MXPA00007829A - Pollen specific promoter - Google Patents

Abstract

A recombinant nucleic acid which comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen. The DNA sequence of the promoter is depicted in figure 5. Expression cassettes and expression systems as well as transformation methods, transformed plants including the promoter sequence of the invention are also claimed. The nucleic acid may be used inter alia in the production of male sterile plants and/or hybrids as well as in the transformation of pollen.

Description

SPECIFIC PROMOTER OF POLLEN DESCRIPTION OF THE INVENTION The present invention relates to a promoter sequence which is specific for pollen, for constructs and transgenic plant cells, as well as plants comprising the promoter as well as methods for transforming pollen and controlling fertility in plants using this promoter. In order to introduce desirable genetic traits of two plants into a single plant, such as a variety of hybrid crossbreeding represents the traditional approach. In order to reliably obtain consistent hybrids, it is necessary to ensure that the self-pollination of the parent plants is not carried out. This can be obtained by ensuring that one of the parental lines is a sterile male. Several techniques for producing male sterility are known and have been proposed in the art. One method involves the removal of male anthers and spikes from the female parent plant, either manually or mechanically. This plant can then only be fertilized by plant pollen > male parent and therefore their progeny will be hybridized. However, such a process requires a lot of work and is not always as reliable as it is possible that some female plants may lose the process of removing male spikes or in some cases, the plants develop secondary male spikes after it has been completed. the elimination of the male spikes. Furthermore, in this system, the male parent is able to self-pollinate so that it is necessary to physically separate the male and female parents to allow hybrid seeds to be harvested easily. This planting in block works well for maize because the pollen is light and is produced in large quantity. However, this solution is not applicable to species with heavier pollen and in species in which the male and female floral organs are contained within a flower, for example, wheat, rice. Chemical methods are also known to prevent the production of pollen. The U.S. patent No. 4,801,326 describes chemical substances which can be applied to the plant or soil to prevent the production of pollen. However, again, such techniques require a lot of work and are not completely reliable. It is essential that sufficient chemical is applied at appropriate time intervals to ensure that pollination does not occur, with the concomitant burden on the environment. In addition, these chemicals are very expensive. Genetic engineering has demonstrated an alternative way by which male sterility can be produced, maintained and / or subsequently restored in plants. Systems have been described in which inducible promoters which allow fertility to be adjuvanted or inactivated depend on the application of variability of an external compound. For example, WO 90/08830 describes the induction of male sterility in a plant by a sequence cascade of genes which express a protein which alters the biosynthesis of pollen. WO 93/18171 describes the use of a GST promoter to indefinitely express chalcone synthase (chs) and restore fertility in a male sterile plant that has become sterile by "removing" the gene, from the endogenous chs genes. In WO 93/25695 (PGS) a different solution is described. This is based on the use of a specific tapeto promoter to express a pollen mortality gene, varnasa, in tapétal cells, which are critical for the development of pollen and therefore interrupt pollen production. A restorer gene can be used to restore fertility (Mariani et al., Nature, vol 357: 384-387). There would be advantages in expressing genes which have an impact on the production of pollen in pollen directly, in a controllable way. Properly, fertility can be restored when desired.

One component of a system for genetically engineering male sterility is the availability of a promoter which specifically drives expression in either pollen or tissue or on which it depends on the production or development of pollen. Many of the characterized genes which are expressed in a specific or elevated way in pollen and pollen in germination, encode proteins that are likely to play a role in cell wall metabolism, for example, those that have homology to genes that code for enzymes involved in the degradation of pectin; polygalacturonase (SM Brown et al., Plant Cell (1990) 2: 263-274, SJ Tebbutt et al., Plant Mol. Biol. (1994) 25: 283-299), pectate lyase (HJ Rogers et al., Plant Mol Biol 20: 493-502 (1992), RA Wing et al., Plant Mol Biol 14: 17-28 (1989)) and pectin methylesterase (PME) JH Mu et al., Plant Mol Biol 25: 539-544 ( 1994)). Other genes highly expressed in pollen include those coding for clostaketal proteins (I. Lopez et al., Proc. Nati, Acad Sci USA 93: 7415-7420 (1996), HJ Roregs et al., Plant J. 4: 875- 882 (1993) and CJ Staiger et al., Plant J. 4: 631-641 (1993)), the putative ascorbate oxidase, a Ku itz protein inhibitor and many others whose function can not be inferred by homology to known genes. . The temporal expression of such genes has been studied and much has been found to be expressed late in the microsporogenesis that reaches a maximum in mature microsporocytes. In some cases, continuous expression has also been demonstrated in the pollen tube (AK Kononowicz et al., Plant Cell 4: 513-524 (1992)). These genes have been termed as "late genes." Most of the expression in this stage is from the vegetative cell instead of a generating cell and it is probable that most of these "late" genes are transcribed from a vegetative nucleus; although this has only been shown for a "late" gene (D. Twell, Plant J2: 887-892 (1992).) It is found that a different class of genes expressed in the anthers have a different expression program, and that it is detectable first so soon after the stage of detection and that it declines in well expression before pollen maturation.It is likely that the main role of these "early" genes may be during the differentiation and development of the microspores instead of the In addition, US Patent No. 5,086,169 (Mascarenhas) describes isolation of the first pollen specific promoter from maize Applicants have isolated an additional promoter which is specifically expressed only in pollen tissue. it is derived from a gene expressed from "late" pollen isolated from maize, ZmC5.

Thus, according to a first aspect of the present invention, there is provided a recombinant nucleic acid sequence which comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen. As used herein, the term "fragment" includes one or more regions of the basic sequence which retain promoter activity. Where the fragments comprise one or more regions, they may be directly joined or separated by additional bases. The term "variant" with reference to the present invention means any substitution, variation of, modification of, replacement of, deletion or addition of one or more nucleotides from or to the nucleic acid sequence that provides a resulting sequence that shows expression pollen promoter. The term also includes a sequence that can substantially hybridize to the nucleic acid sequence. As used herein, the term "ZmC5 gene" refers to a maize gene which codes for a sequence of 563 amino acids as described herein.

A cDNA sequence encoding this sequence is identified in EMBL Y13285. . The promoter sequence of the present invention is constituted within the clone deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 40915 on January 26, 1998. This is a Sal I fragment derived as described in the following. The promoter region is within a region which consists of approximately 2 kb of a sequence towards the 5 'end of the transcription star site of the maize ZmC5 gene, as shown in Figure 1 below. According to a preferred embodiment of the present invention, there is provided a recombinant nucleic acid sequence which comprises a promoter sequence comprising at least a part of the DNA sequence as shown in Figure 5 or at least one part of the sequence encoding a promoter which has an activity substantially similar to the promoter encoded by Figure 5 or a variant or fragment thereof. The term "substantially similar activity" includes DNA sequences which are complementary and hybridize with the DNA of the present invention and which code for a promoter which acts on the pollen. Preferably, such hybridization occurs at or between conditions of low and high stringency. In general terms, low stringency conditions can be defined as 3 x SCC at room temperature between about 60 ° C and about 65 ° C, and high stringency conditions such as 0.1 x SSC ^ at about 65 ° C.

SSC is the name of a 0.015M NaCl buffer, 0.015M trisodium citrate, 3 x SSC is three times as strong as SSC, and so on. The pollen specific promoter of the present invention can be used to engineer male sterility by driving genes capable of interfering with pollen production or viability, or to express genes of interest specifically in pollen grains. According to a second aspect of the present invention, the promoter sequence can be part of an expression cassette in combination with genes whose expression in pollen, and particularly in the late production of pollen, may be desirable. These include genes which have an impact on pollen or pollen production. Such genes can be those involved in the control of male fertility, genes which code for insecticidal toxins (which can then be targeted to insect species, which feed on pollen) or genes which can improve or modify the value Pollen Nutrition In addition, the promoter sequence can be used to boost the expression of a selectable marker for use in pollen transformation. Examples of suitable selectable marker genes include genes that provide resistance to antibiotics such as the kanamycin resistance gene., the hygromycin resistance gene and PAT resistance gene, so as to allow stable transformants to be identified depending on the species, for example maize, rice, wheat. The term "expression cassette" - which is synonymous with terms such as "DNA construct", "hybrid" and "conjugate" - includes a gene directly or indirectly linked to the regulatory promoter, so as to form a cassette . An example of an indirect linkage is the provision of a suitable spacer group such as an intron intermediate sequence to the promoter and the target gene. The DNA sequences can also be in different vectors and therefore not necessarily located in the same vector. The same is valid for the term "merged" in relation to the present invention, in which the direct or indirect connection is included. Such constructs also include plasmids and phages which are suitable for transforming a cell of interest. According to a preferred embodiment, the expression cassettes of the present invention comprise a promoter sequence as described above which is arranged to control the expression of a gene which is harmful to the development of pollen, such as genes coding for barnase, an adenine nucleotide translocator, tubilin mutants, T-urf (as claimed in WO 97/041116) or trehalose phosphate phosphatase (TPP).

For example, WO 93/25695 describes the use of the barnase gene which codes for a cytotoxic protein, which is under the control of a specific tapeto promoter. The expression of barnase in tappetal cells disrupts these cells and leads to the interruption of pollen production. Ribozymes are RNA molecules capable of catalyzing endonucleolytic separation reactions. They can catalyze reactions in the trans position and can be directed to different sequences. Therefore, they are potential alternatives to antisense as a means to modulate gene expression (Hasselhof and Gerlach (1988) Nature Vol. 334: 585-591) or Wegener et al., (1994) Mol Gen Genet 245: (: 465-470) have demonstrated the generation of a trans dominant mutation by expression of a ribozyme gene in plants. If required, the specific promoter of the present invention can be used to control the expression of the ribozymes so that they are specifically expressed in the pollen. Baulcombe (1997) describes a method of silencing or inactivating a gene in transgenic plants via the use of replicable viral RNA vectors (Amplicon ™) which may also be useful as a means to suppress the expression of an endogenous gene. This method has the advantage that it produces a dominant mutation, that is, it is classifiable in the heterozygous state and removes the gene from all the copies of the target gene and can also eliminate the gene from isoforms. This is a clear advantage in wheat, which is hexaploid. Fertility can then be restored by using an inducible promoter to activate the expression of a functional copy of the gene that has been deleted. By including the specific pollen promoter as the elements of the Amplicon ™ vector, expression of the gene would take place specifically in pollen. The use of cytotoxic genes or switches as a means to interrupt pollen production requires the expression of restorative genes to reestablish fertility. Suitably, the construct further comprises a cassette comprising a nucleotide sequence which is capable of overcoming the effect of the deleterious gene, such as a restorer gene such as barstar in the case of barnase or TPS in the case of TPP, or a sequence which codes for a construct in which it is direct or antisense to a harmful gene. An alternative means to control the expression of harmful genes is to use operator sequences. Operator sequences such as lac, tet, 434, etc., can be inserted into the promoter regions, as described in WO 90/08830. Then repressor molecules can be attached to these operator sequences and prevent transcription of the gene towards the 3 rd end? for example, a gene harmful to the development of pollen (Wilde et al., (1992) EMBO J. 11, 1251). In addition, it is possible to engineer operator sequences with improved binding capacity such as the Lac I mutant. His, as described in (Lehming et al., (1987) EMBO 6, 3145-3153). This has an amino acid change from tyrosine to histidine at position 17 which provides tight control of expression. Used in combination with the inducible expression of the repressor this then allows the expression of an inactivating gene to be interrupted. In this way, expression cassettes can be incorporated into expression systems which can be used in the control of plant fertility, as described above. The term "expression system" means that the system defined above can be expressed in an appropriate organism, tissue, cell or medium. The system may comprise one or more expression cassettes and may also comprise additional components that ensure increased expression of the target gene through the use of the regulatory promoter. According to a third aspect of the present invention, there is provided an expression system comprising: (a) a first promoter sequence which is specifically expressed in -polen; (b) a first gene which, when expressed, interrupts pollen biogenesis, under the control of such a pollen specific promoter; (c) a second promoter sequence that responds to the presence or absence of an exogenous chemical inducer; and (d) a second gene which codes for an element which inhibits the expression of the gene or which can inhibit the protein purified by the first gene, operably linked to, and under the control of the second promoter sequence. The elements (a) and (b) and (c) and (d) above can be provided by one or two individual vectors, but preferably they are contained in the same vector to ensure cosegregation. This can be used to transform or cotransform plant cells so that appropriate interaction between the elements is allowed to take place. The second promoter sequence and the second gene provide chemical "communication" to activate or deactivate the first gene. When the second promoter sequence responds to the presence of an exogenous chemical inductor, the application of the chemical inducer to the pollen or to the plant will have the effect of activating the second gene, thus counteracting the effect of the first gene. The absence of the chemical inducer will have a similar effect when the second promoter sequence is active only in the absence of the chemical inducer. The elements (c) and (d) suitably are in the form of an expression cassette comprising a nucleotide sequence which is capable of resolving the effect of the damaging gene, so that the restorer gene such as barstar in the case of barnase or TPS in the case of TPP, or a sequence which codes for a construct which is direct or antisense to a harmful gene, or a gene that codes for a repressor molecule in the case of operator sequences that are being used operatively interconnected with a inducible promoter. The expression system of the present invention may further comprise a selectable marker, such as herbicide resistance genes or antibiotic resistance genes so as to allow stable transformants to be identified depending on the species, e.g., corn, rice, wheat. The presence of a herbicide resistance gene also allows the selection of male sterile progeny in a segregating population. The transformation of a plant with such an expression system will result in the production of male sterile plants and methods to produce such a plant from a fourth aspect of the present invention.

Expression systems according to this embodiment of the present invention, wherein the gene is harmful to viable pollen production, are useful in the production of hybrids but are especially useful when the male sterile line can become homozygous. When "late" promoters such as the ZmC5 promoter described above are used, because gene products are expressed late in pollen development, primary transformants and heterozygotes produce pollen which is segregated 1: 1 by sterility. , that is, 50% of the pollen is fertile and self-pollination can occur which leads to a non-hybridized seed. In order to obtain a homozygous sterile plant, the inducible promoter must be activated to drive the expression of the restorer gene using the appropriate chemical such as ethanol as in the case of the Al cA / R switch, or an insurance substance for the GST switch . This then inactivates the harmful gene, so that it allows self-pollination to occur according to the following model.

MS = Dominant Male Sterility Res = Inducible Restorer Heterozygous Genotype MSRcs Gametos MSRcs Autopolinization after chemical induction provides MSRcs MSRcs MSMSRcsRcs MSRcs MSRcs All pollen from homozygous progeny (MSMSRcsRcs) will be sterile. 50% of the pollen of the heterozygous progeny (MSRcs) will be sterile. The total pollen of the null progeny () will be fertile. The pollen staining of these lines with a vital dye such as DAPI will allow the identification of the plant that produces 100% sterile pollen. Alternatively, the induction and self-pollination of this segregating population can be repeated and the progeny can be analyzed to determine segregation of sterility. Clearly, all the progeny that derives from the self-pollination of a homozygous line and with a sterile male gender, that is, will produce a '100% sterile pollen, while the progeny arising from the self-pollination of a heterozygous line will continue to segregate to determine sterility . Alternatively, the gene that gives rise to male sterility binds to a gene that confers resistance to herbicides, then the progeny can be dispersed with herbicides in an early seedling stage, and herbicide tolerance will be segregated with the herbicide gene. sterility. In this way, male sterile parents can be identified and selected for hybrid production. Once identified, this homozygous sterile line can be used in the production of Fl hybrids through cross-pollination by a non-modified inbred male parent line. See below.

Father in female male father MSMSRcsRcs * * * * * * Gametos MSRcs MSRcs MSRcs MSRcs1 MSRcs *** MSRcs1 MSRcs *** Therefore, all Fl seeds are hybrid and heteroatomic by sterility, which means that 50% of the pollen of each plant is fertile. In a crop species such as corn, this is a highly viable pollen to ensure complete pollination through a field due to the altered volume of pollen produced by each male spike. In wheat, this would also be enough pollen to ensure that there is no loss of yield because self-pollination occurs while the flora is still closed, so wind loss, etc. is reduced. In species in which the vegetative part of the plant is harvested, then the viability of reduced pollen is not a factor to be taken into consideration. There is also an additional benefit to the hybrid seed producer in that the farmer retains the seeds F2 for subsequent planting and will present a loss in yield as a result of the loss of heterosis. See later.

Fl MSRcs *** Gametos MSRcs (not available) *** MSRcs none *** MSRcs F2 any * •*• * * * * These methods can allow the reversal of sterility, for example in the production of hybrids, by activation using an inducible promoter. According to a fifth aspect of the present invention, a method for controlling the fertility of a plant is provided, which comprises transforming the plant with an expression system, as described above, and then restoring fertility, activating the promoter inducible Suitable inducible promoters include those which are controlled by the application of external chemical stimuli, such as herbicide insurers. Examples of inducible promoters include, for example, a two-component system such as the switch promoter system of the AlcA / cR gene described in our published international publication No. WO 93/21334, the ecdysone switching system as described in our international publication WO 96/37609 or the GST promoter, as described in published international patent application Nos. WO 90/08826 and WO 93/031294, the teachings of which are incorporated herein by reference. Such promoter systems, which are referred to herein as "switch promoters". The commutator chemicals used in conjunction with the commutator promoters are agriculturally acceptable chemicals which make this system particularly useful in the method of the present invention. If the pollen-specific promoter of the present invention is used to obtain male sterility, full restoration of fertility may not be available by this method to the extent that the pollen is haploid. This means that only 50% of the pollen produced after the activation of the restorer gene is fertile. This can be counteracted by the fact that the expression using the promoter of the present invention is highly tissue specific. This property makes the use of the promoter of the present invention particularly useful in some of several particular applications. For example, in some cases pollen transformation is required. In this case, the use of the pollen specific promoter may be highly desirable. An example of such an application is known as MAGE LITER (male germline transformation), and is described by Stoger et al., (Plant Cell Reports 14 (1995) 273-278). In this method, pollen is transformed by microprojectile bombardment. A specific pollen promoter is used to drive, for example, a selectable marker gene. The fact that the promoter is specific for pollen confers several advantages. First of all, the marker is expressed only in the pollen, and not in the rest of the plant and in this way the remaining plant tissue contains no unwanted marker. further, having a selectable marker only in the pollen allows the possibility of retransforming the usual methods without having to acquire a different selectable marker, that is, this allows for easier stacking of genes. The reasons why pollen transformation is important is that pollen can be made to undergo sporophytic development, that is, to give rise to duplicated haploid and haploid plants. This means that the homozygous condition is obtained in one stage. Alternatively, the transformed pollen can be used to pollinate wild-type plants and thus generate seeds that present the introduced transgene, again a faster process than the traditional transformation route. The expression systems of the present invention can be introduced into a plant or plant cell via any of the available methods including infection by Agroba cterumum tumefaciens containing the recombinant Ti plasmids, electroporation, microinjection of plant cells and protoplasts, bombardment with microprojectiles, bacterial bombardment, particularly the "fiber" or "mustache" method, and transformation of the pollen tube, depending on the particular plant species that is transformed. The transformed cells, then in suitable cases can be regenerated in whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocotyledonous and dicotyledonous plants can be obtained in this manner. Reference can be made to the literature for full details of the known methods. Such methods form a further aspect of the present invention. The method of the present invention would be useful in the production of a wide range of hybrid plants such as wheat, rice, corn, cotton, sunflower, sugar beet and lettuce, colse oil seed and tomato. Plant cells which contain a plant gene expression system as described above, together with plants comprising these genes, form additional aspects of the invention. According to a ninth aspect of the present invention, a replicable viral DNA vector (Amplicon ™) is provided, which comprises a recombinant nucleic acid as defined above.

According to a tenth aspect of the present invention, there is provided a method for transforming pollen cells with an expression system as described above. The invention will now be described particularly by way of example only with reference to the accompanying drawings, in which: Figure 1 is a diagram showing the alignment of the ZmC5 cDNA with a 2.4 kb fragment from the 5 'region of its corresponding gene. The transcriptional start point is indicated with (*) and the putative translational start is underlined. Sa = Sal I, S = Sma I, Sp = Sph I, H = Hind III, X = Xho I, P = Pst I; Figure 2 shows the result of a Southern blot test of maize genomic DNA (inbred line A188). Each lane contains 15 μg of digested genomic DNA in lane 1 with Eco Rl, in lane 2 with Hind III and in the lane with Bam Hl. The Southern blot test hybridizes with a radiolabelled ZmC5 cDNA probe. Figure 3 shows gels stained with ethidium bromide showing 18S RNAs from various total corn tissue RNAs and the results of the Northern blot test of the same gels probed with the ZmC5 cDNA probe.

(A) The gel is loaded with 10 μg of total RNA from various maize tissues. (B) the gel is loaded with 10 μg of total RNA isolated from shoots, in a stage of development in a series of spicules, pollen and germinative pollen. Figure 4 shows: (A) a physical map of the construct ZmC5 :: uidA and the binding sequence. Residues A by *, t and t correspond to the starting point of transcription ZmC5, maturation T to A to remove the Hind III site (for ease of cloning) and the 3 'part of the promoter region ZmC5, respectively, the translation start uidA is underlined. (B) the histochemical analysis of GUS activity in pollen of transgenic tobacco ZmC5 :: uidA showing segregation of blue staining. (C) promoter activity of ZmC5 in transgenic tobacco tissue from 2 transgenic lines, GC5-2 (white columns) and GC5-7 (black columns). The data of each line represents the average of 2 independent tests, normalized to a non-transgenic control. The stages of the buttons are as follows: button-1 correspond to buttons of 5-8 'mm (microspores in meiosis / tetrad stage), button-2: buttons of 10-12 mm (uninucleated microspores), button-3: 13-15 mm buttons (mitosis of microspores), button-: (17-22 mm) binucleate gametophytes from early to middle, button-5: (27-45 mm) binucleate gametophytes from middle to late stage (Tebbutt et al. ., supra). Figure 5 shows the DNA sequence encoding the promoter sequence of ZmC5 in maize. The underlined A is the putative transcriptional start point and the underlined ATG sequence in bold is the translational start point. Figure 6 shows an expression cassette comprising C5-barnase / barstar-nos.

EXAMPLE 1 ZmC5 cDNA and genomic clones Maize pollen cDNA libraries (inbred line A188) and germinating pollen (HJ Rogers et al., Plant J4 (1993) 875-882) were analyzed by using cDNA probes made for poly (A) RNA. pollen and rods following standard procedures (FM Ausubel et al., Current protocols in Molecular Biology, Wiley, New York (1990).) All clones were taken, totaling 1.101, which showed hybridization to the radiolabeled pollen cDNA, but to the Radiolabeled stem Random colonies were taken and the sequence was established at the 5 'end, and the sequence is compared to current databases A clone which has an 800 bp insert shows a significant sequence identity with known pectin methylterases The pollen cDNA library is reanalyzed with this cDNA insert and the full length of the clone ZmC5 (ZmC5c) is identified and sequenced.A geonomic library of maize (inbred line B73) is analyzed (8 x 106 plates) using a PCR fragment corresponding to the 270 bp 5 'section of the labeled ZmC5c clone cDNA by random priming (Ausubel et al., supra). A positive clone of ZmC5g is purified on plate. Comparison of the sequence of a 2.5 kb Sal I fragment of ZmC5g subcloned in pUC19 with ZmC5c (and deposited as NCIMB 40915) shows that the two sequences overlap and are identical, indicating that the clones represent the same gene. Figure 1 shows a representation of the overlap between the 2.5 kb fragment of the geonomic clone with the cDNA. A transcriptional start point is mapped using two oligonucleotide primers complementary to nucleotides 1 to 21 (5 '-ACCTAGGAGAGCCTTTGCCAT-3') and 56 to 82 (5'-AGCGGGTGACGGTGGCGACCACACCGA-3 ') of the coding sequence (data not shown). The products unequivocally locate the transcriptional start point at nucleotide A in only 15 bases towards the 5 'end of putative ATG (Figure 1). Other pollen-specific genes having long 5 'untranslated sequences have been found (SJ Tebbutt et al., Plant Mol Biol (1994) 25: 283-299). Therefore, this region of ZmC5g seems to be unusually short. The free energy, calculated for this short 5 'UTL, is 0.9 kJ / mol (M. Zuker, Meths Enzymol (1989) 180: 262-288), which makes it unlikely that it will be able to form any stable secondary structure. An open reading frame of 1692 bp was identified and the putative ATG start codon (Figure 1) matches well with the consensus sequences (HA Lutcke et al., EMBO J 6: (1987) 43-48). A putative TATAA motif (CP Joshi, Nucí Acids Res. 15: 6648-6653 (1987)) starts at -32, however, there is no recognizable CAAAT motif in the 60 bp towards the 5 'end from the transcriptional start (figure 1) as has been found in many other plant genes. ZmC5c lacks a clearly recognizable AATAAA polyadenylation site signal. This is not uncommon: 30% of plant genes lack a recognizable AATAAA motif (BD Mogen et al., Plant Cell 2 (1990) 1261-1272). At ZmC5c, a sequence of five A residues of 16 bp towards the 5 'end from the poly (A) addition site may be acting as a polyadenylation signal, although it has been found that other pollen-specific transcripts have greater distances than the average between the AATAAA motif and the poly (A) + addition site (Tebbutt et al., supra).

EXAMPLE 2 Amino acid deduced sequence of ZxaC5 The deduced amino acid sequence of ZmC5 (563 amino acids) is compared with the EMBL and GenBank databases, and show a high degree of homology with both the plant (between 30.9% and 41.4%) as microbial PME (between 18.6% and . 8%). An alignment of the amino acid sequences shows conservation through both the plant and microbial sequences restricted mainly to the C-terminus of the protein which includes 4 regions that are probably the catalytic domains or the active sites of the enzyme (D. Albani et al. al., Plant Mol Biol (1991) 16: 501-513), O Marcovic et al., Protein Sci 1: 1288-1292 (1992)). Mutagenesis in vi tro of A. niger PME (B Duwe et al., Biotechnol Letts 18: 621-626 (1996)) indicates that the histidine residue, which is conserved in ZmC5, within region I can be located in the active site of the enzyme and in TO . Niger is required for enzyme activity. However, this histidine is substituted by other amino acid residues in several PMEs of both plant and fungal origin, suggesting that it is not essential in all PMEs. In a comparison of the plant PME, ZmC5 shows a closer relationship with the "late" pollen of P. inflata that expresses the PPE gene (JH Mu et al., Plant Mol Biol 25: 539-544 (1994)), compared to the "early" pollen of B. napus expressing the Bpl9 gene (D. Albani et al., supra).

EXAMPLE 3 Estimation of the ZmC5 gene number A corn genomic Southern blot test (inbred line A188) containing 15 μg of DNA digested with Bam Hl, Eco Rl or Hind III is probed with a radiolabeled full length ZmC5 cDNA insert. Two strong hybridization bands in each lane of the transfer in Figure 2 suggest the presence of at least two similar genes in the maize genome. Several additional bands with which a much weaker signal is shown suggest that this family of genes may also comprise several less related members.

EXAMPLE 4 Spatial and temporal expression of ZmC5 A Northern blot containing 10 μg of Total RNA from 8 maize tissues is probed with ZmC5 cDNA to determine the program of gene expression. A transcript of approximately 2.0 kb is detected only in the pollen and pollen in germination (figure 3 (A)), which indicates that, within the limits of detection of this technique, the expression of this gene seems to be restricted to these two tissues. There is no detectable signal in leaves, roots, shoots, cobs, endosperm or embryos. The expression program during the development of spicules was also determined. Figure 3 (B) shows a Northern blot containing total RNA of spicules of 0.25, 0.5 and 1.0 cm, mature pollen and germinating pollen. Gels stained with ethidium bromide demonstrate equal charges of RNA in the lanes of each gel. The spicules are classified by staining the anthers with acto-carmin, and the anthers are found to contain cells in the following stages: premeitotic sporogenic cells (0.25 cm), medium prophase I (0.5 cm), maturing pollen grains (1.0 cm). However, certain overlaps between consecutive stages are inevitable due to the variation in the stage of development between the two flowers within the same spicule. This Northern analysis shows that the expression of ZmC5 is restricted to mature dehisced pollen and to germination pollen without detectable expression of any other maize tissue including spicules containing cells in previous stages of microsporogenesis.

EXAMPLE 5 Expression of promoter Z C5 / GUS constructs in transgenic plants Transcriptional fusions were performed between the 5 'region of ZmC5g and the β-glucuronidase indicator gene (FIG. 4A), and were used to transform tobacco by transformation with Agrobacterium. The construct is constructed as follows: the two Sph I sites, one within the fragment Sal I of 2.5 kb which contains 2 kb of the sequence 5 'in relation to the ATG, on one inside the polylinker, were used to remove the 3' end of the position -61 a +403 (figure 1). This was directly substituted by a fragment of PCR digested with Sph I that includes the region -61 to +1, and the additional restriction sites placed at the 3 'end (Bam Hl, Hind III, Sal I) and a mutation to remove the Hind III site placed at the transcriptional start (figures 1 and 4A). The original Sal I 5 'site and the Sal I 3' site introduced later is used to cut the ZmC5 promoter region which is cloned into the Sma I site of pGUS. The transcriptional fusion ZmC5-UID-A promoter is then transferred to the pBin 19 vector (FIG. 4A) and used for transformation of the leaf disk mediated by Agroba cteri um of Nicotiana tabacum var Samsun. The transformants are selected in kanamycin and the primary transgenic plants are regenerated, two of which, positive for transgene expression, are taken in the T2 generation. The pollen grains of the dehisced anthers of the transgenic plants are harvested and stained to determine GUS activity, as described by J.A. Jefferson (Plant Mol Biol (1987) 5: 387-405). Two plants that were positive show approximately 50% blue pollen staining (Figure 4B). No blue coloration was detected in the non-transgenic controls. To investigate the number of integration site, the plants of two transgenic lines were self-pollinated, and the progeny were classified to determine kanamycin resistance. Of the progeny analyzed to determine the transgenic condition the plant GC5-2, 303 were resistant to kanamycin and 8 were sensitive to kanamycin, which gives an average ratio of 38: 1, indicative of at least two integration sites. The progeny of the transgenic plant GC5-7 provides a mean ratio of kanamycin resistance to kanamycin sensitivity of 3.8: 1, indicative of a single integration site (ratio expected for an integration site is 3: 1, for two of 15: 1 and for three of 63: 1). Extracts were produced from a range of tissues including five stages of developing anthers and analyzed fluorometrically for GUS expression (Jefferson, supra). Figure 4 (C) shows GUS activity of two transgenic plants. Only very low levels of expression are detectable in tissues different from those of the dehisced anthers in immature development. In tobacco, the button development stage can be correlated with the length of the buttons (Tebutt et al., Supra) but this depends on the growing conditions. Therefore, i-button corresponds to microspores in the stage of mitosis / tetrada, button-2, uninucleated microspores, button-3 mitosis of microspores, button-4 gametophyte binucleate early to middle stage, button-5 binucleate gametophyte in middle stage to late. The microspore stages are determined by staining with DAPI (data not shown) which indicates that the synchronization of expression of the ZmC5 promoter in tobacco agrees well with its expression in maize, based on the Northern test data (Fig. 3; Figure 4C) therefore, both in the native maize environment and transgenic tobacco, the ZmC5 promoter functions late in pollen development and is virtually inactive before mitosis of microspores. Some variation in the expression between the two transgenic lines can be observed with GC5-2, which shows higher levels of expression in most of the tissues tested. Variation in expression levels is commonly found in transgenic populations and has been ascribed to the insertion site of the 9SLA transgene Hobbs et al., Plant Mol Biol 21: 17-26 (1993)).

EXAMPLE 6 Expression of GUS in pollen driven by the inducible promoter AleA A plant transformation vector comprising the AlcA promoter that drives GUS expression and a CaMV 35S promoter that drives the expression of AlcR have been introduced into tobacco and tomato plants. The expression of GUS can be studied in all tissues before and after induction with ethanol as a flood in the roots.

GUS staining of tomato anthers and pollen shows a clear expression of GUS after induction. The same result is expected from the pollen of other species.

EXAMPLE 7 Preparation of cassette C5-barnase - a cassette of dominant male sterility The only Sal I site from pBluescrip SK + (Stratagene) is replaced with a Notl recognition site by insertion of an oligonucleotide linker MKLINK4 (5'-TGCATTCGGCGGCCGCCGAA-3 ') into the digested Sali site. A Bam HI-Hind III fragment of 0.9 kb carrying the coding region of barnase followed by a coding region of barstar driven by a bacterial promoter is inserted into the corresponding fragment of modified pBluescrip. The terminator we in a Hind III-Notl fragment is inserted into the corresponding fragment of the resulting vector. An unwanted BamHI site is then removed using Stratagene's QuickChange system, following the manufacturer's instructions and using oligonucleotides DAM-3A (51-GGTCGACTCTAGAGGAACCCCGGGTACCAAGC-3 ') and DAM-3S (5 * -GCTTGGTACCCGGGGTTCCTCTAGAGTCGACC-3'). The resulting plasmid (designated pSK-BBN) is digested to completion with BamHI, dephosphorylated with shrimp alkaline phosphatase (37 ° C, 1 hour).

A 1.9 kb BamHI fragment of the 5 'franking region of C5 is ligated therein, followed by digestion with BamHI and PstI to verify the presence and orientation of the insert, respectively. The resulting plasmid is called pSK-C5-BBN (figure 6). The whole cassette is then removed as an EcoRI-Notl fragment to a binary plant transformation vector pVB6. The construct is then introduced into Agroba cteri um tumefa ciens by the method of freezing and reheating. Standard techniques are used to introduce DNA into tobacco.

EXAMPLE 8 Analysis of sterile transgenic plants Primary transformants are selected by growth in kanamycin in tissue culture and this is confirmed by PCR analysis. The plants are grown to maturity in a greenhouse. Pollen is collected from the anthers after dehiscence and vital staining is used to establish whether the pollen is fertile or sterile. It is expected that 50% of the pollen will be sterile. The backcrossing of these plants with wild-type plants (after removal of the anthers) or by allowing self-pollination to occur results in progeny in which 50% of the pollen is sterile. Other modifications to the present invention will be apparent to those skilled in the art, without departing from the scope of the invention. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (27)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A recombinant nucleic acid sequence, characterized in that it comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen.

2. The recombinant nucleic acid sequence according to claim 1, characterized in that it comprises approximately 2 kb towards the 51 end of a transcriptional start site of the maize ZmC5 gene, as shown in Figure 1.

3. The recombinant nucleic acid sequence according to claim 1 or claim 2, characterized in that it comprises a promoter sequence comprising at least part of the DNA sequence as shown in figure 5, or at least part of a sequence which encodes a promoter which has an activity substantially similar to that shown in Figure 5, or a variant or fragment thereof.

4. An expression cassette, characterized in that it comprises a nucleic acid sequence according to any of claims 1 to 3, wherein the cassette is arranged to control a gene which is desired to express the pollen and encodes it for a product capable of having an impact on the production of pollen, insecticide toxins or that improves or modifies the nutritional value of pollen.

5. The expression cassette, according to claim 4, characterized in that the gene comprises a gene which is harmful to the development of pollen.

6. The expression cassette according to claim 5, characterized in that the gene comprises a gene coding for already barnase, adenine nucleotide translocator, mutant tubulins, T-urf or trehalose phosphate phosphatase (TPP) or a ribozyme.

7. The expression cassette, according to claim 4, characterized in that the gene comprises a selectable marker gene.

8. The expression cassette, according to claim 7, characterized in that the selectable marker gene comprises an antibiotic resistance gene.

9. An expression system characterized in that it comprises an expression cassette according to any of claims 4 to 8.

10. The expression system, according to claim 9, characterized in that it comprises a gene which is harmful to pollen, and which further comprises an expression cassette comprising a second nucleic acid sequence which encodes a peptide or protein capable of of overcoming the effect of the harmful gene, operatively interconnected with a chemical inducible promoter.

11. The expression system, according to claim 10, characterized in that the nucleic acid sequence comprises a restorer gene.

12. The expression system, according to claim 11, characterized in that the restoring gene is barstar or TPS.

13. The expression system, according to claim 10, characterized in that the nucleic acid sequence encodes a construct which is direct or antisense to the harmful gene so as to suppress the expression thereof.

14. The expression system, according to claim 10, characterized in that the second nucleic acid sequence codes for a repressor protein for lac, tet, 434, lac-His, which interacts with an operator sequence which is operably linked to a nucleic acid sequence according to any one of claims 1 to 3 or an Amplicon ™, so as to avoid the expression of the first gene which is desired to be expressed in pollen.

15. The expression system, according to any of claims 10 to 14, characterized in that the inducible promoter is Al cA / R, GST or the promoter inducible by Ecdysone.

16. The expression system, according to any of claims 9 to 15, characterized in that it also comprises a selection marker.

17. The expression system according to any of claims 9 to 16, characterized in that the gene which is desired to be expressed in pollen binds to a herbicide resistance gene.

18. An expression system, characterized in that it comprises: (a) a first promoter sequence which is specifically expressed in pollen; (b) a first gene which, when expressed, interrupts pollen biogenesis, under the control of the pollen specific promoter; (c) a second promoter sequence that responds to the presence or absence of an exogenous chemical inducer; and (d) a second gene encoding an element which can inhibit the expression of the first gene, or which can inhibit the first protein, operably linked and under the control of the second promoter sequence.

19. A method for producing a plant, which method is characterized in that it comprises transforming a plant cell with an expression system according to any of claims 9 to 18.

20. A plant cell, characterized in that it comprises an expression system according to any of claims 9 to 18.

21. A plant, which comprises cells according to claim 20.

22. A method for inducing male sterility in plants, which method is characterized in that it comprises transforming a plant with an expression system according to any of claims 10 to 18.

23. A method for controlling the fertility of a plant, characterized in that it comprises transforming the plant with an expression system according to claim 11 or claim 18, and when fertility is required, restoring it by activating an inducible promoter.

24. The method according to claim 23, characterized in that the inducible promoter is activated by application of a chemical substance to the plant.

25. The replicable viral RNA vector (Amplicon ™), characterized in that it comprises a recombinant nucleic acid, according to any of claims 1 to 3.

26. A method for transforming pollen, characterized in that it comprises transforming pollen cells with an expression system according to claim 9 to claim 18.

27. A recombinant nucleic acid, an expression cassette, an expression system or a method, characterized in that it is substantially as described in the foregoing with reference to any of the appended figures.