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WO2005026367A1 - Surexpression de genes etrangers dans les plantes - Google Patents

Surexpression de genes etrangers dans les plantes Download PDF

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Publication number
WO2005026367A1
WO2005026367A1 PCT/IN2004/000294 IN2004000294W WO2005026367A1 WO 2005026367 A1 WO2005026367 A1 WO 2005026367A1 IN 2004000294 W IN2004000294 W IN 2004000294W WO 2005026367 A1 WO2005026367 A1 WO 2005026367A1
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rna polymerase
promoter
gus
expression
plants
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PCT/IN2004/000294
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English (en)
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Vanga Siva Reddy
Huu Tam Nguyen
Sadhu Leelavathi
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International Centre For Genetic Engineering And Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • 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
    • C12N15/8225—Leaf-specific, e.g. including petioles, stomata

Definitions

  • the present invention relates to transcription systems for overexpression of foreign proteins in higher eukaryotic genomes. More particularly, the present invention relates to. transcription systems for overexpression of foreign proteins in higher eukaryotic genomes, such as nuclear genomes of higher organisms. More particularly, • the present invention relates to T5 RNA polymerase, SP6 RNA polymerase or Bacteriophage T7 RNA polymerase based transcription systems for overexpression of foreign proteins in higher eukaryotic genomes, particularly nuclear genomes in plants. In particular, the present invention relates to bacteriophage T5 RNA polymerase, SP6 RNA polymerase or T7 RNA polymerase based transcription systems for use in overexpression of foreign proteins in a tissue specific and inducible manner.
  • the present invention also relates to a method for overexpression of foreign proteins in plants using the novel Bacteriophage T5 RNA polymerase, SP6 RNA polymerase and T7 RNA polymerase based transcription systems.
  • Background of the invention Genetic engineering offers enormous scope to utilize plants as protein production factories. However, commercialization of this important technology is hampered by generally observed low-level expression of recombinant proteins in a desired plant tissue. Plants are increasingly being used as "natural bioreactors" for large-scale production of foreign proteins ' for industrial application (Giddings, G. Transgenic plants as protein factories. Curr Opin Biotechnol. 12: 450-454 (2001)), an approach the success of which is highly dependent on the expression levels achieved for heterologous proteins in plants.
  • chloroplast genetic engineering (Geert De Jaeger, Stanley Scheffer, Anni Jacobs, Mukund Zambre, Oliver Zobell, Alain Goossens, Ann Depicker & Geert Angenon.Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat. Biotechnol 20,1265 - 1268 (2002)).
  • chloroplast transformation has been achieved routinely so far only in tobacco.
  • T7 RNAP The bacteriophage T7 RNA polymerase (T7 RNAP) is the most commonly used transcription system to overproduce recombinant proteins in microbial systems.
  • T7 RNAP The bacteriophage T7 RNA polymerase
  • success has been achieved to establish T7-mediated expression in lower eukaryotic organism Trypanosoma brucei (Fujimoto H, Itoh K, Yamamoto M, Kyozuka J, Shimamoto K. Insect resistant rice generated by introduction of a modified delta- endotoxin gene of Bacillus thuringiensis.
  • T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89 (1990)).
  • T7 RNAP can transcribe a foreign gene integrated stably into higher plant nuclear genome, as plants and animals share similar chromatin organization.
  • Development of a widely applicable regulated and tissue specific high level expression system for foreign genes in transgenic plants will have a profound impact on several currently ongoing plant biotechnology programs and on functional genomic studies. Though considerable increase in trangene expression was achieved through the use of strong viral and tissue specific promoters, protein targeting and codon optimization methods, lack of a generally applicable high level expression to a wide range of crop species in a desired tissue is still a major limiting step.
  • T7 RNA polymerase T7 RNA polymerase based transcription is the used commonly most expression system to overproduce recombinant proteins in microbes (Studier, F. W., Rosenberg, AH., Dunn, J.J., & Dubendorjf, J. W.
  • T7 RNAP based expression has been achieved for transgene in lower eukaryotic organism Trypanosoma brucei recently (Wirtz, E., Hoek, M., & Cross, G.A. Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei. Nucleic Acids Res. 26: 4626-4634 (1998)), it has met with very little success in higher eukaryotic animals. Data from several reports (Hartvig, L., & Christiansen, J.
  • T7 RNAP can transcribe a foreign gene integrated randomly into higher plant nuclear genome, as plant genome is also organized into chromatin structure.
  • the structure of naked DNA in microbial systems in which bacteriophage T7 RNA polymerase (T7 RNAP) has been successfully used to overproduce recombinant proteins is very similar to the chloroplast genome of higher plants. While, it does not automatically' predict that such transcription systems will effectively work to overproduce recombinant proteins in the chloroplast genomes of higher plants, given the unpredictable nature of biotechnological inventions in general, Monsanto, in its recent U.S. Patent teaches overexpression of recombinant proteins in the chloroplast genomes of higher plants.
  • T7 RNAP bacteriophage T7 RNA polymerase
  • the plastid genome is very small [(1.3-1.5) X 10 5 kb] when compared to nuclear genomes of higher plants [1.1 - 4.3 X 10 8 bp], and exists as a double-stranded circular DNA in multiple copies, resembling the genome of prokaryotic organisms in its structure.
  • the nuclear genome is much more complex with a highly organized chromatin and a well-defined nucleus. Therefore, if T7 RNAP is reported to be ineffective to transcribe a foreign gene integrated stably into the chromatin genome of an animal, it would be expected to be equally ineffective to transcribe a foreign gene integrated into a higher plant nuclear genome, as plants and animals share similar chromatin organization.
  • Objects of the invention Accordingly, it is one of the objects of the present invention to effectively transcribe a foreign gene integrated stably into eukaryotic genomes of higher organisms. It is another object of the present invention to provide a method for overexpression of foreign proteins in eukaryotic genomes of higher organisms. It is another object of the present invention to provide a method for overexpression of foreign proteins in eukaryotic genomes, particularly in higher plants.
  • Summary of the invention The above and other objects of the present invention are achieved by the novel Bacteriophage RNA polymerase based transcription systems for overexpression of foreign genes in a higher transgenic organism comprising a foreign gene placed under the control of expression signals and a modified RNA polymerase to specifically transcribe said foreign gene, both said foreign said and said RNA polymerase being located either in cis or trans position with respect to each other in the nuclear genome of a higher organism, said RNA polymerase being selected from T5 RNA polymerase, SP6 RNA polymerase and bacteriophage.
  • T7 RNA polymerase The present invention will be described herein after with reference to T7 RNA polymerase. On the basis of the description provided herein, it will be apparent to a person skilled in the art that the invention will work equally with other polymerases such as T5 RNA polymerase and SP6 RNA polymerase.
  • said modified T7 RNA polymerase is a bacteriophage T7 RNA polymerase.
  • the transgenic organism is a plant.
  • said Bacteriophage T7 RNA polymerase based transcription system is in the form of a construct having a T7 promoter and terminator.
  • the T7 RNA polymerase is preferably expressed with a nuclear localization signal (NLS) under the control of a plant tissue specific promoter to direct the polymerase to the nucleus and to place the transgene under the control of the T7 promoter and terminator in said construct.
  • the transgene is a uidA gene (GUS) and said higher transgenic organism is a monocotyledonous or a dicotyledonous plant.
  • GUS uidA gene
  • the present invention for the first time demonstrates the use of the T7 RNAP to specifically transcribe a foreign gene integrated randomly into the nuclear genomes of plants.
  • GUS uidA gene
  • a commonly used reporter gene in plants Jefferson, R.A., Kavanagh, T.A., & Bevan, M.W. GUS fusions: ⁇ -glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
  • EMBO J. 6: 3901-3907 (1989) integrated randomly into the nuclear genomes of tobacco (a dicot) and rice (a monocot), in a tissue-specific and inducible manner at high levels.
  • a skilled reader of the present specification will be able to appreciate that the present invention can be successfully employed to overexpress any foreign gene in the nuclear genome of any tissue of any plant.
  • T7 RNA polymerase directed tissue specific overexpression of foreign genes in transgenic plants was developed. This was achieved through the transformation of a modified T7 RNA polymerase placed under a tissue specific plant promoter that specifically recognized the transgene (uidA) placed under T7 expression sequences and integrated randomly into tobacco and rice genomes; Results from the use of six different promoters with different tissue specificities indicated that recombinant protein can be expressed at several fold high (3 - 10 times) as compared to transgene expressed directly under these tissue specific promoters. Another important feature of T7 system in plants was found to be the low variations in the transgene expression among independently transformed plants.
  • Each promoter was designed with two constructs, one is control gus gene directly and the other is controlling GUS gene through T7-RNA polymerase system (T7 RNA polymerase with T7-promoter and T7-terminator).
  • LB left border
  • RB right border
  • pro promoter
  • ter terminator
  • pA poly A
  • Hyg hygromycine resistant gene.
  • Figure 1. (C) Northern blot analysis to detect the presence of GUS transcripts in Nt-441-1 (1), Nt-450-2 (2) and Nt-1301-1 (3) using uidA probe. Re-hybridization of the same blot was carried out with ribosomal 16S (16SrRNA) probe to show equal loading of RNA (lower panel).
  • Figure 1. Mapping of the 5' ends of the uidA transcripts by primer extension. ATGC represent partial nucleotide sequence of pITB450 generated by GUS internal primer. Lane 1 shows the extension product using total RNA from wild type
  • Nt.450-2 (not shown) and Nt.450-2 plant.
  • E Histochemical staining with X-gluc indicating the tissues specificity for GUS expression.
  • Top panel The Nt.441-1, Nt.450-2 and Nt.1301-1 plants obtained by transforming pITB441, pITB450 and pCAMBIA1301 constructs, respectively. Lower panel show representative stem and root sections corresponding to the same plants shown in the top panel. Note that while GUS staining was observed in all tissues of Nt-1301-1 plant, it was restricted to green chloroplast containing tissues and totally absent in roots of NT-441-1 and Nt-450-2 plants.
  • Figure 2. In situ analysis of GUS expression. Figure 2.
  • FIG. 3 (J and K). Comparison of GUS expression under rbcS, kinl, cor6.6, pail, pal ⁇ and CaMV 35S promoters directly and through T7-system among independently transformed plants.
  • Figure 3 (A). Gene constructs used for tetracycline inducible expression of GUS under T7 RNAP transcription;
  • the pBin-tetR contained tetracycline repressor gene (tetK) under CaMV35S promoter (35S-pro).
  • the pITB228 contained T7 RNAP under a modified tripleX 35S promoter (12) and GUS under T7 promoter (T7-pro) and terminator.
  • FIG. 3 (B). Northern blot analysis showing the expression of GUS and T7 RNAP upon induction with tetracycline. Blots were probed either with uidA (left panel) or uidA and T7 RNAP together (right panel). UN, uninduced; IN, induced. Re- hybridization of the same blot was carried out with ribosomal 16S (l ⁇ SrRNA) probe to show equal loading of RNA (lower panel).
  • FIG. 3 ( ⁇ )). Kinetics of Tc-induced GUS expression.
  • Figure 4 (A). Histochemical staining to detect the GUS expression in rice leaves. Leaves from wild type (Wt), transformed with pCAMBIAl301 (Os.1301-1) and pITB228 (Os.228-2).
  • Example 1 Tissue specific high level expression
  • the general object of the present invention was to express a modified T7 RNAP with a nuclear localization signal (NLS) (Dunn, J.J., Krippl, B., Bernstein, K.E., Westphal, H, & Studier, F. W. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene 68: 259-266 (1988)), to target the T7 RNAP to nucleus, under a plant tissue specific gene promoter and express the transgene under T7 promoter and terminator in the same construct.
  • NLS nuclear localization signal
  • pITB450, pITB550, pITB650, pITB750 and pITB850 constructs contained uidA placed under T7 promoter and terminator sequences (Fig. 1 A) and the modified T7 RNAP with NLS was placed under the control of the small subunit of ribulose-bisphosphate carboxylase (rZ>cS:3A) (Kuhlemeier, C.
  • the uidA was also placed directly under kinl, cor6.6,pall m ⁇ pallA promoters, in pITB541, pITb641, pITB741 and pITB841 constructs, respectively.
  • pCAMBIA1301 vector containing uidA under the control of a strong cauliflower mosaic virus (CaMV) 35S promoter (Benfey, P.N., and Chua, N.H. The cauliflower mosaic virus 35S promoter: Combinatorial regulation of transcription in plants. Science 250: 959-966 (1990)) that express constitutively in most tissue types was also transformed into tobacco and rice plants for comparison.
  • CaMV cauliflower mosaic virus
  • Tobacco transgenic plants were produced for each of the construct following Agrobacterium mediated transformation (Horsch, R.B. et al A simple and general method for transferring genes into plants: Science 227: 1229-1231 (1985)). Southern hybridization or polymerase chain reaction (PCR) was used to confirm the transformation. Northern blot and real time PCR (data not shown) techniques were used to confirm the transcription of uidA (Fig. 1C). As can be seen from Fig.
  • the uidA transcription under T7-system was 2 - 3 times higher when compared to uidA transcription directly Under rbcS3A promoter and the transcript levels were comparable to uidA transcripts under strong CaMV 35 S promoter in Nt.1301 - 1.
  • primer extension analysis was carried out to authenticate the transcription of uidA by T7 RNAP. It can be seen from figure ID, GUS transcripts initiated from the nucleotide 'G', specific for T7 promoter in Nt.450-2.
  • GUS activity was present in Nt.1301-1 and absent in Nt441-1 and Nt.450-2 plants. Quantification of GUS activity in various tissues further confirmed that the GUS expression under rbcS:3A promoter was highly tissue specific with the highest activity in leaves and lowest in ' roots (Fig. 2A). On the other hand, GUS in all the tissues investigated under 35S promoter expressed and the activity was 3 times high in leaves and 15 times high in roots when compared to GUS expressed under r£cS:3A promoter in leaves and roots, respectively. The pattern of GUS expression was similar in both Nt.441-1 and Nt.450-r2 plants ' with the highest activity in leaf followed by stem and roots.
  • GUS under 35S promoter was uniformly high in all zones of root (Fig. 2D). Within the leaf, as expected, GUS expressed more prominently in the guard cells under cor6.6 (data not shown) and kinl (Fig. 2E) promoters. Analysis of phenylalanine ammonia-lyase (pall) gene promoter from Arabidopsis revealed that the pall promoter is highly tissue specific with maximum activity in the vascular tissue of roots and leaves.
  • a full length (pall, +1 to -832) and a truncated (pallA, +1 to -540) promoters that have same tissue specificity but differ in their strength were used to test the GUS expression under T7- system.
  • uidA vf&s also expressed directly under both /1 wad pall A promoters.
  • expression of GUS was high in vascular tissue of roots (Fig. 2F) and leaves. Similar expression pattern was observed for GUS under pall promoter using T7-system (Fig. 2G). Again, the GUS activity was high in Nt.750-1 when compared to Nt.741-1. Similar results were obtained for pallA promoter.
  • T7-expression system in plants was found to be uniform levels of transgene expression among independently transformed plants, as opposed to large variations found under direct expression of plant promoters. It establishes that very low expression of T7 RNAP is just sufficient to transcribe the transgene at maximum level. This feature will be particularly useful in plants such as legumes, cereals and tree species where it is most difficult to transform and regenerate large number of transgrnic plants required to identify high-expressing plant(s).
  • Example 3 To test the wider application of T7-expression system in plants, the expression of GUS in rice, a monocot plant, with worldwide significance as a major source of staple food was examined. Biolistic mediated transformation (Cao J., Wang Y-C, Klein TM., Sanford JC, Wu R. Transformation of rice and maize using the particle gun method. Pages 21-33 in Plant gene transfer. Lam C.J. and Beachy RN, eds, Wiley- Liss, New York (1990)) was followed to introduce ⁇ CAMBIA1301 and pITB228 constructs into rice genome. A large number of putative transgenic plants, regenerated on hygromycin selection, were screened for the expression of GUS.
  • the non-Tc-treated leaves showed very low activity ( ⁇ 1%) throughout the test period.
  • the GUS activity remained high with no significant differences between treated and un-treated samples.
  • the GUS activity in the Nt.228-1 plant was comparable to the activity observed after 48 - 72 hours of Tc-treated Nt.228+tetR-l plant. No significant difference was observed between the Tc-treated and untreated samples from Nt.1301-1 plant.
  • the co-expression of both T7 RNAP and uidA only after Tc-treatment coupled with the presence of GUS activity clearly demonstrate the highly regulated expression of GUS under T7-system.
  • Hindlll-Ncol (bl ⁇ nt ended) containing CaMV 35S:T7- RNAP:35S polyA cassette from pFF19-T7-35S was cloned into a plant transformation vector pCAMBIA1300 at Hindlll-Smal sites to create pITB239.
  • the PCR amplified uidA gene fragment from pFF19G (using primers forward (5'gattccatggTCCGTCCTGTAGAAACCCCA3' Seq LD 2) and reverse
  • pITB450 was constructed from pITB250 by replacing CaMV 35S promoter with pea rZ>cS:3A promoter.
  • the rbcS:3A promoter (Gene bank Ace. No. M21356) was PCR amplified from pea genomic DNA using primers forward
  • pITB450 (Fig. IB).
  • the plasmid pITB441 was created from pITB450 by removing T7Pro: «/flfA:T7Ter cassette by digesting with Bglll-BamHI and religation.
  • TheT7 RNAP:35SpolyA was replaced with w dA:35S ⁇ ofyA from pFF19G using Ncol-Smal.
  • the plasmid pITB550, pITB650 were created from pITB450 by replacing r ⁇ cS.3A promoter with kinl and cor ⁇ . ⁇ 5-promoters as Smal-Bglll fragments, respectively.
  • the plasmid pITB541 and pITB641 were created from pITB441 by replacing rZ>cS.3A promoter with kinl and cor6.6 promoters as BamHI-Hindlll fragments, respectively.
  • the plasmid pITB750 and pITB850 were created from pITB550 by replacing cor6. d-promoter with pall and pall A promoters as Smal-Bglll fragments, respectively.
  • the plasmid pITB741 and pITB841 were created from pITB541 by replacing cor6. o ' -promoter with pall and pall A promoters, respectively.
  • the pCAMBIA1301 vector containing GUS under CaMV 35S promoter was used for the comparison.
  • the plasmid pBin-tetR was constructed by ligating CaMV 35S:te/R:OCter gene cassette as EcoRI - HindHI fragment into pBin-Hyg in the same sites.
  • the pITB228 construct was created first by cloning the T7 RNAP from pITB250 was cloned as BamHI-Sall fragment into pBinHygTX in the same sites to yield pBin-Hyg-TX-T7.
  • the T7Pro:wtd ⁇ :T7Ter gene cassette from pITB450 was cloned into HindHI site of plasmid pBin-Hyg-TX-T7 to create pITB228. Transformation
  • the LBA4404 strain of Argrobacterium tumefaciens carrying either of the gene constructs was used to transform tobacco (Nicotianatabacum cv. Petit Havana) by leaf disc method.
  • Particle delivery system (PDS lOOHe, BioRad) was used to transform rice.
  • GUS analysis For the detection of GUS expression in various tissues, intact plantlets grown in in vitro or cross sectioned stems or roots were vacuum infiltrated with histochemical staining solution containing 1 mM X-Gluc (5-bromo-4chloro-3-indolyl-b-D-glucuronic acid cyclohexyammonium), 0.1 M NaH2PO4 (pH 7.0), 0.25 M ethylenediaminetetraacetic acid (EDTA), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and incubated at 37°C. After 1 - 12 hours, tissues were treated with 70% ethanol and the GUS activity was visualized under microscope.
  • X-Gluc 5-bromo-4chloro-3-indolyl-b-D-glucuronic acid cyclohexyammonium
  • 0.1 M NaH2PO4 pH 7.0
  • EDTA ethylenediaminetetraacetic acid
  • GUS activity was measured fluorometrically using ImM 4-methylumbelliferyl- ⁇ -D-Glucoronide (MUG) as substrate.
  • UMG ImM 4-methylumbelliferyl- ⁇ -D-Glucoronide
  • Nucleic acid analysis Total genomic DNA isolated from transgenic and wild type plants was digested with relevant restriction endonucleases, resolved on 0.8% agarose gels and transferred on to nylon membrane. About 20 ⁇ g of total RNA isolated from leaf tissue was separated in denaturing formaldehyde agarose gel (1.5%) and blotted on nylon membranes. The membranes were UV crosslinked and then probed with 32P labeled GUS and T7 RNAP coding regions. Standard procedures were followed for nucleic acid hybridization.
  • Transcription start site Primer extension was performed using preamplification kit (Invitrogen) to locate the 5' ends of uidA transcripts. Reaction was carried out with 10 ⁇ g of total RNA using the GUS internal primer (Fig. 1A). Primer was labeled with (gamma 32 P) ATP using T4 polynucleotide kinase (Promega). The size of the extension product was determined by comparison with the DNA sequence generated using the same primer and pITB450 DNA (Seque ⁇ ase II kit, USB). Induction of GUS expression
  • Tetracycline (Tc, l' mg/L) was used for the induction of GUS expression in detached leaves or in in vitro grown plants. Kinetics of induction was followed by real time PCR and by quantifying the GUS activity in the Tc-treated and untreated leaf samples at defined time periods.

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Abstract

L'invention concerne un système de transcription pour la surpexpression de protéines étrangères dans des génomes eucaryotes de plantes transgéniques. On exprime une polymérase ARN modifiée avec un signal de localisation nucléaire sous le contrôle d'un promoteur spécifique au tissu de plante pour orienter la polymérase vers le noyau et placer le transgène sous le contrôle d'un promoteur correspondant et d'un terminateur.
PCT/IN2004/000294 2003-09-18 2004-09-17 Surexpression de genes etrangers dans les plantes WO2005026367A1 (fr)

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WO2024030824A3 (fr) * 2022-08-02 2024-04-18 Syngenta Crop Protection Ag Séquences régulatrices de plantes et cassettes d'expression

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WO2000042206A1 (fr) * 1999-01-18 2000-07-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme de mise au silence de l'expression et ses differentes utilisations

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Publication number Priority date Publication date Assignee Title
WO2000042206A1 (fr) * 1999-01-18 2000-07-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Systeme de mise au silence de l'expression et ses differentes utilisations

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WO2024030824A3 (fr) * 2022-08-02 2024-04-18 Syngenta Crop Protection Ag Séquences régulatrices de plantes et cassettes d'expression

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