Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
• • 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
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

• the present invention relates to the field of molecular biology, more particularly to regulation of gene expression in fungi.
• the novel compositions and methods can be used to improve fungal species by recombinant technologies known to those of skill in the art, such as the improved pathogen, pest, and pesticide resistance, yield quantity and quality, extended produce shelf life, improved culinary, nutritional, and medicinal value, and the like, as well as the commercial production of heterologous proteins.
• bisporus have been only moderately successful and may not be sufficient in the long term. This is because conventional breeding techniques for fungi are highly time consuming, and because the genetic variation in commercially available strains is limited, offering little advancement by selection (Horgen et al. "Homology between mitochondrial DNA of A. bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis" Curr Genet. 1991. 19:495- 502).
• the main obstacle to effective breeding strategies is the rather abnormal life cycle involving the unusual simultaneous segregation of either parental nucleus into one basidiospore. After outgrowth of this basidiospore,
• heterokaryotic mycelium is formed containing nuclei and genetic characteristics that do not differ from those present in the parental mycelium. In addition, only little recombinational activity is observed during meiosis (Summerbell et al. Genetics, Oct. 123(2) 1989 pp. 293- 300).
• the primary promoter used in fungal transformation to date is the glyceraldehyde-3 -phosphate dehydrogenase (gpd) promoter.
• gpd glyceraldehyde-3 -phosphate dehydrogenase
• the gpd promoter is a strong promoter that can be induced by any carbon source and has been widely used in the expression of heterologous proteins in Saccharomyces cerevisiae, Pichia pastoris and other yeasts.
• the gpd genes have also been cloned from basidiomycetous fungi, including Schizophyllum commune, Phanerochaete chrysosporium, Agaricus bisporus (Harmsen et al, 1992), and Lentinula edodes (Hirano et al, 1999).
• basidiomycetous fungi including Schizophyllum commune, Phanerochaete chrysosporium, Agaricus bisporus (Harmsen et al, 1992), and Lentinula edodes (Hirano et al, 1999).
• genetic transformation using homologous gpd promoter was reported to be successful only in A. bisporus, Flammulina velutipes and L. edodes (Hirano et al., 2000, Kuo et al., 2004, van de Rhee et al, 1996).
• heterologous promoters have been used for the expression of drug-resistant marker genes, the genetic transformation is not sufficient to express heterologous genes (Ruiz-Diez, 2002). To sufficiently and effectively express a heterologous gene, it is important for a host cell to recognize the promoter sequence by its transcriptional machinery. Chun-Yi Kuo et al. demonstrated that a heterologous gene, hygromycin B phosphotransferase gene (hpt), can be expressed in F. velutipes (Kuo et al., 2004). However, it was found that although the gpd genes in basidiomycetous fungi are highly similar, these gene differ significantly in their promoter regions.
• a further object of this invention is to provide mechanisms for application of transgenic techniques such as those applied to bacteria, non- filamentous fungi (yeast), plants, and animals to increase yield, disease, and pest resistance, product quality, shelf life, or culinary, nutritional, or medicinal value, to produce heterologous proteins commercially, or other such protocols.
• transgenic techniques such as those applied to bacteria, non- filamentous fungi (yeast), plants, and animals to increase yield, disease, and pest resistance, product quality, shelf life, or culinary, nutritional, or medicinal value, to produce heterologous proteins commercially, or other such protocols.
• polynucleotide constructs for use in such transgenic protocols.
• the invention comprises an isolated regulatory element/promoter comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence comprising the nucleic acid sequence as shown in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and b) a fragment of the polynucleotide sequence of a) capable of regulating transcription of an operably linked polynucleotide molecule in a fungal cell.
• the invention also comprises a polynucleotide construct comprising the promoter of the invention operably linked to a heterologous polynucleotide molecule.
• the invention also includes a transformed cell comprising the construct of the invention as well as methods of transformation using the constructs of the invention and heterologous protein products produced thereby.
• the invention is directed to a promoter from an A. bisporus lectin-encoding gene, useful as a regulatory region and providing for expression of a nucleotide sequence of interest.
• the accumulation of a heterologus protein or nucleotide sequence of interest is driven to the fruit body tissue.
• the invention is further directed to functional fragments that drive expression of a heterologus protein or nucleotide sequence of interest - in the fruit body tissue.
• Expression cassettes that incorporate the promoter driving expression of a nucleotide sequence, plants, fungi or bacterial cells expressing same, and methods of use in modulating expression of nucleotides sequences are within the scope of the invention.
• Figure 1 is a 2-D gel analysis of proteins extracted from Agaricus bisporus fruiting bodies. Protein separation on 2-D SDS PAGE was performed according to the
• Figure 2 is a map of binary vector pAGN-750 which includes the promoter of the invention linked to a reporter gene.
• Figure 3 is a photograph showing expression of a GUS reporter gene in the vegetative mycelium of pAGN-750 events grown on filter paper.
• Figure 4 is a photograph showing GUS staining of fruiting body slices for independent transgenic events of pAGN-750.
• Figure 5 is a map of Binary vector pAGN-755 which includes the promoter of the invention linked to a target gene of interest.
• Figure 6 depicts the sequences of the lectin promoter. 5' end DNA sequences in each vector are underlined and the corresponding vector identified. All vectors share the same 3' sequence as presented. pAGN-755 and pAGN-750 share the identical sequence.
• Figure 7 shows the GUS staining of liquid mycelium culture of the events generated from truncated promoters shown in Table 5.
• compositions of the present invention comprise novel nucleotide sequences for fungal regulatory elements natively associated with the nucleotide sequences coding for A. bisporus lectin protein.
• the regulatory element drives transcription in a fungal tissue or more specifically achieves expression of a protein or transcript of interest in a fungal fruiting body tissue
• said regulatory element comprises a nucleotide sequence selected from the group consisting of: a) sequences natively associated with, and that regulate expression of DNA coding for lectin protein); b) the sequence of SEQ ID NO : 1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; or c) a sequence comprising a functional fragment of the nucleotide sequence set forth in SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
• Further embodiments are to expression cassettes, transformation vectors, fungi, bacteria, and bacterial and fungal cells comprising the above nucleotide sequences.
• the invention is further to methods of using the sequence in plants, fungi, and bacterial tissues and their cells.
• a method for expressing an isolated nucleotide sequence in a fungus using the regulatory sequences disclosed herein comprises transforming a fungal cell with a transformation vector that comprises an isolated nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed fungus from the transformed fungal cell.
• the regulatory sequences are useful for controlling the expression of endogenous as well as exogenous products in a fruiting body tissue.
• a native DNA sequence within the tissues of a fungus it might be desirable to inhibit expression of a native DNA sequence within the tissues of a fungus to achieve a desired phenotype.
• such inhibition might be accomplished, for example, with transformation of a fungus to comprise the promoter operably linked to an antisense nucleotide sequence, hairpin, R A interfering or other nucleic acid molecule, such that expression of the molecule interferes with translation of the mR A of the native DNA sequence or otherwise inhibits expression of same in a subset of the cells of the fungus.
• a sequence of interest Under the regulation of the regulatory element will be a sequence of interest, which will provide for modification of the phenotype of a fungus. Such modification includes modulating the production of an endogenous product, as to amount, relative distribution, or the like, or production of an exogenous expression product to provide for a novel function or product in a fungus.
• Such a promoter is useful for a variety of applications, such as production of transgenic fungi with desired traits, including, for example, altered content, protein quality, cell growth or nutrient quality or preferably for the production of heterologous proteins.
• fruiting body tissue promoter is intended expression that is capable of transcribing an operatively linked nucleotide sequence efficiently and thereby achieving accumulation of a protein or nucleotide sequence of interest at high levels in the described tissues, here the fruiting body tissue cells.
• Tissue can refer to a cell of a particular tissue.
• regulatory element is intended sequences responsible for expression of the linked nucleic acid molecule including, but not limited to, promoters, terminators, enhancers, introns, and the like.
• promoter is intended a regulatory region of DNA capable of regulating the transcription of a sequence linked thereto. It usually comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence.
• a promoter may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate and further include elements that impact spatial and temporal expression of the linked nucleotide sequence. It is recognized that having identified the nucleotide sequences for the promoter region disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5' region upstream from the particular promoter region.
• the promoter region disclosed here may comprise upstream regulatory elements such as those responsible for tissue and temporal expression of the nucleic acid molecule, and may include enhancers, the DNA response element for a transcriptional regulatory protein, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, activator sequence and the like.
• the promoter elements that enable such expression can be identified, isolated, and used with other core promoters to confirm expression in fruiting body tissue.
• core promoter is meant the minimal sequence required to initiate transcription, such as the sequence called the TATA box, which is common to promoters in genes encoding proteins.
• the upstream promoter of SB-LEG can optionally be used in conjunction with its own or core promoters from other sources.
• the promoter may be native or non-native to the cell in which it is found.
• the isolated promoter sequence of the present invention can be modified to provide for a range of expression levels of the isolated nucleotide sequence. Less than the entire promoter region can be utilized and the ability to achieve expression in the fruit body tissue retained. It is recognized that expression levels of mR A can be modulated with specific deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts.
• a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts. Generally, at least about 20 nucleotides of an isolated promoter sequence will be used to drive expression of a nucleotide sequence.
• Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like.
• the promoter of the present invention can be isolated from the 5' region of its native coding region or 5' untranslated region (5' UTR). Likewise the terminator can be isolated from the 3' region flanking its respective stop codon.
• isolated refers to material, such as a nucleic acid or protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment, or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in a cell other than the locus native to the material.
• Methods for isolation of promoter regions are well known in the art. One method is the use of primers and genomic DNA used in conjunction with the Genome Walker Kit.TM. (Clonetech).
• the A. bisporus lectin promoter is set forth in SEQ ID NO: 1 , with functional truncations shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 4.
• the minimal core promoter is 45 nucleotides in length, with the full-length promoter being approximately 135 to 180 nucleotides in length. 160 is 818 base pair nucleotides in length.
• the lectin promoter was isolated from the A. bisporus lectin coding region.
• the regulatory regions of the invention may be isolated from any plant animal or fungi, but is preferably filamentous fungi, including, but not limited to the fungi of the genera Flammulina, Agaricus, Lentinula, and Pleurotus. Promoters isolated from one fungal species can be expected to express in another fungal species.
• Regulatory sequences from other fungi may be isolated according to well-known techniques based on their sequence homology to the coding region of the sequences set forth herein. In these techniques, all or part of the known coding sequence is used as a probe that selectively hybridizes to other sequences present in a population of cloned genomic DNA fragments (i.e. genomic libraries) from a chosen organism. Methods are readily available in the art for the hybridization of nucleic acid sequences.
• Functional variants of the regulatory sequences are also encompassed by the compositions of the present invention.
• Functional variants include, for example, the native regulatory sequences of the invention having one or more nucleotide substitutions, deletions or insertions.
• Functional variants of the invention may be created by site-directed mutagenesis, induced mutation, or may occur as allelic variants (polymorphisms).
• a "functional fragment” is a regulatory sequence variant formed by one or more deletions from a larger regulatory element.
• the 5' portion of a promoter up to the TATA box near the transcription start site can be deleted without abolishing promoter activity, as described by Opsahl-Sorteberg et al, "Identification of a 49-bp fragment of the HvLTP2 promoter directing aleurone cell specific expression” Gene 341 :49-58 (2004).
• Such fragments should retain promoter activity, particularly the ability to drive expression in the select tissue. Activity can be measured by Northern blot analysis, reporter activity measurements when using transcriptional fusions, and the like. See, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• Functional fragments can be obtained by use of restriction enzymes to cleave the naturally occurring regulatory element nucleotide sequences disclosed herein; by synthesizing a nucleotide sequence from the naturally occurring DNA sequence; or can be obtained through the use of PCR technology See particularly, Mullis et al. (1987) Methods Enzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New York).
• a routine way to remove part of a DNA sequence is to use an exonuclease in combination with DNA amplification to produce unidirectional nested deletions of double stranded DNA clones.
• a commercial kit for this purpose is sold under the trade name Exo-Size.TM. (New England Biolabs, Beverly, Mass.). Briefly, this procedure entails incubating exonuclease III with DNA to progressively remove nucleotides in the 3' to 5' direction at 5' overhangs, blunt ends or nicks in the DNA template. However, exonuclease III is unable to remove nucleotides at 3', 4-base overhangs. Timed digests of a clone with this enzyme produces unidirectional nested deletions.
• the entire promoter sequence or portions thereof can be used as a probe capable of specifically hybridizing to corresponding promoter sequences.
• probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
• Such probes can be used to amplify corresponding promoter sequences from a chosen organism by the well-known process of polymerase chain reaction (PCR). This technique can be used to isolate additional promoter sequences from a desired organism or as a diagnostic assay to determine the presence of the promoter sequence in an organism. Examples include hybridization screening of plated DNA libraries (either plaques or colonies; see e.g. Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, eds., Academic Press). Primers used in isolating the promoter of the present invention are shown below.
• terminator sequence a nucleotide sequence that functions as a polyadenylation signal and signals the end of transcription. Functional fragments that retain such activity are within the scope of the invention.
• the regulatory elements disclosed in the present invention are useful in the genetic manipulation of any fungus when operably linked with an isolated nucleotide sequence of interest whose expression is to be controlled to achieve a desired phenotypic response.
• operably linked is intended a functional linkage between a regulatory region and a second sequence, wherein the regulatory sequence initiates and mediates
• the regulatory elements of the invention can be operably linked to the isolated nucleotide sequence of interest in any of several ways known to one of skill in the art.
• the isolated nucleotide sequence of interest can be inserted into a site within the genome which is 3' to the promoter of the invention using site specific integration as described in U.S. Pat. No. 6,187,994.
• nucleotide sequence of interest refers to a nucleic acid molecule (which may also be referred to as a polynucleotide) which can be an RNA molecule as well as DNA molecule, and can be a molecule that encodes for a desired polypeptide or protein, but also may refer to nucleic acid molecules that do not constitute an entire gene, and which do not necessarily encode a polypeptide or protein.
• the promoter when used in a homologous recombination process, the promoter may be placed in a construct with a sequence that targets an area of the chromosome in the fungi but may not encode a protein.
• the nucleotide sequence of interest can be optimized for fungi translation by optimizing the codons used for fungi and the sequence around the translational start site for fungi. Sequences resulting in potential mR A instability can also be avoided.
• the regulatory elements of the invention can be operably linked in expression cassettes along with isolated nucleotide sequences of interest for expression in the desired fungi or plant or animal.
• Such an expression cassette is provided with a plurality of restriction sites for insertion of the nucleotide sequence of interest under the transcriptional control of the regulatory elements.
• a specific result can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or co factors in fungi.
• This down regulation can be achieved through many different approaches known to one skilled in the art, including antisense, co-suppression, use of hairpin formations, or others, and discussed infra. Importation or exportation of a cofactor also allows for control of composition.
• the regulatory elements may be used with their native or other coding sequences to increase or decrease expression of an operably linked sequence in the transformed fungi, plant, seed or animal cell.
• genes of interest for the purposes of the present invention include for example, those 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, and other characteristics. Still other categories of transgenes include genes for inducing expression of exogenous products such as enzymes, cofactors, and hormones. Still others include those encoding an antibody, a secondary metabolite, a therapeutic compound, a biological macromolecule, a medical enzyme; or a gene that confers or contributes to a value-added trait.
• Examples include a secondary metabolite such as lectin, a therapeutic compound such as a vaccine, a biological macromolecule such as an interferon, endostatin or insulin, a medical enzyme such as a thrombolytic or cerebrosidase and a gene that confers resistance to pests, diseases, or herbicides such as a pesticidal compound Bacillus thuringiensis protein (Bt toxin).
• a secondary metabolite such as lectin
• a therapeutic compound such as a vaccine
• a biological macromolecule such as an interferon, endostatin or insulin
• a medical enzyme such as a thrombolytic or cerebrosidase
• a gene that confers resistance to pests, diseases, or herbicides such as a pesticidal compound Bacillus thuringiensis protein (Bt toxin).
• Bacillus thuringiensis protein Bacillus thuringiensis protein
• Means for increasing or inhibiting a protein are well known to those skilled in the art and, include, but are not limited to: transgenic expression, antisense suppression, co- suppression, R A interference, gene activation or suppression using transcription factors and/or repressors; mutagenesis including, but not limited to, transposon tagging; directed and site-specific mutagenesis, chromosome engineering (see Nobrega et. al, Nature 431 :988-993(04)), homologous recombination, TILLING (Targeting Induced Local Lesions In Genomes), and biosynthetic competition to manipulate the expression of proteins.
• RNA sequences are well known to one of skill in the art, including but not limited to knock-outs (such as by insertion of a transposable element such as Mu, Vicki Chandler, The Maize Handbook ch. 118 (Springer- Verlag 1994) or other genetic elements such as a FRT, Lox or other site specific integration site; RNA sequences, including but not limited to knock-outs (such as by insertion of a transposable element such as Mu, Vicki Chandler, The Maize Handbook ch. 118 (Springer- Verlag 1994) or other genetic elements such as a FRT, Lox or other site specific integration site; RNA sequences, including but not limited to knock-outs (such as by insertion of a transposable element such as Mu, Vicki Chandler, The Maize Handbook ch. 118 (Springer- Verlag 1994) or other genetic elements such as a FRT, Lox or other site specific integration site; RNA sequences, including but not limited to knock-outs (such as by insertion of a transposable element such
• the nucleotide sequence operably linked to the regulatory elements disclosed herein can be an antisense sequence for a targeted gene.
• antisense DNA nucleotide sequence is intended a sequence that is in inverse orientation to the 5'-to-3' normal orientation of that nucleotide sequence. When delivered into a cell, expression of the antisense DNA sequence prevents normal expression of the DNA nucleotide sequence for the targeted gene.
• the antisense nucleotide sequence encodes an R A transcript that is complementary to and capable of hybridizing with the endogenous messenger RNA (mRNA) produced by transcription of the DNA nucleotide sequence for the targeted gene.
• mRNA messenger RNA
• production of the native protein encoded by the targeted gene is inhibited to achieve a desired phenotypic response.
• the regulatory sequences disclosed herein can be operably linked to antisense DNA sequences to reduce or inhibit expression of a native protein in fungi.
• co-suppression can be achieved by linking the promoter to a DNA segment such that transcripts of the segment are produced in the sense orientation and where the transcripts have at least 65% sequence identity to transcripts of the endogenous gene of interest, thereby suppressing expression of the endogenous gene in said fungal cell.
• the endogenous gene targeted for co-suppression may be a gene encoding any protein that accumulates in the fungal species of interest.
• RNA interference and promoter silencing involve the silencing of a targeted gene by spliced hairpin RNA's and similar methods also called RNA interference and promoter silencing (see Smith et al. (2000) Nature 407:319-320, Waterhouse and Helliwell (2003)) Nat. Rev. Genet. 4:29-38; Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959- 13964; Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;
• the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene.
• the miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is
• miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the R A
• the regulatory region of the invention may also be used in conjunction with another promoter.
• the fungal selection marker and the gene of interest can be both functionally linked to the same promoter.
• the selection marker and the gene of interest can be functionally linked to different promoters.
• the expression vector can contain two or more genes of interest that can be linked to the same promoter or different promoters.
• the SB-LEG promoter described here can be used to drive the gene of interest and the selectable marker, or a different promoter used for one or the other.
• These other promoter elements can be those that are constitutive or sufficient to render promoter-dependent gene expression controllable as being cell-type specific, tissue-specific or time or developmental stage specific, or being inducible by external signals or agents.
• Promoter elements employed to control expression of product proteins and the selection gene can be any compatible promoter. These can be fungal gene promoters, such as, for example, gpd promoter , or promoters from the tumor-inducing plasmids from Agrobacterium tumefaciens, such as the nopaline synthase, octopine synthase and mannopine synthase promoters (Velten, J. and Schell, J.
• the expression cassette may also include at the 3' terminus of the isolated nucleotide sequence of interest, a transcriptional and translational termination region functional in fungi.
• the termination region can be native with the promoter nucleotide sequence of the present invention, can be native with the DNA sequence of interest, or can be derived from another source.
• any convenient termination regions can be used in conjunction with the promoter of the invention, and are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also: Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell
• the expression cassettes can additionally contain 5' leader sequences.
• leader sequences can act to enhance translation.
• Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region), Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco etch virus), Allison et al. (1986); MDMV leader (Maize dwarf mosaic virus), Virology 154:9-20; human immunoglobulin heavy-chain binding protein (BiP), Macejak et al.
• EMCV leader Engelphalomyocarditis 5' noncoding region
• potyvirus leaders for example, TEV leader (Tobacco etch virus), Allison et al. (1986); MDMV
• the cassette can also contain sequences that enhance translation and/or mRNA stability such as introns.
• the expression cassette can further comprise a coding sequence for a transit peptide.
• transit peptides are well known in the art and include, but are not limited to: the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase, and the like.
• the various DNA fragments can be any DNA fragment.
• adapters or linkers can be employed to join the DNA fragments or other manipulations can be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
• in vitro mutagenesis, primer repair, restriction digests, annealing, and resubstitutions, such as transitions and transversions can be involved.
• Reporter genes can be included in the transformation vectors. Examples of suitable reporter genes known in the art can be found in, for example: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1- 33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J. 9:2517- 2522; Kain et al. (1995) Bio Techniques 19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.
• Selectable marker genes for selection of transformed cells or tissues can be included in the transformation vectors. These can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to: genes encoding resistance to chloramphenicol, Herrera Estrella et al. (1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820; hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5: 103-108; Zhijian et al. (1995) Plant Science 108:219-227; streptomycin, Jones et al. (1987) Mol. Gen. Genet. 210:86-91;
• a promoter of the invention when linking a promoter of the invention with a nucleotide sequence encoding a detectable protein, expression of a linked sequence can be tracked in fungi, thereby providing useful screenable or scorable markers.
• the expression of the linked protein can be detected without the necessity of destroying tissue.
• the promoter can be linked with detectable markers including a ⁇ - glucuronidase, or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known (Jefferson et al, 1986, Proc. Natl. Acad. Sci.
• GFP green fluorescent protein
• a lux gene which encodes a luciferase, the presence of which may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry (Teeri et al. (1989) EMBO J. 8:343); DS-RED EXPRESS (Matz et al.
• the present invention provides vectors capable of expressing genes of interest under the control of the regulatory elements.
• the vectors should be functional in fungal cells.
• Vectors and procedures for cloning and expression in E. coli are discussed in Sambrook et al. (supra). The
• transformation vector comprising the regulatory sequence of the present invention operably linked to an isolated nucleotide sequence in an expression cassette, can also contain at least one additional nucleotide sequence for a gene to be co-transformed into the organism.
• additional sequence(s) can be provided on another transformation vector.
• Vectors that are functional in fungi can be binary plasmids derived from Agrobacterium. Such vectors are capable of transforming fungal cells. These vectors contain left and right border sequences that are required for integration into the host chromosome. At minimum, between these border sequences is the gene to be expressed under control of the regulatory elements of the present invention.
• a selectable marker and a reporter gene are also included.
• a transformation vector comprising the particular regulatory sequences of the present invention, operably linked to an isolated nucleotide sequence of interest in an expression cassette, can be used to transform any fungi.
• genetically modified fungi, fungal cells, fungal tissue, and the like can be obtained. Transformation protocols can vary depending on the type of cell. Suitable methods of transforming fungal cells include microinjection, Crossway et al. (1986) Biotechniques 4:320-334;
• Agrobacterium-rnQdiated transformation see for example, Townsend et al. U.S. Pat. No. 5,563,055; Romaine et al US Pat No. 7,700,439, direct gene transfer, Paszkowski et al.
• the cells that have been transformed can be grown into fungi in accordance with conventional methods. These fungi can then be reproduced with the same transformed strain or different strains. The resulting fungi having constitutive expression of the desired phenotypic characteristic can then be identified. Two or more generations can be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited.
• Example 1 Identification of a highly expressed lectin gene sequence from Agaricus bisporus
• a lectin gene was determined to be highly expressed in the fruiting body of A. bisporus.
• Fruiting bodies commercial intermediate hybrid strain selected were near maturity, but without exposed gills.
• Freshly harvested fruit tissue was frozen in liquid nitrogen, pulverized using a mortar and pestle, and extracted with cold acetone with 20% trichloroacetate and 0.2% dithiothreitol and submitted to Alphalyse Inc. (Palo Alto, CA) for 2-D protein profiling.
• the resulting gel image is presented in Fig. 1.
• FIG. 1 2-D gel analysis of Agaricus bisporus fruiting body proteins.
• Proteins #4, #6 and #7 were among the most abundant and, therefore, were chosen for additional analysis, including peptide mapping and sequencing analysis carried out by Alphalyse Inc.
• the resulting peptide map and partial peptide sequence information of proteins #4, #6, and #7 were blast searched against a protein database created by Alphalyse Inc. and were shown to match the cDNA sequence of the A. bisporus lectin gene (U14936, Crenshaw et al. 1996).
• the three proteins shared an identical amino acid sequence. The observed separation of the lectin protein into three distinct protein spots probably reflects variation in post-translational modifications, such as glycosylation.
• Example 2 Isolation and sequence determination of the 5' upstream DNA region of the lectin gene encompassing the promoter
• genomic 5 ' upstream DNA region of the lectin gene was carried out using genome walking (APAgeneTM Gold Genome Walking Kit, BIO S&T Inc. Montreal, QC, Canada).
• APAgeneTM Gold Genome Walking Kit BIO S&T Inc. Montreal, QC, Canada.
• TTCGTTCAACGGGACGGAAGAAGCCTTT -3 ' TCTGGTAGACGCGAATGCTGATGGTGTA-3 '
• lectin-F2 5 '- TCTGGTAGACGCGAATGCTGATGGTGTA-3 '
• Genomic DNA of A. bisporus was extracted using an AquaGenomic Kit (MultiTarget Pharmaceuticals LLC, Salt Lake City, Utah) and used as the template for genome-walking PCR according to the protocol provided with the APAgeneTM Gold Genome Walking Kit.
• the 15 ⁇ of primary PCR mixture contained 5 ⁇ of 3x APAgeneTM Gold buffer, 0.3 ⁇ of 50 x PCR annealing enhancer, 0.4 ⁇ of 40 mM dNTPs, 0.7 ⁇ of 20 pmol/ ⁇ of lectin-Fl, 1 ⁇ of genomic DNA (-100 ng), 0.2 ⁇ (1 U) of Taq DNA polymerase (Platinum Taq, Invitrogen, Carlsbad, CA) and 7.4 ⁇ of water.
• the PCR program consisted of one cycle at 94 C for 4 min followed by 25 cycles at 94 C for 30 sec, 63 C for 10 sec, ramp to 66 C at 0.1 ° C/sec, and 68 C for 3 min.
• PCR product from A, B, C and D tubes was transferred to tubes labeled Al, Bl, CI, and Dl, respectively.
• a 1 ⁇ aliquot of lOx DRT primer digestion buffer was added to each tube with 0.5 ⁇ of digestion enzyme mix, and 8 ⁇ of water. The digestion mixtures were vortexed and incubated at 37 C for 30 min. The enzyme in the digestion mixtures was then heat-inactivated at 95 C for 5 min. Following the digestion reactions the 5 ' genomic DNA region upstream of the lectin gene was amplified by nested PCR.
• reaction mixture 15 ⁇ of reaction mixture were added to each of four tubes labeled A2, B2, C2, and D2, using 3x APAgene Gold buffer I for tubes A2 and B2, and APAgeneTM Gold buffer II for tubes C2 and D2.
• the 15 ⁇ of reaction mixture for the nested PCR contained 5 ⁇ of 3x APAgeneTM Gold buffer (I or II), 0.3 ⁇ of 50x PCR annealing enhancer, 0.3 ⁇ of 40 mM dNTPs, 0.4 ⁇ of 20 pmol/ ⁇ of lectin-F2 primer, 0.4 ⁇ of Universal Tagging Primer, 0.5 ⁇ of digested PCR product from tubes Al, Bl, Cl, or Dl, and 0.3 ⁇ of Taq DNA polymerase (Invitrogen) and 7.8 ⁇ of water.
• 3x APAgeneTM Gold buffer I or II
• 50x PCR annealing enhancer 0.3 ⁇ of 50x PCR annealing enhancer
• 0.3 ⁇ of 40 mM dNTPs 0.4 ⁇ of 20 pmol/ ⁇ of lectin-F2 primer
• 0.4 ⁇ of Universal Tagging Primer 0.5 ⁇ of digested PCR product from tubes Al, Bl, Cl, or
• PCR products were separated in a 1% agarose gel in lx TBE (1 x
• TRIS/BORATE/EDTA 54 g of Tris base, 27.5 g of boric acid, 20 ml of 0.5 M EDTA (pH 8.0) in 1000 ml ) by gel electrophoresis.
• the PCR products of reactions A2, C2 and D2 ranged in size from -400 bp to -600 bp. Amplicons were recovered from the agarose gel using a Quick Gel Extraction Kit (Invitrogen).
• PCR amplification products of the putative 5' genomic DNA regions upstream of the lectin genes were cloned into a pCR2.1 TOPO vector using a TOPO TA Cloning Kit (Invitrogen) following the manufacturer's protocol. Briefly, 4 ⁇ of PCR product recovered from the agarose gel was added to 6 ⁇ of pCR2.1 ligation reaction. After ligation for 5 min at room temperature, 3 ⁇ of each reaction mixture were used to transform ONE-SHOT DH5a chemical competent cells (Invitrogen).
• the transformed cells were plated and selected on LB (Growcells, Irvine, CA) agar plate containing 50 ⁇ g/ml of kanamycin with 40 ⁇ of 40 mg/ml of X-gal (MP Biomedicals, Solon, Ohio) spread on the surface. After overnight incubation at 37 C in the dark, white colonies were picked for plasmid DNA preparation. Plasmid DNA was digested with EcoRI and separated by 1% agarose gel electrophoresis in lx TBE (described above) to identify clones with the correct insert sizes. The clones containing PCR product from the A2 reaction were designated pAGN-743, those from the C2 reaction as pAGN-744 and those from D2 as pAGN-745.
• the nucleotide sequence of the lectin gene promoter was determined by sequencing the inserts in pAGN-743, pAGN-744 and pAGN745 using an M13R-reverse primer.
• pAGN745 clones (pAGN-745-1, -2 and - 3) contained the longest 5' genomic DNA region upstream of the lectin gene.
• the consensus sequence of the 5 ' genomic DNA region upstream of the lectin gene was generated using sequence information from pAGN-745 clones.
• the overlap of the consensus sequence with the 5 ' end cDNA sequence of lectin gene confirmed that the DNA fragment generated by genome walking represented the promoter region of the lectin gene.
• GenBank+EMBL+DDBJ+PDB sequences failed to show identity with any known sequences, indicating that the isolated sequence was a novel promoter.
• Example 3 Demonstrated ability of the lectin promoter to direct expression of the
• Binary vector pAGN-750 ( Figure 2) was constructed to test the ability of the lectin promoter (designated as pLctn) to drive the expression of a ⁇ -glucuronidase (GUS) reporter gene in A. bisporus.
• pLctn was amplified from pAGN-745 using primers Lctn- Bglll (5 '-AGCTTAGATCTGAACACGCG TCGTTTACCTCC-3') and Lctn-Ncol (5'- GTAAGTCCATGGTCTGCTCAACGTTATGAG TTAGTTG-3 '). Restriction enzyme sites, Bglll and Ncol, were incorporated into primers to facilitate cloning.
• the PCR product was digested with Bglll/Ncol and the digested product was used to replace the Bglll-Ncol fragment in GUS expression vector pAGN-030 to create pAGN-750.
• the pLctn sequence in pAGN-750 was confirmed by sequencing.
• pAGN-750 was introduced into Agrobacterium tumefaciens and then A. bisporus following the transformation procedure described by Chen et al. (2000). Transgenic events generated from pAGN-750 were analyzed for GUS expression (GUS protocol by Sean R. Gallagher, 1991) in the vegetative mycelium and reproductive fruiting body.
• mycelium of transgenic events was allowed to colonize filter paper for 10- 14 days on SM agar plate containing 30 ⁇ g/ml of hygromycin B.
• GUS expression in mycelium was evaluated based on the intensity of blue color present in the mycelial colony exposed to staining buffer on filter paper (Fig. 3). The results of this study showed that pLctn drove GUS expression in the vegetative growth stage of A. bisporus. GUS expression in fruit tissue was also evaluated. Mycelia of transgenic events were used to inoculate growth media for mushroom production.
• A, B and C represent fruiting body A, B, and C, respectively, of the event
• FIG. 4 GUS staining of fruiting body slices for independent transgenic events of pAGN- 750.
• Binary vector pAGN-755 ( Figure 5) was constructed to test the ability of pLctn to effect expression of an Aprotinin target gene.
• Construct pAGN-755 was synthesized by replacing the Bglll-Ncol promoter fragment in aprotinin expression vector pAGN-021 using the Bglll-NcoI-pLctn fragment released from pAGN-750.
• the pLctn sequence in pAGN-755 was confirmed by sequencing.
• pAGN-755 was introduced into A. tumefaciens and then A. bisporus after the transformation procedure described by Chen et al. (2000). Fruiting bodies were produced as described previously for a panel of transgenic events generated using pAGN-755.
• aprotinin expression in fruiting body tissue was analyzed using an enzyme-linked immunosorbent assay (ELISA) and Western blotting. High expression of aprotinin, up to 200 mg/kg of fresh fruiting body tissue, was observed (Table 3, Fig. 4).
• Example 5 Determination of the DNA sequence essential for lectin promoter activity
• the minimal region of the 5 ' genomic DNA region upstream of the lectin gene required for promoter activity was defined by deletion analysis.
• the following PCR primers were designed and synthesized in order to evaluate the capability of individual segments of this sequence to effect gene expression: Lctn-BglII-2, 5'- AGCTTAGATCTGTCCTGGC ACTAGTC ACAG-3 ' .
• Lctn-BglII-6 5'- AGCTTAGATCTTATAATTAGCACATACCC-3 * , Lctn-BglII-7, 5 ' - AGCTTAGATCT CGTCTTC AT AGAT ATGGCC AAGC ATC-3 ' , Lctn-BglII-8, 5 '-AGCTTAGATCT TGCTACTTGTAGATAAGTTTATCTACACC-3 ', Lctn-BglII-9, 5 '-
• Figure 6 shows the Lectin promoter sequences evaluated. 5 ' ends DNA sequence in each vector are underlined and the corresponding vector identified. All vectors share the same 3' sequence presented. pAGN-755 and pAGN-750 share the identical sequence. The constructs incorporating fragments of the 5' region of the lectin gene were transformed into A. tumefaciens and then A. bisporus as described previously. GUS expression driven by the three truncated promoter sequences was compared to that obtained with the full-length 5' genomic DNA region upstream of the lectin gene present pAGN-774. The sequences are shown in Table 5.
• Figure 7 shows mycelium GUS staining of the events generated from the truncated promoters. Mushroom fruit bodies generated from pAGN-1304 and pAGN-1305 also show very weak or no GUS expression in GUS staining (data not shown).

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