BR122014029560B1 - VECTOR OF EXPRESSION - Google Patents

Description

EXPRESSION VECTOR

Divided from patent application 0114199-6, filed September 25, 2001.

BACKGROUND OF THE INVENTION The development of hybrid plant breeding has made possible considerable advances in the quality and quantity of crops produced. Increased yield and combination of desirable characteristics such as disease and insect resistance, heat and drought tolerance, along with variations in plant composition are also possible due to hybridization procedures. These procedures often rely heavily on providing a male parent who contributes pollen to a female parent to produce the resulting hybrid.

Field crops are reproduced by techniques that take advantage of the plant pollination method. A plant is self-pollinated if a flower's pollen is transferred to the same or another flower of the same plant. A plant is cross-pollinated if pollen comes from a flower on a different plant.

[003] In Brassica, the plant is usually self-sterile and can only be cross-pollinated. In self-pollinating species, such as soybeans and cotton, male and female plants are anatomically juxtaposed. During natural pollination, male reproductive organs of a given flower pollinate female reproductive organs of the same flower.

Corn plants (Zea mays L.) present a unique situation in which they can be reproduced by both self-pollination and cross-pollination techniques. Corn has male flowers, located on the fringes, and female flowers, located on the spikes, in the same plant. It can self-pollinate or cross-pollinate. Natural pollination occurs in maize when wind blows pollen from the fringes to silks that extend from the tops of incipient ears.

[005] A reliable method of plant fertility control should offer the opportunity to improve plant breeding. This is especially true for the development of maize hybrids, which is based on some type of male sterility system and where a female sterility system should reduce production costs.

[006] The development of maize hybrids requires the development of homozygous generation lines, the crossing of these lines, and the evaluation of crosses. Pedigree breeding and recurrent selection are two of the breeding methods used to develop population generation lines. Breeding programs combine desirable traits of two or more generation lines from various broad base sources into breeding sets from which new generation lines are developed by selecting desired phenotypes. A hybrid maize variety is the crossing of two generation lines, each of which may have one or more characteristics that are either absent or complement the other. New generations are cross-bred with other generation lines and the hybrids of these crosses are evaluated to determine which one has commercial potential. The first generation hybrid offspring is called Fl. In hybrid development, only F1 hybrid plants are made. Hybrid F1 is more vigorous than its parent. This hybrid vigor, or heterosis, can be manifested in many forms, including increasing vegetative growth and increasing yield.

[007] Hybrid maize seed can be produced by a male sterility system that incorporates manual shredding. To produce hybrid seed, the male fringe is removed from the growing female parent, which can be planted in various row patterns alternating with the male parent. Consequently, as long as there is sufficient isolation from sources of foreign maize pollen, female generation ears will only be fertilized with male generation pollen. The resulting seed is therefore hybrid (F1) and will form hybrid plants.

[008] Environmental variation in plant development can result in fringing plants after manual parental disintegration is completed. Or, a defrancher should not completely remove fringes from a female generation plant. In any event, the result is that the female plant will release pollen successfully and some female plants will be self-pollinated. This will result in female generation seed being harvested together with the hybrid seed that is normally produced. Female generation seed is not as productive as Fl seed. In addition, the presence of female seed may pose a germplasm safety risk to the company that produces the hybrid.

Alternatively, the female generation may be mechanically engineered by machine. Mechanical disassembly is about as reliable as the manual, but is faster and less expensive. However, most blanching machines do more damage to plants than manual blanching. Thus none of the offsets is now fully satisfactory, and there remains a need for alternatives that also reduce production costs and to eliminate female parental self-pollination in hybrid seed production.

[010] A reliable system of male genetic sterility should provide advantages. The laborious process of disintegration can be avoided in some genotypes by using generations of male cytoplasmic sterility (CMS). In the absence of a fertility restorer gene, plants of a generation of CMS are sterile males as a result of factors that result from the cytoplasmic, as opposed to nuclear, genome. Thus, this trait is inherited exclusively through the female parent in maize plants, as only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another generation that is not sterile male. Second generation pollen may or may not contribute genes that make the male hybrid plant fertile. Usually normal-blanched corn seed and CMS-produced seed of the same hybrid should be mixed to ensure adequate pollen loads are available for fertilization when hybrid plants grow and to ensure cytoplasmic diversity.

[011] There may be other inconveniences to CMS. One is the historically observed association of a specific variant of CMS with susceptibility to certain crop diseases. This problem has discouraged the widespread use of that CMS variant in hybrid maize production and has had a negative impact on CMS use in maize in general.

One type of genetic sterility is discussed in U.S. Patent 4,654,465 and 4,727,219 to Brar et al. However, this form of male genetic sterility requires the maintenance of multiple mutant genes at separate locations in the genome and requires a complex marker system to track genes and make use of the convenient system. Patterson also described a gene system of chromosomal translocations that may be effective but complicated. (see U.S. Patent Nos. 3,861,709 and 3,710,511).

[013] Several other attempts have been made to improve these disadvantages. For example, Fabijanski, et al., Have developed various methods of causing male sterility in plants (see EPO 89 / 3010153.8, publication No. 329,308 and application PCT / CA90 / 00037 published as WO 90/08828). One method includes releasing to the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter. Another involves an antisense system in which a fertility critical gene is identified and an antisense to the gene is inserted into the plant. Mariani et al also show various cytotoxic antisense systems. See EP 89 / 401,194. Still other systems use "repressor" genes that inhibit the expression of another gene critical for male sterility. PCT / GB90 / 00102, published as WO 90/08829.

Still further improvement of this system is described in U.S. 5,478,369 (incorporated herein by reference) in which a method of providing controllable male sterility is achieved by silencing a plant-native gene that is critical for male fertility and replacing the native DNA with a promoter-linked male fertility-critical gene. that controls the expression of the gene. The plant is therefore constitutively sterile, becoming fertile only when the promoter is induced in its attached male fertility gene is expressed.

[015] As noted, an essential aspect of much of the work related to male sterility systems is the identification of genes that affect male fertility.

Such a gene may be used in a variety of systems to control male fertility including those described herein. Previously, a male fertility gene was identified in Arabidopsis thaliaraa and used to produce a sterile male plant. Aarts, et al., "Transposon Tagging of a Male Sterility Gene in Arabidopsis", Nature, 363: 715-717 (Jun. 24,1993). U.S. Patent No. 5,478,369 is one such gene that affects male fertility. In the present invention the inventors provide novel DNA molecules and the encoded amino acid sequence that are critical to male fertility in plants. These can be used in any of the systems where fertility control is useful, including those described above.

Therefore, a subject of the invention is to provide a nucleic acid sequence, the expression of which is critical for male fertility in plants.

Another subject of the invention is to provide a DNA molecule encoding an amino acid sequence, the expression of which is critical to male fertility in plants.

Yet another subject of the invention is to provide a promoter of such a nucleotide sequence and its essential sequences.

A further subject of the invention is to provide a method of using such DNA molecules to mediate male fertility in plants.

Additional subjects of the invention will become apparent from the following description and claims.

Summary of the Invention This invention relates to nucleic acid sequences, and specifically to DNA molecules and the amino acid encoded by DNA molecules, which are critical for male fertility. A DNA promoter is identified, as are its essential sequences. It is also related to the use of such DNA molecules to mediate fertility in plants.

Brief Description of the Drawings FIG. 1. is a location map of the male sterility gene SBMu200, where E = EcoRI, X = Xbal, B = BamHI, P = PstI, and S = Saci.

[024] FIG. 2. is a gel from a Southern blot analysis of ECORI digested DNA from a Mu family that secretes for male sterility and hybridized to a Mu 8 marker.

[025] FIG. 3. is a Northern Blot assay gel hybridized to a PstI fragment isolated from clone SBMu2003.1.

FIG 4 is the sequence of SBMu200 (cDNA is Id. Of Seq. No. 1, protein is Id. Of Seq. No. 2).

[027] FIG. 5 is the genomic sequence of SBMu200 (also referred to as Seq. Id. No. 7).

FIG 6 is a comparison of the SBMu200 genomic sequence with the SBMu200 cDNA.

[029] FIG. 7. is a Northern analysis gel showing developmental gene expression in SBMu200 gene microsporogenesis.

[030] FIG. 8 is the full length promoter of SBMu200 (Seq. Id. No. 5).

[031] FIG. 9. is a bar graph showing luciferase activity after deletion of selected regions of the SbMu200 promoter.

[032] FIG. 10 shows an essential region of the SBMu200 promoter (Seq. Id. No. 6).

[033] The coordinates are relative to the assumed TATA box (underlined). P reasons are in italics. Binder scanning mutations that reduce activity to 5% or less are bolded. Mutations with a significant but less pronounced effect are bold and italic.

FIG. 11 is a bar graph showing luciferase activity following substitution by restriction site ligand scanning of small selected regions (9-10bp) of the SBMu200 essential promoter fragment.

FIG 12 is a comparison of SBMu200 sorghum fringe and SBMu200 cDNA 8.1 corn (sorghum DNA is Seq. No. 3 and protein is Id. Of Seq. No. 4).

Discovery of the Invention All references herein are incorporated by reference.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise noted, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

Genetic male sterility results from a mutation, deletion, or other impact to one of the genes critical for a specific step in microsporogenesis, the term applied to the entire pollen formulation process. These genes may be referred to collectively as male fertility genes (or, alternatively, male sterility genes). There are several steps on the general path where genetic function affects fertility. This seems supported by the frequency of genetic male sterility in maize. New alleles of male sterility mutants are uncovered in materials ranging from elite generations to unadapted populations. To date, published genetic male sterility research has been mostly descriptive. Some efforts have been made to establish the sterility mechanism in maize, but few have been satisfactory. This should not be surprising given the number of genes that have been identified as responsible for male sterility. One mechanism is unlikely to apply to all mutations.

[039] In Patent No. No. 5,478,369 describes a method by which a male sterility gene was labeled on maize chromosome 9. Previously, the only male sterility gene described on chromosome 9 was MS2, which was never cloned or sequenced. See Albertsen, M. and Phillips, R. L., "Developmental Cytology of 13 Genetic Male Sterile Loci in Maize" Canadian Journal of Genetics & Cytology 23: 195-208 (Jan. 1981). The only fertility gene cloned before this was the Arabadopsis gene described in Aarts et al., Supra.

[040] The SBMu200 gene described therein is located on maize chromosome 1 and its dominant allele is critical for male fertility. The location map is depicted in Figure 1. It can be used in the systems described above, and other systems that affect male fertility.

The family of maize co-secreting for sterility was named SBMu200 and was found to have an approximately 5.5 Kb EcoRI fragment that hybridizes to a Mu8 marker. A genomic clone of the family containing a Mu8 transposon was isolated. It turned out that a marker made from DNA that borders the transposon hybridizes to the same ~ 5.5 Kb EcoRI fragment. This marker was used to isolate cDNA clones from a fringe cDNA library. The SBMu200 paD cDNA is 1906 bp, and Mu was inserted into exon 1 of the gene. Figure 9 (discussed further below) represents the genomic nucleotide sequence. Expression patterns, as determined by Northern analysis, show spike-specific fringe specificity at the four to four release stages of microsporogenesis.

Plasmids containing the nucleotide sequence of the invention were deposited with the American Type Culture Collection (ATCC) Patent Filer, Manassas, Virginia, received October 16, 1998 and designated ATCC Designation No. 98931. The deposit will be maintained under the terms of the Budapest Treaty in the "International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure". The deposit was made as a convenience only to those skilled in the art and is not a statement that a deposit is required.

Additionally, it will be apparent to one skilled in the art that variations, mutations, derivations including fragments smaller than the entire sequence presented may be used that retain the male sterility control properties of the gene. One of ordinary skill in the art can readily evaluate the variant or fragment by introducing homozygous plants to a stable sterile male allele of MS26, followed by observing a development of male plant tissue.

The invention also includes those nucleotide sequences that selectively hybridize to SBMu200 nucleotide sequences under stringent conditions. In reference to a sequence that "selectively hybridizes" to SBMu200, the term includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to the specified target nucleic acid sequence to a detectably greater degree (e.g., by 2-fold less than background) than its hybridization to a non-target nucleic acid.

The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a label will hybridize to its target sequence at a higher level detectable than other sequences (e.g., at least 2-fold over base). Restricted conditions are sequence dependent and will differ depending on the structure of the polynucleotide. By controlling the severity of hybridization and / or wash conditions, target sequences can be identified that can be up to 100% complementary to a marker (homologous assay). Alternatively, severity conditions may be adjusted to allow some mismatch in sequences so that lower levels of similarity are detected (heterologous assay). Generally, such markers are in the range of about 1000 nucleotides in length to about 250 nucleotides in length.

[046] An extensive guide to nucleic acid hybridization is found in Tjissen, Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "OverView of Principles of Hybridization and the Strategy of Nucleic Acid Probe Assays" ", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). See also Sambrook, et al. (1989) Molecular Cloning: A Mannual Laboratory (2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

In general, sequences that correspond to the nucleotide sequence of the present invention and hybridize to the nucleotide sequence disclosed therein will be at least 50% homologous, 70% homologous, and even 85% homologous or more with the disclosed sequence. That is, the sequence similarity between marker and target may vary, sharing at least about 50%, about 70%, and even about 85% sequence similarity.

[048] Specificity is typically the function of posthybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. Generally, stringent wash temperature conditions are selected to be about 5 ° C to about 2 ° C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow temperature range and represents the disruption of the double helix into its complementary single strands. The process is described by the midpoint transition temperature, Tm, which is also called melting temperature. Formulas are available in the art for determining melting temperatures.

Preferred hybridization conditions for the nucleotide sequences of the invention include hybridization at 42 ° C in 50% (w / v) formamide, 6X SSC, 0.5% (w / v) SDS, 100 (g / ml) ) of salmon sperm DNA. Example low stringency wash conditions include hybridization at 42 ° C in a 2X SSC, 0.5% (w / v) SDS solution for 30 minutes and repeating. Moderate stringency conditions of example include a 2X SSC wash, 0.5% (w / v) SDS at 50 ° C for 30 minutes and repetition. Exemplary high stringency conditions include a 2X SSC wash, 0.5% (w / v) SDS, at 65 ° C for 30 minutes and repeating. Sequences corresponding to the promoter of the present invention may be obtained using all of the above conditions. For purposes of defining the invention, high stringency conditions are used.

Sequence alignment methods for comparison are well known in the art. Gene comparisons may be determined by conducting BLAST searches (Basic Local Alignment Search Tool; Altschul, SF, et al., (1993) J. Mol. Biol. 215: 403-410; see also www.ncbi.nlm.nih .gov / BLAST /) under default parameters for identity to sequences contained in the "GENEMBL" BLAST database. A sequence can be analyzed for identity to all publicly available DNA sequences contained in the GENEMBL database using the BLASTN algorithm under default parameters. Sequence identity of the present invention should mean a nucleotide poly-sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% more preferably at least 85% sequence identity, more preferably at least 90% sequence identity and more preferably at least 95% sequence identity.

Promoter regions can be easily identified by one of ordinary skill in the art. The supposed start codon containing the ATG standard is identified and above the start codon is the presumed promoter. By "promoter" is meant a DNA regulatory region usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter may further comprise other recognition sequences generally positioned above or 5 'to the TATA box, referred to as above promoter elements, which influence the transcription initiation rate. It is recognized that having identified nucleotide sequences for the promoter region disclosed therein, it is within the state of the art to isolate and identify additional regulatory elements in the region above the TATA box of the particular promoter region identified therein. Thus the promoter region disclosed therein is generally also defined as comprising above regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers and the like. Likewise, promoter elements that allow expression in the desired tissue such as male tissue can be identified, isolated, and used with other core promoters to confirm preferred male tissue expression.

The isolated promoter sequence of the present invention may be modified to provide a range of expression levels of the heterologous nucleotide sequence. Unless the entire promoter region may be utilized and the previously preferred expression targeting ability is retained. However, it is recognized that mRNA expression levels can be decreased by deletion of portions of the promoter sequence. Thus, the promoter may be modified to a weak or strong promoter. Generally, by "weak promoter" is meant a promoter that directs the expression of a coding sequence at a low level. By "low level" are levels from about 1 / 10,000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts. Conversely, a "strong promoter" directs expression of a coding sequence at a "high level", or about 1/10 transcripts to about 1/100 transcripts to about 1/1000 transcripts. Generally, at least about 30 nucleotides of an isolated promoter sequence will be used to direct expression of a nucleotide sequence. It is recognized that to increase transcription levels enhancers may be used in combination with the promoter regions of the invention. Enhancers are nucleotide sequences that act to increase expression of a promoter region. Enhancers are well known in the art and include the enhancer region SV40, the enhancer element 35S, and the like.

Sequences that hybridize to the sequences of the present invention are within the scope of the invention. Sequences that correspond to the promoter sequences of the present invention and hybridize to the promoter sequences disclosed therein will be at least 50% homologous, 70% homologous, and even 85% homologous or more with the disclosed sequence.

Smaller fragments may further contain the regulatory properties of the promoter so identified deletion analysis is a method of identifying essential regions. Blanking analysis can occur from both 5 'and 3' ends of the regulatory region. Fragments may be obtained by site-directed mutagenesis, mutagenesis using the polymerase chain reaction and the like. (see, Directed Mutagenesis: A Practical Approach IRL Press (1991)). The 3 'deletions can delineate the essential region and identify the 3' end so that that region can then be operatively linked to a core promoter of choice. Once the essential region is identified, transcription of an exogenous gene can be controlled by the essential region plus a core promoter. The core promoter may be any of the known core promoters such as the Cauliflower Mosaic Virus promoter 35S or 19S (US Patent No. 5,352,605), ubiquitin promoter (US Patent No. 5,510,474) and the core promoter (US Patent No. 5,364,780) or a Figwort mosaic virus promoter (Gruber, et al. "Vectors for Plant Transformation" Methods in Plant Molecular Biology and Biotechnology; Glick et al., CRC Press pp. 89-119 (1993)).

[055] The SBMU200 regulatory region has been identified as including the 1005 bp region above the supposed TATA box. See Figure 8. In addition, using the procedures set forth above, it has been determined that an essential promoter region includes -180 bp above the TATA box and specifically, the -176 to -44 region is particularly essential.

Promoter sequences from other plants may be isolated according to well known techniques based on their sequence homology to the promoter sequence set forth therein. In these techniques, all or part of the known promoter sequence is used as a marker that selectively hybridizes to other sequences present in a population of cloned genomic DNA fragments (ie genomic libraries) of a chosen organism. Methods are readily available in the art for hybridization of nucleic acid sequences.

[057] The entire promoter sequence or portions thereof may be used as a marker capable of hybridizing specifically to the corresponding promoter sequences. To achieve specific hybridization under a variety of conditions, such markers include sequences that are unique and are preferably at least 10 nucleotides in length, and more preferably at least about 20 nucleotides in length. Such markers may be used to amplify corresponding promoter sequences of an organism chosen by the well-known polymerase chain reaction (PCR) process. This technique can be used to isolate additional promoter sequences from a desired organism or as a diagnostic assay to determine the presence of the promoter sequence in an organism. Examples include hybridization classification of plated DNA libraries (plaques or colonies; see, for example, Innis et al., Eds., (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press).

In addition, the promoter of the present invention may be linked with nucleotide sequences other than the SBMu200 gene to express other heterologous nucleotide sequences. The nucleotide sequence for the promoter of the invention, as well as fragments and variants thereof, may be provided in expression cassettes together with heterologous nucleotide sequence for expression in the plant of interest, more particularly in male tissue of a plant. Such an expression cassette is provided with a plurality of restriction sites for nucleotide sequence insertion to be under promoter transcriptional regulation. These expression cassettes are useful in genetically manipulating any plant to achieve a desired phenotypic response. Examples of other nucleotide sequences that may be used as the exogenous gene of an SBMu200 promoter expression vector include complementary nucleotide units such as antisense molecules (RNA antisense callase, RNA antisense barnase, and antisense chalcona synthase). RNA sense, Ms45 antisense RNA), ribozymes and external guide sequences, an aptamero or single-stranded nucleotides. The exogenous nucleotide sequence may also encode auxins, rol B, cytotoxins, diphtheria toxin, DAM methylase, avidin, or may be selected from a prokaryotic regulatory system. By way of example, Mariani, et al., Nature; Vol 347; pp. 737; (1990), showed that expression in Aspergillus oryzae RNase-T1 or a Bacillus amyloliquefaciens RNase, designated "barnase," induced destruction of the tapetum cells resulting in male infertility. Quas, et al., Eur. J. Biochem. Vol. 173: pp. 617 (1988), describes the synthesis and chemistry of RNase-T1, while the nucleotide sequence of the barnase gene is disclosed in Hartley, J. Molec. Biol .; Vol. 202: pp. 913 (1988). The Agrobacterium rhizogenes rolB gene codes for an enzyme that interferes with auxin metabolism by catalyzing the release of indoles free from indoxyl-p-glycosides. Estruch, et al., EMBO J. Vol. 11: pp. 3125 (1991) and Spena, et al., Theor. Appl. Genet .;

Vol 84: pp. 520 (1992), showed that anther-specific expression of the rolB gene in tobacco resulted in plants having wrinkled anthers in which pollen production was severely decreased and the rolB gene is an example of a gene that is useful for production control. pollen Slightom, et al., J. Biol. Chem. Vol 261: pp. 108 (1985), discloses the nucleotide sequence of the rolB gene. DNA molecules encoding the diphtheria toxin gene can be obtained from the American Type Culture Collection (Rockville, MD), ATCC No. 39359 or ATCC No. 67011 and see Fabijanski, et al., E. P. Appl. No. 90902754.2, "Molecular Methods of Hybrid Seed Production" for examples and methods of use. The DAM methylase gene is used to cause sterility in the methods discussed in U.S. Patent No. 5,689,049 and PCT / US95 / 15229 Cigan, A.M. and Albertsen, M.C., "Reversible Nuclear Genetic System for Male Sterility in Transgenic Plants". Also see the discussion of the use of the avidin gene to cause sterility in U.S. Patent No. 5,962,769 "Induction of Male Sterility in Plants by Expression of High Levels of Avidin" by Albertsen et al.

[059] The invention includes vectors with the SBMu200 gene. A vector comprising the SBMu200 gene is prepared, a promoter will direct gene expression in the plant and a terminator region. As noted, the promoter in the construct may be the native promoter or a substituted promoter that will provide expression in the plant. Selection of the promoter will depend on the intended use of the gene. The promoter in the construct may be an inducible promoter, so that expression of the sense or antisense molecule in the construct may be controlled by exposure to the inducer.

[060] Other vector components may be included, also depending on the intended use of the gene. Examples include selectable markers, wash or regulatory sequences, stabilizing or leader sequences, etc. General descriptions and examples of plant expression vectors and reporter genes can be found in Gruber et al., "Vectors for Plant Transformation" in Method in Plant Molecular Biology and Biotechnology. Glick et al eds; CRC Press pp. 89-119 (1993). Selection of an appropriate expression vector will depend on the host and the method of introducing an expression vector into the host. The expression cassette will also include the 3 'terminus of the heterologous nucleotide sequence of interest, a transcriptional and translational functional termination region in plants. The termination region may be native to the nucleotide promoter sequence of the present invention, may be native to the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the A. tumefaciens Ti-plasmid, such as the octopin synthase and nopaline synthase termination regions. See also Guerineau et al Mol. Gen. Genet. 262: 141-144 (1991); Proudfoot Cell 64: 671-674 (1991); Sanfacon et al Genes Dev. 5: 141-149 (1991); Mogen et al Plant Cell 2: 1261-1272 (1990); Munroe et al Gene 91: 151-158 (1990); Ballas et al Nucleic Acids Res. 17: 78917903 (1989); Joshi et al Nucleic Acid Res. 15: 9627-9639 (1987).

Expression cassettes may additionally contain 5 'leader sequences. Such leader sequences may act to improve translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5 'non-coding region), Elroy-Stein et al. Proc. Nat. Acad. Know. USA 86: 6126-6130 (1989); potivirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al; MDMV leader (Maize Dwarf Mosaic Virus), Virology 154: 9-20 (1986); human immunoglobulin heavy chain binding protein (BiP), Macejak et al Nature 353: 90-94 (1991); untranslated leader of the alfalfa mosaic virus cover protein mRNA (AMV RNA 4), Jobling et al Natura 325: 622-625 (1987); leader of tobacco mosaic virus (TMV), Gallie et al (1989) Molecular Biology of RNA, pages 237-256; and leader of the chlorotic spotted maize virus (MCMV) Lommel et al Virology 81: 382-385 (1991). See also Della-Cioppa et al. Plant Physiology 84: 965-968 (1987). The cassette may also contain sequences that improve mRNA translation and / or stability such as introns.

In such cases when it is desirable to have the expressed heterologous nucleotide sequence product directed to a particular organelle, particularly the plastid, amyloplast, or endoplasmic reticulum, or secreted on the cell surface or extracellularly, an expression cassette may also comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, transit peptide for the acyl carrier protein, the small RUBISCO subunit, EPSP plant synthase, and the like. One skilled in the art will readily appreciate the various options available in expressing a product for a particular organelle. For example, the barley alpha amylase sequence is often used to direct expression to the endoplasmic reticulum (Rogers, J. Biol. Chem. 260: 3731-3738 (1985)). The use of transit peptides is well known (for example, see U.S. Patent Nos. 5,717,084; 5,728,925).

In the preparation of an expression cassette, the various DNA fragments may be engineered to provide the DNA sequences in the proper orientation and, as appropriate, in the appropriate reading frame. Toward this end, adapters or linkers may be employed to join DNA fragments or other manipulations may be involved to provide convenient sites and restriction, superfluous DNA removal, site removal and restriction, or the like. To this end, in vitro mutagenesis, primer repair, restriction digestions, stiffening, and replacements, such as transitions and transversions, may be involved.

As noted herein, the present invention provides vectors capable of expressing genes of interest under the control of the promoter. In general, vectors must be functional in plant cells. At times, it may be preferable to have vectors that are functional in E. coli (e.g., protein production to raise antibodies, DNA sequence analysis, insertion construction, nucleic acid yielding). Vectors and procedures for cloning and expression in E. coli are discussed in Sambrook et al. (supra).

The transformation vector comprising the promoter sequence of the present invention operably linked to a heterologous nucleotide sequence in an expression cassette may also contain at least one additional nucleotide sequence for a gene to be co-transformed in the organism. Alternatively, the additional sequence (s) may be provided in another transformation vector.

[066] Reporter genes may be included in transformation vectors. Examples of suitable reporter genes known in the art can be found, for example, in Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin et al (Kluwer Academic Publishers), pp. 133; DeWet et al Mol. Cell. Biol. 7: 725-737 (1987); Goff et al EMBO J. 9: 2517-2522 (1990); Kain et al BioTechniques 19: 650-655 (1995); and Chiu et al Current Biology 6: 325-330 (1996).

[067] Selectable marker genes for selecting transformed cells or tissues can be included in transformation vectors. These may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding chloramphenicol resistance, Herrera Estrella et al. EMBO J. 2: 987-992 (1983); methotrexate, Herrera Estrella et al Nature 303: 209-213 (1983); Meijer et al Plant Mol. Biol. 16: 807-820 (1991); hygromycin, Waldron et al Plant Mol. Biol. 5: 103-108 (1985); Zhijian et al Plant Science 108: 219-227 (1995); streptomycin, Jones et al. Mol. Gen. Genet. 210: 86-91 (1987); spectinomycin, Bretagne-Sagnard et al Transgenic Res. 5: 131-137 (1996); bleomycin, Hille et al Plant Mol. Biol. 7: 171-176 (1990); sulfonamide, Guerineau et al Plant Mol. Biol. 15: 127136 (1990); bromoxynil, Stalker et al Science 242: 419-423 (1988); glyphosate, Shaw et al Science 233: 478-481 (1986); phosphinothricin, DeBlock et al EMBO J. 6: 2513-2518 (1987).

[068] The transformation / transfection method is not critical to the present invention; Various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they can be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to achieve transcription or transcription and translation of the sequence to effect phenotypic changes in organisms. Therefore, any method that provides effective transformation / transfection can be employed.

[069] Methods for introducing plant tissue expression vectors available to a person skilled in the art are varied and will depend on the selected plant. Procedures for transforming a wide variety of plant species are well known and described in the literature. See, for example, Miki et al, "Procedures for Introducing Foreign DNA into Plant" in Methods in Plant Molecular Biotechnology, supra; Klein et al., Bio / Techmology 10: 268 (1992); and Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). For example, the DNA construct may be introduced into the genomic DNA of a plant cell using techniques such as microprojectile mediated release, Klein et al., Nature 327: 70-73 (1987); electroporation, Fromm et al., Proc. Natl. Acad. Know. 82: 5824 1985); precipitation of polyethylene glycol (PEG), Paszkowski et al., EMBO J. 3: 2717-2722 (1984); direct gene transfer WO 85/01856 and EP No. 0 275 069; in vitro protoplast transformation U.S. patent no. 4,684,611; and microinjection of plant cell protoplasts or embryogenic callus. Crossway, Mol. Gen.

Genetics 202: 179-185 (1985). Co-cultivating plant tissue with Agrobacterium tumefaciens is another option, where DNA constructs are placed in a binary vector system. See, for example, U.S. Patent No. 5,591,616; Ishida et al., "High Efficiency Transformation of Maize (Zea mays L.) mediated by Agrobacterium tumefaciens" Nature Biotechnology 14: 745-750 (1996). The virulence functions of the host Agrobacterium tumefaciens will direct insertion of the construct into a plant cell DNA when the cell is infected with the bacterium. See, for example, Horsch et al., Science 233: 496-498 (1984), and Fraley et al., Proc. Natl. Acad. Know. 80: 4803 (1983).

[070] Standard methods for canola transformation are described in Moloney et al. "High Efficiency Transformation of Brassica napus using Agrobacterium Vector" Plant Cell Reports 8: 238-242 (1989). Corn transformation is described by Fromm et al., Bio / Technology 8: 833 (1990) and Gordon-Kamm et al., Supra. Agrobacterium is primarily used in dicotyledons, but certain monocotyledons such as maize can be transformed by Agrobacterium. See supra and U.S. Patent No. 5,550,318. Rice transformation is described by Hiei et al, "Efficient Transformation of Rice (Oryza sativs L.) Mediated by Agrobacterium and Following Analysis of the Boundaries of the T-DNA" The Plant Journal 6 (2): 271-282 (1994) ), Christou et al, Trends in Biotechnology 10: 239 (1992) and Lee et al Proc. Nat'l Acad. Know. USA 88: 6389 (1991). Wheat can be processed by techniques similar to those used for processing corn or rice. Sorghum transformation is described in Casas et al, supra and sorghum by Wan et al, Plant Physicol. 104: 37 (1994). Soybean transformation is described in a variety of publications, including U.S. Patent no. 5,015,580.

[071] Further detailed description is provided below by way of instruction and illustration and is not intended to limit the scope of the invention.

Example 1 - Identification and Co-segregation of SBMu200 [072] Plant families of a mutating (Mu) population that secrete to plants that were primarily sterile males, with no or only a few abnormal anther expelled, none of which had pollen, were identified. gift. Male sterility is separated as a result of these cases where a Mu element was randomly integrated into a gene responsible for some step in microsporogenesis, disrupting its expression. Plants of a segregating F2 family in which the sterile male mutation was designated SBMu200, grew and ranked for male fertility / sterility based on the above criteria. Leaf samples were taken and DNA was subsequently isolated in approximately 20 plants by phenotypic classification, ie male fertility vs. male sterility.

Southern analysis was performed to confirm the association of Mu with sterility. Southern analysis is a technique well known to those skilled in the art. This common process involves isolating plant DNA, cutting with restriction endonucleases, fractionating the cut-to-molecular weight DNA on an agarose gel, and transferring it to nylon membranes to fix the separate DNA. These membranes are subsequently hybridized to a marker fragment that has been radiolabeled with P32P-dCTP, and washed in an SDS solution. Southern, E., "Detection of Specific Sequences Among DNA Fragments by Gel Electrophoresis" J. Mol. Biol. 98: 503-317 (1975). Plants from a segregating F2 SBMu200 family grew and were classified for male fertility / sterility. Leaf samples were taken and subsequently DNA isolation was conducted on approximately 20 plants by phenotypic classification. DNA (~ 7ug) from 5 fertile plants and 12 sterile plants was digested with EcoRI and electrophoresed through a 0.75% agarose gel 1. Digested DNA was transferred to a nylon membrane via Southern blot. The membrane was hybridized to an internal fragment of the Mu8 transposon. Membrane autoradiography revealed co-segregation of a 5.5 Kb EcoRI fragment with the sterility phenotype as shown in Figure 2. This EcoRI band secreted into the fertile plants suggesting a heterozygous wild-type condition for the allele.

Example 2 - Library Construction and Classification The cDNA library classification process is commonly known among those skilled in the art and is described in Sambrook, J., Fritsch, EF, Maniatis T., et al., Molecular Cloning. : A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Lab Press, Plainview, NY (1989). The libraries were created as follows.

DNA from a sterile plant was digested with EcoRI and passed on a preparation gel. DNA with a molecular weight between 5.0 and 6.0 Kb was removed from the gel, electroeluted and ethanol precipitated. This DNA was ligated into the Lambda Zap vector (StratageneTM) using the manufacturer's protocol. Bound DNA was packaged into phage particles using Gigapack Gold (Stratagene ™). Approximately 500,000 PFU were plated and suspended on nitrocellulose membranes. The membranes were hybridized with the Mu8 marker. A pure clone was obtained after 3 rounds of classification. The insert was taken from the phage as a plasmid and designated SBMu200-3. 1. A PstI marginal fragment from this clone was isolated and used to re-mark the EcoRI co-segregation spot. The 5.5 Kb EcoRI fragment is homozygous in all sterile plants, confirming that the correct Mu fragment has been isolated. Three of the fertile plants are heterozygous for the 5.5 Kb EcoRI band and one 4.3 Kb EcoRI band. Two of the fertile plants are homozygous for the 4.3 Kb EcoRI band EcoRI, presumably the wild-type allele.

Example 3 - Expression Analysis and cDNA Isolation

Northern analysis can be used to detect expression of anther developmental gene traits in various microsporogenesis states. Northern analysis is also a commonly used technique known to those skilled in the art and is similar to Southern analysis except that mRNA rather than DNA is isolated and placed in the gel. The RNA is then hybridized to the labeled label. Potter, E., et al., "Thyrotrotropin Releasing Hormone Exerts Rapid Nuclear Effects to Increase Production of the Primary Prolactin in RNA Transcript" Proc. Nat. Acad. Know. USA 78: 6662-6666 (1981), Lechelt, et al., "Isolation & Molecular Analysis of the Plows" Mol. Gen. Genet. 219: 225-234 (1989). The PstI fragment from clone SBMu200-3.1 was used to label a Northern blot containing seed, immature ear, fringe and seeding RNA. A signal was observed only in the fringe RNA at approximately the fourth stage of microsporogenesis, as reported in Figure 3. The transcription is about 2.3 KB in length. The same marker was also used to classify a cDNA library constructed from mRNA isolated from uninucleated later stage meiotic anthers. One clone, designated SBMu200-8.1, was isolated from the library.

Example 4 - Sequence Analysis The genomic clone SBMu200-3.1 and cDNA clone, SBMu200-8.1, were sequenced by Loftstrand Labs Limited. Sanger, F., Nicklen, S., Coulson A. R. (1977) "DNA sequencing with chain terminating inhibitors" Proc. Natl. Acad. Know. USA 74: 54635467. The sequences are shown in Figures 4 and 5 and the comparison is in Figure 6. The cDNA / genomic comparison reveals that five introns are present in the genomic clone. Mu8 insertion occurs in exon 1. Test for codon preference preference non-randomness at the third position of each codon was consistent with the main ORF in cDNA being likely protein-encoding Orf. There is a Met initiation codon assumed at position 1089 in the genomic clone. CDNA homology to the genomic clone begins at nucleotide 1094. Therefore SBMu200-8.1 does not represent a full length clone and is devoid of 5 bases to the Met initiation codon. A database search revealed significant homology to P450 enzymes found in yeast, plants and mammals. P450 enzymes have been widely studied and three characteristic protein domains have been elucidated. A comparison of the predicted SBMu200-8.1 protein for the consensus amino acid sequence of these domains showed 1) 92% identity to the dioxigen binding domain, 2) 85% identity to the tridecapeptide domain (steroid binding), and 3) 100% identity to the C-terminal heme attachment domain. Additional expression studies were done using the SBMu200-8.1 cDNa marker against a northern mRNA containing distinct stages of microsporogenesis. A signal was detected from meiosis II / quarter to late uninucleate, with the maximum signal being observed from early uninucleate through late uninucleate as shown in Figure 7.

Example 5 - Identification of Promoter and Its Essential Regions An alleged TATA box can be identified by primer extension analysis as described by Current Protocols in Molecular Biology, Ausubel, F. M. et al. eds; John Wiley and Sons, New York pp. 4.8.1-4.8.5 (1987).

Anther gene regulatory regions, such as promoters, can be identified in genomic subclones using functional analysis, usually verified by observing reporter gene expression in anther tissue and a lower level or absence of reporter gene expression in non-anther tissue. . The possibility of regulatory regions residing "above" 5 'from the translation initiation site can be tested by subcloning a DNA fragment containing the above region into expression vectors for transient expression experiments. It is expected that smaller subgenomic fragments may contain the regions essential for preferred expression of male tissue. For example, the essentials of CaMV 19S and 35S promoters have been identified in relatively small fragments derived from larger genomic pieces as described in U.S. Pat. No. 5,352,605.

The selection of a suitable expression vector with which functional expression is tested will depend on the host and the method of introducing an expression vector into the host and such methods are well known to those skilled in the art. For eukaryotes, the regions in the vector include regions that control transcription initiation and processing control. These regions are operably linked to a reporter gene such as UidA, which encodes β-glucuronidase (GUS), or luciferase. General descriptions and examples of plant expression vectors and reporter genes can be found in Gruber, et al "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology; Glick et al eds; CRC Press; pp. 89-119; (1993). GUS expression vectors and GUS gene cassettes are commercially available from Clonetech, Palo Alto, CA, while luciferase expression vectors and luciferase gene cassettes are commercially available from Promega Corporation, Madison, WI. Ti plasmids and other Agrobacterium vectors are described in Ishida, Y. et al., Nature Biotechnology; Vol. 14; pp. 745-750; (1996) and in Pat. No. 5,591., 616 "Method for Transforming Monocotyledons" (1994).

Expression vectors containing supposed regulatory regions located in genomic fragments can be introduced into intact tissues such as anthers, embryos or callus. DNA release methods include microprojectile bombardment, DNA injection, electroporation, and Agrobacterium-mediated gene transfer (see Gruber et al., "Vectors for Plant Transformation" in Methods in Plant Molecular Biology and Biotechnology, Glick et al. eds.; CRC Press; (1993); U.S. Pat. No. 5,591,616; and Ishida, Y. et al., Nature Biotechnology; Vol. 14; pp. 745-750 (1996)). General methods of plant tissue culture are found in Gruber et al., Supra and Glick, supra.

[081] For the transient assay system, isolated, staged anthers are immediately placed in fringe culture medium (Pareddy, DR and JF Petelino, Crop Sci. J .; Vol. 29; pp. 15641566; (1989)) with Phytagel 0.5% (Sigma, St. Louis) or other solidifying media. Expression vector DNA is introduced within 5 hours preferably by microprojectile-mediated release into 1.2 µπι particles at 6.89-7.58 MPa. After DNA release, anthers are incubated at 26 ° C under the same fringe culture medium for 17 hours and analyzed by preparation of a complete tissue homogenate and analysis for GUS or luciferase activity (see Gruber et al ., supra).

Above the likely SBMu200 translation initiation codon, 1088 bp of DNA was present in the genomic clone SBMu200-3.1. Translation fusions via a constructed Ncol site were generated with reporter genes encoding luciferase and p-glucuronidase to test whether this DNA fragment had promoter activity in transient bombarded plant tissue expression assays. Activity was demonstrated in anthers rather than coleoptilos, root and callus, suggesting specific or preferred anther promoter activity.

[083] A reasonable TATA box was observed by inspection, about 83-77 bp above the translation initiation codon. The genomic clone SBMu200-3.1 thus includes about 1005 bp above the possible TATA box. For typical plant genes, the start of transcription is 26-36 bp below the TATA box, which would give SBMu200 mRNA a 5 'untranslated leader of about 48-58 nt. The 1088 bp total SBMu200 subgenomic fragment, including untranslated leader, transcription start, TATA box, and sequences above the TATA box, was thus shown to be sufficient for promoter activity. See Figure 8, which is Seq Id. No. 5. The supposed TATA box (TATATCA) is underlined. Therefore, the present invention encompasses a DNA molecule having a nucleotide sequence of Seq Id. No. 5 (or those with sequence identity) and having the function of a preferred male tissue regulatory region.

Deletion analysis can occur from both 5 'and 3' ends of the regulatory region: fragments can be obtained by site-directed mutagenesis, polymerase chain reaction mutagenesis, and the like (Directed Mutagenesis: A Practical Approach IRL Press (1991) The 3 'end of the preferred male tissue regulatory region may be delineated by proximity to the assumed TATA box or by 3' deletions if necessary.The essential region may then be operatively linked to the nucleus promoter. Since the essential region is identified, transcription of an exogenous gene can be controlled by the preferred male tissue region of SBMu200 plus a core promoter.The core promoter can be any of the core promoters such as mosaic virus. of cauliflower 35S or 19S promoter (US Patent No. 5,352,605), ubiquitin promoter (US Patent No. 5,510,474) and IN2 core promoter (Paten te U. S. No. 5,364,780) or a Figwort mosaic virus promoter (Gruber, et al. "Vectors for Plant Transformation" Methods in Plant Molecular Biology and Biotechnology; Glick et al eds, CRC Press pp. 89-119 (1993)). Preferably, the promoter is the core promoter of a preferred male tissue gene or the CaMV 35S core promoter. More preferably, the promoter is a promoter of a preferred male tissue gene and in particular the SBMu200 core promoter.

Further mutation analysis, for example by ligand screening, a method well known in the art, can identify small segments containing sequences necessary for preferred anther expression. Such mutations may introduce functionality modifications such as expression levels, expression time, or expression tissue. Mutations can also be silent and have no observable effect.

[086] Previous procedures were used to identify essential regions of the SBMu200 promoter. after ligating the promoter with the luciferase marker gene the gene deletion analysis was performed on the promoter regions above the assumed TATA box as shown in Figure 9. The x-axis of the bar graph indicates the number of pairs of base just above the supposed TATA box retained in a series of erasure derivatives starting from the 5 'end of the promoter. The y-axis shows normalized luciferase activity as a percentage of full length promoter activity.

As is apparent from the graph, approximately 176 bp immediately above the TATA box was sufficient when paired to the core promoter (TATA box assumed to start transcription), plus 5 'untranslated leader, of transient anther expression. In contrast, luciferase activity was minimal under the additional 5 'end deletion to 91 bp above the assumed TATA box. This end 176 bp of the TATA box assumed through the untranslated leader can be considered a minimal promoter, which is depicted in Figure 10. The TATA box is underlined. Deletion with the full-length promoter from -176 to -92 relative to TATA box reduced about 1% wild-type activity. Deletion from -39 to -8 did not greatly reduce activity. Thus, the -176 to -44 bp region contains an essential region and could constitute an enlarged end element conferring anther expression on the promoter, which is referred to as the "anther box".

Scanning binder analysis was conducted through anther box in 9-10 bp increments. Scanning ligand substitution positions in this region are shown in Figure 10 and the expression level of wild-type sequence related mutants are shown in Figure 11. The most drastic effect on transient anther expression was observed for LS 12 mutants. and LS13, in the region 52-71 bp above the supposed TATA box. A major effect on transient anther expression was also observed for mutants LS06, LS07, LS08 and LS 10, in the 82-131 bp region above the assumed TATA box. Anther box sequences required transient expression wild-type levels and are thus demonstrated in the -52 to -131 region relative to the TAATA box, particularly the -52 to -71 region.

Example 6 - Comparison of SBMu200 from RT-PCR sorghum bangs and SBMu200 corn cDNA [089] As noted above, SBMu200 is a male fertility gene. When it is modified, it will result in male sterility. A SBMu200 counterpart was identified in sorghum. SBMu200 sorghum cDNA was isolated by using SBMu200 maize gene primers in a polymerase chain reaction with a sorghum fringe cDNA as the model. The resulting cDNA fragment was sequenced by methods described above and then compared to the SBMu200 corn cDNA. Nucleotide sequence comparisons are shown in Figure 10 and show 90% identity.

As is evident from the above, the SBMu200 gene is critical for male fertility in plants.

[091] It can therefore be observed that the invention achieves at least all its aims.

Claims (3)

Expression vector, characterized in that it comprises an exogenous gene, wherein the exogenous gene is operably linked to a promoter, wherein it is the promoter is operably linked to a male tissue-specific regulatory element, wherein the regulatory element is is selected from the group comprising: (a) Seq Id. No. 6; (b) 130 contiguous base pairs of -38 and higher above the TATA box of the Seq Id. No. 7; (c) sequences from -44 to -180 above the TATA box of Seq Id. No. 7; (d) sequences -92 to -176 above the TATA box of Seq Id. No. 7; (e) sequences -44 to -89 above the TATA box of Seq Id. No. 7; (f) sequences -52 to -131 above the TATA box of Seq Id. No. 7; (g) sequences -52 to -71 above the TATA box of Seq Id. No. 7; or (h) sequences -82 through -131 above the TATA box of Seq Id. No. 7 Expression vector according to claim 1, characterized in that the promoter is selected from either CaMV35S, SGB6, SBMu200, MS45 or 5126, or the Id. Of Seq. No.5. Expression vector according to claim 1, characterized in that the exogenous gene product disrupts male tissue function.