WO2006089911A2 - Cassettes d'expression pour expression preferentielle d'une semence chez les plantes - Google Patents

Cassettes d'expression pour expression preferentielle d'une semence chez les plantes Download PDF

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WO2006089911A2
WO2006089911A2 PCT/EP2006/060185 EP2006060185W WO2006089911A2 WO 2006089911 A2 WO2006089911 A2 WO 2006089911A2 EP 2006060185 W EP2006060185 W EP 2006060185W WO 2006089911 A2 WO2006089911 A2 WO 2006089911A2
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sequence
sequences
seq
expression
gene
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PCT/EP2006/060185
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WO2006089911A3 (fr
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Elke Duwenig
Linda Patricia Loyall
Oliver Oswald
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Basf Plant Science Gmbh
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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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm

Definitions

  • the present invention relates to expression cassettes comprising transcription regulat- ing nucleotide sequences with seed-preferentiai or seed-specific expression profiles in plants obtainable from the Arabidopsis thahana gene described by the locus AtSg01670 or its orthologs from other plant species (such as Brasstca napus)
  • the transcription regulating nucleotide sequences preferably exhibit strong expression activity especially in seeds and seed tissues
  • Manipulation of plants to alter and/or improve phenotypic characteristics requires the expression of heterologous genes in plant tissues
  • Such genetic manipulation relies on the availability of a means to drive and to control gene expression as required
  • genetic manipulation relies on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant
  • the seed-preferential or seed-specific promoters are useful for expressing genes as well as for producing large quantities of protein, for expressing oils or proteins of interest, e g , antibodies, genes for increasing the nutritional value of the seed and the like St is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment Moreover, the increasing interest Jn cotransformi ⁇ g plants with multiple plant transcription units (PTU) and the potential problems associated with using common regulatory sequences for these purposes merit having a variety of promoter sequences available
  • a first embodiment of the invention relates to an expression cassette for regulating seed-specific or seed-preferential expression in plants comprising i) at least one transcription regulating nucleotide sequence of a gene encoding an aldose reductase-related protein, and functionally linked thereto ii) at least one nucleic acid sequence which ss heterologous in relation to said transcription regulating nucleotide sequence
  • the expression cassette of the invention comprises a) at least one transcription regulating nucleotide sequence of a plant gene, said plant gene selected from the group consisting of
  • the transcription regulating nucleotide sequence may be obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide which a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences i) VGLGTW (SEQ ID NO 27), preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO 28),
  • H YRH(V/I)DTA (SEQ ID NO: 29), in) FVTSK(L ⁇ //I)W (SEQ ID NO 30), IV) CQMEMH (SEQ ID NO 31), preferably VCQMEMHPGW (SEQ ID NO 32), and v) RGTSVIP (SEQ ID NO 33), preferably QRGTSViPKS (SEQ ID NO 34), or
  • b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO 8, 10, 12, 14, 16, 18, and 36
  • the expression cassette comprises a transcription regulating nucleotide sequence, which is obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably an orthologous protein) which a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences i) VGLGTW (SEQ ID NO" 27), preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO 28), i ⁇ ) YRH(V/!DTA (SEQ ID NO' 29), in) FVTSK(L/V/!W (SEQ ID NO 30),
  • a transcription regulating nucleotide sequence which is obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably an orthologous protein) which a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences i) VGLGTW (SEQ ID NO" 27), preferably P(A/G)VGLGTW(R/K
  • CQMEMH (SEQ ID NO" 31), preferably VCQMEMHPGW (SEQ ID NO' 32), and v) RGTSVIP (SEQ ID NO- 33), preferably QRGTSViPKS (SEQ ID NO 34), and b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO 8, 10, 12, 14, 16, 18, and 36
  • said orthologous protein has furthermore the same enzymatic activity than the protein encoded by the Arabidopsis thafiana locus AtSg01670
  • the transcription regulating nucleotide sequence is selected from the group of sequences consisting of ⁇ ) the sequence described by SEQ ID NOs 1 , 2, 3, 4 5, or 6, i!) a fragment of at least 50 consecutive bases of a sequence under ⁇ ),
  • SDS 1 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 0 5 X SSC, 0 1% SDS at 5O 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 5O 0 C with washing in 0 1 X SSC, 0 1% SDS at 50°C, more preferabfy in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 5O 0 C with wash- ing in 0 1 X SSC, 0 1% SDS at 65 0 C) to a transcription regulating nucleotide sequence described by SEQ ID NO 1 , 2, 3, 4, 5, or 6, or the complement thereof, v) a nucleotide sequence capable of hybridizing (preferably under conditions equivalent to hybridization in 7% sodium dodecyl sulfate
  • nucleotide sequence which is the complement or reverse complement of any of the previously mentioned nucleotide sequences under ⁇ ) to v)
  • sequences specified under ⁇ ), in), ⁇ v) v) and v ⁇ ) above are capable to modify transcription in a piant ceil or organism, preferably said sequences have substantially the same transcription regulating activity as the transc ⁇ p- tion regulating nucleotide sequence described by SEQ (D NO 1 2, 3, 4, 5, or 6
  • a nucleotide sequence having substantial similarity to a transcription regulating nucleotide sequence described by SEQ ID NO 1 , 2 3, 4, 5, or 6 has a sequence identity of at least 50% or 60%, preferably at least 70% or 80%, more preferabfy at least 90% or 95%, most preferably at least 98% to a sequence described by SEQ ID NO 1 , 2, 3, 4, 5, or 6
  • hybridization is performed under stringent conditions (including low and high stringency conditions), more preferably under high stringency conditions
  • the expression cassette may be employed for numerous expression purposes such as for example expression of a protein, or expression of a antisense RNA, sense or double-stranded RNA
  • expression of the nucleic acid sequence confers to the plant an agronomically valuable trait.
  • vectors comprising an expression cas- sette of the invention, and transgenic host cell or non-human organism comprising an expression cassette or a vector of the invention.
  • the organism is a plant More preferably a plant used for oil production such as - for example - Brassica nap ⁇ s, Brassica j ⁇ ncea, soybean, or sunflower.
  • Another preferred embodiment of the invention relates to a method for identifying and/or isolating a transcription regulating nucleotide sequence characterized that said identification and/or isolation utilizes a nucleic acid sequence encoding a polypeptide as described by SEQ ID NO' 8, 10, 12, 14, 16, 18, or 36, or a part of at least 15 consecutive nucleotides of said nucleic acid sequence.
  • the nucleic acid se- quence utilized for the isolation is described by SEQ ID NO 7, 9, 11, 13, 15, 17, or 35 or a part of at least 15 consecutive nucleotides thereof More preferably, said identification and/or isolation is realized by a method selected from polymerase chain reaction, hybridization, and database screening
  • I isolating of a transcription regulating nucleotide sequence of a plant gene utilizing at least one nucleic acid sequence encoding a polypeptide described by SEQ ID NO 8, 10, 12, 14, 16, 18, or 36, or a part of at least 15 consecutive nucleotides of said nucleic acid sequence, and
  • Another embodiment of the invention relates to a method for providing a transgenic expression cassette for seed-specific or seed-preferential expression comprising the steps of.
  • nucleic acid sequence utilizing at least one nucleic acid sequence or a part thereof, wherein said sequence is encoding a polypeptide described by SEQ ID NO 8, 10,12. 14, 16, 18, or 36, or a part of at least 15 consecutive nucleotides thereof, and
  • nucleotide sequence utilized for isolation of said transcription regulating nucleotide sequence is encoding a polypeptide comprising at least one sequence motive selected from the group consisting of the amino acid sequences
  • VGLGTW (SEQ ID NO 27), preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO 28), ii) YRH(V/!DTA (SEQ ID NO 29), v) FVTSK(LMI)W (SEQ ID NO 30),
  • CQMEMH SEQ ID NO 31
  • VCQMEMHPGW SEQ ID NO 32
  • RGTSVIP SEQ ID NO 33
  • QRGTSVIPKS SEQ ID NO. 34
  • nucleotide sequence (preferably having transcription regulating activity, more preferably having promoter activity) selected from the group of sequences consisting of a) the sequence as described by SEQ ID NO 1 or 2, and sequences having an identity of at least 99%, preferably 99 5%, more preferably 99 8% to a sequence as de- scribed by SEQ ID NO 1 or 2, and sequences comprising at least 600 consecutive nucleotides, preferably at least 800 consecutive nucleotide, more preferably at least 900 consecutive nucleotides, more preferably at least 1000 consecutive nucleotides of a sequence as described by SEQ ID NO 1 or 2, and b) the sequence as described by SEQ ID NO 5 or 6, and sequences havsng an identity of at least 40% or 50%, preferably 60% or 70%, more preferably 80 or 90%, most preferably 95% or 98% to a sequence as described by
  • Fig 1 Alignment of the putative aldose reductase-related protein (At5g01670 protein) with its orthologs from various species including Brassica napus (two versions Brassica_napus-V1 and Brassica_napus-V2), Bromus-inermis, Xerophyta- viscosa, Avena-fatua, and Hordeum-vulgare
  • genes include coding sequences and/or the regulatory sequences required for their expression
  • gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein and which includes regulatory sequences
  • Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters
  • mutant or wild type gene refers to a gene that is present in the genome of an untransformed cell, i e., a cell not having a known mutation.
  • a "marker gene” encodes a selectable or screenable trast.
  • chimeric gene refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or
  • a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged sn a manner different from that found in nature
  • transgene refers to a gene that has been introduced into the genome by transformation and is stably maintained Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
  • oligonucleotide corresponding to a nucleotide sequence of the invention, e g , for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e g , 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30)
  • primers are upwards of 14 nucleotides in length
  • primers of 16 to 24 nucleotides in length may be preferred Those skilled in the art are well versed in the design of primers for use processes such as PCR !f required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even 1000's of nucleotides in length
  • amino acid sequence or a ' polypeptide sequence
  • amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission
  • the abbreviations used herein are conventional one letter codes for the amino acids A, alanine, B, asparagine or aspartic acid, C, cysteine, D aspartic acid, E, glutamate, glutamic acid, F, phenylalanine, G, glycine, H histidine; I isoleucine, K, lysine, L leucine, M, methson
  • Coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-codmg sequences It may constitute an "uninterrupted coding sequence", i e lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions
  • An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the ceil to create the mature mRNA that can be translated into a protein
  • open reading frame and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codo ⁇ s of a coding sequence
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides ( ' codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation)
  • a “functional RNA” refers to an antisense RNA, ribozyme, or other RNA that is not translated
  • RNA transcript refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence
  • primary transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell
  • cDNA refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA
  • transcription regulating nucleotide sequence each refer to nucleotide sequences influencing the transcription, RNA processing or stability, or translation of the associated (or functionally linked) nucleotide sequence to be transcribed
  • the transcription regulating nucleotide sequence may have various localizations with the respect to the nucleotide sequences to be transcribed
  • the transcription regulating nucleotide sequence may be located up- stream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of the sequence to be transcribed (e g , a coding sequence)
  • the transcription regulating nucleotide sequences may be selected from the group comprising enhancers, promoters, translation leader sequences, introns 5'-untranslated sequences, 3'-untranslated sequences, and polyadenylation signal sequences They include natural and synthetic sequences as well as sequences, which may be a combination of synthetic and natural sequences As is noted above, the
  • 5' non-coding sequence or 'S'-untranslated sequence or "-region” refers to a nucleotide sequence located 5' (upstream) to the coding sequence It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner 1995)
  • 3' non-coding sequence or “3'-untranslated sequence” or “-region” refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression
  • the poiyadenylation signal is usually char- acte ⁇ zed by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon
  • the translation leader sequence may affect processing of the primary transcript to mRNA mRNA stability or translation efficiency
  • Signal peptide refers to the ammo terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway
  • signal sequence refers to a nucleotide sequence that encodes the signal peptide
  • transition peptide refers part of a expressed polypeptide (preferably to the ammo terminal extension of a polypeptide), which is translated in conjunction with the polypeptide formsng a precursor peptide and which is required for its entrance into a cell organelle (such as the plastids (e g , chloroplasts) or mitochondria)
  • transit sequence refers to a nucleotide sequence that encodes the transit peptide
  • Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recog- nition for RNA polymerase and other factors required for proper transcription.
  • Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcrtption initiation, to which regulatory elements are added for control of expression
  • Promoter afso refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers Accordingly, an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter It is capable of
  • the "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 With respect to this site ail other sequences of the gene and its controlling regions are numbered Downstream sequences ( ⁇ e , further protein encoding sequences in the 3 r direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative
  • Promoter elements particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters"
  • minimal or core promoters In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription
  • a "minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e g , a TATA box and/or an initiator
  • Constant expression refers to expression using a constitutive or regulated pro- moter
  • Consditional and regulated expression refer to expression controlled by a regulated promoter
  • Constutive promoter refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant
  • ORF open reading frame
  • Regular promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found m the compilation by Okamuro et a!
  • Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system promoters derived from salicylate-inducible systems, promoters derived from afcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-indu ⁇ ble systems, and promoters derived from ecdysone-inducible systems
  • tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells) These also include promoters that are temporally regulated such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf or at the onset of senescence
  • “Inducible promoter” refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone stress, or a pathogen
  • operably-linked or “functionally linked” refers preferably to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other
  • a regulatory DNA sequence ss said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence ( ⁇ e , that the coding sequence or functional RNA is under the transcriptional control of the promoter) Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation
  • “Expression” refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants
  • expression may refer to the transcription of the antisense DNA only
  • expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA Expression may also refer to the production of protein
  • Specific expression is the expression of gene products which is limited to one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation) It is acknowledged that hardly a true specificity exists 1 promoters seem to be preferably switch on in some tissues, while in other tissues there can be no or only little activity This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues
  • the "expression pattern" of a promoter is the pattern of ex- pression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter.
  • the level of expression of a promoter can be determined by measuring the " steady state ' concentration of a stan- dard transcribed reporter mRNA This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded Therefore, the steady state level is the product of synthesis rates and degradation rates The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.
  • Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of the reporter gene Also frequently observed is the phenomenon that such transformants do not express any detectable product (RNA or protein) The variability in expression is commonfy ascribed to ' position effects ' , although the molecular mechanisms underlying this inactivity are usually not clear
  • “Overexpression” refers to the level of expression in transgenic cells or organisms that exceeds levels of expression m normal or untransformed (non-transgenic) cells or organisms
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene
  • Gene silencing' 1 refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes Gene silencing may be transcriptional, when the sup- pression is due to decreased transcription of the affected genes, or post-transcriptionai, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes (English 1996) Gene silencing includes virus-induced gene silencing (Ruiz et al 1998).
  • heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cefl but has been modified through, for example, the use of DNA shuffling
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position withrn the host cell nucleic acid in which the element is not ordinarily found Exogenous DNA segments are expressed to yield exogenous polypeptides
  • a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host ceil into which tt is introduced.
  • “Homologous to” in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (as described in Haines and Higgtns (eds ), Nucleic Acid Hybridization, IRL Press, Oxford, U K ), or by the comparison of sequence similarity between two nucleic acids or proteins
  • substantially similar refers to nucleotide and ammo acid sequences that represent functional and/or structural equivalents or orthologs of Arabidopsis thaliana or Brassica napus sequences disclosed herein
  • nucleotide sequence is part of a gene which encodes a polypeptide having substantially the same structure and function as a polypeptide encoded by a gene for the reference nucleotide sequence, e g , the nucleotide sequence comprises a promoter from a gene that is the ortholog of the gene corresponding to the reference nucleotide sequence, as well as promoter sequences that are structurally related the promoter sequences particularly exemplified herein, i e , the substantially similar promoter sequences hybridize to the complement of the promoter sequences exemplified herein under high or very high stringency conditions For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode ammo acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences
  • substantially similar also includes nucleotide sequences
  • substantially similar when used herein with respect to polypeptide means that the polypeptide has substantially the same structure and function as the reference polypeptide
  • amino acid sequences that are substantially similar to a particular sequence are those wherein overall amino acid identity is at least 50% or greater to the instant sequences Modifications that result in equivalent nucleotide or ammo acid sequences are well within the routine skill in the art
  • the percentage of amino acid sequence identity between the substantially similar and the reference polypeptide is at least 50% e g , 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82% 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90% or more,
  • Sequence comparisons maybe earned out using a Smith-Waterman sequence alignment algorithm (see e g , Waterman (1995))
  • the localS program, version 1 16 is preferably used with following parameters match 1 , mismatch penalty 0 33, open-gap penalty 2, extended-gap penalty 2
  • nucleotide sequence that is "substantially similar" to a reference nucleotide sequence is said to be “equivalent” to the reference nucleotide sequence
  • equivalent nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under low, moderate and/or stringent conditions (e g , 0 1 X SSC 0 1% SDS, 65°C), with the nucleotide sequences that are within the literal scope of the instant claims
  • “substantially the same activity" when used in reference to a polynucleotide or polypeptide fragment is that the fragment has at least 50%, e g , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%,
  • Target gene refers to a gene on the replicon that expresses the desired target coding sequence, functional RNA, or protein
  • the target gene is not essential for replicon replication
  • target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences.
  • the regulatory sequences in the target gene may come from any source, including the virus.
  • Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed However, target genes do not include native viral genes Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance Proteins encoded by target genes are known as "foreign proteins" The expression of a target gene in a plant will typically produce an altered plant trait
  • altered plant trait means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host
  • Replication gene refers to a gene encoding a viral replication protein
  • the replication gene may also contain other overlapping or non-overlapping ORF(s), as are found in viral sequences in nature While not essential for replication, these additional ORFs may enhance replication and/or viral DNA accumulation Examples of such additional ORFs are AC3 and AL3 m ACMV and TGMV geminiviruses, respectively.
  • Chimeric trans-acting replication gene refers either to a replication gene in which the coding sequence of a replication protein is under the control of a regulated plant promoter other than that in the native viral replication gene, or a modified native viral repli- cation gene, for example, in which a site specific sequence(s) is inserted in the 5' transcribed but untranslated region Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene
  • Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independentSy, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance
  • Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms”
  • Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere 1987) and particle bombardment technology (US 4,945,050) Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced
  • the nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed (Sambrook 1989, Innis 1995; Gelfand 1995, lnnis & Gelfand 1999 Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specsfic primers, vector-specific primers, partially mismatched primers, and the like.
  • “transformed,” “transformant,” and “transgenic” plants or callt have been through the transformation process and contain a foreign gene integrated into their chromosome
  • the term “untransformed” refers to normal plants that have not been through the transformation process
  • Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
  • “Stably transformed” refers to cells that have been selected and regenerated on a selection media following transformation
  • Transient expression refers to expression in cells in which a virus or a transgene is introduced by viral infection or by such methods as Agrobacterium-mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance
  • Genetically stable and “heritable” refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations
  • Primary transformant and “TO generation” refer to transgenic plants that are of the same genetic generation as the tissue which was initially transformed (i e , not having gone through meiosis and fertilization since transformation).
  • “Secondary transformants” and the “T1, T2, T3, etc generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants "Wild-type” refers to a virus or organism found in nature without any known mutation
  • genomic DNA is referring to the heritable genetic information of a host organism
  • Said genomic DNA comprises the DNA of the nucleus (also re- ferred to as chromosomal DNA) but also the DNA of the plastids (e g , chloroplasts) and other cellular organelles (e.g., mitochondria)
  • genome or genomic DNA is referring to the chromosomal DNA of the nucleus
  • chromosomal DNA or "chromosomal DNA-sequence” is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status Chromosomal DNA might therefore be organized in chromosomes or chromatids, they might be condensed or uncoiled An insertion into the chromosomal DNA can be demonstrated and analyzed by various methods known in the art like e g , polymerase chain reaction (PCR) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH), and in situ PCR
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • nucleic acid refers to deoxy ⁇ bonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e g , degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosme residues (Batzer 1991 , Ohtsuka
  • nucleic acid fragment is a fraction of a given nucleic acid molecule
  • deoxyribonucleic acid DNA
  • RNA ribonucleic acid
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers
  • nucleic acid or nu- cleic acid sequence may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene
  • an "isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • an "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid ( ⁇ e , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0 5 kb, or 0 1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived
  • a protein that is substantially free of cellular material includes preparations of protein or
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein
  • nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e g ,
  • Constantly modified variations of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypep- tide For instance the codons CGT, CGC, CGA CGG, AGA, and AGG all encode the amino acid arginine Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein Such nucleic acid variations are "silent variations" which are one species of “conservatively modified variations " Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methion
  • the nucleic acid molecules of the invention can be "optimized" for enhanced expression in plants of interest (see, for example, WO 91/16432, Perlak 1991 ; Murray 1989) In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons (see, for example, Campbell & Gowri, 1990 for a discussion of host-preferred codon usage).
  • the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.
  • Variant nucleotide sequences and proteins also encompass, sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein
  • variants may result from, for example, genetic polymorphism or from human manipulation Methods for such manipulations are generally known in the art
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art
  • amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA.
  • “Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably (inked to a nucleotide sequence of interest, which is - optionally - operably linked to termination signals and/or other regulatory elements
  • An expression cassette may also comprise sequences required for proper translation of the nucleotide sequence
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one, which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression
  • An expression cassette may be as-Lited entirely extracellularly (e g , by recombinant cloning techniques)
  • an expression cassette may also be assembled using sn part endogenous components
  • an expression cassette may be obtained by placing (or inserting) a promoter sequence upstream of an endogenous sequence, which thereby becomes functionally linked and controlled by said promoter sequences
  • a nucleic acid se- quence to be expressed may be placed (or inserted) downstream of an endogenous promoter sequence thereby forming an expression cassette.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus
  • the promoter can also be specific to a particular tissue or organ or stage of development (e.g , the seed-specific or seed-preferential promoters of the invention)
  • such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest
  • Such an expression cassette is preferably provided with a plurality of restriction sites for inser- tion of the gene of interest to be under the transcriptional regulation of the regulatory regions
  • the expression cassette may additionally contain selectable marker genes
  • the cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants The termination region may be na- tive with the transcriptional initiation region, may be native with the DNA sequence of
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be seif transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e g autonomous replicating plasmid with an origin of replication)
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e g higher plant, mammalian, yeast or fungal cells)
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell
  • an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell
  • the vector may be a bi-functional ex- pression vector which functions in multiple hosts In the case of genomic DNA 1 this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell
  • Coding vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector Marker genes typically include genes that provide tetracycline resis- tance, hygromycin resistance or ampicillin resistance
  • a “transgenic plant” is a plant having one or more plant cells that contain an expression vector
  • Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue
  • the plant tissue may be in plants or in organ, tissue or cell culture
  • sequence relationships between two or more nucleic acids or polynucleotides (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantia! identity”
  • reference sequence is a defined sequence used as a basis for sequence comparison
  • a reference sequence may be a subset or the entirety of a specified sequence, for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) com- pared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer Those of skill in the art understand that to avoid a high similarity to a ref- erence sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity
  • Such implementations include, but are not limited to CLUSTAL in the PC/Gene program (available from Intelhgenetics, Mountain View, Calif ), the ALIGN program (Version 2 0) and GAP,
  • HSPs high scoring sequence pairs
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e g Kar- Im & Altschul (1993) One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probabil- ity by which a match between two nucleotide or amino acid sequences would occur by chance
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0 1, more preferably less than about 0 01, and most preferably less than about 0 001
  • Gapped BLAST in BLAST 2 0
  • PSI-BLAST in BLAST 2 0
  • iterated search that detects distant rela- tionships between molecules See Altschul et al , supra
  • Gapped BLAST, PSI-BLAST, the default parameters of the respective programs can be used.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10
  • the BLOSU M62 scoring matrix See http / ⁇ vww ncbi nlm nih gov Alignment may also be performed manually by inspection
  • equivalent program any sequence comparison program that, for any two sequences in question, gen- erates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window
  • sequences differ sn conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution Sequences that differ by such conservative substitutions are said to have "sequence similarity” or “similarity " Means for making this adjustment are well known to those of skill in the art Typically this involves scoring a conservative substitution as a partial rather than a fuil mismatch, thereby increasing the percentage sequence identity Thus, for example, where an identical amino acid is given
  • percentage of sequence identity means the vaiue determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may com- prise additions or deletions ( ⁇ e , gaps) as compared to the reference sequence
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 50%, 50% e g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below) Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1 °C to about 2O 0 C, depending upon the desired degree of stringency as otherwise quali- fied herein
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substa ⁇ tialiy identical if the polypeptides they encode are substantially identical This may occur, e g , when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 50%, e g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e g , total cellular) DNA or RNA
  • B ⁇ nd(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommo- dated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution For DNA-DNA hybrids, the T m can be approximated from the equation of Meinkoth and Wahl, 1984.
  • T m 81 5°C + 16 6 (log 10 M)+0 41 (%GC) - 0 61 (% form) - 500 / L
  • M is the molarity of monovalent cations
  • %GC is the percentage of guanosine and cytosine nucleotides in the DNA
  • % form is the percentage of formamicSe in the hybridization solution
  • L is the length of the hybrid in base pairs T m is reduced by about 1 °C for each 1 % of mismatching
  • T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity
  • the T m can be decreased 10 0 C
  • stringent conditions are selected to be about 5 0 C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH How- ever, severely stringent conditions can utilize a hybridization and/or wash at 1 , 2, 3, or 4 0 C lower than the
  • An example of highly stringent wash conditions is 0 15 M NaCI at 72°C for about 15 mmutes
  • An example of stringent wash conditions is a 0 2 X SSC wash at 65 0 C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer) Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal
  • An example medium stringency wash for a duplex of e g , more than 100 nucleotides is 1 X SSC at 45°C for 15 minutes
  • An example low stringency wash for a duplex of, e g , more than 100 nucleotides is 4 to 6 X SSC at 40°C for 15 minutes
  • stringent conditions typically involve salt concentrations of less than about 1 5 M, more preferably about 0 01 to 1 0 M, Na ion concentration (or other salts) at pH 7 0 to 8 3, and the temperature is typically at least about
  • Very stringent conditions are selected to be equal to the T m for a particular probe
  • An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide e g , hybridization in 50% formamsde, 1 M NaCI, 1 % SDS at 37°C, and a wash in 0 1 x SSC at 60 to 65°C
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 0 M NaCI, 1 % SDS at 37 0 C, and a wash in 0 5
  • a reference nucleotide sequence pref- erably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 2 X SSC, 0.
  • SDS sodium dodecyl sulfate
  • DNA shuffling is a method to introduce mutations or rearrangements, preferably randomly, m a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule
  • the shuffled DNA preferably en- codes a variant polypeptide modified wtth respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA
  • Recombinant DNA molecule * is a combination of DNA sequences that are joined to- gether using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al , 1989
  • plant refers to any plant, particularly to agronomically useful plants (e g , seed plants), and "plant cell” is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ differentiated into a structure that is present at any stage of a plant's development
  • Such structures include one or more plant organs including, but are not limited to, fruit, shoot, stem, leaf, flower petal, etc
  • the term "plant” includes whole plants, shoot vegetative organs/structures (e g leaves, stems and tubers), roots, flowers and flora!
  • organs/structures e g bracts, sepals, petals, stamens, carpels, anthers and ovules
  • seeds including embryo, endosperm, and seed coat
  • fruits the mature ovary
  • plant tissues e g vascular tissue, ground tissue, and the like
  • cells e g guard cells, egg cells, trichomes and the like
  • progeny of same The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyiedonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
  • plants and plant materials of the following plant families Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Composi- tae, C ⁇ curbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Tetragon laceae.
  • Said plant may include - but shall not be limited to - bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses), pteridophytes such as ferns, horsetail and clubmosses, gymnosperms such as conifers, cycads, ginkgo and Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophy- ceae, Bacillariophyceae (diatoms) and Euglenophyceae
  • - bryophytes such as, for example, Hepaticae (hepaticas) and Musci (mosses), pteridophytes such as ferns, horsetail and clubmosses, gymnosperms such as conifers, cycads, ginkgo and Gnetaeae
  • algae such as Chlorophyceae, Pha
  • Plants for the purposes of the invention may comprise the families of the Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas, Euphorhiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias,
  • Gesne ⁇ aceae such as African violet, Balsammaceae such as touch-me-not, Orchida- ceae such as orchids, I ⁇ daceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena, Moraceae such as ficus, Araceae such as philodendron and many others
  • the transgenic plants according to the invention are furthermore selected in particular from among dicotyledonous crop plants such as, for example, from the families of the Leguminosae such as pea, alfalfa and soybean; the family of the Umbelhferae, particu- larly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens var dulce (celery)) and many others, the family of the Solanaceae, particularly the genus Lycopersicon, very particularly the species es- culentum (tomato) and the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine), tobacco and many others, and the genus Capsi- cum, very particularly the species annum (pepper) and many others; the family of the Leguminosae, particularly the genus Glycine, very particularly the species max (soybean) and many others, and the family of
  • trees such as apple, pear, quince, plum, cherry, peach, nectarine, apricot, papaya, mango, and other woody species including coniferous and deciduous trees such as poplar, pine, sequoia, cedar, oak, etc.
  • Ar ⁇ bidopsis thaliana thaliana
  • Nicotiana ta- bacum oilseed rape
  • soybean corn
  • corn corn
  • wheat Linum usitatissimum (linseed and fax)
  • Camelina sativa Brassica juncea
  • “Significant increase” is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater
  • the present invention thus provides for isolated nucleic acid molecules comprising a plant nucleotide sequence that directs seed-preferential or seed-specific transcription of an operably linked nucleic acid fragment in a plant cell.
  • the present invention provides transgenic expression cassettes for regulating seed-specific or seed-preferential expression in plants comprising a) at least one transcription regulating nucleotide sequence of a gene encoding an aldose reductase-related protein, and functionally linked thereto b) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence
  • the expression cassette of the invention comprises a) at least one transcription regulating nucleotide sequence of a plant gene, said plant gene selected from the group consisting of in) the gene described by the GenBank Arabidopsis thaliana genome foci At5g01670, and ⁇ v) orthologous genes of the gene described by the GenBank Arabidopsis thaliana genome loci At5g01670, and functionally linked thereto b) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence
  • the seed-preferential or seed-specific promoters may be useful for expressing genes as well as for producing large quantities of protein, for expressing oris or proteins of interest, e g , antibodies, genes for increasing the nutritional value of the seed and the like
  • seed in the context of the inventions means a seed of a plant in any stage of its development i.e. starting from the fusion of pollen and oocyte continuing over the embryo stage and the stage of the dormant seed, until the germinating seed, ending with early seedling organs, as e g. cotyledons and hypocotyl
  • “Seed-specific transcription” in the context of this invention means the transcription of a nucleic acid sequence by a transcription regulating element in a way that transcription of said nucleic acid sequence in seeds contribute to more than 90%, preferably more than 95%, more preferably more than 99% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant during any of its developmental stage
  • the transcription regulating nucleotide sequences specifically disclosed herein are considered to be seed-specific transcription regulating nucleotide sequences
  • “Seed-preferential transcription” in the context of this invention means the transcription of a nucleic acid sequence by a transcription regulating element in a way that transcription of said nucleic acid sequence in seeds contribute to more than 50%, preferably more than 70%, more preferably more than 80% of the entire quantity of the RNA transcribed from said nucleic acid sequence in the entire plant du ⁇ ng any of its developmental stage
  • “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined Falling within this generic term are the terms “ortholog”, and “paralog” "Paralog” refers to a polynucleotide or polypeptide that within the same species which is functionally similar "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species
  • An orthologous gene means preferably a gene which is encoding a orthologous protein More specifically, the term “ortholog” denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species Sequence differences among orthologs are the result of speciation More preferably "or
  • a transcription regulating nucleotide sequence of the invention comprises at least one promoter sequence of the respective gene ⁇ e.g., a sequence localized upstream of the transcription start of the respective gene capable to induce transcription of the downstream sequences).
  • Said transcription regulating nucleotide sequence may comprise the promoter sequence of said genes but may further comprise other elements such as the 5'-untranslated sequence, enhancer, introns etc.
  • said promoter sequence directs seed-preferential or seed-specific transcription of an opera- bly linked nucleic acid segment in a plant or plant cell e.g , a linked plant DNA comprising an open reading frame for a structural or regulatory gene.
  • Table 1 illustrates the genes from which the promoters of the invention are preferably isolated, the function of said genes, the cDNA encoded by said genes, and the protein (ORF) encoded by said genes.
  • nucleotide sequence preferably having transcription regulating activity, more preferably having promoter activity
  • an isolated nucleotide sequence selected from the group of sequences consisting of: a) the sequence as described by SEQ ID NO: 1 or 2, and sequences having an identity of at least 99%, preferably 99.5%, more preferably 99.8% to a sequence as described by SEQ ID NO.
  • sequences comprising at least 600 consecutive nucleotides, preferably at feast 800 consecutive nucleotide, more preferably at least 900 consecutive nucleotides, more preferably at least 1000 consecutive nucleotides of a sequence as described by SEQ ID NO: 1 or 2, and b) the sequence as described by SEQ ID NO 1 5 or 6, and sequences having an identity of at least 40% or 50%, preferably 60% or 70%, more preferably 80 or 90%, most preferably 95% or 98% to a sequence as described by SEQ ID NO: 5 or 26, and se- quences comprising at least 15 or 20 consecutive nucleotides, preferably at least 50 or 100 consecutive nucleotide, more preferably at least 250 consecutive nucleotides, more preferably at least 500 consecutive nucleotides of a sequence as described by SEQ ID NO 5 or 6,
  • W wordlength
  • E expectation
  • M 5
  • the BLASTP program is used with default parameters with a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Hemkoff, 1989)
  • the BLAST Program version 1 4 7 or later is used
  • the transcription regulating nucleotide sequence is selected from the group of sequences consisting of ⁇ ) the sequence described by SEQ ID NOs 1 , 2, 3, 4 5, or 6, n) a fragment of at least 50 consecutive bases of a sequence under ⁇ ), in) a nucleotide sequence having substantial similarity with a sequence rdentity of at least 50% to a transcription regulating nucleotide sequence described by SEQ (D NO I 1 2, 3, 4, 5, or 6, iv) a nucleotide sequence capable of hybridizing (preferably under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 2 X SSC 0 1 % SDS at 5O 0 C, more desirably in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 1 X
  • SDS 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 0 5 X SSC, 0 1 % SDS at 50 0 C, preferably in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0 1 X SSC, 0 1% SDS at 50 0 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0 5 M NaPO 4 , 1 mM EDTA at 50 0 C with wash- ing in 0 1 X SSC, 0 1 % SDS at 65°C) to a transcription regulating nucleotide sequence described by SEQ ID NO 1 , 2 3, 4, 5, or 6, or the complement thereof, v) a nucleotide sequence capable of hybridizing (preferably under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0
  • a nucleotide sequence having substantial similarity to a transcription regulating nucleotide sequence described by SEQ ID NO 1 , 2, 3, 4, 5, or 6 has a sequence identity of at least 50% or 60%, preferably at least 70% or 80% more preferably at least 90% or 95%, most preferably at least 98% to a sequence described by SEQ ID NO 1 , 2, 3, 4, 5, or 6
  • hybridization is performed under stringent conditions (including low and high stringency conditions), more preferably under high stringency conditions
  • the transcription regulating nucleotide sequence may be obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably from an ortholo- gous gene i e , encoding an orthologous protein) which a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences i) VGLGTW (SEQ ID NO 27), preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO 28), ii) YRH(V/I)DTA (SEQ ID NO 29), in) FVTSK(L ⁇ //I)W (SEQ ID NO 30),
  • CQMEMH (SEQ ID NO 31), preferably VCQMEMHPGW (SEQ ID NO 32), and v) RGTSVIP (SEQ ID NO 33), preferably QRGTSVIPKS (SEQ ID NO 34), or
  • b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO 8, 10, 12, 14, 16, 18, and 36
  • a functional equivalent of the transcription regulating nucleotide sequence can be obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably from an orthologous gene i e , encoding an orthologous protein) which is substantially Similar and preferably has at least 50% or 60%, preferably at least 70% or 80%, more preferably at least 90% or 95%, most preferably at least 97% or 98% amino acid sequence identity to a polypeptide described by any one of SEQ ID NOs 8, 10, 12, 14 16, 18, or 36, respectively, or a fragment of said transcription regulating nucleotide sequence which exhibits promoter activity in a seed-preferential or seed-specific fashion
  • transcription regulating nucleotide sequence of the invention are obtainable from plant genomic DNA from a gene encoding a polypeptide which comprises at least two or three, preferably at least four or five, more pref- erably six, most preferably all of the sequence motives described by SEQ ID NO 27, 28, 29, 30, 31 , 32, 33 or 34 and - preferably - exhibits promoter activity in plants (preferably in a seed-preferential or seed-specific fashion)
  • the expression cassette comprises a transcription regulating nucleotide sequence, which is obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably an orthologous protein) which a) comprises at least one, preferably at least two or three, more preferably all sequence motive(s) selected from the group consisting of the amino acid sequences i) VGLGTW (SEQ ID NO: 27), preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO. 28), n) YRH(V/I)DTA (SEQ ID NO 29), iii) FVTSK(L/V/I)W (SEQ ID NO- 30),
  • a transcription regulating nucleotide sequence which is obtained or is obtainable from plant genomic DNA from a gene encoding a polypeptide (preferably an orthologous protein) which a) comprises at least one, preferably at least two or three, more preferably all sequence motive(s) selected from the group consisting
  • CQMEMH (SEQ ID NO 31), preferably VCQMEMHPGW (SEQ ID NO 32), and v) RGTSViP (SEQ ID NO 33), preferably QRGTSVI PKS (SEQ ID NO 34), and b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO- 8, 10, 12, 14, 16, 18, and 36
  • said orthologous protein has furthermore the same enzymatic activity than the protein encoded by the Arabidopsis thaliana locus At5g01670
  • the activity of a transcription regulating nucleotide sequence is considered equivalent if transcription is initiated in a seed-preferential or seed-specific fashion (as defined above) Such expression profile is preferably demonstrated using reporter genes oper- ably linked to said transcription regulating nucleotide sequence.
  • reporter genes are green fluorescence protein (GFP) (Chui
  • transcription regulating activity of a function equivalent may vary from the activity of its parent sequence, especially with respect to expression level
  • the ex- pression level may be higher or lower than the expression level of the parent sequence
  • Both derivations may be advantageous depending on the nucleic acid sequence of interest to be expressed
  • Preferred are such functional equivalent sequences, which - in comparison with its parent sequence - does, not de ⁇ vate from the expression level of said parent sequence by more than 50%, preferably 25%, more preferably 10% (as to be preferably judged by either mRNA expression or protein (e g., reporter gene) expression)
  • equivalent sequences which demonstrate an increased expression in comparison to its parent sequence preferably an increase my at least 50%, more preferably by at least 100%, most preferably by at least 500%
  • functionaf equivalent of the transcription regulating nucleotide sequence can be obtained or is obtainable from plant genomic DNA from a gene expressing a mRNA described by a cDNA which is substantially similar and preferably at least 50% or 60%, preferably at least 70% or 80%
  • Such functional equivalent of the transcription regulating nucleotide sequence may be obtained from other plant species by using the seed-preferential or seed-specific Arabidopsis thaliana or Brassica napus promoter sequences described herein as probes to screen for homologous structural genes in other plants by hybridization under low, moderate or stringent hybridization conditions Regions of the seed-preferentia!
  • seed-preferential or seed-specific promoter sequences of the present invention which are conserved a- mong species could also be used as PCR primers to amplify a segment from a species other than Arabidopsis or Brassica napus, and that segment used as a hybridization probe (the latter approach permitting higher stringency screening) or in a transcription assay to determine promoter activity
  • the seed-preferential or seed-specific promoter sequences could be employed to identify structurally related sequences in a database using computer algorithms
  • orthologs may be identified or isolated from the genome of any desired organism, pref- erably from another plant, according to well known techniques based on their sequence similarity to the Arabidopsis or Brassica napus nucleic acid sequences, e g , hybridization, PCR or computer generated sequence comparisons.
  • genomic and cDNA libraries may be prepared from any cell or tissue of an organism
  • Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e g , Sambrook 1989) and amplification by PCR using oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g , lnnis 1990)
  • oligonucleotide primers preferably corresponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g , lnnis 1990)
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest
  • Methods for designing PCR primers and PCR cloning are generally known in the art
  • probes for hybridization can be genomsc DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequence of the invention
  • nucleic acid molecules of the invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having a specified amino acid sequence identity or DNA having a specified nucleotide sequence identity Methods of alignment of sequences for comparison are well known in the art and are described hereinabove
  • the isolated nucleic acid molecules of the invention include the orthologs of the Arabidop ⁇ is thaliana or Brassica napus sequences disclosed herein, i e , the corresponding nucleotide sequences in organisms other than Arabidopsis thaliana or Bras- sica napus including, but not limited to, plants other than Arabidopsis thaliana or Brassica napus, preferably dicotyledonous plants, e g , alfalfa, sunflower, soybean, cotton, peanut, tobacco or sugar beet, but also cereal plants such as corn, wheat, rye, turfgrass, sorghum, millet, sugarcane, barley and banana
  • An orthologous gene is a gene from a different species that encodes a product having the same or similar function, e g , catalyzing the same reaction as a product encoded by a gene from a reference organism.
  • an ortholog includes polypeptides having less than, e g , 50% amino acid sequence identity, but which ortholog encodes a polypeptide having the same or similar function Databases such GenBank may be employed to identify sequences related to the Arabidopsis sequences, e g , orthologs in other dicotyledonous plants. Alternatively, recombinant DNA techniques such as hybridization or PCR may be employed to identify sequences related to the Arabidopsis thaliana or Brassica napus sequences or to clone the equivalent sequences from different DNAs
  • the transcription regulating nucleotide sequences of the invention or their functional equivalents can be obtained or isolated from any plant or non-plant source, or produced synthetically by purely chemical means
  • Preferred sources include, but are not limited to the plants defined in the DEFINITION section above
  • another preferred embodiment of the invention relates to a method for identifying and/or isolating a transcription regulating nucleotide sequence (preferably with seed- preferential or seed-specific transcription regulating activity) characterized that said identification and/or isolation utilizes a nucleic acid sequence encoding a polypeptide as described by SEQ ID NO- 8, 10, 12, 14, 16, 18, or 36, or a part of said nucleic acid sequence
  • a nucleic acid sequence encoding a polypeptide as described by SEQ ID NO- 8, 10, 12, 14, 16, 18, or 36, or a part of said nucleic acid sequence
  • Preferred are nucleic acid sequences described by SEQ ID NO 7, 9, 1 1 , 13, 15, 17, or 35 or parts thereof "Part" in this context means a nucleic acid sequence of at least 15 consecutive nucleotides preferably at least 25 consecutive nucleotides, more preferably at least 50 consecutive nucleotides
  • the method for identification and/or isolation can be based on (but is not limited to) the methods described above such as polymerase chain reaction, hybridization or database screening
  • this method of the invention is based on a polymerase chain reaction, wherein said nucleic acid sequence or its part is utilized as oligonucleotide primer
  • iPCR inverse PCR
  • TAIL PCR thermo asymmetric interlaced PCR
  • Another embodiment of the invention relates to a method for providing or produc- ing a transgenic expression cassette for heterologous expression in plants comprising the steps of
  • I isolating of a transcription regulating nucleotide sequence of a plant gene utilizing at least one nucleic acid sequence encoding a polypeptide described by SEQ ID NO 8, 10, 12, 14, 16, 18, or 36, or a part of at least 15 consecutive nucleotides of said nucleic acid sequence and
  • the nucleotide sequence utilized for isolation of said transcription regulating nucleotide sequence is encoding a polypeptide (preferably from an orthologous gene i e , encoding an orthologous protein) comprising at least one sequence motive se- lected from the group consisting of the amino acid sequence i) VGLGTW (SEQ ID NO 27) preferably P(A/G)VGLGTW(R/K/Q)(A/S)G (SEQ ID NO 28), ii) YRH(V/I)DTA (SEQ ID NO 29), in) FVTSK(LMI)W (SEQ ID NO 30), iv) CQMEMH (SEQ ID NO 31 ), preferably VCQMEMHPGW (SEQ ID NO 32), and v) RGTSVIP (SEQ ID NO 33), preferably QRGTSVIPKS (SEQ ID NO 34),
  • the nucleic acid sequence employed for the isolation comprises at least 15 consecutive nucleotides, preferably at least 25 consecutive nucleotides, more preferably at least 50 consecutive nucleotides of a sequence described by SEQ ID NO 7, 9, 11 , 13, 15, 17, or 35
  • the isolation of the seed-preferential or seed-specific transcription regulating nucleotide sequence is realized by a polymerase chain reaction utilizing said nucleic acid sequence as a primer
  • the operable linkage can be realized by standard cioning method known in the art such as ligation-mediated cloning or re- combination-mediated cloning
  • the transcription regulating nucleotide sequences and promoters of the invention include a consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e g 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of any one of SEQ ID NOs 1 , 2, 3, 4, 5, and 6, or the promoter orthologs thereof, which ⁇ n- elude the minimal promoter region
  • said consecutive stretch of about 25 to 2000 including 50 to 500 or 100 to 250 and up to 1000 or 1500, contiguous nucleotides, e g , 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, has at least 50% or 60% preferably at least 70% or 80%, more preferably at least 90% and most preferably at least 95%, nucleic acid sequence identity with a corresponding consecutive stretch of about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500, contiguous nucleotides, e g , 40 to about 743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743, of any one of SEQ ID NOs 1 , 2, 3, 4, 5, and 6, or the promoter orthologs thereof, which include the minimal promoter region
  • the above-defined stretch of contiguous nucleotides preferably comprises one or more
  • the transcription regulating nucleotide sequences of the invention or their functional equivalents are capable of driving seed-preferential or seed-specific expression of a coding sequence in a target cell, particularly in a plant cell.
  • the promoter sequences and methods disclosed herein are useful in regulating seed-preferential or seed- specific expression, respectively of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of that plant.
  • These promoters can be used with combinations of enhancer, upstream elements, and/or activating sequences from the 5' flanking regions of plant expressible structural genes Similarly the upstream element can be used in combination with various plant promoter sequences.
  • the transcription regulating nucleotide sequences and promoters of the invention are useful to modify the phenotype of a plant
  • Various changes in the phenotype of a transgenic plant are desirable, i e , modifying the fatty acid composition in a plant, alter- ing the arntno acid content of a plant, altering a plant's pathogen defense mechanism and the like
  • results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants
  • the results can be achieved by providing for a reduction of expression of one or more en- doge ⁇ ous products, particularly enzymes or cofactors in the plant
  • the transcription regulating nucleotide sequences and promoters of the invention may be employed to express a nucleic acid segment that is operably linked to said promoter such as, for example, an open reading frame, or a portion thereof, an anti-sense sequence, a sequence encoding for a double-stranded RNA sequence, or a transgene in plants
  • An operable linkage may - for example - comprise an sequential arrangement of the transcription regulating nucleotide sequence of the invention (for example a sequence as described by SEQ ID NO 1 , 2, 3, 4, 5, or 6) with a nucleic acid sequence to be expressed, and - optionally - additional regulatory elements such as for example polyade ⁇ ylation or transcription termination elements, enhancers, introns etc, in a way that the transcription regulating nucleotide sequence can fulfill its function in the proc- ess of expression the nucleic acid sequence of interest under the appropriate conditions
  • the term "appropriate conditions" mean preferably the presence of the expression cassette in a plant cell
  • Preferred are arrangements in which the nucleic acid sequence of interest to be expressed is placed down-stream ( ⁇ e , in 3'-d ⁇ rection) of the transcription regulating nucleotide sequence of the invention in a way, that both se- quences are covalently linked
  • additional sequences may be inserted in- between the two sequences Such sequences may
  • an operable linkage in relation to any expression cassette or of the invention may be realized by various methods known in the art, comprising both in vitro and in vivo procedure
  • an expression cassette of the invention or an vector comprising such expression cassette may by realized using standard recombination and cloning techniques well known in the art (see e g , Maniatis 1989, Silhavy 1984, Ausubel 1987)
  • An expression cassette may also be assembled by inserting a transcription regulating nucleotide sequence of the invention (for example a sequence as described by SEQ ID NO 1 , 2, 3, 4, 5, or 6 ) into the plant genome Such insertion will result in an operable linkage to a nucleic acid sequence of interest which as such already existed in the genome
  • the nucleic acid of interest is expressed in a seed-preferential or seed-specific way due to the transcription regulating properties of the transcription regulating nucleotide sequence
  • the insertion may be directed or by chance
  • the insertion is directed and realized by for example homologous recombination
  • a natural promoter may be exchanged against the transcription regulating nucleotide sequence of the invention, thereby modifying the expression profile of an endogenous gene
  • the transcription regulating nucleotide sequence may also be inserted in a way, that antisense mRNA of an endogenous gene is expressed, thereby inducing gene silencing
  • a nucleic acid sequence of interest to be expressed may by inserted into a plant genome comprising the transcription regulating nucleotide sequence in its natural genomic environment ( ⁇ e linked to its natural gene) in a way that the inserted se- quence becomes operably linked to the transcription regulating nucleotide sequence, thereby forming an expression cassette of the invention
  • the expression cassette may be employed for numerous expression purposes such as for example expression of a protein, or expression of a antisense RNA, sense or dou- ble-stranded RNA
  • expression of the nucleic acid sequence confers to the plant an agronomically valuable trait.
  • the open reading frame to be linked to the transcription regulating nucleotide sequence of the invention may be obtained from an insect resistance gene, a disease resistance gene such as, for example, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a scree ⁇ able marker gene, a negative selectable marker, a positive selectable marker, a gene affecting plant agronomic characteristics, i e , yield, stand- ability, and the like, or an environment or stress resistance gene, i e , one or more genes that confer herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, oomycete, or nematode), stress tolerance or resistance (as exemplified by resistance or tolerance to drought,
  • Seed-preferential or seed-specific transcription regulating nucleotide sequences are useful for expressing a wide variety of genes including those which alter metabolic pathways, confer disease resistance, for protein production, e g , antibody production, or to improve nutrient uptake and the like
  • Seed-preferential or seed-specific transcription regulating nucleotide sequences may be modified so as to be regulatable, e g , inducible
  • the genes and transcription regulating nu- cleotide sequences (e g , promoters) described herei ⁇ above can be used to identify orthologous genes and their transcription regulating nucleotide sequences (e g , promoters) which are also likely expressed in a particular tissue and/or development man- ner.
  • orthologous transcription regulating nucleotide sequences are useful to express linked open reading frames
  • novel cis elements can be identified that are useful to generate synthetic transcription regulating nucleotide sequences (e g , promoters)
  • nucleotide sequences specified above may be optionally operably linked to other suitable regulatory sequences, e g , a transcription terminator sequence, operator, repressor-binding site, transcription factor binding site and/or an enhancer
  • the present invention further provides a recombinant vector containing the expression cassette of the invention, and host cells comprising the expression cassette or vector, e g , comprising a plasmid
  • the expression cassette or vector may augment the ge- nome of a transformed plant or may be maintained extra chromosomally.
  • the expression cassette or vector of the invention may be present in the nucleus, chloropla ⁇ t, mitochondria and/or plastid of the cells of the plant.
  • the expression cassette or vector of the invention is comprised in the chromosomal DNA of the plant nucleus
  • the present invention also provides a transgenic plant prepared by this method, a seed from such a plant and progeny plants from such a plant including hybrids and inbreds
  • the expression cassette may be operatively linked to a structural gene, the open reading frame thereof, or a portion thereof
  • the expression cassette may further comprise a Ti plasmid and be contained in an Agrobacte ⁇ um tumefaciens cell, it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell, or it may be contained in a plant cell or protoplast.
  • the expression cassette or vector can be contained in a transformed plant or cells thereof, and the plant may be a dicot or a monocot.
  • the plant may be a dicotyledonous plant.
  • Preferred transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato
  • the invention also provides a method of plant breeding, e g , to prepare a crossed fertile transgenic plant
  • the method comprises crossing a fertile transgenic plant comprising a particular expression cassette of the invention with itself or with a second plant, e g , one lacking the particular expression cassette, to prepare the seed of a crossed fertile transgenic plant comprising the particular expression cassette.
  • the seed is then planted to obtain a crossed fertile transgenic plant
  • the plant may be a monocot or a dicot In a particular embodiment, the plant is a dicotyledonous plant
  • the crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent
  • the second plant may be an inbred plant
  • the crossed fertile transgenic may be a hybrid Also included within the present invention are seeds of any of these crossed fertile transgenic plants
  • the transcription regulating nucleotide sequences of the invention further comprise sequences which are complementary to one (hereinafter "test" sequence) which hybridizes under stringent conditions with a nucleic acid molecule as described by SEQ ID NO 1 , 2, 3, 4, 5, or 6 as well as RNA which is transcribed from the nucleic acid molecule
  • test sequence sequences which are complementary to one (hereinafter "test" sequence) which hybridizes under stringent conditions with a nucleic acid molecule as described by SEQ ID NO 1 , 2, 3, 4, 5, or 6 as well as RNA which is transcribed from the nucleic acid molecule
  • test or nucleic acid molecule of invention is preferably supported, e g , on a membrane or DNA chip.
  • either a denatured test or nucleic acid molecule of the invention JS preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e g., between 55 and 70 0 C, in double strength citrate buffered saline (SC) containing 0.1 % SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration.
  • SC citrate buffered saline
  • SC citrate buffered saline
  • a buffer having a reduced SC concentration are typically single strength SC containing 0 1% SDS, haff strength SC containing 0 1 % SDS and one- tenth strength SC containing 0 1% SDS.
  • More preferably hybridization is carried out under high stringency conditions (as defined above).
  • any DNA composition may be used for delivery to recipient plant cells, e g , dicotyledonous cells, to ultimately produce fertile transgenic plants in accordance with the present invention
  • DNA segments or fragments in the form of vectors and piasmids, or linear DNA segments or fragments, in some instances containing only the DNA element to be expressed in the plant, and the like may be employed.
  • the construction of vectors, which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see, e g , Sambrook 1989, Geivin 1990)
  • Vectors, piasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduce into the cells
  • DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired
  • the DNA segment, fragment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant
  • this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes
  • replication- competent viral vectors include, for example, wheat dwarf virus (WDV) "shuttle" vectors, such as pW1-11 and PW1-GUS (Ugaki 1991 ) These vectors are capable of autonomous replication in maize cells as well as E. coli, and as such may provide increased sensitivity for detecting DNA delivered to transgenic cells
  • WDV wheat dwarf virus
  • PW1-GUS Ugaki 1991
  • a replicating vector may also be useful for delivery of genes flanked by DNA sequences from transposable elements such as Ac, Ds, or Mu.
  • transposable elements would be useful for introducing DNA segments or fragments lacking elements necessary for selection and maintenance of the plasmid vector in bacteria, e g , antibiotic resistance genes and origins of DNA replication It is also proposed that use of a transposable element such as Ac 1 Ds, or Mu would actively promote integration of the desired DNA and hence increase the frequency of stably transformed cells The use of a transposable element such as Ac, Ds, or Mu may actively promote integration of the DNA of interest and hence increase the frequency of stably transformed cells Transposable elements may be useful to allow separation of genes of interest from elements necessary for selection and maintenance of a plasmid vector in bacteria or selection of a transformant By use of a transposable element, desirable and undesirable DNA se- quences may be transposed apart from each other in the genome, such that through genetic segregation in progeny, one may identify plants with either the desirable undesirable DNA sequences.
  • the nucleotide sequence of interest linked to one or more of the transcription regulat- ing nucleotide sequences of the invention can, for example, code for a ⁇ bosomal RNA, an antisense RNA or any other type of RNA that is not translated into protein
  • said nucleotide sequence of interest is translated into a protein product
  • the transcription regulating nucleotide sequence and/or nucleotide sequence of interest linked thereto may be of homologous or het- eroiogous origin with respect to the plant to be transformed.
  • a recombinant DNA molecule useful for introduction into plant cells includes that which has been derived or isolated from any source, that may be subsequently characterized as to structure, size and/or function, chemically altered, and later introduced into plants
  • An example of a nucleotide sequence or segment of interest "derived" from a source would be a nu- cleotide sequence or segment that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form.
  • nucleotide sequence or segment of interest "isolated" from a source, would be nucleotide sequence or segment that is excised or removed from said source by chemical means, e g , by the use of restriction endonucleases, so that it can be further manipulated, e g., amplified, for use in the invention, by the methodology of genetic engineering
  • nucleotide sequence or segment is commonly referred to as "recombinant"
  • a useful nucleotide sequence, segment or fragment of interest includes completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA.
  • the introduced DNA is not originally resident in the plant genotype which ss the recipient of the DNA 1 but it is within the scope of the invention to isolate a gene from a given plant genotype, and to subsequently introduce multiple copies of the gene into the same genotype, e g , to enhance production of a given gene product such as a storage protein or a protein that confers tolerance or resistance to water deficit
  • the introduced recombinant DNA molecule includes but is not limited to, DNA from plant genes, and non-plant genes such as those from bacteria, yeasts, animals or v ⁇ - ruses
  • the introduced DNA can include modified genes, portions of genes, or chimeric genes, including genes from the same or different genotype
  • the term "chimeric gene” or “chimeric DNA” is defined as a gene or DNA sequence or segment comprising at least two DNA sequences or segments from species which do not combine DNA under natural conditions, or which DNA sequences or segments are positioned or linked in a manner which does not normaily occur in the native genome of untransformed plant
  • the introduced recombinant DNA molecule used for transformation herein may be circular or linear, double-stranded or single-stranded.
  • the DNA is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences, which promote the expression of the recombinant DNA present in the resultant plant.
  • the introduced recombinant DNA molecule will be relatively small, i e , less than about 30 kb to minimize any susceptibility to physical, chemical, or enzymatic degradation which is known to increase as the size of the nucleotide molecule increases.
  • the number of proteins, RNA transcripts or mixtures thereof, which is introduced into the plant genome is preferably prese- lected and defined, e g., from one to about 5-10 such products of the introduced DNA may be formed
  • overexpression and underexpression Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression
  • antisense downregulation and “sense downregulation” (sense downregulation is also referred to as “cosuppression")
  • gene silencing Both of these methods lead to an inhibition of expression of the target gene
  • heterologous DNA sequences in a plant host is dependent upon the presence of an oper- ably linked promoter that is functional within the plant host Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed
  • a promoter to potentially improve the utility of the elements for the expression of transgenes in plants.
  • the mutagenesis of these elements can be carried out at random and the mutagenized promoter sequences screened for activity in a trial-by-error procedure.
  • particular sequences which provide the promoter with desirable expression characteristics, or the promoter with expression enhancement activity could be identified and these or similar sequences introduced into the sequences via mutation It is further contemplated that one could mutagenize these sequences in order to enhance their expression of transgenes in a particular species
  • the means for mutage ⁇ izing a DNA segment encoding a promoter sequence of the current invention are well known to those of skill in the art As indicated, modifications to promoter or other regulatory element may be made by random, or site-specific mutagenesis procedures The promoter and other regulatory element may be modified by altering their structure through the addition or deletion of one or more nucleotides from the sequence which encodes the corresponding unmodified sequences.
  • Mutagenesis may be performed in accordance with any of the techniques known in the art, such as, and not limited to, synthesizing an oligonucleotide having one or more mutations within the sequence of a particular regulatory region
  • site- specific mutagenesis is a technique useful in the preparation of promoter mutants, through specific mutagenesis of the underlying DNA
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations by introducing one or more nucleotide sequence changes into the DNA
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA se- quence of the desired mutation as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed
  • a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25
  • the technique of site-specsfic mutagenesis is well known in the art, as exemplified by various publications
  • the technique typically employs a phage vector, which exists in both a single stranded and double stranded form
  • Typical vectors useful in sste-directed mutagenesis include vectors such as the M13 phage
  • These phages are readily commercially available and their use is generally well known to those skilled in the art
  • Double stranded plasmids also are routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a plasmid to a phage
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the promoter
  • An oligonucleotide primer bearing the desired mutated sequence is prepared generally synthetically
  • This primer ts then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation
  • This heteroduplex vector is then used to transform or transfect appropriate cells, such as E coli cells, and cells are selected which include recombinant vectors bearing the mutated sequence arrangement
  • Vector DNA can then be isolated from these cells and used for plant transformation
  • a genetic selection scheme was devised by Kunkel et al
  • oligonucleotide directed mutagenesis procedure refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification
  • oligonucleotide directed mutagenesis procedure also is intended to refer to a process that involves the template-dependent extension of a primer molecule
  • template-dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson and Rarnstad 1987)
  • vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U S Pat No
  • a clone comprising a promoter has been isolated in accordance with the instant invention, one may wish to delimit the essential promoter regions within the clone
  • One efficient, targeted means for preparing mutagenizing promoters relies upon the identification of putative regulatory elements within the promoter sequence This can be initiated by comparison with promoter sequences known to be expressed in similar tsssue- specific or developmental ⁇ unique manner Sequences, which are shared among promoters with similar expression patterns, are likely candidates for the binding of transcription factors and are thus likely elements that confer expression patterns Confirmation of these putative regulatory elements can be achieved by deletion analysis of each putative regulatory region followed by functional analysis of each deletion construct by assay of a reporter gene which is functionally attached to each construct As such, once a starting promoter sequence is provided, any of a number of different deletion mutants of the starting promoter could be readily prepared
  • Functionally equivalent fragments of a transcription regulating nucleotide sequence of the invention can also be obtained by removing or deleting non-essential sequences without deleting the essential one Narrowing the transcription regulating nucleotide sequence to its essential, transcription mediating elements can be realized in vitro by t ⁇ al-and-arrow deletion mutations or in silico using promoter element search routines Regions essential for promoter activity often demonstrate clusters of certain known promoter elements Such analysis can be performed using available computer algorithms such as PLACE ("Plant Cis-acting Regulatory DNA Elements", Higo 1999), the BIOBASE database Transfac" (Biologtsche banken GmbH, Braunschweig, Win- gender 2001) or the database PiantCARE (Lescot 2002)
  • functional equivalent fragments of one of the transcription regulating nu- cleotide sequences of the invention comprises at least 100 base pairs, preferably, at least 200 base pairs, more preferably at least 500 base pairs of a transcription regulating nucleotide sequence as described by SEQ ID NO 1 1 , 2, 3, 4, 5, or 6 More preferably this fragment is starting from the 3'-end of the indicated sequences.
  • transcription regulating nucleotide sequences which are obtained by deleting the region encoding the ⁇ '-untranslated region of the mRNA, thus only providing the (untranscnbed) promoter region
  • the 5'- untranslated region can be easily determined by methods known in the art (such as 5'- RACE analysis) Accordingly, some of the transcription regulating nucleotide se- quences of the invention are equivalent fragments of other sequences (see Table 2 below)
  • deletion mutants, deletion mutants of the promoter of the invention also could be randomly prepared and then assayed With this strategy, a series of constructs are prepared, each containing a different portion of the clone (a subclone), and these constructs are then screened for activity
  • a suitable means for screening for activity is to attach a deleted promoter or intron construct, which contains a deleted seg- ment to a selectable or screenable marker, and to isolate only those cells expressing the marker gene.
  • An expression cassette of the invention may comprise further regulatory elements
  • Regulatory elements may for example modify transcription and/or translation in pro- karyotic or eukaryotic organism
  • the expression cassette of the invention comprised downstream (in 3'-d ⁇ rect ⁇ on) of the nucleic acid sequence to be expressed a transcription termination sequence and - optionally additional regulatory elements - each operably liked to the nucleic acid sequence to be expressed (or the transcription regulating nucleotide sequence)
  • Additional regulatory elements may comprise additional promoter, minimal promoters, or promoter elements, which may modify the expression regulating properties. For ex- ample the expression may be made depending on certain stress factors such water stress, abscisin (Lam 1991) or heat stress ⁇ Schoffl 1989) Furthermore additional promoters or promoter elements may be employed, which may realize expression in other organisms (such as E coll or Agrobacte ⁇ um) Such regulatory elements can be found in the promoter sequences or bacteria such as army and SPO2 or in the promoter sequences of yeast or fungal promoters (such as ADC1 , MFa, AC T P-60, CYC1 , GAPDH, TEF, rp28 and ADH)
  • promoters combining elements from more than one promoter may be useful
  • US 5,491 ,288 discloses combining a Cauliflower Mosaic Virus promoter with a histone promoter
  • the elements from the promoters disclosed herein may be combined with elements from other promoters Promoters, which are useful for plant transgene expression incSude those that are inducible, viral, synthetic, constitutive (Odell 1985), temporally regulated, spatially regu- lated, tissue-specific, and spatial-temporally regulated
  • tissue-specific promoters may be used
  • inducible promoters are the regulatory elements of choice
  • continuous expres- sio ⁇ is desired throughout the cells of a plant constitutive promoters are utilized
  • Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying (evels of expression of heterologous nucleotide sequences in a transgenic plant
  • the 3' nontranslated regulatory DNA sequence preferably includes from about 50 to about 1 000, more preferably about 100 to about 1 ,00O 1 nucleotide base pairs and contains plant transcriptional and transla- tionai termination sequences
  • Approp ⁇ ate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tmf terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacte ⁇ um tumefaciens and the 3' end of the protease inhibitor I or Il genes from potato or tomato, although other 3' elements known to those of skill in the art can also be employed Alternatively, one also could use a gamma coixm, oleosin 3
  • Preferred 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3 1 end of the protease inhibitor I or Il genes from potato or tomato
  • leader sequences are contemplated to include those, which include sequences, predicted to direct opti- mum expression of the attached gene, i e , to include a preferred consensus leader sequence, which may increase or maintain mRNA stability and prevent inappropriate initiation of translation
  • sequences predicted to direct opti- mum expression of the attached gene, i e
  • consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation
  • Preferred regulatory elements also include the 5'-untrans!ated region, intro ⁇ s and the 3'-untranslated region of genes
  • sequences that have been found to enhance gene expression in transgenic plants include intron sequences (e g , from Adh1 , bronzel , actini actin 2 (WO 00/760067), or the sucrose synthase intron, see The Maize Handbook, Chapter 1 16, Freeling and Walbot, Eds , Springer, New York (1994)) and virai leader sequences (e g , from TMV 1 MCMV and AMV, Gaflie 1987)
  • TMV Tobacco Mosaic Virus
  • MCMV Maize ChSorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • Other leaders known in the art include but are not limited to Pi
  • Adh intron 1 (Callis 1987), sucrose syn- thase intron (Vasil 1989) or TMV omega element (Gallie 1989)
  • Adh intron 1 (Callis 1987)
  • sucrose syn- thase intron (Vasil 1989)
  • TMV omega element (Gallie 1989)
  • 5'-untranslated region, introns and the 3'- untranslated region selected from a) the gene described by the GenBank Arabidopsis thaliana genome locii At5gO1670 and b) orthogogous genes of the gene described by the GenBank Arabidopsis thaliana genome locii At5g01670
  • 5'-untranslated sequences comprised in the sequences as described by SEQ ID NO 1 , 3, 5 or 6 (see sequence listing for specification of the localization)
  • Additional preferred regulatory elements are enhancer sequences or polyadenyiation sequences
  • Preferred polyadenyiation sequences are those from plant genes or Agro- bacterium T-DNA genes (such as for example the terminator sequences of the OCS (octopine synthase) or NOS (nopalme synthase) genes)
  • enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis el ai , 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis 1987), the maize shrunken I gene (Vasil 1989), TMV Omega element (Gal- he 1989) and promoters from non-plant eukaryotes (e g yeast, Ma 1988)
  • Vectors for use in accordance with the present invention may be constructed to include the ocs enhancer element This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of ultilane (Ellis 1987), and ts present in at least 10 other promoters (Bouchez 1989)
  • an enhancer element such as the ocs elements and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transforma- tion
  • An expression cassette of the invention may comprise additional functional elements, which are to be understood in the broad sense as all elements which influence construction, propagation, or function of an expression cas- sette or a vector or a transgenic organism comprising them
  • Such functional elements may include origin of replications (to allow replication in bacteria, for the ORf of pBR322 or the P15A on Sambrook 1989), or elements required for Agrobacte ⁇ um T- DNA transfer (such as for example the left and/or rights border of the T-DNA)
  • the most desirable DNA segments for introduction into, for example, a dicot genome may be homologous genes or gene families which encode a desired trait (e g , increased yield per acre) and which are introduced under the control of novel promoters or enhancers, etc , or perhaps even homologous or tissue specific (e g , root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters or control elements
  • a desired trait e g , increased yield per acre
  • tissue specific e g , root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific
  • vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene The resultant transit or signal peptide will transport the protein to a particular intracellular or extracellular destination, respectively, and will then be post- translationalfy removed Transit or signal peptides act by facilitating the transport of protems through intracellular membranes, e g , vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane
  • a particular example of such a use concerns the direction of a herbicide resistance gene, such as the EPSPS gene, to a particular organelle such as the chloroplast rather than to the cytoplasm
  • a herbicide resistance gene such as the EPSPS gene
  • organelle such as the chloroplast rather than to the cytoplasm
  • rbcs transit peptide which confers plastid-specific targeting of proteins
  • these sequences may increase the accumulation of gene product protecting them from proteolytic degradation
  • these sequences also allow for additional mRNA sequences from highfy expressed genes to be attached to the coding sequence of the genes Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product.
  • a chimeric zinc finger protein may include amino acid sequences, which bind to a specific DNA sequence (the zinc finger) and amino acid sequences that activate (
  • nucleotide segment of interest is reflective of the commercial markets and interests of those involved in the development of the crop Crops and markets of interest changes, and as developing nations open up world markets, new crops and technologies will also emerge.
  • General categories of nucleotides of interest include, for example, genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins
  • More specific categories of transgenes include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products
  • Genes of interest include, generally, those involved in starch, oil, carbohydrate, or nutrient metabolism, as well as those affecting kernel size, sucrose loading, zsnc finger proteins, see, e g , US 5,789,538, WO 99/48909,
  • a line which can be crossed with lines carrying different inactive replicons or inactive transgene for activation
  • Other sequences which may be linked to the gene of interest, which encodes a polypeptide are those which can target to a specific organelle, e g , to the mitochondria, nucleus, or plastid within the plant cell Targeting can be achieved by providing the polypeptide with an appropriate targeting peptide sequence, such as a secretory signal peptide (for secretion or cell wali or membrane targeting, a plastid transit peptide, a chloroplast transit peptide, e g , the chlorophyll a/b binding protein, a mitochondrial target peptide, a vacuole targeting peptide,
  • a secretory signal peptide for secretion or cell wali or membrane targeting, a plastid transit peptide, a chloroplast transit peptide, e g , the chlorophyll a/b binding protein, a mitochondria
  • the piastids are major sites of biosynthesis in plants In addition to photosynthesis in the chloroplast, plastids are also sites of lipid biosynthesis, nitrate reduction to ammonium and starch storage And while plastids contain their own circular, genome, most of the proteins localized to the plastids are encoded by the nuclear genome and are imported into the organelle from the cytoplasm
  • Transgenes used with the present invention will often be genes that direct the expres- sion of a particular protein or polypeptide product, but they may also be non- expres ⁇ ible DNA segments, e g , transposons such as Ds that do no direct their own transposition
  • an "expressible gene” is any gene that is capable of being transcribed into RNA (e g mRNA, antisense RNA, etc ⁇ or translated into a protein, expressed as a trait of interest, or the like, etc , and is not limited to selectable, screenable or non-selectable marker genes
  • the invention also contemplates that, where both an expressible gene that is not necessarily a marker gene is employed in combination with a marker gene, one may employ the separate genes on either the same or different DNA segments for transformation In the latter case, the different vectors are delivered concurrently to recipient cells to maximize cotransformation
  • the choice of the particular DNA segments to be delivered to the recipient cells will often depend on the purpose of the transformation
  • One of the major purposes of transformation of crop plants is to add some commercially desirable, agronomically important traits to the plant
  • Such traits include but are not limited to, herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial, fungal, nematode), stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing excessive moisture, salt stress, oxidative stress, increased yields food content and makeup, physical appearance, male sterility, drydown; standability prolificacy, starch properties oil quantity and quality, and the like
  • the present invention contemplates the transformation of a recipient cell with more than one advantageous transgene
  • Two or more transgenes can be supplied in a single transformation event using either distinct transgene- encoding vectors, or using a single vector incorporating two or more gene coding se- que ⁇ ces
  • plasmids bearing the bar and aroA expression units in either convergent, divergent, or colinear orientation are considered to be particularly useful
  • Further preferred combinations are those of an insect resistance gene, such as a Bt gene, along with a protease inhibitor gene such as pinll, or the use of bar in combina- tion with either of the above genes
  • any two or more transgenes of any description such as those conferring herbicide, insect, disease (viral, bacterial, fungal, nematode) or drought resistance, male sterility, drydown, standabihty, prolificacy, starch properties, oil quantity and quality, or those increasing yield or nutritional quality may be employed as desired
  • the genes encoding phosphinoth ⁇ cm acetyltransferase (bar and pat), glyphosate tolerant EPSP synthase genes, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), herbicide resistant (e g , sulfonylurea and imidazolinone) acetolactate synthase, and bxn genes (encoding a nit ⁇ lase enzyme that degrades bromoxynil) are good examples of herbicide resistant genes for use in transformation
  • the bar and pat genes code for an enzyme, phosphsnothricin acetyltransferase (PAT) which inactivates the herbicide phosphinoth ⁇ cin and prevents this compound from inhibiting glutamine synthetase enzymes
  • PAT phosphsnothric
  • Bacillus thuringiensis crystal toxin genes or Bt genes may provide resistance to lepidopteran or coleopteran pests such as European Corn Borer (ECB) and corn rootworm (CRW)
  • Bt toxin genes for use in such embodiments include the CrylA(b) and CrylA(c) genes Endotoxin genes from other species of B thuringiensis, which affect insect growth or development may also be employed in this regard
  • Protease inhibitors may also provide insect resistance (Johnson 1989), and will thus have utility in plant transformation The use of a protease inhibitor I!
  • pinll from tomato or potato is envisioned to be particularly useful Even more advantageous is the use of a pinll gene in combination with a Bt toxin gene, the combined effect of which has been discovered by the present inventors to produce synergistic insecticidal activity
  • Other genes which encode inhibitors of the insects' digestive system, or those that encode enzymes or co-factors that facilitate the production of inhibitors, may also be useful Cystatin and amylase inhibitors, such as those from wheat and barley, may exemplify this group
  • Lectins (originally termed phytohemagglutimns) are multivalent carbohydrate-binding proteins, which have the ability to agglutinate red btood cells from a range of species Lectins have been identified recently as insecticidal agents with activity against weevils, ECB and rootworm (Murdock 1990, Czapla & Lang, 1990) Lectin genes contemplated to be useful include, for example, barley and wheat germ agglutinin (VVGA) and ⁇ ce lectins (Gatehouse 1984), with WGA being preferred
  • Genes controlling the production of large or small polypeptides active against insects when introduced into the insect pests form another aspect of the invention
  • insect pests such as, e g lytic peptides, peptide hormones and toxins and venoms
  • the expression of juvenile hormone esterase, directed towards specific insect pests may also result in insecticida! activity, or perhaps cause cessation of metamorphosis (Hammock 1990)
  • Transgenic plants expressing genes, which encode enzymes that affect the integrity of the insect cuticle form yet another aspect of the invention include those encoding, e g , chitinase, proteases, lipases and also genes for the production of nik- komycin, a compound that inhibits chitin synthesis, the introduction of any of which is contemplated to produce insect resistant maize plants Genes that code for activities that affect insect molting, such those affecting the production of ecdysteroid UDP- glucosyl transferase, also fall within the scope of the useful transgenes of the present invention
  • Genes that code for enzymes that facilitate the production of compounds that reduce the nutritional quality of the host plant to insect pests are also encompassed by the present invention It may be possible, for instance, to confer insecticidal activity on a plant by altering its sterol composition Sterols are obtained by insects from their diet and are used for hormone synthesis and membrane stability Therefore alterations in plant sterol composition by expression of novel genes, e g , those that directly promote the production of undesirable sterols or those that convert desirable sterols into unde- sirable forms, could have a negative effect on insect growth and/or development and hence endow the plant with insecticidal activity Lipoxygenases are naturally occurring plant enzymes that have been shown to exhibit anti-nut ⁇ tional effects on insects and to reduce the nutritional quality of their diet Therefore, further embodiments of the invention concern transgenic plants with enhanced lipoxygenase activity whsch may be resis- tant to msect feeding
  • the present invention also provides methods and compositions by which to achieve qualitative or quantitative changes in plant secondary metabolites
  • One example concerns transforming plants to produce DIMBOA which, it is contemplated, will confer resistance to European corn borer rootworm and several other maize insect pests
  • Candidate genes that are particularly considered for use in this regard include those genes at the bx locus known to be involved in the synthetic DIMBOA pathway (Dunn 1981)
  • the introduction of genes that can regulate the production of maysin, and genes involved in the production of dhur ⁇ n in sorghum is also contemplated to be of use in facilitating resistance to earworm and rootworm, respectively
  • T ⁇ psacum dactyloides is a species of grass that is resistant to certain insects, including corn rootworm It is anticipated that genes encoding proteins that are toxic to insects or are involved in the biosynthesis of compounds toxic to insects will be isolated from Tripsacum and that these novel genes will be useful in conferring resistance to insects It is known that the basis of insect resistance in T ⁇ psa
  • genes encoding proteins characterized as having potential insecticidai activity may also be used as transgenes sn accordance herewith
  • Such genes include, for example, the cowpea trypsin inhibitor (CpTI, Hilder 1987) which may be used as a root- worm deterrent, genes encoding avermectin (Campbell 1989, lkeda 1987) which may prove particularly useful as a corn rootworm deterrent, ⁇ bosome inactivating protein genes, and even genes that regulate plant structures
  • Transgenic maize including anti- insect antibody genes and genes that code for enzymes that can covert a non-toxic insecticide (pro-insecticide) applied to the outside of the plant into an insecticide inside the plant are also contemplated
  • drought resistance and “drought tolerance” are used to refer to a plants increased resistance or tolerance to stress induced by a reduction in water availability, as compared to normal circumstances, and the ability of the plant to function and survive in lower-water environments, and perform in a relatively superior manner
  • a gene encoding the biosynthesis of osmotically active solutes can impart protection against drought
  • osmotically active solutes which are not sugars, include, but are not limited to, proline and glyci ⁇ e- betaine (Wyn-Jones and Storey, 1981).
  • Late Embryogenic Proteins have been assigned based on structural similarities (see Dure 1989) All three classes of these proteins have been demonstrated in maturing (i e , desfccating) seeds. Within these 3 types of proteins, the Type-ll (dehyd ⁇ n-type) have generally been implicated in drought and/or desiccation tolerance in vegetative plant parts (e g. Mundy and Chua, 1988, Piatkowski 1990, Ya- maguchi-Shinozaki 1992). Recently, expression of a Type-Ill LEA (HVA-1 ) in tobacco was found to influence plant height, maturity and drought tolerance (Fitzpatrick, 1993).
  • HVA-1 Type-Ill LEA
  • Improved protection of the plant to abiotic stress factors such as drought, heat or chill can also be achieved - for example - by overexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO 00/00512), Myoxocephaius octodecemspinosus, the Arabidopsis thaliana transcription activator CBF1 , glutamate dehydrogenases (WO 97/12983, WO 98/1 1240), calcium-dependent protein kinase genes (WO 98/26045), calcineu ⁇ ns (WO 99/05902), casein kinase from yeast (WO 02/052012), farnesyltrans- ferases (WO 99/06580, Pei ZM et al (1998) Science 282 287-290), ferritin (Deak M et al (1999) Nature Biotechnology 17 192-196), oxalate oxidase (WO 99/04013, Dunwell JM (1998) Biotechn Genet Eng Rev 15
  • Resistance to viruses may be produced through expression of novel genes.
  • expression of a virai coat protein m a transgenic plant can impart resistance to infection of the plant by that virus and perhaps other closely related viruses (Cuozzo 1988, Hemenway 1988, Abel 1986)
  • expression of antisense genes targeted at essential virai functions may impart re- sistance to said virus
  • an antisense gene targeted at the gene responsible for replication of viral nucleic acid may inhibit said rep ⁇ catfon and lead to resistance to the virus
  • interference with other viral functions through the use of antisense genes may aiso increase resistance to viruses.
  • Peptide antibiotics are polypeptide sequences, which are inhibitory to growth of bacteria and other microorganisms.
  • PR proteins pathogenesis related proteins
  • toxin resistance proteins affecting host-pathogen interactions
  • proteins affecting host-pathogen interactions such as morphological characteristics
  • Peptide antibiotics are polypeptide sequences, which are inhibitory to growth of bacteria and other microorganisms.
  • the classes of peptides referred to as cecropins and magainms inhibit growth of many species of bacteria and fungi
  • expression of PR proteins in plants may be useful in conferring resistance to bacteria! disease.
  • genes are induced following pathogen attack on a host plant and have been divided into at least five classes of proteins (BoI 1990) Included amongst the PR proteins are beta-1 ,3-glucanases, chitinases, and osmottn and other proteins that are believed to function in plant resistance to disease organisms Other genes have been identified that have antifungal properties, e g , UDA (stinging nettle lectin) and hevein (Broakgert 1989, Barkai-Golan 1978).
  • Plant parasitic nematodes are a cause of disease in many plants It is proposed that it would be possible to make the plant resistant to these organisms through the expression of novel genes ft ts anticipated that control of nematode infestations would be accomplished by altering the ability of the nematode to recognize or attach to a host plant and/or enabling the plant to produce nematicidal compounds, including but not limited to proteins
  • a resistance to fungi, insects, nematodes and diseases can be achieved by by targeted accumulation of certain metabolites or proteins
  • proteins include but are not limited to gtucosinolates (defense against herbivores), chitinases or gluca- nases and other enzymes which destroy the cell wall of parasites, ⁇ bosome- mactivating proteins (RlPs) and other proteins of the plant resistance and stress reaction as are induced when plants are wounded or attacked by microbes, or chemically, by, for example, salicylic acid, jasmonic acid or ethylene, or lysozymes from nonplant sources such as, for example, T4-lysozyme or lysozyme from a variety of mammals, msecti ⁇ dal proteins such as Bacillus thu ⁇ ngiensis endotoxin, a-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), lectins such as wheatgerm
  • mycotoxins including aflatoxm and fumonisin
  • fungi associated with plants are a significant factor in rendering the grain not useful
  • These fungal organisms do not cause disease symptoms and/or interfere with the growth of the plant, but they produce chemicals (mycotoxins) that are toxic to animals Inhibition of the growth of these fungi would reduce the synthesis of these toxic substances and, therefore, reduce grain losses due to mycotoxin contamination
  • Novel genes may be introduced into plants that would inhibit synthesis of the mycotoxin without interfering with fungal growth
  • Expression of a novel gene, which encodes an enzyme capable of rendering the mycotoxin nontoxic would be useful in order to achieve reduced mycotoxin con- lamination of grain The result of any of the above mechanisms would be a reduced presence of mycotoxins on grain
  • Genes may be introduced into plants particularly commercially important cereals such as maize, wheat or rice, to improve the grain for which the cereal is primarily grown
  • cereals particularly commercially important cereals
  • wheat or rice to improve the grain for which the cereal is primarily grown
  • a wide range of novel transgenic plants produced in this manner may be envisioned depending on the particular end use of the gram
  • the largest use of maize grain is for feed or food
  • Introduction of genes that alter the composition of the grain may greatly enhance the feed or food value
  • the primary components of maize grain are starch, protein, and oil
  • Each of these primary components of maize grain may be improved by altering its level or composition
  • the protein of many cereal grains is suboptimal for feed and food purposes especially when fed to pigs, poultry, and humans
  • the protein is deficient in several amino acids that are essential in the diet of these species, requiring the addition of supplements to the grain
  • Limiting essential amino acids may include lysine, methionine, tryptophan, threonine, valine, arginme, and histidine
  • Some amino acids become limiting only after the grain is supplemented with other inputs for feed formulations
  • methionine becomes limiting
  • the levels of these essential amino acids m seeds and grain may be elevated by mechanisms which include, but are not limited to, the introduction of genes to increase the biosynthesis of the amino acids, decrease the degradation of the amino acids, increase the storage of the amino acids in proteins, or increase transport of the amino acids to the seeds or grain
  • One mechanism for increasing the biosynthesis of the amino acids is to introduce genes that deregulate the amino acid biosynthetic pathways such that the plant can no longer adequately control the levels that are produced This may be done by deregulat- ing or bypassing steps in the amino acid biosynthetic pathway that are normally regulated by levels of the amino acid end product of the pathway Examples include the introduction of genes that encode deregulated versions of the enzymes aspartokinase or dihydrodipicohnic acid (DHDP)-synthase for increasing lysine and threonine production, and anthranilate synthase for increasing tryptophan production Reduction of the catabolism of the amino acids may be accomplished by introduction of DNA sequences that reduce or eliminate the expression of genes encoding enzymes that catalyse steps in the catabodc pathways such as the enzyme lysme-ketoglutarate reductase
  • DHDP dihydrodipicohnic acid
  • the protein composition of the grain may be altered to improve the balance of amino acids in a variety of ways including elevating expression of native proteins, decreasing expression of those with poor composition, changing the composition of native proteins, or introducing genes encoding entirely new proteins possessing superior composition DNA may be introduced that decreases the expression of members of the zein family of storage proteins.
  • This DNA may encode ribozymes or antisense sequences directed to impairing expression of zein proteins or expression of regulators of zein expression such as the opaque-2 gene product
  • the protein composition of the grain may be modified through the phenomenon of cosuppression, i e , inhibition of expression of an endogenous gene through the expression of an identical structural gene or gene fragment introduced through transformation (Goring 1991) Additionally, the intro- prised DNA may encode enzymes, which degrade zeines.
  • zein expression may be accompanied by increases in proteins with more desirable amino acid composition or increases in other major seed constituents such as starch
  • a chimeric gene may be introduced that comprises a coding sequence for a native protein of adequate amino acid composition such as for one of the globulin proteins or 10 kD zein of maize and a promoter or other regulatory sequence designed to elevate expression of said protein.
  • the coding sequence of said gene may include additional or replacement codons for essential amino acids
  • a coding sequence obtained from another species, or, a partially or completely synthetic sequence encoding a completely unique peptide sequence designed to enhance the amino acid composition of the seed may be employed
  • the introduction of genes that alter the oil content of the grain may be of value Increases in oil content may result in increases in metabohzabie energy content and density of the seeds for uses in feed and food
  • the introduced genes may encode enzymes that remove or reduce rate-limitations or regulated steps in fatty acid or lipid biosynthesis
  • Such genes may include, but are not limited to those that encode acetyl- CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase plus other well- known fatty acid biosynthetic activities
  • Other possibilities are genes that encode proteins that do not possess enzymatic activity such as acyl carrier protein Additional examples include 2-acetyltransferase, oleosin pyruv
  • Genes may be introduced that enhance the nutritive value of the starch component of the grain, for example by increasing the degree of branching, resulting in improved utilization of the starch m cows by delaying its metabolism
  • genes may be introduced that affect a variety of other nutritive, processing, or other quality aspects of the grain as used for feed or food
  • pigmentation of the grain may be increased or decreased
  • Enhancement and stability of yellow pigmentation is desirable in some animal feeds and may be achieved by introduction of genes that result in enhanced production of xanthophylls and carotenes by eliminating rate-limiting steps in their production
  • genes may encode altered forms of the enzymes phytoene synthase, phytoene desaturase, or lycopene synthase
  • unpigmented white corn is desirable for production of many food products and may be produced by the introduction of DNA, which blocks or eliminates steps in pigment production pathways
  • Feed or food comprising some cereal grains possesses insufficient quantities of vita- mins and must be supplemented to provide adequate nutritive value
  • Introduction of genes that enhance vitamm biosynthesis in seeds may be envisioned including, for example vitamins A, E, B 12 , choline, and the like
  • maize grain also does not possess sufficient mineral content for optimal nutritive value
  • Genes that affect the accumulation or availability of compounds containing phosphorus, sulfur, calcium, manganese, zinc, and iron among others would be valuable
  • An example may be the introduction of a gene that reduced phytic acid production or encoded the enzyme phy- tase, which enhances phytic acid breakdown
  • These genes would increase levels of available phosphate in the diet, reducing the need for supplementation with mineral phosphate
  • Improvement of cereals for feed and food purposes might not even necessarily involve the gram, but may for example, improve the value of the grain for silage Introduction of DNA to accomplish this might include sequences that alter lignin production such as those that result in the "brown msdrib" phenotype associated with superior feed value for cattle
  • genes may also be introduced which improve the processing of gram and improve the value of the products resulting from the processing
  • the primary method of processing certain grains such as maize is via wetmilling Maize may be improved though the expression of novel genes that increase the efficiency and reduce the cost of processing such as by decreasing steep- ing time
  • Improving the value of wetmilling products may include altering the quantity or quality of starch, oil, corn gluten meal, or the components of corn gluten feed Elevation of starch may be achieved through the identification and elimination of rate limiting steps in starch biosynthesis or by decreasing levels of the other components of the grain resulting in proportional increases in starch
  • An example of the former may be the introduction of genes encoding ADP-glucose pyropho ⁇ phorylase enzymes with altered regulatory activity or which are expressed at higher level
  • Examples of the latter may include selective inhibitors of, for example, protein or oil biosynthesis expressed during later stages of kernel development
  • the properties of starch may be beneficially altered by changing the ratio of amySose to amylopectin, the size of the starch molecules, or their branching pattern
  • a broad range of properties may be modified which include, but are not limited to, changes in geSatinization temperature, heat of gelatinization, clarity of films and pastes, Theological properties, and the like
  • genes that encode granule-bound or soluble starch synthase activity or branching enzyme activity may be introduced alone or combination DNA such as antisense constructs may also be used to decrease levels of endogenous activity of these enzymes
  • the introduced genes or constructs may possess regulatory sequences that time their expression to specific intervals in starch biosynthesis and starch granule development
  • the covalent attachment of any molecule may be envisioned, limited only by the existence of enzymes that catalyze the
  • Oil is another product of wetmilling of corn and other grains the value of which may be improved by introduction and expression of genes
  • the quantity of oil that can be ex- tracted by wetmilling may be elevated by approaches as described for feed and food above Oil properties may also be altered to improve its performance in the production and use of cooking oil, shortenings, lubricants or other oil-de ⁇ ved products or ⁇ m- provement of its health attributes when used in the food-related applications
  • Novel fatty acids may also be synthesized which upon extraction can serve as starting materials for chemical syntheses
  • the changes in oil properties may be achieved by altering the type, level, or lipid arrangement of the fatty acids present in the oil This in turn may be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids and the lipids possessing them or by increasing levels of native fatty acids while possibly reducing levels of precursors Alternatively DNA sequences may be introduced which slow or block steps in fatty acid biosynthesis resulting in the increase in precursor fatty acid intermediates Gene
  • Improvements in the other major cereal wetmilling products, gluten meal and gluten feed may also be achieved by the introduction of genes to obtain novel plants. Representative possibilities include but are not limited to those described above for improvement of food and feed value
  • the plant be used for the production or manufacturing of useful biological compounds that were either not produced at all, or not produced at the same level, in the plant previously
  • useful biological compounds that were either not produced at all, or not produced at the same level, in the plant previously
  • the novel plants producing these compounds are made possible by the introduction and expression of genes by transformation methods
  • the possibilities include, but are not limited to, any biological compound which is presently produced by any organism such as proteins, nucleic acids, primary and intermediary metabolites, carbohydrate polymers, etc
  • the compounds may be produced by the plant, extracted upon harvest and/or processing, and used for any presently recognized useful purpose such as pharmaceuticals, fragrances, sndus- trial enzymes to name a few
  • nucleic acid sequences that can be combined with the promoter nucleic acid sequence of the present invention and provide improved end- product traits include, without limitation, those encoding seed storage proteins, fatty acid pathway enzymes, tocopherol biosynthetic enzymes, amino a ⁇ d biosynthetic en- zymes, and starch branchtng enzymes
  • a discussion of exemplary heterologous DNAs useful for the modification of plant phenotypes may be found in, for example U S Pat Nos 6, 194 636, 6,207,879 6,232,526, 6,426 446; 6,429,357, 6,433,252, 6,437,217 6,515,201 , and 6,583,338 and PCT Publication WO 02/057471 , each of which is spe- crfically incorporated herein by reference in its entirety
  • Such traits include but are not limited to
  • nucleic acids such as the artificial cDNA which encodes a microbial phytase (GenBank Ace No A19451) or functional equivalents thereof
  • Preferred tocopherol biosynthetic enzymes include tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, MTI 1 tMT2, AANT1 , sir 1737 and an anttsense construct for homogentisic acid dioxygenase (K ⁇ dl et a!
  • nutraceuticals such as, for example, polyunsaturated fatty acids (for example arachidonic acid, eicosape ⁇ taenoic acid or docosahexaenoic acid) by ex- pression of fatty acsd elongases and/or desaturases, or production of proteins with improved nutritional value such as, for example, with a high content of essential amino acids (for example the high-methionine 2S albumin gene of the brazil nut)
  • nucleic acids which encode the Berthoiletia excelsa high-methionine 2S albumin GenBank Ace No AB044391
  • the Physcomitrella patens ⁇ -acyi-liptd de ⁇ aturase GenBank Ace No AJ222980, Girke et al (1998) Plant J 15 39-48
  • nucleic acids are those which encode the Medicago sativa acetyl-CoA carboxylase (ACCase) (GenBank Ace No L25042), or functional equivalents thereof
  • ACCase Medicago sativa acetyl-CoA carboxylase
  • seed storage proteins include zeins (U S Pat Nos 4,886,878 4,885 357, 5,215,912, 5,589,616, 5,508,468, 5,939,599,
  • GLTP ⁇ -gluca ⁇ L-type tuber phosphoryiase
  • GHTP ⁇ -glucan H-type tuber phosphoryiase
  • a potato plant having improved cold-storage characteristics comprising a potato plant transformed with an expres- sion cassette having a TPT promoter sequence operably linked to a DNA sequence comprising at least 20 nucleotides of a gene encoding an ⁇ -glucan phosphoryiase selected from the group consisting of ⁇ -glucan L-type tuber phosphoryiase (GLTP) and ⁇ -glucan
  • the plant to be grown in a particular area is selected for its ability to mature and dry down to harvestable moisture content within the required period of time with maximum possible yield
  • plants of varying maturities are developed for different growing locations Apart from the need to dry down sufficiently to permit harvest is the desirability of having maximal drying take place in the fseld to minimize the amount of energy required for additional drying post-harvest
  • the more readily the grain can dry down the more time there is available for growth and kernel fill Genes that influence maturity and/or dry down can be identified and introduced into plant lines using transformation techniques to create new varieties adapted to different growing locations or the same growing location but having improved yield to moisture ratio at harvest
  • Expression of genes that are involved in regulation of plant development may be especially useful, e g , the liguleless and rough sheath genes that have been identified in plants
  • Genes may be introduced into plants that would improve standability and other plant growth characteristics
  • expression of novel genes which confer stronger stalks, improved root systems, or prevent or reduce ear droppage would be of great value to the corn farmer
  • Introduction and expression of genes that increase the total amount of photoasstmilate available by for example, increasing light distribution and/or interception would be advantageous
  • expression of genes that increase the efficiency of photosynthesis and/or the leaf canopy would further increase gams in productivity
  • male sterility is useful in the production of hybrid seed ft is proposed that male sterility may be produced through expression of nove! genes
  • nove! genes For example, it has been shown that expression of genes that encode proteins that interfere with development of the male inflorescence and/or gametophyte result in male sterility
  • Chimeric ribonucSease genes that express in the anthers of transgenic tobacco and oilseed rape have been demonstrated to lead to male sterility (Ma ⁇ ani 1990)
  • T cytoplasm One mutation in particular, referred to as T cytoplasm, also correlates with sensitivity to Southern corn leaf blight
  • a DNA sequence, designated TURF-13 was identified that correlates with T cytoplasm It would be possible through the introduction of TURF-13 via transformation to separate male sterility from disease sensitivity As it is necessary to be able to restore male fertility for breeding purposes and for grain production, it is proposed that genes encoding restoration of male fertility may also be introduced
  • Non-Protein-Expressing Sequences 1.11.1 RNA-Expressing DNA may be introduced into plants for the purpose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein Two examples are antise ⁇ se RNA and RNA with ⁇ bozyme activity Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes
  • Genes may be constructed or isolated, which when transcribed, produce antisense RNA or double-stranded RNA that is complementary to all or part(s) of a targeted messenger RNA(s).
  • the antisense RNA reduces production of the polypeptide product of the messenger RNA.
  • the polypeptide product may be any protein encoded by the plant genome
  • the aforementioned genes will be referred to as antisense genes
  • An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest
  • the protein may be an enzyme that catalyzes a reaction in the plant Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic acids and the like.
  • the protein may be a storage protein, such as a zein, or a structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively
  • a storage protein such as a zein
  • structural protein the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively
  • RNA is preferably a non- translatable RNA Gene regulation by double-stranded RNA ("double-stranded RNA interference"; dsRNAi) is well known in the arte and described for various organism including plants (e g , Matzke 2000, Fire A et al 1998, WO 99/32619; WO 99/53050, WO 00/68374, WO 00/44914, WO 00/44895; WO 00/49035, WO 00/63364).
  • double-stranded RNA interference double-stranded RNA interference
  • Genes may also be constructed or isolated, which when transcribed produce RNA enzymes, or ribozymes, which can act as endoribonucleases and catalyze the cleavage of RNA molecules with selected sequences The cleavage of selected messenger RNA's can result in the reduced production of their encoded polypeptide products
  • RNA enzymes or ribozymes
  • cleavage of selected messenger RNA's can result in the reduced production of their encoded polypeptide products
  • These genes may be used to prepare novel transgenic plants, which possess them
  • the transgenic plants may possess reduced levels of polypeptides including but not limited to the polypeptides cited above that may be affected by antisense RNA
  • genes may be introduced to produce novel transgenic plants, which have reduced expression of a native gene product, by a mechanism of cosup- pression It has been demonstrated in tobacco, tomato, and petunia (Go ⁇ ng 1991 , Smith 1990; Napoli 1990, van der Krol 1990) that expression of the sense transcript of a native gene wtll reduce or eliminate expression of the native gene in a manner similar to that observed for antisense genes
  • the introduced gene may encode all or part of the targeted native protein but its translation may not be required for reduction of levels of that native protein
  • DNA elements including those of transposable elements such as Ds, Ac, or Mu, may be, inserted into a gene and cause mutations These DNA elements may be inserted in order to inactivate (or activate) a gene and thereby "tag" a particular trait In this instance the transposable element does not cause instability of the tagged mutation, because the utility of the element does not depend on its ability to move in the genome
  • the introduced DNA sequence may be used to clone the corresponding gene, e g , using the introduced DNA sequence as a PCR primer together with PCR gene cloning techniques (Shapiro, 1983, Dellaporta 1988) Once identified, the entire gene( ⁇ ) for the particular trait, including control or regulatory regions where desired may be isolated, cloned and manipulated as desired
  • the utility of DNA elements introduced into an organism for purposed of gene tagging ss inde- pendent of the DNA sequence and does not depend on any biological activity of the DNA sequence, i e , transcription into RNA
  • MAR matrix attachment region ele- ment
  • MAR matrix attachment region ele- ment
  • Stief 1989 chicken lysozyme A element
  • nucleic acid molecules e g , DNA or RNA
  • isolated nucleic acid molecules comprising a plant nucleotide sequence according to the invention comprising an open reading frame that is preferentially expressed in a specific tissue, i e , seed-, root, green tissue (leaf and stem), panicle-, or pollen, or is expressed constitutively
  • a selectable or screenable marker gene is genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker
  • Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ' select ' for by chemical means, i e , through the use of a selective agent (e g , a herbicide, antibiotic, or the like) or whether it is simply a tratt that one can identify through observation or testing, i e , by screening' (e g , the R-locus trait the green fluorescent protein (GFP))
  • a selective agent e g , a herbicide, antibiotic, or the like
  • screening' e g , the R-locus trait the green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • selectable or screenable marker genes are also genes which encode a "secreiable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells
  • markers which encode a se- cretable antigen that can be identified by antibody interaction, or even secretable enzymes, which can be detected by their catalytic activity
  • Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e g , by ELISA, small active enzymes detectable in extracellular solution (e g , alpha-amylase, beta- iactamase, phosphinoth ⁇ cin acetyltransferase), and proteins that are inserted or trapped in the cell wall (e g , proteins that include a leader sequence such as that found in the expression unit of extensm or tobacco PR-S)
  • a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous
  • Such a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the eel! wall and yet accessible to antibodies
  • a normally secreted wall protein modified to include a unique epitope would satisfy all such requirements
  • HPRG hy- droxyproltne rich glycoprotein
  • the maize HPRG (Steifel 1990) molecule is well characterized in terms of molecular biology, expression and protein structure
  • any one of a variety of ultilane and/or glycine- ⁇ ch wall proteins (Keller 1989) could be modified by the addition of an antigenic site to create a screenable marker
  • a secretable screenable marker concerns the use of a maize sequence encoding the wall protein HPRG, modified to include a 15 residue epitope from the pro-region of murine mterleukin, however, virtually any detectable epitope may be employed in such embodiments, as selected from the extremely wide va- ⁇ ety of antigen-antibody combinations known to those of skill in the art
  • the unique extracellular epitope can then be straightforwardly detected using antibody labeling in conjunction with chromoge ⁇ ic or fluorescent adjuncts
  • markers are known in the art suitable for plant transformation Such markers may include but are not limited to
  • Negative selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e g , 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e g , kanamycin, G 418, bleomycin or hygromycin) or herbicides (e g , phosphinoth ⁇ ci ⁇ or glyphosate)
  • a metabolic inhibitor e g , 2-deoxyglucose-6-phosphate, WO 98/45456
  • antibiotics e g , kanamycin, G 418, bleomycin or hygromycin
  • herbicides e g phosphinoth ⁇ ci ⁇ or glyphosate
  • Transformed plant material e g , cells, tissues or plantlets
  • a corresponding selection compound e g , antibiotic or herbicide
  • Especially preferred negative selection markers are those, which confer resistance to herbicides Examples, which may be mentioned, are - Phosphin
  • EPSP 5-enolpyruvylshik ⁇ mate-3-phosphate synthase
  • Glyphosate ® degrading enzymes (Glyphosate ® oxidoreductase, gox), Dalapon ® inactivating dehalogenases (deh) sulfonylurea- and/or imidazolinone-inactivating acetolactate synthases (ahas or ALS, for example mutated ahas/ALS variants with, for example, the S4, XI12,
  • Kanamycin- or geneticin (G418) resistance genes (NPTII, NPT or neo, Potrykus 1985) coding e g for neomycin phosphotransferases (Fraley 1983, Nehra 1994) - 2-Desoxyglucose-6-phosphate phosphatase (DOG R 1 -Gene product, WO 98/45456, EP 0 807 836) conferring resistance against 2-desoxyglucose (Randez- GiI 1995) hygromycin phosphotransferase (HPT), which mediates resistance to hygromycin (Vanden Elzen 1985) - altered dihydrofolate reductase (Eichholtz 1987) conferring resistance against methotrexat (Thillet 1988), mutated anthranilate synthase genes that confers resistance to 5-methyl tryptophan
  • Additional negative selectable marker genes of bacterial origin that confer resistance to antibiotics include the aadA gene, which confers resistance to the antibiotic spectino- myci ⁇ , gentamycin acetyl transferase, streptomycin phosphotransferase (SPT), ami- noglycoside-3-adenyl transferase and the bleomycin resistance determinant (Hayford 1988, Jones 1987, Svab 1990, HiIIe 1986)
  • negative selection markers that confer resistance against the toxic effects imposed by D-amino acids like e g , D-alanine and D-serine (WO 03/060133 E ⁇ kson 2004)
  • negative selection marker in this contest are the dao ⁇ gene (EC 1 4 3 3 GenBank Ace -No U60066) from the yeast Rhodotorula gracilis (Rhodospondium torul ⁇ ides) and the E col/ gene dsdA (D-serine dehydratase (D-se ⁇ ne deaminase) [EC 4 3 1 18, GenBank Ace -No J01603)
  • Transformed plant material e g , cells, embryos, tissues or plantlets
  • a corresponding selection compound e g , antibiotic or herbicide
  • the resulting plants can be bred and hy- b ⁇ dtzed in the customary fashion Two or more generations should be grown in order to ensure that the genomic integration is stable and hereditary Corresponding methods are described (Jenes 1993, Potrykus 1991)
  • reporter genes can be employed to allow visual screening, which may or may not (depending on the type of reporter gene) require supplementation with a substrate as a selection compound
  • ⁇ elec- tion is preferably applied throughout callus induction phase for about 4 weeks and beyond at least 4 weeks into regeneration
  • Such a selection scheme can be applied for all selection regimes. It is furthermore possible (although not explicitly preferred) to remain the selection also throughout the entire regeneration scheme including rooting
  • phosphinot ⁇ cin resistance gene (bar) as the selective marker phosphinotricin at a concentration of from about 1 to 50 mg/l may be incSuded in the medium
  • phosphinotricin at a concentration of from about 1 to 50 mg/l may be incSuded in the medium
  • D-serine or D- alanine at a concentration of from about 3 to 100 mg/l may be included in the medium
  • Typical concentrations for selection are 20 to 40 mg/l
  • PURSUIT TM at a concentration of from about 3 to 100 mg/l may be included in the medium
  • Typical concentrations for selection are 20 to 40 mg/l
  • positive selection marker can be employed Genes like tsopentenyltrans- ferase from Agrobacterium tumefaciens (strain PO22, Genbank Ace -No AB025109) may - as a key enzyme of the cytokinin biosynthesis - facilitate regeneration of transformed plants (e g , by selection on cytokin in-free medium) Corresponding selection methods are described (Ebinuma 2000a b) Additional positive selection markers, which confer a growth advantage to a transformed plant in comparison with a non- transformed one are described e g , in EP-A 0 601 092 Growth stimulation selection markers may include (but shall not be limited to) ⁇ -Glucuronidase (in combination with e g , a cytokinin gSucuronide), mannose-6-phos ⁇ hate isomerase (in combination with mannose), UDP-galactose-4-ep ⁇ merase (in combination with e g , galactose), wherein mannose-6-
  • Counter-selection markers are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek 1999)
  • Examples for counter- selection marker comprise thymidin kinases (TK), cytosine deaminases ⁇ Gleave 1999, Per- era 1993, Stougaard 1993), cytochrom P450 proteins (Koprek 1999), haloalkan deha- iogenases (Naested 1999), saaH gene products (Sundaresan 1995), cytosine deami- nase codA (Schlaman & Hooykaas 1997), tms2 gene products (Fedoroff & Smith 1993), or ⁇ -naphtha!ene acetamide (NAM, Depicker 1988)
  • Counter selection markers may be useful in the construction of transposon tagging lines For example, by marking an autonomous transposable element such as Ac, Master Mu, or En/Spn with a counter selection marker, one
  • Screenable markers that may be employed include but are not limited to, a beta- glucuromdase (GUS) or uidA gene which encodes an enzyme for which various chro- mogenic substrates are known, an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Deflaporta 1988), a beta-lactamase gene (Sutcliffe 1978), which encodes an enzyme for which various chromogenic substrates are known (e g , PADAC, a chromogenic cephalosporin), a xylE gene (Zukowsky 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols, an ⁇ -amylase gene (Ikuta 1990), a tyrosinase gene (Katz 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn con
  • R gene complex encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue
  • a gene from the R gene complex was applied to maize transformation, because the expression of this gene in transformed cells does not harm the cells
  • an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector
  • a maize line is dominant for genes encoding the en- zymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1 , A2, Bz 1 and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation
  • Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR1 12, a K55 derivative which is r-g, b, P1 Alternatively any genotype of maize can be utilized if
  • R gene regulatory regions may be employed in chimeric constructs in order to provide mechanisms for controlling the expression of chimeric genes More diversity of phenotypic expression is known at the R locus than at any other locus (Coe 1988) It is contemplated that regulatory regions obtained from regions 5' to the structural R gene would be valuable in directing the expression of genes, e g , insect resistance, drought resistance, herbicide tolerance or other protein coding regions.
  • any of the various R gene fam- ily members may be successfully employed (e g , P, S, Lc, etc ) However, the most preferred will generally be Sn (particularly Sn bol3).
  • Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R
  • a further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene
  • the presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell lumi- nometry It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
  • a screenable marker gene such as lux or GFP
  • benefit may be realized by creating a gene fusion between the screenable marker gene and a selectable marker gene, for example, a GFP-NPTlI gene fusion This could allow, for example, selection of transformed cells foliowed by screening of transgenic plants or seeds
  • the invention provides an isolated nucleic acid molecule, e g , DNA or RNA, comp ⁇ s- ing a plant nucleotide sequence comprising an open reading frame that is preferentially expressed in a specific plant tissue, i e., in seeds, roots, green tissue (leaf and stem), panicles or pollen, or is expressed constitutively, or a promoter thereof.
  • a specific plant tissue i e., in seeds, roots, green tissue (leaf and stem), panicles or pollen, or is expressed constitutively, or a promoter thereof.
  • promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters
  • Promoter sequences are known to be strong or weak A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression.
  • An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or de- velopmental stimulus
  • a bacterial promoter such as the P tac promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells
  • An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to aliow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired
  • the first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene.
  • the core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence that defines the transcription start point for the structural gene.
  • the presence of the core promoter region defines a sequence as being a promoter if the region is absent, the promoter is non-functional Furthermore, the core promoter region is insufficient to provide full promoter activity.
  • a series of regulatory sequences upstream of the core constitute the remainder of the promoter
  • the regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemscals, hormones)
  • Regulated expression of the chimeric transacting viral replication protein can be further regulated by other genetic strategies For example, Cre-mediated gene activation as described by Odell et al.
  • a DNA fragment containing 3' regulatory sequence bound by lox sites between the promoter and the replication protein coding sequence that blocks the expression of a chimeric replication gene from the promoter can be removed by Cre-mediated excision and result in the expression of the transacting replication gene
  • the chimeric Cre gene, the chimeric trans-acting replication gene, or both can be under the control of tissue- and developmental-specific or inducible promoters
  • An alternate genetic strategy is the use of tRNA suppressor gene
  • the regulated expression of a tRNA suppressor gene can condi- tionally control expression of a trans-acting replication protein coding sequence containing an appropriate termination codon as described by Ulmasov et al 1997.
  • either the chimeric tRNA suppressor gene, the chimeric transacting replication gene, or both can be under the control of tissue- and developmental-specific or inducible promoters
  • RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
  • a DNA segment representing the promoter region is removed from the 5' region of the gene of interest and operabiy linked to the coding sequence of a marker (reporter) gene by recombinant DNA techniques well known to the art
  • the reporter gene is operabiy finked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene
  • Reporter genes generally encode proteins, which are easily measured, including but not limited to, chloramphenicol acetyl transferase (CAT), beta-glucuronsdase (GUS), green fluorescent protein (GFP), beta-galactosidase (beta-GAL), and luciferase
  • the construct containing the reporter gene under the control of the promoter ss then introduced into an appropriate cell type by transfection techniques well known to the art
  • cell lysates are prepared and appropriate assays, which are well known in the art, for the reporter protein are performed
  • CAT were the reporter gene of choice
  • the lysates from cells transfected with constructs containing CAT under the control of a promoter under study are mixed with iso- topicafly labeled chloramphenicol and acetyl-coenzyme A (acetyl-CoA)
  • the CAT en- zyme transfers the acetyl group from acetyl-CoA to the 2- or 3-pos ⁇ t ⁇ on of chloramphenicol
  • the reaction is monitored by thin-layer chromatography, which separates acetylated chloramphenicol from unreacted material
  • the reaction products are then visualized by autoradiography
  • the level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression from the promoter of interest
  • This level of expression can be compared to other promoters to determine the relative strength of the promoter under study
  • the level of the reporter mRNA can be measured directly such as by Northern blot analysis
  • mutational and/or deletional analyses may be employed to determine the minimal region and/or sequences required to initiate transcription
  • sequences can be deleted at the 5' end of the promoter region and/or at the 3' end of the promoter region, and nucleotide substitutions introduced These constructs are then introduced to cells and their activity determined
  • the promoter may be a gamma zein promoter, an oleosin ole16 promoter, a globulins promoter, an actm I promoter, an actin ct promoter a sucrose synthetase promoter, an INOPS promoter, an EXM5 promoter, a globulin2 promoter, a b-32 ADPG-pyrophosphorylase promoter, an Ltpl promoter, an Lfp2 promoter, an oleosin ole17 promoter, an oleosin ole18 promoter, an actin 2 promoter, a pollen- specific protein promoter, a pollen-specific pectate lyase promoter, an anther-specific protein promoter, an anther-specific gene RTS2 promoter, a poHen-specific gene pro- moter, a tapeturn-specific gene promoter, tapeturn-specific gene RAB24 promoter, a anthran
  • Plant species may be transformed wsth the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art
  • tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis may be transformed with a vector of the present invention
  • organogenesis means a process by which shoots and roots are de- veloped sequentially from me ⁇ stematic centers
  • embryogenesis means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyis, megagametophytes, callus tissue, existing meristematic tissue (e g , apical me ⁇ stems, axillary buds, and root tne ⁇ stems) and induced me ⁇ stem tissue (e g , cotyledon me ⁇ stem and uttilane me ⁇ stem)
  • Plants of the present invention may take a variety of forms
  • the plants may be chime- ras of transformed cells and non-transformed cells, the plants may be clonal transfor- mants ⁇ e g , ail cells transformed to contain the expression cassette), the plants may comprise grafts of transformed and untransformed tissues (e g , a transformed root stock grafted to an untransfom ⁇ ed scion in citrus species)
  • the transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques For example, first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques
  • a dominant selectable marker (such as npt H) can be associated with the expression cassette to assist in breeding
  • transgenic plants of the present invention are crop plants and in particular cereals (for example corn, alfalfa, sunflower, rice, Brassica, canoia, soybean barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, Linum usitatissimum (linseed and fax), Camelina sativa Brassica juncea, etc ), and even more preferably corn, rice and soybean
  • cereals for example corn, alfalfa, sunflower, rice, Brassica, canoia, soybean barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, Linum usitatissimum (linseed and fax), Camelina sativa Brassica juncea, etc
  • Other embodiments of the invention are related to cells cell cultures, tissues, parts (such as plants organs leaves, roots, etc ) and propagation materia! (such as seeds) of such plants
  • the transgenic expression cassette of the invention may not only be comprised in plants or plant cells but may advantageously also be containing in other organisms such for example bacteria
  • another embodiment of the invention relates to transgenic cells or non-human, transgenic organisms comprising an expression cassette of the invention
  • Preferred are prokaryotic and eukaryotsc organisms
  • Both microorganism and higher organisms are comprised
  • Preferred microorganisms are bacteria, yeast, algae, and fungi Preferred bacteria are those of the genus Escherichia, Erwmia, Agro- bacterium, Ravobacte ⁇ um, Alcaligenes, Pseudomonas, Bacillus or Cyanobacte ⁇ m such as - for example - Synechocystis and other bacteria described in Brock Biology of Microorganisms Eighth Edition (pages A-8, A-9, A10 and A1 1)
  • microorganisms capable to infect plants and to transfer DNA into their genome especially bacteria of the genus Agrobacte ⁇ um, preferably Agrobac- terium tumefa ⁇ ens and rhszogenes
  • Preferred yeasts are Candida, Saccharomyces, Hansenula and Pichia
  • Preferred Fungi are Aspergillus T ⁇ choderma, Ashbya, Neuro- spora, Fusa ⁇ um, and Beauveria
  • Transformation of piants can be undertaken wtth a single DNA molecule or multiple DNA molecules ( ⁇ e co-transformation), and both these techniques are suitable for use with the expression cassettes of the present invention
  • Numerous transformation vec- tors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors The selection of vector will de- pend upon the preferred transformation technique and the target species for transformation
  • Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells
  • expression vectors are introduced into intact tissue
  • General methods of cuituring plant tissues are provided for example by Maki et al , (1993), and by Phillips et al (1988)
  • expression vectors are introduced into maize or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporatson and the like More preferably expression vectors are introduced into plant tissues using the microprojectile media delivery with the bioiistic device See, for example, Tomes et al (1995)
  • the vectors of the invention can not only be used for expression of structural genes but may also be used in exon-trap clonsng or promoter trap procedures to detect differential gene expression m varieties of tissues (Lindsey 1993, Auch & Reth 1990)
  • the binary type vectors of Ti and Ri plasmids of Agro- bacterium spp Ti-denved vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton rape, tobacco, and rice (Pacciotti 1985 Byrne 1987, Sukhapinda 1987, Lorz 1985, Potrykus, 1985, Park 1985 Hsei 1994)
  • the use of T-DNA to transform plant ceils has received extensive study and is amply described (EP 120516, Hoekema, 1985, Knauf, 1983, and An 1985)
  • the chimeric genes of the invention can be inserted into binary vectors as described in the examples
  • transforming plant cells include, but are not limited to, microinjec- tion (Cro ⁇ sway 1986), electroporation ⁇ Riggs 1986), Agrobacte ⁇ um-mediated transformation (Hinchee 1988), direct gene transfer (Paszkowski 1984), and ballistic particle acceleration using devices available from Agracetus, lnc , Madison, Wis.
  • a nucleotide sequence of the present invention is directly transformed into the plastid genome Plastic) transformation technology is extensively described in US 5,451 ,513, 5,545,817, and 5,545,818, in PCT application no WO 95/16783, and tn McBride et al , 1994
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e g , using biolistics or protoplast transformation (e g , calcium chloride or PEG mediated transformation)
  • the 1 to 1 5 kb flanking regions termed targeting sequences, facilitate orthologous recom- bination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome
  • point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab
  • Plastid expression in which genes are inserted by orthologous recombination into all of the several thousand copies of the circular pSastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein
  • a nucleotide sequence of the present invention is inserted into a plastid-targeting vector and transformed into the pSastid genome of a desired plant host Plants homoplastic for piastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • Agrobacte ⁇ um tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid are useful in methods of making transformed plants.
  • Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant ceil
  • Numerous Agrobacte ⁇ um vector systems useful in carrying out the present invention are known
  • Agrobacte ⁇ um strains can be employed, preferably disarmed Agrobactenum tumefa ⁇ ens or rhizogenes strains
  • Agrobacte ⁇ um strains for use in the practice of the invention include octopine strains, e g , LBA4404 or ag- ropine strains, e g EHA101 or EHA105
  • Suitable strains of A tumefaciens for DNA transfer are for example EHA101 [ ⁇ EHA101] (Hood 1986), EHA105[pEHA105] (Li 1992), LBA4404[pAL4404] (Hoekema 1983) C58C1[pMP90] (Koncz & Schell 1986), and C58C1 [pGV2260] (Deblaere 1985)
  • Other suitable strains are Agrobacte ⁇ um tumefaciens C58, a nopaline strain
  • Other suitable strains are A tumefaciens C58C
  • the method of the invention can also be used in combination with particular Agrobacte ⁇ um strains, to further increase the transformation efficiency, such as Agrobactenum strains wherein the vir gene expression and/or induction thereof is altered due to the presence of mutant or chimeric virA or vsrG genes (e g. Hansen 1994, Chen and Wi- nans 1991 , Scheeren-Groot 1994)
  • Agrobactenum strains wherein the vir gene expression and/or induction thereof is altered due to the presence of mutant or chimeric virA or vsrG genes
  • Preferred are further combinations of Agrobacte- ⁇ um tumefaciens strain LBA4404 (Hiei 1994) with super-virulent plasmids are preferably pTOK246-based vectors (Ishida 1996)
  • a binary vector or any other vector can be modified by common DNA recombination techniques, multiplied in E coli and introduced into Agrobacte ⁇ um by e g , electropo- ration or other transformation techniques (Mozo & Hooykaas 1991)
  • Agrobactenum is grown and used in a manner similar to that described in Ishida (1996)
  • the vector comprising Agrobacte ⁇ um strain may, for example, be grown for 3 days on YP medium (5 g/l yeast extract, 10 g/l peptone, 5 g/l NaCI, 15 g/l agar, pH 6 8) supplemented with the appropriate antibiotic (e g , 50 mg/l spectinomycin) Bacteria are collected with a loop from the solid medium and resuspe ⁇ ded
  • Agrobactenum cultures are started by use of aljquots frozen at - 80 0 C
  • the transformation of the target tissue (e g , an immature embryo) by the Agrobactenum may be carried out by merely contacting the target tissue with the Agrobacte ⁇ um
  • concentration of Agrobactenum used for infection and co-cultivation may need to be varied
  • a cell suspension of the Agrobacte ⁇ um having a population density of approximately from 10 5 - 10 11 preferably 10 6 to 10 10 , more preferably about 10 8 cells or cfu / ml is prepared and the target tissue is immersed in this suspension for about 3 to 10 minutes
  • the resuiting target tissue is then cultured on a solid medium for several days together with the Agrobacte ⁇ um
  • the bacterium is employed in concentration of 10 ⁇ to 10 10 cfu/ml
  • concentration of 10 ⁇ to 10 10 cfu/ml In a preferred embodiment for the co-cultivation step about 1 to 10 ⁇ l of a suspension of the soil-borne bacterium (e g , Agrobacte ⁇ a) in the co-cultivation medium are directly ap- plied to each target tissue explant and air-dried This is saving labor and time and is reducing unintended Agrobacterium-mediated damage by excess Agrobacterium usage
  • the bacteria are resuspended in a plant compatible co- cultivation medium Supplementation of the co-culture medium with antioxidants (e g silver nitrate), phenol-absorbing compounds (like polyvinylpyrrolidone, Perl 1996) or thiol compounds (e g , dithiothreitol, L-cysteine, OShoft 2001) which can decrease tissue necrosis due to plant defence responses (like phenolic oxidation) may further improve the efficiency of Agrobacterium-mediated transformation
  • the co-cultivation medium of comprises least one thiol compound preferably selected from the group consisting of sodium thiolsulfate, dithiot ⁇ etol (DTT) and cysteine
  • the concentration is between about 1 mM and 1OmM of L- Cysteme, 0.1 mM to 5 mM DTT and/or 0 1 mM to 5 mM sodium thiolsulfate
  • the medium preferably the concentration is between about 1 mM
  • Various vector systems can be used in combination with Agrobacte ⁇ a Preferred are binary vector systems
  • Common binary vectors are based on "broad host range"- plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson 1985) derived from the P-type plasmid RK2 Most of these vectors are derivatives of pBIN19 (Bevan 1984)
  • Various binary vectors are known some of which are commercially available such as, for ex- ample pBI101 2 or pBIN19 (Clontech Laboratories, lnc USA) Additional vectors were improved with regard to size and handling (e g pPZP, Hajdukiewicz 1994) Improved vector systems are described also in WO 02/00900
  • a selectable marker which may provide resistance to an antibiotic (e g , kanamycin, hygromycin or methotrexate) or a herbicide (e g , phosphinothricin)
  • antibiotic e g , kanamycin, hygromycin or methotrexate
  • herbicide e g , phosphinothricin
  • Selection markers used routinely in transformation include the nptl! gene which confers resistance to kanamycin and related antibiotics (Messing & Vierra 1982, Bevan 1983), the bar gene which confers resistance to the herbicide phosphinoth ⁇ cin (White 1990, Spencer 1990), the hph gene which confers resistance to the antibiotic hygromyc ⁇ n (Blochlinger & Diggelmann), and the dhfr gene which confers resistance to methotrexate (Bourouis 1983)
  • Transgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells, which are then grown to callus Shoots are grown from callus Plantlets are generated from the shoot by growing in rooting medium
  • the various con- structs normally will be joined to a marker for selection in plant cells
  • the marker may be resistance to a biocide (particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like)
  • the particular marker used will allow for selection of transformed cells as compared to cells lacking the DNA, which has been introduced
  • Components of DNA constructs including tran- sc ⁇ ption cassettes of this invention may be prepared from sequences, which are native (endogenous) or foreign (exogenous) to the host By "foreign" it is meant that the sequence is not found sn the wild-type host into which the construct JS introduced Heterologous constructs will contain at least one region, which is not native to the gene from which the transc ⁇ ption
  • assays include, for example, "molecular biological” assays well known to those of skill in the art such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT- PCR or TaqMan, "biochemical” assays, such as detecting the presence of a protein product, e g , by immunological means (ELISAs and Western blots) or by enzymatic function plant part assays, such as seed assays, and also by analyzing the phenotype of the whole regenerated plant, e g , for disease or pest resistance
  • “molecular biological” assays well known to those of skill in the art such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT- PCR or TaqMan
  • biochemical assays, such as detecting the presence of a protein product, e g , by immunological means (ELISAs and Western blots)
  • DNA may be isolated from cell lines or any plant parts to determine the presence of the preselected nucleic acid segment through the use of techniques well known to those skilled in the art Note that sntact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell
  • nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR) Using these technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration of the intro-uted preselected nucleic acid segment into the host cell genome In addition, it is not possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the, genome, i e , whether transformants are of independent origin It is contemplated that using PCR techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced preselected DNA segment Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified Hence the Southern hyb ⁇ di- zation pattern of a given transformant
  • RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues PCR techniques may also be used for detection and quantitation of RNA produced from intro- scored preselected DNA segments In this application of PCR it is fsrst necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product Further information about the nature of the RNA product may be obtained by Northern blotting This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species
  • Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures Examples are as varied as the enzyme to be ana- lyzed
  • bioassays Very frequently the expression of a gene product is determined by evaluating the phe- notypic results of its expression
  • these assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physio- logical properties of the plant Morphological changes may include greater stature or thicker stalks Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays
  • an expression cassette of the invention may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques
  • Particuiariy preferred plants of the invention include the agronomicaily important crops listed above
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction and can thus be maintained and propagated in progeny plants
  • the present invention also relates to a transgenic plant ceil, tissue, organ seed or plant part obtained from the transgenic plant
  • transgenic descendants of the plant as well as transgenic plant cells tissues, organs, seeds and plant parts obtained from the de- scendants
  • the expression cassette in the transgenic plant is sexually transmitted
  • the coding sequence is sexually transmitted through a complete normal sexual cycle of the RO plant to the R1 generation
  • the expression cassette is expressed in the cells, tissues, seeds or plant of a transgenic plant in an amount that is different than the amount in the cells, tissues, seeds or plant of a plant, which only differs in that the expression cassette is absent
  • the transgenic plants produced herein are thus expected to be useful for a variety of commercial and research purposes
  • Transgenic plants can be created for use in traditional agriculture to possess traits beneficial to the grower (e g , agronomic traits such as resistance to water deficit, pest resistance, herbicide resistance or increased yield), beneficial to the consumer of the grain harvested from the plant (e g , improved nutritive content in human food or animal feed, increased vitamin, amino acid, and antioxidant content, the production of antibodies (passive immunization) and nut ⁇ ceuticais), or beneficial to the food processor (e g improved processing traits)
  • the grower e g ,
  • Transgenic plants may also find use in the commercial manufacture of proteins or other molecules where the molecule of interest is extracted or pu ⁇ fsed from plant parts, seeds, and the like Cells or tissue from the plants may also be cultured, grown in vitro, or fermented to manufacture such molecules The transgenic plants may also be used in commercial breeding programs, or may be crossed or bred to plants of related crop species Improvements encoded by the expression cassette may be transferred, e g , from maize cells to cells of other species, e g , by protoplast fusion
  • the transgenic plants may have many uses in research or breeding, including creation of new mutant plants through insertional mutagenesis, in order to identify beneficial mutants that might later be created by traditional mutation and selection.
  • An example would be the introduction of a recombinant DNA sequence encoding a transposable element that may be used for generating genetic variation
  • the methods of the invention may also be used to create plants having unique "signature sequences" or other marker sequences which can be used to identify proprietary lines or varieties
  • the transgenic plants and seeds according to the invention can be used in plant breeding, which aims at the development of plants with improved properties conferred by the expression cassette, such as tolerance of drought, disease, or other stresses
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or select- ing appropriate descendant plants Depending on the desired properties different breeding measures are taken
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multilane breeding variety blend, interspecific hybridization, aneuploid techniques etc
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means Cross-pollination of a male sterile plant with pollen of a different line assures that the genome of the male steriSe but female fertile plant will uniformly obtain properties of both parental lines
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, which for example
  • chemicais and reagents in the Examples were obtained from Sigma Chemical Company (St Louis, MO), restriction endonucleases were from New England Biolabs (Beverly, MA) or Roche (Indianapolis, IN) oligonucleotides were synthesized by MWG Biotech lnc (High Point NC), and other modifying enzymes or kits regarding biochemscals and molecular biological assays were from Clontech (PaSo Alto, CA), Pharmacia Biotech (Piscataway, NJ), Promega Corporation (Madison, Wl), or Stratagene (La JoIIa, CA) Materials for cell culture media were obtained from Gibco/BRL (Gaithersburg, MD) or DIFCO (Detroit, Ml) The cloning steps carried out for the purposes of the present invention, such as, for example, restriction cleavages, aga- rose gel electrophoresis, purification of DNA fragments transfer of nucleic acids to nitrocellulose and nylon membranes
  • Example 1 Generation of transgenic plants
  • Transgenic linseed plants can be generated for example by the method of Bell et al , 1999, In Vitro Cell Dev. Biol -Plant 35(6) 456-465 by means of particle bombardment Agrobacte ⁇ a-mediated transformations can be generated for example by the method of Mlynarova et al (1994), Plant Cell Report 13 282-285.
  • Oilseed rape can be transformed by petiole, cotyledon or hypocotyi transformation (Moloney 1989, De Block 1989)
  • the use of antibiotics for the selection of Agrobacte ⁇ a and plants depends on the binary vector and the Agrobacterium strain used for the transformation
  • the selection of oilseed rape is generally carried out using kanamyci ⁇ as selectable plant marker
  • oilseed rape can be transformed as follows Agrobacterium strains transformed with the plasmid of interest s grown in 50 m L YEB medium at 28°C overnight.
  • the Agrobacterium solution is mixed with liquid co-cultivation medium (double concentrated MSB5 salts (Duchefa), 30 g/L sucrose (Duchefa), 3 75 mg/Ll BAP (6-benzylarmno purine, Duchefa), 0 5 g/L MES (Duchefa), 0 5 mg/L GA3 (Gibberellic Acid, Duchefa); pH5 2) untsl OD 65 o of 0 5 is reached.
  • liquid co-cultivation medium double concentrated MSB5 salts (Duchefa), 30 g/L sucrose (Duchefa), 3 75 mg/Ll BAP (6-benzylarmno purine, Duchefa), 0 5 g/L MES (Duchefa), 0 5 mg/L GA3 (Gib
  • Petiols of 4 days old seedlings of Brassica napus cv Westar grown on growth medium B (MSB5 salts (Duchefa), 3% sucrose (Duchefa), 0 8% oxoidagar (Oxoid GmbH), pH 5,8) are cut Petiois are dipped for 2-3 seconds in the Agrobacterium solution and afterwards put into solid medium for co-cultivation (co-cultivation medium supplemented with 1 6% Oxoidagar) The co- cultivation lasts 3 days (at 24°C and about 50 ⁇ Mol/m 2 s light intensity) Afterwards petiols are transferred to co-cultivation medium supplemented with the appropriate selection agent (18 mg/L kanamycin (Duchefa) for plants comprising the nptll marker kanamycin for plants carrying the nptll resistance marker, or 0 3 to 30 mM D-amino acids, as described below) for plants comprising an expression cassette for the dao1 gene from
  • Transformed petioles are incubated on the selection medium for four weeks at 24°C This step is repeated until shoots appear
  • Shoots are transferred to A6 medium (MS salts (Sigma Aldrich), 20 g/L sucrose, 100 mg/L myo-inositol (Duchefa), 40 mg/L ade- nmesulfate (Sigma Aldrich), 500 mg/L MES, 0 0025 mg/L BAP (Sigma), 5 g/L oxoida- gar (Oxoid GmbH), 150 mg/L timetin (Duchefa), 0 1 mg/L IBA (indol butyric acid, Duchefa), pH 5,8) supplemented with the appropriate selection agent (18 mg/L kanamycin (Duchefa) for plants comprising the nptll marker kanamycin for plants carrying the nptll resistance marker, or 0 3 to 30 mM D-amino acids, as described below) until they elongated
  • EXAMPLE 2 Growth conditions for plants for tissue-specific expression analysis
  • seedlings are grown at a 16 h light / 8 h dark cycle (Philips 58W/33 white light) at 22°C and 120 rpm and harvested after 3 weeks
  • seeds are sown on standard soil (Type VM 1 Manna-ltalia, Via S.
  • the regenerated transgenic linseed and rape seed plants are tested in tissue culture for early leakiness.
  • 3 individual plants per single or multi insertion line (5-15 lines in total per construct) are analyzed in the T1 to T3 generation regarding the potential expression of the promoter candidates in all non-seed tissues as well as in different phases of seed development - Mature seeds
  • embryos from e g linseed capsule and rapeseed siliques are isolated from different stages of fruit development based on visual parameters and sorted to the following stages of embryo development " early, young, medium, late and mature The promoters are also checked for their inducibility by biotic and abiotic stress via ABA spraying on [eaves
  • EXAMPLE 3 Demonstration of expression profile
  • a promoter of the useful to operably link the promoter or its fragments to a reporter gene which can be employed to monitor its expression both qualitatively and quantitatively
  • bacterial ⁇ -glucuromdase is used (Jefferson 1987)
  • ⁇ -glucuronidase activity can be monitored in planta with chromogenic substrates such as 5-bromo-4-Chloro-3- ⁇ ndolyl- ⁇ -D- glucuronic acid during corresponding activity assays (Jefferson 1987)
  • chromogenic substrates such as 5-bromo-4-Chloro-3- ⁇ ndolyl- ⁇ -D- glucuronic acid during corresponding activity assays (Jefferson 1987)
  • tissue specificity plant tissue is dissected embedded, stained and analyzed as described (e g , Baumlein 1991)
  • MUG methylumbelliferyl glucuronide
  • MU methylumbeiliferone
  • genomic DNA is isolated from Arabidopsis thaliana (ecotype Columbia) or Brassica napus as described (Galbiati 2000) The isolated genomic DNA is employed as matrix DNA for a polymerase chain reaction (PCR) mediated amplification using the oligonucleotide primers and protocols indicated below (Table 3)
  • Amplification is carried out as follows 100 ng genomic DNA 1X PCR buffer 2,5 mM MgCI 2 , 200 ⁇ M each of dATP dCTP dGTP und dTTP 10 pmol of each oligonucleotide primers 2 5 Units Pfu DNA Polymerase (Stratagene) in a final volume of 50 ⁇ l
  • the resulting PCR-products are digested with the restriction endonucleases specified in the Table above (Table 3) and cloned into the vector pSUN0301 (SEQ ID NO 147) (pre ⁇ d ⁇ gested with the same enzymes) upstream and in operable linkage to the g!u- curonidase (GUS) gene Following stable transformation of each of these constructs into Arabidopsis thahana tissue specificity and expression profile was analyzed by a histochemical and quantitative GUS-assay, respectively
  • EXAMPLE 5 Expression profile of the various promoter: :GUS constructs in stably transformed A. thaliana, rapeseed and linseed plants
  • the transcription regulating sequences of the invention are seed-specific promoters in both Arabidopsis rapeseed and linseed
  • the GUS expression pattern investigated in Arabidopsis, rapeseed and linseed In general, no to very weak GUS expression was detected in early and young embryos Middle strong to strong GUS expression was found in middle, late and mature embryos
  • the promoter BnAK700 shows medium expression only or preferably in the coleoptile In the later stages medium to strong expression is found in the entire embryo
  • BnAK700 only slight ieakiness has been found in a few events (in roots, stem, leaf flower, capsule) In the majority of events no Ieakiness is observed
  • AtAK especially SEQ ID NO 1
  • BnAK400 BnAK400
  • Vectors used for expression of full-length "candidate genes" of interest in plants are designed to overexpress the protein of interest and are of two general types, biolistic and binary, depending on the plant transformation method to be used
  • biolistic transformation For biolistic vectors, the requirements are as follows.
  • E coll a backbone with a bacterial selectable marker (typically, an antibiotic resistance gene) and origin of replication functional in Escherichia coil (E coll , e g , CoIEI), and
  • a plant-specific portion consisting of a a gene expression cassette consisting of a promoter (eg ZmUBIint MOD), the gene of interest (typically, a full-length cDNA) and a transcriptional terminator (e g , Agrobacterium tumefaciens nos terminator), b a plant selectable marker cassette, consisting of a suitable promoter, selectable marker gene (e g D-amino acid oxidase, daol ) and transcriptional terminator (eg nos terminator)
  • a promoter eg ZmUBIint MOD
  • the gene of interest typically, a full-length cDNA
  • a transcriptional terminator e g , Agrobacterium tumefaciens nos terminator
  • b a plant selectable marker cassette, consisting of a suitable promoter, selectable marker gene (e g D-amino acid oxidase, daol ) and transcriptional terminator (eg nos terminator
  • Vectors designed for transformation by Agrobacterium tumefaciens consist of
  • Vectors designed for reducing or abolishing expression of a single gene or of a family or related genes are also of two general types corresponding to the methodology used to downregulate gene expression antisense or double- stranded RNA interference (dsRNAi)
  • a full-length or partial gene fragment (typically, a portion of the cDNA) can be used in the same vectors described for full-length expression, as part of the gene expression cassette
  • a full-length or partial gene fragment (typically, a portion of the cDNA)
  • the coding region of the gene or gene fragment will be in the opposite o ⁇ en- tation relative to the promoter, thus mRNA will be made from the non-coding (an- tisense) strand in planta
  • dsRNAi vectors a partial gene fragment (typically, 300 to 500 basepairs long) is used in the gene expression cassette and is expressed in both the sense and an- tssense orientations, separated by a spacer region (typically, a plant intron eg the OsSHI intron 1 , or a selectable marker, eg conferring kanamycin resistance) Vectors of this type are designed to form a double-stranded mRNA stem, resulting from the basepairing of the two complementary gene fragments in planta
  • a spacer region typically, a plant intron eg the OsSHI intron 1 , or a selectable marker, eg conferring kanamycin resistance
  • Biolistic or binary vectors designed for overexpression or knockout can vary in a number of different ways including eg the selectable markers used in plant and bacteria, the transcriptional terminators used in the gene expression and plant seiectabfe marker cassettes, and the methodologies used for cloning in gene or gene fragments of inter- est (typically, conventional restriction enzyme-mediated or GatewayTM recombmase- based cloning)

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Abstract

L'invention concerne des cassettes d'expression comprenant des séquences nucléotidiques de régulation de transcription avec des profils spécifiques à une semence ou préférentiels d'une semence dans des plantes pouvant être obtenues à partir du gène Arabidopsis thahana décrit par le locus At5g01670 ou ses orthologues à partir d'autres espèces de plantes (telles que Brassica napus). Les séquences nucléotidiques de régulation de transcription présentent, de préférence, une forte activité d'expression, en particulier, chez les semences et les tissus de semence.
PCT/EP2006/060185 2005-02-23 2006-02-22 Cassettes d'expression pour expression preferentielle d'une semence chez les plantes WO2006089911A2 (fr)

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WO2010000708A2 (fr) * 2008-07-01 2010-01-07 Basf Plant Science Gmbh Promoteurs de brassica napus pour l'expression de gènes spécifique de graines
WO2010000708A3 (fr) * 2008-07-01 2010-04-22 Basf Plant Science Gmbh Promoteurs de brassica napus pour l'expression de gènes spécifique de graines
US8853383B2 (en) 2008-07-01 2014-10-07 Basf Plant Science Gmbh Promoters from Brassica napus for seed specific gene expression
EP2826864A3 (fr) * 2008-07-01 2015-05-06 BASF Plant Science GmbH Promoteurs dérivés de brassica napus pour l'expression génique spécifique de semences

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