EP0455666A1 - Regulation de l'expression de genes vegetaux - Google Patents

Regulation de l'expression de genes vegetaux

Info

Publication number
EP0455666A1
EP0455666A1 EP90901856A EP90901856A EP0455666A1 EP 0455666 A1 EP0455666 A1 EP 0455666A1 EP 90901856 A EP90901856 A EP 90901856A EP 90901856 A EP90901856 A EP 90901856A EP 0455666 A1 EP0455666 A1 EP 0455666A1
Authority
EP
European Patent Office
Prior art keywords
gene
dna
plant
operator
binding protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP90901856A
Other languages
German (de)
English (en)
Inventor
Ian George Bridges
Simon William Jonathan Bright
Andrew James Greenland
Wolfgang Walter Schuch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Ltd
Original Assignee
Zeneca Ltd
Imperial Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zeneca Ltd, Imperial Chemical Industries Ltd filed Critical Zeneca Ltd
Publication of EP0455666A1 publication Critical patent/EP0455666A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/72—Expression systems using regulatory sequences derived from the lac-operon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8237—Externally regulated expression systems
    • 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
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible

Definitions

  • This invention relates to the regulation of plant gene expression. More particularly, the invention is concerned with the regulation of plant gene expression by the use of repressor molecules. The invention also provides materials for use as repressors and methods for the production thereof.
  • Traditionally the improvement of crop plant species involves the introduction of desired traits by genetic crosses. However, although these breeding techniques are highly successful, they provide no means of controlling the expression of the newly acquired traits. Recent advances in technology are now allowing the genes responsible for determining plant structure and the productivity and quality of the crop to be identified and isolated. A major aim in the field of improvement is therefore to be able to manipulate complex developmental processes genetically in order to improve crop performance. Essential to this objective is the determination of strategies which allow the expression of specific plant genes to be regulated at will.
  • CMS cytoplasmic male sterility
  • Control of gene expression in both prokaryotes and eukaryotes relies primarily on the interaction of regulatory proteins with specific DNA sequences. Depending on the nature of these interactions, transcription from the cognate promoters may either be repressed or activated. Indeed, in some cases the same protein may either reduce or enhance transcription according to the nature of the contacts made. Furthermore, the ability of some regulatory proteins to bind their target sequences is modulated by the binding of ligands or by specific proteolytic cleavage. Such mechanisms may be exploited in order to include inducibility amongst strategies for plant gene regulation.
  • the best characterised regulatory systems are those of bacteria in which the interactions between the DNA-binding proteins (repressors) and the target DNA sequences (operators) are understood in great detail.
  • a comparison of the best understood systems, including repressor and cro proteins of bacteriophage ⁇ and 434, the Lacl repressor and the catabolite gene-activating protein (CAP) reveals several factors in common.
  • These regulatory proteins bind as di ers or tetramers to short operators that exhibit a high degree of dyad symmetry. In most cases the domain responsible for DNA-recognition, which is separate from that concerned with oli.gomerisation of the monomers, contains a conserved helix-turn-helix structure.
  • a specific helix within this structure in each monomer, the recognition helix, is aligned with the major groove of the DNA and only if specific contacts are formed between the amino acids of this recognition helix and the bases of the adjacent DNA can a functional repressor/operator complex be formed. Such interactions are highly specific, and the high-affinity complexes are formed with extremely rapid kinetics.
  • Eukaryotic regulatory systems appear to exhibit a greater diversity of structure and a higher degree of complexity than their prokaryotic counterparts. For instance, control of transcription from eukaryotic promoters is thought to involve the interaction of many proteins (perhaps in the order of tens) with the regulatory DNA. Furthermore, at least three different protein structures (the helix-turn-helix, the zinc-finger and the leucine zipper) have been implicated in the specificity of DNA-binding by various eukaryotic regulatory factors.
  • DNA-binding protein constitute a class of proteins characterised by their ability to bind to DNA of genes to give the effect of either repressing or activating the gene to which they bind. Unless the context otherwise requires, such DNA-binding proteins are hereinafter referred to for convenience simply as "repressors" .
  • An object of the present invention is to provide means for the control of expression of specific plant genes.
  • a recombinant plant gene comprising a repressor gene of bacterial origin and a promoter which operates in plants for driving expression of the repressor gene, said gene encoding a repressor protein capable of interaction with an operator sequence associated with a selected target plant gene so that on expression of the repressor protein expression of the target plant gene is inhibited.
  • the invention also provides stably transformed cells containing the said recombinant plant gene.
  • the invention further provides a vector, preferably the plasmid, designated p35Slacl, containing the said DNA, which has been deposited in an E.coli, strain TG-2, host with the National Collection of Industrial and Marine Bacteria Limited, Aberdeen, United Kingdom, on 12th December 1988, under the Accession Number NCIB 40092.
  • the invention provides a plant transformation vector comprising Agrobacterium tumefaciens, harbouring the plasmid aforesaid.
  • a bacterial lad operator system is utilised to regulate gene expression. Lac repression can be relieved by iso-propyl thiogalactoside (IPTG) and other sugar analogues.
  • IPTG iso-propyl thiogalactoside
  • the method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed.
  • Transgenic plants are obtained by regeneration from the transformed cells.
  • lac repressor lacl ⁇
  • the lac repressor (lacl ⁇ ) is available on plasmid pMJR 156.
  • the translation initiation codon GTG
  • suitable restriction sites Hindlll and Pstl
  • a Cfr 10 restriction site is located at position 134.
  • pMJR was cut with Cfr 10 and a synthetic DNA fragment which reconstitutes the N-terminus of the lad gene, the altered translational start codon ATG, a plant consensus sequence for efficient translational initiation and a BamHI restriction site were inserted into pJRl.
  • the sequence of this synthetic fragment was: BamHI consensus
  • the PCR reaction was carried out under the prescribed conditions.
  • the product was cut with BamHI (at the newly introduced site) and Pstl.
  • the resulting fragment was clones into pJRl cut with BamHI and Pstl. Recombinants were identified by hybridisation and restriction analysis using standard protocols. One of the resulting clones was characterised by DNA sequence analysis.
  • this vector was achieved by replacing the CaMV promoter in p35SlacI with the maize CAB promoter, the DNA sequence of which is given in Figure 3 herewith, which is found in vector pCAB48.1.
  • the CaMV promoter was removed by restriction of p35SlacI with EcoRI and BamHI using standard conditions.
  • the CAB promoter was isolated from pCAB48.1 by restriction with Xbal and Sau3A using partial restriction conditions for Sau3A. This promoter fragment was then inserted into the promoter-less p35SlacI.
  • This vector, designated pCABlacI has been characterised by restriction mapping and DNA sequence analysis.
  • the expression modules from the vectors described above were transferred to BIN19 and then to tobacco using leaf disc transformation following standard protocols.
  • the plasmids were transferred to Agrobacterium using triparental mating.
  • Agrobacteria were purified, and used in leaf disc transformation experiments. Thirty-seven plants containing the CaMV-lad expression module and thirty-eight plants containing the BAB-lacl construct were regenerated and analysed for the relative expression of lad. 1.4 Analysis of transgenic plants for lad expression
  • the expression of the lad gene was monitored using Western analysis of extracted proteins. Extracts were prepared, proteins recovered on polyacrylamide gels and prepared for Western analysis. The analyses confirmed the expression of the lad gene construct in the transformed plants. Different levels of la gene expression were observed in different independent transformants. The results for plants transformed with the CaMV-lad construct are given in the following Table
  • the maize CAB promoter can be found in plasmid pCAB48.1 and we have found that this promoter can drive expression of foreign genes in a transient tobacco expression system and in stably transformed plants. This gene, therefore, is an excellent target to demonstrate control through lad as high levels of expression can be obtained both i_n vitro and iji vivo.
  • the CAB promoter from other systems have been extensively analysed in detail and reported in the literature. The published information facilitates the selection of suitable sites for operator insertion.
  • pCAB48.1 is a maize promoter and the use of this system is important to demonstrate the applicability of this invention to monocotyledonous plants such as maize, heat, barley and sorghum.
  • the restriction enzyme PvuII recognises a single site within the 2.8 kb Pstl fragment containing the CAB gene.
  • the site lies between the TATA element and the transcription start point (TSP) of the CAB promoter.
  • TTP transcription start point
  • the vector pCAB48.1 contains numerous PvuII sites (within pUCl9). Therefore, the 2.8 kb Pstl fragment was cloned into the standard cloning vector pATl53 (which lacks a PvuII site) to give pCABPl.
  • pUCl ⁇ was digested with Hindlll and BamHI and the promoter fragment from (i) above was inserted to give pCABP2. Digestion of pUC18 with BamHI removes the single Sphl site from the polylinker. Therefore, pCABP2 contains a unique Sphl site into which operators can be inserted.
  • the second oligonucleotide overlaps the Ncol site and contains the operator sequence shown below.
  • a slightly different approach which eliminates the intermediate cloning step into pCABPl may also be used. This involves using an oligonucleotide which overlaps the unique Xbal site in the CAB promoter together with the operator nucleotides outlined previously. Digestion of PCR DNA with Xbal/Ncol results in a fragment which can be directly cloned into pCGl and pCG2. However the Xbal to Ncol fragment from the PCR reaction is much larger than the PvuII to Ncol fragment obtained from the previous strategy. Operator insertion between the CAAT and TATA This is effected using PCR.
  • a promoter-less 35S vector is an excellent receptor for the insertion of activating sequences.
  • the lac operator can be inserted into this vector, p- ⁇ -35S, and once inserted the 35S enhancer is cloned 5' upstream of the lac operator.
  • (3) Control of gene expression by lac repressor (a) Control of target gene expression in a transient expression system Plants which express lad constitutively transformed with p351acl may be prepared from protoplasts and, using methods described above) they may be tested for expression of the lad protein. The target gene constructs may then be introduced into the protoplasts using standard methods and protocols. Protoplasts from untransformed plants can serve as control. Further control may be provided by protoplasts from plants expressing the GUS marker gene under the control of the CAB promoter without the operator insertions.
  • IPTG can be used to overcome repression by the lac repressor.
  • a switchable gene system there is formed a switchable gene system.
  • Lac repressor/operator interactions can down-regulate marker gene expression in plants to different levels. This is an important effect in that there may be situations where a different degree of down-regulation may be required.
  • suitable operator insertion constructs may be transferred to tobacco plants by the methods described above.
  • the regenerated plants may be crossed with the lad expressing plants described above, which express the lac repressor under control of the constitutive CaMV35S promoter.
  • Plants may also be constructed which express the lad gene under control of the light-inducible maize CAB promoter. The expression of the lad gene in these plants will then be light-inducible. These plants may be crossed with plants which contain the GUS marker gene from the CaMV promoter containing the lad operator insertion. Insertion of multiple operators into the CAB promoter
  • multiple operators can be inserted into the target promoter. This can either be by the insertion of multiple copies of the operator at one site, or the combination of fragments of the promoter in which the operator is inserted at different positions in the promoter, this yielding vectors in which the multiple operators are located at multiple locations in the promoter.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Communicable Diseases (AREA)
  • Botany (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Un gène végétal de recombinaison permettant la régulation de l'expression de gènes, comporte un gène de protéine de répression d'origine bactérienne associé à un opérateur reconnu par la protéine de répression bactérienne, et régulant l'expression d'un gène structurel étranger.
EP90901856A 1989-01-26 1990-01-25 Regulation de l'expression de genes vegetaux Withdrawn EP0455666A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB898901674A GB8901674D0 (en) 1989-01-26 1989-01-26 Regulation of plant gene expression
GB8901674 1989-01-26

Publications (1)

Publication Number Publication Date
EP0455666A1 true EP0455666A1 (fr) 1991-11-13

Family

ID=10650614

Family Applications (1)

Application Number Title Priority Date Filing Date
EP90901856A Withdrawn EP0455666A1 (fr) 1989-01-26 1990-01-25 Regulation de l'expression de genes vegetaux

Country Status (6)

Country Link
EP (1) EP0455666A1 (fr)
AU (1) AU644783B2 (fr)
CA (1) CA2008696A1 (fr)
GB (1) GB8901674D0 (fr)
WO (1) WO1990008829A1 (fr)
ZA (1) ZA90607B (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5202422A (en) * 1989-10-27 1993-04-13 The Scripps Research Institute Compositions containing plant-produced glycopolypeptide multimers, multimeric proteins and method of their use
US7005560B1 (en) 1989-10-27 2006-02-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
DE4100594A1 (de) * 1991-01-08 1992-07-09 Inst Genbiologische Forschung Neue plasmide zur zeitlichen und oertlichen kontrollierten expression eines heterologen produktes in pflanzen
CA2316036A1 (fr) * 1999-08-27 2001-02-27 Keqiang Wu Regulation de l'expression genetique chez des vegetaux
US7517975B2 (en) 2000-09-26 2009-04-14 Pioneer Hi-Bred International, Inc. Nucleotide sequences mediating male fertility and method of using same
EP2333084A1 (fr) 2003-12-16 2011-06-15 Pioneer Hi-Bred International Inc. Transgène de suppression de gène dominant et leurs procédés d'utilisation
MX2010001347A (es) 2007-08-03 2010-03-01 Pioneer Hi Bred Int Secuencias de nucleotidos msca1 que afectan la fertilidad vegetal masculina y metodo para usar las mismas.
AU2012333207A1 (en) 2011-06-21 2014-01-23 E.I. Du Pont De Nemours And Company Methods and compositions for producing male sterile plants
AU2018325363A1 (en) 2017-08-29 2020-04-09 Global Crop Innovations Pty. Ltd. Improved BLue Aleurone and other segregation systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9008829A1 *

Also Published As

Publication number Publication date
AU4945390A (en) 1990-08-24
WO1990008829A1 (fr) 1990-08-09
GB8901674D0 (en) 1989-03-15
ZA90607B (en) 1990-10-31
CA2008696A1 (fr) 1990-07-26
AU644783B2 (en) 1993-12-23

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