CA2521497A1 - Modulation of cytokinin activity in plants - Google Patents

Modulation of cytokinin activity in plants Download PDF

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CA2521497A1
CA2521497A1 CA002521497A CA2521497A CA2521497A1 CA 2521497 A1 CA2521497 A1 CA 2521497A1 CA 002521497 A CA002521497 A CA 002521497A CA 2521497 A CA2521497 A CA 2521497A CA 2521497 A1 CA2521497 A1 CA 2521497A1
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cytokinin
promoter
plant
transgenic plant
expression cassette
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CA2521497C (en
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Jeffrey E. Habben
Chris Zinselmeier
Dwight T. Tomes
Shane E. Abbitt
Timothy G. Helentjaris
Xiaomu Niu
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Pioneer Hi Bred International Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract

This invention relates generally to the field of plant molecular biology. More specifically, this invention relates to methods and reagents for the temporally- and/or spatially-regulated expression of genes that affect metabolically effective levels of cytokinins in plants, particularly in plant seeds and related female reproductive tissue. This invention further relates to transgenic plants having enhanced levels of cytokinin expression wherein the transgenic plant exhibits useful characteristics, such as improved seed size, decreased tip kernel abortion, increased seed set during unfavorable environmental conditions, or stability of yield. The present invention also provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions comprise novel nucleotide sequences for seed-preferred promoters known as eep1 and eep2. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one of the promoters of the present invention and regenerating a stably transformed plant from the transformed plant cell.

Claims (65)

1. A method for producing transgenic plants capable of the regulated expression of a cytokinin-modulating gene in developing seed or related female reproductive tissue comprising:
transformation of plant host cells with a genetic construct capable of temporally- or spatially-regulated expression of a cytokinin modulating gene in developing seed or related female reproductive tissue; and regenerating and recovering said transgenic plants.
2. The method according to Claim 1 wherein the transformation is carried out by a process selected from the group consisting of electroporation, PEG
poration, particle bombardment, silicon fiber delivery, microinjection, and Agrobacterium-mediated transformation.
3. The method according to Claim 2 wherein said process of transformation is particle bombardment.
4. The method according to Claim 2 wherein said process of transformation is Agrobacterium-mediated transformation.
5. The method according to Claim 1 wherein said genetic construct comprises a promoter directing temporal or spatial gene expression in developing seed or related female reproductive tissue, operably linked to a cytokinin-modulating gene.
6. The method according to Claim 5 wherein said promoter is selected from the group consisting of zag2.1, zap, tb1, eep1, eep2, F3.7, thxH, Zm40, ESR, PCNA2, lec1, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4, and ZmCkx5.
7. The method according to Claim 1 wherein the cytokinin modulating gene is selected from the group consisting of genes encoding cytokinin biosynthetic enzymes, cytokinin catabolic enzymes, cytokinin catabolic enzyme antagonists and cytokinin biosynthetic enzyme agonists.
8. A transgenic plant comprising a genetic construct stably integrated into the genome thereof, said construct comprising a promoter operably linked to a cytokinin-modulating gene, wherein said promoter directs temporal or spatial expression in developing seed or related female reproductive tissues of said plant.
9. The plant according to Claim 8 wherein said promoter is selected from the group consisting of zag2.1, zap, tb1, eep1, eep2, F3.7, thxH, Zm40, ESR, PCNA2, lec1, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4, and ZmCkx5.
10. The plant according to Claim 9 wherein the cytokinin modulating gene is selected from the group consisting of genes encoding cytokinin biosynthetic enzymes, cytokinin catabolic enzymes, cytokinin catabolic enzyme antagonists and cytokinin biosynthetic enzyme agonists.
11. An isolated recombinant DNA comprising a genetic construct comprising a promoter directing temporal or spatial gene expression in developing plant seed or related female reproductive tissue operably linked to a cytokinin modulating gene.
12. Host cells having stably introduced therein the genetic construct of Claim 11.
13. A method for improving stress tolerance and yield stability in plants comprising:
transformation of plant host cells with a genetic construct driving preferential expression of cytokinin-modulating genes in developing seed and related female reproductive tissue;
and regenerating and recovering tranformed plants from said cells.
14. The method according to Claim 13 wherein said preferential expression occurs from about 14 days prior to about 25 days after pollination.
15. The method according to Claim 13 wherein said preferential expression occurs from about 0 to about 6 days after pollination.
16. The method according to Claim 13 wherein said preferential expression occurs from about 0 to about 12 days after pollination.
17. The method according to Claim 13 wherein said preferential expression occurs from about 4 to about 21 days after pollination.
18. A transgenic plant comprising a recombinant expression cassette stably integrated into the genome thereof, said cassette capable of effecting an increase in cytokinin activity, wherein said transgenic plant displays enhanced vigor without significant detrimental effects of increased cytokinin activity.
19. Seeds of the transgenic plant of Claim 18.
20. The transgenic plant of Claim 18, wherein said enhanced vigor is expressed in the presence or absence of abiotic stress.
21. A method of developing a maize plant utilizing the plant of Claim 18 as a source of genetic material in a breeding program.
22. The method of Claim 21 further comprising one or more techniques selected from the group consisting of: recurrent selection, mass selection, bulk selection, backcross, pedigree, development of a synthetic, and open pollination.
23. The transgenic plant of Claim 18, wherein said recombinant expression cassette comprises a polynucleotide encoding a protein involved in cytokinin biosynthesis.
24. The transgenic plant of Claim 18, wherein said recombinant expression cassette comprises a polynucleotide encoding isopentenyl transferase.
25. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the zag2.1 promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
26. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the eep1 promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
27. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the eep2 promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
28. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the zap promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
29. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the tb1 promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
30. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the ckx1-2 promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis.
31. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises a promoter which drives low-level constitutive expression of an operably-linked polynucleotide encoding a protein involved in cytokinin biosynthesis.
32. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises the F3.7 promoter.
33. The transgenic plant of Claim 23, wherein said recombinant expression cassette comprises (1) a reproductive-tissue-preferred promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis and (2) one or more promoters or enhancer elements of a highly-expressed gene.
34. The transgenic plant of Claim 33 wherein the enhancer element comprises the 35S enhancer of cauliflower mosaic virus.
35. The transgenic plant of Claim 34 wherein the 35S enhancer comprises SEQ
ID NO: 4.
36. The transgenic plant of Claim 33 wherein the recombinant expression cassette comprises (1) the zag2.1 promoter operably linked to a polynucleotide encoding ipt and (2) the cauliflower mosaic virus 35S
enhancer.
37. The transgenic plant of Claim 36 wherein the recombinant expression cassette comprises (1) SEQ ID NO: 3 operably linked to the coding region of SEQ ID NO: 1 and (2) SEQ ID NO: 4.
38. The transgenic plant of Claim 18, wherein said recombinant expression cassette comprises a first polynucleotide involved in silencing of a gene encoding a protein involved in decreasing the active cytokinin pool.
39. The transgenic plant of Claim 38, wherein said protein involved in decreasing the active cytokinin pool is cytokinin oxidase.
40. The transgenic plant of Claim 38, further comprising a promoter preferentially driving expression in developing seed or related female reproductive tissues, operably linked to a second polynucleotide encoding a protein involved in cytokinin biosynthesis.
41. The transgenic plant of Claim 38, wherein said first polynucleotide comprises an antisense sequence for a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
42. The transgenic plant of Claim 38, wherein said first polynucleotide comprises a sequence effective in cosuppression of a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
43. The transgenic plant of Claim 38, wherein said first polynucleotide comprises a sequence effective in RNAi of a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
44. A method of modulating cytokinin activity in a plant, wherein modulated cytokinin activity enhances plant vigor without significant detrimental effects, comprising stably transforming said plant to result in an increase in cytokinin activity.
45. The method of Claim 44 wherein said plant is stably transformed with a recombinant expression cassette capable of effecting an increase in cytokinin activity, wherein said transgenic plant displays enhanced vigor without significant detrimental effects of increased cytokinin activity.
46. The method of Claim 45, wherein said enhanced vigor is expressed in the presence or absence of abiotic stress.
47. The method of Claim 45, wherein said recombinant expression cassette comprises a polynucleotide encoding a protein involved in cytokinin biosynthesis.
48. The method of Claim 45, wherein said recombinant expression cassette comprises a polynucleotide encoding isopentenyl transferase.
49. The method of Claim 45, wherein said recombinant expression cassette comprises a promoter selected from the group consisting of zag2.1, zap, tb1, eep1, eep2, F3.7, thxH, Zm40, ESR, PCNA2, lec1, ZmCkx1-2, ZmCkx2, ZmCkx3, ZmCkx4, and ZmCkx5.
50. The method of Claim 45, wherein said recombinant expression cassette comprises (1) a female reproductive-tissue-preferred promoter operably linked to a polynucleotide encoding a protein involved in cytokinin biosynthesis and (2) one or more promoters or enhancer elements of a highly-expressed gene.
51. The method of Claim 50 wherein the enhancer element comprises the 35S
enhancer of cauliflower mosaic virus.
52. The method of Claim 51 wherein the 35S enhancer comprises SEQ ID NO: 4.
53. The method of Claim 50 wherein the recombinant expression cassette comprises (1) the zag2.1 promoter operably linked to a polynucleotide encoding ipt and (2) the cauliflower mosaic virus 35S enhancer.
54. The method of Claim 53 wherein the recombinant expression cassette comprises (1) SEQ ID NO: 3 operably linked to the coding region of SEQ ID
NO: 1 and (2) SEQ ID NO: 4.
55. The method of Claim 45, wherein said recombinant expression cassette comprises a first polynucleotide involved in silencing of a gene encoding a protein involved in decreasing the active cytokinin pool.
56. The method of Claim 55, wherein said protein involved in decreasing the active cytokinin pool is cytokinin oxidase.
57. The method of Claim 55, wherein said plant is further stably transformed with a promoter preferentially driving expression in developing seed or related female reproductive tissues, operably linked to a second polynucleotide encoding a protein involved in cytokinin biosynthesis.
58. The method of Claim 55, wherein said first polynucleotide comprises an antisense sequence for a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
59. The method of Claim 55, wherein said first polynucleotide comprises a sequence effective in cosuppression of a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
60. The method of Claim 55, wherein said first polynucleotide comprises a sequence effective in RNAi of a polynucleotide encoding a protein involved in decreasing the active cytokinin pool.
61. An isolated promoter capable of driving transcription in a seed-preferred manner, wherein the promoter comprises a nucleotide sequence selected from the group consisting of:
a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO: 7 or 18; and the nucleotide sequence set forth in SEQ ID NO: 7 or 18.
62. The isolated promoter of Claim 61 capable of driving transcription in a seed-preferred manner, wherein said promoter comprises a fragment of the nucleotide sequence set forth in SEQ ID NO: 7 or 18.
63. The isolated promoter of Claim 61 capable of driving transcription in a seed-preferred manner, wherein said promoter comprises the nucleotide sequence set forth in SEQ ID NO: 7 or 18.
64. A recombinant expression cassette comprising a promoter and a nucleotide sequence operably linked to the promoter, wherein the promoter comprises a nucleotide sequence selected from the group consisting of:
a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO: 7 or 18; and the nucleotide sequence set forth in SEQ ID NO: 7 or 18.
65. A plant stably transformed with an expression cassette comprising a maize promoter and a nucleotide sequence operably linked to the promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of:
a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO: 7 or 18; and the nucleotide sequence set forth in SEQ ID NO: 7 or 18.
CA2521497A 2003-04-04 2004-04-02 Modulation of cytokinin activity in plants Expired - Fee Related CA2521497C (en)

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US46071803P 2003-04-04 2003-04-04
US60/460,718 2003-04-04
PCT/US2004/010064 WO2004090143A2 (en) 2003-04-04 2004-04-02 Modulation of cytokinin activity in plants

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CN (3) CN101220358A (en)
AR (1) AR045420A1 (en)
AU (1) AU2004227360B2 (en)
BR (1) BRPI0409178A (en)
CA (1) CA2521497C (en)
NZ (1) NZ543070A (en)
WO (1) WO2004090143A2 (en)
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AU2004227360A1 (en) 2004-10-21
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