WO2007112326A1 - Promoter, promoter control elements, and combinations, and uses thereof - Google Patents
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- WO2007112326A1 WO2007112326A1 PCT/US2007/064848 US2007064848W WO2007112326A1 WO 2007112326 A1 WO2007112326 A1 WO 2007112326A1 US 2007064848 W US2007064848 W US 2007064848W WO 2007112326 A1 WO2007112326 A1 WO 2007112326A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
Definitions
- PROMOTER PROMOTER CONTROL ELEMENTS, AND COMBINATIONS
- the present invention relates to promoters and promoter control elements
- promoter control elements can be included in polynucleotide constructs, expression cassettes,
- vectors or inserted into the chromosome or as an exogenous element, to modulate in vivo and
- Host cells including plant cells, and organisms,
- polynucleotides comprising the promoters and promoter control elements of the present
- This invention relates to promoter sequences and promoter control element
- An introduced gene is generally a chimeric gene composed of the coding region that confers the desired trait and regulatory sequences.
- One regulatory sequence is the
- promoter which is located 5' to the coding region. This sequence is involved in regulating the pattern of expression of a coding region 3' thereof.
- the promoter sequence binds RNA polymerase complex as well as one or more transcription factors that are involved in
- coding region derived from a different source than is the coding region. It may be from a different gene of
- the promoter may confer broad expression as in the case of the widely-used
- cauliflower mosaic virus (CaMV) 35S promoter The promoter may confer tissue-specific expression as in the case of the seed-specific phaseolin promoter.
- the promoter may confer a
- the promoter may be induced by an applied chemical compound, or by an environmental condition applied to the plant.
- the promoter that is used to regulate a particular coding region is determined by the desired expression pattern for that coding region, which itself is determined by the desired resulting phenotype in the plant. For example, herbicide resistance is desired throughout the plant so the 35S promoter is appropriate for expression of an herbicide-
- a seed-specific promoter is appropriate for changing the oil content of soybean seed.
- An endosperm-specific promoter is appropriate for changing the starch
- a root-specific promoter can be important for improving water or nutrient up-take in a plant. Control of expression of an introduced gene by the promoter is
- plants, mammals, yeast, and prokaryotes having particular desired characteristics or traits.
- This technology permits one or more polynucleotides from a source different than the organism of choice to be transcribed by the organism of choice. If desired,
- the present invention is directed to isolated polynucleotide sequences that
- promoters and promoter control elements from plants, especially Arabidopsis thaliana, and other promoters and promoter control elements functional in plants.
- promoter sequences comprise, for example
- Promoter or promoter control element sequences of the present invention are identical to promoter or promoter control element sequences of the present invention.
- the present promoter control elements are capable of serving as or fulfilling the function, for example, as a core promoter, a TATA box, a
- polymerase binding site an initiator site, a transcription binding site, an enhancer, an inverted repeat, a locus control region, or a scaffold/matrix attachment region.
- the first promoter control element is a promoter control element sequence as discussed above, and the second
- promoter control element is heterologous to the first control element; wherein, the first and
- second control elements are operably linked. Such promoters may modulate transcript levels
- the present isolated polynucleotide comprises a
- promoter control element is operably linked to a polynucleotide to be transcribed.
- the promoter and promoter are identical to [0014] in another embodiment of the present invention.
- control elements of the instant invention are operably linked to a heterologous polynucleotide
- the host cell can include, for instance, bacterial, yeast, insect, mammalian, and plant.
- the host cell can include, for instance, bacterial, yeast, insect, mammalian, and plant.
- Such a promoter or promoter control element exogenous to the genome.
- a promoter or promoter control element exogenous to the genome.
- the host cell is a plant cell capable of
- This method comprises providing a polynucleotide or vector according to the
- the polynucleotide or vector in another embodiment of the present method, the polynucleotide or vector
- Table 1 consists of the Expression Reports for each promoter of the invention providing the nucleotide sequence for each promoter and details for expression
- Each row of the table begins with heading of the data to be found in the
- FIGURE 1 - pNewbin4-HAPl-GFP
- Figure l is a schematic representation of a vector that is useful to insert promoters of the invention into a plant.
- HAPlUAS the upstream activating sequence for HAPl
- 5ERGFP the green fluorescent protein gene that has been optimized for localization to the endoplasmic reticulum
- OCS2 the terminator sequence from the octopine synthase 2 gene
- OCS the terminator sequence from the octopine synthase gene
- p28716 (a.k.a 28716 short) - promoter used to drive expression of the PAT (BAR) gene
- PAT (BAR) - a marker gene conferring herbicide resistance
- TrfA Spec - a marker gene conferring spectinomycin resistance TrfA - transcription repression factor gene
- the invention disclosed herein provides promoters capable of driving the
- promoters is one object of this invention.
- the promoter sequences, SEQ ID NOs: 1 - 22, are
- Chimeric The term “chimeric” is used to describe polynucleotides or genes
- Promoters referred to herein as "broadly Expressing Promoter Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter: Promoters referred to herein as "broadly Expressing Promoter"
- promoters actively promote transcription under most, but not necessarily all, environmental conditions and states of development or cell differentiation.
- broadly expressing promoters include the cauliflower mosaic virus (CaMV) 35S transcript initiation region and the 1 ' or 2' promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes, such as the maize ubiquitin-1
- Domains are fingerprints or signatures that can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can be used to characterize protein families and/or parts of proteins
- each domain has been associated with either a conserved primary sequence or a sequence
- a domain can be any length, including the entirety of the polynucleotide to be transcribed. Examples of domains include, without limitation, AP2,
- Endogenous refers to any polynucleotide, polypeptide or protein sequence which is a natural part
- endogenous coding region or “endogenous cDNA” refers to the coding region that is naturally operably linked to the promoter.
- Enhancer/Suppressor An “enhancer” is a DNA regulatory element that
- Enhancers can increase the steady state level of a transcript, usually by increasing the rate of transcription initiation. Enhancers usually exert their effect regardless of the distance, upstream or downstream location, or orientation of the enhancer relative to the start site of
- a "suppressor” is a corresponding DNA regulatory element that decreases the steady state level of a transcript, again usually by affecting the rate of transcription initiation.
- the essential activity of enhancer and suppressor elements is to bind
- Such binding can be assayed, for example, by methods described below.
- the binding is typically in a manner that influences the steady state level of a transcript in a cell or in an in vitro transcription extract.
- Exogenous is any polynucleotide
- T 0 for the primary transgenic plant and Tj for the first generation.
- exogenous as used herein is also
- Heterologous sequences are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from
- corn is considered heterologous to an Arabidopsis coding region sequence. Also, a promoter
- Regulatory element sequences such as UTRs
- expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.
- This similarity may be in only a fragment of the sequence and often represents a functional domain such as, examples including without limitation a DNA binding domain or a domain
- Inducible Promoter An "inducible promoter" in the context of the current
- invention refers to a promoter, the activity of which is influenced by certain conditions, such as light, temperature, chemical concentration, protein concentration, conditions in an organism,
- a typical example of an inducible promoter, which can be utilized with the polynucleotides of the present invention, is PARSKl, the promoter from an Arabidopsis
- Misexpression refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to the
- This term also encompasses expression and/or translation of a gene or coding region
- coding region from a different plant species or from a non-plant organism.
- transcription describes the biological activity of a promoter sequence or promoter control
- Such modulation includes, without limitation, up- and down-regulation of initiation of transcription, rate of transcription, and/or transcription levels.
- Operable Linkage is a linkage in which a promoter
- sequence or promoter control element is connected to a polynucleotide sequence (or
- Two DNA sequences (such as
- a polynucleotide to be transcribed and a promoter sequence linked to the 5' end of the polynucleotide to be transcribed are said to be operably linked if induction of promoter
- a promoter sequence would be operably linked to a polynucleotide sequence if the promoter was capable of effecting transcription of that polynucleotide sequence.
- Percentage of sequence identity refers to the degree of identity between any given query sequence and a subject sequence.
- a subject sequence typically has a length that is from about 80 percent to
- a query nucleic acid or amino acid sequence is
- ClustalW calculates the best match between a query and one or more
- Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments.
- scoring method percentage; number of top diagonals: 4; and gap penalty: 5.
- gap opening penalty 10.0
- gap extension penalty 5,0
- weight transitions yes.
- word size 1 ; window
- the output is the percent identity of the subject sequence with respect to
- the percent identity value can be rounded to the nearest
- 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
- Plant Promoter A "plant promoter” is a promoter capable of initiating
- transcription in plant cells can modulate transcription of a polynucleotide.
- promoters need not be of plant origin.
- promoters derived from plant viruses such as the CaMV35S promoter or from Agrobacterium tumefaciens such as the T-DNA promoters,
- ubiquitin-1 maize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.
- Plant Tissue The term “plant tissue” includes differentiated and
- the plant tissue may be in plants or in organ, tissue or cell culture.
- Preferential Transcription is defined as transcription that occurs in a particular pattern of cell types or developmental times or in response to specific stimuli or combination thereof. Non-limitive examples of preferential
- transcription include: high transcript levels of a desired sequence in root tissues; detectable transcript levels of a desired sequence in certain cell types during embryogenesis; and low transcript levels of a desired sequence under drought conditions.
- preferential transcription can be determined by measuring initiation, rate, and/or levels of transcription.
- Promoter is a DNA sequence that directs the transcription of
- a promoter is located in the 5' region of a polynucleotide to be
- promoters are defined as the region upstream of the first exon; more typically, as a region
- downstream of the preceding gene and upstream of the first of multiple transcription start sites are downstream of the preceding gene and upstream of the first of multiple transcription start sites; more typically, the region downstream of the polyA signal and upstream of the first of
- the promoters of the invention comprise at least a core promoter as
- promoters are capable of directing transcription of genes located on
- promoters exhibit bidirectionality and can direct transcription of a downstream gene when
- promoter may also include at least one control element such as an upstream element.
- control elements include UARs and optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element.
- Promoter control elements include transcriptional regulatory sequence determinants such as, but not limited to,
- enhancers scaffold/matrix attachment regions, TATA boxes, transcription start locus control regions, UARs, URRs, other transcription factor binding sites and inverted repeats.
- Public sequence refers to any sequence that has been deposited in a publicly accessible
- amino acid and nucleotide sequences are publicly accessible, for example, on the BLAST databases on the NCBI FTP web site (accessible via the internet).
- GenBank GenBank
- EMBL GeneBank
- DBBJ DNA Database of Japan
- PDB Brookhaven Protein Data Bank
- regulatory region refers to nucleotide
- sequences that, when operably linked to a sequence, influence transcription initiation or translation initiation or transcription termination of said sequence and the rate of said
- operably linked refers to positioning of a regulatory region and said
- Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Regulatory regions can be classified in two categories, promoters and other regulatory regions.
- regulatory sequence The term "regulatory sequence,” as used in the
- transcript or polypeptide product refers to any nucleotide sequence that influences transcription or translation initiation and rate, or stability and/or mobility of a transcript or polypeptide product.
- Regulatory sequences include, but are not limited to, promoters, promoter control elements,
- protein binding sequences 5' and 3' UTRs, transcriptional start sites, termination sequences, polyadenylation sequences, introns, certain sequences within amino acid coding sequences
- promoters refers to a subset of promoters that have a high preference for modulating
- temporal and/or tissue or organ specific promoters of plant origin that can be used with the polynucleotides of the present invention, are:
- PTA29 a promoter which is capable of driving gene transcription specifically in tapetum and only during anther development (Koltonow et al. (1990) Plant Cell 2: 1201 ; RCc2 and RCc3,
- tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues or organs, such as root, ovule, fruit, seeds, or flowers.
- Other specific promoters include those from genes encoding seed storage proteins or the lipid body membrane protein, oleosin. A few root-specific promoters are noted above. See
- Stringency is a function of nucleic acid molecule probe length, nucleic acid molecule probe composition (G + C content), salt
- Stringency is typically measured by the parameter T m , which is the temperature at
- N is the number of nucleotides of the nucleic acid molecule probe.
- T 111 81.5+16.6 log ⁇ [Na + ]/(l+0.7[Na + ]) ⁇ + 0.41(%G+C)-500/L 0.63(%formamide) (II)
- T m of Equation II is affected by the nature of the hybrid: for DNA-RNA hybrids, T m is 10-15 0 C higher than calculated; for RNA-RNA hybrids, T 111 is 20-25 0 C higher. Because the
- T 111 decreases about 1 0 C for each 1 % decrease in homology when a long probe is used
- stringency conditions can be adjusted to favor detection of identical genes or related family members.
- Equation II is derived assuming the reaction is at equilibrium. Therefore,
- hybridizations according to the present invention are most preferably performed under
- hybridization buffer that includes a hybridization accelerator such as dextran sulfate or another high volume polymer.
- Stringency can be controlled during the hybridization reaction, or after
- wash solution stringencies lie within the ranges stated above;
- high stringency is 5-8°C below T 111
- medium or moderate stringency is 26-29°C below T 111 and
- low stringency is 45-48 0 C below T m .
- T 0 refers to the whole plant, explant or callus tissue
- T 1 refers to either the progeny of the T 0 plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue
- T 2 refers to the progeny of the Ti plant. T 2 progeny are the result of self-fertilization or cross-pollination of a Ti plant.
- T 3 refers to second generation progeny of the plant that is the
- T 3 progeny are the result of self-fertilization or
- TATA to start shall mean the distance, in number of nucleotides, between the primary TATA motif and the start of transcription.
- Transgenic plant A "transgenic plant” is a plant having one or more plant
- translational start site is usually an ATG or AUG in a transcript, often the first ATG or
- a single protein encoding transcript may have multiple translational start
- Transcription start site "Transcription start site” is used in the current
- This point is typically located about 25 nucleotides downstream from a TFIID binding site, such as a TATA box.
- Upstream Activating Region An "Upstream Activating Region" or
- UAR is a position or orientation dependent nucleic acid element that primarily directs tissue, organ, cell type, or environmental regulation of transcript level, usually by affecting the rate of transcription initiation.
- Upstream Repressor Regions or "URR”s.
- the essential activity of these elements is to bind a protein factor. Such binding can be assayed by methods
- the binding is typically in a manner that influences the steady state level of
- a transcript in a cell or in vitro transcription extract is a transcript in a cell or in vitro transcription extract.
- UTR Untranslated region
- nucleotide bases that is transcribed, but is not translated.
- a 5' UTR lies between the start site
- UTRs can be any sequence of the transcript and the translation initiation codon and includes the +1 nucleotide.
- a 3' UTR lies between the translation termination codon and the end of the transcript. UTRs can be any sequence of the transcript.
- 3' UTRs include, but are not limited to polyadenylation signals and
- the promoters and promoter control elements of this invention are capable of modulating transcription. Such promoters and promoter control elements can be used in
- regulatory sequences to modulate transcription and/or translation.
- promoters and control elements of the invention can be used to modulate transcription of a desired polynucleotide, which includes without limitation: a) antisense; b) ribozymes; c) coding sequences; or
- the promoter also can modulate transcription in a host genome in cis- or in
- PCR polymerase chain reaction
- genomic sequences can be constructed according to Sambrook et ah, Molecular
- tail-PCR 5' rapid amplification of cDNA ends
- Promoter Reports in Table 1 (SEQ. ID. Nos. 1 - 22) can be chemically synthesized according
- sequence identity to SEQ. ID. Nos. 1 - 22 namely that exhibits at least 80% sequence identity
- sequence identity compared to SEQ. ID. Nos. 1 - 22.
- sequence identity can be calculated by the algorithms and computers programs described above.
- Promoters of the invention were tested for activity by cloning the sequence
- sequences of the invention inserted into a vector suitable for transformation of plant cells.
- the construct can be made using standard recombinant DNA techniques (Sambrook et al. 1989) and can be introduced to the species of interest by Agrobacterium-medi ⁇ Aed transformation or by other means of transformation as referenced below.
- the vector backbone can be any of those typical in the art such as plasmids,
- the construct comprises a vector containing a promoter sequence
- the promoter was identified as a promoter by the expression of the marker gene.
- the vector may also comprise a marker gene that confers a selectable phenotype on plant cells.
- the marker may encode
- Biocide resistance particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or phosphinotricin.
- Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc.
- the desired preferential transcription can be combined with the promoters of the invention.
- transcription can be determined using the techniques or assays described above.
- Promoters can contain any number of control elements.
- a promoter can contain multiple transcription binding sites or other control elements.
- element may confer tissue or organ specificity; another element may limit transcription to
- promoters will contain at least a basal or core promoter
- fragment comprising a basal or "core" promoter can be fused with another fragment with any number of additional control elements.
- Topoisomerase II pea; see Reddy et al. (1999) Plant MoI Biol 41 : 125-37), chalcone synthase (soybean; see Wingender et al. (1989) MoI Gen Genet 218:315-22) mdm2 gene (human
- MipB iceplant; Yamada et al. (1995) Plant Cell 7:1129-42) and SUCS (root nodules; broadbean; Kuster et al. (1993)
- Still other promoters are affected by hormones or participate in specific physiological processes, which can be used in combination with those of present invention.
- Some examples are the ACC synthase gene that is induced differently by ethylene and brassinosteroids (mung bean; Yi et al. (1999) Plant MoI Biol 41 :443-54), the TAPGl gene that is active during abscission (tomato; Kalaitzis et al. (1995) Plant MoI Biol 28:647-56),
- the binding sites are spaced to allow each factor to bind without steric hindrance.
- the spacing between two such hybridizing control elements can be as small
- two protein binding sites can be adjacent to each other when the proteins bind at different times during the transcription
- the spacing between two such hybridizing control elements can be
- the spacing is no smaller than 5 bases; more typically, no smaller than 8; more typically, no smaller than 15 bases; more typically, no smaller than 20 bases; more typically, no smaller than 25 bases; even more typically, no smaller than 30, 35, 40 or 50 bases.
- the fragment size in no larger than 5 kb bases; more usually, no larger
- Such spacing between promoter control elements can be determined using
- inventions may be introduced into plants by any plant transformation method. Methods and
- materials for transforming plants by introducing a plant expression construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated
- present promoters and/or promoter control elements may be delivered to
- promoter or promoter control element may range from simply introducing the promoter or promoter control element by itself randomly into a cell to integration of a cloning vector containing the present promoter or
- a vector need not be limited to a DNA molecule such as a plasmid, cosmid or bacterial phage that has the capability of replicating autonomously in a host cell. All other manner of delivery of the promoters and promoter control elements of the invention are envisioned.
- the various T-DNA vector types are a preferred vector for use with
- genes that provide antibiotic resistance such as tetracycline resistance
- selectable marker genes may be used to confer resistance to herbicides such as glyphosate, glufosinate or broxynil (Comai et al. (1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; and Stalker et al. (1988) Science 242: 419-423).
- herbicides such as glyphosate, glufosinate or broxynil
- the promoter or promoter control element of the present invention may be any promoter or promoter control element of the present invention.
- promoter operably linked to a polynucleotide to be transcribed.
- promoter control element may modify transcription by modulating transcript levels of that
- the promoter or promoter control element need not be linked, operably or otherwise, to a polynucleotide to be transcribed.
- the promoter or promoter control element may be inserted alone into the genome in front of a polynucleotide already present in the genome. In this manner, the promoter or
- promoter control element may modulate the transcription of a polynucleotide that was already present in the genome.
- This polynucleotide may be native to the genome or inserted at an
- the promoter or promoter control element may be inserted into a genome alone to modulate transcription. See, for example, Vaucheret, H et al. (1998) Plant
- the promoter or promoter control element may be simply inserted into a genome or maintained extrachromosomally as a way to divert transcription resources of the
- This approach may be used to downregulate the transcript levels of a group
- polynucleotide to be transcribed is not limited. Specifically, the polynucleotide may include sequences that will have activity as RNA as well
- sequences that result in a polypeptide product may include, but are not
- RNAi sequences RNAi sequences
- ribozyme sequences ribozyme sequences
- spliceosomes amino acid coding sequences, and fragments thereof.
- Specific coding sequences may include, but are
- Constructs of the present invention would typically contain a promoter
- constructs may include but are not limited to additional regulatory nucleic acid molecules from the 3'-untranslated region (3' UTR) of plant
- genes e.g., a 3' UTR to increase mRNA stability of the mRNA, such as the PI-II termination
- Constructs may include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid
- non-translated 5' leader nucleic acid molecules derived from heat shock protein genes have been demonstrated to enhance gene expression in plants (see for example, U.S. Pat. No. 5,659,122 and U.S. Pat. No. 5,362,865, all of which are hereby incorporated by reference).
- additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or
- one embodiment of the invention is a promoter such as provided in
- SEQ ID NOs: 1 - 22 operably linked to a transcribable nucleic acid molecule so as to direct
- transcribable nucleic acid molecule transcription of said transcribable nucleic acid molecule at a desired level or in a desired tissue or developmental pattern upon introduction of said construct into a plant cell.
- the transcribable nucleic acid molecule comprises a protein-coding region of a gene, and the promoter provides for transcription of a functional mRNA molecule that is translated
- Constructs may also be constructed for transcription of antisense RNA molecules or other similar inhibitory RNA in order to inhibit expression of a
- RNA molecule of interest in a target host cell.
- transcribable nucleic acid molecules for incorporation into constructs of the present invention include, for example, nucleic acid molecules or genes from
- nucleic acid molecule is intended to refer to any gene or nucleic acid molecule that is introduced into a recipient cell.
- the type of nucleic acid molecule included in the exogenous nucleic acid molecule can include a nucleic acid molecule that is already present in the plant cell, a nucleic acid molecule from another plant, a nucleic acid molecule from a different organism, or a nucleic acid molecule generated externally, such as a nucleic acid molecule containing an antisense message of a gene, or a nucleic acid molecule encoding an artificial or modified version of a
- the promoters of the present invention can be incorporated into a construct
- marker gene refers to any gene expression in stable plant systems.
- marker gene refers to any gene expression in stable plant systems.
- transcribable nucleic acid molecule whose expression can be screened for or scored in some
- tissue sources or particle bombardment of specific tissues of interest The present invention
- plant tissues envisioned to test in transients via an appropriate delivery system would include
- leaf base tissues but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral
- tissue pollen, and epidermal tissue.
- Such processes include, but are not limited to,
- genes, transcripts and peptides or polypeptides participating in these processes which can be modulated by the present invention: are tryptophan decarboxylase (tdc) and strictosidine synthase (strl), dihydrodipicolinate synthase (DHDPS) and aspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma- zeins, ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus
- thuringiensis Bacillus subtilis
- CpTI cowpea trypsin inhibitor
- asparagine synthetase asparagine synthetase
- nitrite reductase nitrite reductase
- these peptides and polypeptides by incorporating the promoters in constructs for antisense use, co-suppression use or for the production of dominant negative mutations.
- polynucleotide to be transcribed, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine.
- the vector of the present invention may contain additional components.
- an origin of replication allows for replication of the vector in a host cell.
- homologous sequences flanking a specific sequence allow for specific
- T-DNA sequences also allow for insertion of a specific sequence randomly into a target genome.
- the vector may also be provided with a plurality of restriction sites for insertion of a polynucleotide to be transcribed as well as the promoter and/or promoter
- the vector may additionally contain selectable
- the vector may also contain a transcriptional and translational initiation region, and a transcriptional and translational termination region functional in the host cell.
- termination region may be native with the transcriptional initiation region, may be native with the polynucleotide to be transcribed, or may be derived from another source. Convenient
- termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) MoI.
- the polynucleotide to be transcribed may be optimized for increased expression in a certain host cell.
- the polynucleotide can be synthesized using preferred codons for improved transcription and translation. See U.S.
- the G-C content of the polynucleotide may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
- sequence may be modified to avoid hairpin secondary mRNA structures.
- GFP vectors are available from Aurora Biosciences.
- the promoters according to the present invention can be inserted into a host
- a host cell includes but is not limited to a plant, mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.
- the method of insertion into the host cell genome is chosen based on
- the insertion into the host cell genome may either be
- the promoters of the present invention can exist autonomously or
- Vectors of these types are known in the art and include, for example, certain type of non-integrating viral vectors, autonomously replicating plasmids, artificial chromosomes, and the like. [00107] Additionally, in some cases transient expression of a promoter may be desired.
- inventions may be transformed into host cells. These transformations may be into protoplasts.
- expression vectors are introduced into intact tissue.
- General methods of culturing plant tissues are provided for example by Maki et al. (1993) "Procedures for Introducing Foreign DNA into Plants” In Methods in Plant Molecular
- Methods of introducing polynucleotides into plant tissue include the direct infection or co-cultivation of plant cell with Agrobacterium tumefaciens, Horsch et al. (1985)
- polynucleotides are introduced into plant cells or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably polynucleotides are introduced into
- oats A vena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue
- turfgrass species e.g. species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum
- wheat Triticum aestivum
- switchgrass Panicum vigatum
- alfalfa Medicago sativa
- expression constructs can be used for gene expression in callus culture for the purpose of expressing marker genes
- a promoter that is operatively linked to a polynucleotide to be transcribed is transformed into
- callus-inducing media If the transformation is conducted with leaf discs, for example, callus will initiate along the cut edges. Once callus growth has initiated, callus cells can be transferred to callus shoot- inducing or callus root-inducing media. Gene expression will occur in the callus cells
- callus root-inducing promoters will be activated on callus root-inducing media, etc.
- transformation markers include, but are not limited to barstar, glyphosate, chloramphenicol
- CAT acetyltransferase
- kanamycin kanamycin
- spectinomycin streptomycin or other antibiotic
- GFP green fluorescent protein
- GUS ⁇ -glucuronidase
- tissues or organs are somatic embryos, cotyledon, hypocotyl, epicotyl, leaf, stems, roots,
- Integration into the host cell genome also can be accomplished by methods
- callus formation somatic embryo formation, shoot formation or root formation can be used to generate a callus formation, somatic embryo formation, shoot formation or root formation.
- the vectors of the invention can be used not only for expression of coding
- Entrapment vectors first described for use in bacteria (Casadaban and Cohen
- Promoter or gene trap vectors often contain a reporter gene, e.g., lacZ, lacking its own
- promoter gene traps contain a
- reporter gene with a splice site but no promoter. If the vector lands in a gene and is spliced into the gene product, then the reporter gene is expressed.
- the DNA constructs are inserted into a bacterial strain otherwise lacking the metabolic energy
- the method contain constructs that are selectively induced only during infection of the host.
- the IVET approach can be modified for use in plants to identify genes induced in either the bacteria or the plant cells upon pathogen infection or root colonization.
- nucleic acid molecule as shown in
- SEQ ID NOs: 1 - 22 is incorporated into a construct such that a promoter of the present
- transcribable nucleic acid molecule that is a gene of
- agronomic interest refers to a transcribable nucleic acid molecule that includes but is not limited to a gene that provides a desirable characteristic associated with plant morphology, physiology, growth and
- genetic elements comprising herbicide resistance, increased yield, insect control, fungal
- transcribable nucleic acid molecule can effect the above
- RNAi inhibitory RNA
- RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product.
- a catalytic RNA molecule i.e., a ribozyme
- modulation of genes, transcripts, and/or polypeptides in response to oxidative stress can protect cells against damage caused by oxidative agents, such as hydrogen peroxide and other free radicals.
- Drought induction of genes, transcripts, and/or polypeptides are useful to increase the viability of a plant, for example, when water is a limiting factor.
- Drought induction of genes, transcripts, and/or polypeptides are useful to increase the viability of a plant, for example, when water is a limiting factor.
- water is a limiting factor.
- genes, transcripts, and/or polypeptides induced during oxygen stress can help the flood
- the promoters and control elements of the present invention can modulate
- VuPLDl rought stress; Cowpea; see Pham-Thi et al. (1999) Plant MoI Biol 39:1257-65), pyruvate
- Promoters and control elements providing preferential transcription during wounding or induced by methyl jasmonate can produce a defense response in host cells or
- polypeptides under such conditions is useful to induce a defense response to mechanical wounding, pest or pathogen attack or treatment with certain chemicals.
- Promoters and control elements of the present invention also can trigger a response similar to those described for cf9 (viral pathogen; tomato; see O'Donnell et al.
- HAI-I hepatocyte growth factor activator inhibitor type 1
- Cytochem 47: 673-82 copper amine oxidase (CuAO), induced during ontogenesis and wound healing (wounding; chick-pea; Rea et al. (1998) FEBS Lett 437: 177-82), proteinase
- VspA methyl jasmonate; tomato; see Farmer and Ryan (1990) Proc Natl Acad Sci USA 87: 7713-7716
- VspB wounding, jasmonic
- genes, transcripts, and/or polypeptides that increase oxidative are examples of genes, transcripts, and/or polypeptides that increase oxidative,
- promoter or control elements which provide preferential
- transcript levels that
- elements produce transcript levels that are above background of the assay.
- induced by light exposure can be utilized to modulate growth, metabolism, and development;
- guard cells to control the size of stomata in leaves to prevent water loss
- the promoters and control elements of the present invention also can trigger responses similar to those described in: abscisic acid insensitive3 (ABI3) (dark-grown
- genes, transcripts, and/or polypeptides that increase drought or light tolerance may require up-regulation of transcription.
- promoter or control elements which provide preferential
- promoter and control elements produce transcript levels that are above background of the assay. 7.5. Dark Induced Preferential Transcription
- Promoters and control elements providing preferential transcription when
- polypeptides in response to dark is useful, for example,
- promoter or control elements which provide preferential
- genes, transcripts, and/or polypeptide in a leaf is useful, for example,
- genes, transcripts, and/or polypeptides that increase growth for example, genes, transcripts, and/or polypeptides that increase growth, for
- example may require up-regulation of transcription.
- promoter or control elements which provide preferential
- transcript levels that are expressed in the cells, tissues, or organs of a leaf, produce transcript levels that are expressed in the cells, tissues, or organs of a leaf.
- elements produce transcript levels that are above background of the assay.
- genes, transcripts, and/or polypeptides that increase or decrease growth may require up-regulation of transcription.
- promoter or control elements which provide preferential
- transcript levels that are statistically significant as compared to other cells, organs or tissues.
- elements produce transcript levels that are above background of the assay.
- a plant for example, preferential modulation of genes, transcripts, and/or polypeptide in a stem or shoot, is useful, for example, (1) to modulate stem/shoot size, shape, and development; or (2) to modulate energy or nutrient usage in relation to other organs and tissues
- Up-regulation and transcription down-regulation is useful for these applications.
- example may require up-regulation of transcription.
- promoter or control elements which provide preferential
- transcript levels that are statistically significant as compared to other cells, organs or tissues.
- elements produce transcript levels that are above background of the assay.
- silique or fruit can time growth, development, or maturity; or modulate fertility; or modulate
- seeds such as, storage molecules, starch, protein,
- genes, transcripts, and/or polypeptides that increase or decrease growth may require up-regulation of transcription.
- promoter or control elements which provide preferential transcription in the cells, tissues, or organs of siliques or fruits, produce transcript levels that
- elements produce transcript levels that are above background of the assay.
- Promoters and control elements providing preferential transcription in a callus can be useful to modulating transcription in dedifferentiated host cells.
- transformation for example, preferential modulation of genes, transcripts, in callus is useful to modulate transcription of a marker gene, which can facilitate selection of cells that are transformed with exogenous polynucleotides.
- Up-regulation and transcription down-regulation is useful for these
- genes, transcripts, and/or polypeptides that increase marker gene are applications. For instance, genes, transcripts, and/or polypeptides that increase marker gene
- detectability for example, may require up-regulation of transcription.
- elements produce transcript levels that are above background of the assay.
- flowers can modulate pigmentation; or modulate fertility in host cells or organisms.
- pigmentation for example, may require up-regulation of transcription
- promoter or control elements which provide preferential
- immature bud or inflorescence can time growth, development, or maturity; or modulate fertility or viability in host cells or organisms.
- a plant for example, preferential
- genes, transcripts, and/or polypeptides that increase or decrease growth, for example, may require up-regulation of transcription
- promoter or control elements which provide preferential
- transcript levels that are statistically significant as compared to other cell types, organs or tissues.
- Promoters and control elements providing preferential transcription during senescence can be used to modulate cell degeneration, nutrient mobilization, and scavenging of free radicals in host cells or organisms. Other types of responses that can be modulated
- SAG senescence associated genes
- polypeptides during senescencing is useful to modulate fruit ripening.
- genes, transcripts, and/or polypeptides that increase or decrease scavenging of free radicals may require up-regulation of transcription.
- promoter or control elements which provide preferential
- transcript levels that are expressed in cells, tissues, or organs during senescence, produce transcript levels that are
- promoter and control elements produce transcript levels that are above background of the assay. 7.14. Germination Preferential Transcription
- Promoters and control elements providing preferential transcription in a germinating seed can time growth, development, or maturity; or modulate viability in host cells or organisms.
- growth for example, may require up-regulation of transcription.
- promoter or control elements which provide preferential transcription in a germinating seed, produce transcript levels that are statistically significant as compared to other cell types, organs or tissues.
- promoter and control elements For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
- genomic DNA were conducted.
- the resulting product was isolated, cleaved with BstXI and cloned into the BstXI site of an appropriate vector, such as pNewBin4-HAPl-GFP (see
- WS plants are transformed with Ti plasmids containing nucleic acid sequences to be
- a Ti plasmid vector useful for these constructs, CRS 338, contains
- Horticulture, Ltd., Bellevue, WA is mixed with 16L Therm-O-Rock vermiculite (Therm-O- Rock West, Inc., Chandler, AZ) in a cement mixer to make a 60:40 soil mixture.
- Therm-O-Rock vermiculite Therm-O- Rock West, Inc., Chandler, AZ
- OSMOCOTE ® 14-14-14 Hummert, Earth City, MO
- 1 Tbsp Peters fertilizer 20-20-20 J. R. Peters, Inc., Allentown, PA
- Pots are then covered with 8-inch squares of nylon netting.
- Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown
- infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 ⁇ L 2 mg/ml
- Tissues are dissected by eye or under magnification using INOX 5 grade
- Tl Mature These are the Tl plants resulting from independent transformation events. These are screened between stage 6.50-6.90 (i.e. the plant is flowering
- the plants are initially imaged under UV with a Leica Confocal microscope to allow examination of the plants on a global level. If expression is present, they
- T2 Seedling Progeny are collected from the Tl plants giving the same expression pattern and the progeny (T2) are sterilized and plated on agar-solidified medium containing M&S salts. In the event that there is no expression in the Tl plants, T2 seeds are planted from all lines. The seedlings are grown in Percival incubators under continuous light at 22°C for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, and the shoot
- T2 Mature The T2 mature plants were screened in a similar manner to the
- Tl plants The T2 seeds were planted in the greenhouse, exposed to selection and at least one plant screened to confirm the Tl expression pattern. In instances where there were any subtle
- T3 Seedling This was done similar to the T2 seedlings except that only the plants for which we are trying to confirm the pattern are planted.
- Tl Mature expression Broadly expressed GFP expression. High GFP expression throughout mature tissues. High GFP expression at the inflorescence meristem and flowers. High GFP expression in epidermis, cortex, vascular, vascular bundles and parenchyma cells of stem. High
- GFP expression in epidermis, mesophyll and vasculature of leaf High GFP expression in anther wall. Not expressed in pollen. High GFP expression in heart to mature stage embryos.
- T2 Seedling expression High GFP expression in epidermis, mesophyll and vasculature of cotyledons and rosette leaves. High GFP expression in root at transition zone decreasing toward root tip. GFP expressed in meristem cells at root tip. Low GFP expression in root hairs.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- Marker Type GFP-ER Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
- Herbicide resistance antibiotic resistance, insect resistance, virus resistance, fungal resistance, nematode resistance, abiotic stress resistance, nutrient utilization, delayed senescence, protein synthesis, chemical synthesis, modulating gene expression, antibiotic resistance gene expression, herbicide resistance gene expression, transformation efficiency, plant biomass, plant architecture, organ number, organ size, photosynthesis, source strength, seed number, seed size, seed yield, modulate flowering time, modulate flower number.
- Tl mature High guard cell expression throughout all organs.
- T2 seedling Low GFP expression in root epidermis, vascular and lateral root initial cells-
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- Tl mature Guard cell expression throughout inflorescence apex and carpels in early flower buds.
- T2 seedling GFP expression specific within cortex cells overlaying lateral root primordia and root hair producing epidermal cells.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- Event-01 5/6
- Event-02 4/6
- Tl mature Guard cell expression throughout stem and pedicels.
- T2 seedling Guard cell expression throughout seedling.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- Tl mature GFP expression specific to embryo. Highest expression at root cap in heart stage through mature embryo.
- T2 seedling Low GFP expression in epidermis of hypocotyl.
- T2 mature GFP expression in embryo confirmed.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- This promoter sequence could be useful to improve: timing of seed germination, efficiency of germination, faster root growth and seedling establishment, seed tolerance to cold, and seed tolerance to desiccation and drought, seed composition.
- T2 Seedling expression Low GFP expressed in cortex cells of seedling root.
- T2 Mature expression Low GFP expression in root detected.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- This promoter sequence could be useful to improve: nitrogen and water loading and vasculature in limiting and non-limiting conditions, drought tolerance, biomass, protein content and composition.
- T2 Seedling expression High GFP expression specific to root cortex cells.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
- This promoter sequence could be useful to improve: water uptake and conductivity to vasculature and shoot, tolerance to drought and low soil water conditions, nitrogen uptake and utilization efficiency in limiting and non-limiting conditions.
- Tl Mature expression High GFP expression in developing pollen and tapetum cells of anthers.
- T2 Seedling expression Low GFP expression in root epidermis, and cotyledon vasculature, mesophyll and epidermis.
- Source Promoter Organism Arabidopsis thaliana, Columbia (Col) ecotype
Abstract
The present invention is directed to promoter sequences and promoter control elements, polynucleotide constructs comprising the promoters and control elements, and methods of identifying the promoters, control elements, or fragments thereof. The invention further relates to the use of the present promoters or promoter control elements to modulate transcript levels in plants, and plants containing such promoters or promoter control elements.
Description
PROMOTER, PROMOTER CONTROL ELEMENTS, AND COMBINATIONS,
AND USES THEREOF
[0001] This application claims priority to U.S. Provisional Application No.
60/785,794 filed March 24, 2006, the entire contents of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to promoters and promoter control elements
that are useful for modulating transcription of a desired polynucleotide. Such promoters and
promoter control elements can be included in polynucleotide constructs, expression cassettes,
vectors, or inserted into the chromosome or as an exogenous element, to modulate in vivo and
in vitro transcription of a polynucleotide. Host cells, including plant cells, and organisms,
such as regenerated plants therefrom, with desired traits or characteristics using
polynucleotides comprising the promoters and promoter control elements of the present
invention are also a part of the invention.
BACKGROUND OF THE INVENTION
[0003] This invention relates to promoter sequences and promoter control element
sequences which are useful for the transcription of polynucleotides in a host cell or
transformed host organism.
[0004] The introduction of genes into plants has resulted in the development of plants having new and useful phenotypes such as pathogen resistance, higher levels of
healthier types of oils, novel production of healthful components such as beta-carotene
synthesis in rice. An introduced gene is generally a chimeric gene composed of the coding region that confers the desired trait and regulatory sequences. One regulatory sequence is the
promoter, which is located 5' to the coding region. This sequence is involved in regulating the pattern of expression of a coding region 3' thereof. The promoter sequence binds RNA polymerase complex as well as one or more transcription factors that are involved in
producing the RNA transcript of the coding region.
[0005] The promoter region of a gene used in plant transformation is most often
derived from a different source than is the coding region. It may be from a different gene of
the same species of plant, from a different species of plant, from a plant virus, or it may be a
composite of different natural and/or synthetic sequences. Properties of the promoter
sequence generally determine the pattern of expression for the coding region that is operably linked to the promoter. Promoters with different characteristics of expression have been
described. The promoter may confer broad expression as in the case of the widely-used
cauliflower mosaic virus (CaMV) 35S promoter. The promoter may confer tissue-specific expression as in the case of the seed-specific phaseolin promoter. The promoter may confer a
pattern for developmental changes in expression. The promoter may be induced by an applied chemical compound, or by an environmental condition applied to the plant.
[0006] The promoter that is used to regulate a particular coding region is determined by the desired expression pattern for that coding region, which itself is determined by the desired resulting phenotype in the plant. For example, herbicide resistance is desired
throughout the plant so the 35S promoter is appropriate for expression of an herbicide-
resistance gene. A seed-specific promoter is appropriate for changing the oil content of soybean seed. An endosperm-specific promoter is appropriate for changing the starch
composition of corn seed. A root-specific promoter can be important for improving water or nutrient up-take in a plant. Control of expression of an introduced gene by the promoter is
important because it is sometimes detrimental to have expression of an introduced gene in non-target tissues.
[0007] One of the primary goals of biotechnology is to obtain organisms, such as
plants, mammals, yeast, and prokaryotes having particular desired characteristics or traits.
Examples of these characteristics or traits abound and may include, for example, in plants,
virus resistance, insect resistance, herbicide resistance, enhanced stability or additional nutritional value. Recent advances in genetic engineering have enabled researchers in the field
to incorporate polynucleotide sequences into host cells to obtain the desired qualities in the
organism of choice. This technology permits one or more polynucleotides from a source different than the organism of choice to be transcribed by the organism of choice. If desired,
the transcription and/or translation of these new polynucleotides can be modulated in the
organism to exhibit a desired characteristic or trait. Alternatively, new patterns of
transcription and/or translation of polynucleotides endogenous to the organism can be
produced.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to isolated polynucleotide sequences that
comprise promoters and promoter control elements from plants, especially Arabidopsis thaliana, and other promoters and promoter control elements functional in plants.
[0009] It is an object of the present invention to provide isolated polynucleotides
that are promoter or promoter control sequences. These promoter sequences comprise, for
example,
(1) a polynucleotide having a nucleotide sequence according to SEQ. ID. Nos. 1 -
22;
(2) a polynucleotide having a nucleotide sequence having at least 80% sequence
identity to a sequence according to SEQ. ID. Nos. 1 - 22; and
(3) a polynucleotide having a nucleotide sequence which hybridizes to a sequence
according to SEQ. ID. Nos. 1 - 22 under a condition establishing a Tm-5°C.
[0010] Promoter or promoter control element sequences of the present invention are
capable of modulating preferential transcription.
[0011] In another embodiment, the present promoter control elements are capable of serving as or fulfilling the function, for example, as a core promoter, a TATA box, a
polymerase binding site, an initiator site, a transcription binding site, an enhancer, an inverted repeat, a locus control region, or a scaffold/matrix attachment region.
[0012] It is yet another object of the present invention to provide a polynucleotide that includes at least a first and a second promoter control element. The first promoter
control element is a promoter control element sequence as discussed above, and the second
promoter control element is heterologous to the first control element; wherein, the first and
second control elements are operably linked. Such promoters may modulate transcript levels
preferentially in a particular tissue or under particular conditions.
[0013] In another embodiment, the present isolated polynucleotide comprises a
promoter or a promoter control element as described above, wherein the promoter or
promoter control element is operably linked to a polynucleotide to be transcribed.
[0014] In another embodiment of the present invention, the promoter and promoter
control elements of the instant invention are operably linked to a heterologous polynucleotide
that is a regulatory sequence.
[0015] It is another object of the present invention to provide a host cell comprising
an isolated polynucleotide or vector as described above or fragment thereof. Host cells
include, for instance, bacterial, yeast, insect, mammalian, and plant. The host cell can
comprise a promoter or promoter control element exogenous to the genome. Such a promoter
can modulate transcription in cis- and in trans-.
[0016] In yet another embodiment, the host cell is a plant cell capable of
regenerating into a plant.
[0017] It is yet another embodiment of the present invention to provide a plant
comprising an isolated polynucleotide or vector described above.
[0018] It is another object of the present invention to provide a method of
modulating transcription in a sample that contains either a cell-free system of transcription or
host cell. This method comprises providing a polynucleotide or vector according to the
present invention as described above, and contacting the sample of the polynucleotide or
vector with conditions that permit transcription.
[0019] In another embodiment of the present method, the polynucleotide or vector
preferentially modulates, depending upon the function of the particular promoter, constitutive
transcription, stress induced transcription, light induced transcription, dark induced transcription, leaf transcription, root transcription, stem or shoot transcription, silique transcription, callus transcription, flower transcription, immature bud and inflorescence
specific transcription, senescing induced transcription, germination transcription or drought
transcription.
[0020] Other and further objects of the present invention will be made clear or
become apparent from the following description.
BRIEF DESCRIPTION OF THE TABLES AND FIGURES
[0021] Table 1 consists of the Expression Reports for each promoter of the invention providing the nucleotide sequence for each promoter and details for expression
driven by each of the nucleic acid promoter sequences as observed in transgenic plants. The
results are presented as summaries of the spatial expression, which provides information as to
gross and/or specific expression in various plant organs and tissues. The observed expression
pattern is also presented, which gives details of expression during different generations or different developmental stages within a generation. Additional information is provided
regarding the source organism of the promoter, and the vector and marker genes used for the
construct. The following symbols are used consistently throughout the Table:
- Tl : First generation transformant
- T2: Second generation transformant
- T3 : Third generation transformant
- (L): low expression level
- (M): medium expression level
- (H): high expression level
[0022] Each row of the table begins with heading of the data to be found in the
section. The following provides a description of the data to be found in each section:
[0023] Figure l is a schematic representation of a vector that is useful to insert promoters of the invention into a plant. The definitions of the abbreviations used in the vector
map are as follows: Ori - the origin of replication used by an E. coli host
RB - sequence for the right border of the T-DNA from pMOG800
BstXl - restriction enzyme cleavage site used for cloning
HAPl VP 16 - coding sequence for a fusion protein of the HAPl and VP 16 activation domains NOS - terminator region from the nopaline synthase gene
HAPlUAS - the upstream activating sequence for HAPl
5ERGFP - the green fluorescent protein gene that has been optimized for localization to the endoplasmic reticulum
OCS2 - the terminator sequence from the octopine synthase 2 gene
OCS - the terminator sequence from the octopine synthase gene
p28716 (a.k.a 28716 short) - promoter used to drive expression of the PAT (BAR) gene
PAT (BAR) - a marker gene conferring herbicide resistance
LB - sequence for the left border of the T-DNA from pMOG800
Spec - a marker gene conferring spectinomycin resistance TrfA - transcription repression factor gene
RK2-OriV - origin of replication for Agrohacterium
DETAILED DESCRIPTION OF THE INVENTION
[0024] The following definitions and methods are provided to better define the
present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional
usage by those of ordinary skill in the relevant art.
[0025] The invention disclosed herein provides promoters capable of driving the
expression of an operably linked transgene. The design, construction, and use of these
promoters is one object of this invention. The promoter sequences, SEQ ID NOs: 1 - 22, are
capable of transcribing operably linked nucleic acid molecules in particular plant tissues/organs or during particular plant growth stages, and therefore can selectively regulate
expression of transgenes in these tissues/organs or at these times of plant development.
1. Definitions
[0026] Chimeric: The term "chimeric" is used to describe polynucleotides or genes,
or constructs wherein at least two of the elements of the polynucleotide or gene or construct,
such as the promoter and the polynucleotide to be transcribed and/or other regulatory sequences
and/or filler sequences and/or complements thereof, are heterologous to each other.
[0027] Broadly Expressing Promoter: Promoters referred to herein as "broadly
expressing promoters" actively promote transcription under most, but not necessarily all, environmental conditions and states of development or cell differentiation. Examples of broadly expressing promoters include the cauliflower mosaic virus (CaMV) 35S transcript initiation region and the 1 ' or 2' promoter derived from T-DNA of Agrobacterium tumefaciens,
and other transcription initiation regions from various plant genes, such as the maize ubiquitin-1
promoter, known to those of skill.
[0028] Domain: Domains are fingerprints or signatures that can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can
comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three- dimensional conformation. A similar analysis can be applied to polynucleotides. Generally,
each domain has been associated with either a conserved primary sequence or a sequence
motif. Generally these conserved primary sequence motifs have been correlated with specific
in vitro and/or in vivo activities. A domain can be any length, including the entirety of the polynucleotide to be transcribed. Examples of domains include, without limitation, AP2,
helicase, homeobox, zinc finger, etc.
[0029] Endogenous: The term "endogenous," within the context of the current invention refers to any polynucleotide, polypeptide or protein sequence which is a natural part
of a cell or organism(s) regenerated from said cell. In the context of promoter, the term "endogenous coding region" or "endogenous cDNA" refers to the coding region that is naturally operably linked to the promoter.
[0030] Enhancer/Suppressor: An "enhancer" is a DNA regulatory element that
can increase the steady state level of a transcript, usually by increasing the rate of transcription initiation. Enhancers usually exert their effect regardless of the distance, upstream or downstream location, or orientation of the enhancer relative to the start site of
transcription. In contrast, a "suppressor" is a corresponding DNA regulatory element that decreases the steady state level of a transcript, again usually by affecting the rate of
transcription initiation. The essential activity of enhancer and suppressor elements is to bind
a protein factor(s). Such binding can be assayed, for example, by methods described below.
The binding is typically in a manner that influences the steady state level of a transcript in a cell or in an in vitro transcription extract.
[0031] Exogenous: As referred to within, "exogenous" is any polynucleotide,
polypeptide or protein sequence, whether chimeric or not, that is introduced into the genome
of a host cell or organism regenerated from said host cell by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and
include Agrobacterium-mediated transformation (of dicots - e.g. Salomon et al. (1984)
EMBO J. 3:141 ; Herrera-Estrella et al (1983) EMBO J. 2:987; of monocots, representative
papers are those by Escudero et al. (1996) Plant J. 10:355), Ishida et al. (1996) Nature Biotech 14:745, May et al. (1995) Bio/Technology 13:486), biolistic methods (Armaleo et al.
(1990) Current Genetics 17:97), electroporation, in planta techniques, and the like. Such a
plant containing the exogenous nucleic acid is referred to here as a T0 for the primary transgenic plant and Tj for the first generation. The term "exogenous" as used herein is also
intended to encompass inserting a naturally found element into a non-naturally found location.
[0032] Heterologous sequences: "Heterologous sequences" are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from
corn is considered heterologous to an Arabidopsis coding region sequence. Also, a promoter
from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor. Regulatory element sequences, such as UTRs
or 3 ' end termination sequences that do not originate in nature from the same gene as the coding
sequence, are considered heterologous to said coding sequence. Elements operatively linked in
nature and contiguous to each other are not heterologous to each other. On the other hand,
these same elements remain operatively linked but become heterologous if other filler sequence is placed between them. Thus, the promoter and coding sequences of a corn gene
expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.
[0033] Homologous: In the current invention, a "homologous" polynucleotide
refers to a polynucleotide that shares sequence similarity with the polynucleotide of interest.
This similarity may be in only a fragment of the sequence and often represents a functional domain such as, examples including without limitation a DNA binding domain or a domain
with tyrosine kinase activity. The functional activities of homologous polynucleotides are not
necessarily the same.
[0034] Inducible Promoter: An "inducible promoter" in the context of the current
invention refers to a promoter, the activity of which is influenced by certain conditions, such as light, temperature, chemical concentration, protein concentration, conditions in an organism,
cell, or organelle, etc. A typical example of an inducible promoter, which can be utilized with the polynucleotides of the present invention, is PARSKl, the promoter from an Arabidopsis
gene encoding a serine-threonine kinase enzyme, and which promoter is induced by
dehydration, abscissic acid and sodium chloride (Wang and Goodman (1995) Plant J. 8:37). Examples of environmental conditions that may affect transcription by inducible promoters
include anaerobic conditions, elevated temperature, the presence or absence of a nutrient or other chemical compound or the presence of light.
[0035] Misexpression: The term "misexpression" refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to the
wild-type. This term also encompasses expression and/or translation of a gene or coding region
or inhibition of such transcription and/or translation for a different time period as compared to the wild-type and/or from a non-natural location within the plant genome, including a gene or
coding region from a different plant species or from a non-plant organism.
[0036] Modulate Transcription Level: As used herein, the phrase "modulate
transcription" describes the biological activity of a promoter sequence or promoter control
element. Such modulation includes, without limitation, up- and down-regulation of initiation of transcription, rate of transcription, and/or transcription levels.
[0037] Operable Linkage: An "operable linkage" is a linkage in which a promoter
sequence or promoter control element is connected to a polynucleotide sequence (or
sequences) in such a way as to place transcription of the polynucleotide sequence under the
influence or control of the promoter or promoter control element. Two DNA sequences (such
as a polynucleotide to be transcribed and a promoter sequence linked to the 5' end of the polynucleotide to be transcribed) are said to be operably linked if induction of promoter
function results in the transcription of mRNA encoding the polynucleotide and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a
frame-shift mutation, (2) interfere with the ability of the promoter sequence to direct the expression of the protein, antisense RNA, RNAi or ribozyme, or (3) interfere with the ability
of the DNA template to be transcribed. Thus, a promoter sequence would be operably linked to a polynucleotide sequence if the promoter was capable of effecting transcription of that polynucleotide sequence.
[0001] Percentage of sequence identity As used herein, the term "percent sequence identity" refers to the degree of identity between any given query sequence and a subject sequence. A subject sequence typically has a length that is from about 80 percent to
250 percent of the length of the query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100,
105, 1 10, 1 15, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 percent of the length of the query sequence. A query nucleic acid or amino acid sequence is
aligned to one or more subject nucleic acid or amino acid sequences using the computer
program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment).
Chenna el al. (2003) Nucleic Acids Res. 31(13):3497-500.
[0002] ClustalW calculates the best match between a query and one or more
subject sequences, and aligns them so that identities, similarities and differences can be
determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic
acid sequences, the following default parameters are used: word size: 2; window size: 4;
scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For an
alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5,0; and weight transitions: yes. For fast pairwise
alignment of protein sequences, the following parameters are used: word size: 1 ; window
size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: GIy, Pro, Ser, Asn, Asp, GIn, GIu, Arg, and Lys; residue-specific gap penalties: on. The output is a sequence alignment that reflects the relationship between sequences.
ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website and at the European Bioinformatics Institute website on the World Wide Web.
[0038] To determine a percent identity for polypeptide or nucleic acid sequences between a query and a subject sequence, the sequences are aligned using Clustal W and the
number of identical matches in the alignment is divided by the query length, and the result is
multiplied by 100. The output is the percent identity of the subject sequence with respect to
the query sequence. It is noted that the percent identity value can be rounded to the nearest
tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[0039] Plant Promoter: A "plant promoter" is a promoter capable of initiating
transcription in plant cells and can modulate transcription of a polynucleotide. Such
promoters need not be of plant origin. For example, promoters derived from plant viruses, such as the CaMV35S promoter or from Agrobacterium tumefaciens such as the T-DNA promoters,
can be plant promoters. A typical example of a plant promoter of plant origin is the maize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.
[0040] Plant Tissue: The term "plant tissue" includes differentiated and
undifferentiated tissues or plants, including but not limited to roots, stems, shoots, cotyledons,
epicotyl, hypocotyl, leaves, pollen, seeds, tumor tissue and various forms of cells in culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture.
[0041] Preferential Transcription: "Preferential transcription" is defined as transcription that occurs in a particular pattern of cell types or developmental times or in
response to specific stimuli or combination thereof. Non-limitive examples of preferential
transcription include: high transcript levels of a desired sequence in root tissues; detectable transcript levels of a desired sequence in certain cell types during embryogenesis; and low transcript levels of a desired sequence under drought conditions. Such preferential transcription can be determined by measuring initiation, rate, and/or levels of transcription.
[0042] Promoter: A "promoter" is a DNA sequence that directs the transcription of
a polynucleotide. Typically a promoter is located in the 5' region of a polynucleotide to be
transcribed, proximal to the transcriptional start site of such polynucleotide. More typically, promoters are defined as the region upstream of the first exon; more typically, as a region
upstream of the first of multiple transcription start sites; more typically, as the region
downstream of the preceding gene and upstream of the first of multiple transcription start sites; more typically, the region downstream of the polyA signal and upstream of the first of
multiple transcription start sites; even more typically, about 3,000 nucleotides upstream of the ATG of the first exon; even more typically, 2,000 nucleotides upstream of the first of multiple transcription start sites. The promoters of the invention comprise at least a core promoter as
defined above. Frequently promoters are capable of directing transcription of genes located on
each of the complementary DNA strands that are 3' to the promoter. Stated differently, many
promoters exhibit bidirectionality and can direct transcription of a downstream gene when
present in either orientation (i.e. 5' to 3' or 3' to 5' relative to the coding region of the gene).
Additionally, the promoter may also include at least one control element such as an upstream element. Such elements include UARs and optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element.
[0043] Promoter Control Element: The term "promoter control element" as used
herein describes elements that influence the activity of the promoter. Promoter control elements include transcriptional regulatory sequence determinants such as, but not limited to,
enhancers, scaffold/matrix attachment regions, TATA boxes, transcription start locus control regions, UARs, URRs, other transcription factor binding sites and inverted repeats.
[0044] Public sequence: The term "public sequence," as used in the context of the instant application, refers to any sequence that has been deposited in a publicly accessible
database prior to the filing date of the present application. This term encompasses both
amino acid and nucleotide sequences. Such sequences are publicly accessible, for example, on the BLAST databases on the NCBI FTP web site (accessible via the internet). The
database at the NCBI FTP site utilizes "gi" numbers assigned by NCBI as a unique identifier
for each sequence in the databases, thereby providing a non-redundant database for sequence
from various databases, including GenBank, EMBL, DBBJ (DNA Database of Japan) and PDB (Brookhaven Protein Data Bank).
[0045] Regulatory Regions: The term "regulatory region" refers to nucleotide
sequences that, when operably linked to a sequence, influence transcription initiation or translation initiation or transcription termination of said sequence and the rate of said
processes, and/or stability and/or mobility of a transcription or translation product. As used
herein, the term "operably linked" refers to positioning of a regulatory region and said
sequence to enable said influence. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional
start sites, termination sequences, polyadenylation sequences, and introns. Regulatory regions can be classified in two categories, promoters and other regulatory regions.
[0046] Regulatory Sequence: The term "regulatory sequence," as used in the
current invention, refers to any nucleotide sequence that influences transcription or translation initiation and rate, or stability and/or mobility of a transcript or polypeptide product.
Regulatory sequences include, but are not limited to, promoters, promoter control elements,
protein binding sequences, 5' and 3' UTRs, transcriptional start sites, termination sequences, polyadenylation sequences, introns, certain sequences within amino acid coding sequences
such as secretory signals, protease cleavage sites, etc.
[0047] Specific Promoters: In the context of the current invention, "specific
promoters" refers to a subset of promoters that have a high preference for modulating
transcript levels in a specific tissue or organ or cell and/or at a specific time during
development of an organism. By "high preference" is meant at least 3-fold, preferably 5-fold,
more preferably at least 10-fold still more preferably at least 20-fold, 50-fold or 100-fold increase in transcript levels under the specific condition over the transcription under any other
reference condition considered. Typical examples of temporal and/or tissue or organ specific promoters of plant origin that can be used with the polynucleotides of the present invention, are:
PTA29, a promoter which is capable of driving gene transcription specifically in tapetum and only during anther development (Koltonow et al. (1990) Plant Cell 2: 1201 ; RCc2 and RCc3,
promoters that direct root-specific gene transcription in rice (Xu et al. (1995) Plant MoI. Biol.
27:237; TobRB27, a root-specific promoter from tobacco (Yamamoto et al. (1991) Plant Cell 3:371). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues or organs, such as root, ovule, fruit, seeds, or
flowers. Other specific promoters include those from genes encoding seed storage proteins or the lipid body membrane protein, oleosin. A few root-specific promoters are noted above. See
also "Preferential transcription."
[0048] Stringency: "Stringency," as used herein is a function of nucleic acid molecule probe length, nucleic acid molecule probe composition (G + C content), salt
concentration, organic solvent concentration and temperature of hybridization and/or wash conditions. Stringency is typically measured by the parameter Tm, which is the temperature at
which 50% of the complementary nucleic acid molecules in the hybridization assay are
hybridized, in terms of a temperature differential from T111. High stringency conditions are
those providing a condition of Tm - 5°C to Tm - 1O0C. Medium or moderate stringency
conditions are those providing T111 - 2O0C to T111 - 290C. Low stringency conditions are those
providing a condition of Tm - 400C to Tm - 48°C. The relationship between hybridization
conditions and T111 (in 0C) is expressed in the mathematical equation:
Tm = 81.5 -16.6(1OgI0[Na+]) + 0.41(%G+C) - (600/N) (I)
[0049] where N is the number of nucleotides of the nucleic acid molecule probe. This
equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence. The equation below, for T111 of DNA-DNA hybrids, is useful for probes having
lengths in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide):
T111 = 81.5+16.6 log {[Na+]/(l+0.7[Na+])}+ 0.41(%G+C)-500/L 0.63(%formamide) (II)
[0050] where L represents the number of nucleotides in the probe in the hybrid (21). The Tm of Equation II is affected by the nature of the hybrid: for DNA-RNA hybrids, Tm is
10-150C higher than calculated; for RNA-RNA hybrids, T111 is 20-250C higher. Because the
T111 decreases about 10C for each 1 % decrease in homology when a long probe is used
(Frischauf et al. (1983) J MoI Biol, 170: 827-842), stringency conditions can be adjusted to favor detection of identical genes or related family members.
[0051] Equation II is derived assuming the reaction is at equilibrium. Therefore,
hybridizations according to the present invention are most preferably performed under
conditions of probe excess and allowing sufficient time to achieve equilibrium. The time
required to reach equilibrium can be shortened by using a hybridization buffer that includes a hybridization accelerator such as dextran sulfate or another high volume polymer.
[0052] Stringency can be controlled during the hybridization reaction, or after
hybridization has occurred, by altering the salt and temperature conditions of the wash solutions. The formulas shown above are equally valid when used to compute the stringency
of a wash solution. Preferred wash solution stringencies lie within the ranges stated above;
high stringency is 5-8°C below T111, medium or moderate stringency is 26-29°C below T111 and
low stringency is 45-480C below Tm.
[0053] To: The term "T0" refers to the whole plant, explant or callus tissue,
inoculated with the transformation medium.
[0054] T1: The term T| refers to either the progeny of the T0 plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue
transformation.
[0055] T2: The term T2 refers to the progeny of the Ti plant. T2 progeny are the result of self-fertilization or cross-pollination of a Ti plant.
[0056] T3: The term T3 refers to second generation progeny of the plant that is the
direct result of a transformation experiment. T3 progeny are the result of self-fertilization or
cross-pollination of a T2 plant.
[0057] TATA to start: "TATA to start" shall mean the distance, in number of nucleotides, between the primary TATA motif and the start of transcription.
[0058] Transgenic plant: A "transgenic plant" is a plant having one or more plant
cells that contain at least one exogenous polynucleotide introduced by recombinant nucleic
acid methods.
[0059] Translational start site: In the context of the present invention, a
"translational start site" is usually an ATG or AUG in a transcript, often the first ATG or
AUG. A single protein encoding transcript, however, may have multiple translational start
sites.
[0060] Transcription start site: "Transcription start site" is used in the current
invention to describe the point at which transcription is initiated. This point is typically located about 25 nucleotides downstream from a TFIID binding site, such as a TATA box.
Transcription can initiate at one or more sites within the gene, and a single polynucleotide to be transcribed may have multiple transcriptional start sites, some of which may be specific for transcription in a particular cell-type or tissue or organ. "+1 " is stated relative to the transcription start site and indicates the first nucleotide in a transcript.
[0061] Upstream Activating Region (UAR): An "Upstream Activating Region" or
"UAR" is a position or orientation dependent nucleic acid element that primarily directs tissue, organ, cell type, or environmental regulation of transcript level, usually by affecting the rate of transcription initiation. Corresponding DNA elements that have a transcription
inhibitory effect are called herein "Upstream Repressor Regions" or "URR"s. The essential activity of these elements is to bind a protein factor. Such binding can be assayed by methods
described below. The binding is typically in a manner that influences the steady state level of
a transcript in a cell or in vitro transcription extract.
[0062] Untranslated region (UTR): A "UTR" is any contiguous series of
nucleotide bases that is transcribed, but is not translated. A 5' UTR lies between the start site
of the transcript and the translation initiation codon and includes the +1 nucleotide. A 3' UTR lies between the translation termination codon and the end of the transcript. UTRs can
have particular functions such as increasing niRNA message stability or translation attenuation. Examples of 3' UTRs include, but are not limited to polyadenylation signals and
transcription termination sequences.
2. Use Of the Promoters of the Invention
[0063] The promoters and promoter control elements of this invention are capable of modulating transcription. Such promoters and promoter control elements can be used in
combination with native or heterologous promoter fragments, control elements or other
regulatory sequences to modulate transcription and/or translation.
[0064] Specifically, promoters and control elements of the invention can be used to modulate transcription of a desired polynucleotide, which includes without limitation:
a) antisense; b) ribozymes; c) coding sequences; or
d) fragments thereof.
[0065] The promoter also can modulate transcription in a host genome in cis- or in
trans-.
[0066] In an organism, such as a plant, the promoters and promoter control elements
of the instant invention are useful to produce preferential transcription which results in a
desired pattern of transcript levels in a particular cells, tissues, or organs, or under particular conditions.
4. Identifying and Isolating Promoter Sequences of the Invention
[0067] The promoters and promoter control elements of the present invention are
presented in the Promoter Reports of Table 1 and were identified from Arabidopsis thaliana. Isolation from genomic libraries of polynucleotides comprising the sequences of the promoters and promoter control elements of the present invention is possible using known techniques. For example, polymerase chain reaction (PCR) can amplify the desired
polynucleotides utilizing primers designed from SEQ ID NOs: 1-22. Polynucleotide libraries
comprising genomic sequences can be constructed according to Sambrook et ah, Molecular
Cloning: A Laboratory Manual 2nd Ed. (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY), for example.
[0068] Other procedures for isolating polynucleotides comprising the promoter sequences of the invention include, without limitation, tail-PCR, and 5' rapid amplification of
cDNA ends (RACE). See, for tail-PCR, for example, Liu et al. (1995) Plant J 8(3): 457-463;
Liu et al. (1995) Genomics 25: 674-681 ; Liu et al. (1993) Nucl. Acids Res. 21(14): 3333- 3334; and Zoe et al. (1999) BioTechniques 27(2): 240-248; for RACE, see, for example, PCR
Protocols: A Guide to Methods and Applications, (1990) Academic Press, Inc.
[0069] In addition, the promoters and promoter control elements described in the
Promoter Reports in Table 1 (SEQ. ID. Nos. 1 - 22) can be chemically synthesized according
to techniques in common use. See, for example, Beaucage et al. (1981) Tet. Lett. 22: 1859 and U.S. Pat. No. 4,668,777. Such chemical oligonucleotide synthesis can be carried out
using commercially available devices, such as, Biosearch 4600 or 8600 DNA synthesizer, by Applied Biosystems, a division of Perkin-Elmer Corp., Foster City, California, USA; and
Expedite by Perceptive Biosystems, Framingham, Massachusetts, USA.
[0070] Included in the present invention are promoters exhibiting nucleotide
sequence identity to SEQ. ID. Nos. 1 - 22 namely that exhibits at least 80% sequence identity,
at least 85%, at least 90%, and at least 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ. ID. Nos. 1 - 22. Such sequence identity can be calculated by the algorithms and computers programs described above.
5. Testing of Promoters
[0071] Promoters of the invention were tested for activity by cloning the sequence
into an appropriate vector, transforming plants with the construct and assaying for marker gene expression. Recombinant DNA constructs were prepared which comprise the promoter
sequences of the invention inserted into a vector suitable for transformation of plant cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al. 1989)
and can be introduced to the species of interest by Agrobacterium-mediεAed transformation or by other means of transformation as referenced below.
[0072] The vector backbone can be any of those typical in the art such as plasmids,
viruses, artificial chromosomes, BACs, YACs and PACs and vectors of the sort described by (a) BAC: Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89: 8794-8797; Hamilton et
al. (1996) Proc. Natl. Acad. Sci. USA 93: 9975-9979;
(b) YAC: Burke et al. (1987) Science 236:806-812;
(c) PAC: Sternberg N. et al (1990) Proc Natl Acad Sci U S A. 87(l): 103-7;
(d) Bacteria- Yeast Shuttle Vectors: Bradshaw et al (1995) Nucl Acids Res 23: 4850-
4856;
(e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al (1983) J. MoI Biol 170: 827-842; or Insertion vector, e.g., Huynh et al (1985) In: Glover NM (ed) DNA
Cloning: A practical Approach, Vol.l Oxford: IRL Press; T-DNA gene fusion vectors
:Walden et al. (1990) MoI Cell Biol 1 : 175-194; and
(g) Plasmid vectors: Sambrook et al, infra.
[0073] Typically, the construct comprises a vector containing a promoter sequence
of the present invention operationally linked to any marker gene. The promoter was identified as a promoter by the expression of the marker gene. Although many marker genes
can be used, Green Fluroescent Protein (GFP) is preferred. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker may encode
biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or
phosphinotricin. Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc.
6. Constructing Promoters with Control Elements
6.1 Combining Promoters and Promoter Control Elements
[0074] The promoter and promoter control elements of the present invention, both
naturally occurring and synthetic, can be used alone or combined with each other to produce
the desired preferential transcription. Also, the promoters of the invention can be combined
with other known sequences to obtain other useful promoters to modulate, for example, tissue transcription specific or transcription specific to certain conditions. Such preferential
transcription can be determined using the techniques or assays described above.
[0075] Promoters can contain any number of control elements. For example, a promoter can contain multiple transcription binding sites or other control elements. One
element may confer tissue or organ specificity; another element may limit transcription to
specific time periods, etc. Typically, promoters will contain at least a basal or core promoter
as described above. Any additional element can be included as desired. For example, a
fragment comprising a basal or "core" promoter can be fused with another fragment with any number of additional control elements.
[0076] The following are promoters that are induced under stress conditions and can
be combined with those of the present invention: ldhl (oxygen stress; tomato; see Germain and Ricard (1997) Plant MoI Biol 35:949-54), GPx and CAT (oxygen stress; mouse; see Franco et al. (1999) Free Radic Biol Med 27:1122-32), ci7 (cold stress; potato; see Kirch et ah (1997) Plant MoI Biol. 33:897-909), Bz2 (heavy metals; maize; see Marrs and Walbot
(1997) Plant Physiol 113:93-102), HSP32 (hyperthermia; rat; see Raju and Maines (1994) Biochim Biophys Acta 1217:273-80); MAPKAPK-2 (heat shock; Drosophila; see Larochelle and Suter (1995) Gene 163:209-14).
[0077] In addition, the following examples of promoters are induced by the
presence or absence of light can be used in combination with those of the present invention:
Topoisomerase II (pea; see Reddy et al. (1999) Plant MoI Biol 41 : 125-37), chalcone synthase (soybean; see Wingender et al. (1989) MoI Gen Genet 218:315-22) mdm2 gene (human
tumor; see Saucedo et al. (1998) Cell Growth Differ 9:119-30), Clock and BMALl (rat; see
Namihira et al. (1999) Neurosci Lett 271 : 1-4, PHYA (Arabidopsis; see Canton and Quail
(1999) Plant Physiol 121 :1207-16), PRB-Ib (tobacco; see Sessa et al. (1995) Plant MoI Biol 28:537-47) and YprlO (common bean; see Walter et al. (1996) Eur J Biochem 239:281-93).
[0078] The promoters and control elements of the following genes can be used in
combination with the present invention to confer tissue specificity: MipB (iceplant; Yamada et al. (1995) Plant Cell 7:1129-42) and SUCS (root nodules; broadbean; Kuster et al. (1993)
MoI Plant Microbe Interact 6:507-14) for roots, OsSUTl (rice ; Hirose et al. (1997) Plant Cell Physiol 38: 1389-96) for leaves, Msg (soybean; Stomvik et al. (1999) Plant MoI Biol
41 :217-31) for siliques, cell (Arabidopsis; Shani et al (1997) Plant MoI Biol 34(6):837-42) and ACTI l (Arabidopsis; Huang et al. (1997) Plant MoI Biol 33:125-39) for inflorescence.
[0079] Still other promoters are affected by hormones or participate in specific physiological processes, which can be used in combination with those of present invention. Some examples are the ACC synthase gene that is induced differently by ethylene and brassinosteroids (mung bean; Yi et al. (1999) Plant MoI Biol 41 :443-54), the TAPGl gene
that is active during abscission (tomato; Kalaitzis et al. (1995) Plant MoI Biol 28:647-56),
and the l-aminocyclopropane-l-carboxylate synthase gene (carnation; Jones et al. (1995) Plant MoI Biol 28:505-12) and the CP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol
Reprod 57: 1467-77), both active during senescence.
[0080] Spacing between control elements or the configuration or control elements
can be determined or optimized to permit the desired protein-polynucleotide or polynucleotide interactions to occur.
[0081] For example, if two transcription factors bind to a promoter simultaneously
or relatively close in time, the binding sites are spaced to allow each factor to bind without steric hindrance. The spacing between two such hybridizing control elements can be as small
as a profile of a protein bound to a control element. In some cases, two protein binding sites can be adjacent to each other when the proteins bind at different times during the transcription
process.
[0082] Further, when two control elements hybridize the spacing between such elements will be sufficient to allow the promoter polynucleotide to hairpin or loop to permit
the two elements to bind. The spacing between two such hybridizing control elements can be
as small as a t-RNA loop, to as large as 10 kb.
[0083] Typically, the spacing is no smaller than 5 bases; more typically, no smaller than 8; more typically, no smaller than 15 bases; more typically, no smaller than 20 bases; more typically, no smaller than 25 bases; even more typically, no smaller than 30, 35, 40 or 50 bases.
[0084] Usually, the fragment size in no larger than 5 kb bases; more usually, no larger
than 2 kb; more usually, no larger than 1 kb; more usually, no larger than 800 bases; more usually, no larger than 500 bases; even more usually, no more than 250, 200, 150 or 100 bases.
[0085] Such spacing between promoter control elements can be determined using
the techniques and assays described above.
6.2 Vectors Used to Transform Cells/Hosts
[0086] A plant transformation construct containing a promoter of the present
invention may be introduced into plants by any plant transformation method. Methods and
materials for transforming plants by introducing a plant expression construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated
methods including electroporation (U.S. Pat. No. 5,384,253); microprojectile bombardment
(U.S. Pat. No. 5,015,580; U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861 ; and U.S. Pat. No. 6,403,865); Agrobacterium-mediated transformation (U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,591 ,616; U.S. Pat. No. 5,981,840;
and U.S. Pat. No. 6,384,301); and protoplast transformation (U.S. Pat. No. 5,508,184).
[0087] The present promoters and/or promoter control elements may be delivered to
a system such as a cell by way of a vector. For the purposes of this invention, such delivery
may range from simply introducing the promoter or promoter control element by itself randomly into a cell to integration of a cloning vector containing the present promoter or
promoter control element. Thus, a vector need not be limited to a DNA molecule such as a plasmid, cosmid or bacterial phage that has the capability of replicating autonomously in a host cell. All other manner of delivery of the promoters and promoter control elements of the
invention are envisioned. The various T-DNA vector types are a preferred vector for use with
the present invention. Many useful vectors are commercially available.
[0088] It may also be useful to attach a marker sequence to the present promoter and promoter control element in order to determine activity of such sequences. Marker sequences
typically include genes that provide antibiotic resistance, such as tetracycline resistance,
hygromycin resistance or ampicillin resistance, or provide herbicide resistance. Specific
selectable marker genes may be used to confer resistance to herbicides such as glyphosate, glufosinate or broxynil (Comai et al. (1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2: 603-618; and Stalker et al. (1988) Science 242: 419-423). Other marker
genes exist which provide hormone responsiveness.
[0089] The promoter or promoter control element of the present invention may be
operably linked to a polynucleotide to be transcribed. In this manner, the promoter or
promoter control element may modify transcription by modulating transcript levels of that
polynucleotide when inserted into a genome.
[0090] However, prior to insertion into a genome, the promoter or promoter control
element need not be linked, operably or otherwise, to a polynucleotide to be transcribed. For example, the promoter or promoter control element may be inserted alone into the genome in front of a polynucleotide already present in the genome. In this manner, the promoter or
promoter control element may modulate the transcription of a polynucleotide that was already present in the genome. This polynucleotide may be native to the genome or inserted at an
earlier time.
[0091] Alternatively, the promoter or promoter control element may be inserted into a genome alone to modulate transcription. See, for example, Vaucheret, H et al. (1998) Plant
J 16: 651-659. Rather, the promoter or promoter control element may be simply inserted into a genome or maintained extrachromosomally as a way to divert transcription resources of the
system to itself. This approach may be used to downregulate the transcript levels of a group
of polynucleotide(s).
[0092] The nature of the polynucleotide to be transcribed is not limited. Specifically, the polynucleotide may include sequences that will have activity as RNA as well
as sequences that result in a polypeptide product. These sequences may include, but are not
limited to antisense sequences, RNAi sequences, ribozyme sequences, spliceosomes, amino acid coding sequences, and fragments thereof. Specific coding sequences may include, but are
not limited to endogenous proteins or fragments thereof, or heterologous proteins including
marker genes or fragments thereof.
[0093] Constructs of the present invention would typically contain a promoter
operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs may include but are not limited to additional regulatory nucleic acid molecules from the 3'-untranslated region (3' UTR) of plant
genes (e.g., a 3' UTR to increase mRNA stability of the mRNA, such as the PI-II termination
region of potato or the octopine or nopaline synthase 3' termination regions). Constructs may include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid
molecule which can play an important role in translation initiation and can also be a genetic component in a plant expression construct. For example, non-translated 5' leader nucleic acid molecules derived from heat shock protein genes have been demonstrated to enhance gene
expression in plants (see for example, U.S. Pat. No. 5,659,122 and U.S. Pat. No. 5,362,865, all of which are hereby incorporated by reference). These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or
heterologous with respect to the other elements present on the promoter construct.
[0094] Thus, one embodiment of the invention is a promoter such as provided in
SEQ ID NOs: 1 - 22, operably linked to a transcribable nucleic acid molecule so as to direct
transcription of said transcribable nucleic acid molecule at a desired level or in a desired tissue or developmental pattern upon introduction of said construct into a plant cell. In some
cases, the transcribable nucleic acid molecule comprises a protein-coding region of a gene, and the promoter provides for transcription of a functional mRNA molecule that is translated
and expressed as a protein product. Constructs may also be constructed for transcription of antisense RNA molecules or other similar inhibitory RNA in order to inhibit expression of a
specific RNA molecule of interest in a target host cell.
[0095] Exemplary transcribable nucleic acid molecules for incorporation into constructs of the present invention include, for example, nucleic acid molecules or genes from
a species other than the target gene species, or even genes that originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods
rather than classical reproduction or breeding techniques. Exogenous gene or genetic element
is intended to refer to any gene or nucleic acid molecule that is introduced into a recipient cell. The type of nucleic acid molecule included in the exogenous nucleic acid molecule can include a nucleic acid molecule that is already present in the plant cell, a nucleic acid molecule from another plant, a nucleic acid molecule from a different organism, or a nucleic acid molecule generated externally, such as a nucleic acid molecule containing an antisense
message of a gene, or a nucleic acid molecule encoding an artificial or modified version of a
gene.
[0096] The promoters of the present invention can be incorporated into a construct
using marker genes as described, and tested in transient analyses that provide an indication of
gene expression in stable plant systems. As used herein the term "marker gene" refers to any
transcribable nucleic acid molecule whose expression can be screened for or scored in some
way. Methods of testing for marker gene expression in transient assays are known to those of
skill in the art. Transient expression of marker genes has been reported using a variety of
plants, tissues, and DNA delivery systems. For example, types of transient analyses can
include but are not limited to direct gene delivery via electroporation or particle bombardment
of tissues in any transient plant assay using any plant species of interest. Such transient
systems would include but are not limited to electroporation of protoplasts from a variety of
tissue sources or particle bombardment of specific tissues of interest. The present invention
encompasses the use of any transient expression system to evaluate promoters or promoter
fragments operably linked to any transcribable nucleic acid molecules, including but not
limited to selected reporter genes, marker genes, or genes of agronomic interest. Examples of
plant tissues envisioned to test in transients via an appropriate delivery system would include
but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral
tissue, pollen, and epidermal tissue.
[0097] Promoters and control elements of the present invention are useful for
modulating metabolic or catabolic processes. Such processes include, but are not limited to,
secondary product metabolism, amino acid synthesis, seed protein storage, oil development,
pest defense and nitrogen usage. Some examples of genes, transcripts and peptides or
polypeptides participating in these processes, which can be modulated by the present invention: are tryptophan decarboxylase (tdc) and strictosidine synthase (strl), dihydrodipicolinate synthase (DHDPS) and aspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma- zeins, ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus
thuringiensis (Bt) insecticidal protein, cowpea trypsin inhibitor (CpTI), asparagine synthetase and nitrite reductase. Alternatively, expression constructs can be used to inhibit expression of
these peptides and polypeptides by incorporating the promoters in constructs for antisense use, co-suppression use or for the production of dominant negative mutations.
[0098] As explained above, several types of regulatory elements exist concerning
transcription regulation. Each of these regulatory elements may be combined with the present
vector if desired. Translation of eukaryotic mRNA is often initiated at the codon that encodes the first methionine. Thus, when constructing a recombinant polynucleotide according to the present invention for expressing a protein product, it is preferable to ensure that the linkage
between the 3' portion, preferably including the TATA box, of the promoter and the
polynucleotide to be transcribed, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine.
[0099] The vector of the present invention may contain additional components. For example, an origin of replication allows for replication of the vector in a host cell.
Additionally, homologous sequences flanking a specific sequence allow for specific
recombination of the specific sequence at a desired location in the target genome. T-DNA sequences also allow for insertion of a specific sequence randomly into a target genome.
[00100] The vector may also be provided with a plurality of restriction sites for insertion of a polynucleotide to be transcribed as well as the promoter and/or promoter
control elements of the present invention. The vector may additionally contain selectable
marker genes. The vector may also contain a transcriptional and translational initiation region, and a transcriptional and translational termination region functional in the host cell. The
termination region may be native with the transcriptional initiation region, may be native with the polynucleotide to be transcribed, or may be derived from another source. Convenient
termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) MoI.
Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes
Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91 : 151-158; Ballas et al. (1989) Nucleic Acids Res . 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[00101] Where appropriate, the polynucleotide to be transcribed may be optimized for increased expression in a certain host cell. For example, the polynucleotide can be synthesized using preferred codons for improved transcription and translation. See U.S.
Patent Nos. 5,380,831, 5,436, 391; see also and Murray et al. (1989) Nucleic Acids Res. 17:477-498.
[00102] Additional sequence modifications include elimination of sequences
encoding spurious polyadenylation signals, exon intron splice site signals, transposon-like
repeats, and other such sequences well characterized as deleterious to expression. The G-C content of the polynucleotide may be adjusted to levels average for a given cellular host, as
calculated by reference to known genes expressed in the host cell. The polynucleotide
sequence may be modified to avoid hairpin secondary mRNA structures.
[00103] A general description of expression vectors and reporter genes can be found in Gruber, et al. (1993) "Vectors for Plant Transformation" In Methods in Plant Molecular
Biology & Biotechnology, Glich et al. Eds. pp. 89-119, CRC Press. Moreover GUS expression vectors and GUS gene cassettes are available from Clonetech Laboratories, Inc., Palo Alto, California while luciferase expression vectors and luciferase gene cassettes are
available from Promega Corp. (Madison, Wisconsin). GFP vectors are available from Aurora Biosciences.
6.3 Polynucleotide Insertion Into A Host Cell
[00104] The promoters according to the present invention can be inserted into a host
cell. A host cell includes but is not limited to a plant, mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.
[00105] The method of insertion into the host cell genome is chosen based on
convenience. For example, the insertion into the host cell genome may either be
accomplished by vectors that integrate into the host cell genome or by vectors which exist independent of the host cell genome.
[00106] The promoters of the present invention can exist autonomously or
independent of the host cell genome. Vectors of these types are known in the art and include, for example, certain type of non-integrating viral vectors, autonomously replicating plasmids, artificial chromosomes, and the like.
[00107] Additionally, in some cases transient expression of a promoter may be desired.
[00108] The promoter sequences, promoter control elements or vectors of the present
invention may be transformed into host cells. These transformations may be into protoplasts
or intact tissues or isolated cells. Preferably expression vectors are introduced into intact tissue. General methods of culturing plant tissues are provided for example by Maki et al. (1993) "Procedures for Introducing Foreign DNA into Plants" In Methods in Plant Molecular
Biology & Biotechnology, Glich et al. Eds. pp. 67-88 CRC Press; and by Phillips et al. (1988) "Cell-Tissue Culture and In-Vitro Manipulation" In Corn & Corn Improvement, 3rd Edition
Sprague et al. eds., pp. 345-387, American Society of Agronomy Inc. et al.
[00109] Methods of introducing polynucleotides into plant tissue include the direct infection or co-cultivation of plant cell with Agrobacterium tumefaciens, Horsch et al. (1985)
Science, 227:1229. Descriptions of Agrobacterium vector systems and methods for
Agrobacterium-mediated gene transfer provided by Gruber et al. supra.
[00110] Alternatively, polynucleotides are introduced into plant cells or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably polynucleotides are introduced into
plant tissues using the microprojectile media delivery with the biolistic device. See, for
example, Tomes et al., "Direct DNA transfer into intact plant cells via microprojectile bombardment" In: Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture:
Fundamental Methods, Springer Verlag, Berlin (1995).
[00111] Methods for specifically transforming dicots are well known to those skilled in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, cotton (Gossypium hirsutum), soybean
(Glycine max), peanut (Arachis hypogaea), and members of the genus Brassica.
[00112] Methods for transforming monocots are well known to those skilled in the
art. Transformation and plant regeneration using these methods have been described for a
number of crops including, but not limited to, barley (Hordeum vulgarae); maize (Zea mays);
oats (A vena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue
(Festuca arundinacea); turfgrass species (e.g. species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticum aestivum), switchgrass (Panicum vigatum) and alfalfa (Medicago sativa). It is apparent to those of skill in the art that a number of
transformation methodologies can be used and modified for production of stable transgenic
plants from any number of target crops of interest.
[00113] In another embodiment of the current invention, expression constructs can be used for gene expression in callus culture for the purpose of expressing marker genes
encoding peptides or polypeptides that allow identification of transformed plants. Here, a promoter that is operatively linked to a polynucleotide to be transcribed is transformed into
plant cells and the transformed tissue is then placed on callus-inducing media. If the transformation is conducted with leaf discs, for example, callus will initiate along the cut edges. Once callus growth has initiated, callus cells can be transferred to callus shoot- inducing or callus root-inducing media. Gene expression will occur in the callus cells
developing on the appropriate media: callus root-inducing promoters will be activated on
callus root-inducing media, etc. Examples of such peptides or polypeptides useful as
transformation markers include, but are not limited to barstar, glyphosate, chloramphenicol
acetyltransferase (CAT), kanamycin, spectinomycin, streptomycin or other antibiotic
resistance enzymes, green fluorescent protein (GFP), and β-glucuronidase (GUS), etc. Some
of the promoters provided in SEQ ID NOs: 1-22 will also be capable of sustaining expression
in some tissues or organs after the initiation or completion of regeneration. Examples of
these tissues or organs are somatic embryos, cotyledon, hypocotyl, epicotyl, leaf, stems, roots,
flowers and seed.
[00114] Integration into the host cell genome also can be accomplished by methods
known in the art, for example, by the homologous sequences or T-DNA discussed above or
using the cre-lox system (A.C. Vergunst et al. (1998) Plant MoI. Biol. 38:393).
7. Uses of the Promoters of the Invention
7.1 Use of the Promoters to Study and Screen for Expression
[00115] The promoters of the present invention can be used to further understand
developmental mechanisms. For example, promoters that are specifically induced during
callus formation, somatic embryo formation, shoot formation or root formation can be used to
explore the effects of overexpression, repression or ectopic expression of target genes, or for
isolation of trans-acting factors.
[00116] The vectors of the invention can be used not only for expression of coding
regions but may also be used in exon-trap cloning, or promoter trap procedures to detect
differential gene expression in various tissues (see Lindsey et al. (1993) Transgenic Research 2:3347. Auch and Reth (1990) Nucleic Acids Research 18: 6743).
[00117] Entrapment vectors, first described for use in bacteria (Casadaban and Cohen
(1979) Proc. Nat. Aca. Sci. U.S.A. 76: 4530; Casadaban et al (198O) J. Bacteriol. 143: 971) permit selection of insertional events that lie within coding sequences. Entrapment vectors
can be introduced into pluripotent ES cells in culture and then passed into the germline via
chimeras (Gossler et al. aaa91989) Science 244: 463; Skarnes (1990) Biotechnology 8: 827). Promoter or gene trap vectors often contain a reporter gene, e.g., lacZ, lacking its own
promoter and/or splice acceptor sequence upstream. That is, promoter gene traps contain a
reporter gene with a splice site but no promoter. If the vector lands in a gene and is spliced into the gene product, then the reporter gene is expressed.
[00118] Recently, the isolation of preferentially-induced genes has been made
possible with the use of sophisticated promoter traps (e.g. IVET) that are based on conditional auxotrophy complementation or drug resistance. In one IVET approach, various bacterial
genome fragments are placed in front of a necessary metabolic gene coupled to a reporter
gene. The DNA constructs are inserted into a bacterial strain otherwise lacking the metabolic
gene, and the resulting bacteria are used to infect the host organism. Only bacteria expressing the metabolic gene survive in the host organism; consequently, inactive constructs can be
eliminated by harvesting only bacteria that survive for some minimum period in the host. At the same time, broadly active constructs can be eliminated by screening only bacteria that do not express the reporter gene under laboratory conditions. The bacteria selected by such a
method contain constructs that are selectively induced only during infection of the host. The IVET approach can be modified for use in plants to identify genes induced in either the
bacteria or the plant cells upon pathogen infection or root colonization. For information on
IVET see the articles by Mahan et al. (1993) Science 259:686-688, Mahan et al. (1995) Proc. Natl. Acad. ScL USA 92:669-673, Heithoff et al. (1997) Proc. Natl. Acad. Set USA 94:934- 939, and Ψanget αl. (1996) Proc. Nαtl. Acαd. Sci USA 93: 10434.
7.2 Use of the Promoters to Transcribe Genes of Interest
[00119] In one embodiment of the invention, a nucleic acid molecule as shown in
SEQ ID NOs: 1 - 22 is incorporated into a construct such that a promoter of the present
invention is operably linked to a transcribable nucleic acid molecule that is a gene of
agronomic interest. As used herein, the term "gene of agronomic interest" refers to a transcribable nucleic acid molecule that includes but is not limited to a gene that provides a desirable characteristic associated with plant morphology, physiology, growth and
development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. The expression of a gene of agronomic interest is desirable in order to
confer an agronomically important trait. A gene of agronomic interest that provides a beneficial agronomic trait to crop plants may be, for example, including, but not limited to
genetic elements comprising herbicide resistance, increased yield, insect control, fungal
disease resistance, virus resistance, nematode resistance, bacterial disease resistance, starch
production, modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, biopolymers,
environmental stress resistance, pharmaceutical peptides, improved processing traits, improved digestibility, industrial enzyme production, improved flavor, nitrogen fixation,
hybrid seed production, and biofuel production. The genetic elements, methods, and transgenes described in the patents listed above are hereby incorporated by reference.
[00120] Alternatively, a transcribable nucleic acid molecule can effect the above
mentioned phenotypes by encoding a RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense, inhibitory RNA (RNAi), or
cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product. Thus, any nucleic acid molecule that encodes a protein or mRNA that expresses a phenotype or morphology
change of interest may be useful for the practice of the present invention.
7.3. Stress Induced Preferential Transcription
[00121] Promoters and control elements providing modulation of transcription under
oxidative, drought, oxygen, wound, and methyl jasmonate stress are particularly useful for
producing host cells or organisms that are more resistant to biotic and abiotic stresses. In a
plant, for example, modulation of genes, transcripts, and/or polypeptides in response to oxidative stress can protect cells against damage caused by oxidative agents, such as hydrogen peroxide and other free radicals.
[00122] Drought induction of genes, transcripts, and/or polypeptides are useful to increase the viability of a plant, for example, when water is a limiting factor. In contrast,
genes, transcripts, and/or polypeptides induced during oxygen stress can help the flood
tolerance of a plant.
[00123] The promoters and control elements of the present invention can modulate
stresses similar to those described in, for example, stress conditions are VuPLDl (drought stress; Cowpea; see Pham-Thi et al. (1999) Plant MoI Biol 39:1257-65), pyruvate
decarboxylase (oxygen stress; rice; see Rivosal et al. (1997) Plant Physiol 114(3): 1021-29), chromoplast specific carotenoid gene (oxidative stress; capsicum; see Bouvier et al (1998) J
Biol Chem 273: 30651-59).
[00124] Promoters and control elements providing preferential transcription during wounding or induced by methyl jasmonate can produce a defense response in host cells or
organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or
polypeptides under such conditions is useful to induce a defense response to mechanical wounding, pest or pathogen attack or treatment with certain chemicals.
[00125] Promoters and control elements of the present invention also can trigger a response similar to those described for cf9 (viral pathogen; tomato; see O'Donnell et al.
(1998) Plant J 14(1): 137-42), hepatocyte growth factor activator inhibitor type 1 (HAI-I),
which enhances tissue regeneration (tissue injury; human; Koono et al. (1999) J Histochem
Cytochem 47: 673-82), copper amine oxidase (CuAO), induced during ontogenesis and wound healing (wounding; chick-pea; Rea et al. (1998) FEBS Lett 437: 177-82), proteinase
inhibitor II (wounding; potato; see Pena-Cortes et al. (1988) Planla 174: 84-89), protease
inhibitor II (methyl jasmonate; tomato; see Farmer and Ryan (1990) Proc Natl Acad Sci USA 87: 7713-7716), two vegetative storage protein genes VspA and VspB (wounding, jasmonic
acid, and water deficit; soybean; see Mason and Mullet (1990) Plant Cell 2: 569-579).
[00126] Up-regulation and transcription down-regulation are useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase oxidative,
flood, or drought tolerance may require up-regulation of transcription.
[00127] Typically, promoter or control elements, which provide preferential
transcription in wounding or under methyl jasmonate induction, produce transcript levels that
are statistically significant as compared to cell types, organs or tissues under other conditions.
[00128] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.4. Light Induced Preferential Transcription
[00129] Promoters and control elements providing preferential transcription when
induced by light exposure can be utilized to modulate growth, metabolism, and development;
to increase drought tolerance; and decrease damage from light stress for host cells or
organisms. In a plant, for example, modulation of genes, transcripts, and/or polypeptides in
response to light is useful
(1) to increase the photosynthetic rate;
(2) to increase storage of certain molecules in leaves or green parts only, e.g.
silage with high protein or starch content;
(3) to modulate production of exogenous compositions in green tissue, e.g. certain
feed enzymes;
(4) to induce growth or development, such as fruit development and maturity,
during extended exposure to light;
(5) to modulate guard cells to control the size of stomata in leaves to prevent water loss, or
(6) to induce accumulation of beta-carotene to help plants cope with light induced stress.
[00130] The promoters and control elements of the present invention also can trigger responses similar to those described in: abscisic acid insensitive3 (ABI3) (dark-grown
Arabidopsis seedlings, see Rohde et al. (2000) Plant Cell 12: 35-52), asparagine synthetase
(pea root nodules, see Tsai and Coruzzi (1990) EMBO J 9: 323-32), mdm2 gene (human tumor, see Saucedo et al. (1998) Cell Growth Differ 9: 119-30).
[00131] Up-regulation and transcription down-regulation are useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase drought or light tolerance may require up-regulation of transcription.
[00132] Typically, promoter or control elements, which provide preferential
transcription in cells, tissues or organs exposed to light, produce transcript levels that are
statistically significant as compared to cells, tissues, or organs under decreased light exposure (intensity or length of time).
[00133] For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
7.5. Dark Induced Preferential Transcription
[00134] Promoters and control elements providing preferential transcription when
induced by dark or decreased light intensity or decreased light exposure time can be utilized
to time growth, metabolism, and development, to modulate photosynthesis capabilities for
host cells or organisms. In a plant, for example, modulation of genes, transcripts, and/or
polypeptides in response to dark is useful, for example,
(1) to induce growth or development, such as fruit development and maturity,
despite lack of light;
(2) to modulate genes, transcripts, and/or polypeptide active at night or on cloudy
days; or
(3) to preserve the plastid ultra structure present at the onset of darkness.
[00135] The present promoters and control elements can also trigger response similar
to those described in the section above.
[00136] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease
growth and development may require up-regulation of transcription.
[00137] Typically, promoter or control elements, which provide preferential
transcription under exposure to dark or decrease light intensity or decrease exposure time,
produce transcript levels that are statistically significant.
[00138] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.6. Leaf Preferential Transcription
[00139] Promoters and control elements providing preferential transcription in a leaf
can modulate growth, metabolism, and development or modulate energy and nutrient
utilization in host cells or organisms. In a plant, for example, preferential modulation of
genes, transcripts, and/or polypeptide in a leaf, is useful, for example,
(1) to modulate leaf size, shape, and development;
(2) to modulate the number of leaves ; or
(3) to modulate energy or nutrient usage in relation to other organs and tissues
[00140] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for
example, may require up-regulation of transcription.
[00141] Typically, promoter or control elements, which provide preferential
transcription in the cells, tissues, or organs of a leaf, produce transcript levels that are
statistically significant as compared to other cells, organs or tissues.
[00142] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.7. Root Preferential Transcription
[00143] Promoters and control elements providing preferential transcription in a root
can modulate growth, metabolism, development, nutrient uptake, nitrogen fixation, or
modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or in a leaf, is useful
(1) to modulate root size, shape, and development;
(2) to modulate the number of roots, or root hairs; (3) to modulate mineral, fertilizer, or water uptake;
(4) to modulate transport of nutrients; or
(4) to modulate energy or nutrient usage in relation to other organs and tissues.
[00144] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease growth, for example, may require up-regulation of transcription.
[00145] Typically, promoter or control elements, which provide preferential
transcription in cells, tissues, or organs of a root, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[00146] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.8. Stem/Shoot Preferential Transcription
[00147] Promoters and control elements providing preferential transcription in a stem
or shoot can modulate growth, metabolism, and development or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptide in a stem or shoot, is useful, for example, (1) to modulate stem/shoot size, shape, and development; or
(2) to modulate energy or nutrient usage in relation to other organs and tissues
[00148] Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for
example, may require up-regulation of transcription.
[00149] Typically, promoter or control elements, which provide preferential
transcription in the cells, tissues, or organs of a stem or shoot, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[00150] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.9. Fruit and Seed Preferential Transcription
[00151] Promoters and control elements providing preferential transcription in a
silique or fruit can time growth, development, or maturity; or modulate fertility; or modulate
energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential
modulation of genes, transcripts, and/or polypeptides in a fruit, is useful
(1) to modulate fruit size, shape, development, and maturity;
(2) to modulate the number of fruit or seeds;
(3) to modulate seed shattering;
(4) to modulate components of seeds, such as, storage molecules, starch, protein,
oil, vitamins, anti-nutritional components, such as phytic acid;
(5) to modulate seed and/or seedling vigor or viability;
(6) to incorporate exogenous compositions into a seed, such as lysine rich
proteins;
(7) to permit similar fruit maturity timing for early and late blooming flowers; or
(8) to modulate energy or nutrient usage in relation to other organs and tissues.
[00152] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease growth, for example, may require up-regulation of transcription.
[00153] Typically, promoter or control elements, which provide preferential transcription in the cells, tissues, or organs of siliques or fruits, produce transcript levels that
are statistically significant as compared to other cells, organs or tissues.
[00154] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.10. Callus Preferential Transcription
[00155] Promoters and control elements providing preferential transcription in a callus can be useful to modulating transcription in dedifferentiated host cells. In a plant
transformation, for example, preferential modulation of genes, transcripts, in callus is useful to modulate transcription of a marker gene, which can facilitate selection of cells that are transformed with exogenous polynucleotides.
[00156] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase marker gene
detectability, for example, may require up-regulation of transcription.
[00157] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.11. Flower Specific Transcription
[00158] Promoters and control elements providing preferential transcription in
flowers can modulate pigmentation; or modulate fertility in host cells or organisms. In a
plant, for example, preferential modulation of genes, transcripts, and/or polypeptides in a
flower, is useful,
(1) to modulate petal color; or
(2) to modulate the fertility of pistil and/or stamen.
[00159] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease
pigmentation, for example, may require up-regulation of transcription
[00160] Typically, promoter or control elements, which provide preferential
transcription in flowers, produce transcript levels that are statistically significant as compared
to other cells, organs or tissues.
[00161] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.12. Immature Bud and Inflorescence Preferential Transcription
[00162] Promoters and control elements providing preferential transcription in a
immature bud or inflorescence can time growth, development, or maturity; or modulate fertility or viability in host cells or organisms. In a plant, for example, preferential
modulation of genes, transcripts, and/or polypeptide in a fruit, is useful,
(1) to modulate embryo development, size, and maturity;
(2) to modulate endosperm development, size, and composition;
(3) to modulate the number of seeds and fruits; or
(4) to modulate seed development and viability.
[00163] Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease growth, for example, may require up-regulation of transcription,
[00164] Typically, promoter or control elements, which provide preferential
transcription in immature buds and inflorescences, produce transcript levels that are statistically significant as compared to other cell types, organs or tissues.
[00165] For preferential up-regulation of transcription, promoter and control
elements produce transcript levels that are above background of the assay.
7.13. Senescence Preferential Transcription
[00166] Promoters and control elements providing preferential transcription during senescence can be used to modulate cell degeneration, nutrient mobilization, and scavenging of free radicals in host cells or organisms. Other types of responses that can be modulated
include, for example, senescence associated genes (SAG) that encode enzymes thought to be involved in cell degeneration and nutrient mobilization (Arabidopsis; see Hensel et al. (1993)
Plant Cell 5: 553-64), and the CP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol
Reprod 57: 1467-77), both induced during senescence.
[00167] In a plant, for example, preferential modulation of genes, transcripts, and/or
polypeptides during senescencing is useful to modulate fruit ripening.
[00168] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease scavenging of free radicals, for example, may require up-regulation of transcription.
[00169] Typically, promoter or control elements, which provide preferential
transcription in cells, tissues, or organs during senescence, produce transcript levels that are
statistically significant as compared to other conditions.
[00170] For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
7.14. Germination Preferential Transcription
[00171] Promoters and control elements providing preferential transcription in a germinating seed can time growth, development, or maturity; or modulate viability in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts,
and/or polypeptide in a germinating seed, is useful,
(1) to modulate the emergence of they hypocotyls, cotyledons and radical; or
(2) to modulate shoot and primary root growth and development;
[00172] Up-regulation and transcription down-regulation is useful for these
applications. For instance, genes, transcripts, and/or polypeptides that increase or decrease
growth, for example, may require up-regulation of transcription.
[00173] Typically, promoter or control elements, which provide preferential transcription in a germinating seed, produce transcript levels that are statistically significant as compared to other cell types, organs or tissues.
[00174] For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
8. GFP EXPERIMENTAL PROCEDURES AND RESULTS PROCEDURES
[00175] The polynucleotide sequences of the present invention were tested for promoter activity using Green Fluorescent Protein (GFP) assays in the following manner.
[00176] Approximately 1-3 kb of genomic sequence occurring immediately upstream
of the ATG translational start site of the gene of interest was isolated using appropriate primers tailed with BstXI restriction sites. Standard PCR reactions using these primers and
genomic DNA were conducted. The resulting product was isolated, cleaved with BstXI and cloned into the BstXI site of an appropriate vector, such as pNewBin4-HAPl-GFP (see
Figure 1).
Agrobacterium-Mediated Transformation of Arabidopsis
[00177] Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija
(WS) plants are transformed with Ti plasmids containing nucleic acid sequences to be
expressed, as noted in the respective examples, in the sense orientation relative to the 35S promoter in a Ti plasmid. A Ti plasmid vector useful for these constructs, CRS 338, contains
the Ceres-constructed, plant selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.
[00178] Ten independently transformed events are typically selected and evaluated
for their qualitative phenotype in the Ti generation.
[00179] Preparation of Soil Mixture: 24L Sunshine Mix #5 soil (Sun Gro
Horticulture, Ltd., Bellevue, WA) is mixed with 16L Therm-O-Rock vermiculite (Therm-O- Rock West, Inc., Chandler, AZ) in a cement mixer to make a 60:40 soil mixture. To the soil
mixture is added 2 Tbsp Marathon 1 % granules (Hummert, Earth City, MO), 3 Tbsp
OSMOCOTE® 14-14-14 (Hummert, Earth City, MO) and 1 Tbsp Peters fertilizer 20-20-20 (J. R. Peters, Inc., Allentown, PA), which are first added to 3 gallons of water and then added
to the soil and mixed thoroughly. Generally, 4-inch diameter pots are filled with soil mixture.
Pots are then covered with 8-inch squares of nylon netting.
[00180] Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated. 25
drops are added to each pot. Clear propagation domes are placed on top of the pots that are
then placed under 55% shade cloth and subirrigated by adding 1 inch of water.
[00181] Plant Maintenance: 3 to 4 days after planting, lids and shade cloth are
removed. Plants are watered as needed. After 7-10 days, pots are thinned to 20 plants per pot using forceps. After 2 weeks, all plants are subirrigated with Peters fertilizer at a rate of 1 Tsp per gallon of water. When bolts are about 5-10 cm long, they are clipped between the
first node and the base of stem to induce secondary bolts. Dipping infiltration is performed 6
to 7 days after clipping.
[00182] Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL each
of carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stock concentration).
Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown
to an ODόoo of approximately 1.0) and inoculated one culture vessel per construct by transferring 1 mL from appropriate well in the starter block. Cultures are then incubated with
shaking at 27°C. Cultures are spun down after attaining an OD60O of approximately 1.0
(about 24 hours). 200 mL infiltration media is added to resuspend Agrobacterium pellets. Infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/ml
benzylaminopurine to 900 ml water.
[00183] Dipping Infiltration: The pots are inverted and submerged for 5 minutes so that the aerial portion of the plant is in the Agrobacterium suspension. Plants are allowed to grow normally and seed is collected.
[00184] High-throughput Screening of Ti Transgenic Plants: Seed is evenly
dispersed into water-saturated soil in pots and placed into a dark 40C cooler for two nights to
promote uniform germination. Pots are then removed from the cooler and covered with 55%
shade cloth for 4-5 days. Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis, Paris, France) is sprayed on plants (3 ml FINALE® diluted into 48 oz. water) and repeated every 3-4 days until only transformants remain.
GFP Assay
[00185] Tissues are dissected by eye or under magnification using INOX 5 grade
forceps and placed on a slide with water and coversliped. An attempt is made to record images of observed expression patterns at earliest and latest stages of development of tissues
listed below. Specific tissues will be preceded with High (H), Medium (M), Low (L)
designations.
Flower Pedicel, receptacle, nectary, sepal, petal, filament,t anther, pollen, carpel, style, papillae, vascular, epidermis, stomata, trichome
Silique Stigma, style, carpel, septum, placentae, transmitting tissue, vascular, epidermis, stomata, abscission zone, ovule
[00186] Tl Mature: These are the Tl plants resulting from independent transformation events. These are screened between stage 6.50-6.90 (i.e. the plant is flowering
and 50-90% of the flowers that the plant will make have developed), which is 4-6 weeks of
age. At this stage the mature plant possesses flowers, siliques at all stages of development,
and fully expanded leaves. The plants are initially imaged under UV with a Leica Confocal microscope to allow examination of the plants on a global level. If expression is present, they
are re-imaged using scanning laser confocal micsrocopy.
[00187] T2 Seedling: Progeny are collected from the Tl plants giving the same expression pattern and the progeny (T2) are sterilized and plated on agar-solidified medium containing M&S salts. In the event that there is no expression in the Tl plants, T2 seeds are planted from all lines. The seedlings are grown in Percival incubators under continuous light
at 22°C for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, and the shoot
meristem region of individual seedlings were screened until two seedlings were observed to have the same pattern. In general, the same expression pattern was found in the first two seedlings. However, up to 6 seedlings were screened before "no expression pattern" was recorded. All constructs are screened as T2 seedlings even if they did not have an expression
pattern in the Tl generation.
[00188] T2 Mature: The T2 mature plants were screened in a similar manner to the
Tl plants. The T2 seeds were planted in the greenhouse, exposed to selection and at least one plant screened to confirm the Tl expression pattern. In instances where there were any subtle
changes in expression, multiple plants were examined and the changes noted in the tables.
[00189] T3 Seedling: This was done similar to the T2 seedlings except that only the plants for which we are trying to confirm the pattern are planted.
IMAGE DATA:
[00190] Images are collected by scanning laser confocal microscopy. Scanned images
are taken as 2-D optical sections or 3-D images generated by stacking the 2-D optical sections collected in series. All scanned images are saved as TIFF files by imaging software, edited in
Adobe Photoshop, and labeled in Powerpoint specifying organ and specific expressing tissues.
RESULTS
[00191] The Promoter Expression Reports of Table 1 present the results of the GFP assays as reported by their corresponding construct number and line number.
Promoter Expression Report #324.PT1026.CC
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H pedicel H receptacle H nectary H sepal H petal H filament L anther H carpel
H style H papillae H vascular H epidermis H stomata L trichome H silique Silique H stigma H style H carpel H septum H placentae H funiculus H vascular
H epidermis H stomata H abscission zone H ovule Ovule Pre-fertilization: H outer integument H funiculus H chalaza
Post-fertilization: H funiculus H inner integument H outer integument
H seed coat H chalaza
Embryo H heart H torpedo H late H mature H radicle H cotyledons
Stem H epidermis H cortex H interfascicular region H vascular H xylem
H phloem
H pith H stomata
Leaf H petiole H mesophyll H vascular H epidermis L trichome H stomata
Shoot apical H Shoot apical meristem H Flower primordium meristem
Hypocotyl H epidermis H cortex H vascular H xylem H phloem H stomata Cotyledon H mesophyll H vascular H epidermis Rosette Leaf H mesophyll H vascular H epidermis H petiole H primordia Primary Root H epidermis H cortex H endodermis H vascular H xylem H phloem H pericycle H quiescent L root hairs
Observed expression pattern:
Tl Mature expression: Broadly expressed GFP expression. High GFP expression throughout mature tissues. High GFP expression at the inflorescence meristem and flowers. High GFP expression in epidermis, cortex, vascular, vascular bundles and parenchyma cells of stem. High
GFP expression in epidermis, mesophyll and vasculature of leaf. High GFP expression in anther wall. Not expressed in pollen. High GFP expression in heart to mature stage embryos.
T2 Seedling expression: High GFP expression in epidermis, mesophyll and vasculature of cotyledons and rosette leaves. High GFP expression in root at transition zone decreasing toward root tip. GFP expressed in meristem cells at root tip. Low GFP expression in root hairs.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAP 1 -GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=l
Guard cell expression observed
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve:
Herbicide resistance, antibiotic resistance, insect resistance, virus resistance, fungal resistance, nematode resistance, abiotic stress resistance, nutrient utilization, delayed senescence, protein synthesis, chemical synthesis, modulating gene expression, antibiotic resistance gene expression, herbicide resistance gene expression, transformation efficiency, plant biomass, plant architecture, organ number, organ size, photosynthesis, source strength, seed number, seed size, seed yield, modulate flowering time, modulate flower number.
2. Jagdeep S. Sandhu, Carl I. Webster, John C. Gray, A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants, Plant Molecular Biology, Volume 37, Issue 5, JuI 1998, Pages 885 - 896
Construct: PT 1026
Promoter candidate I.D: 22205547
Events expressing: -02, -04, -05, -07, -08
Promoter Expression Report #137.PT0513 1
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H stomata
Silique H stomata
Stem H stomata
Leaf H stomata
Hypocotyl L vascular
Primary Root L epidermis L vascular
Lateral root L initials
Observed expression pattern:
Tl mature: High guard cell expression throughout all organs.
T2 seedling: Low GFP expression in root epidermis, vascular and lateral root initial cells-
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n= 2 Events Expressing: n= 2
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= 3 Events Expressing: n= 3
Table 4. Promoter utility
Utility: Drought tolerance, nitrogen use efficiency, nitrogen uptake, and seedling establishment
Construct: PT0513
Promoter candidate LD: 11768790
Lines expressing: -01, -02, -03
Promoter Expression Report #152.PT590.CC
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H stomata
Silique H stomata
Rosette Leaf H stomata H stipule
Primary Root L epidermis L trichoblast H cortex L root hairs
Observed expression pattern:
Tl mature: Guard cell expression throughout inflorescence apex and carpels in early flower buds.
T2 seedling: GFP expression specific within cortex cells overlaying lateral root primordia and root hair producing epidermal cells.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling DT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=2
X Flower Dpedicel Dreceptacle Dnectary Dsepal Dpetal Dfilament Danther □pollen Dcarpel □ style Dpapillae Dvascular Depidermis H stomata Dtrichome □ silique
X Silique Dstigma D style Dcarpel Dseptum Dplacentae Dtransmitting tissue Dvascular Depidermis H stomata Dabscission zone Dovule
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=2 Events Expressing: n=2
Seedlings expressing / Seedlings screened
Event-01 : 5/6 Event-02: 4/6
X Rosette Leaf Dmesophyll Dvascular Depidermis Otrichome Dpetiole Dprimordia H stomata H stipule Dmargin Dhydathode
X Primary Root L epidermis L trichoblast Datrichoblast H cortex Dendodermis
Dvascular Dxylem Dphloem Dpericycle Dquiescent
D columella D root cap L root hairs
Table 3. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve:
Drought tolerance, nitrogen use efficiency, nitrogen uptake, and seedling establishment
Construct: PT0590
Promoter candidate I .D: 11768848
Lines expressing: PT0590 -03, -04
Promoter Expression Report #191.PT0850.CC
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Flower M stomata
Stem M stomata
Hypocotyl M stomata
Cotyledon M stomata
Rosette Leaf M stomata
Observed expression pattern:
Tl mature: Guard cell expression throughout stem and pedicels.
T2 seedling: Guard cell expression throughout seedling.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling DT2 Mature DT3 Seedling
Table 3. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve:
Drought tolerance, nitrogen use efficiency, nitrogen uptake, and seedling establishment
Trait Area: Water use efficiency, nutrients and light quality
Construct: PT0850
Promoter candidate I .D: 15224215
Lines expressing: PT0850 -02, -04
Promoter Expression Report #212.PT0723
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Ovule Post-fertilization: H suspensor H embryo
Embryo H suspensor H heart H torpedo H late H mature H hypophysis H radicle
H cotyledons Hypocotyl L epidermis
Observed expression pattern:
Tl mature: GFP expression specific to embryo. Highest expression at root cap in heart stage through mature embryo.
T2 seedling: Low GFP expression in epidermis of hypocotyl.
T2 mature: GFP expression in embryo confirmed.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=4 Events Expressing: n=2
X Ovule Pre-fertilization: Dprimordia Dinner integument D outer integument D embryo sac D funiculus Dchalaza Dmicropyle Dgametophyte Post-fertilization: D zygote H suspensor D embryo sack D funiculus □ inner integument ϋ outer integument D endothelium 3 seed coat Dprimordia Dchalaza Dmicropyle Dearly endosperm D mature endosperm H embryo
X Embryo H suspensor Dpreglobular D globular H heart H torpedo H late H mature D pro vascular H hypophysis H radicle H cotyledons D hypocotyl
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n=3
X Hypocotyl L epidermis Dcortex Dvascular Dxylem Dphloem Dstomata
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=2 Events Expressing: n=2
X Embryo Dsuspensor Dpreglobular D globular D heart D torpedo D late H mature
Dprovascular Dhypophysis Dradicle Dcotyledons Droot meristem D shoot meristem
X Aerial organs D inflorescence meristem D shoot apical meristem D flower primordium Dsilique D ovule H embryo D stem D leaf
Table 3. Induction Screens
Drought: No drought expression was observed
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: timing of seed germination, efficiency of germination, faster root growth and seedling establishment, seed tolerance to cold, and seed tolerance to desiccation and drought, seed composition.
Construct: PT0723
Promoter candidate I .D: 15371692
Lines expressing: 01, 02, 03, 05
Promoter Expression Report#258.PT0769 [
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Primary Root L cortex
Mature root L mature root
Observed expression pattern:
Tl Mature expression: None observed.
T2 Seedling expression: Low GFP expressed in cortex cells of seedling root.
T2 Mature expression: Low GFP expression in root detected.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=0
X No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Table 3 . T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=2
X Root L mature root
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: nitrogen and water loading and vasculature in limiting and non-limiting conditions, drought tolerance, biomass, protein content and composition.
Notes: Peumans WJ, Van Damme EJ1 Barre A, Rouge P. Classification of plant lectins in families of structurally and evolutionary related proteins. Adv Exp Med Biol. 2001 ;491 :27-54. Review.
PMID: 14533788
Barre A, Bourne Y1 Van Damme EJ, Peumans WJ, Rouge P. Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process. Biochimie. 2001
Jul;83(7):645-51. Review. PMID: 11522393
Bouckaert J1 Hamelryck T, Wvns L1 Loris R. Novel structures of plant lectins and their complexes with carbohydrates. Curr Opin Struct Biol. 1999 Oct;9(5):572-7. Review. PMID: 10508764
Van Damme EJ1 Zhang W1 Peumans WJ. Induction of cytoplasmic mannose-binding jacalin- related lectins is a common phenomenon in cereals treated with jasmonate methyl ester. Commun
Agric Appl Biol Sci. 2004;69(l):23-31. PMID: 15560260
Construct: PT0769
Promoter candidate LD: 15371914
Events expressing: 02, 03, 05, 06
Promoter Expression Report #293.PT0614
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Primary Root H cortex
Observed expression pattern:
Tl Mature expression: No expression observed.
T2 Seedling expression: High GFP expression specific to root cortex cells.
T2 Mature expression: No expression detected.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=0
X No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n=6
X Primary Root D epidermis Dtrichoblast Datrichoblast H cortex D endodermis
D vascular Dxylem Dphloem Dpericycle Dquiescent
D columella D root cap D root hairs
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= 2 Events Expressing: n=0
X No GFP Expression Detected
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: water uptake and conductivity to vasculature and shoot, tolerance to drought and low soil water conditions, nitrogen uptake and utilization efficiency in limiting and non-limiting conditions.
Construct: PT0614
Promoter candidate I.D: 13148301
Events expressing: 01-06
Promoter Expression Report #294.PT0621
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H anther H tapetum H pollen
Leaf L epidermis
Cotyledon L mesophyll L vascular L epidermis
Primary Root L epidermis
Observed expression pattern:
Tl Mature expression: High GFP expression in developing pollen and tapetum cells of anthers.
Low expression in leaf epidermis.
T2 Seedling expression: Low GFP expression in root epidermis, and cotyledon vasculature, mesophyll and epidermis.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: male sterility, outbreeding, crossing and development of hybrids, CO2 capture, sucrose loading and biomass, photosynthetic efficiency, growth rate, nitrogen assimilation.
Notes: Evaluation and classification of RTNG-finger domains encoded by the Arabidopsis genome.
Kosarev P, Mayer KF, Hardtke CS. Genome Biol. 2002; 3(4): research0016.l-research0016.12. published online before print March 14, 2002 PMCID: 1 15204 Abstract Full Text PDF-182K
Supplemental Data
Construct: PT0621
Promoter candidate LD: 13148309
Events expressing: 01, 05, 06
Promoter Expression Report #295.PT0693
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H anther
Leaf L vascular L hydathode
Cotyledon L vascular H epidermis H hydathode
Primary Root H epidermis H root cap
Observed expression pattern:
Tl Mature expression: High GFP expression specific to anthers in flowers. No pollen expression. Low GFP expression in vascular tissues of leaf.
T2 Seedling expression: High GFP expression in epidermal cells at root transition zone decreasing toward root tip.
T2 mature expression: GFP expressed in anthers and leaf vascular tissues.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: crossing and outbreeding, male sterility, seedling establishment and growth, tolerance to low soil water content and drought, nitrogen use efficiency and uptake of nitrogen in limiting and non-limiting conditions.
Construct: PT0693
Promoter candidate I •D: 15371530
Events expressing: 03, 04, 05, 06, 08
Promoter Expression Report#303.PT0761
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Primary Root H epidermis H root cap
Observed expression pattern:
Tl Mature expression: No expression observed.
T2 Seedling expression: High GFP expression throughout seedling root epidermis.
T2 Mature expression: No expression observed.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAP 1 -GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature X T2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=0
No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n=3
X Primary Root H epidermis Dtrichoblast Datrichoblast D cortex D endodermis
D vascular Dxylem Dphloem Dpericycle Dquiescent
D columella H root cap D root hairs
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=2 Events Expressing: n=0
X No GFP Expression Detected
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: nitrogen and mineral ion uptake and loading to cortex and vasculature, water uptake and loading to cortex and xylem, tolerance of plants to low nitrogen and drought conditions, and protection against root nematodes and pathogens.
Construct: PT0761
Promoter candidate LD: 15371875
Events expressing: -01,-03,-06
Promoter Expression Report #322.PT1016
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H receptacle H sepal H vascular Hypocotyl L vascular Cotyledon L vascular L hydathode Primary Root H epidermis Root H mature root
Observed expression pattern:
Tl Mature expression: High GFP expression in vasculature of flowers at abscission zone.
T2 Seedling expression: High GFP expression in epidermis. Low GFP expression in vasculature of hypocotyl and cotyledons.
T2 Mature expression: High GFP expression in roots and in vasculature of flowers at abscission zone.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=2
X Flower D pedicel H receptacle D nectary H sepal D petal □ filament D anther Dtapetum πpollen Dcarpel Dstyle Dpapillae H vascular Depidermis Dstomata Dtrichome Dsilique
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: development and structure of flowers, carpel and seed number, seed yield, breeding biology (outcrossing), water and nitrogen uptake from soil, water and nitrogen transport and use efficiency, flower, fruit and seed abscission, and post-harvest maturation of fruit and seed.
Construct: PT1016
Promoter candidate LD: 22204494
Events expressing: 02-06
Promoter Expression Report #211.PT0695
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Embryo L late L mature
Hypocotyl L epidermis
Cotyledon H epidermis H petiole
Observed expression pattern:
Tl mature: Embryo specific GFP expression. GFP expressed in developing and mature embryo.
T2 seedling: GFP expressed in epidermis of cotyledon.
T2 mature: Embryo expression confirmed. No GFP expression detected in other organs.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 matureDT3 Seedling
Inductions < .ompleted.
Treatment Age: Gen: Time points: Events Screened Response
• / Response
1. Drought 4 wks T2 8 days ~ 1.0% moisture 3/0 No
Inducible expression summary: Treatment: Time point induced : Organs induced: Tissues induced:
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n= 2 Events Expressing: n= 2
X Embryo Dsuspensor Dpreglobular Dglobular Dheart Dtorpedo L late L mature Dprovascular Dhypophysis Dradicle Dcotyledons Dhypocotyl
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n= 3 Events Expressing: n= 1
Seedlings expressing / Seedlings screened
Event-01 : 0/6 Event-02: 0/6 Event-03: 3/6
GFP Expression Detected
X Hypocotyl L epidermis Dcortex Dvascular Dxylem Dphloem Dstomata
X Cotyledon Dmesophyll Dvascular H epidermis D margin H petiole Dstomata Dhydathode
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= l Events Expressing: n= 1
X No GFP Expression Detected
T2 mature scan. Embryo expression confirmed. No germination in second event.
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: size, seed size, seed weight, seed yield, seed composition, germination timing, germination efficiency, germination rate, root growth, leaf angle, light capture and source strength in shade and low light.
Construct: PT0695
Promoter candidate LD: 15371536
Lines expressing: 01, 03
Promoter Expression Report #225.PT0879 1
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Embryo H late H mature H suspensor H provascular H hypophysis H radicle H cotyledons
Observed expression pattern:
Tl mature: Embryo specific GFP expression. High GFP expression throughout mature embryo.
GFP preferentially expressed at root cap.
T2 seedling: No expression observed.
T2 mature: Embryo specific expression confirmed.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling XT2 Mature D T3 Seedling
Inductions completed.
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n= 6 Events Expressing: n=3
X Embryo H suspensor Dpreglobular Dglobular πheart Dtorpedo H late H mature H provascular H hypophysis H radicle H cotyledons
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=2 Events Expressing: n=0
No GFP Expression Detected
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=2 Events Expressing: n=2
X Embryo Dsuspensor Hpreglobular tlglobular Hlheart Dtorpedo H late H mature
D provascular D hypophysis □ radicle D cotyledons 3 root meristem D shoot meristem
Table 3. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: embryo size, seed size, seed yield, seed composition, more efficient germination, faster germination, faster seedling emergence and establishment, faster seedling growth, and more robust seedlings.
Construct: PT0879
Promoter candidate I .D: 15371602
Lines expressing: 02, 05, 06
Promoter Expression Report #246.PT0738 I
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H pedicel H receptacle
Primary Root H epidermis H cortex H endodermis H vascular H pericycle
Observed expression pattern:
Tl Mature: None observed.
T2 Seedling: High GFP expression throughout seedling root. Highest GFP expression at transition zone decreasing toward root tip.
T2 Mature: High GFP expression in pedicels of flowers at the inflorescences.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=0
No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=3 Events Expressing: n=3
Seedlings expressing / Seedlings screened
Event-01 : 4/6 Event-02: 3/6 Event-02: 5/6
X Primary Root H epidermis Dtrichoblast Datrichoblast H cortex H endodermis H vascular Oxylem D phloem H pericycle □ quiescent □ columella D root cap D root hairs
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=2 Events Expressing: n=2
X Flower H pedicel H receptacle D nectary D sepal D petal O filament D anther
Dpollen Dcarpel Dstyle πpapillae Dvascular Depidermis Dstomata
Dtrichome
Dsilique
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: nitrogen use efficiency and nitrogen loading for transport in low and non-limiting nitrogen conditions, enhanced water uptake in drought and non-limiting water environments, and water loading to vasculature and transport to shoot, floral morphology, flower structure, pollination and breeding biology, senescence, flowers and fruit abscission and flower and fruit drop,
Construct: PT0738
Promoter candidate LD: 15371758
Events expressing: 01, 02, 03
Promoter Expression Report #247.PT0834
Promoter Tested In; Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Flower H anther
Leaf L mesophyll L epidermis
Root H mature root
Primary Root H epidermis H root hairs
Observed expression pattern:
Tl Mature: High GFP expression in anther walls and roots. Low GFP expression in leaf epidermis and mesophyll.
T2 Seedling: High GFP expression in epidermis of roots.
T2 Mature: GFP expression detected in anthers. Low root GFP expression.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=2 Events Expressing: n=2
Seedlings expressing / Seedlings screened
Event 01 : 3/6 Event 02: 4/6
X Primary Root H epidermis Dtrichoblast Datrichoblast D cortex D endodermis
D vascular Dxylem Dphloem Dpericycle Dquiescent
D columella D root cap H root hairs
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Table 3. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: male sterility, outbreeding, crossing, CO2 capture, sucrose loading of seeds, leaf area and photosynthetic capacity, growth rate and biomass.
Construct: PT0834
Promoter candidate LD: 15371521
Events expressing: 01, 02
Promoter Expression Report #254.PT0746
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Primary Root L epidermis L cortex H pericycle
Mature root L epidermis L cortex L vascular H pericycle
Observed expression pattern:
Tl Mature expression: High GFP expression in mature root. GFP expression observed in all cells of lower root near root tip. GFP specific to pericycle cells in upper roots.
T2 Seedling expression: High GFP expression in pericycle cells at transition zone increasing expression in epidermis and cortex toward root tip.
T2 Mature: High GFP expression in roots.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n= 6 Events Expressing: n= > 1
X Root L epidermis Dtrichoblast Datrichoblast L cortex D endodermis
L vascular Dxylem Dphloem H pericycle Dquiescent
D columella D root cap D root hairs
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n=2
Seedlings expressing / Seedlings screened
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= 2 Events Expressing: n= 2
X Root H mature root
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: uptake and loading to the vasculature in shoot, water capture from soil, and nitrogen uptake and loading, all in limiting and non-limiting environments.
Construct: PT0746
Promoter candidate LD: 15371812
Events expressing: 01, 05
Promoter Expression Report #299.PT0607
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Ovule Post-fertilization: H outer integument H endothelium H seed coat
Root H mature root
Observed expression pattern:
Tl Mature expression: High GFP expression in seed coat of developing seed. Seed coat specific
GFP expression.
T2 Seedling expression: None observed.
T2 Mature expression: GFP expressed in seed coat and roots.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAP 1 -GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling X T2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=3
X Ovule Pre-fertilization: Dprimordia Dinner integument D outer integument
D embryo sac D funiculus Dchalaza Dmicropyle Dgametophyte Post-fertilization: D zygote Dsuspensor D embryo sack □ funiculus Dinner integument H outer integument H endothelium H seed coat Dprimordia Dchalaza Dmicropyle Dearly endosperm Dmature endosperm D embryo
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n= 0
No GFP Expression Detected
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=2 Events Expressing: n=2
X Root H mature root
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: seed size, seed weight, seed shape, seed germination timing, germination efficiency, resistance to seed fungal and bacterial pathogens, nitrogen and water utilization and tolerance to low nitrogen and drought environments.
Construct: PT0607
Promoter candidate I. D: 13148287
Events expressing: 02, 05, 06
Promoter Expression Report#300.PT0861
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Flower H vascular H pedicel H receptacle
Hypocotyl H vascular Cotyledon H vascular Rosette Leaf H vascular Primary Root H vascular H xylem H phloem H pericycle H endodermis Root H mature root
Observed expression pattern:
Tl Mature expression: High GFP expression specific to vascular tissues at inflorescences and root.
T2 Seedling expression: High GFP expression in vascular cells throughout organs of seedling.
T2 Mature expression: High GFP expression specific to vascular tissues at inflorescences and root.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=2
X Flower H pedicel H receptacle Dnectary Dsepal Dpetal Dfilament Danther Dtapetum Dpollen Dcarpel Dstyle Dpapillae H vascular Depidermis Dstomata Dtrichome Dsilique
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: water loading from root to shoot, water transport to shoot and flower, tolerance to drought, loss of flowers, fruits and seeds to wilting, drooping and abscission, nitrogen translocation from root to shoot, nitrogen uptake and loading, and fruit and seed yield under limiting and non-limiting soil water and nitrogen environments.
Construct: PT0861
Promoter candidate LD: 15371947
Events expressing: 01-06
Promoter Expression Report#318.PT0760
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Primary Root H epidermis
Observed expression pattern:
Tl Mature expression: None observed.
T2 Seedling expression: Seedling specific root expression. High GFP expression specific to epidermal cells of root.
T2 Mature expression: None detected.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAP 1 -GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=0
No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=3 Events Expressing: n=2
Seedlings expressing / Seedlings screened
Event-01 : 5/6 Event-02: 4/6
X Primary Root H epidermis Dtrichoblast Datrichoblast D cortex D endodermis
D vascular Dxylem Dphloem Dpericycle Dquiescent
D columella H root cap D root hairs
Table 3. T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= 6 Events Expressing: n= 0
No GFP Expression Detected
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: Water uptake, nitrogen uptake, loading of water and nitrogen to cortex and vasculature, tolerance to drought and low levels of soil nitrogen, and protection against soil nematodes and fungal and bacterial pathogens.
Construct: PT0760
Promoter candidate I.D: 15371872
Events expressing: 01, 03
Promoter Expression Report#319.PT0878
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Primary Root H epidermis H cortex H endodermis
Root H mature root
Observed expression pattern:
Tl Mature expression: None observed.
T2 Seedling expression: High GFP expression in ground cells - epidermis, cortex and endodermis of seedling root. Root specific GFP expression. Not in vascular bundle.
T2 mature: High GFP expression in roots.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Table 1. Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n=3 Events Expressing: n=0
No GFP Expression Detected
Table 2. T2 Seedling Expression Tissues Screened
Events Screened: n=6 Events Expressing: n=2
X Primary Root H epidermis Dtrichoblast Datrichoblast H cortex H endodermis D vascular Dxylem Dphloem Dpericycle Dquiescent Dcolumella D root cap D root hairs
Table 3 . T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=6 Events Expressing: n=5
X Root H mature root
Table 4. Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: nitrogen use efficiency in lower/non-limiting nitrogen environments, enhanced water uptake in drought and non-limiting water environments, and protection against soil-borne nematodes, root worms, fungal and bacterial pathogens.
Construct: PT0878
Promoter candidate I.D: 15371944
Events expressing: 01, 02, 03, 04, 06
Promoter Expression Report #519.YP2532
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary: Flower H stomata H nectary H petal
Silique H stomata
Stem H stomata
Leaf H stomata
Primary Root H cortex H epidermis H root hairs L root cap
Observed expression pattern:
Tl Mature expression: GFP expressed in guard cells throughout aerial organs. High GFP expression in flowers stems and leaves. In flowers, GFP also highly expressed in nectary and petals.
T2 Seedling expression: High GFP expression in root epidermis cells including root hairs.
T2 Mature expression: GFP expressed in root epidermis.
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAP 1 -GFP
Marker Type: GFP-ER
Generation Screened: X Tl Mature X T2 Seedling X T2 Mature DT3 Seedling
T2 Seedling Expression Tissues Screened
Events Screened: n=5 Events Expressing: n=3 (02, 04,05)
X Primary Root H epidermis Dtrichoblast Datrichoblast H cortex D endodermis D vascular Dxylem Dphloem Dpericycle D quiescent center D root meristem D columella L root cap H root hairs
T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n=5 Events Expressing: n= 3 (01, 02, 05)
X Root H mature root
Promoter utility
Utility: Among other uses this promoter sequence could be useful to improve: the uptake of water and nutrients and to provide a more rapid establishment of seedlings by increasing nutrient uptake.
Construct: YP2532
Promoter candidate I.D: 40983432
Events expressing: 01, 02, 05
Promoter Expression Report #696.YP2573
Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype
Spatial expression summary:
Drought-induced expression in roots and flowers.
ABA-indueed expression in seedlings.
Observed expression pattern: Tl mature: No expression observed T2 seedling: No expression observed T2 Mature: No expression observed
Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype
Vector: pNewbin4-HAPl-GFP
Marker Type: GFP-ER
Generation Screened: XTl Mature XT2 Seedling XT2 Mature DT3 Seedling
Tl Mature Plant Expression Organs/Tissues screened
Events Screened: n= 5 Events Expressing: n= 0
No GFP Expression Detected
D Flower Dpedicel Dreceptacle Dnectary Dsepal Dpetal Dfilament ϋanther
Dpollen Dcarpel Dstyle Dpapillae Dvascular Hepidermis □ stomata
Dtrichome
Dsilique
D Silique Dstigma Dstyle Dcarpel Dseptum Dplacentae Dtransmitting tissue D vascular D epidermis D stomata D abscission zone D ovule D Ovule Pre-fertilization: Dprimordia Dinner integument D outer integument D embryo sac D funiculus Dchalaza Dmicropyle Dgametophyte Post-fertilization: D zygote Dsuspensor D embryo sack Dinner integument D outer integument D endothelium D seed coat Dprimordia Dchalaza Dmicropyle Dearly endosperm Dmature endosperm Dembryo
D Embryo Dsuspensor Dpreglobular Dglobular Dheart Dtorpedo Dlate Dmature Dprovascular Dhypophysis Dradicle Dcotyledons Dhypocotyl
T2 Mature Plant Expression Organs/Tissues screened
Events Screened: n= 5 Events Expressing: n= 0
No GFP Expression Detected
Inductions Table Organs/Tissues screened
100 μM ABA Treatment
Events Screened: n= 5 Events Expressing: n=4 (01, 02, 04, 05)
Drought Induction 2% Soil Moisture Whole Plant
Events Screened: n= 4 Events Expressing: n=2 (01, 05)
Promoter utility
Utility: Among other things, this promoter could be engineered to promote drought-tolerance in plants by increasing water uptake or reducing water loss during drought-stress.
Construct: YP2573
Promoter candidate LD: 29223801 cDNA LD: 36536957
Lines expressing: 01,02,04,05
[00192] The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the
invention as defined by the following claims.
[00193] Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.
Claims
1. An isolated nucleic acid molecule that shows at least 80% sequence identity to any one of SEQ ID Nos. 1-22, wherein said nucleic acid molecule comprises a regulatory region that directs transcription of an operably linked heterologous polynucleotide.
2. The nucleic acid of claim 1, wherein said regulatory region shows at least 85 percent sequence identity to any one of SEQ ID NOs: 1-22.
3. The nucleic acid of claim 1 , wherein said regulatory region has at least 90 percent sequence identity to any one of SEQ ID NOs: 1-22.
4. The isolated nucleic acid molecule of claim 1, wherein said regulatory region comprises at least one member selected from the group consisting of a promoter, an enhancer and an intron.
5. A vector construct comprising: a) a first nucleic acid molecule according to claim 1 ; and b) a second nucleic acid molecule to be transcribed, wherein said first and second nucleic acid molecules are heterologous to each other and are operably linked.
6. The vector construct according to claim 5, wherein said first nucleic acid consists of the nucleic acid molecule set forth in any one of SEQ ID NOs: 1-
22.
7. A vector according to claim 5, wherein said second nucleic acid molecule comprises a nucleic acid sequence that encodes a polypeptide.
8. A vector according to claim 7, wherein said second nucleic acid molecule is operably linked to said first nucleic acid molecule in the sense orientation.
9. A vector according to claim 8, wherein said second nucleic acid molecule is transcribed into an RNA molecule that expresses the polypeptide encoded by said second nucleic acid molecule.
10. A vector according to claim 7, wherein said second nucleic acid molecule is operably linked to said first nucleic acid molecule in the antisense orientation.
1 1. A vector according to claim 10, wherein said second nucleic acid molecule is transcribed into an antisense RNA molecule.
12. A vector according to claim 5, wherein said second nucleic acid molecule is transcribed into an interfering RNA against an endogenous gene.
13. A plant or plant cell transformed with: a) a nucleic acid molecule according to claim 1 that is operably linked to a heterologous polynucleotide, or b) a vector construct according to claim 5.
14. The plant or plant cell of claim 13, wherein said first nucleic acid molecule consists of the sequence set forth in any one of SEQ ID NOs: 1-22.
15. A plant or plant cell transformed with a vector construct according to any one of claims 7, 8, 9, 10 or 1 1.
16. A method of directing transcription by combining, in an environment suitable for transcription: a) a first nucleic acid molecule according to claim 1 ; and b) a second nucleic acid molecule to be transcribed; wherein said first and second nucleic acid molecules are heterologous to each other and operably linked.
17. The method of claim 16, wherein said first nucleic acid molecule consists of a sequence according to any one of SEQ ID NOs: 1-22.
18. The method of claim 16 or 17, wherein said operably linked first and second nucleic acid molecules are inserted into a plant cell and said plant cell is regenerated into a plant.
19. A plant comprising a vector according to claim 5.
20. A plant according to claim 19, wherein said second nucleic acid molecule codes for a polypeptide of agronomic interest.
21. A transgenic plant according to claim 20, wherein said nucleic acid molecule and said transcribable nucleic acid molecule are heterologous to each other.
22. A seed of a plant according to claim 20 or 21.
23. A method of expressing an exogenous coding region in a plant comprising:
(a) transforming a plant cell with a vector of claim 8;
(b) regenerating a stably transformed plant from the transformed plant cell of step (a); and (c) selecting plants containing a transformed plant cell, wherein expression of the vector gene results in production of a polypeptide encoded by said second nucleic acid.
24. A method of altering the expression of a gene in a plant comprising: a) transforming a plant cell with a nucleic acid molecule according to claim
1 that is operably linked to a heterologous polynucleotide, and b) regenerating stably transformed plants from said transformed plant cell.
25. A plant prepared according to the method of claim 23 or 24.
26. Seed from a plant according to claim 25.
27. A method of producing a transgenic plant, said method comprising:
(a) introducing into a plant cell:
(i) an isolated polynucleotide comprising a nucleic acid according to claim 1 that is operably linked to a heterologous polynucleotide, or (ii) a vector according to claim 5, and
(b) growing a plant from said plant cell.
28. The method of claim 28, wherein said heterologous polynucleotide comprises a nucleic acid sequence encoding a polypeptide.
29. The method of claim 28, wherein said heterologous polynucleotide is operably linked to said regulatory region in the antisense orientation.
30. The method of claim 28, wherein said heterologous polynucleotide is transcribed into an interfering RNA.
Priority Applications (6)
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US12/294,418 US20100170002A1 (en) | 2006-03-24 | 2007-03-23 | Promoter, promoter control elements, and combinations, and uses thereof |
US12/698,056 US20100299784A1 (en) | 2006-03-24 | 2010-02-01 | Promoter, promoter control elements, and combinations, and uses thereof |
US13/664,313 US20130117881A1 (en) | 2003-10-14 | 2012-10-30 | Promoter, promoter control elements, and combinations, and uses thereof |
US15/967,437 US10851383B2 (en) | 2003-10-14 | 2018-04-30 | Promoter, promoter control elements, and combinations, and uses thereof |
US16/938,557 US11634723B2 (en) | 2003-09-11 | 2020-07-24 | Promoter, promoter control elements, and combinations, and uses thereof |
US16/938,550 US11739340B2 (en) | 2003-09-23 | 2020-07-24 | Promoter, promoter control elements, and combinations, and uses thereof |
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US60/785,794 | 2006-03-24 |
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US12/294,418 A-371-Of-International US20100170002A1 (en) | 2003-10-14 | 2007-03-23 | Promoter, promoter control elements, and combinations, and uses thereof |
US12/698,056 Continuation US20100299784A1 (en) | 2003-09-11 | 2010-02-01 | Promoter, promoter control elements, and combinations, and uses thereof |
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CN112011566A (en) * | 2009-08-31 | 2020-12-01 | 巴斯夫植物科学有限公司 | Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants to promote enhanced polyunsaturated fatty acid synthesis |
US11708578B2 (en) | 2009-08-31 | 2023-07-25 | Basf Plant Science Company Gmbh | Regulatory nucleic acid molecules for enhancing constitutive gene expression in plants |
CN112011566B (en) * | 2009-08-31 | 2023-12-05 | 巴斯夫植物科学有限公司 | Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants to promote enhanced polyunsaturated fatty acid synthesis |
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US20100299784A1 (en) | 2010-11-25 |
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