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WO2002042450A1 - Promoteurs du virus bacilliforme de la canne a sucre isoles a partir de cultivars de canne a sucre - Google Patents

Promoteurs du virus bacilliforme de la canne a sucre isoles a partir de cultivars de canne a sucre Download PDF

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Publication number
WO2002042450A1
WO2002042450A1 PCT/AU2001/001499 AU0101499W WO0242450A1 WO 2002042450 A1 WO2002042450 A1 WO 2002042450A1 AU 0101499 W AU0101499 W AU 0101499W WO 0242450 A1 WO0242450 A1 WO 0242450A1
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Prior art keywords
nucleic acid
seq
fragment
promoter
plant
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PCT/AU2001/001499
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WO2002042450A8 (fr
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Kathryn Susan Braithwaite
Robert Jason Geijskes
Grant Richard Smith
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Bureau Of Sugar Experiment Stations
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Priority to AU2002223272A priority Critical patent/AU2002223272A1/en
Publication of WO2002042450A1 publication Critical patent/WO2002042450A1/fr
Publication of WO2002042450A8 publication Critical patent/WO2002042450A8/fr

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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
    • 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
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8229—Meristem-specific, e.g. nodal, apical

Definitions

  • THIS INVENTION relates to plant virus promoters. More particularly, this invention relates to promoters isolated from sugarcane baciUiform virus (SCBV), plasmid vectors incorporating these plant viral promoters, methods of plant transformation, transgenic plants and parts thereof.
  • SCBV sugarcane baciUiform virus
  • Genetic regulatory elements typically include 5' untranslated sequences comprising transcription factor and RNA polymerase binding sites, enhancer/silencer elements, a TATA box and a CAAT box together with 3' polyadenylation sequences, transcription stop signals, translation start and stop signals, splice donor/acceptor sequences and the like.
  • a “promoter” or “promoter-active region” is an untranslated region of a gene usually 5' or upstream of the gene which functions to initiate transcription of a transcribable nucleic acid component of the gene.
  • a promoter typically includes at least an RNA polymerase binding site together with one or more transcription factor binding sites which modulate transcription in response to occupation by transcription factors.
  • promoters used in this fashion are CaMV35S promoter (Nagy et al. In: Biotechnology in plant science: relevance to agriculture in the eighties. Eds. Zaitlin et al. Academic Press, Orlando, 1985.), maize ubiquitin promoter (Ubi; Christensen & Quail, 1996, Transgenic Research 5 213) and the Emu promoter (Last et al., 1991, Theor. Appl. Genet. 81 581).
  • CaMV35S promoter CaMV35S promoter
  • maize ubiquitin promoter Ubi; Christensen & Quail, 1996, Transgenic Research 5 2183
  • Emu promoter Last et al., 1991, Theor. Appl. Genet. 81 581).
  • One problem associated with promoters used in plant genetic engineering is that very few are useful in monocotyledonous plants.
  • Monocots include commercially important plants such as cereals, sugarcane, bananas and pineapples. Clearly, many of these commercially-important monocots are the subjects of intensive genetic engineering to introduce desired traits such as disease and pest resistance, salt tolerance, sugar content and regulated flowering and fruit ripening, for example.
  • SCBV characteristically infects Saccharum or related genera (Lockhart & Olszewski, In: The Encyclopaedia of Virology Eds. Webster & Granoff (Academic Press ⁇ Y, 1994) and is a member of the badnavirus (BV) group of viruses characterized by non-enveloped, baciUiform shaped circular dsD ⁇ A viruses.
  • Badnaviruses also include Commelia yellow mottle virus
  • a feature of badnaviruses is that in an infected plant cell, a positive strand is transcribed from the genome, with formation of a negative strand (and hence genome replication) achieved via a BN- encoded reverse transcriptase. It is postulated that the process of reverse transcription introduces sequence variation into the BN genome.
  • BSN as being useful in the production of transgenic monocots.
  • the invention is therefore broadly directed to a promoter nucleic acid isolated from a badnavirus.
  • the invention provides an isolated nucleic acid selected from the group consisting of:-
  • the first aspect includes an isolated homolog, fragment or variant of an isolated nucleic acid selected from the group consisting of SEQ ID NOS: 1- 4.
  • the isolated fragment is selected from the group consisting of: (a) a Hindlll/Spel fragment of SEQ ID NO. 1 comprising nucleotides 856-1952;
  • the isolated nucleic acid fragment is a promoter- active fragment.
  • the invention provides an expression vector comprising an isolated nucleic acid of the first aspect.
  • the invention provides a chimeric gene comprising an isolated nucleic acid of the first aspect and an operably linked transcribable nucleic acid.
  • the invention provides an expression construct comprising the chimeric nucleic acid of the third aspect.
  • the invention provides a method of producing a transgenic plant including the step of transforming a plant cell or tissue with an isolated nucleic acid according to the first aspect.
  • the isolated nucleic acid of the fifth aspect is an expression construct according to the fourth aspect.
  • the transgenic plant is a dicotyledon.
  • the dicotyledon is tobacco.
  • the tobacco is Nicotiana tabacum.
  • the transgenic plant is a monocotyledon.
  • the monocotyledon is sugarcane. More preferably, the sugarcane is Saccharum spp. hi a sixth aspect, the invention provides a plant cell or tissue transformed with an isolated nucleic acid according to the first aspect.
  • the isolated nucleic acid of the sixth aspect is an expression construct according to the fourth aspect.
  • the plant cell or tissue is from a dicotyledon.
  • the dicotyledon is tobacco.
  • the tobacco is Nicotiana tabacum.
  • the plant cell or tissue is from a monocotyledon.
  • the monocotyledon is sugarcane.
  • the sugarcane is Saccharum spp.
  • the invention provides a transgenic plant transformed with an isolated nucleic acid according to the first aspect.
  • the isolated nucleic acid is an expression construct according to the fourth aspect.
  • the transgenic plant is a dicotyledon.
  • the dicotyledon is tobacco.
  • the tobacco is Nicotiana tabacum.
  • the transgenic plant is a monocotyledon.
  • the monocotyledon is sugarcane. More preferably, the sugarcane is Saccharum spp.
  • the invention provides a plant cell, fruit, leaf, root, shoot, flower, seed, cutting and other reproductive material useful in sexual or asexual propagation, progeny plants inclusive of FI hybrids, male-sterile plants and all other plants and plant products derivable from a transgenic plant produced in accordance with the invention.
  • the invention provides a method for isolating a plant viral promoter-active nucleic acid including the step of hybridizing a nucleic acid primer or probe derived from SEQ ID NOS. 1-4 to a target nucleic acid.
  • the nucleic acid primer or probe is SEQ ID NO. 5 or 6.
  • the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
  • the relative level of GUS expression is indicated by the number of +'s. -, +, ++, +++ and ++++ which correspond to no, low, low to medium, medium to high and high blue colour production respectively.
  • the construct providing the highest expression is determined first and all other samples at that time point are rated relative to that. Ratings are not comparable across the time points. 7 Note that a low level of endogenous GUS activity was detected in some negative control callus.
  • GUS expression in different sugarcane tissues after bombardment of embryogenic callus with constructs comprising SCBV-IM-derived promoters Relative GUS expression rating is based on an average of all individual GUS expression scores for the samples or plants transformed with each construct. Data from the original transformed callus and secondary callus is pooled. GUS expression patterns are rated by the number of +'s. -, +, ++, +++ and ++++ correspond to no, low, low to medium, medium to high and high blue colour production respectively. 2 The number of samples or plants positive for GUS out of the total number examined. Percent is given in brackets. Data from the original transformed callus and secondary callus is pooled.
  • Promoter activity is determined by measuring the NPTII levels by ELISA. Results are expressed as ng NPTII per mg total protein extracted. Callus, shoot and root results are based on an average of two samples. Leaf results are based on an average of four samples. Duplicate ELISA readings were taken for all tissues except roots. The strength of each promoter in each tissue relative to the Ubi promoter is also shown. 1 Result is based on only one callus sample.
  • Promoter activity is determined by measuring the NPTII levels by ELISA. Results are expressed as ng NPTII per mg total protein extracted. Results for the negative controls are based on an average of data from six "unshot” and "no DNA” plants. Results for all other constructs are based on an average of data from eight plants. The strength of each promoter in each tissue relative to the Ubi promoter is also shown. 1 Note that high background NPTII levels were detected in some "unshot” and "no DNA" controls.
  • Promoter activity is determined by measuring the NPTII levels by ELISA.
  • Results are expressed as ng NPTII per mg total protein extracted.
  • Results for the negative controls are based on an average of data from seven "unshot” and "no DNA plants”.
  • pEKN results are based on an average of data obtained from nine plants.
  • pIMPS-kn results are based on an average of data obtained from seven plants.
  • pIMNS-kn results are based on an average of data obtained from six plants.
  • Results for all other constructs are based on an average of data obtained from eight plants. The strength of each promoter in each tissue relative to Ubi is also shown.
  • FIG. 1 Nucleotide sequence of SCBV promoter [SEQ ID NO. 1] isolated from virion DNA purified from the noble sugarcane Ireng Maleng. The length of the promoter is 1952 bp if 5' Pstl and 3' Spel "overhang" nucleotides are included, or 1942 bp if these nucleotides are excluded. Fragments of the promoter (1097 bp Hindlll-Spel; 1699 bp Pstl-Sspl; 1243 bp Sacl-Sspl; 844 bp Hindl ⁇ l-Sspl; and 454 bp Ncol-Sspl fragments) were isolated and also tested for promoter activity. Fragment sizes comprise entire nucleotide sequence for respective restriction enzyme recognition site.
  • FIG. 2 Nucleotide sequence [SEQ ID NO. 2] of a PCR product amplified from SCBV DNA isolated from Ireng Maleng-infecting virions. A 416 bp fragment was also used in a gus expression construct as shown in FIG. 6.
  • FIG. 3 Nucleotide sequence [SEQ ID NO. 3] of a PCR product amplified from SCBV DNA isolated from IJ76-465-infecting virions. The entire PCR product was 949 bp [SEQ ID NO. 3], although only a 785 bp fragment or a 440 bp fragment was used in expression constructs as shown in FIG 6.
  • FIG. 4 Nucleotide sequence [SEQ ID NO. 4] of a PCR product amplified from SCBV DNA isolated from IJ76-468-infecting virions. A 418 bp fragment was also used in a gus expression construct as shown in FIG. 6.
  • FIG. 5 Cloning strategies used to prepare SCBV-DVI promoter-g ⁇ .5 expression constructs.
  • pSCBVIM12 a clone comprising most of the SCBV-DVI genome, was subcloned with Pstl and Spel and the promoter fragment [SEQ ID NO. 1] fused upstream of the gus reporter gene and nos terminator to give pIMPS- gn.
  • pIMPS-gn was further subcloned by digesting with Hindlll to give pIMHS- gn, which is a fragment of SEQ ID NO. 1.
  • the first nucleotide of respective original restriction enzyme recognition sites in the SCBV-IM genome are shown at the top of the figure.
  • FIG. 6 Cloning strategies used to prepare three SCBV promoters based on
  • SEQ ID NOS. 2-4 obtained by PCR amplification from three virion preparations using the primers F8/R8, followed by cloning into pCR2.1. Two fragment lengths of each promoter were excised from pCR2.1 and fused upstream of the gus reporter gene and NOS terminator.
  • p465BB-gn comprises a fragment of SEQ ID NO. 3 inclusive of nucleotides 165 to 949.
  • a smaller fragment was obtained by PCR amplification from the nested deletion p465-21-7G using the primers T7 (modified to contain a Pstl site) and M13R, followed by restriction with Pstl and Spel to give p465PS-gn.
  • p465PS-gn comprises a fragment of SEQ ID NO. 3 inclusive of nucleotides 510 to 949.
  • pLMXB-gn comprises full-length SEQ ID NO. 2
  • p468XB-gn comprises full-length SEQ ID NO. 4.
  • Promoter fragments were respectively excised with Spel to give pDVISS-gn (SEQ ID NO. 2 fragment) and p468SS-gn (SEQ ID NO. 4 fragment) respectively.
  • Grey boxes represent the T7 and M13R priming sites within the vector and the hatched boxes represent SCBV priming sites.
  • F8 is within the RNase coding region and R8 corresponds to the tRNA met priming site for minus- strand replication. Sizes of each SCBV promoter fragment are shown above each construct. (P, Pstl; S, Spel; X, Xbal; B, BamHT).
  • FIG. 7 Cloning strategies used to prepare four SCBV-EVI promoter-npt/7 expression constructs using pEVlPS-gn, which comprises SEQ ID NO. 1, as the source of SCBV-EVI DNA.
  • pIMPS-gn was subcloned by digesting with Pstl and Sspl, Sacl and Sspl, Hindlll and Sspl, or Ncol and Sspl thereby producing fragments of SEQ ID NO. 1.
  • the SCBV fragments were fused upstream of the nptll gene and nos terminator to give pEVLPS-kn, pIMSS-kn, plMHS-kn and pEVINS-kn respectively.
  • the first nucleotide of respective original restriction enzyme recognition sites in the SCBV-EVI genome are shown at the top of the figure. Numbering of the plus-strand sequence follows badnavirus convention and
  • FIG. 8 Histochemical analysis of GUS expression driven by SCBV-IM promoters in transformed sugarcane. Constructs pIMPS-gn and pIMHS-gn were used throughout. Photographs A and B are of cultured tissue; photographs C-F are of glasshouse plants.
  • A transient GUS activity in callus
  • B stable GUS activity in shoots regenerating from callus
  • C base of youngest unfurled leaf (transverse view)
  • D another leaf showing activity in vascular bundles (transverse view)
  • ⁇ meristem
  • F leaf whorl.
  • FIG. 9 GUS staining in tobacco.
  • A Expression of pEViHS-gn in a small tobacco shoot at 3 weeks following cocultivation with Agrobacterium strain LBA4404 containing pPZP-IMHSgn;
  • B Transient expression of pMPS-gn;
  • C pIMHS-gn in tobacco cv. Ti68 leaf tissue at 48 h after bombardment.
  • the present invention is predicated, at least in part, by the discovery of promoter-active regions of sugarcane baciUiform virus (SCBV) isolates obtained from a number of different sugarcane cultivars and accessions.
  • SCBV sugarcane baciUiform virus
  • the isolated promoters displayed considerable sequence diversity between isolates, and when compared to prior art badnavirus promoters such as hereinbefore described.
  • the present inventors have sought to utilize the promoters of the invention in generating transgenic plants, and in doing so, have demonstrated considerable variation in promoter activity between isolates, between full-length promoters and fragments thereof, and with respect to activity in different plant tissues and stages of plant development.
  • the present invention may therefore enable the use of particular BV promoters to effectively "tailor" expression of transgenes according to the particular BV promoter isolate used.
  • Promoter nucleotide sequences The present invention provides isolated nucleic acids as set forth in
  • FIGS 1-4 are examples of "BV promoters" of the invention. Also provided are promoter-active fragments of SEQ ID NOS. 1-4 as described herein.
  • the BV promoters of the invention are capable of initiating, regulating or otherwise controlling transcription of a transcribable nucleic acid operably linked thereto, in a plant.
  • the plant may be of any taxonomic group, including dicots, monocots, ferns, lichens, gymnosperms and angiosperms.
  • the plant is of the genus Saccharum. In another embodiment, the plant is of the genus Nicotiana.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • polypeptide is also meant “protein”, either term referring to an amino acid polymer.
  • a “peptide” is a protein having no more than fifty (50) amino acids.
  • Proteins, polypeptides and peptides may comprise natural and/or non-natural amino acids as are well known in the art.
  • nucleic acid designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
  • a "transcribable nucleic acid' is an isolated nucleic acid which, when operably linked to a BV promoter of the invention, can be transcribed in an appropriate biochemical or cellular environment to produce an RNA copy thereof.
  • a "chimeric gene” comprises a transcribable nucleic acid operably linked to a BV promoter of the invention. It will be appreciated that transcription may occur in in vitro systems or in plant cells, for example.
  • Transcribable nucleic acids preferably in the form of double-stranded DNA, encompass nucleic acids that encode proteins, and hence comprise a coding region or open reading frame (ORF) as are well understood in the art.
  • transcribable nucleic acid may encode a transcript complementary to an endogenous or virally-encoded mRNA transcript, such as used in "antisense" expression.
  • ribozymes are also contemplated. Anti-sense regulation and the use of ribozymes and co-suppression in plants are well known in the art. However, the skilled person is referred to United States Patent 5,759,829 for an example of antisense technology and to U.S.
  • mRNA and "transcript” are used interchangeably when referring to a transcribed copy of a transcribable nucleic acid.
  • transcribable nucleic acid may be referred to as a "transgene”.
  • a "polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides.
  • a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
  • a “primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template- dependent fashion by the action of a DNA polymerase such as Taq polymerase,
  • RNA-dependent DNA polymerase or SequenaseTM.
  • primers used herein include primers F8/R8 [SEQ ID NOS. 5 and 6].
  • variable means a BV promoter of the invention, the nucleotide sequence of which has been mutagenized or otherwise altered so as to display substantially the same, or a modified, promoter activity.
  • nucleotide sequence alterations may be introduced so as to modify promoter activity. These alterations may include deletion or addition of one or more bases of BV promoter nucleotide sequence, or involve non-conservative substitution of one base for another. Such alterations can have profound effects upon promoter activity, either increasing or decreasing activity as required.
  • promoter mutagenesis may be performed in a random fashion or by site-directed mutagenesis in a more "rational" manner.
  • a nucleic acid "fragment” comprises a nucleotide sequence that constitutes less than 100% of a nucleic acid of the invention.
  • a fragment includes a polynucleotide, oligonucleotide, probe, primer and an amplification product, eg. a PCR product.
  • a fragment may be orientated either as sense or antisense relative to nucleic acids of the invention. Examples of fragments are primers set forth in SEQ ID NOS: 5 and 6.
  • a "biologically-active fragment” also referred to as a "promoter-active fragment” is a BV promoter fragment which retains BV promoter activity notwithstanding deletion of bases from the BV promoter nucleotide sequence.
  • the biologically-active fragment retains at least 1% BV promoter activity, preferably at least 10%, more preferably at least 25% and even more preferably at least 75% of BV promoter activity.
  • Examples of a biologically-active fragment include a 1097 bp Hindlll-Spel fragment, a 1699 bp Pstl-Sspl fragment, a 1243 bp Sacl-Sspl fragment, a 844 bp Hind l-Sspl fragment and a 454 bp Ncol-Sspl fragment of the BV promoter sequence set forth in SEQ ID NO. 1 as shown in FIG. 1. It will also be appreciated that a biologically-active fragment may have greater activity than a "full-length" BV promoter from which the fragment is derived, as is the case with pEVIHS-gn expression construct.
  • the present invention also contemplates promoters obtained from homologs of the BV promoters as set forth in [SEQ ED NOS. 1-4] and shown in FIGS 1-4, which sequences are embodiments of a "reference nucleic acid'.
  • a “homolog” shares a definable nucleotide sequence relationship with a reference nucleic acid.
  • a homolog shares at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% sequence identity with a reference nucleic acid.
  • sequence relationships between respective nucleic acids include “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleic acids/polypeptides may each comprise (1) only one or more portions of a complete nucleic acid/polypeptide sequence that are shared by the nucleic acids/polypeptides, and (2) one or more portions which are divergent between the nucleic acids/polypeptides, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically at least 6 contiguous residues (as for example applicable to FASTA) that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by fritelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • sequence identity is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity may be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).
  • a homolog hybridizes to a reference nucleic acid under at least medium stringency conditions or, preferably, under high stringency conditions.
  • Hybridize and Hybridization is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid.
  • Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between purine bases (adenine, guanine) and pyrimidine bases (thymine or uracil. cytosine).
  • Modified purines for example, inosine, methylinosine and methyladenosine
  • modified pyrimidines for example, thiouridine and methylcytosine
  • Stringency refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences. “Stringent conditions” designates those conditions under which only a nucleic acid having a high frequency of complementary bases will hybridize to a reference nucleic acid.
  • BSA Bovine Serum Albumin
  • High stringency conditions include and encompass:- (i) from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about
  • the T m of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases.
  • complementary nucleotide sequences are identified by blotting techniques that include a step whereby nucleotides are immobilized on a matrix (preferably a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary
  • DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences are well known by those skilled in the art, and have been described in Ausubel et ⁇ /., .swpr , at pages 2.9.1 through 2.9.20. According to such methods, Southern blotting involves separating DNA molecules according to size by gel elecfrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence.
  • DNA samples are directly applied to a synthetic membrane prior to hybridization as above.
  • An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic
  • DNA library such as through the process of plaque or colony hybridization.
  • Other typical examples of this procedure are described in Chapters 8-12 of Sambrook et al, supra which are herein incorpoated by reference.
  • nucleic acids are blotted/transferred to a synthetic membrane, as described above.
  • a wild type nucleotide sequence of the invention is labeled as described above, and the ability of this labeled nucleic acid to hybridize with an immobilized nucleotide sequence analyzed.
  • radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10 8 dpm mg to provide a detectable signal.
  • a radiolabeled nucleotide sequence of specific activity 10 s to 10 9 dpm/mg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA, usually 10 ⁇ g.
  • Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization can also increase the sensitivity of hybridization (see Ausubel et al, supra at 2.10.10).
  • an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization
  • a sufficient amount of the labeled nucleic acid must be hybridized to the immobilized nucleic acid following washing. Washing ensures that the labeled nucleic acid is hybridized only to the immobilized nucleic acid with a desired degree of complementarity to the labeled nucleic acid.
  • a microarray also uses hybridization-based technology that, for example, may allow detection and/or isolation of a nucleic acid by way of hybridization of complementary nucleic acids.
  • a microarray provides a method of high throughput screening for a nucleic acid in a sample that may be tested against several nucleic acids attached to a surface of a matrix or chip. In this regard, a skilled person is referred to Chapter 22 of CURRENT PROTOCOLS IN
  • homologs of the invention may be prepared according to the following procedure:
  • Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, supra, and Chapter 2 of PLANT MOLECULAR BIOLOGY A Laboratory Manual, supra which are incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252 which is incorporated herein by reference; rolling circle amplification (RCA) as for example described in Liu et al, 1996, J. Am. Chem. Soc.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • RCA rolling circle amplification
  • nucleic acid sequence-based amplification as for example described by Sooknanan et al, 199 , Biotechniques 17 1077) which is incorporated herein by reference; ligase chain reaction (LCR) as for example described in International Application WO89/09385 which is incorporated by reference herein; and Q- ⁇ replicase amplification as for example described by Tyagi et al, 1996, Proc. Nafi.
  • NASBA nucleic acid sequence-based amplification
  • LCR ligase chain reaction
  • Q- ⁇ replicase amplification as for example described by Tyagi et al, 1996, Proc. Nafi.
  • an "amplification product” refers to a nucleic acid product generated by nucleic acid amplification techniques.
  • the nucleic acid sequence amplification technique is PCR.
  • the nucleic acid extract used at step (i) may be obtained from any badnavirus as hereinbefore defined.
  • the badnavirus is sugarcane baciUiform virus (SCBV).
  • SCBV sugarcane baciUiform virus
  • an "expression vector" comprises at least a BV promoter of the invention, and may further comprise elements such as a polylinker, selection marker gene, bacterial origin of replication and antibiotic resistance gene and the like as will be discussed in more detail hereinafter.
  • the expression vector is a plasmid, although other vectors such as bacteriophage. Bacterial Artificial Chromosomes (BAG) and cosmids are contemplated.
  • an "expression construct” comprises an expression vector and a transcribable nucleic acid, operably linked to the BV promoter component of the expression vector.
  • expression constructs of the invention include the pEVIPS, pEVISS, pEVIHS and pIMNS series of constructs to be described in more detail hereinafter. Summarized examples of expression construct preparation are provided in FIGS. 5-7.
  • linketf is meant that transcription of the transcribable nucleic acid is initiated, regulated or otherwise controlled by the BV promoter.
  • the expression vector of the invention includes a polylinker downstream and adjacent to the BV promoter which facilitates directional cloning of the transcribable nucleic acid into the vector so that the promoter and transcribable nucleic acid are operably linked.
  • transgenic expression of a polypeptide when transgenic expression of a polypeptide is required, the correct orientation of the encoding nucleic acid is 5'- 3' relative to the BV promoter, for example. However, where antisense expression is required, the transcribable nucleic acid is oriented 3'-*5'. Both possibilities are contemplated by the expression construct of the present invention, and directional cloning for these purposes is assisted by the presence of a polylinker.
  • the expression construct includes a selection marker nucleic acid to allow selective propagation of plant cells and tissues transformed with an expression construct of the invention.
  • the selection marker is included in a separate selection construct, h either case, one or more regulatory elements, as herein described, may be provided to direct expression of the selection marker nucleic acid.
  • Suitable selection markers include, but are not limited to, neomycin phosphotransferase II which confers kanamycin and geneticin/G418 resistance (nptll; Raynaerts et al, In: Plant Molecular Biology Manual A9:l-16. Gelvin & Schilperoort Eds (Kluwer, Dordrecht, 1988), bialophos/phosphinothricin resistance (bar; Thompson et al, 1987, EMBO J. 6 1589), streptomycin resistance (aadA; Jones et al, 1987, Mol. Gen. Genet. 210 86) paromomycin resistance (Mauro et al, 1995, Plant Sci.
  • the expression vector of the present invention may also comprise other gene regulatory elements, such as a 3' non-translated sequence.
  • a 3' non- translated sequence refers to that portion of a gene that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
  • the polyadenylation signal is characterized by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
  • the 3' nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequences.
  • suitable 3' non-translated sequences are the 3' transcribed non-translated regions containing a polyadenylation signal from the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan et al, 1983, Nucl. Acid Res., 11 369) and the terminator for the T7 transcript from the octopine synthase (ocs) gene of Agrobacterium tumefaciens.
  • a nopaline synthase (nos) terminator is utilized.
  • suitable 3' non-translated sequences may be derived from plant genes such as the 3' end of the protease inhibitor I or II genes from potato or tomato, the soybean storage protein genes and the pea E9 small subunit of the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene, although other 3' elements known to those of skill in the art can also be employed.
  • 3' non-translated regulatory sequences can be obtained de novo as, for example, described by An, 1987, Methods in Enzymology, 153 292, which is incorporated herein by reference.
  • the expression vector of the present invention may further include an enhancer.
  • Enhancers are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences.
  • the initiation codon must be in phase with the reading frame of the coding sequence relating to the transcribable nucleic acid to ensure translation of the entire sequence.
  • the translation control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • Translational initiation regions may be provided from the source of the transcriptional initiation region, or from the foreign or endogenous DNA sequence.
  • the sequence can also be derived from the source of the promoter selected to drive transcription, and can be specifically modified so as to increase translation of the mRNA.
  • transcriptional enhancer elements include, but are not restricted to, elements from the CaMV 35S promoter and octopine synthase (ocs) genes as for example described in U.S. Patent No. 5,290,924, which is incorporated herein by reference. It is proposed that the use of an enhancer element such as the ocs element, 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 transformation.
  • ocs octopine synthase
  • a nucleotide sequence (for example a "leader sequence") inserted between the transcription initiation site and an amino acid coding portion of a transcribable nucleic acid can influence expression of the transcribable nucleic acid.
  • leader sequences include those that comprise sequences selected to direct optimum expression of the foreign or endogenous DNA sequence.
  • leader sequences include a preferred consensus sequence which can increase or maintain mRNA stability and prevent inappropriate initiation of translation as for example described by Joshi, 1987, Nucl. Acid Res., 15 6643, which is incorporated herein by reference.
  • leader sequences e.g., the leader sequence of RTBV
  • other leader sequences have a high degree of secondary structure that is expected to decrease mRNA stability and/or decrease translation of the mRNA.
  • leader sequences (i) .that do not have a high degree of secondary structure, (ii) that have a high degree of secondary structure where the secondary structure does not inhibit mRNA stability and/or decrease franslation, or (iii) that are derived from genes that are highly expressed in plants, will be most preferred.
  • sucrose synthase infron as, for example, described by Vasil et ⁇ /.,1989, Plant Physiol., 91 5175
  • Adh intron I as, for example, described by Callis et /.,1987
  • Genes & Development 1 1183 or the TMV omega element as, for example, described by Gallie et al, 1989
  • Plant Cell 1 301 can also be included where desired.
  • Other such regulatory elements useful in the practice of the invention are known to those of skill in the art.
  • targeting sequences may be employed to target a protein product of the transcribable nucleic acid to an intracellular compartment within plant cells or to the extracellular environment.
  • a DNA sequence encoding a transit or signal peptide sequence may be operably linked to a sequence encoding a desired protein such that, when translated, the transit or signal peptide can transport the protein to a particular intracellular or extracellular destination, respectively, and can then be post-translationally removed.
  • Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.
  • the transit or signal peptide can direct a desired protein to a particular organelle such as a plastid (e.g., a chloroplast), rather than to the cytoplasm.
  • the expression construct can further comprise a plastid transit peptide encoding DNA sequence operably linked between a promoter region or promoter variant according to the invention and transcribable nucleic acid.
  • a promoter region or promoter variant for example, reference may be made to Heijne et ⁇ l, 1989, Eur. J. Biochem. 180 535 and Keegsfra et /., 1989, Ann. Rev. Plant Physiol. Plant Mol. Biol. 40 471, which are incorporated herein by reference.
  • the expression vector of the invention is a plasmid and includes additional elements commonly present in plasmids for easy selection, amplification, and transformation of the transcribable nucleic acid in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pBluescript-derived vectors, pGEM-derived vectors. Additional elements include those which provide for autonomous replication of the vector in bacterial hosts (examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19 and the ColEl replicon which function in many E. coli.
  • the vector may also include an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.
  • the vector may be integrated into the host cell genome when introduced into a host cell.
  • the vector may rely on the foreign or endogenous DNA sequence or any other element of the vector for stable integration of the vector into the genome by homologous recombination.
  • the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell.
  • the additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location in the chromosome.
  • the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding nucleic acid sequences. Promoter activity assays
  • Reporter assays involve preparation of an expression construct where a promoter of interest is operably linked to a transcribable nucleic acid in the form of a
  • reporter gene The expression construct is then used to transform plant cells or tissues. Promoter activity may be measured in transient assays (such as a few days after transformation) or in plants selectively propagated or regenerated from transformed cells or tissue. Reporter genes are well known in the art and include chloramphenicol acetyl transferase (cat; Lindsey & Jones, 1987, Plant Mol. Biol. 10 43), green fluorescent protein and various derivatives thereof (gfp; Haseloff & Amos, 1995, Trends Genet. 11 328), neomycin phosphofransferase (nptll; Reiss et al, 1984, Gene 30 211), ⁇ -galactosidase (lacZ; Helmer et al, 1984,
  • Plant transformation Other aspects of the present invention relate to transgenic plants and a method of producing transgenic plants.
  • the method of producing a transgenic plant includes the steps of:-
  • step (ii) selectively propagating a transgenic plant from the plant cell or tissue transformed in step (i).
  • the expression construct may comprise a homolog, promoter active fragment, variant or derivative of a BV promoter of the invention.
  • the plant cell or tissue used at step (i) may be leaf disk, callus, meristem, root, leaf spindle or whorl, leaf blade, stem, shoot, petiole, axillary bud, shoot apex, internode, flower stalk or inflorescence tissue.
  • the tissue is callus.
  • the plant cell or tissue may be obtained from any plant species including monocots, dicots, ferns and gymnosperms such as conifers, without being limited thereto.
  • the plant is a monocotyledon or dicotyledon.
  • the monocotyledon is a graminaceous monocotyledon.
  • the monocotyledon is Saccharum spp.
  • the dicotyledon is tobacco.
  • the tobacco is Nicotianna tabacum.
  • transformation methods are applicable to the method of the invention, such as -4grob-.ctert-.m-mediated (Gartland & Davey, 1995, Agrobacterium Protocols (Human Press Inc. NJ USA); United States Patent No. 6,037,522; WO99/36637), microprojectile bombardment (Franks & Birch, 1991, Aust. J. Plant. PhysioL, 18 471; Bower et al, 1996, Molecular Breeding, 2 239; Nutt et al, 1999, Proc. Aust.
  • Example 14 tobacco leaf disk transformation is shown in Example 14 herein.
  • Other dicots may likewise be transformed as discussed in (Horsch efal, 1985, Science 227 1229; Fry et al, 1987, Plant Cell Rep. 6 321) which are incorporated herein by reference.
  • microprojectile bombardment is preferable for monocotyledons
  • microprojection and Agrobacterium transformation are also useful for transforming dicotyledons.
  • microprojectile bombardment is used at transformation step (i).
  • this is the preferred method for monocot transformation, as some monocot species have proven refractory to transformation by methods such as -4grob cterz--m-mediated transformation.
  • certain monocots see for example United States Patent No. 6,037,522 in relation to cereals and WO99/36637 in relation to pineapples), incorporated herein by reference, so that Agrobacterium-mediated transformation of monocots is contemplated by the present invention.
  • selective propagation at step (ii) is performed in a selection medium which includes geneticin as selection agent.
  • the expression construct further comprises a selection marker nucleic acid in the form of an nptll gene.
  • a separate selection construct is included at step (i), which comprises a selection marker nucleic acid in the form of an nptll gene.
  • selection agents useful according to the invention, the choice of selection agent being determined by the selection marker nucleic acid used in the expression construct or provided by a separate selection construct.
  • transgenic status of transgenic plants of the invention may be ascertained by measuring transgenic expression of a polypeptide encoded by the transcribable nucleic acid. This can be performed using the aforementioned methods applicable to measuring promoter activity.
  • transgene expression can be detected by antibodies specific for the encoded polypeptide:
  • the aforementioned protein-based detection methods may take advantage of "fusion partners” such as glutathione-S-transferase (GST), Fc portion of human IgG, maltose binding protein (MBP) and hexahistidine (HIS 6 ), which are particularly useful for isolation of the fusion polypeptide by affinity chromatography.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • HIS 6 hexahistidine
  • relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively.
  • Many such matrices are available in “kit” form, such as the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners and the Pharmacia GST purification system.
  • Fusion partners also include "epitope tags", which are usually short peptide sequences for which a specific antibody is available.
  • epitope tags for which specific monoclonal antibodies are readily available include c-myc, influenza virus haemagglutinin and FLAG tags.
  • transgenic plants of the invention may be screened for the presence of mRNA corresponding to a transcribable nucleic acid and/or a selection marker nucleic acid. This may be performed by RT-PCR and/or Northern hybridization. Southern hybridization and/or PCR may be employed to detect DNA (the BV promoter, transcribable nucleic acid and/or selection marker) in the transgenic plant genome.
  • PCR is a technique well known in the art and the aforementioned incorporated references provide exemplary PCR methods applicable to the present invention.
  • RNA isolation and Northern hybridization methods For examples of RNA isolation and Northern hybridization methods, the skilled person is referred to Chapter 3 of PLANT MOLECULAR BIOLOGY: A Laboratory Manual, supra, which is herein incorporated by reference. Southern hybridization is described, for example, in Chapter 1 of PLANT MOLECULAR BIOLOGY: A Laboratory Manual, supra, which is herein incorporated by reference.
  • the extraction method was based on that of Lockhart, 1990, Phytopathology 80 127 and Bouhida et al, 1993, J. Gen. Virol. 74 15, which are incorporated herein by reference, and was used to extract virions from SCBV-infected Ireng Maleng, IJ76-465 and IJ76-468 sugarcane cultivars and accessions.
  • the midrib was removed from sugarcane leaf tissue and the remaining leaf was cut into pieces approximately 2-4cm in length which was either used fresh, or sealed in plastic bags and stored at -70°C for up to four weeks before use.
  • Virions were extracted from 400 to 450 grams of tissue using the method of Bouhida et al, 1993, supra modified as described below.
  • the aqueous phase (top layer) was placed onto a 30% sucrose cushion and centrifuged at 225,000g in a Beckman 75Ti rotor at 4°C for VA hours. The pellets were resuspended in a total of 1 mL of TE buffer pH 8.0. Presence of SCBV particles was confirmed by immunosorbent electron microscopy (ISEM) using 35x SCBV antisera. This virion preparation was then used for the extraction of viral DNA.
  • IEM immunosorbent electron microscopy
  • Sugarcane nucleic acids were removed from the extracted virions by adding DNase I (50 units), 10 ⁇ L of 2M MgCl 2 , and 2 ⁇ L of RNase A (10 mg/niL) per 1 mL of virion preparation and incubated at 37°C for lhour. 20 ⁇ L of 0.5M EDTA pH 8.0 was added to inactivate DNase. Following Dnase/Rnase treatment, 10 ⁇ L of 50 mg/mL Proteinase K was added for every 1 mL of virion preparation and incubated at 50°C for 10 minutes. The digest was incubated for a further 30 minutes following the addition of 20 ⁇ L of 10% SDS for every 1 mL of virion preparation.
  • Viral proteins were removed by two successive phenol:chloroform:isoamyl alcohol (25:24:1) extractions followed by a chloroform soamyl alcohol (24:1) extraction.
  • the aqueous phase from the phenol/chloroform extracted material was placed into dialysis tube (50,000 MW cut off) and dialysed against 1 L of sterile water for 1-2 hours, then 1 L of sterile
  • the viral DNA was precipitated with one tenth volume 3 M sodium acetate pH 5.2 and two volumes 100% ethanol at -20°C for 2-3 hours.
  • the DNA was centrifuged at 20,000g in a Sorvall SS-34 rotor at 4°C for 30minutes. The DNA pellet was washed with 70% ethanol and left to air dry.
  • a clone (pSCBV-EVI12) comprising an insert of approximately 7.5kb was selected for characterization.
  • Deletion mutants were made from pSCBV-EVI12, using Erase-a-base deletion mutant kit (Promega Co ⁇ oration, Wisconsin, USA). Sequencing was performed in duplicate for each strand using the ABI prism sequencing kit (Perkin-Elmer Co ⁇ oration, California, USA) on either an ABI373 or an ABI377 automated sequencer. Specific primers were selected from the obtained sequences to sequence regions not present in the deletion mutants. A small section of the complete sequence was not present in the pSCBV-EVI12 clone.
  • the clone pSCBVEVI-F5 contained the sequence required and was obtained by PCR using specific primers designed from the pSCBV-EVI12 clones.
  • the final sequence of 7687 bp, referred to as SCBV-EVI represents the entire genomic sequence of an SCBV isolate from Ireng Maleng.
  • the SCBV-EVI promoter sequence set forth in SEQ ED NO. 1 as shown in FIG. 1 was obtained by digesting pSCBV-EVI12 with Estl and Spel and ligating the resultant 1952 bp fragment into the Pstl and Xbal sites of pUC19-gn (see Example 5) to give pEVfPS-gn.
  • SCBV-EVI promoter constructs were made by subcloning this plasmid (ie pIMHS-gn, pIMPS-kn, pLVISS-kn, pIMHS-kn and pIMNS-kn).
  • a region of approximately 1 kb was PCR amplified from respective SCBV DNA preparations isolated from IJ76-465, IJ76-468 and Ireng Maleng using the primer pair F8 (5'-AGCTGACATCTTGTCCAG-3') [SEQ 3D NO. 5] and R8 (5'-
  • F8 targets a conserved region in the RNase H coding region of open reading frame III, while R8 targets the tRNA met binding site. These conserved regions were identified by aligning all badnaviruses, with the final primer sequences based on pSCBV20 (Bouhida et al, 1993, supra). These primers [SEQ ED NOS. 5 and 6] were designed to enable the isolation of BV promoters from a wide range of SCBV infected sugarcane germplasm.
  • the PCR amplification mix (25 ⁇ L) contained 200 ⁇ M each dATP, dCTP, dGTP and dTTP, 4 mM MgCl 2 , 1 unit Taq DNA polymerase (Biotech International), buffer supplied by the manufacturer, 200 nM of each primer and 1 ⁇ L purified viral template. Cycling conditions began with a hot start at 95°C for 7 min, then 40 cycles of 95 °C for 30 sec, 45 °C for 30 sec and 72 °C for 1 min, followed by a final extension of 72 °C for 7 min. PCR products were cloned using the pCR2.1 cloning vector and TA cloning kit (Invitrogen).
  • Ligations were transformed into E.coli strain DH5 or other competent cells provided with the kit.
  • the PCR products comprising each promoter were sequenced, and the respective nucleotide sequences are set forth in SEQ ID NOS. 2-4 as shown in FIGS 2-4.
  • Expression constructs using BV promoters corresponding to the 1952 bp Pstl-Spe ⁇ fragment of FIG. 1 [SEQ J-D NO. 1] and the 1097 bp Hindlll-Spel fragment of FIG. 1 [SEQ ID NO. 1] were operably linked to a ⁇ -glucuronidase (gus) reporter gene and a nopaline synthase (nos) terminator in pUC19-gn to form the respectively named constructs pEVIPS-gn and pEVIHS-gn (schematically described in FIG. 5).
  • the gus and nos elements had been excised from pBI221 (Clontech Laboratories) using Xbal and EcoRl restriction enzymes and inserted into pUC19.
  • the resulting plasmid, pUC19-gn was used as a promoterless gus construct and formed the basis for most promoter-reporter gene constructs.
  • BV promoters prepared by PCR amplification from SCBV DNA isolated from Ireng Maleng (SEQ ED NO. 2; FIG.2), IJ76-465 (SEQ ID NO. 3; FIG. 3) and IJ76-468 (SEQ ED NO. 4; FIG. 4) were operably linked to the gus reporter gene in pUC19-gn.
  • Each promoter was used to prepare two gus fusion constructs: approximately the full length of the promoter and approximately half-length as schematically described in FIG. 6.
  • the IJ76-465 sequence was excised from pCR2.1 by digestion with BamHl and the resulting 785 bp of promoter DNA inserted into pUC19-gn (p465BB-gn).
  • the IJ76-465 PCR product was subcloned by preparing nested deletions with an Erase-a-base Kit (Promega).
  • One deletion subclone, (p465-21-7G) was used to make the shorter promoter construct by PCR amplification with primers targeted to the T7 and M13R promoters.
  • neomycin phosphofransferase resistance gene (nptll) was used as both a selection marker gene and as a reporter gene.
  • a promoterless construct p_KN was prepared by removing the Emu promoter from pEKN (which includes an nptll reporter gene and nos terminator) with Hindlll and Xbal followed by the addition of an Xbal linker to the Hindlll end.
  • SCBV-EVI fragments (of the genome sequence shown in FIG.
  • Microprojectile bombardment of callus, selection and growth of transformed plants were essentially as described by ⁇ utt et al, 1999 supra and
  • Bombarded callus was retained up to 4 hr on MS medium plus 0.2 M mannitol and 0.2 M sorbitol then rested for 3-4 days on MS medium with 3 mg/L 2,4-D in the dark, followed by 6-8 weeks in the dark on MS medium with 3 mg/L 2,4-D and 60 ⁇ g/mL geneticin.
  • the selected callus tissue was then introduced to light on MS medium and 60 ⁇ g/mL geneticin until shoots (approx. 5 days) and roots (approx. 6 weeks) appeared, then onto ! strength MS medium prior to transfer of plantlets to a glasshouse.
  • BV promoter-g-.-? expression constructs were ' co-transformed with pUKN to provide antibiotic selection.
  • pUKN contains a neomycin phosphofransferase (nptll) gene, which provides resistance to geneticin and kanamycin, a nos terminator, and a maize ubiquitin-1 promoter (Ubi-1; Christensen & Quail, 1996, supra).
  • the plasmid pEMU-gn (referred to as pEmuGN in Last et al, 1991, supra) was generally used as a positive confrol and contains the recombinant Emu (also referred to as pEMU) promoter, gus gene and the nos terminator.
  • GUS protein assays PCR isolated promoters
  • Transformed plant tissue samples were taken at various stages of tissue culture and plant growth. Root samples taken from the soil were surface sterilized in 1% bleach followed by rinsing in sterile water prior to assaying. Tissue pieces were submerged in GUS substrate (1.15 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D- glucuronidase (X-Gluc) dissolved in DMSO, 50 mM Na Phosphate buffer pH7.6, 20% v/v methanol, 0.3% v/v Triton X-100) and vacuum infiltrated for 5 min. Samples were kept at 37°C for 24 to 48 hours, cleared in several changes of 80% ethanol:20% methanol and then assessed visually for number of blue spots, size of blue spots or intensity of blue.
  • GUS substrate 1.15 mM 5-bromo-4-chloro-3-indolyl- ⁇ -D- glucuronidase (X-Gluc) dissolved in DMSO, 50
  • Transient activity was assessed in callus two days after bombardment. All BV promoters (and fragments) were able to drive GUS activity in transient assays with the substantially full-length promoters almost as active as pEMU. Several pieces of callus bombarded with pUC19-gn gave positive results. This may be due to insertion of the plasmid behind an endogenous sugarcane promoter. A similar result was observed when stable activity in callus was assessed two months after bombardment while the callus was still on antibiotic selection (Table 2). The substantially full-length promoters displayed more activity than the shorter fragments. Once again, several pieces of callus shot with pUC19-gn gave positive results.
  • Sugarcane callus transformed with pIMPS-gn, pIMHS-gn, pEMU-gn or pUC19- gn was sampled for GUS activity one day after microprojectile bombardment.
  • GUS activity was assayed in callus and shoots. Plants were regenerated and established in the glasshouse as described above. After 2-3 months growth, approximately half the plants derived from the secondary callus were assayed for GUS activity. For each replicate, roots, meristem and young leaf samples were taken. The remaining glasshouse plants were grown until mature (7- 8 months old) and then harvested. The meristem, leaf whorl and young leaf were sampled for GUS activity.
  • GUS activity in all sugarcane tissues is shown in Table 3. Results from both the initial transformed callus and regenerated plants and secondary callus and regenerated plants have been combined. No GUS activity in any tissue was detected in plants transformed with pUC19-gn. pEMU-gn drives very high GUS expression in callus. However this activity begins to decline once shoots regenerate on the callus. Some activity is also observed in the meristematic region of mature plants. This is a region of high cell division. Both pIMPS-gn and pEVIHS-gn are active in all tissues except roots. However pEVIHS-gn is clearly more active that pEVIPS-gn, both in the intensity of activity and in the proportion of plants showing measurable activity.
  • GUS activity during different stages of sugarcane development is shown in Table 4. hi this table only the tissue showing highest GUS activity at each stage of development is shown.
  • pEMU-gn is very active in callus but this activity declines once leaves develop. Activity is only observed in rapidly dividing cells eg meristems. However this decline in activity is not permanent. Promoter activity is restored in callus initiated from mature plants that were showing low levels of activity. Again the activity declines as the plants develop.
  • pIMPS-gn also shows a decline in GUS intensity and in the proportion of positive plants as plants develop from callus and this activity is resorted when callus is reinitiated. However the decline in GUS activity as plants develop is not apparent in plants transformed with pEVIHS-gn.
  • pEVIHS-gn shows the highest activity in mature plants.
  • the general conclusions drawn from these studies were that:- a. the pEVIHS-gn expression construct (comprising the shorter SCBV- EVI promoter) generated the highest levels of GUS activity; b. the pEVIHS-gn expression construct generated the highest frequency of GUS-expressing transgenic plants. c. activity was higher in mature plants when compared to young plants; d. highest GUS activity was generally observed in meristematic tissues; and e. no GUS activity was detected in the roots of young plants. Furthermore, GUS activity in the secondary plants generally mirrored the activity in primary plants, especially when considering pEMU-gn and pEVIPS-gn.
  • GUS activity was high in tissue culture, lower in mature plants, higher again in secondary culture and lower again in secondary plants, hi contrast, pEVIHS-gn-driven GUS activity remained relatively constant.
  • Representative examples of GUS expression by plant tissues at various stages after transformation are provided in FIG. 8.
  • promoter activity was determined quantitatively by measuring NPTII activity with NPTII ELISA kits supplied as 5 Prime-3 Prime or Eppendorf-5 Prime kits. Crude protein was extracted from transgenic plant tissue in lx PBS according to Nagel et al, 1992, Plant Molecular Biology Reporter 10 263, which is inco ⁇ orated herein by reference, and protein was estimated using the Bio-Rad reagents based on Bradford, 1976, Anal. Biochem. 72 248. The ELISAs were performed according to the method supplied with the kits and the modifications described by Nagel et al, 1992, supra. Protein samples were diluted until activity could be detected on the standard curve.
  • SCBV-EVI-nptll promoter constructs provided levels of NPTII expression comparable to or better than that produced by the UBi promoter.
  • all SCBV-EVI promoters displayed greater average activity than Ubi in all tissues examined.
  • Tables 6 and 7 were obtained from up to 8 replicate samples taken from plants regenerated from sugarcane callus transformed as described above. Replicate plants transformed with the same expression construct showed a wide range of NPTII activity, fri all tissues, the average NPTII activity driven by the SCBV-EVI promoters was higher than the average activity driven by Ubi.
  • Ubi and the SCBV-EVI promoters did not shut down in activity as the plants developed.
  • the SCBV-EVI promoters drove stable NPTII expression in transgenic sugarcane callus, shoots, leaves and roots (in tissue culture) and leaves, roots, leaf whorls and meristems (in plants);
  • D ⁇ A was extracted using the Rapid Release Prep which is based on Thomson & Henry, 1995, BioTechniques 19 394, but modified for sugarcane. The modifications include (i) initially grinding the tissue either fresh with a power pestle or frozen in liquid ⁇ 2 with a mortar and pestle, and (ii) a chloroform isoamyl alcohol extraction at the end. PCR was performed using gus and ALS control primers.
  • GUS 1000 5'-TTT GCA AGT GGT GAA TCC CGA CCT-3' [SEQ ID NO. 7];
  • GUS1600 5'-AGT TTA CGC GTT GCT TCC GCC AGT-3' [SEQ ID NO. 8].
  • the PCR reaction mix was the same as that used for nptll (see below) except that 0.2 ⁇ M of each primer was used.
  • the PCR program was 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 sec, 60 °C for 30 sec, 72 °C for 1 min, followed by 72 °C for 7 min.
  • ALS -1 and ALS-2 primers were used as a PCR positive control.
  • the ALS PCR test amplifies acetolactate synthase, a sugarcane gene, and serves as a positive internal control.
  • the ALS primers were: ALS-1 : 5'-CGA CGA CCC ACT GTC ACT GC-3' [SEQ ID NO. 9]; and
  • ALS-2 5'-TCC TCC CAC TGC ACC ACC AT-3' [SEQ ID NO. 10].
  • Untransformed callus was used as a negative confrol.
  • the ALS PCR mix and program are the same as the test gus PCR reaction, but was performed in separate PCR tubes (ie not multiplexed).
  • the primer sequences are as described in Fromm et al, 1990, Bio/Technology 8 833.
  • the PCR data confirmed that the lack of gus staining in certain samples was not due to lack of, or poor, transformation. Almost all transformed samples were GUS DNA-positive.
  • NPTII primers were: NPTII-F: 5'-CGG CTA TGA CTG GGC ACA ACA GAC-3' [SEQ ID NO.11]; and
  • NPTII-R 5'-CAT AGA AGG CGG CGG TGG AAT CGA-3' [SEQ ID NO.12].
  • the PCR cycling program was 95 °C for 30 sec, followed by 36 cycles of 95 °C for 40 sec, 65 °C for 40 sec, 72 °C for 80 sec, followed by 72 °C for 10 min.
  • the PCR mix contained lx buffer supplied by the manufacturer, 100 ⁇ M each dNTP, 1.5 mM MgCl 2 , 0.15 ⁇ M each primer, 0.625 units Taq polymerase (Geneworks) and 1 ⁇ L of a 1/5 dilution of the rapid release prep in a 25 ⁇ L reaction.
  • the ALS PCR mix and program were the same as the test npt-7 PCR reaction, but was performed in separate PCR tubes (ie not multiplexed).
  • PCR analysis to detect the nptll reporter transgene confirmed that all tissue culture samples (except two p_KN samples) transformed with an nptll expression construct were positive for nptll reporter gene DNA.
  • Tobacco leaf disks were fransformed using Agrobacterium tumefaciens carrying binary vectors including badnavirus (BV) promoter-gM5 expression cassettes from Sugarcane baciUiform badnavirus (SCBV).
  • BV promoter expression was determined by staining the regenerated tobacco shoots for GUS followed by light microscope visualisation.
  • pIMPS-gn and pIMHS-gn were used for the construction of binary vectors for -4grob ⁇ ctert ⁇ .m-mediated transformation of Nicotiana tabacum.
  • pEVIPS-gn and pEVIHS-gn were digested with Pst I and Hind III, respectively, followed by partial digestion with EcoR I.
  • Pstl and EcoEI digestion of p ⁇ VIPS-gn releases a 1952 bp BN promoter comprising S ⁇ Q ⁇ D NO. 1, gus reporter gene and nos terminator, while Hindlll and EcoRI digestion of p ⁇ VIHS-gn releases a 1097 bp BN promoter fragment of S ⁇ Q ID NO.
  • pPZP-111 is an Agrobacterium binary vector comprising T-DNA borders, multiple cloning site, chloramphenicol and kanamycin resistance genes.
  • the resulting binary vectors carrying the BN promoter-expression cassettes were termed pPZP- ⁇ VfPS-gn and pPZP-IMHS-gn. Plasmid D ⁇ A isolated from recipient E. coli cultures was analysed to determine that the correct promoter-gus fusions were present. The binary constructs pPZP- ⁇ VIPS-gn and pPZP-IMHS-gn in E.
  • the triparental matings were carried out on non-selective LB media for 2 days at
  • Tobacco (Nicotiana tabacum var. Ti68) was transformed according to a modification of the leaf-disk method (Horsch et al, 1985, Science 227 1229), inco ⁇ orated herein by reference. Seeds were surface sterilised by submersion in 70% ethanol followed by 5% bleach solution. Seeds were washed 5. times in sterile distilled water before gemination on MSO culture plates comprising MS salts, MS vitamins, 3% sucrose and 0.7% agar, pH 5.7. Single tobacco seedlings were transferred to vented-lid jars comprising MSO medium one week after germination. Growth conditions were 23°C under cool white fluorescent lamps for approximately 8 weeks.
  • Agrobacterium strains comprising binary vectors were selected on LB media comprising rifampicin (50 ⁇ g/ml) and chloramphenicol (20 ⁇ g/ml).
  • the disks were washed in sterile water, blotted with sterile filter paper and placed adaxial side up on MS9 media plates comprising MS salts; MS vitamins; 1 mg/1 BAP; 0.5 mg/1 IAA; 150 mg/1 TimentinTM ; 50 mg/1 kanamycin.
  • Leaf disks were subcultured every two weeks on to the same media. Callus and shoots began regenerating within two weeks on selective media.
  • Transient expression of the BN promoters was assessed within Nicotiana tabacum var. Ti68 by microprojectile bombardment of tobacco leaves and callus with plasmids pIMPS-gn and pEVIHS-gn (described in Example 5). Tobacco callus and leaves grown in sterile conditions (see Example 14) were placed adaxial side up on MSO medium for microprojectile bombardment. The method was essentially as described previously in Bower et al, 1996, supra, inco ⁇ orated herein by reference. Helium (4000 kPa) was used to deliver microprojectiles with a 0.1 msec solenoid opening duration, and 4 ⁇ l of D ⁇ A-coated microprojectiles were used per shot.
  • Tungsten microparticles (l-1.2 ⁇ m dia.) were sterilized in a volume of ethanol (lO ⁇ l/mg tungsten), vortexed, centrifuged for approximately 10 sees and the ethanol replaced with an equal volume of sterile dH 2 O. The washing step was repeated twice prior to resuspending in the same volume of water. Microprojectiles were coated using the following conditions: volume

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Abstract

L'invention concerne des acides nucléiques de promoteurs viraux de plantes et des fragments de ceux-ci isolés à partir du virus bacilliforme de la canne à sucre (SCBV). Par ailleurs, l'invention concerne des produits recombinés d'expression contenant lesdits acides nucléiques des promoteurs viraux de plantes et des marqueurs moléculaires, à savoir, gus et nptII respectivement. Lesdits produits recombinés d'expression ont été transformés en plantes de la classe des monocotylédones (canne à sucre) et de celle des dicotylédones (tabac).
PCT/AU2001/001499 2000-11-21 2001-11-20 Promoteurs du virus bacilliforme de la canne a sucre isoles a partir de cultivars de canne a sucre WO2002042450A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110283377A1 (en) * 2010-05-10 2011-11-17 Mirkov T Erik Compositions, organisms, systems, and methods for expressing a gene product in plants
US20120054908A1 (en) * 2010-08-30 2012-03-01 Agrigenetics, Inc. Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
WO2013101344A1 (fr) * 2011-12-30 2013-07-04 Dow Agrosciences Llc Procédé et construction pour un promoteur bidirectionnel synthétique de plante scbv
WO2013130813A1 (fr) * 2012-02-29 2013-09-06 Dow Agrosciences Llc Activateur du virus bacilliforme de la canne à sucre (scbv) et son utilisation en génomique végétale fonctionnelle
CN103409414A (zh) * 2013-07-15 2013-11-27 福建农林大学 甘蔗基因组内标基因乙酰乳酸合酶基因pcr引物序列及扩增方法
US20150096080A1 (en) * 2010-08-30 2015-04-02 Dow Agrosciences Llc Activation tagging platform for maize, and resultant tagged populations and plants
AU2015224510B2 (en) * 2010-08-30 2017-11-16 Dow Agrosciences Llc Activation tagging platform for maize, and resultant tagged population and plants
CN114574492A (zh) * 2022-03-25 2022-06-03 广东省科学院南繁种业研究所 一种来自甘蔗杆状病毒的组成型启动子pscbv-chn1及其应用

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WO1999009190A1 (fr) * 1996-08-09 1999-02-25 Regents Of The University Of Minnesota Promoteur du virus bacilliforme de la canne a sucre

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DATABASE GENBANK 15 March 1999 (1999-03-15), "Musa x paradisiaca clone Musa6 banana streak virus sequence" *
SCHENK P.M. ET AL.: "A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monoot and dicot plants", PLANT MOLECULAR BIOLOGY, vol. 39, 1999, pages 1221 - 1230 *
TZAFRIR I. ET AL.: "The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots", PLANT MOLECULAR BIOLOGY, vol. 38, 1998, pages 347 - 356 *

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US8710207B2 (en) 2010-05-10 2014-04-29 The Texas A&M University Systems Compositions, organisms, systems, and methods for expressing a gene product in plants using SCBV expression control sequences operable in monocots and dicots
WO2011143204A3 (fr) * 2010-05-10 2012-01-05 The Texas A & M University System Compositions, organismes, systèmes, et procédés pour exprimer un produit génique dans des plantes
AU2011252008B2 (en) * 2010-05-10 2016-03-17 The Texas A & M University System Compositions, organisms, systems, and methods for expressing a gene product in plants
CN107090453A (zh) * 2010-05-10 2017-08-25 德克萨斯A&M大学系统 用在植物中表达基因产物的组合物、生物体、体系和方法
CN103052712A (zh) * 2010-05-10 2013-04-17 德克萨斯A&M大学系统 用于在植物中表达基因产物的组合物、生物体、体系和方法
US10202612B2 (en) 2010-05-10 2019-02-12 The Texas A&M University System Compositions, organisms, systems, and methods for expressing a gene product in plants
US20140208462A1 (en) * 2010-05-10 2014-07-24 The Texas A&M University System Compositions, organisms, systems, and methods for expressing a gene product in plants
US20110283377A1 (en) * 2010-05-10 2011-11-17 Mirkov T Erik Compositions, organisms, systems, and methods for expressing a gene product in plants
US9896692B2 (en) * 2010-08-30 2018-02-20 Dow Agrosciences Llc Sugarcane bacilliform viral enhancer-based activation tagging platform for maize, and resultant tagged populations and plants
US10227598B2 (en) 2010-08-30 2019-03-12 Dow Agrosciences Llc Sugarcane bacilliform viral (SCBV) enhancer and its use in plant functional genomics
JP2013538571A (ja) * 2010-08-30 2013-10-17 ダウ アグロサイエンシィズ エルエルシー サトウキビ桿状ウイルス（ｓｃｂｖ）エンハンサーおよび植物機能ゲノミクスにおけるその使用
US8785612B2 (en) 2010-08-30 2014-07-22 Agrigenetics, Inc. Sugarcane bacilliform viral (SCBV) enhancer and its use in plant functional genomics
CN103201389A (zh) * 2010-08-30 2013-07-10 陶氏益农公司 甘蔗杆状病毒(scbv)增强子及其在植物功能基因组中的用途
US20140289899A1 (en) * 2010-08-30 2014-09-25 Dow Agrosciences Llc Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
AU2015224510B2 (en) * 2010-08-30 2017-11-16 Dow Agrosciences Llc Activation tagging platform for maize, and resultant tagged population and plants
US20150096080A1 (en) * 2010-08-30 2015-04-02 Dow Agrosciences Llc Activation tagging platform for maize, and resultant tagged populations and plants
WO2012030711A1 (fr) * 2010-08-30 2012-03-08 Agrigenetics, Inc. Activateur du virus bacilliforme de la canne à sucre (scbv) et son utilisation en génomique végétale fonctionnelle
US20120054908A1 (en) * 2010-08-30 2012-03-01 Agrigenetics, Inc. Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
WO2013101344A1 (fr) * 2011-12-30 2013-07-04 Dow Agrosciences Llc Procédé et construction pour un promoteur bidirectionnel synthétique de plante scbv
US9453235B2 (en) 2011-12-30 2016-09-27 Dow Agrosciences Llc Method and construct for synthetic bidirectional SCBV plant promoter
WO2013130813A1 (fr) * 2012-02-29 2013-09-06 Dow Agrosciences Llc Activateur du virus bacilliforme de la canne à sucre (scbv) et son utilisation en génomique végétale fonctionnelle
JP2015508668A (ja) * 2012-02-29 2015-03-23 ダウ アグロサイエンシィズ エルエルシー サトウキビ桿状ウイルス（ｓｃｂｖ）エンハンサーおよび植物機能ゲノミクスにおけるその使用
CN104411827A (zh) * 2012-02-29 2015-03-11 陶氏益农公司 甘蔗杆状病毒(scbv)增强子及其在植物功能基因组学中的用途
US10077450B2 (en) * 2012-02-29 2018-09-18 Dow Agrosciences Llc Sugarcane bacilliform viral (SCBV) enhancer and its use in plant functional genomics
US20150059021A1 (en) * 2012-02-29 2015-02-26 Dow Agrosciences Llc Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics
CN103409414B (zh) * 2013-07-15 2015-07-08 福建农林大学 甘蔗基因组内标基因乙酰乳酸合酶基因pcr引物序列及扩增方法
CN103409414A (zh) * 2013-07-15 2013-11-27 福建农林大学 甘蔗基因组内标基因乙酰乳酸合酶基因pcr引物序列及扩增方法
CN114574492A (zh) * 2022-03-25 2022-06-03 广东省科学院南繁种业研究所 一种来自甘蔗杆状病毒的组成型启动子pscbv-chn1及其应用
CN114574492B (zh) * 2022-03-25 2023-09-19 广东省科学院南繁种业研究所 一种来自甘蔗杆状病毒的组成型启动子pscbv-chn1及其应用

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