AU5424499A

AU5424499A - Modified synthetic dna sequences for improved insecticidal control

Info

Publication number: AU5424499A
Application number: AU54244/99A
Authority: AU
Inventors: Anne Kanerva; Kimmo Koivu; Viktor Kuvshinov; Eija Pehu
Original assignee: Unicrop Ltd
Current assignee: Unicrop Ltd
Priority date: 1998-08-24
Filing date: 1999-08-24
Publication date: 2000-03-14
Also published as: FI981809A0; CN1326464A; WO2000011025A1; EP1107984A1; CA2341278A1

Description

WO00/11025 PCT/F1I99/00698 1 Modified Synthetic DNA Sequences for Improved Insecticidal Control The Technical Field of the Invention 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. The Background of the Invention The use of crystalline endotoxins (Cry protein) 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. In addition to the selectivity and specificity, Bt toxins are considered to be the most effective bioinsecticides used in agriculture. Generally, the target insects develop resistance to Bt toxins much slower than to chemical insecticides. For example, 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 WO00/11025 PCTIF199/00698 2 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 (Vaeck, M., et al., (1987) Nature 328:33-37). Hence, different 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 (Barton, K.A., et al., (1987) Plant Physiol. 85:1103-1109; Vaeck, M., et al., (1987) Nature 328:33-37; Carozzi, N.B., et al., (1992) Plant Mol. Biol. 20:539-548), tomato (Fischhoff, D.A., et al., (1987) Bio/Technology 5:807-813; Delannay, X., et al., (1989) Bio/Technology 7: 1265-1269 and cabbage (Bai, Y.Y., et al., (1993) Current Plant Science and Biotechnology in Agriculture 15: 156-159). Summarising these data it can be stated that in laboratory experiments the expression of the Bt toxins has been sufficient for controlling insects, but field experiments have not been successful enough (Warren, G.W., et al., (1992), J. Econ. Entomol. 85 (5): 1651-1659) to fulfill the expectations. The problem in the field experiments is mainly the variability in the level of expressed toxins. In order to enhance the expression of the toxins, strong promoter sequences such as the 35S CaMV promoter and untranslated leader-sequences from cauliflower mosaic virus CaMV and translational fusions with nptII, have been used in tomato (Vaeck, M., et al., (1987) Nature 328: 33-37) and potato (Sticklen, M.B., et al., (1993) In: Yoy, C.B., et al., (eds.) Biotechnology in Agriculture. Kluwer Academic Publishers, Netherlands, 233-236). With such manipulations it has been possible to increase the expression of the toxin genes and to stabilize the protein production to some degree, but not enough to allow stable insect control. mRNA instability and differences in bacterial and plant codon usage have been suggested to be the main reasons for the lack of Bt toxin expression in transgenic plants. In the late eighties several research groups started to work WO 00/11025 PCT/FI99/00698 3 actively on plant gene expression. As a result codon preference tables of plant genes were composed (Murray, E.E., et al., (1989) Nucl. Acad. Res. 17:477-497). Collectively, the codon usage analysis of different plant genes showed that there is a strong avoidance of XCG codons in dicot plants and XTA in monocot plants (Grantham, R., et al., (1986) In: Oxford Surveys in Evol. Biol. 3:48-81) as well as avoidance of some other minor codons. It was also shown that putative splicing (Goodball, G J., and Filipowics, W., (1989) Cell 58:473-483) and polyadenylation (Dean, C., et al., (1986) Nucl. Acid. Res. 14(5) :2229-2240; Joshi, C.P., (1987) Nucl. Acid Res. 14(5): 2229-2240) sites of plant genes were characterized by AT-rich regions. Cry gene sequences are rich in AT content and are far from the preferred AT/GC ratio of plant genes. Also a repeated AUUUA sequence has been shown to be responsible for mRNA degradation in eucaryotes (Ohme-Takagi, M., (1993) Proc. Natl. Acad. Sci. USA, 90:11811-11815). Furthermore, a conserved context or motif has been found in the vicinity of the translational start codon AUG in plant genes. As a conclusion, in 1986-1989 features reducing the expression level of some bacterial genes in plants were elucidated. It was shown that extra AT content leads to spontaneous formation of putative splicing and polyadenylation sites and occurrence of minor (for plant codon preference) codons in the context of the gene interfering with its expression. Partial modification of the crylAb gene at the sites of putatively aberrant mRNA processing was shown to lead to 10-fold increased gene expression (Perlak, F.J., et al., (1991) Proc. Natl. Acad. Sci. USA 88: 3324-3328; van der Salm, T., et al.; (1994) Plant Mol. Biol. 26: 51-59). Reports on the three first synthesised cry genes appeared in the early nineties. Perlak, F.J., et al., 1991 (Proc. Natl. Acad. Sci. USA 88: 3324-3328) reported hyperexpression of fully modified crylA(b) and crylA(c) genes transformed in tobacco and tomato plants. Resynthesised gene sequences produced 100 times more protein than wild type truncated versions of the genes. Similar results were reported for cryIIIA (Adang, M.J. et al., (1993), WO00/11025 PCTIFI99/00698 4 Plant Mol. Biol. 21:1131-1145) and crylA(b) (Fujimoto, H., et al., (1993) Bio/Technology 11:1151-1153). Adang, M.J., et al., resynthetised the sequence of cryllIA 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 crylA(b) gene sequence using high fidelity PCR. Seven synthesised fragments were ligated into an entire gene sequence which was then transformed in rice plants (Fujimoto, H., et al., (1993) Bio/Technology 11:1151-1155). All of these three articles described the resynthesis of the toxin gene lacking undesirable processing signal sites and a change in the codon context from bacterial to plant preference. Modification strategies of the DNA sequence of Bt-toxins including monocot and dicot preference were disclosed in US patent 5,380,831 and US patent 5,500,365. Resynthesised crylAb (Koziel, M.J., et al., (1993) Bio/Technology 11: 194-200), cryIIIA (Perlak, F.J., et al., (1993) Plant Molecular Biology 22: 313-321) and crylAc (Perlak, M.J., et al., (1991) Proc. Natl. Acad. Sci. USA 88: 3324-3328) were highly expressed in different plants in laboratory and field trials. Interestingly, high expression of the unmodified truncated crylA(c) gene was achieved using site directed transformation of tobacco chloroplasts (MacBrige, K. E., et al., (1995) Bio/Technology 13:362-365. Transportation of the crylA(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., (1994) Plant Mol. Biol. 26: 51-59. As disclosed above point mutated cryl genes (Lepidopteran active) have been modified and especially synthetic crylA(c), crylA(b) and cryIllA (Coleopteran active) genes have been WO 00/11025 PCT/FI99/00698 5 prepared and used for transforming plant cells. In 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. In other words, 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 Summary of the Invention 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. Thus, the present invention is related to modified synthetic WO00/11025 PCT/F1I99/00698 6 DNA sequences of the truncated DNA sequence of the cry9Aa gene encoding a protein characterized by an amino acid sequence (SEQ ID NO:I:) 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. For example, 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. In the experiments 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 WO00/11025 PCT/FI99/00698 7 other CryI 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 cryIC 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. Preferably, 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.

WO00/11025 PCT/F1I99/00698 8 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 CryI or Bt toxins. The transgenic plants of the present invention were tested with wild insects and the results were recorded using bioassays described below. Two CryIAc and CryIC resistant lines of diamond back moth (Plutella xylostella) were used in the cross-resistance bioassays. The results obtained, confirmed the originality of Cry9Aa toxin, which killed the larvae of insects resistant to CryIAc and CryIC. Also transgenic plant cell lines capable of expressing Cry9Aa toxin killed the larvae of resistant Plutella xylostella strains. The Brief Description of the Drawings 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.

WO00/11025 PCT/FI99/00698 9 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 syn thesized 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 cry9Aa 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; nptII neomycin phosphate transferase - II gene; hpt - hygromycin phosphate transferase gene uidA (GUS) - S glucuronidase gene. The other names shown are restriction enzymes beside the pBIN and pHTT plant transformation vectors. Figure 6 depicts bioassays of cauliflower transgenic plants against P. xylostella 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 WO00/11025 PCT/F1I99/00698 10 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. Figure 7A depicts a Northern blot of total RNA. Samples containing 3 yg 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 gg 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 gg 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 p~g of total RNA. The average expression of cry9Aa mRNA is 2 pg/pg 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 WO00/11025 PCTIFI99/00698 11 control protein is 30 amino acids longer than the protein expressed in the plants because of the additional LacZ leader protein. According to this Western blotting 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 and 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 Ag/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 pg 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/bg of total RNA and the average expression is about 0.2 pg/Ag 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. The T-G/G-10...21-lines of tobacco transformed with truncated native cry9Aa sequence fused in a translational frame with uidA (GUS) gene. C - RNA of non-transformed tobacco plants. 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/pg of total RNA and WO00/11025 PCTIFI99/00698 12 average expression is about 0.3 pg/pg 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 Ag 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 pg 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. Figure 8D depicts a Western blot of T-GS-8 and non-trans formed 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.

WO00/11025 PCTIFI99/00698 13 Figure 9A depicts a Northern blot of total RNA. Samples containing 3 pg 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. It means that expression is about 3 pg per 1 pg of total RNA. The P-GS 5, 7 and 8 lines show the signal at about 3 pg, meaning that the expression is about 1 pg per 1 pg of total RNA. The average expression of cry9Aa mRNA is 2 pg/pg of total RNA. Figure 9B depicts a Western blot of potato plants cv. Pito transformed with synthetic cry9Aa. Samples containing 40 pg 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. According to this Western 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.

WO00/11025 PCTIFI99/00698 14 Samples containing 3 Ag 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 Ag 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 Ag of total RNA. Figure 10B depicts Western blot of cauliflower cv.Asterix. Samples containing 50 Ag 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. Samples containing 3 pg 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 Ag of total

RNA.

WO00/11025 PCT/FI99/00698 15 Donor organisms, promoters, leader sequences, vectors, bacterial and insects used in the present invention Below is a non-exhaustive list of materials used in the present invention. However, the present invention is not restricted to the use of the listed materials. One skilled in the art can easily think of other suitable readily available sources of material and organisms and can also apply them in the present invention in order to accomplish the same result. Cry-gene donor organisms Bacillus thuringiensis ssp. galleria The Russian Institute for Genetics of Industrial Microorganisms Moscow, Doroznyi proezd 1. Plant promoters 35S promoter of CaMV (Odell, J.T., et al., Nature v. 313 28 February 1985, p 810-812; Qun Zhu, et al., Bio/technology v. 12, 1994, p. 807-812). Leader sequence AMV leader (Datla, R.S.S., 1993 Plant Sci. 94:139-149). Plant Transformation Binary Ti vector PGPTV vectors (Becker, et al., 1992, Plant Mol. Biol. 20:1195-1197). pBIN vector (Firsch, D.A., et al., 1995, Plant Mol. Biol. 27: 405-409). Plant transformation cointegrative Ti vector pHTT (Elomaa, P., et al., (1993) Bio/Technol. 11:508-511) Bacterial strains Agrobacterium tumefaciens strains C58C1 with pGV3850 (Zambryski, et al., 1983 EMBO J 2:2143-2150), C58C1 with WO00/11025 PCT/F199/00698 16 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). Insect Strains Plutella xylostella wild type collected from the Viikki Experimental Farm. Two CryIAc and CryIC 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. The Detailed Description of the Invention In the present invention most terms used have the same meaning as they generally have in the fields of recombinant DNA techniques, molecular biology as well as in plant production and entomology related sciences. Some terms are, however, used in a somewhat different way and are explained in more detail below. The term "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 (SEQ ID NO:3:) obtained from the cry9Aa gene by changing the codon bias to prefer the codon usage of selected higher plant(s), preferably dicots, such as Brassica, or combinations thereof by using compiled tables indicating dicot, monocot and/or selected higher plant, e.g. Brassica preference. 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. The term "modifications 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 WO00/11025 PCT/F1I99/00698 17 higher plant(s), e.g. Brassica. In other words the modified sequences comprise all synthetic DNA sequences in which at least 10-20, preferably 15, but not all codons have been changed. Furthermore, the term "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). The only prerequisite for the modified synthetic DNA sequences of the present invention is that they 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. galleria (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. The term "cry9Aa" (in H6fte 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. The terms "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 cryg9Aa (crylG) gene, but can differ WO00/11025 PCT/F1I99/00698 18 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:l:) 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. In active domains of the protein 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. These truncations and replacements can be carried out with per se known methods. However, it is important that said 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. Especially, the minor truncations and/or replacements should not make the endotoxin less effective than the native Cry9Aa endotoxin characterized by having SEQ ID NO:l: or Figure 1. The term "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. In order to apply said strategies new DNA sequences and/or WO00/11025 PCT/F1I99/00698 19 genes with unique properties are required. The term "improved mRNA processing" means that the modified synthetic DNA sequences convey successful processing of mRNA of the foreign gene in a transgenic plant. The term "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. The term "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 WO00/11025 PCT/FI99/00698 20 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 expressed effective amount should have a desired insecticidal dietary effect of at least

LC

5 0 = 60, preferably 80, most preferably 100 ng/ml diet against Plutella xylostella (Diamond back moth) and Pieris brassica (cabbage butterfly) and potato tuber moth in laboratory conditions. These numbers are applicable for a time of two days. Generally, it can be said that the longer the effective time is the smaller is the amount needed. Due to the diversity in the genomes of wild insects as well as the natural variation in gene expression in the plants, 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. The term "codon 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.

WO00/11025 PCTIFI99/00698 21 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. The term "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. The term "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. For example the toxin selected for the present invention has essentially no cross-resistance, which is a typical property in other insecticidal CryI proteins. This property is assumed to be an indication of a different receptor binding pattern or mode of action. The term "capable of demonstrating properties, which are substantially similar to the properties of a selected domain of a selected insecticidal protein" means that the insecticidal proteins encoded by the modified synthetic DNA sequences of the present invention should have an insecticidal WO00/11025 PCT/F1I99/00698 22 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. The term "higher plants" means especially flowering plants and includes according to taxonomic classification systems both angiosperms and gymnosperms. The term "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. General Description of the Invention 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 WO 00/11025 PCT/FI99/00698 23 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 complicated mechanisms of action and high receptor binding specificity makes the toxins harmless to all other organisms except the insects with the correct receptor molecules in the midgut. Usually the resistance of insects against a delta-endotoxin of Bacillus thuringiensis is assumed to be a result of absence, removal or alteration of the receptor molecule. For example Cry9Aa, unlike CryIAc and CryIC (truncated or native) is insecticidal against Plutella xylostella and Pieris brassica as well as against potato tuber moth, but not very effective against European corn borer (Ostrinia nublialis). In order to provide improved insecticidal resistance and to solve the problem of Bt toxin tolerance development or resistance in target insects the present inventors WO00/11025 PCT/F1I99/00698 24 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 CryI-proteins as determined by a similarity test with a computer program. In the context of resistance management, the 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. As a novel insect toxin, 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. As the CryIG (now Cry9Aa) has an entirely different amino acid sequence in the toxic domain than other known Cry proteins of Bacillus thuringiensis, it was considered to be an indication that the gene has its own receptor recognition system. 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 (Btg) has several delta-endotoxin genes (Shevelev, et al., Mol. Biol. (Rus). 28: 3(1), 388-393). Two of them are identified as CryIG (Cry9Aa in WO 00/11025 PCTIFI99/00698 25 present nomenclature (Smulevich, et al., (1991) FEBS Lett. 293:1-2, 25-28) and CryIX (Shevelev, et al., FEBS Lett. (1993) 336: 79-82) and have been cloned in the Institute of Microbial Genetics, Moscow, Russia. According to the classification by H6fte and Whiteley (1989), Microbiological Review 52: 24- 55, CryIG 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 CryIG delta-endotoxin protein differs significantly from other Bt toxins. It is only related to the above mentioned protein CryIX. Trypsin resistant, insecticidally active N-terminal part of the CryIG consists of a peptide comprising 632 amino acids. 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. Gleave, A.P., et al. (1998), (Mol. Breed. 4:459-472) reported that potato lines transformed with the crylAc gene had a greater impact on larval growth and survival than the cry9A2 gene, but they seemed to have overlooked the potential of Cry9Aa in resistance management strategies. In fact, in the cry9Aa2 gene only 4.2 % of the nucleotides of the native nucleotide sequence were replaced using site-directed mutagenesis. The transgenic tobacco plants carrying said site-mutated gene revealed insecticidal properties, but the WO00/11025 PCT/F1I99/00698 26 mortality was not high. Only in one transgenic line 100 % of the larvae died in 9 days, whereas 20 - 50 % of the larvae in the others lines of tobacco were alive. Unfortunately, the report contains no data about mRNA and protein expression, which makes the comparison of results difficult. The data shown by Gleave, A.P., et al., (1998) correlate with those of Perlak, F.J.,et al., (1991) (Proc. Natl. Acad. Sci. USA 88:3324-3328), in which it is concluded that partial modification of the cry gene makes transgenic plants insecticidal, but partial modification does not provide a sufficiently high expression of Bt toxin, to provide sustainable results in field trials. However, in the present invention it was shown that, larvae of P. xylostella (independent of stage of development) died in two days when fed with transgenic cauliflower expressing the synthetic cry9Aa DNA sequence having a 23 % altered sequence. The data of the bioassays suggests that plants harboring the modified synthetic DNA sequences of the present invention express much more of the insecticidal protein, because potato tuber moth has LC50 = 80 ng per ml of diet in 5 days of feeding and diamond back moth has LC50 = 120 ng per ml of diet in 6 days of feeding. Accordingly, 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 same could also be concluded from unpublished data according to which the cry9Aa gene has a target species profile which differs from other cryl genes and has less activity against the European corn borer in comparison to CryAb. The results also confirmed the idea that the protein encoded by the cry9Aa has its own binding receptor site and/or mode of action.

WO00/11025 PCT/F1I99/00698 27 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 Cry9Aa-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:l:) and Figure 1 or alterations therof still having an insecticidal action which is substantially similar to that of the insecticidal protein Cry9Aa. Methods for transforming truncated cry genes have been described in several publications and said methods are applicable also to provide the transformants and transgenic plants of the present invention. Transformation of tobacco has been described by Barton, K.A., et al., (1987) Plant Physiol. 85:1103-1109; Vaeck, M., et al., (1987) Nature 328:33-37; Carozzi, N.B., et al., (1992) Plant Mol. Biol. 20:539-548), tomato by Fischhoff, D.A., et al., (1987) Bio/Technology WO00/11025 PCT/F1I99/00698 28 5:807-813; Delannay, X., et al., (1989) Bio/Technology 7: 1265-1269 and cabbage by Bai, Y.Y., et al., (1993) In Biotechnology in Agriculture. Proceedings of the First Asia-Pacific Conference on Agricultural Biotechnology, Beijing, China, 20-24 August 1992. In Current Plant Science and Biotechnology in Agriculture, 15, 156 - 159). The present inventors performed modifications of DNA sequence encoding insecticidal active N-terminal domain of cry9Aa gene. In principle 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. Expression of the gene was improved on the level of translation (protein synthesis from mRNA molecule). 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. Further improvements were carried out mainly on the mRNA processing level by removal of putative polyadenylation and splicing signal sites (sequences/motifs/contexts) based on the coding sequence of the native gene. The start codon vicinity was changed to higher plant preference (this can not be species preference) and all ATTTA mRNA destabilising motifs were removed. Removing of the sites means changing some nucleotide(s) in the sequence so that signal motif disappears. The enhanced cry9Aa gene expression obtained by improved mRNA processing and mRNA stability and translation (codon preference and start codon vicinity) 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 WO00/11025 PCT/F1I99/00698 29 the amino acid sequence was change only by truncation to give the active toxin protein. However, it is self-evident to those skilled in the art of protein engineering that the amino acids can be replaced or removed to a certain degree without loosing the activity. Thus, 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. the Nos promoter from Agrobacterium tumnefaciens, the 35S promoter from Cauliflower Mosaic Virus small subunit of ribulose-l,5-biphosphate carboxylase (Rubisco) promoter to mention a few examples. 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 WO00/11025 PCT/F1I99/00698 30 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 Pieris brassica indicated that the synthetic sequence could produce at least 50 times more protein than the native. Thus, the results obtained proved the validity of the working hypothesis of the inventors, i.e. that the cry9Aa gene would provide improved insecticidal control and provide a useful tool in target insect resistance management strategies. For those skilled in the art 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. The invention is described in more detail below. The examples and experiments are disclosed to provide more detailed guidance for those skilled in the art. Even if the examples and experiments were carried out with a modified synthetic DNA sequence demonstrating the closest preference to Brassica, the examples should not be interpreted as limiting the scope of the protection of higher plants. The examples indicate how to proceed to obtain the desired result when combatting tolerance or resistance development in target insects. Example 1 Origin, structure and function of the native cry9Aa gene. Bacillus thuringiensis ssp. galleria (Btg) delta-endotoxins have been described in the report Protein Chemistry of WO00/11025 PCTIFI99/00698 31 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 (CryIG) toxin. 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 (Ostrinia nubialis). Example 2 Cross-resistance investigations in bioassays 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. In order to clarify originality of the gene the present inventors performed bioassays on Plutella xylostella lines resistant to well known Bt toxins CryIAc and CryIC (according WO00/11025 PCTIFI99/00698 32 to old classification). The mechanisms of the insect resistance are not known, but it is supposed that mutations in the binding receptor are possible. In Table 1 the results of bioassays with crystal Cry9Aa protoxin protein of B.thuringiensis ssp. galleria are shown. Suspensions of purified crystals were dissolved in lysis-loading buffer and ran in PAGE for quantification of the Cry9Aa protein. The protein crystal stock with quantified Cry9Aa protoxin was used for insect tests. The crystal suspension was mixed in 6 mg/ml solution of casein for better adhesion. About 100 p1l of the mixture, with a known quantity of the protoxin were spread and dried on the surface of cauliflower 500 mg leaf sheet. We calculate that the toxin was 5 times diluted in the feed. Leaves coated with the Cry9Aa protoxin suspension were exchanged with fresh ones every 2 days of feeding. 5 second instar larvae of P. xylostella were assessed in each point of the experiment. The results of the experiments are shown in Table 1. In the results of the bioassays, the originality of the Cry9Aa toxin was confirmed. The larvae resistant to CrylAc as well as to CryIC toxins were susceptible to Cry9Aa. CryIC resistant larvae had partial resistance to Cry9Aa toxin. But they also died, if they were fed with toxin in concentrations corresponding to concentration expressed in transgenic plants carrying the synthetic Bt toxin gene (1-2 pg/g of leaf tissue). Example 3 Improvements in the gene structure. Codon preference tables. 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).

WO00/11025 PCT/F1I99/00698 33 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 - SphI. The 5 terminus primer comprises an introduced BglII restriction site before the stop codon; the stop TAA codon and the XmaI site after the stop codon. Central part of the cloned PCR product (limited by BbvI and NcoI 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. Synthetic DNA sequence of the cry9Aa gene (Figure 2) was compiled on the basis of the active toxin context so, that synthetic gene protein sequence was identical to the native one (Figure 3). Full modification of the gene sequence implied the following changes. 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 NcoI 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). The table consists of columns with codon preferences of higher plants: dicots, monocots and Brassica plants. The present inventors compiled the codon usage tables for coding sequences of genes of Brassica oleracea, B. campestris 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.

WO00/11025 PCT/F199/00698 34 The sequence was checked for the presence of undesirable sequences: putative signal sites responsible for splicing (Goodball, G.J. and Filipowicz, W., (1989) Cell 58: 473-483), polyadenylation (Dean, C., et al., (1986) Nuc. Acid Res. 14(5): 2229-2240; Joshi, C.P., (1987) Nuc. Acid Res. 15(23): 9627-9640) and mRNA destabilising sequences (Shaw, G. and Kamen, R. (1986) Cell, 46: 659-667; Ohme-Takagi, M., et al., (1993) Proc. Natl. Acad. Sci. USA, 90: 11811-11815) being listed below. Two signal sites for splicing, 6 for polyadenylation and 14 mRNA destabilising sequences were found and removed from the sequence. Finally the gene sequence was checked for possible folding. 13 putative sites of loop formation were removed from the sequence. Splicing:

CAN

7 -9AGTNNA Polyadenylation: 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) mRNA destabilising: ATTTA To compare the expression of the native and synthetic cry genes in crop plants we have made two different versions of the cry9Aa gene. First, we truncated the gene (Figure 3-4) near the trypsin processing sites to express only the insecticidally active N-terminal part of the toxin in plants (Figure 1). We cut the DNA sequence corresponding to the protein sequence site at 12 amino acids before the N-terminal trypsin processing site and 9 amino acids after the C-terminal processing site (Figure 4). The synthetic DNA sequence of the cry9Aa gene shown in Figure 2 and Figure 3 was compiled on the basis of the active toxin WO00/11025 PCT/F1I99/00698 35 so, that the protein product of the synthetic gene would be identical to the native protein (Figure 4). Full modification of the gene sequence implied the changes shown in Table 2. Example 4 Synthesis of the cry9Aa gene sequence. 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. Finally, 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. Example 5 Cloning of cry9Aa genes and transformation of plants. The synthetic sequence of 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 WO00/11025 PCTIFI99/00698 36 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. Example 6 Gene transfers and expression analysis. 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 C1C58pGV3850 (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. Example 7 Western blot of synthetic Cry9Aal protein product The cry9Aal coding sequence was placed in translational frame under LacZ promoter of pBluescript SK(+) and expressed in XL1 WO00/11025 PCT/F1I99/00698 37 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). Example 8 Transformation of tobacco. 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. Regenerants were tested for expression of the mRNA product. Total RNA (3 ptg) 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 (Boeringer Mannheim) indicated that most of transgenic plants transformed with the synthetic gene expressed mRNA of cry9Aa gene from 0.3 to 5 pg per 1 ug of total RNA, the average being 2 pg per 1 pg of total RNA (Figure 7A). Plants transformed with truncated WO00/11025 PCTIFI99/00698 38 native gene sequence as well as the GUS fusion express mRNA of cry9Aa gene from 0.03 to 2 pg per 1 pg 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 Ag) 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. Expression of the 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 (1g) was ground in liquid nitrogen and mixed with 2 ml of the buffer, then heated to 600C 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 -200C. The dried precipitate was resuspended and dissolved in a loading buffer as 1 p~g of the precipitate in 40 pl 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 WO00/11025 PCT/F1I99/00698 39 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 XLI) protein for 15 minutes at ambient temperature. As shown on the Figure 7B, the expression of Cry9Aal protein expression using a synthetic transgene construct was from 0.6 to 1.44 gg per 1 g of leaf tissue, or from 0.15 to 0.3 % of soluble protein, the average being 1 gg and 0.2 %. In order to compare protein product expression, 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 gg/gg 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). Samples of tobacco transgenic lines expressing native truncated and GUS fusion mRNA were loaded in the same Western as 50 Ag of total protein. None of the samples showed a clearly detectable signal of Cry9Aa protein (Figure 8C). The results indicate that the synthetic sequence of the cry9Aa gene produces at least 50 times more protein product in transgenic tobacco than the native sequences. Example 9 Potato transformation 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 WO00/11025 PCT/F1I99/00698 40 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 (Boehringer Mannheim) shows that most of the transgenic potato plants transformed with the synthetic gene express cry9Aa gene mRNA from 1 to 3 pg per 1 jg of total RNA, the average being 2 pg/Ag (Figure 9A). The transgenic inserts in the genome (data not shown) 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 Ag per 1 g of leaf material or 0.03 % of soluble protein (Figure 9B). Example 10 Cauliflower transformation Cauliflower plants cv. Asterix were transformed with A. tumefaciens LBA4404 comprising the pGPTV-HPT plasmid carrying the synthetic cry9Aal gene sequence (SEQ ID NO:2:). Hypocotyl segments were cultivated for 1 day on hormonal medium and co-cultivated with Agrobacterium for 2 days. After that the explants were placed on selection medium. Of the plants 5 regenerated after transformation, 2 showed a positive signal in a Northern blot performed as described for tobacco. The cry9Aa mRNA product was expressed as 2 pg (line A-0) and 0.5 pg (line A-10) per 1 gg of total RNA (Figure 10A). 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). In general, 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, WO00/11025 PCT/F1I99/00698 41 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. More specifically, 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 CryIAc and CryIC 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 CryIAc resistant larvae after 8 - 10 days. Transgenic line A-10 did not kill all larvae resistant to CryIC 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. Example 11 Turnip rape transformation. 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 pg of total RNA (Figure 11). While the Southern analysis confirmed transformation, the protein product was undetectable on Western blot partially due to a high background on the membrane. Nevertheless, two transgenic lines had an insecticidal impact on Plutella xylostella. The V-12.1 and V-14.3 lines killed larvae after 3 - 6 days of feeding (Table 4). The expression of the cry9Aa gene in Brassica plants was WO00/11025 PCT/FI99/00698 42 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. Conclusions. 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. In cauliflower, an expression of 100 ng of Cry9Aa protein per 1 g of leaf tissue led to a high insecticidal impact on Plutella xylostella, 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 CryIAc and CryIC resistant strains of P. xylostella. This fact confirms the conclusion that Cry9Aa toxin has its own binding receptor mechanism. The CrylAc and CryIC resistant insects used in bioassays did not demonstrate cross-resistance to Cry9Aa toxin. Tables Table 1. The results of bioassays with crystal Cry9Aa protein fed to second instar larvae of Plutella xylostella strains, susceptible wild, and resistant to CryIAC and CryIC toxins. Number of alive (a) or dead (d) larvae as well as number of pupas (p) are shown for each day of the feeding.

WO 00/11025 PCT/FI99/00698 43 Table 1 P.xylostella Days of feeding stain strain 2 3 4 8 Fina Iresults 8 - 10 pg/g of Cry9Aa protein in feed Wild 3 d 2 d 5 d 2 a CryiAc 2 d 3 d 5 d 3 a CryIC 2 d 3 d 5 d 3 a 3.5 - 4 Ag/g of Cry9Aa protein in feed Wild 3d 2 d 5 d CrylAc 5 a 3 d 2 d 5 d 2 a CryIC 5 a. 2 d 2 d 1 5 5 d :. 3 a I a 1.5 - 2 f Cry9Aa protein in feed Wild 2 d 3 d 5 d 3 a CrylAc 5 a 5 5 ad 5 d CrylC 5 a d 2 d 2 d 5 d 4 a 2 a 0.7 - 1 4g/g of Cry9Aa protein in feed Wild 5 a 3 d 2 d 5d 2 a CrylAC 5 a 2 d 1 d 2 d 5 d 3 a 2 a CryIC 5 a 5 a i d d p id 3 d 4 a 3a 2a i p 2p 6 ng/ml casein in feed Wild Sa 5a Sa 1p 1 P P 5 'D 4 a 3a CrylAc S a 5 a 5 a 4p a p 5 p 1 a CryIC 5 a 5 a 5 a 3 p 2 p 5 p 2 a WO00/11025 PCT/F1I99/00698 44 Table 2: Proportion of codons used in coding sequences of monocots, dicots and Brassica plants. Aminoacid Codon Monocots Dicots Brassicas Gly GGG 0.25 0.13 0.20 Gly GGA 0.19 0.38 0.32 Gly GGT 0.14 0.35 0.32 Gly GGC 0.42 0.14 0.16 Glu GAG 0.76 0.51 0.58 Glu GAA 0.24 0.49 0.42 Asp GAT 0.24 0.64 0.62 Asp GAC 0.76 0.36 0.38 Val GTG 0.40 0.27 0.26 Val GTA 0.08 0.13 0.12 Val GTT 0.16 0.40 0.35 Val GTC 0.36 0.20 0.26 Ala GCG 0.28 0.12 0.17 Ala GCA 0.16 0.25 0.26 Ala GCT 0.19 0.45 0.36 Ala GCC 0.37 0.19 0.22 Arg AGG 0.24 0.24 0.27 Arg AGA 0.11 0.33 0.33 Ser AGT 0.07 0.16 0.17 Ser AGC 0.23 0.15 0.14 Lys AAG 0.83 0.56 0.55 Lys AAA 0.17 0.44 0.45 Asn AAT 0.23 0.46 0.43 Asn AAC 0.77 0.54 0.57 Met ATG 1.00 1.00 1.00 Ile ATA 0.12 0.20 0.23 Ile ATT 0.24 0.43 0.38 Ile ATC 0.63 0.38 0.39 Thr ACG 0.24 0.13 0.19 Thr ACA 0.17 0.28 0.29 Thr ACT 0.18 0.35 0.26 WO00/11025 PCT/FI99/00698 45 Aminoacid Codon Monocots Dicots Brassicas Thr ACC 0.41 0.24 0.26 Trp TGG 1.00 1.00 1.00 End TGA 0.62 0.48 0.35 Cys TGT 0.24 0.54 0.59 Cys TGC 0.76 0.46 0.41 End TAG 0.21 0.19 0.29 End TAA 0.17 0.33 0.35 Tyr TAT 0.22 0.45 0.43 Tyr TAC 0.78 0.55 0.57 Leu TTG 0.14 0.23 0.22 Leu TTA 0.04 0.12 0.07 Phe TTT 0.26 0.47 0.37 Phe TTC 0.74 0.53 0.63 Ser TCG 0.18 0.09 0.13 Ser TCA 0.14 0.19 0.21 Ser TCT 0.14 0.27 0.21 Ser TCC 0.24 0.14 0.14 Arg CGC 0.17 0.08 0.10 Arg CGA 0.08 0.10 0.12 Arg CGT 0.10 0.18 0.14 Arg CGC 0.30 0.07 0.05 Gln CAG 0.44 0.46 0.51 Gln CAA 0.56 0.54 0.49 His CAT 0.34 0.57 0.52 His CAC 0.66 0.43 0.48 Leu CTG 0.31 0.11 0.11 Leu CTA 0.08 0.10 0.11 Leu CTT 0.12 0.26 0.30 Leu CTC 0.31 0.18 0.19 Pro CCG 0.29 0.15 0.15 Pro CCA 0.37 0.36 0.38 Pro CCT 0.14 0.36 0.33 Pro CCC 0.21 0.13 0.14 WO00/11025 PCT/F199/00698 46 Accession numbers of used DNA sequences in GenBank ???. Brassica oleracea: U16751, U18995, M87514, L36926, L36927, M76647, Y00286. Brassia campestris: L41355, M64631, D38563, U17098, L21896, L21897, L23554. Brassica napus: U04945, U15604, U17987, U20179, U21849, U00443, M99415, L25406, L15303, L12395, L29214, L19879, L08608, L08607, L12393, M97667. Species and gene number used in the column of dicot codon preference. Arabidopsis thaliana-854 coding sequence (John Morris john.morris@frodo.mgh.harvard.edu), Pisum sativa - 37 sequences, Petunia sp. -18 genes, Phaseolus vulgaris - 26 genes, Solanum tuberosum 21 genes, Glycine max - 59, Nicotiana tabacum - 21, Lycopersicon esculentum - 41 genes (J. Michael Cherry cherry@frodo.mgh.harvard.edu) Species and gene number used in the column with monocot codon preference. Hordeum vulgare - 36 genes, Zea mays - 71, Oryza sativa - 16, Triticum aestivum - 45 genes (J. Michael Cherry cherry@frodo.mgh.harvard.edu) WO 00/11025 PCT/FI99/00698 47 Table 3. Bioassays of transgenic cauliflower leaves fed to first instar larvae of Plutella xylostella strains (susceptible wild; and resistant to CrylAC and CryIC toxins). Number of alive (a) or dead (d) larvae as well as number of pupas (p) are shown for successive days of the feeding. P.xylostella Days of feeding line ne 2 4 6 8 10 14 Final results A-0 line of cauliflower Wild 5 d 5 d CrylAc 2d 3 d 5d 3 a CryIC 2 d I d I d I d 5 d 3 a 2 a I a A-10 line of cauliflower Wild 5a 5 a 3 d 2 d 5 d 2 a CryIAc 5a 5 a 5 a 3 d 2 d 5 d 2 a CryiC 5 a 5 a 5 a 5 a 1 p 2p 3 4 a 2 d 2 -d Control line of cauliflower cv. Asterix Wild 5 a 5 a 1 p 4 p 5 p 4 a CryIAc 5 a 5 a 2 p 3 p 5 p 3 a CrylC 5 a 5a 2p 2 p 1 p 5 p 3 a 1 a WO00/11025 PCTIF199/00698 48 Table 4. Bioassays of transgenic turnip rape leaves fed to second - third instar larvae of Plutella xylostella wild type. Number alive or died larvae as well as number of pupas are shown for successive days of feeding. Turnip rape Days of feeding line 2 3 4 5 6 8 Final results V-12.1 5 a 5 a 2 d 3 d 5 d V-14.3 l d 2d 2a I d i d 5d 4a 2a la Control 5 a 5a 5 a 2 p 2 p i p 5 p 3a la WO 00/11025 PCT/FI99/00698 49 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: UniCrop Ltd (B) STREET: Helsinki Science Park, Viikinkaari 6 (C) CITY: HELSINKI (E) COUNTRY: FINLAND (F) POSTAL CODE (ZIP): FIN-00710 (ii) TITLE OF INVENTION: MODIFIED SYNTHETIC DNA SQUENCES FOR IMPROVED INSECTICIDAL CONTROL (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (EPO) (D) SOFTWARE: patentIn Release 1.0, Verson #.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: FI 981809 (B) FILING DATE: 24-AUG-1998 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 624 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: galleria (C) INDIVIDUAL ISOLATE: 11-67 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Pro Leu Ala Asn Asn Pro Tyr Ser Ser Ala Leu Asn Leu Asn Ser Cys 15 10 15 Gln Asn Ser Ser Ile Leu Asn Trp Ile Asn Ile Ile Gly Asp Ala Ala 20 25 30 Lys Glu Ala Val Ser Ile Gly Thr Thr Ile Val Ser Leu Ile Thr Ala 35 40 45 WO00/11025 PCT/FI99/00698 5sO Pro Ser Leu Thr Gly Leu Ile Ser Ile Val Tyr Asp Leu Ile Gly Lys 50 55 60 Val Leu Gly Gly Ser Ser Gly Gin Ser Ile Ser Asp Leu Ser Ile Cys 65 70 75 80 Asp Leu Leu Ser Ile Ile Asp Leu Arg Val Ser Gin Ser Val Leu Asn 85 90 95 Asp Gly Ile Ala Asp Phe Asn Gly Ser Val Leu Leu Tyr Arg Asn Tyr 100 105 110 Leu Glu Ala Leu Asp Ser Trp Asn Lys Asn Pro Asn Ser Ala Ser Aia 115 120 125 Glu Giu Leu Arg Thr Arg Phe Arg Ile Ala Asp Ser Glu Phe Asp Arg 130 135 140 Ile Leu Thr Arg Gly Ser Leu Thr Asn Gly Gly Ser Leu Ala Arg Gin 145 150 155 160 Asn Ala Gin Ile Leu Leu Leu Pro Ser Phe Ala Ser Ala Ala Phe Phe 165 170 175 His Leu Leu Leu Leu Arg Asp Ala Thr Arg Tyr Gly Thr Asn Trp Gly 180 185 190 Leu Tyr Asn Ala Thr Pro Phe Ile Asn Tyr G1n Ser Lys Leu Val Glu 195 200 205 Leu Ile Glu Leu Tyr Thr Asp Tyr Cys Val His Trp Tyr Asn Arg Gly 210 215 220 Phe Asn Glu Leu Arg Gin Arg Gly Thr Ser Ala Thr Ala Trp Leu Glu 225 230 235 240 Phe His Arg Tyr Arg Arg Glu Met Thr Leu Met Val Leu Asp Ile Val 245 250 255 Ala Ser Phe Ser Ser Leu Asp Ile Thr Asn Tyr Pro Ile Glu Thr Asp 260 265 270 Phe Gin Leu Ser Arg Val Ile Tyr Thr Asp Pro ile Gly Phe Val His 275 280 285 Arg Ser Ser Leu Arg Giy Glu Ser Trp Phe Ser Phe Val Asn Arg Ala 290 295 300 Asn Phe Ser Asp Leu Glu Asn Ala Ile Pro Asn Pro Arg Pro Ser Trp 305 310 315 320 Phe Leu Asn Asn Met Ile Ile Ser Thr Gly Ser Leu Thr Leu Pro Val 325 330 335 Ser Pro Ser Thr Asp Arg Ala Arg Val Trp Tyr Gly Ser Arg Asp Arg 340 345 350 WO00/11025 PCTIFI99/00698 51 Ile Ser Pro Ala Asn Ser Gin Phe Ile Thr Glu Leu Ile Ser Gly Gin 355 360 365 His Thr Thr Ala Thr Gin Thr Ile Leu Gly Arg Asn Ile Phe Arg Val 370 375 380 Asp Ser Gin Ala Cys Asn Leu Asn Asp Thr Thr Tyr Giy Val Asn Arg 385 390 395 400 Ala Val Phe Tyr His Asp Ala Ser Glu Gly Ser Gin Arg Ser Val Tyr 405 410 415 Glu Gly Tyr Ile Arg Thr Thr Gly Ile Asp Asn Pro Arg Val Gin Asn 420 425 430 Ile Asn Thr Tyr Leu Pro Gly Glu Asn Ser Asp Ile Pro Thr Pro Glu 435 440 445 Asp Tyr Thr His Ile Leu Ser Thr Thr Ile Asn Leu Thr Gly Gly Leu 450 455 460 Arg Gin Val Ala Ser Asn Arg Arg Ser Ser Leu Val Met Tyr Gly Trp 465 470 475 480 Thr His Lys Ser Leu Ala Arg Asn Asn Thr Ile Asn Pro Asp Arg Ile 485 490 495 Thr Gin Ile Pro Leu Thr Lys Val Asp Thr Arg Gly Thr Gly Val Ser 500 505 510 Tyr Val Asn Asp Pro Gly Phe Ile Gly Gly Ala Leu Leu Gin Arg Thr 515 520 525 Asp His Gly Ser Leu Gly Val Leu Arg Val GIn P"e Pro Leu His Leu 530 535 540 Arg Gin Gln Tyr Arg Ile Arg Val Arg Tyr Ala Ser Thr Thr Asn ile 545 550 555 560 Arg Leu Ser Val Asn Gly Ser Phe Gly Thr Ile Ser Gin Asn Leu Pro 565 570 575 Ser Thr Met Arg Leu Gly Glu Asp Leu Arg Tyr Gly Ser Phe Ala ile 580 585 590 Arg Glu Phe Asn Thr Ser Ile Arg Pro Thr Ala Ser Pro Asp Gin l1e 595 600 605 Arg Leu Thr Ile Glu Pro Ser Phe Ile Arg Gin Glu Val Tyr Val Asp 610 615 620 WO 00/11025 PCTIFI99/00698 52 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1953 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACCATGGGAA ACTGTGGATG TGCTTCTGAT GATGTTGCTA AGTACCCATT GGCTAACAAC 60 CCATACTCAT CTGCTCTTAA CCTTAACTCT TGCCAGAACT CTTCTATCCT TAACTGGATC 120 AACATCATTG GAGATGCTGC TAAGGAGGCT GTGTCTATTG GAACTACCAT TGTCTCTCTT 180 ATCACTGCTC CATCTCTTAC TGGATTGATC TCAATTGTGT ACGATCTTAT TGGAAAGGTT 240 CTTGGAGGTT CTTCTGGACA GTCTATCTCT GATCTTTCTA TCTGTGATCT TCTTTCTATC 300 ATTGATCTTA GGGTTTCTCA GTCTGTTTTG AACGATGGAA TTGCTGATTT CAACGGTTCT 360 GTTCTTTTGT ACAGGAACTA CTTGGAGGCT CTTGATTCTT GGAACAAGAA CCCAAACTCT 420 GCTTCTGCTG AGGAGCTTAG GACTAGGTTC AGGATCGCTG ATTCTGAGTT CGATAGGATC 480 TTGACCAGGG GATCTCTTAC CAACGGAGGA TCT'TGGCTA GGCAGAACGC TCAGATCCTT 540 TTGCTTCCAT CTTTTGCATC TGCTGCTTTC TTCCACTTG TGCTCCTTAG GG.ATGCTAC 600 AGGTACGGTA CTAACTGGGG ACTTTACAAC GCTACCCCAT TTATCAACTA CCAAAGCAAG 660 CTGGTTGAGC TTA.TTGAGCT TTACACTGAT TACTGTGTTC ACTGGTACAA CAGGGGATTC 720 AACGAGCTTA GACAGAGGGG AACCTCTGCT ACGGCTTGGC TTGA" TTCCA CAGATACCGC 780 AGAGAGATGA CCTTGATGGT TCTTGATATT GTTGCTTCTT TCTCTT CCT TGATATCACC 840 AACTACCCTA TTGAGACTGA TTTCCAGTTG TCTAGGGTTA TCTACACTGA TCCTATTGGA 900 TTCGTTCACA GGTCCTCTCT TAGGGGAGAG TC T TGGTTCT CTTTCGTTAA CAGGGCTAAC 960 TTCTCTGATT TGGAGAACGC TATCCCAAAC CCAAGACCAT CTTGGTTCCT TAACAACATG 1020 ATCATCTCTA CTGGATCTCT TACCTTGCCA GTTTCTCCAT CTACTGATAG AGCTAGGGTT 1080 TGGTATGGAT CTAGGGATAG GATCTCTCCA GCTAACTCTC AGTTCATCAC TGAGCTTATC 1140 TCTGGACAGC ACACCACTGC TACCCAGACC ATCTTGGGAA GGAATATCTT CAGGGTTGAT 1200 WO 00/11025 PCT/FI99/00698 53 TCTCAAGCTT GCAACTTGAA CGATACTACT TATGGTGTGA ACAGGGCTGT TTTCTACCAT 1260 GATGCTTCTG AGGGTAGCCA AAGGTCTGTT TACGAGGGTT ACATCAGGAC CACTGGAATT 1320 GATAACCCAA GGGTTCAGAA CATCAACACC TACTTGCCAG GAGAGAACTC CGATATTCCA 1380 ACTCCTGAGG ATTACACCCA CATCTTGTCT ACCACCATCA ACTTGACTGG AGGACTTAGA 1440 CAGGTTGCTT CTAACAGGAG GTCATCTTTG GTTATGTACG GATGGACTCA CAAGTCTCTT 1500 GCTAGGAACA ACACCATCAA CCCAGATAGA ATCACCCAGA TCCCATTGAC CAAGGTTGAT 1560 ACCAGAGGAA CTGGAGTTTC TTACGTGAAC GATCCAGGAT TCATTGGAGG AGCTCTTCTT 1620 CAGAGGACTG ACCACGGATC TCTTGGAGTT TTGAGGGTTC AGTTCCCACT TCACTTGAGA 1680 CAGCAGTACA GGATCAGGGT TAGGTACGCT TCTACCACTA ACATCAGGTT GTCTGTGAAC 1740 GGATCTTTCG GAACTATCTC TCAGAACCTT CCATCTACCA TGAGATTGGG AGAGGATTTG 1800 AGATACGGAT CTTTTGCTAT CAGAGAATTC AACACCTCTA TCAGACCAAC TGCTTCTCCA 1860 GATCAGATCA GGTTGACTAT TGAGCCATCT TTCATCAGAC AGGAGGTTTA CGTTGATAGA 1920 ATTGAGTTCA TCCCAGTTAA CCCAGATCTT TAA 1953 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1989 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "truncated plasmid segment' (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringlensis (B) STRAIN: galleria (C) INDIVIDUAL ISOLATE: 11:67 (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: truncated-plasmid segment (B) MAP POSITION: DNA coding for N-terminal part of cry9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGCGGATCCA ACAATGGGCA TGCCCAATTG TGGTTGTGCA TCTGATGATG TTGCGAAATA 60 TCCTTTAGCC AACAATCCAT ATTCATCTGC TTTAAATTTA AATTCTTGTC AAAATAGTAG 120 TATTCTCAAC TGGATTAACA TAATAGGCGA TGCAGCAAAA GAAGCAGTAT CTATTGGGAC 180 WO 00/11025 PCTIFI99/00698 54 AACCATAGTC TCTCTTATCA CAGCACCTTC TCTTACTGGA TTAATTTCAA TAGTATATGA 240 CCTTATAGGT AAAGTACTAG GAGGTAGTAG TGGACAATCC ATATCAGATT TGTCTATATG 300 TGACTTATTA TCTATTATTG ATTTACGGGT AAGTCAGAGT GTTTTAAATG ATGGGATTGC 360 AGATTTTAAT GGTTCTGTAC TCTTATACAG GAACTATTTA GAGGCTCTGG ATAGCTGGAA 420 TAAGAATCCT AATTCTGCTT CTGCTGAAGA ACTCCGTACT CGTTTTAGAA TCGCCGACTC 480 AGAATTTGAT AGAATTTTAA CCCGAGGGTC TTTAACGAAT GGTGGCTCGT TAGCTAGACA 540 AAATGCCCAA ATATTATTAT TACCTTCTTT TGCGAGCGCT GCATTTTTCC ATTTATTACT 600 ACTAAGGGAT GCTACTAGAT ATGGCACTAA TTGGGGGCTA TACAATGCTA CACCTTTTAT 660 AAATTATCAA TCAAAACTAG TAGAGCTTAT TGAACTATAT ACTGATTATT GCGTACATTG 720 GTATAATCGA GGTTTCAACG AACTAAGACA ACGAGGCACT AGTGCTACAG CTTGGTTAGA 780 ATTTCATAGA TATCGTAGAG AGATGACATT GATGGTATTA GATATAGTAG CATCATTTTC 840 AAGTCTTGAT ATTACTAATT ACCCAATAGA AACAGATTTT CAGTTGAGTA GGGTCATTTA 900 TACAGATCCA ATTGGTTTTG TACATCGTAG TAGTCTTAGG GGAGAAAGTT GGTTTAGCTT 960 TGTTAATAGA GCTAATTTCT CAGATTTAGA AAATGCAATA CCTAATCCTA GACCGTCTTG 1020 GTTTTTAAAT AATATGATTA TATCTACTGG TTCACTTACA TTGCCGGTTA GCCCAAGTAC 1080 TGATAGAGCG AGGGTATGGT ATGGAAGTCG AGATCGAATT TCCCCTGCTA ATTCACAATT 1140 TATTACTGAAk CTAATCTCTG GACAACATAC GACTGCTACA CAAACTATTT TAGGGCGAAA 1200 TATATTTAGA GTAGATTCTC AAGCTTGTAA TTTAAATGAT ACCACATATG GAGTGAATAG 1260 GGCGGTATTT TATCATGATG CGAGTGAAGG TTCTCAAAGA TCCGTGTACG AGGGGTATAT 1320 TCGAACAACT GGGATAGATA ACCCTAGAGT TCAAAATATT AACACTTATT TACCTGGAGA 1380 AAATTCAGAT ATCCCAACTC CAGAAGACTA TACTCATATA TTAAGCACAA CAATAAATTT 1440 AACAGGAGGA CTTAGACAAG TAGCATCTAA TCGCCGTTCA TCTTTAGTAA TGTATGGTTG 1500 GACACATAAA AGTCTGGCTC GTAACAATAC CATTAATCCA GATAGAATTA CACAGATACC 1560 ATTGACGAAG GTTGATACCC GAGGCACAGG TGTTTCTTAT GTGAATGATC CAGGATTTAT 1620 AGGAGGAGCT CTACTTCAAA GGACTGACCA TGGTTCGCTT GGAGTATTGA GGGTCCAATT 1680 TCCACTTCAC TTAAGACAAC AATATCGTAT TAGAGTCCGT TATGCTTCTA CAACAAATAT 1740 TCGATTGAGT GTGAATGGCA GTTTCGGTAC TATTTCTCAA AATCTCCCTA GTACAATGAG 1800 ATTAGGAGAG GATTTAAGAT ACGGATCTTT TGCTATAAGA GAGTTTAATA CTTCTATTAG 1860 WO 00/11025 PCT/FI99/0069 8 55 ACCCACTGCA AGTCCGGACC AAATTCGATT GACAATAGAA CCATCTTTTA TTAGACAAGA 1920 GGTCTATGTA GATAGAATTG AGTTCATTCC AGTTAATCCA GATCTATAAC CCGGGCTCGA 1980 GGTACCGCG 1989 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3837 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "plasmid DNA1 (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: galleria (C) INDIVIDUAL ISOLATE: 11:67 viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: plasmid DNA (B) MAP POSITION: full length DNA encoding Cry9Aa toxin (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CAGAAATCAA CTTACAAGTA TTAGGGATTT CCCCGTTCGA CCCGTTTTAT ACAAACTAG 60 TATACTGCGA TGGGTAGATA TTCGTCTATT TCCTTAAGTT TTTAAGATGC AGTATGTATA 120 GATCATATTT TAAAATATAG CGTTCATCAA CTACTATATT ATGTAATTGA CGGTAATCAT 180 ATTCAAGTGC GTGATTTACA AATCAAATTA ATGAAAGAAC ATGCTCAATC TGCTCAAATA 240 TCTGTTTTTT ATTTATGAGT AAAAACCGAA GTTTGTG-AA CGTAAACTGT AATAAACGTA 300 ATGGCGAAGA TATATAGATG TCAATATAAA AAkGTT-AACCC AAATAATGTT TTAAAATTTT 360 AAALATAATG TAGGAGGAAA AATTATG-AAT C-AAAATAAAC ACGGAATTAT TGGCGCTTCC 420 AATTGTGGTT GTGCATCTGA TGATGTTGCG A ATATCCTT TAGCCAAC-AA TCCATATTCA 480 TCTGCTTTA-A ATTTAAATTC TTGTCAAAAT AGTATATTC TCAACTGGAT TAACATAATA 540 GGCGATGCAG CAAAAGAAGC AGTATCTATT GGGACAACCA TAGTCTCTCT TATCACAGCA 600 CCTTCTCTTA CTGGATTAAT TTCAATAGTA TATGACCTTA TAGGTAAAGT ACTAGGAGGT 660 AGTAGTGGAC AATCCATATC AGATTTGTCT ATAT!GTGACT TATTATCTAT TATTGATTTA 720 CGGGTAAGTC AGAGTGTTTT AAATGATGGG ATTGCAGATT TTAATGGTTC TGTACTCTTA 780 WO 00/11025 PCT/FI99/00698 56 TACAGGAACT ATTTAGAGGC TCTGGATAGC TGGAATAAGA ATCCTAATTC TGCTTCTGCT 840 GAAGAACTCC GTACTCGTTT TAGAATCGCC GACTCAGAAT TTGATAGAAT TTTAACCCGA 900 GGGTCTTTAA CGAATGGTGG CTCGTTAGCT AGACAAAATG CCCAAATATT ATTATTACCT 960 TCTTTTGCGA GCGCTGCATT TTTCCATTTA TTACTACTAA GGGATGCTAC TAGATATGGC 1020 ACTAATTGGG GGCTATACAA TGCTACACCT TTTATAAATT ATCAATCAAA ACTAGTAGAG 1080 CTTATTGAAC TATATACTGA TTATTGCGTA CATTGGTATA ATCGAGGTTT CAACGAACTA 1140 AGACAACGAG GCACTAGTGC TACAGCTTGG TTAGAATTTC ATAGATATCG TAGAGAGATG 1200 ACATTGATGG TATTAGATAT AGTAGCATCA TTTTCAAGTC TTGATATTAC TAATTACCCA 1260 ATAGAAACAG ATTTTCAGTT GAGTAGGGTC ATTTATACAG ATCCAATTGG TTTTGTACAT 1320 CGTAG-TAGTC TTAGGGGAGA AAGTTGGTTT AGCTTTGTTA ATAGAGCTAA TTTCTCAGAT 1380 TTAGAAAATG CAATACCTAA TCCTAGACCG TCTTGGTTTT TAAATAATAT C-TTATATCT 1440 ACTGGTTCAC TTACATTGCC GGTTAGCCCA AGTACTGATA GAGCGAGGGT ATGGTATGGA 1500 AGTCGAGA.TC GAATTTCCCC TGCTAATTCA CAATTTATTA CTGACTAAT CTCTGGACAA 1560 CATACGACTG CTACACAAAC TATTTTAGGG CGAAATATAT TTAGAGTAGA TTCTCAAGCT 1620 TGTAATTTAA ATGATACCAC ATATGGAGTG AATAGGGCGG TATTTTATCA TGATGCGAGT 1680 GAAGGTTCTC AAAGATCCGT GTACGAGGGG TATATTCGAA CAACTGGGAT AGATAACCCT 1740 AGAGTTCAAA ATATTAACAC TTATTTACCT GGAGAAAATT CAGA.TATCCC AACTCCAGAA 1800 GACTATACTC ATATATTAAG CACAACAATA AATTTAACAG GAGGACTTAG ACAAGTAGCA 1860 TCTAATCGCC GTTCATCTTT AGTAATGTAT GGTTGGACAC ATAAAAGTCT GGCTCGTAAC 1920 AATACCATTA ATCCAGATAG AATTACACAG ATACCATTGA CGAAGGTTGA TACCCGAGGC 1980 ACAGGTGTTT CTTATGTGAA TGATCCAGGA TTTATAGGAG GAGCTCTACT TCAAAGGACT 2040 GACCATGGTT CGCTTGGAGT ATTGAGGGTC CAATTTCCAC TTCACTTAAG ACAACAATAT 2100 CGTATTAGAG TCCGTTATGC TTCTACAACA AATATTCGAT TGAGTGTGAA TGGCAGTTTC 2160 GGTACTATTT CTCAAAATCT CCCTAGTACA ATGAGATTAG GAGAGGATTT AAGATACGGA 2220 TCTTTTGCTA TAAGAGAGTT TAATACTTCT ATTAGACCCA CTGCAAGTCC GGACCAAATT 2280 CGATTGACAA TAGAACCATC TTTTATTAGA CAAGAGGTCT ATGTAGATAG AATTGAGTTC 2340 ATTCCAGTTA ATCCGACGCG AGAGGCGAAA GAGGATCTAG AAGCAGCAAA AAAAGCGGTG 2400 GCGAGCTTGT TTACACGCAC AAGGGACGGA TTACAAGTAA ATGTGAAAGA TTATCAAGTC 2460 WO 00/11025 PCT/F199/00698 57 GATCAAGCGG CAAATTTAGT GTCATGCTTA TCAGATGAAC AATATGGGTA TGACAAAAAG 2520 ATGTTATTGG AAGCGGTACG TGCGGCAAAA CGACTTAGCC GAGAACGCAA CTTACTTCAG 2580 GATCCAGATT TTAATACAAT CAATAGTACA GAAGAAAATG GATGGAAAGC AAGTAACGGC 2640 GTTACTATTA GTGAGGGCGG GCCATTCTAT AAAGGCCGTG CAATTCAGCT AGCAAGTGCA 2700 CGAGAAAATT ACCCAACATA CATCTATCAA AAAGTAGATG CATCGGAGTT AAAGCCGTAT 2760 ACACGTTATA GACTGGATGG GTTCGTGAAG AGTAGTCAAG ATTTAGAAAT TGATCTCATT 2820 CACCATCATA AAGTCCATCT TGTGAAAAAT GTACCAGATA ATTTAGTATC TGATACTTAC 2880 CCAGATGATT CTTGTAGTGG AATCAATCGA TGTCAGGAAC AACAGATGGT AAATGCGCAA 2940 CTGGAAACAG AGCATCATCA TCCGATGGAT TGCTGTGAAG CAGCTCAAAC ACATGAGTTT 3000 TCTTCCTATA TTGATACAGG GGATTTAAAT TCGAGTGTAG ACCAGGGAAT CTGGGCGATC 3060 TTTAAAGTTC GAACAACCGA TGGTTATGCG ACGTTAGGAA ATCTTGAATT GGTAGAGGTC 3120 GGACCGTTAT CGGGTGAATC TTTAGAACGT GACAAAGGG ATAATACAAA ATGGAGTGCA 3180 GAGCTAGGAA GAAAGCGTGC AGAAACAGAT CGCGTGTATC AAGATGCCAA ACAATCCATC 3240 AATCATTTAT TTGTGGATTA TCAAGATCAA CAATTAAATC CAGAATAGG GATGGCAGAT 3300 ATTATGGACG CTCAAAATCT TGTCGCATCA ATTTCAGATG TATATAGCGA TGCCGTACTG 3360 CAAATCCCTG GAATTAACTA TGAGATTTAC ACAGAGCTGT CCAATCGCTT ACAACAAGCA 3420 TCGTATCTGT ATACGTCTCG A.AATGCGGTG CAAAATGGGG ACTTTAACAA CGGGCTAGAT 3480 AGCTGGAATG CAACAGCGGG TGCATCGGTA CAACAGGATG GCAATACGCA TTTCTTAGTT 3540 CTTTCTCATT GGGATGCACA AGTTTCTCAA CAATTTAGAG TGCAGCCGAA TTGTAAATAT 3600 GTATTACGTG TAACAGCAGA GAAAGTAGGC GGCGGAGACG GATACGTGAC TATCCGGGAT 3660 GATGCTCATC ATACAGAAAC GCTTACATTT AATGCATGTG ATTATGATAT AAATGGCACG 3720 TACGTGACTG ATAATACGTA TCTAACAAAA GAAGTGGTAT TCCATCCGGA GACACAACAC 3780 ATGTGGGTAG AGGTAAATGA AACAGAAGGT GCATTTCATA TAGATAGTAT TGAATTC 3837

Claims

1. Modified synthetic DNA sequences for improved insect control, comprising synthetic DNA sequences modified from the truncated cry9Aa gene of Bacillus thuringiensis ssp. galleria, encoding an insecticidal protein characterized by having the amino acid sequence (SEQ ID NO:1:) or alterations thereof having substantially the same properties as the insecticidally active N-terminal domain of the selected Cry 9Aa protein.

2. The modified synthetic DNA sequences according to claim 1, wherein the modified synthetic DNA sequence comprise modifications of SEQ ID NO:3:.

3. The modified synthetic DNA sequences according to claim 1, wherein the modified synthetic DNA sequences is SEQ ID NO:2: and substantially similar sequences.

4. The modified synthetic DNA sequences according to claim 1, wherein the modifications of SEQ ID NO:3: comprise selected changes of codon preference.

5. The modified synthetic DNA sequences of claim 1, wherein the nucleotide sequence changes comprise removal of the putative polyadenylation sequence.

6. The modified synthetic DNA sequences of claim 1, wherein the nucleotide sequence changes comprise removal of or changes in the splicing and mRNA destabilising signal sequences.

7. The modified synthetic DNA sequences of claim 1, wherein the nucleotide sequence changes comprise one or more optional changes in the vicinity of the start codon to increase its com patibility to the selected higher plant.

8. A DNA construct for cloning and/or transforming procaryotic or eucaryotic organisms, comprising the modified, WO00/11025 PCTIFI99/00698 59 synthetic DNA sequences according to claims 1-7.

9. A procaryotic or eucaryotic host comprising the modified, synthetic DNA sequences according to claims 1-7.

10. A method for preparing modified synthetic DNA sequences according to claims 1-7, comprising the steps of: (a) selecting a DNA sequence encoding an insecticidal protein characterized by having the SEQ ID NO:l: and the unique properties of the Cry9Aa protein and differing substantially from other CryI proteins; (b) providing a synthetic DNA sequences encoding the protein characterized by having (SEQ ID NO:l) which is encoded by the truncated DNA sequence (SEQ ID NO:3:) obtainable from the native cry9Aa gene (SEQ ID NO:4:) by trypsin cleavage; (c) improving the translation of SEQ ID NO:3: by changing its codon preference in selected direction, in order to obtain modifications of SEQ ID NO:3: still encoding an insecticidal protein comprising the amino acid sequence (SEQ ID NO:l:) or alterations thereof, said alterations still having substantially similar insecticidal action as the insecticidal protein encoded by the native cry9Aa gene of Bacillus thuringiensis ssp. galleria.

11. The method according to claim 10, wherein the modifications in the synthetic DNA sequences comprise changing the codon preference of SEQ ID NO:3: in selected direction by methods consisting of removal of putative polyadenylation, splicing and mRNA destabilising signal sequences and/or changes of the vicinity of the start codon by modification of one or more nucleotides in order to increase the compatibility of the synthetic DNA sequence with that of the selected higher plant.

12. The method according to claim 10, wherein the modified synthetic DNA sequences comprise SEQ ID NO:2: and substantially similar modifications thereof. WO 00/11025 PCTIF199/00698 60

13. A method for providing higher plants for improved insecticidal control comprising the steps of incorporating the modified synthetic DNA sequences according to claims 1-7 into a DNA construct in order to functionally incorporate said modified DNA sequences into a plant genome.

14. The use of the modified, synthetic DNA sequence according to claims 1-7, for producing the unique insecticidal protein characterized by having the amino acid sequence (SEQ ID NO:1:) or alterations thereof having substantially the same proper ties as the N-terminal domain of the insecticidal protein encoded by the native cry9Aa gene of Bacillus thuringiensis ssp. galleria.

15. The use of the modified, synthetic DNA sequence according to claims 1-7, wherein the improved properties comprise enhanced expression through improved mRNA processing, stability, and/or translation providing improved tolerance against target insects.

16. The use of the modified, synthetic DNA sequence according to claims 1-7, for producing transgenic plants with improved properties, said transgenic plant being capable of expressing effective amounts of an insecticidal protein being substantial ly similar with the amino acid sequence SEQ ID NO:1: and alterations therof having substantially the same properties as the N-terminal domain of the insecticidal protein encoded by the native cry9Aa gene of Bacillus thuringiensis ssp. galleria.

17. The use of the modified, synthetic DNA sequences according to claims 1-7 for improving insect resistance in higher plants.

18. The use of the modified, synthetic DNA sequences according to claims 1-7 as an implement in resistance management strategies.