DNA constructs that contain Helianthus annuus Hahb-10 gene coding sequence, method for generating plants with a shortened life cycle and a high tolerance to herbicidal compounds and transgenic plants with that sequence

Abstract

The present invention refers to a gene from Helianthus annuus encoding a transcription factor that comprises a homeodomain associated with a leucine zipper. This gene is named Hahb-10. The transcription factor Hahb-10 can be used in DNA constructs to transform host cells and plants. Transgenic plants that overexpress this transcription factor are more tolerant to herbicides, and have a shorter life cycle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

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

FIG. 1 shows that Hahb-10 is mainly expressed in mature leaves.

To perform expression analysis, we have used a specific probe that contains the 5′ portion of Hahb-10. Northern blot analysis using this probe with total RNA extracted from different sunflower organs showed a high expression level in 30-day-old leaves and lower levels in seedlings, stems and cotyledons (FIG. 1). Lower but detectable levels of the transcript were observed in roots, carpels and fertile flowers. Quantitation of the signals indicated that expression in mature leaves is 4 to 5-fold the level of that found in seedlings. The results indicate that this transcription factor may have a function during vegetative/reproductive developmental states in photosynthetic tissues.

Total RNA samples (10 μg each) extracted from 4-day-old seedlings (1), 14-day-old stems (2), 14-day-old leaves (3), or 30-day-old leaves (4), carpels (5) or fertile flowers (6) were analyzed by electrophoresis, transferred onto nylon membranes and hybridized with a ³²P-labeled Hahb-10 cDNA specific probe (upper panel). The same filter was hybridized with an rRNA probe as a control for RNA loading and transfer (lower panel). Spots obtained with the specific probe were quantified in reference to their rRNA using Image Pro Plus software. The graphic in the lower panel shows Hahb-10 transcript levels relative to the level in seedlings.

FIGS. 2A-2B show that Hahb-10 expression is strongly induced in etiolated seedlings.

2A: Total RNA samples (20 μg each) extracted from 4-day-old seedlings subjected to different treatments as described later were hybridized with a ³²P-labeled Hahb-10 cDNA specific probe (A, upper panel). The same filter was hybridized with an rRNA probe as a control for RNA loading and transfer. Spots obtained with the specific probe were quantified in reference to their rRNA using Image Pro Plus software. The graphic shown in the lower panel shows Hahb-10 transcript levels relative to the level measured in heat shock treated seedlings. Quantitation of the signals has been done taking as standard value the signal obtained in these seedlings because the signal obtained in control seedlings was almost not detectable by the software used for this purpose.2B: Total RNA samples (20 μg each) extracted from 7-day-old seedlings grown in normal illumination conditions (lane 1), in the dark (lane 2), or under continuous illumination at 40 cm from the light source applied to 4-day-old seedlings germinated in the dark during 3 additional days (lane 3) were analyzed by electrophoresis, transferred onto nylon membranes and hybridized with a ³²P-labelled Hahb-10 cDNA specific probe (upper panel). The same filter was hybridized with an rRNA probe as a control for RNA loading and transfer (lower panel).

FIG. 3 shows the expression of sunflower Hahb-10 gene in Arabidopsis transgenic plants.

Northern blot analysis of transgenic Arabidopsis plants. Total RNA (10 μg) was extracted from wild-type (WT) and three independent transgenic plants (TG-A, B and C) overexpressing Hahb-10. Probes specific for Hahb-10 or rRNA were used as described above.

FIGS. 4A-4D show the phenotype of 35S:Hahb10 transgenic plants.

Comparison between transformed and control plants. 5A: top view of cotyledons; 5B: side view of cotyledons, 5C: 14-day-old-leaves; 5D: top view of 21-day-old plants of the four genotypes. (WT: wild-type plants; TG: transgenic plants from lines-A, -B, -C).

FIGS. 5A-5B shows that 35S:Hahb10 transgenic plants are less affected by changes in illumination intensity than their wild-type counterparts.

5A: Comparison between transformed and control 7-day-old seedlings grown at 35 μE m-2 s-1 (upper panel) and between 14-day-old plants grown at 57 μm-2 s-1 (lower panel).

5B: Hypocotyl length in 5-day-old seedlings grown on soil with different light qualities. This is a representative experiment done with 20 plants of each genotype. Hypocotyl length was measured with a ruler. Plants were grown in normal illumination conditions or subjected to red enriched light or far-red enriched light. Controls were done with normal illumination as described later. (WT: wild-type plants; TG-A, -B and -C: three independent over expressing Hahb-10 transgenic lines).

FIGS. 6A-6B shows that 35S:Hahb10 transgenic plants develop faster than non-transformed ones.

6A: comparison of developmental state between transformed and control 21-day-old plants grown on soil (four plants per 8 cm diameter pot).

6B: difference in the developmental state between transgenic and non transformed plants in a typical experiment done with twenty plants per 8 cm diameter pot.

FIG. 7 shows that stem elongation is almost unaffected in gibberellin treated transgenic plants.

Stem height was measured with a ruler in non transformed (1) or 35S-Hahb10 transgenic plants (2: line A; 3: line B and 4: line C). Grey bars represent three transgenic lines and white bars represent wild-type plants. Right series in each panel represent GA3 treated plants while left series represent their non-treated counterparts. The first panel represents the observed results in 20-day-old plants while the second panel shows the results observed after a second hormone treatment in 30-day-old plants.

FIG. 8 shows that the expression of the PsbS gene is reduced in Hahb-10 overexpressing transgenic plants.

For Northern blot analysis of non-transformed and transgenic Arabidopsis plants, total RNA (10 μg) was extracted from 4-day-old seedlings of wild-type (WT) or transgenic plants overexpressing Hahb-10 grown under normal illumination conditions or subjected to 8 h of darkness as described later. Probes specific for PsbS, CAB2, CHS or rRNA were used.

FIGS. 9A-9E shows the effect of the herbicide Paraquat 2 days after application. (WT: wild-type plants; TG-A, -B and -C: three independent overexpressing Hahb-10 transgenic lines).

9A: control plants were grown in normal conditions for 30 days.

9B: plants treated with 10 μM methyl viologen.

9C: plants treated with 20 μM methyl viologen.

9D: plants treated with 30 μM methyl viologen.

9E: upper view of 21-day-old transgenic and wild-type plants treated with 10 μM methyl viologen.

FIG. 10 shows that Hahb-10 transgenic plants are less susceptible to methyl viologen than their wild-type counterparts. Plants were treated with 10 μM methyl viologen and observed 48 h later. (WT: wild-type plants; TG-A, -B and -C: Hahb-10 transgenic lines).

FIG. 11 shows the pBI-Hahb-10 expression vector map used in the present invention, which comprises

Kanamicin R: Kanamycin resistance gene.

nos-P: nopaline synthase gene promoter.nos-T: nopaline synthase gene terminator.

Hahb-10: sunflower Hahb-10 gene coding sequence.

LB: left end.

RB: right end.

CaMV35S: cauliflower mosaic virus 35S promoter.

Ori RK2: prokaryotic origin of replication.

SacI: SacI restriction site.

BamHI: BamHI restriction site.

FIG. 12 shows the Hahb-10 nucleotide sequence used in the constructs.

The sequence expressed in the construct corresponds to bases 35 to 745 of SEQ ID NO:1. The start and stop codon are indicated.

Claims

1. A method for obtaining a genetically modified plant having a shorter life cycle, the method comprising the steps of: (a) designing and constructing a DNA construct comprising a promoter operably linked to a nucleic acid sequence encoding the transcription factor Hahb-10, or an active fragment, deletion or genetic variant thereof,(b) introducing said construct into a host cell to produce a transformed host cell, and(c) regenerating the transformed host cell to obtain a stably transformed complete plant expressing the transcription factor Hahb-10 and displaying a shorter life cycle and a higher tolerance to herbicides as compared to wild-type plants.

2. The method of claim 1, wherein the DNA construct further comprises a 3′UTR.

3. The method of claim 2, wherein the promoter is the cauliflower mosaic virus 35S promoter.

4. A plant expression cassette comprising: (a) a promoter functional in plants, and(b) Hahb-10 coding sequence, or an active fragment, deletion or genetic variant thereof.

5. The cassette of claim 4, further comprising: (c) a 3′UTR linked 5′-3′.

6. The cassette of claim 5, wherein the promoter is the cauliflower mosaic virus 35S promoter.

7. A plant expression vector comprising the expression cassette of claim 4.

8. The vector of claim 7, further comprising a 3′ UTR linked 5′-3′.

9. A host cell and any descendant thereof, transformed by the method of claim 1.

10. A cell and any descendant thereof, transformed with the vector of claim 7.

11. The cell of claim 10, wherein the cell is a bacterium.

12. The cell of claim 10, wherein the cell is a plant cell.

13. The cell of claim 10, wherein the cell expresses Hahb-10 coding sequence, or an active fragment, deletion or genetic variant thereof.

14. A plant cell transformed with the cassette of claim 4, wherein the cell has the cassette stably integrated in its genome.

15. A transgenic plant and any descendant thereof, wherein the transgenic plant is regenerated from the plant cell of claim 14.

16. The transgenic plant of claim 15, wherein at least one of its cells expresses the Hahb-10 gene coding sequence under control of the promoter comprised in the expression cassette of claim 4.

17. The plant of claim 16, wherein the plant is a monocot plant.

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

19. A plant of claim 15, wherein the plant has a shorter life cycle as compared to wild-type plants.

20. The plant of claim 19 expressing in at least one of its cells the Hahb-10 coding sequence, or an active fragment, deletion or genetic variant thereof, under control of a promoter functional in plants, wherein the plant belongs to a species of a group consisting of rice, maize, wheat, alfalfa, soy, tobacco and cotton.

21. A method for expressing at least one protein of interest in a host cell, comprising: (a) introducing the cassette of claim 4 into the host cell, and allowing the host cell to produce the protein of interest.

22. A method for obtaining a plant with high tolerance to herbicides which generate oxidative stress, wherein the method comprises: (a) constructing a DNA construct comprising a functional promoter linked to a nucleic acid sequence encoding the transcription factor Hahb-10, or an active fragment, deletion or genetic variant thereof,(b) introducing said construct into a host cell to produce a transformed host cell, and(c) regenerating the transformed host cell to obtain a stably transformed complete plant expressing the transcription factor Hahb-10 and displaying a shorter life cycle and a higher tolerance to herbicides as compared to wild-type plants.

23. The method of claim 22, wherein the DNA construct further comprises a 3′UTR.

24. A plant obtained by the method of claim 22, wherein the plant has high tolerance to herbicides that act through generating oxidative stress as compared to wild-type plants.

25. An isolated DNA molecule, wherein said molecule encodes the transcription factor Hahb-10.

26. A molecule of claim 25, wherein the molecule comprises the sequence SEQ NO:1 or a fragment, deletion or genetic variant thereof that conserves the function of enhancing the tolerance to herbicides acting through the production of oxidative stress in plant cells when it is located under the control of a promoter functional in plants.

27. A molecule of claim 25, wherein the molecule comprises the sequence SEQ No 1 or a fragment, deletion or genetic variant thereof that conserves the function of reducing the life cycle of a plant when it is located under the control of a promoter functional in plants.

28. A fragment, genetic variant or deletion of the DNA molecule of claim 26, wherein the molecule includes at least 100 consecutive bases with an homology of at least 80% to the sequence of cDNA between base pairs 1 and 989 SEQ ID NO:1.

29. A fragment, genetic variant or deletion of the DNA molecule of claim 27, wherein the molecule includes at least 100 consecutive bases with an homology of at least 80% to the sequence of cDNA between base pairs 1 and 989 of SEQ ID NO:1.