CN101914549B - Transcription factor gene induced by dehydration condition and abscisic acid in sunflower, promoter and transgenic plant - Google Patents

Abstract

The present invention characterizes a novel transcription factor-encoding gene in sunflower that is induced by water deficiency or abscisic acid and has a homology domain associated with a leucine zipper. The transcription factor is advantageously cloned in a DNA construct for transformation of host cells and plants. Transgenic plants including the transcription factor gene are tolerant and resistant to adverse environmental conditions such as water stress and high salinity. The present invention also provides a nucleic acid promoter sequence, wherein the sequence is induced by dehydration and abscisic acid. The invention provides constructs, host cells and transgenic plants comprising the transcription factor gene.

Description

Transcription Factor Genes, Promoters and Transgenic Plants Induced by Water Deficiency Conditions and Abscisic Acid in Sunflower

technical field

The present invention relates to a novel gene encoding a transcription factor in sunflower (Helianthus annuus) induced by water deficiency and abscisic acid, which has a homology domain associated with a leucine zipper. It is advantageous to clone the transcription factor in a DNA construct of a transformed host cell or plant. Transgenic plants including this transcription factor are resistant to adverse environmental conditions such as water stress and high salinity. Nucleic acid promoter sequences are also provided, as well as constructs, host cells and transgenic plants comprising the sequences, wherein the sequences are inducible by water deficiency or abscisic acid.

Background technique

Homeodomains are 60 amino acid motifs that occur in many eukaryotic transcription factors involved in developmental processes (Gehring, Science 236, 1245-1252, 1987). Genes containing homeoboxes have been isolated from many eukaryotes including fungi, mammals and plants (Gehring, W.J., et al., Annu. Rev. Biochem. 63, 487-526, 1994). The homeoboxes of plants can be divided into many families according to the sequence conservation and structure inside and outside the homeodomain (Chan, R.L., et al. Biochim. Biophys. Acta 1442(1), 1-19, 1998). Members of one of these families have a unique feature: because they include a homology domain associated with a helix-helix leucine zipper involved in dimerization, they encode a protein called Hd-Zip. Hd-Zip binds efficiently only to DNA in dimeric form (Sessa, G., et al., EMBO J.12, 3507-3517, 1993; Palena C.M., et al., Biochem J.341, 81-87, 1999). It has been suggested that these proteins may be involved in the regulation of developmental processes associated with plant responses to environmental conditions (Chan, R.L., et al. Biochim. Biophys. Acta 1442(1), 1-19, 1998, Carabelli, M., et al. , Plant J.4, 469-479, 1993; Schena, M., et al., Genes Devel. 7, 367-379, 1993). Water scarcity is one of the most common environmental stresses faced by plants. While many seeds tolerate extreme water scarcity, very few vegetative parts of plants are tolerant. Plants respond to water stress by expressing a specific series of genes, which allow them to adapt to changing environmental conditions (Bray, E.A. Trends Plant Sci. 2, 48-54, 1997 y Shinozaki, K.andYamaguchi-Shinozaki, K.Plant Physiol .115, 327-334, 1997). The hormone abscisic acid (ABA) plays an important role in a subset of these responses (Shinozaki, K. and Yamaguchi-Shinozaki, K. Plant Physiol. 115, 327-334, 1997 y Leung, J. and Giraudat, J. Ann Rev. Plant Physiol. Plant Mol. Biol. 49, 199-222, 1998).

Characterization of the promoter regions of genes involved in water stress tolerance and resistance suggested the presence of an ABA-responsive element, ABRE, and a drought-responsive element, DRE.

等已经公开了(描述)脱落酸和缺水诱导的阿布属(Arabidopsis)基因(两种)ATHB-7和ATHB-6(

E.et al.，The PlantJournal 10：375-381，1996 and Soderman E.et al.Plant Molecular Biology40：1073-1083，1999)。作者没有说明这些基因的超表达提供缺水耐受性。

have published (described) abscisic acid and water deficit-induced Arabidopsis genes (two species) ATHB-7 and ATHB-6 (

E. et al., The Plant Journal 10:375-381, 1996 and Soderman E. et al. Plant Molecular Biology 40:1073-1083, 1999). The authors do not state that overexpression of these genes confers water deficit tolerance.

US NO. 5,981,729 discloses a novel gene induced by water deficiency and abscisic acid and encoding a transcription factor of Arabidopsis thaliana (A. Thaliana). This patent does not disclose any reference to transgenic plants carrying the genes of the present invention and resistant to water stress conditions.

Contents of the invention

Therefore, the object of the present invention is to provide a nucleic acid molecule of an isolated coding transcription factor Hahb-4, its functionally active fragment or variant, the isolated nucleic acid molecule has a nucleic acid sequence of SEQ ID No.1 or a fragment thereof, wherein, The nucleic acid molecule is derived from sunflower (Helianthus annuus), and it can be the mRNA or cDNA of SEQ ID No.2, wherein the molecule can be combined with the drought of 5'-CAAT(A/T)ATTG-3'DNA sequence or plant species Transcription regulatory region.

Another object of the present invention is to provide a vector comprising a promoter operably linked to a nucleic acid sequence selected from the group comprising SEQ ID No.1, SEQ ID No.2 and fragments thereof, wherein the vector drives Expression of transcription factor Hahb-4, its functionally active fragment or variant, and wherein said transcription factor Hahb-4, its functionally active fragment or variant is capable of binding to a drought transcriptional regulatory region of a plant variety, wherein, with the host Expression of the vector in a host cell increases the tolerance of the cell to environmental stress, such as water stress, compared to the wild-type variety of the cell.

A further object of the present invention is to provide a transgenic plant stably transformed with a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2 and fragments thereof, wherein the nucleic acid molecule encodes a transcriptional Factor Hahb-4, a functionally active fragment or variant thereof, and wherein the plant has increased resistance to environmental stresses such as drought, salt, osmotic and other, preferably water stress, compared to a wild-type variety of the plant Tolerance, wherein the plant, which may be a monocotyledon, a dicot or any other agricultural plant, is tolerant to water stress through the binding of the transcription factor Hahb-4, a functionally active fragment or variant thereof, to the drought transcriptional regulatory region of the plant sexual.

Another object of the present invention is to provide a plant seed stably transformed with a nucleic acid molecule having a sequence selected from the group comprising SEQ ID No.1, SEQ ID No.2 and fragments thereof, wherein the nucleic acid molecule sequence encodes a transcription factor Hahb-4, functionally active fragments or variants thereof.

Another object of the present invention is to provide a host cell stably transformed by a nucleic acid molecule having a sequence selected from the group comprising SEQ ID No.1, SEQ ID No.2 and fragments thereof, wherein the nucleic acid molecule encodes the transcription factor Hahb -4. A functionally active fragment or variant thereof, wherein the host cell is selected from the group comprising bacterial, fungal, insect, plant and animal cells, preferably, it is a plant cell.

Another object of the present invention is to provide a method for producing water stress-tolerant transgenic plants, which method comprises stably transforming with a nucleic acid sequence selected from the group comprising SEQ ID No.1, SEQ ID No.2 and fragments thereof The step of planting a cell or cell line, and regenerating the cell or cell line into a plant.

Another object of the present invention is to provide an isolated nucleic acid molecule selected from the group comprising the following nucleic acid molecules:

(a) have the nucleic acid molecule of nucleotide sequence SEQ ID No.3;

(b) have the nucleic acid molecule of nucleotide sequence SEQ ID No.10;

(c) a nucleic acid molecule having a nucleotide sequence of 805 to 1221 nucleotides of SEQ ID No.3;

(d) a nucleic acid molecule having a nucleotide sequence of 904 to 1221 nucleotides of SEQ ID No.3;

(e) a nucleic acid molecule having a nucleotide sequence of 1011 to 1221 nucleotides of SEQ ID No.3;

(f) a nucleic acid molecule having a nucleotide sequence of 15 to 622 nucleotides of SEQ ID No.3;

(g) a nucleic acid molecule having a nucleotide sequence of 15 to 409 nucleotides of SEQ ID No.10;

(h) a nucleic acid molecule having a nucleotide sequence complementary to the nucleic acid molecule of (a), (b), (c), (d), (e), (f), or (g); and

(i) has at least 150 nucleotides in length, and has at least A nucleic acid molecule with 80% sequence identity, wherein said nucleic acid molecule is capable of promoting the expression of a heterologous nucleic acid molecule in a transformed cell or tissue selected from the group consisting of bacterial, fungal, insect, plant or animal cells , embryonic tissue, plant callus and plant seeds.

Another object of the present invention is to provide a nucleic acid construct containing a first nucleic acid molecule selected from the group comprising the following nucleic acid molecules:

(a) have the nucleic acid molecule of nucleotide sequence SEQ ID No.3;

(b) have the nucleic acid molecule of nucleotide sequence SEQ ID No.10;

(c) a nucleic acid molecule having a nucleotide sequence of 805 to 1221 nucleotides of SEQ ID No.3;

(d) a nucleic acid molecule having a nucleotide sequence of 904 to 1221 nucleotides of SEQ ID No.3;

(e) a nucleic acid molecule having a nucleotide sequence of 1011 to 1221 nucleotides of SEQ ID No.3;

(f) a nucleic acid molecule having a nucleotide sequence of 15 to 622 nucleotides of SEQ ID No.3;

(g) a nucleic acid molecule having a nucleotide sequence of 15 to 409 nucleotides of SEQ ID No.10;

(h) a nucleic acid molecule having a nucleotide sequence complementary to the nucleic acid molecule of (a), (b), (c), (d), (e), (f), or (g); and

(i) has at least 80% homology or a length of at least A nucleic acid molecule of 150 nucleotides, wherein the first nucleic acid molecule is operably linked to the second nucleic acid molecule encoding the protein of interest and the 3' untranslated region. Preferably, the nucleic acid molecule is a promoter having SEQ ID No.3 or SEQ ID No.10. Also, host cells and transgenic plants stably transformed with at least one of the above constructs are provided.

Another object of the present invention is to provide a method for expressing at least one protein of interest in a host cell, the method comprising introducing one of the above-mentioned constructs into the host cell and causing the host cell to produce the protein of interest, wherein the host cell is selected from Self includes the group of bacteria, fungi, insect, plant and animal cells.

Another object of the present invention is to provide a method for obtaining transgenic plants expressing at least one protein of interest, the method comprising stably transforming plant cells or cell lines with one of the above-mentioned nucleic acid constructs, and then regenerating the cells or cell lines is a whole plant expressing at least one protein, wherein the transgenic plant is selected from the group comprising monocots and dicots.

Another object of the present invention is to provide a transgenic plant stably transformed by at least one of the above constructs, wherein the protein of interest is a nucleic acid having a nucleic acid selected from the group consisting of SEQ ID No.1, SEQ ID No.2 and fragments thereof The transcription factor Hahb-4 of the sequence, and wherein the plant is selected from the group comprising monocotyledonous and dicotyledonous plants, said plant is environmental stress tolerance to conditions such as drought, high salinity, high osmotic pressure and others, preferably , the plant is resistant and tolerant to lack of water. Most preferably, the plant is water stress tolerant through the combination of the transcription factor Hahb-4, its functionally active fragment or variant, and the drought transcriptional regulatory region of the plant, the drought transcriptional regulatory region of the plant being 5' -CAAT(A/T)ATTG-3' DNA sequence.

The above and other objects, features and advantages of the present invention can be better understood with reference to the accompanying drawings and description.

Description of drawings

The invention is explained by way of example in the following drawings, in which:

Figure 1 shows the genome sequence encoding the sunflower Hahb-4 of the present invention. The nucleotide sequence below represents the deduced protein sequence of the open reading frame. Homology domains are indicated in red; leucines in leucine zippers are indicated in bold and underlined. The lower part of the figure shows the identity of the Hd-Zip region of Hahb-4 with the Hd-Zip region of Athb-1, -6, 7 and 12. Shaded boxes indicate identical amino acids.

Figure 2a is a schematic diagram of the Hahb-4 cDNA showing the length and polarity of the RNA riboprobes (arrowheads) and the protected region of the Hahb-4 mRNA (+81 to +429; shaded box).

Figure 2b shows the expression of Hahb-4 induced by water stress subjected to different treatments. Four-day-old seedlings were treated as follows: 100 μΜ ABA, 24 hours; water stress, 2 hours; 4°C, 24 hours; 42°C, 2 hours; 0.5M mannitol, 4 hours.

Figure 2c shows the expression of Hahb-4 induced by water stress for 2 h at different organs, analyzing embryos and dry seeds.

Figure 3 shows the time-dependence of induction of Hahb-4 by water stress at roots, stems and leaves. Seedlings were subjected to water stress treatment for 0.5 or 1 h, or seeds were rehydrated at various times after the 1 h drought treatment. The same filter paper was hybridized with the rRNA probe as a control for RNA loading and transfer (lower panel).

Figure 4 shows the time dependence of Hahb-4 response to ABA. Total RNA (20 μg) was isolated from untreated seedlings, or seedlings treated with 100 μM ABA for different times, as indicated in the individual lanes. In the lower panel, the same filter paper was hybridized with the rRNA probe as a control for RNA loading and transfer.

Figure 5 shows the time-dependence of induction of Hahb-4 by water stress at roots, stems and leaves. RNA was prepared from different organs of plants treated with control, 30 min (1), 60 min (2) or 90 min (3) water stress. The same filter paper was hybridized with the rRNA probe as a control for RNA loading and transfer (right panel).

Figure 6 shows that ABA and water stress induce the expression of nucleoproteins that specifically bind the Hahb-4 DNA target sequence. Lane 0, DNA only; Lane 1, control plants; Lane 2, ABA-treated plants; Lane 3, water stress-treated plants; A and B represent two different migration bands observed using nuclear extracts.

Figure 7 is a diagram illustrating the strategy used to clone the cDNA encoding Hahb-4 under the control of the CaMV35S promoter. Plasmid p35SHB4 was constructed in E. coli, which was then used to transform the CV2260 line of A. tumefaciens. Agrobacterium tumefaciens harboring a plasmid comprising the Hahb-4 cDNA under the control of the CaMV35S promoter was designated ATH4. BD and BI: right and left margin; Pnos: nopaline synthase gene promoter; Tnos: nopaline synthase termination sequence; Km ^r : kanamycin resistance gene; P35S: 35ScaMV promoter, gus: β- glucuronidase gene.

Figure 8 is a graph illustrating the germination time of transformed plants of the invention (black bars) and untransformed control plants (white bars) as percentage of germinated plants (n=22) after 48 hours of treatment at 4°C.

Figure 9 is a graph illustrating stem length measured in millimeters for transformed plants of the invention (black bars) and non-transformed control plants (white bars) from days 28 to 33 from germination. The two terminal bars show the stem length of the transgenic plants subjected to the water stress test, where there were no viable shoots in the group of control plants.

Figure 10 is a bar graph illustrating the amount of siliques formed during the development of plants, ie non-transformed plants (white bars) and transgenic plants (black bars). The two terminal bars illustrate the amount of siliques of the transgenic plants subjected to the water stress test, where there were no viable shoots in the group of control plants. Plants were watered at the end of the period and, once rehabilitated, the parameters of interest were recorded.

Figure 11 is a bar graph illustrating the percentage of non-transformed control plants (white bars) and transgenic plants (black bars) that germinated at different times after breaking dormancy in the presence of 50 mM mannitol.

Figure 12 is a bar graph illustrating the percentage of non-transformed control plants (white bars) and transgenic plants (black bars) that germinated at various times after breaking dormancy in the presence of 200 mM mannitol.

Figure 13 is a bar graph illustrating the percentage of non-transformed control plants (white bars) and transgenic plants (black bars) that germinated at various times after breaking dormancy in the presence of 300 mM mannitol.

Figure 14 is a bar graph illustrating the percentage of non-transformed control plants (white bars) and transgenic plants (black bars) that germinated at different times after breaking dormancy in the presence of 50 mM NaCl.

Figure 15 is a bar graph illustrating the percentage of non-transformed control plants (white bars) and transgenic plants (black bars) that germinated at different times after breaking dormancy in the presence of 150 mM NaCl.

Figure 16 is a bar graph illustrating the percentage of non-transformed control surviving plants (white bars) and transgenic plants (black plants) in three independent experiments. In the first experiment (1 ), the stress occurred when the plants were mature (reproductive stage). In the second test (2), the plants were in a favorable growth stage (full petals). In the third experiment (3), the stress occurred at germination.

Figure 17 shows the phenotypes of control and transgenic plants. (a) Phenotypes of transgenic plants (first and second row), and non-transformed plants (third and fourth row) are observable. The plants were water-stressed at maturity and watered again at the end of the life cycle. In b, the phenotypes of transgenic (left) and non-transformed (right) plants are observable. This group of plants was subjected to water stress treatment while in the growth phase. In c, phenotypes are observable in transgenic plants (left) and non-transformed control plants (right) that were water-treated stress from germination and then re-watered. (d) In d, the phenotype of transgenic plants subjected to extreme drought during the growth phase is shown.

Figure 18 shows the nucleotide sequence of the promoter region of the Hahb-4 gene, in which the sequence corresponding to the TATA box, the elements responding to water stress/low temperature, the ABRE region and the sequence representing the recognition sites of Myb and Myc are marked.

Figure 19 is a schematic diagram of the pBI101.3 vector used to clone multiple fragments of the Hahb-4 promoter. The β-glucuronidase gene is indicated, as well as the gene conferring resistance to kanamycin in E. coli and A. tumefaciens. The origins of replication for E. coli and A. tumefaciens are also noted.

Figure 20 shows the comparison of promoters carrying 0～-400 sequence (805～1221 nucleotides of SEQ ID No.3) (left side) and 0～-1015 sequence (small allele) (right side) respectively Photographs of the expression levels of the gus gene as determined by histochemistry in 10-day-old seedlings of the constructs.

Figure 21 shows the expression of gus in Arabidopsis roots. (a) shows the expression of the gus gene in 20-day-old roots of plants transformed with the 0-400 construct. (b) shows the expression of the gus gene in 20-day-old roots of plants transformed with the 0-1015 construct. (c) shows the expression of the gus gene in 10-day-old roots of plants transformed with the 0-1015 construct.

Figure 22 shows the effect of ABA treatment in 20 day old seedlings transformed with constructs from 0 to -400. Control plants are shown on the left and ABA-treated seedlings are shown on the right.

Figure 23 shows a schematic diagram of the Hahb-4 gene structure. Upper: major allele, lower: minor allele. Isolation of the promoter region and construction of the recombinant plasmid, and oligonucleotides used for plant transformation are shown.

Detailed ways

Now, explaining the present invention in detail, the characterization of the novel gene Hd-Zip refers to the same, ie, the sunflower Hahb-4 gene responsive to water stress conditions. In the homology domain, Hahb-4 shows partial homology to two related proteins of Arabidopsis, Athb-7 and Athb-12. The protein Hahb-4 has a homology domain located immediately to the N-terminus.

The present invention discloses a clone from a cDNA library. This clone represents a member of the Htd-Zip family designated Hahb-4. The sequences corresponding to the 5' and 3' ends were obtained by PCR. The whole cDNA sequence obtained by this method is 674bp in length (SEQ ID No.2), and contains an open reading frame of 177 amino acids (Figure 1).

Comparison of the encoded protein with other Hd-Zip protein sequences indicated that it may belong to subfamily I of Hd-Zip proteins, and that the encoded protein is in the same group as other members of this subfamily except Athb-7 and Athb-12. The source domain has about 50% identical amino acids, with Athb-7 and Athb-12 having 60% and 53% identity in this region, respectively (Figure 1). It is also noteworthy that the Hahb-4 homology domain is located almost at the amino terminus of the protein (17-76 amino acids). Thus, Hahb-4 lacks the acidic region adjacent to the N-terminus of the homology domain that is present in other members of the Hd-Zip protein family. This feature is also the same as Athb-7 and Athb-12. To study the gene structure of the Hahb-4 gene, genomic DNA (genomic DNA is SEQ ID No. 1) has been amplified with multiple oligonucleotides including the full cDNA. A single intron of 101 bp was found between nucleotides 381 and 382 of the cDNA (amino acids 108 and 109).

Northern dot analysis indicated that the Hahb-4 gene of the present invention was expressed at very low levels in sunflower grown under controlled normal environmental conditions. Only weak signals were obtained from total RNA extracted from various tissues and developmental stages.

The expression of Hahb-4 under different environmental conditions (i.e. water, osmotic, salt, cold, heat, and oxidative stress) was analyzed by RNAse protection, which is more sensitive than the Northern technique (Figure 2) . Figure 2b illustrates that Hahb-4 was not detected in 4-day-old seedlings grown under normal conditions. However, in water-stressed seeds, a strong signal was detected. Mannitol also induced Hahb-4 expression, albeit at a lower level, possibly reflecting the reduction in water activity caused by this compound (Fig. 2b). The same result was obtained with NaCl.

Since ABA regulates many responses to water stress, the effect of this hormone on expression was also analyzed. As shown in Fig. 2b, induction was observed 24 h after treatment of watered seedlings with 100 μM ABA. A small but significant increase in transcript levels was also observed with 10 μM ABA. No detectable effect was observed when the seedlings were subjected to cold (4°C) or heat (42°C) stress conditions (Fig. 2b). These results suggest that the effect of water stress is specific.

Figure 2c illustrates that the response to water stress conditions was also observed in roots, stems and leaves of older (21 days old) plants. Induction levels at the airborne parts of the plants were similar to those observed in seedlings. In contrast, roots exhibited considerably lower transcript levels under water stress conditions.

Since ABA is also involved in seed development during embryogenesis involving later stages of the drought process, the transcript levels of Hahb-4 were analyzed in sunflower embryos (20 days after pollination) and dry seeds. No signal was obtained in the RNase protection assay (Fig. 2c), suggesting that the response of Hahb-4 to water stress and ABA is characteristic of the anagen phase of development.

The high level of induction observed under water stress conditions enabled the analysis of time-dependent increases in transcript levels by Northern experiments. Figure 3 illustrates that seedlings exposed to drought significantly increased their Hahb-4 transcript levels after only 30 minutes; the response reached a maximum after 1 hour of water deprivation. After this time, no further increase in Hahb-4 transcript levels was observed. Rehydration of the seedlings reduced the transcript level of Hahb-4 more slowly, only about 50% after 2 hours (Fig. 3, lanes 4 and 5). The effect of ABA treatment was also time-dependent. As shown in Figure 4, the response to ABA was detected within 1 hour and reached a maximum after 3-6 hours. Afterwards, transcript levels decreased slowly, but were still very high after 24 h of treatment. Upon rehydration of the sunflower plants, the high Hahb-4 transcript levels of the present invention were restored, thus again suggesting that the gene is involved in the response to water stress.

The response of Hahb-4 to water stress was also rapid at different organs of plants grown hydroponically for 3 weeks. In roots, transcript levels reached a maximum after 60 min of stress treatment (Fig. 5).

At leaves and stems, the maximum response was observed after longer treatment. This may be related to the fact that the initial perception of water stress is done by the roots, which synthesize ABA and then transfer ABA to the airborne parts of the plant. This result, combined with the results shown above in Figures 2-4 most likely suggest that Hahb-4 expression correlates with endogenous ABA levels in plants under water stress.

Sunflower was analyzed by electrophoretic mobility shift assays using a synthetic double-stranded oligonucleotide comprising the sequence 5′-CAAT(A/T)ATTG-3′ that binds Hahb-4 expressed in bacteria in vitro. Emergence of functional DNA bound to proteins in the nucleus. Nuclear extracts prepared from 4-day-old seedlings showed distinct migratory bands, suggesting the presence of at least two distinct protein complexes that bind this sequence (Fig. 6). The amount of both complexes was significantly increased in extracts obtained from plants treated with ABA. In contrast, only the slower migrating complexes increased when plants were subjected to water stress. An excess of the same unlabeled DNA almost completely stops the formation of both complexes, but an equal amount of similar DNA containing the 5'-CA C T(A/T)A G TG-3' sequence that does not bind Hahb-4 DNA sequences could not (Fig. 6). This result strongly suggests that at least one functional protein with the same DNA binding specificity as Hahb-4 is synthesized and transported to the nucleus in the presence of ABA, or under water stress conditions.

Strong Hahb-4 expression was detected only in plants subjected to water stress, or treated with ABA. Heat, cold and oxidative stress did not induce this expression. Salt and osmosis treatments produced only small increases in Hahb-4 transcript levels. The water stress effect was completely reversed when the plants were rehydrated. These features demonstrate that Hahb-4 expression of the present invention responds directly to the hydration of vegetative cells and that the induction is not the result of disruption or stress in general.

As shown in Example 3 and Figure 7, in order to obtain transgenic plants of Arabidopsis overexpressing the sunflower Hahb-4 of the present invention, the vector pBI121 and the "flower dipping" method were used. As a result, a number of independent lines of transgenic plants were obtained, in which the PCR-positive reaction using the specific oligonucleotide, plants grown in kanamycin-containing medium were tolerant or resistant. The plants stably expressing the transgene (Hahb-4 gene of the present invention) were selected from the obtained independent lines. As a result, the homozygous line F3 was selected for phenotype analysis.

As shown in Figure 8, Arabidopsis transgenic plants carrying the gene of the present invention germinated faster than untransformed control plants. After 14 hours, the ratio of germination rate of transformed plants according to the present invention to non-transformed plants was 85%:58%.

Furthermore, the stems of transformed plants of the invention grew slower and had a maximum height of 85% of the height of control plants grown under the same conditions (providing normal water access - Figure 9). Moreover, the transgenic plants subjected to water stress achieved the same shoot height as the transgenic plants with normal water availability, indicating that water stress did not affect the shoot growth of the transgenic plants of the present invention ( FIG. 9 ). The transgenic plants of the present invention are not only tolerant to water stress, but can also grow normally under water-deficient conditions without significant changes in observed phenotypes.

The number of siliques formed did not change significantly between wild-type and transgenic plants, and, despite shortened pedicel, the number was slightly higher in transgenic plants (Fig. 10). When the transgenic plants grown under water stress conditions were compared to the same plants grown under standard conditions, the number of siliques formed was higher in the stressed transgenic plants (Figure 10). Furthermore, as shown in Table 1, the transformed plants according to the present invention produced seeds with a total weight about 15% higher than that of untransformed control plants.

Table 1

transgenic plants control plants average seed weight 0.0964 0.0803 SD 0.0350 0.0224 number of plants 12 12

Since the Hahb-4 product acts as a transcription factor and its expression at the transcriptional level appears to be regulated by the availability or presence/absence of water, it has been determined that transformed plants overexpressing the sunflower Hahb-4 gene of the present invention are sensitive to water. The study of stress tolerance.

First, the germination process was analyzed under simulated water deficit conditions such as the presence of mannitol, and other conditions that produced salt stress. Figures 11, 12 and 13 show the germination time of plants transformed according to the invention compared to control plants. The presence of mannitol delayed germination. This effect was more pronounced at higher carbohydrate concentrations. However, the transformed plants maintained excellent germination rates even at high concentrations of mannitol.

Similar results were obtained as in the case of mannitol when germination experiments were performed in the presence of different concentrations of NaCl. As shown in Figures 14 and 15, overexpression of Hahb-4 resulted in higher germination ability of transformed plants in saline medium.

It is noteworthy that the roots of the tested or experimental transgenic plants grown under different stress conditions were larger than those of the control plants, indicating a phenotype that is tolerant to many stress conditions.

Subsequently, drought tests were performed at various stages of plant development in soil. Figure 16 shows three trials of water stress survival. Clearly, a higher tolerance to water stress was observed in transformed plants of the invention when compared to non-transformed control plants.

When the plants are subjected to water stress at different developmental stages, the transformed plants show stronger tolerance to the stress conditions. Figure 17 shows the state of plants subjected to water deficit conditions at different growth stages. No matter the plants suffer from water stress at any stage, the transgenic plants carrying the Hahb-4 gene of the present invention have strong tolerance and resistance to the above conditions.

In short, the present invention discloses the obtaining of Arabidopsis transformed plants that overexpress the sunflower Hahb-4 gene of the present invention under the control of the 35S cauliflower mosaic virus promoter, that is, the overexpression is as shown in Figure 7 of constructs. The gene of the present invention, originally isolated from sunflower, encodes a Hd-Zip protein with a homeodomain-type protein domain associated with a leucine zipper. Those of ordinary skill in the art will recognize that any promoter or nucleic acid construct that drives expression of the genes of the present invention can be used without altering the spirit and scope of the present invention. Nucleic acid constructs that enable expression of the genes of the present invention in any host cell such as bacteria, yeast, fungi, animal and plant cells can also be prepared. In preferred embodiments of the invention, the genes are expressed in plant cells and tissues.

Transformed plants expressing Hahb-4 generally have shorter stems under normal growth conditions. As shown by histological studies, it appears that this feature is mainly due to the inhibition of cell expansion, but not of cell division.

In addition to the above discussion, the adult leaves of transgenic plants are rounder and shorter than the adult leaves of non-transformed plants. Combining the two features seems to indicate that the gene product acts as a suppressor of cell swelling and elongation. These phenotypic traits (short stems and rounded leaves) as a "water saving" mechanism should be directly related to the transgenic plants' ability to tolerate and resist water scarcity or scarcity. In contrast, the roots of transgenic plants of the present invention are longer compared to the roots of non-transformed plants. This should account for the emergence of another favorable mechanism for water acquisition.

The transgenic plants of the present invention exhibit significant tolerance to water stress at different growth stages, at the germination stage and at the early and late growth stages, and at the reproductive stage. When both transgenic and non-transformed plants were subjected to drought conditions, the survival of transgenic plants was higher than that observed in non-transformed control plants.

Through all these results, it can be concluded that the Hahb-4 gene is involved in the plant's response to water stress, and its specific function can produce phenotypic changes that enhance the tolerance of plants to water shortage.

Apparently, these phenotypic changes did not adversely affect plant yield and germination. In contrast, the seed yield (measured by weight) of transgenic plants was higher than that of non-transformed plants.

Commercially interesting transgenic plants can be produced using the Hahb-4 of the present invention, wherein the transgenic plants have specific tolerance to water stress. The yield of said agriculturally valuable plants subjected to water stress is expected to be the same as that of untransformed varieties not subjected to such stress conditions. As examples, the agriculturally valuable plants may include sunflower, wheat, barley, soybean, potato, corn, sugar cane or rice, but are not limited thereto.

It is important to note that there are not a large number of overexpressed genes when plants are subjected to water stress, and there is no specific reference to said genes being able to confer drought tolerance in plants like Hahb-4 of the present invention.

According to the present invention, the inventors of the present invention have isolated and characterized the sequence of the promoter region of sunflower Hahb-4. Isolation of the Hahb-4 promoter region was performed in three stages by inverse PCR (inverse PCR) technology using the information obtained at one stage for the design of new oligonucleotide pairs used in the next stage. The fragment obtained by the PCR reaction was cloned into pGEM.-T easy vector (Promega), and the sequence of the promoter region was manually determined by overlapping the repeat region of each construct.

The sequence of the Hahb-4 promoter (SEQ ID No. 3) corresponds to the 1221 bp sequence, including the TATA box located 24 bp upstream of the transcription initiation site. Alignment of this sequence with sequences present in databases revealed the presence of regions of homology to sequences believed to be involved in the response to various environmental factors such as light, abscisic acid and hormones. As an example, Figure 18 shows putative ABA response elements of the ABRE type, and drought stress or low temperature response elements of the DRE type.

Consensus sequences of transcription factors jointly involved in transcription factor responses have also been identified, such as proteins of the Myb and Myc families (Abe et al., Plant cell 9:1859-1868, 1997; Shinozaki and Yamaguchi-Shinozaki, Plant Physol. 115:327- 334, 1997, incorporated herein by reference).

After the Hahb-4 promoter was isolated and sequenced, sequences involved in driving gene expression were searched for. In particular, sequences that are organ specific and responsive to water stress and the presence of ABA are provided. To this end, Arabidopsis plants are transformed with a construct comprising a reporter gene (gus gene) under the control of the entire sequence of the promoter or part of said sequence.

The full sequence of the promoter is cloned into one construct. To this end, two specific oligonucleotides were designed that hybridized to the ends of the promoter region and used in a PCR reaction using the sunflower genomic DNA as template. As a product of the amplification reaction, two bands of about 1000 and 1200 bp were obtained. Considering that the growing material used for these experiments was isolated from a hybrid (contiflor 15), the presence of two bands seems to indicate the presence of two different alleles in the genome.

Both PCR products were cloned into pGEM-Teasy vector (Promega). As shown in Example 4, the PCR product was cut with restriction enzymes BamHI and HindIII and cloned into vector pBI101.3 ( FIG. 19 ). Two constructs were obtained, each including one of the alleles of the promoter driving the expression of the gus gene.

Then, the different promoter fragments including the transcription start site were cloned into the vector pBI101.3. For cloning of promoter fragments farther from the transcription start site, a modified pBI vector carrying a minimal promoter including a TATA box (-90CaMV35S) was used. The resulting clones are described below:

Clone 416: a fragment containing the 0-400 region (from the transcription start site up to IPCR4) cloned into the promoter on HindIII/BamHI of pBI101.3 (SEQ ID No. 4). Clone 416 includes nucleotides 805-1221 of SEQ ID No.3.

Clone 1015: cloned into SalI/BamHI of pBI101.3 containing the 0～-1015 promoter region obtained with nucleotides IPCR10 and IPCR8 (SEQ ID No.5 and SEQ ID No.6 respectively) (Xiao et al fragment of the gene).

Clone 1221: cloned into SalI/BamHI of pBI101.3 containing the 0-1221 promoter region (large alleles).

Clone 318: the 0～-300 region (from Transcription start site up to fragment of IPCR6 (SEQ ID No. 7)). Clone 318 includes nucleotides 904-1221 of SEQ ID No. 3.

Clone 211: the 0～-211 region (from Transcription start site up to fragment of IPCR7 (SEQ ID No. 8). Clone 211 includes nucleotides 1011-1221 of SEQ ID No. 3.

Clone 608: 608 bp corresponding to the 5' promoter region (amplified with IPCR5/IPCR10 (SEQ ID No.9/SEQ ID No.5)) cloned into SalI of pBI-90 with a minimal promoter Fragment (from large allele). Clone 608 includes nucleotides 15-622 of SEQ ID No. 3.

Clone 407: 407 bp corresponding to the 5' promoter region (amplified with IPCR5/IPCR10 (SEQ ID No.9/SEQ ID No.5)) cloned into SalI of pBI-90 with a minimal promoter Fragment (from minor allele). Clone 407 includes nucleotides 15-409 of SEQ ID No. 10.

Arabidopsis plants were then transformed with the constructs described above, transformed plants were selected, and multiple independent lines were picked for isolation of homozygous progeny in the third generation. Selected homozygous plants were used to characterize the allelic and distinct regions of the Hahb-4 promoter by histochemical and fluorometric assays.

The 0-400 region of the promoter was sufficient to drive the expression of the gus gene in the cotyledons of 2-day-old germinated seedlings. This expression was not detected in young seedlings germinated for 10 days.

Expression of the gus gene in the cotyledons of 2-day-old germinated seedlings was also observed when constructs including the complete fragment of the minor allele were used. However, unlike the 0 to -400 region, the gus gene was strongly expressed in leaves and roots of young seedlings (Fig. 20). Finally, during the breeding phase, no expression of any gus gene was detected.

These results illustrate that although expression occurs in the same organ, the intensity of this expression varies. Although the sequences necessary to drive specific expression in cotyledons and leaves are in the 0-400 region, there may be sequences important for transcriptional activation in the -400--1015 region.

When analyzing the expression of the gus gene in the roots of transgenic plants, it was found that expression in the roots of 20 day old plants driven by the 0-400 construct was consistently stronger in the lateral root primordia and in the middle region of developed lateral roots. Furthermore, it was found that in the roots of transgenic plants transformed with the 0-400 construct, the expression of the gus gene was also detected in the cells of the vascular column (Fig. 21). 10 days after germination, strong expression was observed at all primary and lateral root sites, and a stronger expression in the root zone (Fig. 21c).

The results obtained by the fluorescence assay indicated that the construct including the whole fragment had a 10-fold higher priming activity than that of constructs 0-400. Also, as shown in Figure 22, ABA was able to induce constructs 0 to -400.

Analysis of multiple independent lines of plants transformed with constructs including the 0 to -300 region or the 0 to -200 region showed that the gus gene was expressed at different developmental stages in all organs studied and that ABA did not induce the the boot area described above.

The present inventors also disclosed that only one intron occurs in the region interrupting the leucine zipper coding sequence. This intron is 101 bp in length, and is specifically located between the sequences encoding the sixth and seventh heptaploids (108 and 109 amino acids) of the leucine zipper domain, and correspondingly occurs in introns of other organisms The 5'-GT.......AG-3' rule. A schematic diagram of the location and sequence of the intron is shown in FIG. 1 .

Therefore, the present invention includes the Hahb-4 promoter (SEQ ID No. 1) and the cDNA (SEQ ID No. 2) of said gene, wherein said gene encodes a protein of the Hd-Zip family of sunflower. This promoter has two alleles that have been cloned and sequenced, with a region around -900 nucleotides that differs or is not conserved. The large allele comprises a sequence of 1221 bp, and the minor allele comprises a sequence of 1015 bp (SEQ ID No.3 and SEQ ID No.10, respectively).

Analysis of the promoter nucleotide sequence revealed regions of homology to different sequences involved in various environmental factors, such as light, abscisic acid, and hormone responses. The promoters of the present invention include a putative ABA response element, generally designated ABRE, and an element responsive to water-deficiency or cold-induced stress, designated DRE. Consensus sequences linking transcription factors involved in environmental factor responses were also identified.

When the activity of different regions of the promoter of the present invention was analyzed, the results obtained indicated that despite having two putative elements ABRE between the transcription start site and 300 nucleotides upstream, this fragment was unable to drive the reporter gene gus activity, indicating that although this fragment is necessary, it is not sufficient to initiate transcription. In contrast, by using the first 400 nucleotides adjacent to position +1, expression of the gus gene has been observed in cotyledons and roots 2 days after germination. Expression is not very strong, but specific and can be induced by ABA.

On the other hand, the full fragment of the promoter region produces an expression level at least 10-fold higher than that produced by the 0 to -400 fragment. In contrast, the expression was evident at later stages of development (plants germinated up to 20 days old) and in the central region of the root.

The promoter (both alleles), fragments or parts thereof of the invention can be used to drive the expression of any gene of interest. Preferably, the promoters (both alleles or fragments thereof) of the invention may be used in nucleic acid constructs useful for transforming host cells, wherein said promoters drive the expression of any protein of interest. Host cells can be bacteria, yeast, animal cells or plant cells.

It is also possible to construct a vector in which the promoter of the present invention drives the expression of the Hahb-4 gene of the present invention in plant cells. The constructs are useful for obtaining transgenic plants tolerant to water stress. The transgenic plant carrying the Hahb-4 gene of the present invention and the promoter of the present invention may be commercial crops including sunflower, wheat, barley, soybean, potato, corn, sugarcane or rice.

The present invention can be better understood according to the following examples, but these examples do not limit the protection scope of the present invention. On the contrary, it must be clearly understood that those of ordinary skill in the art can propose many other embodiments, modifications and equivalent changes of the content of the present invention after reading the specification of the present invention without departing from the essence and/or additional aspects of the present invention. Scope of Claims.

Example

Example 1: Isolation and identification of genes encoding proteins belonging to the Hd-Zip I family A new gene Hahb-4 in sunflower

A. Isolation of Hahb-4 gene cDNA

To isolate partial cDNA clones containing homeobox sequences, as described above (González and Chan, Trends in Genetics 9:231-232, 1993, cited here as a reference), the sunflower stem cDNA library constructed in λgt10 was used. Total DNA was subjected to a polymerase chain reaction (PCR)-based strategy. The 3' of the Hahb-4 transcript was obtained by PCR using the λgt10 sequencing primer and the specific primer H41 (5'-GGCGGATCCAACAGAAACAACCACCAGG-3' (SEQ ID No.11)) matching 81-100 nucleotides of the cDNA sequence. The sequence of the end (SEQ ID No.2 and Fig. 1). According to Frohman (Frohman Cloning PCR products. In The Polymerase Chain Reaction, eds. K.B. Mullis, F. Fré, & R.A. Gibas, pages 14-37. Birkhauser, Boston, MA, USA, 1994, cited here as a reference), Using specific oligonucleotide IPCRO 5'-GGCGGATCCCCTGGTGGTTGTTTCTGTT-3' (SEQ ID No.12) and primers Qt and Qo (respectively SEQ ID No.13 and 14), RNA and polyA from water-stressed sunflower stems Rapid amplification of cDNA ends (RACE) was performed to obtain the 5' ends of the transcripts.

B. Isolation of the Hahb-4 Genomic Sequence

According to Ochman, Ayala and Hartl (In Methods of Enzymology (ed R. Wu) Vol 218, pages 309-321. Academic Press, San Diego, CA. USA, 1993), the 5' non-specificity of Hagb-4 was determined using inverse PCR. Characterization of transcribed regions. Sunflower genomic DNA was partially digested with Sau3A or HindII under controlled conditions. Following digestion and purification, DNA circularization was performed overnight in the presence of 5 U T4 DNA ligase (Promega Corp., Madison, WI, USA) according to the manufacturer's instructions. The primer pair used for amplification is 5'-GGCGGATCCCCTGGTGGTTGTTTCTGTT-3'(SEQ ID No.12) and 5'-GCCGAATTCAGATTGAGCAAGAGTATAAC-3'(SEQ ID No.15), or 5'-ACCTTTATAAAGACCACTC-3'(SEQ ID No. .16) and 5'ACGCAATGGTGAGTTGTAC-3' (SEQ ID No.17).

C. DNA sequence analysis

The PCR product was cloned into pUC119 or pGEM-Teasy (Promega Corp.). The sequence of the insert was obtained by the chain termination method using the fmol sequencing system (Promega Corp.).

Example 2: Experiments illustrating the induction of Hahb-4 by water deficiency

A. Plant Material, Growth Conditions, and Water Stress Treatment

For sunflower (Helianthus annuus L.) (sunflower cultivar contiflor 15, from Zeneca, Balcarce, Argentina; or cultivar Sunweed, from Lyon, France - Poulenc) seeds were surface sterilized and grown on filter paper in Petri dishes for 4 days. Seedlings are then transferred to plastic carriers containing Hoagland's medium and grown until they have 6 leaves (approximately 3 weeks). Water stress was applied by transferring 4-day-old seedlings to Petri dishes with dry filter paper, or by removing plants from hydroculture. The processing time is shown in the graph.

B. RNase Protection Assay

For RNase protection assays, total RNA ( ¹⁵ μg) prepared above (Almoguera C., et.al.; Plant Molecular Biology 19:781-792, 1992) hybridized with a specific Hahb-4 RNA probe synthesized by in vitro transcription.

A template including the insert corresponding to the coding region between +81 and +429 (Figure 1 and SEQ ID No. 1) was cloned into the SpeI/BamHI site of pBlue-script SK-. The BamHI site absent in the cDNA was derived from amplification using oligonucleotide H41 as described above. The template DNA was restricted with EcoRI to allow transcription of a 411-nucleotide RNA probe containing 63 nucleotides of the vector (from the T3 promoter to the SpeI site of the multisite linker, and from BamHI to the EcoRI site) . The conditions for RNA probe preparation, hybridization, digestion using RNase A, and subsequent electrophoretic analysis of protected RNA fragments are described above (Coca M: A: et.al.; Plant Molecular Biology 31:863-876, 1996 , cited here as a reference).

C.Northern Analysis

Total RNA (20 μg) was denatured using formamide and formaldehyde essentially as described by Sambrook, Fritsch and Maniatis (1989), separated on a 1.5% (w/v) agarose/(6%) formaldehyde gel, and separated The denatured RNA of the sample was loaded onto a nylon membrane (Hybond N; Amersham-Pharmacia, Buckinghamshire, UK).

At 65°C, in 6×SSC (1×SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) bovine serum albumin , 0.1% (w/v) Ficoll (water-soluble polysucrose) (Ficoll), 0.5% (w/v) sodium dodecylsulfonate (SDS) for overnight hybridization. The 3' non-coding region of Hahb-4 cDNA and the last 177 nucleotides corresponding to the coding region were labeled with [ ³² P]dATP (1×10 ⁸ dpm μg ^-1 ) by random labeling (Sambrook et al. 1989) The SpeI/EcoRI fragment excluding the Hd-Zip region was used as a probe. Overnight autoradiography was performed on filter paper using Bio-Max membranes and transscreens (Eastman Kodak, Rochester, NY, USA). To determine the amount of total RNA added in each lane, the filter paper was then reprobed with 25Sr RNA from Vicia faba under conditions similar to those described above, except that the hybridization was performed at 62°C.

D. Preparation of nuclei:

According to the technique (Methods in Plant Molecular Biology. A Laboratory Course Manual, pages 233-260. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1995, cited by reference) described by Maliga et al. , or ABA-treated seedlings (4 days old) to prepare sunflower cores and core extracts. Protein profiles were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) as described by Sedmak J. et al. (Analytical Biochemistry 79:544-552, 1977), and total protein concentrations were determined.

E. DNA binding test

For electrophoretic mobility shift assay (EMSA), an aliquot (30 μg) of purified nucleoprotein was mixed with complementary oligonucleotides 5'-AATTCAGA TCTCAATAATTGAGAG-3' and 5'-GATCCTCTCAATTATTG GATCTG-3' (SEQ ID No.18 and SEQID No.19) (Hahb-4 binding site is marked underlined) hybridization of double-stranded DNA (0.3 ~ 0.6ng, 30000c.pm, by using the Klenow fragment of DNA polymerase in 3 ' end was labeled by adding [ ³² P]dATP) incubation. Contains 20mM HEPES-NaOH (pH7.6), 40mM NaCl, 0.2mM ethylenediaminetetraacetic acid (EDTA), 1.0mM dithiothreitol (DTT), 0.5% Triton X-100, 20% glycerol, and 1.5μg The poly(dI-dC) binding reaction (20 μL) was incubated at 25°C for 20 minutes, supplemented with 2.5% (w/v) water-soluble polysucrose (Ficoll), and immediately added to the electrophoresis gel (dissolved in 0.5× TBE and 5% acrylamide, 0.08% diacrylamide in 2.5% glycerol; 1 x TBE on 90 mM Tris-boronic acid, pH 8.3, 2 mM EDTA). Gels were electrophoresed in 0.5X TBE at 30 mA for 1.5 hours and dried prior to autoradiography.

Embodiment 3: Make Arabidopsis thaliana carrying sunflower Hahb-4 of the present invention have water stress tolerance Receptivity test

A. Biomaterials

The E. coli line DH5α and the Agrobacterium tumefaciens line GV2260 were used. For the transformed plant experiments, seeds of the Arabidopsis ecotype Columbia-0 were used.

B. Molecular cloning

和Willmitzer(

and Willmitzer，Nucleic Acid Research 16：9977，1998)公开的方法，将质粒DNA导入根瘤农杆菌中。具有其中gus基因已由Hahb-4取代的pBI121质粒的根瘤农杆菌命名为ATH4。In order to clone Hahb-4 under the regulation of the CaMV35S promoter, by using the clone corresponding to the cDNA as a template (Gago et al., Plant Cell & Environment 25:633-640, 2002, cited here as a reference), and using two A specific oligonucleotide T1: 5'-GCGGGATCCACCATGTCTCTTTCAACAAGTA-3' hybridized to the two ends of the coding region; (SEQ ID No.20) and T2: 5'-GCCGAGCCTTTTAGAACTCCCAACCACCFTTTG-3' (SEQ ID No.21) were performed PCR reaction. In this approach, the 3' and 5' regions that do not encode messenger RNA are removed, thus reducing possible post-transcriptional regulatory effects. The oligonucleotides were designed to enable the introduction of fragments amplified using the oligonucleotides at the BamHI and SacI sites of the plasmid. In addition, a sequence determined to be consistent with optimal translation was added at the oligonucleotide (oligonucleotide T1) at the 5' cDNA end. Purify the PCR reaction product, digest the PCR reaction product with the above-mentioned enzymes, and use Escherichia coli for transformation, and clone the enzyme-digested PCR reaction product into pBI 121 vector for transformation. Once the desired clone is obtained, the

and Willmitzer (

and Willmitzer, Nucleic Acid Research 16:9977, 1998) to introduce plasmid DNA into Agrobacterium tumefaciens. Agrobacterium tumefaciens with the pBI121 plasmid in which the gus gene has been replaced by Hahb-4 was named ATH4.

Cloning and detection techniques were employed in Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

C. Arabidopsis Transformation

The method used for transformation of Arabidopsis was that described by Clough and Bent using dipping (floral dipping) (Clough and Bent, Plant J. 16:735-743, 1998). The seeds obtained from the transformation experiments were sterilized and cultured in petri dishes containing MS medium supplemented with 40 mg/L kanamycin. Resistant plants (F1) were transferred to soil and grown to produce seeds. The resulting line (F2) was analyzed for the presence of the transgene (Hahb-4 gene) using PCR and the expression of the corresponding transcript was also analyzed by Northern. Lines expressing the transgene are propagated until homozygous daughter lines are obtained.

According to the methods published by Carpenter and Simon (Carpenter, C. and Simon, A. (1998) Preparation of RNA. EnIn: Methods in Molecular Biology, vol 82: Arabidopsis Protocols. J.M.Martinez-Zapater and J.Salinas (Eds.), Humana Press Inc., Totowa, New Jersey) prepared total RNA from Arabidopsis. For Northern analysis, published methods were performed (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1983) Current Protocols in Molecular Biology. John Wiley & Sons , N.Y.).

For analysis of transformed Arabidopsis plants by PCR technique, total DNA was prepared using the rapid method published by Li and Chory (Li, J. and Chory, J. (1998) Preparation of DNA from Arabidopsis. In: Methods in Molecular Biology, vol. 82: Arabidopsis Protocols. J.M. Martinez-Zapater and J. Salinas (Eds.), Humana Press Inc., Totowa, New Jersey).

D. Phenotyping

D1. Analysis in Petri dishes

Seeds of Arabidopsis thaliana were sterilized by washing with 70% (v/v) ethanol for 1 minute, chlorine 5%-SDS 1% for 15 minutes and sterile distilled water 3 times. Then, the seeds were suspended in 8 ml of 0.1% agar, and sowed in a 150 mm petri dish containing MS medium supplemented with vitamins produced by Gamborg. The petri dish was placed at 4°C for 2 days, and then placed in a light-adjusted and temperature-regulated incubator (24°C light for 16 hours, 21°C dark for 8 hours). The required lighting conditions (150-200 μE/m ² ) were obtained artificially by keeping the plants at a distance of 25 cm from 6 fluorescent tubes, and inserting white light and Grolux tubes (purchased from Sylvania) in adjacent positions.

Manipulation of plant material was performed under sterile conditions. MS medium was sterilized in an autoclave, and vitamins were then added by filtration.

D2: Tests in soil

The tests in soil were carried out in pots with a diameter of 12 cm and a height of 10 cm. According to the experiment, about 1 to 3 Arabidopsis seeds were planted in each pot, and they were distributed equidistantly. Cover the pots with clear plastic material until emergence, then remove the plastic. 16 pots each were placed in plastic trays and the plants were grown in an incubator under the same light conditions as disclosed above. Watering water was added to plastic trays.

For the water stress test, 1, 1.5 or 2 liters of water were initially added to the pans. In all cases no water was added until the end of the reproductive cycle. These watering conditions kept the incubator at very low humidity conditions to put the plants under water stress when the plants grew 2 pairs of leaves, full petals or developed flowers respectively. Water stress can be observed by drought and fissures in the soil, loss of leaf swelling, and eventual death of the plant.

Example 4: Hahb-4 promoter, constructs containing different fragments of the promoter and Isolation and Characterization of Transgenic Plants of Arabidopsis Containing the Construct.

A. Plant Material, Culture, and Handling Conditions

Sunflower seeds (Helianthus annuus L.) (Zeneca) of Contiflor 15 were used. For plant transformation experiments Arabidopsis ecotype Columbia-0 was used.

Seeds of Arabidopsis thaliana were sterilized by washing with 70% (v/v) ethanol for 1 min, chlorine 5%-SDS 1% for 15 min and sterile distilled water 3 times. Then, the seeds were suspended in 8 ml of 0.1% agar, and sowed in a 150 mm Petri dish containing MS medium supplemented with vitamins produced by Gamborg. The dishes were kept at 4°C for 2 days, and then placed in a light and temperature controlled incubator (24°C light for 16 hours, 21°C dark for 8 hours).

Arabidopsis plants were cultured in an incubator with adjustable light and temperature (24°C light for 16 hours and 21°C dark for 8 hours). The required lighting conditions (150-200 μE/m ² ) were obtained artificially by keeping the plants at a distance of 25 cm from 6 fluorescent tubes, and inserting white light and Grolux tubes in adjacent positions.

B. Purification of Plant DNA

To extract the total DNA of the plant, the method disclosed by Doyle, J.J. and Doyle, J.L. (Doyle, J.J. and Doyle, J.L. (1987). A rapid DNA isolation process (Phytochemical Bulletin 19, 11-15 ).

C. Plasmids, strains and molecular cloning methods used:

Plasmid pGEM.-T easy (Promega) was used to clone the product of the amplification reaction using Taq DNA polymerase (Promega).

The pBI101 plasmid (Jefferson et al., EMBO J. 6:3901-3907, 1987), which is a derivative of the pBIN19 binary vector, was also used. It contains the gene (gus) encoding E. coli beta-glucuronidase with a polyadenylation signal (nos) for nopaline synthase. The pBI101 plasmid also includes the nptII gene that confers kanamycin resistance to plants. Other related sequences of the vector include the gene for making the bacteria resistant to kanamycin, and the bacterial replication origin RK2. This plasmid was used to clone different fragments of the sunflower Hahb-4 promoter in Arabidopsis with a single restriction site located upstream of the gus gene.

和Willmitzer描述的方法(

，R.andWillmitzer，L.(1988)Storage of competent Agrobacterium cells fortransformation was carried out according to.Nucleic Acids Res.16，1977)。By first using E. coli DH5α competent host cells to clone the construct with T-easy or pBI vector, then carry out the classical transformation method or electroporation as described by Sambrook (Sambrook, J., Fritsch, EF and Maniatis, T. (1989 ) Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). For the preparation of Agrobacterium tumefaciens competent cells and their further transformation, use

and the method described by Willmitzer (

, R. and Willmitzer, L. (1988) Storage of competent Agrobacterium cells for transformation was carried out according to. Nucleic Acids Res. 16, 1977).

D. Molecular cloning of DNA fragments

In order to clone the promoter region of the gene of the present invention, the inverse PCR strategy described by Ochman et al. (Ochman, H., Ayala, F.J. and Hartl, D.L. (1993) Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. In: Methods in Enzimology vol 218. R. Wu (Ed.), Academic Press, San Diego, CA) proceeded as follows.

Sunflower genomic DNA was extracted as described above, taking 5 μg of DNA, and using 1-3 U of SauIIIA or HindIII (Promega) per μg of DNA. After enzymatic digestion has been checked (by adding aliquots to a 0.7% (p/v) agarose gel), then by adding 1/10 volume of 3M NaAc (pH 5.2) and 2 volumes of anhydrous Ethanol precipitated fragments.

To facilitate religation of restriction fragments, reactions were performed in a final volume of 100 μL, and 2, 10 and 20 ng of fragments obtained in each reaction and 5 U of T4 DNA ligase (Promega) were added. The reaction was carried out at 14°C for 16 hours. The fragments were precipitated and purified for use as templates in PCR reactions.

-Teasy载体(Promega)中。检测克隆后，测定对应的序列，并且设计下一步所需要的寡核苷酸。在下一克隆步骤中所用的寡核苷酸的序列和位置如图23所示。With the recovered DNA of the fragment digested with SauIIIA as a template, oligonucleotides IPCR0/IPCR1 (SEQ ID No.12/SEQ ID No.15) were used, and oligonucleotides IPCR2/IPCR3 (SEQ ID No. 15) were used when DNA was digested with HindIII. ID No.16/SEQ ID No.17) for the PCR reaction in the first step. According to the manufacturer's suggested method, the obtained fragments were cloned into

- in Teasy vector (Promega). After detecting the clones, determine the corresponding sequence, and design the oligonucleotides required for the next step. The sequences and positions of the oligonucleotides used in the next cloning step are shown in FIG. 23 .

IPCR0[5'-GGCGGATCCCCTGGTGGTTGTTTCTGTTG-3']

IPCR1 [5′-GCCGAATTCAGATTGAGCAAGAGTATAAC-3′]

IPCR2 [5′-ACCTTTATAAAGACCACTC-3′]

IPCR3 [5′-ACGCAATGGTGAGTTGTAC-3′]

IPCR4 [5′-GCGAAGCTTGATGCGAACGAGTGGTTTA]

IPCR5 [5′-ATTTCGCAAGTAGTCCATT-3′]

IPCR6 [5′-CCCAAGCTTAACCTAAGTCCGCCTTTG-3′]

IPCR7 [5′-GGCAAGCTTATCTCAACCGAAAGTGAC-3]

Finally, since this technique is suitable for fragment cloning, using sunflower genomic DNA as a template, using polymerase chain reaction, and two oligonucleotides designed to hybridize to the corresponding ends (IPCR10: GCGGTCGACACCTGGCACATCGTATCT (SEQ ID No. 5) and IPCR8: CGCGGATCCGAGGGTTTGATAAGTGAT (SEQID No. 6)) amplifies the sequence knowledge of the complete fragment. The amplified product was cloned into the pGEM-T easy vector, and then its sequence was determined.

The strategy used for inverse PCR and construction of recombinant plasmids for transformed plants is shown in Figure 23.

The different fragments of the promoter including the transcription initiation site were then cloned into the 101pBI.3 vector. Alternatively, to study promoter fragments farther from the transcription start site, a modified pBI vector carrying a minimal promoter (-90CaMV35S) including a TATA box was used. This vector also contains the gus gene as an indicator, so that the promoter activity of each cloned fragment can be determined.

E. Cloning of the Hahb-4 intron:

In order to clone the Hahb-4 intron, by using sunflower genomic DNA as a template and using oligonucleotides IPCR1 (5'-GCCGAATTCAGATTGAGCAAGAGTATAAC-3 (SEQ ID No.15) and N1 (5'-GCGGGATCCGTCTGGCAGTTGTTCTTC-3'SEQ ID No.22)) PCR reaction was carried out. The resulting product was digested with EcoRI and BamHI (sites provided by nucleotides) and subsequently cloned into pUC119 plasmid which had been digested with the same enzymes. After verifying the presence of introns of the expected size in some of the resulting clones which were white, their sequences were determined as described below.

F. Transformation of Arabidopsis plants with the constructs obtained in the above steps.

The method used to transform Arabidopsis plants was the dip method (floral dip) as described by Clough and Bent (Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743).

For each construct, 10-12 pots with a diameter of 10 cm were filled with soil and covered with fabric. Fix the covering cloth so that it adheres well to the soil surface. The seeds were then well dispersed in the soil and the pots were placed in trays covered with transparent nylon paper. The plants were grown under the above conditions; after one week, the nylon paper was removed and the strongest plants were selected.

The plants are grown until flowering (approximately 4 weeks). When the flower stalk is exposed (petal body of 1 to 2 cm), be careful not to damage the stem and leaves and cut off the inflorescence. 4-6 days after the above-mentioned cutting, new inflorescences grow. Wait until all inflorescences have at least 4 unopened flowers before proceeding with transformation.

In order to prepare a transformant, Agrobacterium was cultured with stirring at 28° C. for 24 hours in three flasks containing 10 ml of LB medium supplemented with 50 mg/L rifampicin and 50 mg/L kanamycin. These cultures were inoculated in 3 Erlenmeyer flasks containing 200 ml of the same medium and grown to stationary stage (stirred at 28°C for 12-16 hours). Cells were harvested by centrifugation at 5500 xg for 20 minutes. The pellet obtained by centrifugation was resuspended in 1 liter of 5% sucrose solution containing 300 μSilwet L-77 (OSI Specialties, Inc.), and the suspension was placed in a sedimentation cup with a magnetic stirrer. Soak the plants in it for 10-60 seconds while preventing the liquid from touching the soil. Then, place the pots horizontally on a tray, cover them with nylon paper, and place them in an incubator. The next day, the pots were placed in their normal position, water was added to the trays, and the plants were grown until the seeds matured (4-5 weeks).

Finally, the seeds from each pot were collected separately, and the siliques and soil were washed by hand. Seeds were kept in the refrigerator for further analysis.

For selection of transformed plants, seeds collected in transformation experiments were sterilized and seeded in Petri dishes containing MS medium supplemented with 40 mg/L kanamycin as described above. Within the first day of cultivation in the incubator, most of the seeds germinated (95-99%).

At about 10 days, the cotyledons of susceptible plants turned yellow, while those of transformed plants remained green. The plates were left in the incubator for a further 7 days, during which time very green true leaves could only be observed in transformed plants. Non-transformed plants died. Transfer resistant plants to pots with soil. To prevent a sudden drop in humidity, place the pots in a tray with water and cover them with transparent nylon paper for a week. After this stage, the paper covering the pots was removed and the plants were grown until the seeds matured, collected and placed in a refrigerator for further analysis. In addition to selecting plants by their resistance to kanamycin, the presence of the transgene was detected by specific PCR.

Finally, homozygous lines of third generation plants were obtained by growing in plates containing kanamycin and observing 100% tolerance of multiple progeny lines.

G. Histochemical Analysis of β-Glucuronidase Activity of Transgenic Plants of the Invention

The Arabidopsis plants of the present invention obtained through kanamycin resistance selection and PCR reaction identification are subjected to a histochemical test for β-glucuronidase activity. Embryos and organs used for the experiment were washed with 50 mM _Na2HPO4 buffer, pH ₇ . It was then transferred to a solution of 50 mM _{Na2HPO4 pH7} , 0.1% Triton X-100, 2 mM X-gluc (5-bromo-4-chloro-3-indole-β-D-glucuronide), Vacuum was applied for 5 minutes and incubated at 37°C in the dark for 2-12 hours. After incubation, they were placed in 10% formaldehyde solution, 50% ethanol, and 5% acetic acid for 10 minutes at room temperature. Remove formaldehyde, add 70% ethanol, and store at 4°C.

Histochemical analysis was performed on 2-, 10- and 20-day-old seedlings grown in Petri dishes containing MS medium and 0.8% agar, and adult plants grown in pots containing soil.

H. Fluorescent analysis of the β-glucuronidase activity of transformed plants of the present invention:

Transformed plants identified as positive by PCR reaction for antibiotic resistance and expressed β-glucuronidase by histochemical assay were used to study the regulation of the promoter region by fluorescence detection. 30-50 seeds of each line were cultured in 30 mm Petri dishes with MS-agar medium. After proper growth, plants were transferred to tubes with MS liquid medium with or without ABA added and incubated for different times as detailed below. After incubation, the plants were placed in liquid nitrogen until use.

Protein extracts are obtained by homogenizing plant material (approximately 2-5 mg) into a fine powder. Then 500 μl extraction buffer (50 mM Na ₂ HPO ₄ pH 7, 10 mM EDTA, 0.1% SDS, 10 mM β-mercaptoethanol, 1% Tritón X-100) was added. Transfer the suspension to a microcentrifuge tube and centrifuge at 13,000 x g for 10 min at 4 °C. Discard the supernatant and place the microcentrifuge tube containing the pellet on ice.

Fluorescent reactions were performed according to the Jefferson method (Jefferson, RA, Kavanagh, TA and Bevan, MW (1987) Gus fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907). 100 μl protein extract was added to 100 μl methanol and 300 μl substrate MUG (4-methylumbelliferyl β-D-glucuronide). A 100 [mu]l aliquot was pipetted and the fluorescence assay was performed immediately at time 0. The remaining 400 μl was incubated in a 37°C water bath and 100 μl aliquots were removed at 30, 60 and 120 minutes. To stop the reaction, 0.9 ml of 0.2M Na ₂ CO ₃ was used. The value of the fluorescence response is expressed as product per minute (pmol)/total protein (mg) as a function of the concentration of the product 4-MU (7-hydroxy-4 methylumbelliferone) according to the RFU plot.

Fluorescence measurements were performed in 1 ml discs using Bio-Rad's VersaFuor ^™ Fluorescence System.

I. Quantification of Total Protein

Protein extraction was determined by using the method published by Sedmak and Grossberg (Sedmak, J. and Grossberg, S. (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G-250. Anal. Biochem. 79, 544-552) The concentration of soluble protein in the substance. Bovine serum albumin (BSA) was used as a standard sample.

J. Determination and Analysis of Sequences

To determine the DNA sequence of the obtained construct, a commercially available device T7 sequencing kit (Amersham Biosciences), based on the Sanger method (Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors.Proc.Natl.Acad.Sci.USA 74,5463-5467), combined with DNA elongation/termination in only one step. Furthermore, these sequences were verified with an automated sequencer (Service given High Complexity Laboratory, INTA, Castelar, Bs. As. Argentina).

In order to identify regulatory sequences within the promoter region, the database PLACE (Higo, K., Ugawa, Y., Iwamoto, M. and Korenaga, T. (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res.27, 297-300) and PlantCARE (Rombauts, S., Dehais, P., Van Montagu, M. and Rouze, P. (1999) PlantCARE, a plant cis-acting regulatory element database. Nucleic Acids Res.27, 295 -296).

When the technique used is not specified, the method described by Sambrook, J., Fritsch, E.F., and Maniatis, T. ((1989) Molecular Cloning: A Laboratory Manual. Secondedition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Ausubel was used. , F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. ((1983) Current Protocols in Molecular Biology. John Wiley & Sons, N.Y.).

While the preferred embodiments of the invention have been illustrated and described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the scope of the invention as defined in the appended claims.

Claims (6)

1. An isolated nucleic acid molecule selected from the group consisting of the following nucleic acid molecules: (a) a nucleic acid molecule shown by the nucleotide sequence SEQ ID No.3; (b) a nucleic acid molecule shown by the nucleotide sequence SEQ ID No.10; and (c) a nucleic acid molecule shown in the nucleotide sequence of 805 to 1221 nucleotides of SEQ ID No.3, Wherein, the nucleic acid molecule can promote the expression of the heterologous nucleic acid molecule in the transformed cell. 2. A nucleic acid construct comprising the first nucleic acid molecule selected from the following nucleic acid molecules: (a) a nucleic acid molecule shown by the nucleotide sequence SEQ ID No.3; (b) a nucleic acid molecule shown by the nucleotide sequence SEQ ID No.10; and (c) a nucleic acid molecule shown in the nucleotide sequence of 805 to 1221 nucleotides of SEQ ID No.3, Wherein, the first nucleic acid molecule is operably linked to the second nucleic acid molecule encoding the protein of interest and the 3' untranslated region. 3. A host cell stably transformed with the nucleic acid construct of claim 2, wherein the host cell is not a plant cell. 4. The host cell of claim 3, wherein the host cell is selected from bacterial cells and fungal cells. 5. A method for expressing at least one protein of interest in a host cell, the method comprising: Introducing the nucleic acid construct of claim 2 into a host cell and causing the host cell to produce the protein of interest. 6. A method for obtaining a transgenic plant expressing at least one protein of interest, the method comprising: Stable transformation of plant cells or cell cultures with the nucleic acid construct of claim 2; The cell or cell culture is regenerated into a whole plant expressing at least one protein.

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