dicot plant.

32. An isolated DNA molecule comprising the introns of claim 1 in at least one of the following sequences: COX5c-1 gene exon 1-intron-exon 2, SEQ No 2-3-4;COX5c-2 gene exon 1-intron-exon 2, SEQ No 6-7-8;COX5c-3 gene exon 1-intron-exon 2, SEQ No 10-11-12, or a portion thereof.

33. The isolated DNA molecule of claim 32, wherein: the COX5c-1 gene exon 1-intron-exon 2, SEQ No 2-3-4 is from nucleotides 1 to 741 from the start codon of Cox5C-1,the COX5c-2 gene exon 1-intron-exon 2, SEQ No 6-7-8 is from nucleotides 1 to 640 from the start codon of Cox5C-2,the COX5c-3 gene exon 1-intron-exon 2, SEQ No 10-11-12 is from nucleotides 1 to 1123 from the start codon of Cox5C-3,and wherein said portion comprises, a fragment, genetic variant or deletion of at least one of said sequences keeping the capability of enhancing gene expression in plant cells when it is located in a construction under control of a non-related promoter and upstream of at least one coding sequence of interest.

34. A fragment, genetic variant or deletion of the DNA molecule of claim 33, wherein the molecule includes at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 741 when the sequence is SEQ ID No 2-3-4; at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 640 when the sequence is SEQ ID No 6-7-8; and at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence between nucleotides 1 and 1123 when the sequence is SEQ ID No 10-11-12.

35. An expression cassette for enhancing the expression levels of at least one coding sequence of a gene of interest, wherein the cassette comprises at least one of the sequences exon 1-intron-exon 2 of claim 32 associated to a coding sequence and to non-related regulatory elements of gene expression.

36. A plant expression vector comprising the cassette of claim 35.

37. The plant expression vector of claim 36, wherein the effect of said expression cassette is produced in the whole plant.

38. A cell transformed with the cassette of claim 35, and any descendant thereof.

39. The cell of claim 38, wherein the cell is a bacterium.

40. The cell of claim 38, wherein the cell is a plant cell.

41. The plant cell of claim 40 expressing a coding sequence under control of a non-related promoter comprised in the expression cassette.

42. The cell of claim 38 comprising the expression cassette stably integrated in its genome.

43. The cell of claim 38 comprising the vector stably integrated in its genome.

44. A transgenic plant and any seed and descendant thereof, regenerated from the plant cell of claim 40.

45. The transgenic plant of claim 44 expressing in at least one of its cells a coding sequence under control of the expression cassette.

46. The plant of claim 45, wherein the plant is a monocot plant.

47. The plant of claim 45, wherein the plant is a dicot plant.

48. An isolated DNA molecule comprising at least one of the following sequences: COX5c-1 promoter-exon 1-exon 2, SEQ No:1-2-4;COX5c-2 promoter-exon 1-exon 2, SEQ No:5-6-8; andCOX5c-3 promoter-exon 1-exon 2, SEQ No:9-10-12, or a portion thereof

49. The molecule of claim 48, wherein: the COX5c-1 promoter-exon 1-exon 2 sequence, SEQ No:1-2-4, is from base 1 to 21 upstream of the start codon of COX5c-1 linked to the sequence from base 699 to 1211 downstream of the start codon of COX5c-1;the COX5c-2 promoter-exon 1-exon 2 sequence, SEQ No:5-6-8, is from base 1 to 11 upstream of the start codon of COX5c-2 linked to the sequence from base 592 to 1153 of the start codon of COX5c-2;the COX5c-3 promoter-exon 1-exon 2 sequence, SEQ No:9-10-12, is from base 1 to 24 upstream of the start codon of COX5c-3 linked to the sequence from base 1059 to 2205 upstream of the start codon of COX5c-3;and wherein said portion comprises a fragment, genetic variant or deletion of at least one of said sequences keeping the capability of enhancing tissue-specific gene expression in plant cells when some of these sequences is located upstream of at least one heterologous coding sequence of a gene of interest.

50. A fragment, genetic variant or deletion of the DNA molecule of claim 49, wherein the molecule includes at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence SEQ ID No 1-2-4; at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence SEQ ID No 5-6-8; and at least 100 consecutive bases with a homology of 80% with 100 consecutive bases of the sequence SEQ ID No 9-10-12.

51. An expression cassette for enhancing the expression levels of at least one coding sequence of a gene of interest, wherein the cassette comprises at least one of the sequences promoter-exon 1-exon 2 of claim 48 associated to a coding sequence.

52. A plant expression vector comprising the cassette of claim 51.

53. The plant expression vector of claim 52, wherein the effect produced by said expression cassette is tissue-specific.

54. The vector of claim 53, wherein the specific tissue is pollen.

55. A cell transformed with the cassette of claim 51, and any descendant thereof.

56. The cell of claim 55, wherein the cell is a bacterium.

57. The cell of claim 55, wherein the cell is a plant cell.

58. The plant cell of claim 57 expressing a coding sequence under control of at least one of the sequences promoter-exon 1-exon 2 comprised in the expression cassette.

59. The cell of claim 55, comprising the expression cassette stably integrated in its genome.

60. The cell of claim 55, comprising the vector stably integrated in its genome.

61. A transgenic plant and any seed and descendant thereof, regenerated from the plant cell of claim 57.

62. The transgenic plant of claim 61 expressing in at least one of its cells a coding sequence under control of at least one of the sequences promoter-exon 1-exon 2 and comprising the expression cassette.

63. The plant of claim 62, wherein the plant is a monocot plant.

64. The plant of claim 62, wherein the plant is a dicot plant.

65. The isolated DNA molecule of claim 1, wherein it is from Arabidopsis thaliana.

66. The isolated DNA molecule of claim 17, wherein it is from Arabidopsis thaliana.

67. The isolated DNA molecule of claim 33, wherein it is from Arabidopsis thaliana.

68. The isolated DNA molecule of claim 49, wherein it is from Arabidopsis thaliana.

69. A DNA molecule capable of hybridizing in astringent conditions with at least one of the sequences of claim 2.

70. A DNA molecule capable of hybridizing in astringent conditions with at least one of the sequences of claim 18.

71. A DNA molecule capable of hybridizing in astringent conditions with at least one of the sequences of claim 33.

72. A DNA molecule capable of hybridizing in astringent conditions with at least one of the sequences of claim 49.