Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Patent
1994-11-03
1999-09-14
Robinson, Douglas W.
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
204456, 435 912, 4353201, 435419, 435468, 536 2371, 800279, A01H 500, C12N 514, C12N 1532, C12N 1582
Patent
active
059525479
DESCRIPTION:
BRIEF SUMMARY
This invention provides a modified gene, such as a Bacillus thuringiensis ("Bt") gene (the "modified Bt ICP gene") encoding all or an insecticidally-effective portion of a Bt insecticidal crystal protein ("ICP"). A plant, transformed with the modified gene, shows higher expression levels of the encoded protein.
BACKGROUND OF THE INVENTION
Plant genetic engineering technology has made significant progress during the last 10 years. It has become possible to introduce stably foreign genes into plants. This has provided exciting opportunities for modern agriculture. Derivatives of the Ti-plasmid of the plant pathogen, Agrobacterium tumefaciens, have proven to be efficient and highly versatile vehicles for the introduction of foreign genes into plants and plant cells. In addition, a variety of free DNA delivery methods, such as electroporation, microinjection, pollen-mediated gene transfer and particle gun technology, have been developed for the same purpose.
The major aim of plant transformations by genetic engineering has been crop improvement. In an initial phase, research has been focussed on the engineering into plants of useful traits such as insect-resistance. In this respect, progress in engineering insect resistance in transgenic plants has been obtained through the use of genes, encoding ICPs, from Bt strains (Vaeck et al, 1987). A Bt strain is a spore forming gram-positive bacterium that produces a parasporal crystal which is composed of crystal proteins which are specifically toxic against insect larvae. Bt ICPs possess a specific insecticidal spectrum and display no toxicity towards other animals and humans (Gasser and Fraley, 1989). Therefore, the Bt ICP genes are highly suited for plant engineering purposes.
For more than 20 years, Bt crystal spore preparations have been used as biological insecticides. The commercial use of Bt sprays has however been limited by high production costs and the instability of crystal proteins When exposed in the field (Vaeck et al, 1987). The heterogeneity of Bt strains has been well documented. Strains active against Lepidoptera (Dulmage et al, 1981), Diptera (Goldberg and Margalit, 1977) and Coleoptera (Krieg et al, 1983) have been described.
Bt strains produce endogenous crystals upon sporulation. Upon ingestion by insect larvae, the crystals are solubilzed in the alkaline environment of the insect midgut giving rise to a protoxin which is subsequently proteolytically converted into a toxic core fragment or toxin of 60-70 Kda. The toxin causes cytolysis of the epithelial midgut cells. The specificity of Bt ICPs can be determined by their interaction with high-affinity binding sites present on insects' midgut epithelia.
The identification of Bt ICPs and the cloning and sequencing of Bt ICP genes has been reviewed by Hofte and Whiteley (1989). The Bt ICP genes share a number of common properties. They generally encode insecticidal proteins of 130 kDa to 140 kDa or of about 70 kDa, which contain toxic fragments of 60.+-.10 kDa (Hofte and Whiteley, 1989). The Bt ICP genes have been classified into four major groups according to both their structural similarities and insecticidal spectra (Hofte and Whiteley, 1989): Lepidoptera-specific (CryI), Lepidoptera- and Diptera-specific (CryII), Coleoptera-specific (CryIII) and Diptera-specific (CryIV) genes. The Lepidoptera-specific genes (CryI) all encode 130-140 kDa proteins. These proteins are generally synthesized as protoxins. The toxic domain is localized in the N-terminal half of the protoxin. Deletion analysis of several CryI genes confirm that 3' portions of the protoxins are not absolutely required for toxic activity (Schnepf et al, 1985). CryII genes encode 65 kDa proteins (Widner and Whiteley, 1985). The CryIIA proteins are toxic against both Lepidoptera and Diptera while the CryIIB proteins are toxic only to Lepidopteran insects. The Coleoptera-specific genes (CryIII) generally encode proteins with a molecular weight of about 70 kDa (Hofte and Whiteley, 1989). The CryIIIA gene expressed in E. coli directs the s
REFERENCES:
patent: 5380831 (1995-01-01), Adang et al.
patent: 5550365 (1996-08-01), Fischhoff et al.
Vaeck et al. (1987) Nature vol. 328: pp. 33-37 Jul. 1987.
Proceedings of the National Academy of Sciences of USA, "Modification of the Coding Sequence Enhances Plant Expression of Insect Control Protein Genes", Perlack, et al., vol. 88, Apr. 1991, pp. 3324-3328.
Plant Molecular Biology, "Analysis of Unstable RNA Transcipts on Insecticidal Crystal Protein Genes of Bacillus thuringiensis in Transgenic Plants and Electroportated Protoplasts", Murray et al., vol. 16, Jun. 1991, pp. 1035-1050.
Nature, "Transgenic Plants Protected from Insect Attack", Vaeck, et al., vol. 328, Jul. 1987, pp. 33-37.
Cornelissen Marc
Dockx Jan
Soetaert Piet
Stam Maike
Van Aarssen Roel
Nelson Amy J.
Plant Genetic Systems N.V.
Robinson Douglas W.
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