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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
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8239—Externally regulated expression systems pathogen inducible
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
  • C07K14/8114—Kunitz type inhibitors
• • 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

Definitions

• the present invention is the first report of the successful use of a monocot intron being used to enhance the activity of a dicot promoter in a transgenic monocot.
• “monocot” refers to plants belonging to the subclass Monocotyledoneae
• “dicot” refers to plants belonging to the subclass Dicotyledoneae.
• the commercially important monocots which may be transformed with the wound inducible promoter according to the present invention are rice, wheat, barley, oats, sorghum, and maize.
• the use of rice is meant to be descriptive of the monocot subclass of plants; the present invention should not be considered to be limited to only rice.
• foreign gene of interest refers to a gene encoding for the expression of proteins having natural insecticidal or antiviral activities in transgenic monocot plants, and also encompasses a plurality of genes having mixed insecticidal and antiviral activities.
• the insecticidal protein genes include the potato protease inhibitor II and inhibitor I genes, cowpea trypsin inhibitor gene (CPTi), and various Bacillus thurigiensis endotoxin genes.
• the antiviral genes include genes which express proteins which control insect-transmitted viral diseases, including but not limited to tungro virus, dwarf virus, black-streaked dwarf virus, and hoja blanca virus infection, and are based upon viral coat protein, antisense RNA, or an enzymatic requirement for viral infection, replication, or release.
• the foreign gene of interest may be substituted in place of the Gus coating regions, or inserted at an appropriate site within the expression vectors, depicted in the accompanying drawings.
• the important insect pests of rice include yellow stemborer, striped stemborer, leaffolder, gall midge, brown planthopper, whitebacked planthopper, and green leafhopper. It is also known that several devastating viral diseases are transmitted by insects as, for example, tungro virus, dwarf virus, black-streaked dwarf virus, and hoja blanca virus, all of which are known to be transmitted by rice planthoppers or leafhoppers. Most insects have trypsin or chymotrypsin-like proteases in their digestive systems. Thus, introducing the appropriate protease inhibitor genes into rice plants should make the plants insect tolerant - the premise upon which the research that went into the making of the present invention was conducted.
• the different B.t. toxins have been used to kill insects by spraying plants with the crude B.t. toxins.
• the B.t. toxins are specific for lepidopteran insects and exhibit no harmful activity against humans, other vertebrates, and beneficial insects.
• the genes of these insect-killing proteins are attractive candidates for genetic engineering of crop plants for protection against lepidopteran pests.
• Proteinase inhibitors provide natural defenses against insect attack by acting on the primary digestive enzymes in certain insects. Proteinase inhibitors present in dicot plants have been found to be active against a wide range of insects [see BioEssays 10:20 (1989)].
• the potato gene family encoding the serine-type proteinase inhibitor II (Pinll) is by far one of the best analyzed systems of systemic gene activation in response to insect attack [see EMBO 8:1323 (1989); and Proc. Natl. Acad. Sci. USA 87:7205 (1990)]. Pinll is constitutively expressed in the tubers and young floral buds of normal greenhouse grown potato plants.
• the potato protease inhibitor II inhibits both trypsin and chymotrypsin-like proteases, it was selected as the first choice for introducing Pinll-containing constructs according to the present invention into a monocot.
• Rice is a major crop in the world and the the techniques have been developed and used for many years in transforming and regenerating this monocot.
• rice can easily be grown under greenhouse conditions, has a relatively short germination to maturity period, and a number of plants can be grown in a relatively small area. Thus, rice was selected for the initial research leading to the present invention.
• Purified B.t. endotoxins have had a long history of safe use as insecticides because they are non-toxic to non-target organisms and are rapidly biodegraded in the environment [see
• B.t. endotoxins display a narrow range of target insects.
• Several genes encoding different B.t. toxins have been cloned and the expression of individual B.t. toxin genes has been widely studied in transgenic dicot plants [see Plant Physiol. 85:1103 (1989)].
• the present invention was approached with the goal that if the production of B.t. endotoxin could be expressed in transgenic rice plants, this should preclude the need for repeated spraying of field crops with expensive insecticides, and kill the target insects before significant damage could occur.
• the major potential disadvantage to expressing B.t. endotoxins in transgenic plants, whether monocots or dicots is that insect resistance might develop over a period of time.
• transgenic plants expressing one or a variety of natural insecticides When using transgenic plants expressing one or a variety of natural insecticides, the aim should be to keep selection pressure for the insects as low as possible in order to minimize the development of resistant pests. Restricting the expression of such natural insecticides to those plant tissues which are most susceptible to pest damage should decrease selection pressure while still providing adequate protection. Constitutive and high- level expression of a foreign gene in plants may have undesirable effects on the growth and development of the plants.
• an aspect of the research leading to the present invention was to produce transgenic plants that express foreign genes of interest, and mainly natural insecticides such as B.t. endotoxins or cow pea trypsin inhibitor, only in direct response to specific insect associated damage.
• Figure 1 is a depiction of various genetic constructs made in accordance with the present invention
• Figure 2 is a depiction of various expression vectors according to the present invention.
• the initial step in making the present invention was to determine whether the Pinll promoter from a dicot plant could be incorporated into a monocot plant, and if so, whether the promoter would function and respond to wounding.
• the 5' intron of the rice actin 1 gene (Act1 )
• the actin 1 gene when inserted between the cauliflower mosaic virus CaMV35S gene promoter and the ⁇ -glucoronidase (GUS) reporter gene greatly stimulated GUS activity in transformed rice protoplasts
• a construct of the Act1 intron inserted between the Pinll promoter and Gus reporter (which is replaced by a foreign gene of interest in accordance with the ultimate uses of the present invention) gene was constructed to determine if GUS activity would be stimulated.
• the use of the 5' intron from the rice Act1 gene to increase foreign gene expression from the promoter , of the potato wound inducible protease inhibitor II gene (Pinll) in transgenic rice was found to significantly enhance the activity of the potato Pinll promoter in transient assays of transformed protoplast as shown in the following table: Construct Structure Mean GUS Specific
• Nos 3' refers to nopaline synthase gene 3" noncoding regions.
• rice protoplasts (4-10 X 10 6 ) were co-transformed with a Pinll-based fusion plasmid (80 ⁇ g) and a selectable Act1 -bar (glufosinate-ammonium resistant gene) construct (20 ⁇ g) using techniques described in United States Patent Application 07/762,680 and those well known in the art [see Theor. App. Genet. 76:835 (1988)].
• Resistant calli were selected on 4 mg/l glufosinate ammonium using well known techniques.
• Each individual transformation produced between 400 and 600 resistant calli. Plantlet regeneration efficiency from resistant calli was between 10% and 20%, and the mean co- expression frequency was 65% for Act1 5' intron-containing Pinll-based fusion constructs.
• the wound inducible expression of natural insecticides at high levels in monocot plants using this modified Pinll promoter system should provide adequate protection against insect attack.
• the insecticide is not expressed until such time as the plant is wounded or attacked by insects, and as the life of these insecticides in the plant is relatively short - measured in hours - the use of the promoter system should reduce the selection pressure on the development of resistant insects.
• Pinll-based vectors were developed using techniques which have become well known and conventional in the art.
• the structure of the various Pinll-based expression vectors are shown in Figure 2, The strategy for the construction of these vectors was (1) to isolate the Pinll 3' end as a 1 kbp Xbal-Sphl restriction fragment from pDXP3, and to clone this into the plasmid pCOR116 [see Mol. Gen. Genet.
• Plasmids pDX227, pDX202, pDX216, and pDX217 each contain the Pinll promoter and Act15' intron followed by a polylinker sequence into which the foreign gene of interest (with its own 3' transcription terminator region) may be cloned.
• Plasmids pDX427, pDX402, pDX416 and pDX417 each contain a Pinll 3' transcription terminator region following their Pinll promoter and Act1 5' intron with a polylinker between the intron and 3'-end regions into which the coding region of the foreign gene of interest may be cloned.
• Different expression vectors according to the present invention each containing several different restriction sites facilitate the cloning of useful foreign genes and subsequent manipulation of the resulting recombinant DNA molecules.
• Each plasmid pDX227/pDX427, pDX202/pDX402, and pDX216/pDX416 contains an Act1 translation initiation codon in their polylinker region. These plasmids can be used for the creation of translation fusion constructs. Modification of the translation initiation codon region of the foreign genes of interest to create a Ncol restriction site facilitates the creation of fusion constructs using the pDX202/pDX402 and pDX216/pDX416 plasmids.
• each of the translation fusion vectors according to the present invention contains a unique blunt-ended restriction site into which any suitably prepared foreign gene of interest may be cloned.
• blunt ended restriction sites EcoRV or Smai
• EcoRV or Smai are found in all three possible reading frames permitting the creation of translational fusion constructs with any suitably treated foreign gene of interest.
• the transcriptional fusion vectors pDX217/pDX417 can be used to clone foreign genes that cannot tolerate N-terminal amino acid additions.
• Each vector contains no translation initiation codon.
• restriction sites in their respective polylinker region they may each contain a unique blunt-ended restriction site (REcoRV, underlined) for the cloning of any suitable foreign gene of interest.
• viral translational enhancers such as the alfalfa mosaic virus (AMV) enhancer [see Nature 325:622 (1987)] may also be included in the Pinll expression vectors according to the present invention.
• AMV alfalfa mosaic virus
• Specific chimeric plasmids according to the present invention may be constructed along a number of lines.
• the constructs include:
• CPTi gene Agricultural Genetics Company
• Pinll-Act1 5' intron B.t. toxin-Nos 3' constructs were also prepared.
• a B.t. toxin gene (Agracetus) was used which contains a truncated delta endotoxin HD-1 gene preceded by an AMV translational enhancer, and the B.t. toxin gene and the AMV enhancer was placed downstream from the Pinll- Act1 5' intron.
• Another construct was prepared using a B.t. toxin (KC) gene was tested for insect killing activity and found to have a relatively broad host range against different species of insects.
• KC B.t. toxin
• the wound inducible promoter system according to the present invention can be expanded to include the control of insect-transmitted viral diseases.
• the tungro virus infection of rice plants requires that participation of an insect vector.
• the constructs according to the present invention may easily be modified by adding foreign DNA to the construct, by adding the anti-tungro viral DNA to a basic construct according to the present invention (Pinll-Act1 5' intron-Pinll gene-Nos 3' region), and transform rice with this construct, the regenerated transgenic plants will synthesize very little Pinll protein and anti-viral DNA.
• the anti-viral DNA is produced to inhibit virus replication or production.
• MOLECULE TYPE DNA
• SEQUENCE DESCRIPTION SEQ ID NO:3: GTAG G ATG GCT GAC GCC GAG GAT GGG GGA TCC CC 36
• MOLECULE TYPE DNA
• SEQUENCE DESCRIPTION SEQ ID N0:1 :

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• 1992
  • 1992-09-30 EP EP92921633A patent/EP0666921A4/fr not_active Withdrawn
  • 1992-09-30 JP JP5507051A patent/JPH07503126A/ja active Pending
  • 1992-09-30 WO PCT/US1992/008359 patent/WO1993006713A1/fr not_active Application Discontinuation

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