EP1107984A1 - Sequences d'adn synthetiques modifiees pour une meilleure action insecticide - Google Patents

Sequences d'adn synthetiques modifiees pour une meilleure action insecticide

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Publication number
EP1107984A1
EP1107984A1 EP99940216A EP99940216A EP1107984A1 EP 1107984 A1 EP1107984 A1 EP 1107984A1 EP 99940216 A EP99940216 A EP 99940216A EP 99940216 A EP99940216 A EP 99940216A EP 1107984 A1 EP1107984 A1 EP 1107984A1
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EP
European Patent Office
Prior art keywords
synthetic dna
dna sequences
modified
cry9aa
seq
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EP99940216A
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German (de)
English (en)
Inventor
Viktor Kuvshinov
Anne Kanerva
Kimmo Koivu
Eija Pehu
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UniCrop Ltd
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UniCrop Ltd
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is related to synthetic DNA sequences comprising modifications of the truncated gene encoding the N-terminal domain of the Cry9Aa endotoxin of Bacillus thuringiensis ssp. galleria characterized by having the amino acid sequence (SEQ ID NO : 1 : ) or alterations thereof with substantially similar structure and properties.
  • a method for preparing said modified synthetic DNA-sequence is disclosed in the present invention as well as the use of them. Said modified DNA-sequences are useful for improved insecticidal control and as a tool in resistance management strategies.
  • crystalline endotoxins of Bacillus thuringiensis (Bt toxins) for insecticidal control has been the target for intensive research programs because the endo-toxins are non-toxic to non-target organisms, including mammals and human beings.
  • the endo-toxins have a very specific and selective effect on insects, especially during their larval stage.
  • Bt toxins are considered to be the most effective bioinsecticides used in agriculture.
  • the target insects develop resistance to Bt toxins much slower than to chemical insecticides.
  • Plutella xylostella is known to develop resistance to Cryl group of toxins in 30 to 100 generations in laboratory conditions.
  • the bacterial Bt toxins as well as their modes of action have been studied intensively and some of these endotoxins have been used as insecticides by administering culture broth containing the bacteria or by administering more or less purified endotoxin. Because of their insecticidal impact the Bt toxins encoded by the cry genes have also been used to produce transgenic plants. The first attempts to transform plants using the long, native sequences of the genes failed
  • cry genes have been truncated to contain just the DNA sequence encoding the active toxin protein situated in the protoxin between the trypsin cleavage sites . Truncated cry genes have been transformed into many plant species including tobacco
  • crylllA (Adang, M.J. et al . , (1993), Plant Mol. Biol. 21:1131-1145) and cry ⁇ A (b) (Fujimoto, H., et al., (1993) Bio/Technology 11:1151-1153).
  • Adang, M.J., et al . resynthetised the sequence of crylllA gene using ligation of oligonucleotides of 13 different fragments (Adang, M.J., et al., (1993) Plant Mol. Biol. 21:1131-1145). The fragments were then ligated into a full gene sequence which was transformed into potato. Fuijmoto, H., et al . , (1993) resynthetised the cry ⁇ A (b) gene sequence using high fidelity PCR.
  • crylAb Resynthesised crylAb (Koziel, M.J., et al . , (1993) Bio/Technology 11: 194-200), crylllA (Perlak, F.J., et al . ,
  • crylAc Perlak, M.J., et al . , (1991) Proc. Natl. Acad. Sci. USA 88: 3324-3328
  • high expression of the unmodified truncated cry ⁇ A (c) gene was achieved using site directed transformation of tobacco chloroplasts (MacBrige, K. E., et al . , (1995) Bio/Technology 13:362-365.
  • cry ⁇ A (c) gene product expressed in the nucleus into chloroplasts was shown to enhance the amplification of the protein by 10 to 20 fold (Wong, E.Y., et al . , (1992) Plant Mol. Biol. 20:81-93) .
  • Pyramidal expression of two cry genes with different binding receptors was shown to reduce the incidence of resistant insects (van der Salm, T., et al . ,
  • cryl genes Lepidopteran active
  • cry ⁇ A (c) cry ⁇ A (b)
  • crylllA Cold-dextrative active
  • a recent report (Conner, A.J., et al . , Fifth International Symposium on the Molecular Biology of the Potato, August 2-6, 1998, Bogensee, Germany) it was shown that potato lines transformed with truncated crylAc gene had a greater impact on larval growth and survival than a sequence altered by site-directed mutagenesis of truncated, native cry9Aa2. Said results confirmed the observation that partial modification does not result in sufficiently high expression level in plants, disclosed also by other research groups.
  • the main objective of the present invention is to provide plants with an increased toxicity against target insects, such as lepidopteran larvae in higher plants by providing a substantially different insecticidal protein encoded by DNA sequences, which simultaneously convey enhanced expression through improved mRNA processing and stability as well as enhanced translation, which together bring about increased tolerance of higher plants against attacks of target insects.
  • the objectives of the present invention are to obtain transgenic plants highly expressing a synthetic DNA sequence encoding a unique Bt -toxin having a specific insecticidal action, thus providing a means for delaying development of insect tolerance to the toxin and to overcome problems related to cross-resistance phenomena. The latter enables the use of the synthetic DNA sequences of the present invention for implementing resistance management strategies.
  • the objectives of the invention were achieved by providing novel synthetic DNA sequences by modifying part of the gene encoding the N-terminal end of the native Cry9Aa protein, the toxicity of which is based on an insecticidal action and/or binding receptor mechanism differing substantially from that of other Cryl toxins .
  • the present invention is related to modified synthetic DNA sequences of the truncated DNA sequence of the cry9Aa gene encoding a protein characterized by an amino acid sequence (SEQ ID NO:l:) or alterations thereof, which still have substantially the same structure and insecticidal action as the N-terminal domain of the Cry9Aa endotoxin of Bacillus thuringiensis ssp. galleria .
  • the modified synthetic DNA sequences are characterized by conveying improved properties, such as enhanced expression through increased mRNA procession and stability as well as translation capacity in transgenic plants, while the encoded (expressed) delta-endotoxin is still maintaining substantially the same insecticidal action as the delta-endotoxin encoded by the native cry9Aa gene.
  • Said modified synthetic DNA sequence can be inserted into suitable DNA constructs to enable their transfer into desired procaryotic or eucaryotic hosts.
  • the improved mRNA processing and stability as well as translation of the corresponding toxin protein is obtainable by modifying the codon preference as well as providing other modifications in the synthetic DNA sequence of the truncated cry9Aa gene to prefer selected higher plants.
  • the codon bias can be changed to Brassica preference, but also more generally to dicot preference, alteration to monocot preference being the least preferred alternative.
  • the start codon vicinity can also be changed to be more desirable for higher plants.
  • tobacco, turnip rape, cauliflower and potato plants were used, but it is evident that other plants could be transformed as well.
  • the synthetic DNA sequences of the present invention are capable of expressing in procaryotic or eucaryotic organisms an insecticidal protein characterized by having an amino acid sequence (SEQ ID NO : 1 : ) or alterations thereof capable of demonstrating properties, which are substantially similar to the properties of the selected domain of the selected insecticidal protein, i.e. Cry9Aa toxin, which does not demonstrate the cross-resistance phenomena encountered in insects resistant to known insecticidal proteins, such as other Cryl toxins .
  • the modified synthetic DNA sequences encode substantially the same protein as the DNA sequence of the truncated cry9Aa gene encoding the N-terminal domain of the insecticidal protein of Bacillus thuringiensis ssp. galleria .
  • the most preferred embodiment of said DNA sequences are synthetic DNA sequences having a substantial similarity, meaning a similarity comprising not more than 25 % nucleotide changes compared with SEQ ID NO : 2 : and capability of encoding a protein substantially similar with SEQ ID NO : 1 : or a protein having substantially the same properties.
  • the modified DNA sequences of the present invention preferably comprise less than 50 %, more preferably less than 25 %, most preferably more than 5 % nucleotide changes as compared to SEQ ID NO : 3 : .
  • the modified DNA sequences of the present invention preferably comprise up to 20 % or more changes as compared to SEQ ID NO : 2 : .
  • the modified synthetic DNA sequences should encode a protein substantially different from the known insecticidal proteins, e.g. the crylAc and the crylC genes.
  • the modified synthetic DNA sequences of the present invention can be produced by changing the codon bias in the direction of the selected higher plant, preferably to dicot but most preferably to Brassica preference.
  • the putative polyadenylation, splicing and mRNA destabilising signal sequences are removed and the vicinity of the start codon is altered to increase the compatibility to higher plants.
  • the modified synthetic DNA sequences are characterized by having improved expression and stability.
  • the method for preparing modified synthetic DNA sequences of the present invention comprise the steps of selecting a synthetic DNA sequence from a gene encoding an insecticidal protein having properties differing substantially from known insecticidal proteins, which are characterized by conveying resistance, especially preventing development of cross-resistance in target insects.
  • the synthetic DNA sequences can be inserted into suitable DNA constructs, vectors and plasmids, which in turn can be introduced into desired hosts.
  • the present invention also discloses the use of the modified synthetic DNA sequences with a different specificity and expression level and mRNA stability, which confers to the higher plant an improved toxicity to target insects.
  • the modified synthetic DNA sequences of the present invention provide transgenic plants for improved insecticidal control with increased toxicity to target insects . Said transgenic plants are capable of expressing effective amounts of the desired insecticidal protein and of overcoming the onset of the cross-resistance phenomenon currently observed in connection with the use of other Cryl or Bt toxins.
  • transgenic plants of the present invention were tested with wild insects and the results were recorded using bioassays described below.
  • Two CrylAc and CrylC resistant lines of diamond back moth ( Plutella xylostella) were used in the cross-resistance bioassays.
  • transgenic plant cell lines capable of expressing Cry9Aa toxin killed the larvae of resistant Plutella xylostella strains.
  • Figure 1 depicts the amino acid sequence of insecticidal active N-terminal domain of Cry9Aa delta-endotoxin of Bacillus thuringiensis ssp. galleria .
  • Figure 2 depicts synthetic DNA sequence for high expression in higher plants encoding the insecticidal, active N-terminal domain of the Cry9Aa delta-endotoxin of Bacillus thuringiensis ssp. galleria .
  • Figure 3 depicts alignment of native (SEQ ID NO : 4 : ) , truncated (SEQ ID NO:3:) and synthesized (SEQ ID NO : 2 : ) cry9Aa gene sequences .
  • the sites of the start and the termination codons as well as the restriction sites introduced in truncated or synthetic sequences are marked by shadowed boxes. Changed nucleotides are underlined in the synthetic and truncated sequences. The fragments within the restriction sites were synthesized separately. After that they were verified to avoid mistakes and fused together into a synthetic sequence.
  • Figure 4 depicts alignment of native, truncated and synthesized Cry9Aa protein sequences. The changed amino acids are marked by underlining in the truncated and the synthetic gene protein sequences.
  • Figure 5 depicts plant transformation constructs containing the native truncated sequence of cry9Aa gene (A) , the synthetic crySAa gene (B) and translational fusion with uidA gene (C) .
  • the abbreviations in the boxes are as follows: RB, LB - right and left borders of T-DNA from Ti plasmid; pAnos, pAg7 , pAocs - polyadenylation signal sites from different sources; AMV - untranslated leader from CaMV (cauliflower mosaic virus); 35S: Sp - double 35S promoter from CaMV; nosp - promoter from nopalin synthetase gene; nptll - neomycin phosphate transferase - II gene; hpt - hygromycin phosphate transferase gene uidA (GUS) - ⁇ glucuronidase gene.
  • the other names shown are restriction enzymes beside the pBIN
  • Figure 6 depicts bioassays of cauliflower transgenic plants against P. xylos tella insects.
  • the cauliflower plants were placed in a cage with P. xylostella cultures. The plants were grown in the cage for 10 days. After that the photographic picture was taken. The two upper plants are untransformed controls. Two lower plants are transgenic lines of cauliflower with low or moderate (A-10) and/or average level of (A-0) expression of Cry9Aa toxin. The plant of the A-10 line has some injures caused by larvae of all stages transferred from control plants. The plant of the A-0 line has only very small traces of insect bites. Both control plants were fully damaged by the insect attack and died in the next few days .
  • Figure 7 depicts molecular analysis of synthetic cry9Aa gene expression in transgenic tobacco.
  • FIG. 7A depicts a Northern blot of total RNA.
  • Samples containing 3 ⁇ g of total RNA of tobacco plants were loaded in the gel .
  • Northern blot was performed according to the instructions of the supplier Boehringer Mannheim.
  • Samples shown are as follows: C - RNA of non-transformed control plant; and T-GS 1 - 9 are lines of transgenic tobacco:
  • Control RNA synthesized from the gene sequence with T3 RNA polymerase was loaded in the gel in series comprising 0.6 pg - 1.6 pg - 5.0 pg - 15 pg .
  • the T-GS 1, 2 and 4 lines show a signal at about 15 pg RNA. It means that the expression is about 5 pg per 1 ⁇ g of total RNA.
  • the T-GS 3 and 7 lines show the signal at about 3 pg, meaning that the expression is about 1 pg per 1 ⁇ g of total RNA.
  • the T-GS 9 line shows the signal at about 1 pg, meaning that the expression is about 0.3 pg per 1 ⁇ g of total RNA.
  • the average expression of cry9Aa mRNA is 2 pg/ ⁇ g of total RNA.
  • Figure 7B depicts Western blot of tobacco plants transformed with synthetic cry9Aa .
  • Samples containing 10 ⁇ g of fully soluble protein of tobacco plants transformed with synthetic cry9Aa are loaded in 8 % denaturating PAGE, run and blotted in semidry blotting conditions on nitrocellulose membrane. The membrane was blocked with 1 % BSA. Rabbit antibody serum raised against crystal proteins of B . thuringiensis and conjugated with acetone precipitated protein powder from tobacco, cauliflower and turnip rape was used for incubation.
  • the samples are tobacco TGS-2..9 transgenic lines, C - control non-transformed tobacco, Cry9Aa 10 and 25 ng - the protein expressed in E. coli from the synthetic sequence of cry9Aa .
  • the size of this control protein is 30 amino acids longer than the protein expressed in the plants because of the additional LacZ leader protein.
  • the T-GS-2 line expressed Cry9Aa protein as soluble protein 0.2 % or 600 ng/g of leaf tissue the T-GS-4 line expresses 0.15 % of soluble protein or 1020 ng/g of leaf tissue
  • the T-GS-8 line expresses 0.3 % of soluble protein or 1440 ng/g of leaf tissue
  • the T-GS-7 line expresses 0.2 % of soluble protein or 1020 ng/g of leaf tissue.
  • Average Cry9Aa expression in tobacco plants is 1 ⁇ g/g of leaf tissue or 0.2 % of soluble protein.
  • Figure 8 depicts molecular analysis of expression of native truncated and GUS fused constructs of cry9Aa gene.
  • Figure 8A depicts a Northern blot of total RNA of transgenic tobaccos transformed with a truncated native sequence.
  • Samples containing 3 ⁇ g of total RNA of tobacco plants were loaded in the gel.
  • Northern blotting was performed according to the instructions of the supplier Boehringer Mannheim. The samples are as follows: The T-GT-3...11 - lines of tobacco transformed with truncated native cry9Aa gene; and the positive control 0.2 - 5 pg of cry9Aa RNA produced on the pBluescript with T3 RNA polymerase.
  • the signal on the tobacco lanes is seen at 0.1 to 3 pg. It means that mRNA expression is 0.03 - 1 pg/ ⁇ g of total RNA and the average expression is about 0.2 pg/ ⁇ g of total RNA.
  • Figure 8B depicts a Northern blot of total RNA of transgenic tobaccos transformed with a truncated native sequence translationally fused with uidA (GUS) gene.
  • GUS uidA
  • Positive controls are 1 - 10 - 20 pg of cry9Aa RNA produced on the pBluescript with T3 RNA polymerase.
  • the signal on the tobacco lane is from 0.6 to 1.5 pg of cry9Aa mRNA. It means that mRNA expression is 0.2 - 0.5 pg/ ⁇ g of total RNA and average expression is about 0.3 pg/ ⁇ g of total RNA.
  • Figure 8C depicts a Western blot of tobacco plants transformed with truncated native (T-GT) as well as with GUS fused (T-G/G) cry9Aa gene sequence .
  • Samples containing 50 ⁇ g of total soluble protein from tobacco plants transformed with truncated and GUS fused native cry9Aa gene were loaded in 8 % denaturating PAGE, run and blotted in semidry blotting conditions on nitrocellulose membrane. The membrane was blocked with 1 % BSA. Rabbit antibody serum raised against crystal proteins of B . thuringiensis and conjugated with acetone precipitated protein powder of tobacco, cauliflower and turnip rape was used for incubation.
  • the samples used are as follows: Tobacco T-GT-3..11 lines transformed with the truncated native sequence; T-GS-4 (tobacco line transformed with the synthetic cry9Aa sequence) 50 ⁇ g of total soluble protein containing 75 ng Cry9Aa peptide as positive control; and T-G/G 14a...21 - tobacco lines transformed with truncated native Cry9Aa-GUS translational fusion construct. There is no detectable Cry9Aa signal on the lines of tobacco plants transformed with the truncated native or GUS fused cry9Aa gene.
  • FIG. 8D depicts a Western blot of T-GS-8 and non-transformed NTS lines of tobacco mixed in different proportions. Samples of soluble proteins from tobacco were mixed and loaded in the order shown on the photographic picture. The gel and membrane were developed in the same Western procedure as the samples of the Figure 8C. The last track can be used as a negative control for Figure 8C.
  • This Western blot shows that the T-GS-8 sample diluted 50 times still shows a detectable Cry9Aa signal. It means that the native cry9Aa gene construct produces at least 50 times less of the protein product than the synthetic sequence.
  • Figure 9 depicts molecular analysis of the synthetic cry9Aa gene expression in transgenic potato cv . Pito.
  • Figure 9A depicts a Northern blot of total RNA.
  • Samples containing 3 ⁇ g of total RNA of potato plants were loaded in the gel.
  • the samples shown are as follows: C - RNA of non-transformed control potato plant; and P-GS 1-8 are lines of transgenic tobacco.
  • the control RNA synthesized from the gene sequence with T7 RNA polymerase was loaded in the gel in series of 0.6 pg - 1.6 pg - 5.0 pg - 15 pg .
  • the P-GS 1, 2 and 4 lines show a signal at about 10 pg RNA.
  • cry9Aa mRNA is 2 pg/ ⁇ g of total RNA.
  • Figure 9B depicts a Western blot of potato plants cv. Pito transformed with synthetic cry9Aa .
  • Samples containing 40 ⁇ g of total soluble protein from potato plants transformed with synthetic cry9Aa were loaded in 8 % denaturating PAGE, run and blotted in semidry blotting conditions on nitrocellulose membrane. The membrane was blocked with 1 % BSA. Rabbit antibody serum raised against crystal proteins of B . thuringiensis and conjugated with acetone precipitated protein powder from tobacco, cauliflower and turnip rape was used for incubation. The samples used are the potato PGS-1...8 transgenic lines; C - control non-transgenic tobacco, Cry9Aa 5 and 15 ng - the protein expressed in E. coli from the synthetic sequence of cry9Aa .
  • This control protein is 30 amino acids longer than that expressed in the plants because it contains an additional LacZ leader peptide.
  • the P-GS-1 and P-GS-8 lines show a Cry9Aa signal of about 10 ng , which corresponds to an expression of 0.03 % of soluble protein or 300 ng/g leaf tissue.
  • the P-GS-2 and 5 lines show a lower expression.
  • Figure 10 depicts molecular analysis of synthetic cry9Aa expression in transgenic cauliflower cv. Asterix.
  • Figure 10A depicts a Northern blot of total RNA.
  • Samples containing 3 ⁇ g of total RNA from cauliflower plants were loaded in the gel.
  • the samples shown are as follows: Nucleic acid (NA) of non-transformed control plant; the A-0 and A-10 lines of transgenic cauliflower revealed insecticidal properties.
  • the control RNA synthesized from the gene sequence with T3 RNA polymerase was loaded in the gel in series of 1.25 pg - 2.5 pg - 5.0 pg.
  • the A-0 line shows a signal at about 2 pg RNA, meaning that the expression is about 0.7 pg per 1 ⁇ g of total nucleic acid.
  • A-10 line has the signal about 0.6 pg, meaning that the expression is about 0.2 pg per 1 ⁇ g of total RNA.
  • FIG. 10B depicts Western blot of cauliflower cv.Asterix.
  • Samples containing 50 ⁇ g of total soluble protein of cauliflower are used in the Western.
  • the samples are as follows: C is a non-transformed plant control; and A-0 a cauliflower line transformed with the synthetic cry9Aa sequence. 5 and 20 ng of Cry9Aa protein expressed in E. coli represents the positive control.
  • the A-0 lane shows a positive Cry9Aa signal at a concentration of about 5 ng of the toxin protein. It means that the expression is about 100 ng of Cry9Aa protein per 1 g of leaf tissue or 0.01 % of soluble protein.
  • Figure 11 depicts Northern blot analysis of cry9Aa mRNA expression in turnip rape plants cv. Valtti transformed with the synthetic sequence of cry9Aa gene.
  • RNA samples containing 3 ⁇ g of total RNA from turnip rape plants were loaded in the gel.
  • the samples shown are as follows: C - RNA of untransformed control plant; V-GS-12.1 and V-GS-14.3 lines of transgenic turnip rape revealing insecticidal properties.
  • Control RNA synthesised from the gene sequence with T3 RNA polymerase was loaded in the gel in series of 1.25 pg - 2.5 pg - 5.0 pg . Both lines show a signal at about 0.6 pg RNA, meaning that expression is about 0.2 pg per 1 ⁇ g of total RNA.
  • AMV leader (Datla, R.S.S., 1993 Plant Sci. 94:139-149).
  • Agrobacter ium tumefaciens strains C58C1 with pGV3850 (Zambryski, et al . , 1983 EMBO J 2:2143-2150), C58C1 with pGV2260 (Deblaere, et al . , 1985 Nucl. Acids Res. 13:4777-4788), EHA105 (Hood, et al . , 1993 Transgenic research 2:208-218) and LBA4404 with pAL4404 helper plasmid (Hoekema, et al., 1983 Nature (London) 303:179-180).
  • Two CryTAc and CrylC resistant P. xylostella strains were received from Professor T. Shelton at Cornell University, but such strains are available and obtainable from other sources as well.
  • modified synthetic DNA sequence means DNA sequences prepared by synthetic means, such as nucleotide sequencing and/or by replacing nucleotides in the truncated DNA sequence
  • the modified DNA sequences of the present invention encode an insecticidal protein characterized by the amino acid sequence
  • SEQ ID NO : 1 also shown in Figure 1 or alterations therof .
  • the most preferred synthetic DNA sequence is SEQ ID NO : 2 : also shown in Figure 2 or modifications thereof .
  • modified sequences thereof means that at least 5 %, preferably more of the nucleotides of SEQ ID NO : 3 : have been changed so that they are more compatible with the selected higher plant(s), e.g. Brassica .
  • the modified sequences comprise all synthetic DNA sequences in which at least 10-20, preferably 15, but not all codons have been changed.
  • modifications thereof means that the putative polyadenylation, splicing and mRNA destabilising signal sequences have been removed from the DNA sequences (SEQ ID NO:3:) to provide the modified synthetic DNA sequence (SEQ ID NO:2:) shown in Figure 2. It also means that the start codon vicinity has been made more compatible with the selected higher plant (s) .
  • modified synthetic DNA sequences of the present invention should still encode endo-toxins which have "substantially similar” or “essentially identical” properties and/or activities as the truncated insecticidal protein encoded by the cry9Aa gene of Bacillus thuringiensis ssp. gall eria (Btg) . It is important to note that native genes and site directed mutations of it are not sufficient to provide the highly expressed, modified, synthetic DNA sequences of the present invention. In order to get synthetic DNA sequences having the desired properties of the present invention it is necessary to have a synthetic DNA sequence and to alter it in a skilled fashion based on knowledge and experience as described below.
  • cry9Aa (in H ⁇ fte and Whiteley (1989) Microbiology Review 52: 242-255) classification crylG) , encompasses the genes cry9Aal and cry9Aa2 , because both encode the same toxin region and have the same DNA and amino acid sequence.
  • the "cry9Aa” gene was previously called the crylG gene.
  • substantially similar means "essentially identical” or that the amino acid sequences encoded by the DNA sequences of the present invention have a structure which is substantially the same as the structure of the N-terminal, trypsin sustaining, part of the lepidopteran active delta-endotoxin of Bacillus thuringiensis ssp. galleria encoded by the native cry9Aa ( crylG) gene, but can differ somewhat from said delta-endotoxin with the prerequisite that the altered or different sequences still have essentially the same insecticidal action and properties as the amino acid sequence encoded by the native cry9Aa gene.
  • the selected amino acid sequence (SEQ ID NO : 1 : ) also shown in Figure 1 with trypsin cleavage site, can be altered using minor truncations in the N-terminal and/or C-terminal end or minor replacements in the intermediate regions.
  • the size of the C-terminal or N-terminal truncations as well as the number of replacements can be different depending upon the domain in which the truncations or replacements occurs.
  • the replacements should be no more than 10, preferably no more than 5 and most preferably less than 2 amino acid residues whereas amino acid residues in domains less relevant for the insecticidal action can comprise more replacements.
  • truncations or replacements should not alter the properties, especially the insecticidal action of the endotoxins as determined by the bioassays disclosed in the present invention.
  • the minor truncations and/or replacements should not make the endotoxin less effective than the native Cry9Aa endotoxin characterized by having SEQ ID NO : 1 : or Figure 1.
  • implementing resistance management strategies includes for example the following tactics for deploying insect resistance: gene strategies using strongly expressing single genes, multiple genes, e.g. pyramiding and/or chimeric genes; gene promoter strategies using constitutive, tissue specific and/or inducible promoters, e.g. wounding; gene expression strategies using high dose, low dose and/or mixtures; but above all field strategies using uniform single gene tactics, mixtures of genes, gene rotation, mosaic planting and/or spatial or temporal refuges.
  • improved mRNA processing means that the modified synthetic DNA sequences convey successful processing of mRNA of the foreign gene in a transgenic plant.
  • improved properties means above all that the toxin protein is sufficiently unique and expressed in sufficient amounts to enable effective and sustainable insect control and improved resistance management development, but the term also includes other improved tailor-made properties, such as improved production in plant tissues with consequent improved insecticidal action of the protein in field trials.
  • the improved properties are obtainable by selecting the amino acid sequence encoded by the unique cry9Aa gene and using a truncated synthetic DNA sequence as the basis when preparing modified synthesized DNA sequences and providing said sequences with tailor-made modifications prepared with experienced knowledge.
  • the modified, synthetic DNA sequences include removal of the putative polyadenylation, splicing and mRNA destabilising signal sequences as well as removal of hairpin loops with per se known methods .
  • the "improved properties" also are obtainable by changing the codon preference in the direction of a selected higher plant, preferably dicot preference, most preferably Brassica preference or combinations thereof. Monocot preference is a less preferred choice. Additionally, “improved properties” are obtainable by making the start codon vicinity of the synthetic DNA sequence more compatible or desirable to higher plants.
  • improved expression means that the selected plants are capable of expressing elevated amounts of endotoxin which are sufficiently effective to control target insects. This means that the toxin is expressed in amounts capable of killing larvae when compared to previously used constructions.
  • the term "capable of expressing effective amounts of the protein” means that the transformed plant should express the insecticidal protein in at least such amounts that the plant material contains enough endotoxin to kill a substantial part of the target insects.
  • the average dietary effect required should preferably be somewhat higher in field trials. Consequently, it is desirable that the amount toxin expressed in field experiments should also be somewhat higher than the amount required in laboratory experiments .
  • cognate preference or “codon bias” means that the nucleotide codons have been selected based on the highest frequency used for a particular amino acid in the coding sequence of the active genes of the selected species.
  • the term "removal of putative polyadenylation” means removal of the poly-A like signal sequence (s) attached to the end of eucaryotic genes occurring in the cry genes inside the sequence leading to truncation of the coding sequence during mRNA processing. These sequences are removed in constructing the synthetic cry gene sequence.
  • the term "removal of splicing and mRNA destabilising signal sequences” means that branch- sequences commonly present in the intron sequences of eucaryotic genes involved in splicing of the gene and destabilizing sequences having repeated ATTTA motifs are removed by nucleotide replacement in the synthetic cry sequence .
  • the term “altered start codon vicinity” means that the start codon vicinity (i.e. sequences around the start codon) has been changed to be more compatible with a higher plant.
  • removal means that nucleotides are changed or replaced in the DNA sequence so that signal motif (s) disappear while the DNA sequences encode proteins with substantially similar amino acids as the native Cry9Aa toxin.
  • an insecticidal protein differing essentially from known insecticidal proteins means that the amino acid sequence in the N-terminal insecticidal part of the Cry9Aa toxin should be substantially different, preferably differing more than 50 % from that of those insecticidal proteins encoded by other commercially available cryl genes. It also means that the selected cry gene should show essentially no cross-resistance to insects resistant to known, more frequently used insecticidal proteins. In other words it should be able to overcome the problem connected with cross-resistance .
  • the term "capable of demonstrating properties, which are substantially different” means that the properties of the selected domain of the selected insecticidal protein should have a substantially similar effect as the native Cry9Aa toxin, but simultaneously it should bind to receptors, which are substantially different from those of the other known toxins.
  • the toxin selected for the present invention has essentially no cross-resistance, which is a typical property in other insecticidal Cryl proteins. This property is assumed to be an indication of a different receptor binding pattern or mode of action.
  • insecticidal proteins encoded by the modified synthetic DNA sequences of the present invention should have an insecticidal action or effect, which is as good as or better than the insecticidal action or effect of the endotoxin encoded by the native Cry9Aa gene.
  • higher plants means especially flowering plants and includes according to taxonomic classification systems both angiosperms and gymnosperms .
  • DNA constructs means any DNA constructs, vectors and plasmids comprising the modified synthetic DNA sequences of the present invention in combination with other DNA sequences or fragments, useful for cloning, transforming, expressing, secreting, etc., and which include for example promoters, enhancers, signal sequences, terminators, etc., selected from per se known vectors, plasmids or fragments thereof, which are applicable for cloning, transforming of procaryotes or eucaryotes, respectively.
  • the hosts can be selected from bacteria, yeasts, fungi, plant and animal cells, with special emphasis on plant cells and enhanced expression of said insecticidal proteins in transgenic plants.
  • the present invention is related to delta-endotoxins or Bt toxins produced by Bacillus thuringiensis, which are known to have a very high level of toxicity to the host insects .
  • the so called Cry or Bt toxins are responsible for the insecticidal action of the bacterium. These proteins form crystals in the spore while the bacterium sporulates . It is also known that the toxicity varies depending upon the target insect. As described above several types of endotoxins exist and it is also known that these endotoxins have different properties and modes of action.
  • the present inventors selected an endotoxin, which is known to be unique and with properties differing essentially from those of other more frequently used endotoxins to study whether a modified DNA sequence encoding a substantially different type of endotoxin could be used in insecticidal control and provide a tool in resistance management systems .
  • the present inventors started with the hypothesis that if modified synthetic DNA sequences were provided, they would then incorporated into higher plants, provide improved tolerance to target insect attacks including increased specificity, efficacy, toxicity and stability of the toxicity trait.
  • Deployed resistance development in insects can be obtained by sound resistance management systems including gene strategies, gene promoter strategies, and field tactics, such as annual crop rotation with plants carrying different Bt-genes, Bt-gene mixtures in composite seeds, mosaic planting and/or spatial or temporal refuges. These strategies allow insects susceptible of developing resistance to copulate with non-resistant or wild-types, which increases the incidence of sensitive insects and delays the development of tolerance and resistance in insect populations.
  • the present inventors realized that in order to provide sustainable resistance management strategies to combat cross-resistance phenomena, new DNA sequences and/or genes with unique properties would be needed and advantageous in order to develop new transgenic plants producing toxins having substantially different and/or unique modes of action.
  • the present inventors constructed new fully modified synthetic DNA sequences encoding the protein, which was known to be as different as possible from those protein in commercial use and against which target insects have developed tolerance. Due to its unique sequence and high potential for pyramiding Bt toxin genes in plants, the inventors selected for their investigations and studies the insecticidal Cry9Aa protein of Bacillus thuringiensis ssp. galleria (Btg) , which has an amino acid percent identity less than 34 % when compared with other conventional Cryl -proteins as determined by a similarity test with a computer program.
  • Btg Bacillus thuringiensis ssp. galleria
  • cry9Aal gene seemed a good choice and was selected, because it was known to be a potent toxin, the properties of which differed essentially from the properties of the most frequently used and commercially available endotoxins to which the target insects had shown increasing tolerance.
  • Cry9Aa can be used to slow down the development of toxin tolerance in crops.
  • the present inventors wanted to study a Cry toxin with a specific mechanism for acting in the gut of the insect, for example a specific and/or selective receptor target in the digestive tracks of the target insects.
  • CrylG now Cry9Aa
  • Cry9Aa has an entirely different amino acid sequence in the toxic domain than other known Cry proteins of Bacillus thuringiensis
  • the same could also be concluded from unpublished data according to which this gene has significantly less activity against the European corn borer in comparison to CryAb . This also confirmed the idea that the protein encoded by this gene has its own binding receptor and/or mode of action.
  • Bacillus thuringiensis ssp. galleria has several delta-endotoxin genes (Shevelev, et al . , Mol. Biol. (Rus) . 28: 3(1), 388-393) . Two of them are identified as CrylG (Cry9Aa in present nomenclature (Smulevich, et al . , (1991) FEBS Lett. 293:1-2, 25-28) and CrylX (Shevelev, et al . , FEBS Lett. (1993) 336: 79-82) and have been cloned in the Institute of Microbial Genetics, Moscow, Russia.
  • CrylG protein belongs to the lepidopteran active Cryl group of delta-endotoxins of Bt forming bipyramidal crystals in the bacterial spore.
  • the amino acid sequence of the CrylG delta-endotoxin protein differs significantly from other Bt toxins. It is only related to the above mentioned protein CrylX. Trypsin resistant, insecticidally active N-terminal part of the CrylG consists of a peptide comprising 632 amino acids .
  • the Bt toxin In the gut of the insects the Bt toxin has been shown to be processed by trypsin to provide a N-terminal 630 - 675 amino acids long insecticidally active peptide, the toxicity of which is manifested by its binding to specific receptor molecules in the insect gut, with consequent formation of ion channels in the epithelium. This action leads to ion efflux and paralysis of the intestinal function, which causes death of the insect. It is generally believed, that the binding receptor site and target insect specificity are correlated and also determine, which group of insects are sensitive and which are not sensitive. It is further believed that the lack of or a change in the specific receptor molecule in the gut of the insect leads to tolerance or resistance development to the toxin.
  • the present inventors successfully demonstrated that transgenic plants transformed with the modified synthetic DNA sequences of the cry9Aa gene, express more toxin product than plants transformed with the partially modified gene.
  • the elevated expression suffices to enable sustainable field trials .
  • the modified synthetic DNA sequences of the present invention have improved properties especially when expressed in higher plants.
  • the improved properties include enhanced expression through improved mRNA processing, stability as well as translation, whereas the encoded endotoxin has substantially the same efficacy as the native Cry9Aa endotoxins.
  • the synthetic DNA sequences of the present invention can be used to provide improved insecticidal control and are useful tools in insect resistance management programs. When incorporated into higher plants, the modified synthetic DNA sequences provide improved tolerance to target insect attacks including increased specificity, efficacy, toxicity and stability.
  • the truncated DNA sequence (SEQ ID NO : 3 : ) of the cry9Aa gene was used because attempts to transform plants with the long, native sequences of the genes had previously failed (Vaeck, M., et al. (1987) Nature 328 : 33 -37) .
  • the present inventors carried out truncations of the cry9Aa toxin genes at different sites and accordingly they also started with a truncated form of endotoxin encoded by the Cry3 ⁇ a-gene .
  • the improvements were achieved by providing a synthetic DNA sequence of the truncated DNA sequence (SEQ ID NO : 3 : ) of the cry9Aa gene (SEQ ID NO : 4 : ) encoding a protein characterized by having an amino acid sequence (SEQ ID NO : 1 : ) and Figure 1 or alterations therof still having an insecticidal action which is substantially similar to that of the insecticidal protein Cry9Aa .
  • the present inventors performed modifications of DNA sequence encoding insecticidal active N-terminal domain of cry9Aa gene.
  • the encoded protein or amino acid sequence should be unchanged or the same as that of the native gene and the alterations in the nucleotides were performed mainly so that codon was changed to another codon coding the same amino acid.
  • Codon preference was changed to be compatible with higher plants, preferably to dicots and most preferably to Brassica plants. Modifications of the codon preference improved the gene expression in a range of 20 - 5 times, approximately 10 times.
  • the enhanced cry9Aa gene expression obtained by improved mRNA processing and mRNA stability and translation led to higher toxicity of transgenic plants and consequently improved tolerance to insect attacks due to higher level of the toxin.
  • the specificity of the toxin was not changed, because the specificity is a natural property of the selected gene product.
  • the stability of the protein was not changed, because the amino acid sequence was change only by truncation to give the active toxin protein.
  • amino acid sequences provided by recombinant DNA techniques or protein engineering having substantially the same structure and having substantially the same insecticidal effect as the native Cry9Aa toxin are included within the scope of the present invention.
  • the transcription or mRNA synthesis need not be improved in all embodiments of the inventions.
  • One preferred embodiment of the invention comprises removal of the putative transcription end signals, but this is not necessary for enabling the present invention.
  • the transcription level is regulated with the promoter and other untranslated sequences. In one embodiment of the invention only the amino acid sequence of the coding region of the gene was modified.
  • Suitable restriction sites can be introduced into the coding region of the DNA sequence, in order to enable the division of the sequence into one or more conveniently sized DNA fragments.
  • Corresponding fragments can be synthesized using for example high fidelity PCR, using two or more, contradirected primers.
  • the synthetic fragments can be ligated and the fused construct cloned into a plant transformation vector operating under one of the multitude of presently available plant expression promoters.
  • the constructs could be expressed under both constitutive or inducible promoters, e.g.
  • Inducible promoters can be selected from a group of light dependent promoters, such as the small subunit of Rubisco promoter for expression in green leaves or other selected tissues, or of promoters acting in certain tissues, such as the patatin promoter useful for expression in tubers of potato. Those skilled in the art have a multitude of other available choices.
  • the constructs comprise the synthesised cry9Aa gene sequence and are transformed into tobacco, turnip rape, cauliflower and potato plants.
  • the level of the gene expression was verified by Western or Northern blot analysis.
  • the insect bioassays were carried out and results compared to the toxicity of the native truncated cry9Aal gene and to the translational fusions of the native truncated sequence and uidA (GUS) gene.
  • Northern blot analyses and bioassays against Pi eris brassi ca indicated that the synthetic sequence could produce at least 50 times more protein than the native.
  • cry9Aa gene would provide improved insecticidal control and provide a useful tool in target insect resistance management strategies.
  • it is self-evident based on the disclosure of the present invention how to obtain other insecticidal proteins for other target insects in other higher plants, in order to solve the problem of tolerance and resistance in target insects.
  • Btg Bacillus thuringiensis ssp. galleria (Btg) delta-endotoxins have been described in the report Protein Chemistry of Microorganisms of Russian Institute for Selection and Genetics of Industrial Microorganisms (Shevelev, et al . , (1994) Mol. Biol. (Rus) . 28: 3(1), 388-393). Seven Bt toxin-like motifs have been found in the genome of the Btg bacterium. Btg forms bipyramidal protein crystals during sporulation. The main protein component of the crystal consists of Cry9Aa (CrylG) toxin .
  • CrylG Cry9Aa
  • Cry9Aa toxin belongs to the Cryl lepidopteran active class of endotoxins forming bipyramidal protein crystals.
  • the toxin is a 120 kDa protein encoded by a 3,4 kb DNA sequence.
  • Cry9Aa protoxin has trypsin cleavage sites producing a 65 kDa N-terminal insecticidally active toxin-peptide .
  • the trypsin sensitive C-terminal part contains conserved amino acid contexts responsible for crystal formation.
  • the Cry9Aa toxin is very original compared to the other cryl toxins in terms of its protein structure. It is likely that it is bound by a unique receptor molecule in the insect gut. In bioassays, Cry9Aa toxin showed good insecticidal impact on Plutella xylostella , Pieris brassica and potato tuber moth (Phthorimaea operculella) . Cry9Aa is not very effective against European corn borer ( Os trinia nubialis) .
  • Trypsin processed active toxin binds to receptor protein molecule situated in the membrane of the gut cells in the insect . Binding to the surface of insect gut the toxin molecules form ion channels in the cell membrane of the gut . The ion channel formation leads to the efflux of ions, paralysis of intestinal functions and death of the host insect .
  • Transformation of plants by native cry gene sequences does not provide a sufficient protein amplification in plant cells.
  • Two different versions of Cry9Aa gene modifications were prepared.
  • the sequence ( Figure 3) near the trypsin processing sites were truncated in order to express in the plants only the insecticidally active N-terminal part of the toxin ( Figure 1) .
  • the protein sequence was cut at a site 12 amino acids before the N-terminal trypsin processing site and at a site 9 amino acids after the C-terminal processing site.
  • Truncation was performed using high fidelity PCR. Restriction enzyme sites were introduced before start codon - BamHI and around ATG codon - Sphl .
  • the 5 terminus primer comprises an introduced Bglll restriction site before the stop codon,- the stop TAA codon and the Xmal site after the stop codon.
  • Central part of the cloned PCR product (limited by Bbvl and Ncol restriction sites) was exchanged back to the native sequence to exclude possible mismatches during PCR. Terminal parts of the truncated sequence were sequenced to check gene context .
  • Start ATG codon context was formed as ACCATGG, which contains ACC conserved context before start and G base thereafter (Kozak, M., (1987), J. Mol. Biol. 196:947-950. Simultaneously, a .Ncol restriction site was introduced.
  • the coding sequence of the gene was modified to change codon usage from bacterial to higher plants, preferably dicot and most preferably Brassica plant preference. Codon exchange was performed manually according to a compiled codon usage table (Table 2) .
  • Table 2 consists of columns with codon preferences of higher plants: dicots, monocots and Brassi ca plants.
  • the present inventors compiled the codon usage tables for coding sequences of genes of Brassica oleracea, B . campes tris and B. napus . Summary codon frequency tables were prepared for Brassica plants. Dicot and monocot preference columns were made on the basis of available tables for different plants. The sequence was checked for the presence of undesirable sequences: putative signal sites responsible for splicing
  • AATAAA, AATAAT and their variations AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT and AAAATA (the more conserved of the A nucleotides are marked by bold font)
  • the modified gene sequence was screened for the presence of desirable restriction sites also with one mismatch in the site.
  • the gene sequence was divided in five (350 - 430 bases long) parts delimited by introduced restriction sites ( Figure 3) .
  • the sites were designed without change of the amino acid sequence.
  • the amino acid sequences of native and synthetic genes were compared in an alignment program to check that the synthetic gene codes the same amino acid context as the native.
  • Each of the five parts was synthesised by 3 - 4 high fidelity PCR cycles using 6-8 50-80 bp long oligonucleotides purified in PAGE.
  • the oligonucleotides were ordered from DNAgensy and Operone Technologies USA. Each PCR product was cut with appropriate restriction enzymes and cloned into a vector.
  • Sequenced fragments were ligated into the entire gene sequence, which also was sequenced to avoid mistakes in the DNA sequence.
  • the synthesised sequence was situated under LacZ promoter in BamHI site of pUC19.
  • the synthesised sequence of cry9Aa gene, situated in translation frame, was expressed in E. coli cells. Protein product of the expressed gene was identified in Western blotting against antibody serum specific to protein crystals of Bacillus thuringiensis ssp . galleria .
  • cry9Aa gene was cloned under double 35S promoter of CaMV linked to AMV UTR ( (Datla, R.S.S., et al., (1993) Plant Sci. 94:139-149). This construct was transferred into pGPTV-HPT and pGPTV-KAN pBIN19 based vectors (Becker, et al . , 1992, Plant Mol. Biol. 20:1195-1197). The vectors were transformed in Agrobacterium tumefaciens strains LBA4404, EHA105 and C1C58 (helper pGV3850) . A truncated version was placed under the same double 35S promoter and transformed in the same A . tumefaciens strains.
  • the truncated GUS translation fusion gene was cloned under the same promoter cointegrative pHTT (Elomaa, P., et al . , (1993) Bio/Technol. 11:508511) vector and transformed in C1C58 (pAVS850) A . tumefaciens .
  • Vectors and strains mentioned are generally available and known by those skilled in the art and can be replaced by other plasmids, vectors strains with properties compatible for transforming the selected host.
  • Tobacco plants were transformed with both native truncated and truncated-GUS translational fusion as well as synthetic sequence of the cry9Aa gene.
  • Potato, cauliflower and turnip rape plants were transformed with the synthetic sequence only.
  • the constructs used were truncated native cry9Aal gene as well as uidA gene fusion under 35S:S promoter in a pHTT cointegrative vector and in a binary pGPTV (pBIN19 based) vector.
  • Agrobacterium strains used for transformation were ClC58pGV3850 (as a cointegrative) and LBA4404pLA4404 (as a binary helper vector) .
  • Synthetic cry9Aal was cloned in pGPTV-HPT.
  • the above mentioned constructs were used for transformation of tobacco, turnip rape, potato and cauliflower plant. Presence of the transgene was verified by Southern analysis and the expression level of the modified toxin genes could be shown by Western and Northern analysis.
  • cry9Aal coding sequence was placed in translational frame under LacZ promoter of pBluescript SK(+) and expressed in XL1 strain of E . coli . Analysis was performed using rabbit antiserum raised against crystal proteins of Bacillus thuringiensis ssp. galleria and visualized by alkaline phosphatase reaction. Further the expressed protein served as positive control in Western analysis of the transgenic plants ( Figures 7-10) .
  • Tobacco Nicotiana tabaccum cultivar Samsung plants were used in genetic transformation. Tobacco leaf discs were cultivated for 1 day and inoculated with the Agrobacterium at the second day. The cocultivation period was 2 days. After that the leaf discs were washed off the Agrobacterium and cultivated on selection medium. The native truncated, GUS - fused and synthetic sequences of cry9Aa ( Figure 7) were used for tobacco transformation. About 20 antibiotic resistant regenerates were collected for analysis of expression. mRNA expression in transformed plants were studied with Northern blot hybridization. After that the RNA positive plants were studied with Southern and Western analysis. From each the transgene versions 6 typical transgenic lines were collected for demonstrating expression.
  • RNA was run in an agarose gel, blotted on a positive charged nylon membrane on a vacuum blotter. Hybridization and luminiscent detection was performed with a digoxigenin UTP labeled RNA probe according to a protocol of Boehringer Mannheim. The probe was synthesized with T7 RNA polymerase based on the full synthetic or truncated native sequence of cry9Aa cloned in pBluescriptSKII ⁇ .
  • the Northern blot developed with luminiscent reaction indicated that most of transgenic plants transformed with the synthetic gene expressed mRNA of cry9Aa gene from 0.3 to 5 pg per 1 ⁇ g of total RNA, the average being 2 pg per 1 ⁇ g of total RNA ( Figure 7A) .
  • Plants transformed with truncated native gene sequence as well as the GUS fusion express mRNA of cry9Aa gene from 0.03 to 2 pg per 1 ⁇ g of total RNA, the average being 0,2 - 0,3 pg ( Figure 8A) .
  • the analysis of cry9Aa mRNA expression shows that the synthetic sequence expresses on average 7 - 10 times more mRNA than the native gene.
  • the transgenic lines showing mRNA expression were studied as transformation and number of the transgene inserts.
  • Samples of total plant DNA (5 ⁇ g) isolated from green house grown plants were run in agarose gel and blotted on a positively charged nylon membrane. The membrane was hybridized with a digoxigenin dUTP labelled probe. The probe was amplified in PCR on the vector template containing the cry9Aa (synthetic or native) sequence. DNA samples were digested with restriction enzymes, which cut cry9Aa gene from the genome or only on one (left or right) side of the transgene insert. Southern blot analysis gave information that about half of the transgenic plants contained one insert. The inventors could not find any correlation between the number of inserts and mRNA or protein expression.
  • Cry9Aa protein product was analysed with a Western blot. Proteins were extracted in a buffer, containing 50 mM Tris (base), NaOH (up to pH 12), 0,4 M urea, 0,1 M thiourea, 2 mM Dithiotreitol , 0,5 % Tween 20, 0,5 % Triton X100 and 4 % mercaptoethanol (MerEtOH) .
  • Leaf material (Ig) was ground in liquid nitrogen and mixed with 2 ml of the buffer, then heated to 60°C and refrozen in liquid nitrogen. This procedure was repeated 2 times . Debris was removed by centrifugation.
  • the supernatant was precipitated and washed two times with 4 volumes of acetone -20 °C.
  • the dried precipitate was resuspended and dissolved in a loading buffer as 1 ⁇ g of the precipitate in 40 ⁇ l buffer and boiled in a water bath for 10 min.
  • the debris was centrifuged and the supernatant used in the analysis.
  • the concentration of total protein was measured in a Bradford assay.
  • the samples were loaded in polyacrylamide gel (PAGE) . Samples were run in 0.75-1.5 mm thick 8 % PAGE and blotted on a nitrocellulose membrane in a semidry blotter.
  • the membrane was hybridized with polyclonal antiserum raised against B . thuringiensis ssp. galleria protein crystals and purified by conjugation with 1 % of acetone precipitated powder of tobacco plant protein and E. coli (strain XL1) protein for 15 minutes at ambient temperature .
  • the expression of Cry9Aal protein expression using a synthetic transgene construct was from 0.6 to 1.44 ⁇ g per 1 g of leaf tissue, or from 0.15 to 0.3 % of soluble protein, the average being 1 ⁇ g and 0.2 %.
  • the sample T-GS-8 (well expressed Cry9Aa) was mixed with the negative control tobacco protein sample NTS [Nicotiana tabaccum cv. Samsung) in series comprising the following proportions of (T-GS8/NTS ⁇ g/ ⁇ g of total protein): 50/0, 5/50, 3/50, 1.5/50, 1/50 and 0/50. These samples were loaded and run in PAGE. The Western blot of the gel showed that Cry9Aal protein signal is well detectable in all dilutions (even in 1/50) compared with the NTS control ( Figure 8C) .
  • Potato plants cv.Pito were transformed with A . tumefaciens LBA4404 harbored pGPTV-HPT plasmid carrying synthetic cry9Aa gene sequence. The transformation was performed according to the protocol published earlier (Koivu, K. , et al . , 1995 Acta Agric. Scand. Sect.B, Soil and Plant Sci. 45:78-87). Eight hygromycin resistant lines were analysed for gene expression. The plants were analysed for expression of mRNA product expression in the same way as tobacco plants.
  • the Northern blot developed with a luminescent reaction shows that most of the transgenic potato plants transformed with the synthetic gene express cry9Aa gene mRNA from 1 to 3 pg per 1 ⁇ g of total RNA, the average being 2 pg/ ⁇ g ( Figure 9A) .
  • transgenic inserts in the genome were proved with Southern analysis.
  • Western blot was made according to the method described for tobacco plants .
  • the expression of the Cry9Aa protein product in potato was on average 0.3 ⁇ g per 1 g of leaf material or 0.03 % of soluble protein (Figure 9B) .
  • the Western blot shows expression of the Cry9Aa protein in A-0 at the level of 100 ng protein per 1 g of leaf tissue or 0.01 % of soluble protein (Figure 10B) .
  • protein measurements of transgenic plants can result in the detection of low levels of the toxin, because of insolubility of the Cry9Aa toxin when collected in a pH lower than 9.0.
  • the transgenic cauliflower plants especially the A-0 line, had a very high stability of the toxicity in a bioassay of overexpressed target insect attack, where plants were placed in a cage with high density cultures of P . xylostella for 10 days.
  • the results are presented in Figure 6.
  • bioassays with diamond back moth Plutella xylostella show a high insecticidal capacity of the A-0 line, which killed the wild larvae in 1 -2 days of feeding.
  • This transgenic line also killed CrylAc and CrylC resistant larvae of P . xylostella .
  • Line A-10 also had an insecticidal effect. But the wild larvae died after 6 - 8 days of feeding and CrylAc resistant larvae after 8 - 10 days.
  • Transgenic line A-10 did not kill all larvae resistant to CrylC toxin, but they had longer development period and were smaller in size than larvae fed with control plants (Table 3) .
  • This cross-resistance bioassay can serve as a confirmation of the unique properties of Cry9Aa toxin and the possibility to use it in different resistance management strategies .
  • the turnip rape Brassica rapa var . oleifera was transformed with A . tumefaciens LBA4404 comprising the pGPTV-HPT plasmid carrying the synthetic cry9Aa gene sequence according to the protocol (Kuvshinov, V., et al . , 1999 Plant Cell Reports 18 : 773-777) .
  • Two transgenic lines of turnip rape expressed mRNA product at a low level 0.5 pg per 1 ⁇ g of total RNA ( Figure 11) .
  • cry9Aa gene in Brassica plants was 10 - 100 times smaller than in tobacco or potato plants and the expression was not steady. Many Brassica transgenic lines lacked expression when the plants matured. This result might be caused by the use of the 35S promoter or AMV leader of Cauliflower Mosaic Virus, which is known for its unsteady expression. Especially, it can have aberrations in Brassica plants, which are natural hosts of the virus. The fact that suppression of the expression happened on the transcriptional level confirmed the thought that the promoter works but promotes only a low level of mRNA production.
  • cry9Aa The synthetic sequence of cry9Aa with removed transcriptional aberrations express the desired protein product more than 50 times better than the native truncated or GUS fused constructions.
  • cauliflower an expression of 100 ng of Cry9Aa protein per 1 g of leaf tissue led to a high insecticidal impact on Plu tella xylos tella , while tobacco plants expressed the protein in 10 times more according to the Western analysis.
  • Transgenic plants expressing sufficient amounts of cry9Aa gene were lethal to CrylAc and CrylC resistant strains of P . xylostella . This fact confirms the conclusion that Cry9Aa toxin has its own binding receptor mechanism.
  • the CrylAc and CrylC resistant insects used in bioassays did not demonstrate cross-resistance to Cry9Aa toxin .
  • Table 1 The results of bioassays with crystal Cry9Aa protein fed to second instar larvae of Plutella xylostella strains, susceptible wild, and resistant to CrylAC and CrylC toxins. Number of alive (a) or dead (d) larvae as well as number of pupas (p) are shown for each day of the feeding. Table 1
  • Table 2 Proportion of codons used in coding sequences of monocots, dicots and Brassica plants.
  • Brassica napus Brassica napus :
  • Arabidopsis thai iana- 854 coding sequence John Morris john.morris@frodo.mgh.harvard.edu
  • Pisum sativa - 37 sequences Petunia sp . -18 genes
  • Phaseolus vulgaris - 26 genes Solanu tuberosum 21 genes
  • Glycine max - 59 Nicotiana tabacum - 21, Lycopersicon esculentum - 41 genes (J. Michael Cherry cherry@frodo .mgh . harvard. edu)
  • Hordeuin vulgar e - 36 genes Zea mays - 71, Oryza sativa - 16, Trit cum aestivum - 45 genes (J. Michael Cherry cherry@ rodo . mgh. harvard. edu)
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • ORGANISM Bacillus thuringiensis
  • ATCACTGCTC CATCTCTTAC TGGATTGATC TCAATTGTC-T ACGATCTTAT TGGAAAGGTT 240
  • ORGANISM Bacillus thuringiensis
  • AAATGCCCAA ATATTATTAT TACCTTCTTT TGCGAGCGCT GCATTTTTCC ATTTATTACT 600
  • ORGANISM Bacillus thuringiensis
  • GAAGAACTCC GTACTCGTTT TAGAATCGCC
  • GAAGGTTCTC AAAGATCCGT GTACGAGGGG TATATTCGAA CAACTGGGAT AGATAACCCT 1740

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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  • Pest Control & Pesticides (AREA)
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Abstract

Cette invention se rapporte à des séquences d'ADN synthétiques modifiées, permettant une meilleure action insecticide, en raison de la plus grande maturation de l'ARNm, de leur stabilité et de leur translation accrue dans les plantes supérieures. Ces séquences d'ADN synthétiques sont des outils utiles pour gérer la résistance, en particulier pour surmonter les problèmes de résistance croisée. On obtient des améliorations en effectuant des modifications synthétiques de la séquence d'ADN tronquée (numéro d'identification de séquence 3) du gène cry9Aa, de préférence du numéro d'identification de séquence 2 codant pour une protéine se caractérisant par une séquence d'acides aminées de numéro d'identification de séquence 1, ou pour des altérations de celle-ci, produisant pour ainsi dire la même action insecticide que la protéine insecticide codée par le gène cry9Aa du Bacillus thuringiensis ssp. gallieria (Btg). Cette invention concerne également l'utilisation de ces séquences d'ADN synthétiques modifiées pour produire une telle protéine insecticide et pour produire des plantes transgéniques exprimant des quantités efficaces de ces protéines insecticides.
EP99940216A 1998-08-24 1999-08-24 Sequences d'adn synthetiques modifiees pour une meilleure action insecticide Withdrawn EP1107984A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FI981809 1998-08-24
FI981809A FI981809A0 (fi) 1998-08-24 1998-08-24 Muunnetut synteettiset DNA-sekvenssit, jotka koodaavat cry-geeniin perustuvaa proteiinia
PCT/FI1999/000698 WO2000011025A1 (fr) 1998-08-24 1999-08-24 Sequences d'adn synthetiques modifiees pour une meilleure action insecticide

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EP1107984A1 true EP1107984A1 (fr) 2001-06-20

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EP (1) EP1107984A1 (fr)
CN (1) CN1326464A (fr)
AU (1) AU5424499A (fr)
CA (1) CA2341278A1 (fr)
FI (1) FI981809A0 (fr)
WO (1) WO2000011025A1 (fr)

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Publication number Priority date Publication date Assignee Title
US7629504B2 (en) 2003-12-22 2009-12-08 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry9 nucleic acids
UY34014A (es) * 2011-04-15 2012-11-30 Dow Agrosciences Llc Genes sintéticos para expresar proteínas en células de maíz, construcciones, plantas transgénicas, métodos para controlar pestes y composiciones
CN102353726B (zh) * 2011-06-17 2013-12-18 中国计量科学研究院 CryIAc蛋白标准物质定值的方法
CN103923204B (zh) * 2014-04-04 2016-06-08 湖北省生物农药工程研究中心 杀南方根结线虫的苏云金芽胞杆菌的活性物质及其应用

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Publication number Priority date Publication date Assignee Title
US6369213B1 (en) * 1996-07-01 2002-04-09 Mycogen Corporation Toxins active against pests
JP2001507208A (ja) * 1996-07-01 2001-06-05 マイコーゲン コーポレーション 害虫に対して有効な毒素

Non-Patent Citations (1)

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Title
See references of WO0011025A1 *

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Publication number Publication date
CA2341278A1 (fr) 2000-03-02
FI981809A0 (fi) 1998-08-24
WO2000011025A1 (fr) 2000-03-02
CN1326464A (zh) 2001-12-12
AU5424499A (en) 2000-03-14

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