CZ219495A3 - Insect control method - Google Patents

Abstract

Patatins control insects, primarily by stunting growth of larvae, thereby preventing maturation and reproduction. Genes encoding for one or more of these proteins may be cloned into vectors for transformation of plant-colonizing microorganisms or plants, thereby providing a method of controlling insect infestation.

Description

(57) Palatins control insects, primarily by larvae deformation, thus preventing maturity and reproduction. Genes encoding one or more of these proteins can be cloned into vectors for transforming plant or plant populating microorganisms. This gives a way of controlling insect infestation.

Insect control method

Technical field

The invention relates to a method for controlling insect infestation by providing a protein which can be applied directly to the plant or produced by microorganisms, or by genetically modifying the plant to produce a protein, as well as microorganisms and plants useful in the method. BACKGROUND OF THE INVENTION

The use of natural products, including proteins, is a well known method of controlling many insect pests. For example, Bacillus thuringiensis (BT) endotoxins are used to control both lepidopteran and coleopteran insect pests. The genes producing these endotoxins have been introduced and expressed in many different plants, including cotton, tobacco and tomatoes. However, there are a number of commercially important insect pests that are not sensitive to BT endotoxins. Examples of such important pests are Anthonomus grandis (KG) and Diabrotica spp. In addition, it is important, if not vital, for insect control to have different gene products to cope with resistance.

Many other known insecticidal proteins are found in plants. These include lectins, amylase inhibitors, and protease inhibitors that can affect insect growth and development when eaten at high doses (Boulter et al., 1989, Broadway and Duffey, 1986, Czapla and Lang, 1990, Ga2 tehouse et al., 1986, Heusing et al., 1991, Ishimoto and K. Kitamura, 1989, Murdock et al., 1990, Schukle and Murdock, 1983), however, do not report acute mortality due to BT proteins.

It is an object of the present invention to provide proteins capable of controlling AG, D and other insect pests and genes useful in the production of such proteins. Another object of the present invention is to provide genetic constructs and methods for introducing such genetic material into microorganisms and plant cells. It is another object of the present invention to provide transformed microorganisms and plants containing such genetic material.

SUMMARY OF THE INVENTION

It has been discovered that palatins, the main potato germ stock protein, will control various insects including Diabrotica virgifera (DV), Diabrotica undecipunctata (DU) and Anthonomus grandis (AG). Palatins are deadly to some larvae, and I distort the survivors' growth to prevent maturity or to delay so much that there is no reproduction. These proteins, which are known to have esterase (lipid acyl hydrolase) activity, can be applied directly to plants or introduced by other methods, such as by application of plant-settling microorganisms that have been transformed to produce enzymes or by the plants themselves after similar transformation.

Palatins are a group of proteins found in potatoes (Gaillaird, 1971, Racusen, 1984, Andrews et al., 1988) and other plants, especially in solanaceous plants (Ganal et al., 1991, Vancanneyt et al., 1989). In potatoes, the palatins are mainly found in the germ, but also in much smaller levels in other plant organs (Hofgen and Willmitzer, 1990). The specificities of esterase substrates of many palatine isozymes have been studied (Hofgen and Willmitzer, 1990, Racusen, 1986).

The genes encoding palatins were previously isolated by Mignery et al., 1984, Mignery et al., 1988, Stiekema et al., 1988 and others. Rosahl et al., 1987 introduced them into tobacco plants and observed the expression of palatine. This demonstrates that the palatine genes can be expressed heterogeneously by plants.

Similarly, the palatine genes can be isolated and inserted into suitable transformation vector cassettes, which are then (1) used to transform plant-settling microorganisms which, when applied to plants, express the palatine-producing genes thereby providing insect control, or (2) the genome of the plant, which then protects itself against insect attack by expressing the gene and producing palatin. In addition, the plant can also be transformed or cultured to simultaneously express one or more BT genes that code for insect control proteins. This would give plants that are either (1) protected against most pests, or (2) have two options of action against some pests, an important tool for managing resistance. Examples of plants transformed to express BT genes are described in European Patent Publication No. 0 385 962, which corresponds to US Serial Number 476,661, filed February 12, 1990 (Fischhoff et al.), Which is incorporated herein by reference. In addition, plants can be transformed or grown to simultaneously express one proteinase inhibitor gene, such as those encoding a potato papain inhibitor (Rodis and Hoff, 1983) or soybean trypsin inhibitor (reviewed by Ryan, 1990), which potentiate as a proteinase inhibitor activity of other insecticidal proteins.

In achieving this, there is provided in accordance with one aspect of the present invention a method of controlling insect infestation of plants comprising providing an effective amount of insecticidal palatine for insect ingestion. This method can be performed by providing a plant inhabiting microorganisms that have been transformed to express the palatin gene and which are fed to the plant, express such a gene, and give an insecticidally effective amount of palatine. The method can also be performed by genetic transformation of a plant to be protected by a DNA molecule comprising (i) a promoter whose function in the plant causes the production of an RNA chain, (ii) a structural coding chain that encodes palatin, (iii) a 3 'untranslated region in plant cells, the addition of polyadenylate nucleotides to the 3 'end of the RNA chain is effected, wherein said promoter is different from said structural coding strand and wherein said promoter is operably linked to said structural coding strand which is again operatively linked to said untranslated region. Preferably, the plant will express palatin at a level of about 0.1-0.5 of total protein.

Genetically transformed insect resistant maize, cotton, tomato and potato plants are also provided by the present invention.

As used herein, the term insect infestation control means a reduction in the number of insects that reduce the beneficial yield, whether by death, growth retardation or reduced reproductive efficiency.

As used herein, the term insecticidal means capable of reducing the number of insects that reduce beneficial yield, whether by death, growth retardation (deformation) or reduced reproductive efficiency.

As used herein, the term "structural coding strand" means a DNA strand that encodes a polypeptide that can be produced by a cell upon transcription of DNA into mRNA, followed by translation into the desired polypeptide.

As used herein, the term "palatin" means a plant protein having 75% or greater homology to the protein encoded by SEQ ID NO; 31, shown below, or more preferably at least 80% homology, or even more preferably at least 85% homology. This term also includes proteins behind synthetic DNA strands that have been designed for improved expression in monocotyledons.

Palatins are a group of esterases found in potatoes (Gaillaird, 1971, Racusen, 1984, Andrews et al., 1988) and other plants, especially in solanaceous plants (Ganal et al., 1991, Vancanneyt et al., 1989). In potatoes, palatins are found mainly in the germ, but also at much lower levels in other plant organs (Hofgen and Willmitzer, 1990, Racusen, 1986). These enzymes have been shown to have limited substrate requirements. The use of all plant derived palatines and their equivalents, both described herein in detail, whether derived from natural DNA strands or synthetic DNA strands, for the purpose of controlling insect infestation is within the scope of this invention.

Crude palatine preparations are commercially available. For example, Sigma Chemical Company, St. Petersburg. St. Louis, MO, offers potato protein preparations designated as Sigma as acid phosphatase (P-1146 and P-3752) or apyrase (A-9149). Potato sprouts can also be obtained and protein extracts can be prepared by methods described in the literature (Racusen and Foote, 1980, Park et al., 1983).

Examples

Biological activity tests

Artificial diet tests

Activity tests against larvae of Leptinotarsa decemlineata (LD) and Ostrinia nubilalis (ON) were performed by superimposing a test sample on an agar diet similar to that described by Marrone et al., 1985. Test samples were prepared by solubilizing the protein in 4-5 ml 10 mM HEPES, pH 7.5, followed by dialysis in the same buffer by arranging a pipe separating 3500 molecular weight. Freshly hatched larvae are fed on a modified diet at 26 ° C and mortality and growth distortions are evaluated after 5 or 6 days. The results of P-3752 tests (Sigma) are shown in Table 1. These crude palatine preparations showed a broad spectrum of insecticidal activity.

Table 1

Dose_% Mortality and Deformation **

DU_AG_LD_ON

ο, οιχ 0 11 0 0 0,03X 0 * 20 * 0 13 ο, ιοχ 19 ** 20 ** 6 0 * 0,30X 6 ** - *** 46 *** 13* 6 * Ι, ΟΟΧ 6 *** 73 *** 13** 6 **

a * = slight deformation (approximately 30-40% size reduction) ** = moderate deformation (approximately 50-80% size reduction) *** = severe deformation (over 90% size reduction)

Accurate quantitative mass measurements of DU (Table 2) were performed after 5 days of exposure and ON (Table 3) after 5 days of exposure and are listed below. The development of larvae fed diet containing p-3752 showed a 92% weight loss compared to controls, and ON larvae showed a 62% weight loss compared to controls.

Table 2

Treatment Mean weight (SEM)% reduction% mortality

Tris 4 mg (0.60) “control level

P-3752 0.30 mg (0.03) “92 6“ Mean survival weight is significantly different to 95% (one ANOVA) (SEM) = standard deviation

Table 3

Treatment Mean weight (SEM)% reduction% mortality

Tris 5.39 mg (0.49) “control level

P-3752 2.05 mg (0.27) “62 7“ Mean survival weight is significantly different to 95% (one ANOVA) (SEM) = standard deviation

The protein character of the insecticidal component P-3752, which is active against Diabrotica virgifera (DV) and Anthonomus grandis (kG), was determined by thermal lability, ammonium sulfur precipitation, molecular weight fractionation, and protease sensitivity experiments.

To confirm that the effects of P-3752 are due to the direct effect of ingested palatine and not to the indirect effect caused by resistance to ingestion, an ON and DU diet selection study was conducted. The results of this diet selection study showed that both Tris and P-3752 treated diets were taken without any overriding preference. There did not appear to be resistance to the P-3752 diet compared to the Tris diet.

A long-term experiment (25 days) against DU used second phase larvae and many transmissions of surviving insects to a freshly adjusted diet. At the end of the study, all control larvae puped. In contrast, 50% of treated larvae were dead and an additional 50% increased body weight by only 16% of initial weight (2.48 mg vs 2.14 mg). This demonstrates that larval development stops and not only slows down. This has important implications from the insect control standpoint, as larvae do not develop into adulthood. Thus, the number of insects in the next generation will decrease.

The larvae of Diabrotica virgifera (DV) can only be used in laboratory experiments in the second phase of larvae. A parallel P-3752 test against DV in the second phase of larvae was proposed. Treatment with P-3752 resulted in only a 13% and 11% increase in body weight of the second phase of the larvae of DU and DV. Control DUs increased weight by 474% and DVs grew by 200% within 7 days. This suggests that the activity of palatine against DV is roughly equivalent to that of DU.

P-3752 was weakly active against Heliothis virescens (CKV), Spondea exigua (SE), Helicoverpa zea (HZ), Ectinophora gossypiella (EG) and Manduca sexta (MS) with deformation ratios of 1 to 1.5 at the same concentration to achieve a ratio of de9 formation 3 at DU. P-3752 gave a strain ratio of 2.5 for Agrotis ipsilon. (Deformation ratios are defined above in Table 1). It was inactive against Mysus persicae at the concentrations tested.

Plant Tissue Experiments (1) Potato: one gram of crude P-3752 was dissolved in 4 ml of 25 mM Tris pH 7.5 buffer and then dialyzed and filtered through a 0.2 micron membrane. Triton (R) X-100 was added to give a 0.1% solution. The potato leaves were immersed in the enzyme preparation and placed on moistened filter paper in Petri dishes. LD larvae were added and plates were incubated at 27 ° C for 3 days. Treatment of potato P-3752 resulted in deformation and reduced feeding of LD larvae. At the conclusion of the experiment, significantly less leaf tissue remained on the control leaves compared to the P-3752 treated leaves.

(2) Maize and cotton: Mexican Mexican corn (CMS) callus or cotton callus was removed from agar plates and transferred to 50 ml centrifuge tubes. Callus was fed and centrifuged in an IEC clinical centrifuge for 5 minutes at setting 8. The supernatant was decanted. To a 50 ml tube containing 15 ml callus pellets was added 30 ml liquid 2% agar. After thorough mixing, the diet was pipetted into the experimental insect bioassay arena. Dialyzed P-3752 was added as a icing diet (20% by volume) and the assay was performed as described above.

The excised corn roots and shoots were vacuum infiltrated (Inflt) with crude P-3752 or 25 mM Tris pH 7.5 buffer. The control sample was tissue immersed in the Tris buffer. Approximately 10-15 pieces of root tissue or 3 pieces of shoot tissue were placed in the wells of a 24-well tissue culture plate and replicated 4 times. Four freshly hatched DU larvae were placed in each well. The experiment was incubated at 26 ° C for 4 days, at which point mortality and mean weight of larvae were observed. The results of these experiments are shown in Table 4.

Table 4

Tissue_Insects% mortality% weight loss

Inflt corn roots DU 90 44 Inflt corn sprouts DU 51 52 Modified callus CMS DU 24 51 Modified callus CMS DV 0 23 Modified callus CMS HE 0 33 Modified cotton callus AG 60 no data Insecticidal activity against all four insects (DU, AG, LD and ON) therefore preserves even if P-3752 is eaten

along with plant tissue. These dietary studies demonstrate that palatins are insecticidally active when tested in diets whose nutrients are only in plant tissues (roots, shoots, callus or leaves).

Study of the action mode

The following studies show that palatin, the insecticidally active ingredient of P-3752, has a direct effect on insects alone and that this activity demonstrated in the experiments described above cannot be ascribed to the effect (s) of the active ingredient on the insect diet prior to ingestion.

Study of the effects of diet

One gram of P-3752 was dissolved in 10 ml of 25 mM Tris pH

7.6 stand and then dialyzed in the MWCO 12-14000 pipeline against the same buffer. After 0.2 micron filtration, 50 ml aliquots were added to diet plates on two plates four days before the insect was added. Both plates were incubated at 27 ° C for 4 days. After incubation, one plate was heated at 80 ° C for 1 hour to inactivate the enzymes. 50 ml aliquots were added to the third plate. Thus, plates incubated, incubated + heat and non-incubated were used for the bioassay with DU.

Activity against DU was as follows: non-incubated P-3752 6 *** incubated P-3752 0 ** incubated, heated P-3752 0.

While part of the activity was lost during the incubation of the diet, complete loss of activity was due to heating. This figure is consistent with the direct effect after ingestion. When assessed in relation to experiments with plant tissues and variability to various insects, it shows that protein activity on DU and other insects is not via a dietary effect.

Identification of proteins

The insecticidally active component of P-3752 was purified, partially sequenced and characterized. The active agent (a) was identified as palatin, a group of lipid acyl hydrolase from potatoes.

Four different protocols were used to purify the bioactive component against DU of P-3752.

Purification of DU activity by anion exchange chromatography

The insecticidally active anti-DU component of P-3752 was purified by anion exchange chromatography (IEC) Q-Sepharose (Pharmacia) followed by anion exchange chromatography MONO-Q (HR 5/5, Pharmacia). Protein levels in the active fractions against DU indicated that the observed distortions were achieved with a protein concentration of 31 ppm in the diet. SDS-PAGE indicated that three major protein bands were present in the active fractions (Μ * 42 42000, about 26000 and about 16000).

Five-stage DU cleaning

Five sequential purification steps were used to purify the active ingredient against DU of P-3752. These were membrane size sizing, ammonium sulfate precipitation, Q-Sepharose IEC, S-Sepharose IEC, and P-200 SEC. SDS-PAGE of the purest DU-active fractions showed protein bands at M + 42000, about 26000 and about 16000.

Purification of bioactivity by isoelectric focusing

DU bioactive potato proteins were purified by two sequential runs on an RF3 protein fractionator (Rainin) according to the manufacturer's instructions. The SDS-PAGE profile of the active fractions against DU was very similar to that observed with the active fractions from the five-step purification and anion exchange chromatography. The IEF gel showed that proteins fractionate from pH 4.6 to 5.1, consistent with the published pi range for palatin (Racusen and Foote, 1980).

Sequential isoelectric focusing on RF3 in a narrow pH range (pH 4-5) was used in an attempt to determine palatine isozymes. As expected, peak bioactivity was observed for proteins with pi 4.6 - 5.1. These fractions have a distinct pattern of isozymes and different levels of bioactivity. Bioactivity in fractions varied from mortality 0 with * - ** deformations at doses of 80-512 ppm. Some bioactive fractions have only 2 major isozymes, demonstrating that a complex isozyme pattern is not necessary for bioactivity.

Purification of native PAGE

P-3752 was electrophoresed under native conditions and esterase-active triplet bands were isolated (alpha-naphthyl acetate was used as substrate). The gel purified esterase positive material was active against DU and gave 1.5 * deformation. SDS-PAGE of this material showed major bands at M-42000, about 26000 and about 16000, the profile observed previously with other purifications. This is further confirmation that palatin is an insecticidal component of potatoes.

Amino acid sequence

The amino acid sequence of the NH ₂ terminus was obtained for all protein bands (M 42 42000, about 26000 and about 16000) in the DU-active chromatographic fractions from anion exchange and five-step purification chromatography. In total, sequence data of all bands of active fractions were generated. Most bands showed homology over 85% for a sequence of 15 amino acids at either the ΝΗ ₌ end (SEQ ID NO: 1) or internal sequence (SEQ ID NO: 7) of the palatine isozyme (Stiekema et al., 1988). One of the 17 kD bands showed 75% homology with the initial eight amino acids of the published sequence at the konci _χ end of palatine. Another 17 kD band showed homology over 85% with the initial eight amino acids of the published internal sequence. These bands represent the proteolysed products of palatine. The presence of isozymes is clearly indicated by amino acid variability at positions 1 and 3 at both the NH terminus and the internal sequence.

⁹

N-terminal amino acid sequence

Published sequence KLEEMVTVLSIDGGG (SEQ ID NO: 1) Passport 1 (42 Kd) XLGEMVTVLSIDGGG (SEQ ID NO: 2) Passport 2 (28 Kd) TLGEMVTVLSIDGGG (SEQ ID NO: 3) Passport 3 (26 Kd) TLGEMVTVLSIDGGG (SEQ ID NO: 4) Passport 4 (24 Kd) KLXEMVTVLSIDGGG (SEQ ID NO: 5) Passport 5a (17 Kd) XXEEMVTV (SEQ ID NO: 6)

Internal amino acid sequence (amino acid position 224)

Published sequence SLDYKQMLLLSLGTG SEQ ID NO: 7) Passport 5b (17 Kd) SLDYKQML (SEQ ID NO: 8 Passport 6 (16 Kd) SLXYKOMLLLSLGTG (SEQ ID NO: 9) Passport 7 (15 Kd) SLNYKQMLLLSLGTG (SEQ ID NO: 10)

Esterase activity

Numerous experiments have been carried out to test the esterase activity of the active fractions against DU.

Alpha-naphthyl acetate substrate

SDS-PAGE was used to determine whether active fractions against DU (from a five step purification) showed esterase activity (Racusen, 1984). The two halves of the gel were loaded with heated and unheated active fractions against DU. A single esterase-active band was observed in the unheated sample at 55000. The heated sample showed the original 42000 band and the simultaneous absence of 55000 band. This result is consistent with the literature on the electrophoretic mobility of palatine esterase activity (Racusen, 1984). The M5 55000 band was not observed in the heated sample, indicating that heat treatment in SDS eliminates esterase activity. In the absence of 55000 bands, the initially observed 42000 band with coo mass staining was observed.

Study of specificity of p-nitrophenyl substrate

A series of p-nitrophenyl esters (C-2, C-4, C-6, C-8, C-10,

C-12, C-14 and C-16 esters) were assayed to determine substrate specificity. The β-NP C-8 and C-10 esters were consistently the best substrates for the esterase activity of most palatines tested compared to other esters.

Lipid ester substrates

The purified active fraction against DU (from a five-step purification) was tested for its ability to hydrolyze a variety of lipids. Each lipid was dissolved and incubated with a proportion of purified active fraction against DU. Samples were analyzed by TLC using a three-solvent development system (Pernes et al., 1980). Four lipids showed marked modifications in TCL. These were oleoyl lysolethicin, dioleoyl L-alpha-phosphatidylcholine, 1-monolinoleoyl-rac-glycerol and diolein (Sigma). Identified new TLC spot at R _f 0.37 in the organic extract of the reaction mixture, the active lipid fraction in comparison with standards of linoleic and oleic fatty acid. Thus, the anti-DU active material exhibits esterase activity on these four lipid esters. The medial intestines of the third stage of DV larvae fed with corn roots were removed. The median intestinal lipids were extracted, dissolved and incubated at the medial intestinal pH (pH 6.55) with the purified active fraction against DU. Samples were analyzed by TLC using the above method. The purified active fraction against DU exhibited esterase activity on the phosphoripids of the median intestines of DV at the median intestinal pH. This illustrates the possible mode of action for the insecticidal activity of palatine.

Alternative sources of palatine

Since the initial experiments were carried out with P-3752, a commercially available enzyme preparation (Sigma) from Minnesota potato sprouts Russet var. Kranz, it was desirable to demonstrate that insecticidally active palatins can be obtained from fresh potato sprout tissues. Sprout extracts were prepared as described in the literature (Racusen and Foote, 1980, Park et al., 1983). Three commercially available cultivars of S. tuberosum (Russet, Desiree and La Chipper) and seven wild species (S. kurtzianum, S. berthaultii, S. tarijense, S. acaule, S. demissum, S. cardiophyllum and S. raphanofolium were analyzed, all available from Inter-Regional Potato Introduction Station (USDA, ARS, Sturgeon Bay, WI). All extracts were positive for palatin by SDS-PAGE and Western blot tests, all were positive for esterase in the C-10 esterase assay, and all were insecticidally active against DU, for example having a strain ratio of 2-3, see Table 5. This demonstrates that insecticidally active palatins can be isolated from the germ of multiple varieties and that many members in this whole class of proteins should have insecticidal properties.

Table 5

Variety protein delta O.D./min*ml DU " mcr / ml IX 0.1X S. kurtzianum 25.0 2700 2.5 1 S. berthau1tii 23.1 29 3 1 S. tarijense 27.2 1008 2.5 1 S. acaule 26.6 82500 2 1 S. demissum 35.3 375000 3 1.5 S. cardiophyllum 14.6 89 2. 5 1.5 S. raphanofolium 33.7 3725 3 2

activity against DU is expressed in terms of larvae deformation, 1 = slight deformation (30-40% size reduction) 2 = moderate deformation (50-80% size reduction) 3 = severe deformation (over 90% size reduction).

Extracts of S. berthaultii, S. kurtzianum and S. tarijense were bioassayed against two other target insects, LD and ON. Bioassay data are summarized below in Table 6. Little activity was observed in these extracts against LD, but the ON larvae were moderately to severely deformed at the IX dose. However, the ON larvae appear to be slightly less sensitive to these potato extracts than the DU larvae, indicating a complete absence of activity at 0.1X against ON.

Table 6

Variety_LD_ON

IX

0.1X

IX

0.1X

S. berthaultii 00 30

S. kurtzianum 0 0 2.5 o

S. tarijense 00 30 “larvae deformation

Genomic DNA from nine different plants was tested by Southern analysis for proteins homologous to palatine. Southern blots, tested with the alpha- ³² P-labeled sample of SEQ ID NO: 11, showed that there are homologous sequences in many other plant species. Strong signals were obtained in corn, tomatoes, sugar beet, rice and potatoes. In this experiment it was not possible to separate the individual bands, but the size of the spots and their intensity were similar in all these species. Weaker signals were also obtained in courgettes, soybeans and canola and appeared as a small number of discrete bands in DNA of each species. Cucumbers and Arabidopsis do not show detectable hybridization to a sample of palatines under the conditions used in this experiment, possibly for smaller amounts of DNA loaded, as seen in the ethidium bromide spot of the gel.

Those skilled in the art can readily obtain and insert the DNA sequences of these homologous proteins into plants or other organisms by known means. The insecticidal properties of such proteins are best tested by different expression, for example from baculovirus or E. coli. Thus, the proteins that can be used in the methods of the invention can be obtained with a normal number of experiments using known methods, and then used to obtain insect-resistant plants. GENETIC IDENTIFICATION

Palatine genes have been cloned by many researchers. The sequence described by Mignery et al., 1984, was designated GM203. It has an incomplete signal sequence. Migners et al., 1988, have identified a genomic clone, designated PS20, comprising GM203 and containing the complete signal sequence. SEQ ID NO: 11 was constructed with the PS20 signal sequence and cDNA encoding part of GM203, hereinafter referred to as Pat A +. It also contains an NcoI restriction site and an EcoRI restriction site immediately following the translation termination codon.

Solanum tuberosum cultivar Russet Burbank

Twenty cDNAs were isolated from the germ of the Russet Burbank potato cultivar and sequenced. The deduced amino acid sequences indicate that these cDNAs encode eleven different palatine isozymes. These eleven proteins are from about 82 to 100% identical compared to PatA +, SEQ ID NO: 11, with differences occurring at multiple positions in the length of the cDNA. These sequences for eleven different palatine isozymes are labeled as shown in Table 7. cDNAs were constructed by PCR procedures using primers SEQ ID NO: 26 and SEQ ID NO: 27, corresponding to 5 'nucleotides encoding the first few codons of the signal sequence and 3' the end of the coding sequence for further cloning manipulation. The + symbol indicates that a native signal coding sequence is included. Some cDNAs do not contain the full native signal coding sequence and only the adult protein coding sequence was obtained by a similar PCR procedure using primers SEQ ID NO: 32 and SEQ ID NO: 27. These are denoted by subscript.

Table 7

Isozvm_Identification number of the sequence

PatA + SEQ ID NO: 11 Heel xn SEQ ID NO: 14 PatB + SEQ ID NO: 16 PatC + SEQ ID NO: 17 PatD xn SEQ ID NO: 18 PatE + SEQ ID NO: 19 Dec PatE xn SEQ ID NO: 20 May PatF xn SEQ ID NO: 21 PatG + SEQ ID NO: 22nd PatH xn SEQ ID NO: 15 Dec Fifth xn SEQ ID NO: 23 PatL + SEQ ID NO: 24 PatM + SEQ ID NO: 25

Solarium berthaultii

Diploid potato palatine S, berthaultii was isolated by reverse transcription of mDNA germs followed by PCR using primers SEQ ID NO: 26 and SEQ ID NO: 27 as described above. Multiple independent PCR reactions were performed to prevent isolation of duplicated clones due to the amplification process. In total, 14 palatine cDNAs were partially sequenced. All fourteen cDNAs (designated Pat 1 to Pat 14) appear to have a single nucleotide sequence, indicating that at least fourteen different palatine mRNAs are expressed in S. berthaultii germs. The sequence for Pat3 + is SEQ ID NO: 28. The sequence for Pat10 + is SEQ ID NO: 29. The deduced amino acid sequences indicate that the 14 cDNA encodes at least eleven different proteins. In general, cDNA sequences from S. berthaultii sprouts were very similar. Only 12 positions out of a total of 267 residues (3%) showed sequence variability. The amino acid residues present at each of these positions are shown in Table 8. At five of these positions, there was only one variant clone with a unique residue. These changes could reflect the real differences between mRNAs or result from errors made during the PCR process. At all other seven positions there was greater variability, at least two cDNAs had an alternative amino acid. Each of the nine different groups of amino acid sequences had a unique pattern of residues at these seven positions. In some cases, the changes were conservative, such as a change in Thr to Ser at position 164. In other cases, the differences were more dramatic, such as the introduction of proline at position 148.

Table 8 cDNA_Amino acid difference position

89 96 106 113 120 123 148 164 187 200 PAT 3+ GLN LEU GLN TYR GLU WALL ALA ALA THR ASP ASP PAT4 + GLN SER ASP HIS GLU WALL ALA FOR SER ASP WALL PAT5 + GLN SER HIS GLU WALL ALA FOR THR ASP ASP PAT7 + GLN LEU GLN TYR GLU WALL ALA ALA THR ASN ASP PAT8 + LYS SER GLY TYR LYS WALL ALA FOR THR ASP ASP PAT9 + LYS SER ASP TYR LYS WALL ALA FOR THR ASP ASP PAT10 + GLN SER ASP HIS GLU WALL THR FOR THR ASP ASP PAT11 + GLN SER ASP HIS GLU ALA ALA ALA THR ASP ASP PAT12 + GLN SER GLY HIS GLU WALL ALA ALA THR ASP ASP PATA + HIS SER ——— TYR GLU WALL ALA ALA THR GLU ASP

Solanum cardiophvllum

Ten cDNA clones were generated by PCR using mRNA isolated from Solanum cardiophyllum as described above. Nucleotide sequences were obtained at least 75% of the length of each clone. The full length sequence of the clone designated Pat 17+ is SEQ ID NO: 30. SEQ ID NO: 31 is the adult engineered form, Pat 17. S. cardiophyllum clones were almost identical to the only random nucleotide sequence changes that could be actual PCR differences or errors. However, at positions 54 and 519, it was observed that many columns had identical changes, suggesting that they were not due to the amplification process. Nucleotide patterns at these positions indicate that at least four different mRNAs are present. mRNAs from the other two groups were isolated only once in this set of cDNA clones.

The deduced amino acid sequences of S. cardiophyllum clones were extremely similar. There were 8 unique groups of amino acid sequences, each differing from other sequences by only one residue. cDNA clones encoding the amino acid sequence identical to the Pat 17+ sequence were obtained twice and the other seven cDNAs (Pat 18+, 19+, 20+, 21+, 22+, 23+ and 24+) contained a single unique residue.

GENETIC TRANSFORMATION

As discussed above, palatine genes can be isolated from various plant sources. One or more of these genes can be used to transform bacterial or plant cells to become capable of producing palatin and performing the methods of the invention. Examples of how this can be done with different palatine sequences are given below.

Engineering cDNA for palatin

In order to include the palatin gene in vectors suitable for expressing palatine in host cells, it was necessary to introduce suitable restriction sites near the end of the gene. The aim of this mutagenesis was to create cassettes containing a protein coding sequence with minimal non-coding branch sequences and to include useful restriction sites to mobilize these cassettes. Cassettes have been designed to allow the mobilization of an intact coding sequence, including a signal peptide, or only an adult coding sequence. Two mutagenesis primers were designed for PatA _m to generate these cassettes. Mutagenesis with SEQ ID NO: 12 replaced two amino acids (methionine-alanine) with lysine at the N terminus of the adult protein and introduced the NcoI site, and SEQ ID NO: 13 added a second termination codon at the EcoRI site.

The resulting modified sequence was identified as PatA _{m of} SEQ ID NO: 14. Similar modifications were introduced for all other cDNAs, and restriction sites were introduced by PCR and either primers SEQ ID NO: 26 and SEQ ID NO: 27 or primers SEQ ID NO: 32 and SEQ ID NO: 27 as described previously. Expression of palatine in E. coli

The DNA coding sequence for PatA ^ (SEQ ID NO: 14) was inserted into pMON5766, an E. coli expression vector derived from pBR327 (Soberon et al., 1980) with the recA promoter and the G10 leader (Olins et al., 1989). The resulting vector, pMON19714, was mobilized in E. coli strain JM101, which then produced PatA4, as confirmed by Western blot analysis and esterase activity using p-nitrophenyl ester C-10.

DNA coding sequences for both Pat17 _m and PatA _m were each inserted into pMON6235 with the AreBAD promoter (inducible when cells grow in arabinose) and the G10 leader and ampicillin resistance gene. The resulting vectors, pMON25213 containing Pat17 _m and pMON25216 containing PatA _m, were introduced into E. coli strain JM101.

Palatin was expressed in transformed E. coli but is divided into breaking bodies (RB). Intact cells and solubilized RB were used in DU assays. The results are shown in Table 9.

Table 9

Sample_Rep (pMON)

19714 A 1m

25216 A 1m

25213 17 1 per

Intact cells activity against DU ^X

2.5

1.5

1.0

3.0

1.5

Solubilized RB activity against DU ¹ not tested

1.0

1.0

3.0 ²

0.5 ¹ activity against DU is expressed in terms of larvae deformation, 1 = slight deformation (30-40% size reduction) 2 = moderate deformation (50-80% size reduction) 3 = severe deformation (over 90% size reduction).

² mortality in this sample was 81%.

Expression of palatine in plant-settling bacteria

For insect control, it may be desirable to express one or more palatins in the plant-settling bacteria and then apply the bacteria to the plant. When insects feed on the plant, they eat a toxic dose of palatine produced by the plant colonizers. Plant colonizers may be either those populating the surface of the plant, such as Pseudomonas or Agrobacterium species, or endophytes, which populate the vasculature of the invention, such as Clavibacter species. In surface colonizers, the palatin gene can be placed in a wide variety of host vectors capable of replicating these gram-negative hosts. Examples of such vectors are pKT231 from the IncQ intolerance group (Bagdasarian et al., 1981) or pVK100 from the Incp intolerance group (Knauf, 1982). In endophytes, the palatin gene can be placed in the chromosome by homologous recombination or by gene binding to a suitable transposon capable of chromosome insertion in these endophyte bacteria.

Expression of palatine in Baculovirus

The palatine genes were cloned into the baculovirus donor vector pMON14327, described in copending U.S. patent application Ser. No. 07 / 941,363, filed Sep. 4, 1992, which is incorporated herein by reference, as NcoI / EcoRI fragments. The donor vector pMON14327 contains the ampicillin resistance gene, the left and right branches of the Tn7 transposon, and between these branches the gentamicin resistance gene, the strong baculovirus polyhedrin promoter and the polylinker. The baculovirus shuttle vector or bacmid consists of a mini-attTn7 site in frame with the lacZ gene and the kanamicin resistance gene recombined into the AcNPV viral genome. Recombinant AcNPV virus was produced using the helper plasmid pMON7124 resistant to tetracycline by transposing the palatine or GUS genes and marker genes into the viral genome (Luckow et al., 1993). The following genes were inserted into pMON14327: the genes listed in Table 7 together with Pat3 +,

Pat10 + and Pat17 +.

According to the procedures of US Patent Application Serial No. 07 / 941,363 and Luckow et al., The above-mentioned amounts of palatine were produced from the genes. The presence of palatine was confirmed by Western blot analysis and esterase activity using p-nitrophenyl ester C-10. With the exception of Pat A, each isozyme mnom lived at least twice for DU. While Pat E + and Pat E _m fermentations consistently showed little or no palatin, all other isozymes appeared to be expressed in amounts acceptable for bioassays. However, the nature of the post-translational processing of palatine proteins in baculovirus, unlike potatoes, has not been established. The bioactivity of baculovirus-expressed isozymes against DU was observed in Pat17 +, PatB +, PatD +, Pat I, PatL + and Pat3 +.

xn.

The effects of many isozymes (produced by baculovirus) on insect growth and development were determined. Aliquots of eleven Russet isozymes were combined into one sample for bioassays against DU, ON, AI and HV. Ten to fifteen mg of each isozyme purified on Q-Sepharose was combined with the exception of PatD ®, which was only 1.7 mg. This mixture resulted in 100% mortality in the HV tests and a 93% weight loss in ON. Then each isozyme was tested separately against HV and ON. Compared to the control larvae block, the HV and ON larvae fed a PatC +, PatL +, and Fifth Isozyme diet showed significant deformations (over 75%) xn and / or mortality. PatB + and PatD also deformed HV to 69 xn and 78%.

Plant gene construction

Expression of a plant gene existing in the double form of DNA requires transcription of messenger RNA (mRNA) from a single DNA helix by an RNA polymerase enzyme and subsequent processing of the primary mRNA transcript into the nucleus. This processing requires a 3 'untranslated region that adds polyadenylate nucleotides to the 3' end of the RNA strand. Transcription of DNA into mRNA is regulated by a region of DNA commonly referred to as a promoter. This promoter region contains a base sequence that signals RNA polymerase to attach to DNA and initiate transcription of the mRNA using one of the DNA helices as a form to form the corresponding RNA band.

A number of promoters that are active in plant cells have been described in the literature. Such promoters have been obtained from plants or plant viruses and include, but are not limited to, nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are transmitted to tumors acting on Agrobacterium tumefaciens plasmids, 19S and 35S mosaic cauliflower virus (CaMV) promoters. ), a light-induced promoter of a small portion of ribulose 1,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the 35S mosaic Figwort virus (FMV) promoter, all of which were also used to create various types of DNA constructs It could also be desirable to limit expression to certain parts of plants that are susceptible to insect attack. For example, a root-specific promoter can be used to limit expression to roots or a root-enhancing promoter can be used to levels of active proteins in the roots, which is preferred in insect-eating roots.

Certain plant promoters are also effective in monocotyledons. For example, the actin rice promoter described in WO 91/09948 is effective for expression in maize. The ubiquitin corn promoter described in EP 0 342 926 can also be used in monocotyledons.

Promoters used in DNA constructs (e.g., chimeric plant genes) of the invention can be modified if desired to affect their control characteristics. For example, the CaMV35S promoter can be linked to a portion of the ssRUBISCO gene that suppresses the expression of ssRUBISCO in the absence of light to produce a promoter that is active in leaves but not in the roots. The resulting chimeric promoter can be used as described herein.

Thus, for the purpose of this description, the phrase CaMV35S 'promoter includes variations of the CaMV35S promoter, for example, promoters obtained by binding to operator regions, random and controlled mutagenesis, etc. Promoters can be altered to include multiple enhancer sequences to promote increased gene expression. Examples of such enhancement sequences have been reported by Kay et al., (1987).

The selected promoter should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective amount of palatins. The preferred promoter is the CaMV 35S promoter (enhanced CaMV35S).

The DNA produced by the DNA construct of the invention may also comprise a 5 'untranslated leader region. This sequence can be derived from a promoter selected to express the gene and can be specifically modified to increase translation of mRNA. The 5 'untranslated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from synthetic gene sequences. The present invention is not limited to constructions wherein the untranslated region is derived from the 5 'untranslated region that accompanies the promoter sequence.

As noted above, the 3 'untranslated region of the chimeric plant genes of the invention comprises a polyadenylation signal whose function in plants is to effect the addition of adenyl nucleotides to the 3' end of RNA. Examples of preferred 3 'regions are (1) 3' transcribed, untranslated regions containing the polyadenylation signal of Agrobacterium tumor-acting plasmids (Ti) such as the nopaline synthase (NOS) gene, and (2) plant genes such as soybean storage protein genes and bean ssRUBISCO E9 gene (Fischhoff et al.).

Localization

The vectors containing the palatine cassettes described above express the active protein in the cytoplasm or vacuoles of the plant cell. It may be desirable to introduce most or all of the palatine into the secretory pathways of the plant. To achieve this, it may be advantageous to use a signal sequence formed from a bacterial or plant gene, but it is contemplated that a plant gene will be preferred. Examples of such signal sequences are sequences from the endoproteinase B gene (Koehler and Ho) and the tobacco PR1b gene (Cornelissen et al.) PMON10824, described in EP publication 0 385 962, is a plant transformation vector designed to express active B. t. Kurstaki protein (BTK). ) of lepidopteran. In pMON10824, the coding sequence is linked to the PR1b signal sequence plus 10 amino acids from the adult PR1b signal sequence. To produce a vector in which the PR1b signal is linked to the palatin gene, pMON10824 is cut with BglII and NcoI and a small fragment of BglII-NcoI containing the PRlb signal. In the coupling reaction, a small BglII-NcoI fragment of pMON10824 was mixed with the 1.0 kb NcoI-EcoRI fragment of pMON19714 and BamHIEcoRI eaten from pMON19470 (Brown et al.). This reaction constructs a plasmid in which the palatin coding sequence is linked to a secretion signal from the PR1b gene and driven by the CaMV35S promoter and intron from monocotyledon expression.

A similar reaction can be performed to express the dicotyledon gene. The NotI-NotI fragment of a dicotyledonous expression vector can be inserted into a dicotyledonous transformation vector as described below and mobilized into a disarmed Agrobacterium host and used to transform dicotyledons. Thus, plants that produce palatin can be made, which is secreted into the intercellular space.

The NotI-NotI fragment of this monocotyledon plasmid can be inserted into a maize transformation vector (such as pMON 18181 described above) to produce a maize plant that secretes palatin.

It may be advantageous to target the localization of palatine to another cell formation, the chloroplast. Proteins can be directed into the chloroplast at their N terminus by a chloroplast transit peptide (CTP). One CTP that has been involved in localizing foreign proteins to the chloroplast is derived from the small subunit of the RUBISCO gene from Arabidopsis, designated atslA. A variation of this transit peptide that encodes a transit peptide, a 23 amino acid adult RUBISCO sequence plus reiteration of the transit peptide cleavage site was constructed to successfully localize the BTK protein to the chloroplast. pMON19643 (described by Brown et al.) contains the atslA Arabidopsis transit peptide linked to the GOX gene and can be used as a basis for constructing a vector for localizing palatine to the chloroplast. Complete uptake of EcoRI and partial uptake of NcoI from pMON19643 was performed and a large fragment (4.0 kb) was isolated. In the coupling reaction, the NcoI-EcoRI fragment of pMON19714 is mixed with the large fragment of pMON 19643. This reaction constructs a plasmid in which the palatine coding sequence is linked to an Arabidopsis transit peptide of 23 amino acids of adult RUBISCO and driven by the CaMV35S promoter. Alternatively, a similar plasmid can be constructed to replace the FMV35S promoter. Such plasmids are mobilized into a disarmed Agrobacterium host and used to transform dicotyledons. Alternatively, the NotI Not I fragment is cloned into a maize transformation vector as described above. Thus, plants that produce palatin can be made to localize to the chloroplast.

Transformation and expression in the plant

A chimeric plant gene comprising a structural coding sequence of the invention can be inserted into the plant genome by any suitable method. Suitable transformation vectors include those derived from the Ti plasmid of Agrobacterium tour faciens as well as those described, for example, by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO publication 0 120 516 (Schilperoort et al.). In addition, alternative methods for introducing DNA constructs of the invention into plant cells can be used in plant transformation vectors derived from Ti or introducing into the roots (Ri) of Agrobacterium plasmids. Such methods may include, for example, the use of liposomes, electroporation, chemicals that enhance DNA uptake, free delivery of DNA by microprojectile bombardment, and transformation by viruses or pollen.

Transient expression of palatine in tobacco cells

A particularly useful plasmid cassette vector for transformation of dicotyledonous plants is pMON11794. The expression cassette pMON 11794 consists of the CaMV35S promoter, the 5 'untranslated leader of Hsp70 from petunia, and the 3' end comprising polyadenylate signals from the NOS gene. pMON11794 contains the NcoI-EcoRI sites for insertion of the coding sequence and the NotI-NotI sites flanking the gene expression cassette.

PatA + (SEQ ID NO: 11), PatB + (SEQ ID NO: 16), PatC + (SEQ ID NO: 17) and PatG + (SEQ ID NO: 22) were all inserted into pMON11794 to produce pM0N19745, pMON19742, pMON19743 and pMON19744. Each of these vectors was electroporated into tobacco protoplasts. Expression of palatine by transformed tobacco cells was confirmed by Western blot analysis. Permanent transformation of dicotyledons

Permanent transformation of the dicotyledonous gene with palatine was reported by Rosahl et al. Palatin commented.

The NcoI-EcoRI fragment from pMON19745 was inserted into pMON 17227, the Ti plasmid vector described in Barry et al., WO 92/04449, which is incorporated herein by reference to produce pMON22566. This vector contains the glyphosate resistance gene described by Barry for selection of transformed plants. Similarly, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 22 were used to produce the vectors pMON22563, pMON22564 and pMON

22565.

These vectors were introduced into disarmed Agrobacterium ABI and used to transform tomato plants into tissue culture. After selection for glyphosate resistance and plant regeneration, whole plants expressing the palatine gene were obtained. Expression of the palatine gene was confirmed by Western blot analysis and preliminary results show expression at a level of about 0.1-0.5% of total protein. Bioassays with insect larvae are in progress.

Transient expression of palatine in maize cells kb The NcoI-EcoRI fragment of pMON19729, described above, was inserted into pMON 19433, which is disclosed in WO 93/19189 and copending U.S. patent application Ser. No. 07 / 855,857, filed March 19, 1992 (Brown et al.). .), which is incorporated herein by reference. The resulting plasmid, pMON19731, was digested with NotI, and the resulting fragment was inserted into pMON 10081, as also described by Brown et al., Giving pMON19740. This plasmid was electroporated into maize leaf protoplasts as described by Sheen, 1991. Expression of palatine by transformed maize protoplasts was confirmed by Western blot analysis.

To achieve cytoplasmic expression of palatine, the NcoI-EcoRI fragment from pMON19714 was inserted into pMON 19433, yielding pMON19730. The NotI fragment from pMON19730 was inserted into pMON 10081 and the resulting plasmid was electroporated into protoplasts of maize leaves that produced palatin, as confirmed by Western blot analysis.

Pat17, with or without target signals, can also be expressed in maize protoplasts. PMON19761 was constructed by inserting a 1.1 kb NcoI-EcoRI fragment (SEQ ID NO: 30) encoding the Pat177 protein (adult Pat17 protein and its own vacuole targeting signal sequence) into pMON19648. pMON19761 contains the CaMV E35S promoter, the Hsp 70 intron, the Pat17 + gene, and the NOS terminator for expression in maize cells.

To obtain a vector for cytoplasmic expression, Pat17 + in pMON19761 was replaced with the NcoI-EcoRI fragment encoding the Pat17 protein (SEQ ID NO: 31) of pMON25213, giving

IQ.

pMON25223.

pMON25224 was made by inserting two fragments, a 0.3 kb XbaI-NcoI fragment containing the chloroplast transit peptide (CTP) from Arabidopsis thaliana la gene (Timko et al.) from pMON19643 (Brown et al.) and a 1 kb NcoI-EcoRI fragment Pat17 _m of pMON25213, inserted to pMON19761 (XbaI-Ncol). Thus, pMON 25224 contains the CaMV E35S promoter, the Hsp 70 intron, the CPT / Pat 17 coding sequence, and the NOS terminator.

For expression in the extracellular targets, the 5 'end of the endoproteinase B cDNA (Koehler and Ho) encoding the extracellular signal peptide of the secreted protein was joined to the Pat17 gene of pMON25213. The BglII-EcoRI fragment containing the chimeric gene was made by the overlap extension digestion technique (Horton et al.) And inserted into pMON19761 (BamHI-EcoRI), giving pMON25225. All of these constructs were electroporated into protoplasts of maize leaves and expression Patl7 _m confirmed by Western blot analysis.

Permanent transformation of maize with the palatine gene

The maize transformation vector pM0N18181 was constructed from pMON19653 and pMON19643 (Brown et al.). This construct contains the CaMV E35S promoter cassette, Hsp 70 intron, glyphosate selection marker and NOS terminator, CaMV E35S promoter cassette, Hsp 70 intron, GOX glyphosate selection marker and NOS terminator, and a single NotI insertion cassette for gene expression containing the palatine gene. SEQ ID NO: 11 and SEQ ID NO: 30 were both inserted as NotI-NotI fragments into pMON 18181, resulting in PMON19746 and pMON19764.

These vectors were introduced by bombardment of embryonic cells in tissue culture using the biolistic particle cannon described in Brown et al. Transformed cells were selected for glyphosate resistance and whole plants were regenerated. Plant resistance is confirmed by expression of a gene of about 0.1-0.5% of total protein by Western blot analysis, esterase activity assay, or insect resistance assays.

Synthetic genes for better expression in monocotyledons

Modification of the coding sequences has been shown to improve expression of other insecticidal protein genes, such as the Bacillus thuringiensis delta endotoxin sequence (Fischhoff and Perlak, WO 93/07278, Ciba-Geigy). Thus, a modified coding sequence has been proposed to improve the expression of palatine in plants, especially in maize. The modified Pat + sequence is shown as SEQ ID NO: 33. The DNA fragment containing SEQ ID NO: 33 will be synthesized and inserted into a maize expression cassette vector such as pMON19470 (Brown et al.) The maize expression cassette is then inserted. into pMON18181 or another maize plant transformation vector containing an optional maize transformation marker gene to obtain whole plants expressing Pat177 +.

It will be apparent from the foregoing that the present invention will be well adapted to achieve all the objects of the objects outlined above, together with the advantages which are obvious and inherent in the invention. It is understood that certain features and subcombinations are useful and can be used without reference to other features and subcombinations. This is intended by the invention and is within the scope of the invention. Since many embodiments of the invention are possible without departing from the scope thereof, it is to be understood that everything is to be interpreted in an illustrative and not restrictive sense.

All publications and patents mentioned in this specification are incorporated herein by reference, as if each individual publication or patent were specifically and individually named and incorporated by reference.

REFERENCE

Andrews et al., Biochem. J., 252: 199-206 (1988).

Bagdasarian et al., Gene, 16: 237-41, 1981.

Barry et al., WO 92/04449.

Bevan M. et al., Nature, 304: 184, 1983.

D. Boulter, A. M. R. Gatehouse, V. Hilder, Biotech. Adv., 7: 489 (1989).

Broadway and Duffey, J. Insect. Physiol., 23: 827,1986.

Brown et al., WO 93/19189 and copending U.S. patent application Ser. No. 07 / 855,857, filed March 19, 1992.

Cornelissen BJC et al., EMBO Journal, 5: 37-40, 1986.

Czapla and Lang, J. Econ. Entomol., 83: 2480 (1990).

Fischhoff D.A., Perlak F.J., European Patent Application No. 0 385 962, 1990.

Fischhoff D.A., Bio / Technol. 5: 807 (1987).

Gaillaird, T., Biochem. J., 121: 379-390 (1971).

Ganal et al., Mol. Gene. Genetics, 225: 501-509 (1991).

Gatehouse et al., J. Sci. Agric. 37: 727 (1986).

Herrera-Estrella L. et al., Nature, 303: 209, 1983.

Heusing et al., Plant Physiol. 96: 993 (1991).

Hofgen R., Willmitzer L., Plant Science, 66: 221-230, 1990. Horton et al., Gene, 77: 61-68, 1989.

Ishimoto and K. Kitamura, Appl. Ent. Zool., 24: 281,1989.

Kay R. et al., Science, 236: 1299-1302, 1987.

Klee H. J. et al., Bio / Technol. 3: 637-642 (1985).

Knauf, V. C., Nester E., Plasmid, 8: 43-54, 1982.

Koehler S., Ho T.-H. D., Plant Cell, 2: 769-783, 1990.

Luckow, V.A. et al., J. Virol., 67: 4566-4579, 1993.

Marrone P. G. et al.,

J. Econ. Entomol., 78: 290-3, 1985.

Mignery et al., Nucleic Acids Research, 12: 7987-8000, 1984. Mignery et al., Gene, 62: 27-44, 1988.

Murdock et al., Phytochemistry 29: 85, 1990.

Olins P. et al., J. Biol. Chem., 264: 16973-16976 (1989).

Park W. D. et al., Plant Physiol. 71: 156-160,1983.

Pernes, J. F. et al., J. Chromatography, 181: 254-258, 1980 Racusen, D. Can. J. Bot. 62: 1640-1644 1984. Racusen, D. Can. J. BOt. 64: 2104-2106 1986.

Racusen and Foote, J. Food Biochem., 4: 43-52, 1980.

Rodis P., Hoff J. E., Plant Physiol. 96: 993,1983.

Rosahl et al., EMBO Journal, 6 (5): 1155-1159, 1987.

Ryan, Ann. Roar. Phytopath, 28: 425-449 (1990).

Schilperoort et al., European Patent Application No. 0 120 516.

Sheen Jen, Plant Cell, 3: 225-245 (1991).

Schukle and Murdock, Environ. Ent. 12: 787,1983.

Soberon et al., Gene, 9: 287-305, 1980.

Stiekema et al., Plant Mol. Biol., 11: 255-269 (1988).

Timko et al., The Impact of Chemistry on Technology, ACS Books, 1988, 279-295.

Vancanneyt et al., Plant Cell, 1: 533-540, 1989.

List of strings

SEQ ID NO: 1

Characteristics of the chain:

i) A) Length: 15 amino acids B) Type: amino acid D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 1

10 15

Lys-Leu-Glu-Met-Val-Thr-Val-Leu-Ser-Ile-Asp-Gly-Gly-Gly

The string information of SEQ ID NO: 2

Characteristics of the chain:

i) A) Length: 15 amino acids B) Type: amino acid D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 2

10 15

Xaa-Le-Gly-Glu-Met-Val-Thr-Val-Le-Ser-Ile-Asp-Gly-Gly-Gly

The string information of SEQ ID NO: 3

Characteristics of the chain:

i) A) Length: 15 amino acids B) Type: amino acid D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 3

10 15

Thr-Leu-Gly-Glu-Met-Val-Thr-Val-Leu-Ser-Ile-Asp-Gly-Gly-Gly

The string information of SEQ ID NO: 4

Characteristics of the chain:

i) A) Length: 15 amino acids B) Type: amino acid D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 4

10 15

Thr-Leu-Gly-Glu-Met-Val-Thr-Val-Leu-Ser-Ile-Asp-Gly-Gly-Gly

The string information of SEQ ID NO: 5

Characteristics of the chain:

i) A) Length: 15 amino acids B) Type: amino acid D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 5

10 15

Lys-Leu-Xaa-Glu-Met-Val-Thr-Val-Le-Ser-Ile-Asp-Gly-Gly-Gly

Chain Information SEQ ID NO: 6 Chain Characteristics: i) A) Length: 8 amino acids

B) Type: amino acid

D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 615

Xaa-Xaa-Glu-Met-Val-Thr-Val

The string information of SEQ ID NO: 7

Characteristics of the chain:

i) A) Length: 15 amino acids

B) Type: amino acid

D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 7

10 15

Ser-leu-asp-tyr-lys-gln-met-leu-leu-leu-ser-leu-gly-thr-gly

Chain Information SEQ ID NO: 8 Chain Characteristics: i) A) Length: 8 amino acids

B) Type: amino acid

D) Topology: linear (ii) Type of molecule: peptide

- 41 (xi) Chain Description: SEQ ID NO: 8 1 5

Lys-Leu-Asp-Tyr-Lys-Gln-Met-Leu

Chain Information SEQ ID NO: 8 Chain Characteristics: i) A) Length: 15 amino acids

B) Type: amino acid

D) Topology: linear (ii) Type of molecule: peptide (xi) Chain description: SEQ ID NO: 8

10 15

Ser-leu-xaa-tyr-lys-gln-met-leu-leu-leu-ser-leu-gly-thr-gly

Chain Information SEQ ID NO: 9 Chain Characteristics: i) A) Length: 15 amino acids

B) Type: amino acid

D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 9

10 15

Ser-leu-xaa-tyr-lys-gln-met-leu-leu-leu-ser-leu-gly-thr-gly

Chain Information SEQ ID NO: 10 Chain Characteristics: i) A) Length: 15 amino acids

B) Type: amino acid

D) Topology: linear (ii) Molecule type: peptide (xi) Chain description: SEQ ID NO: 10

10 15

Ser-leu-asn-tyr-lys-gln-met-leu-leu-leu-ser-leu-gly-thr-gly

Asn-Ser-Tyr-Arg-Lys-Val-Leu

CCATGGCAAC TACTAAATCT TTTTTAATTT tattttttat GATATTAGCA ACTACTAGTT 60 CAACATGTGC TAAGTŤGGAA GAAÁTGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 gtttattgac TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360 TTATTGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540 TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATTACTTTA 600 TTACTCATAC TAGTAATGGT GATATATATG AGTTCAATCT TGTTGATGGT GGTGTTGCTA 660 CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCACA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TGATAGAATT C

1171

The string information of SEQ ID NO: 11

Characteristics of the chain:

i) A) Length: 1171 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 11

Chain Information SEQ ID NO: 12 Chain Characteristics: i) A) Length: 37 pairs

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: DNA (synthetic) (xi) Chain description: SEQ ID NO: 12 CATGTGCTCT AGAAGATCTC CACCATGGCG TTGGAAG

Chain Information SEQ ID NO: 13 Chain Characteristics: i) A) Length: 26 pairs

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: DNA (synthetic) (xi) Chain description: SEQ ID NO: 13

GCTTCTTTT GTGTTC GGTC

The string information of SEQ ID NO: 14

Characteristics of the chain:

i) A) Length: 1105 base pairs B) Type: nucleic acid (C) Coil: single D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 14

CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60 TCATTCCAGC TATCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120 AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180 TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 240 TTGTACCCTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTATTG 300 GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360 GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420 CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480 ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATTAC TTTATTACTC 540 ATACTAGTAA TGGTGATATA TATGAGTTCA ATCTTGTTGA TGGTGGTGTT GCTACTGTTG 600 GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660 TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720 CTAATTCAGA GTTTGATAAA ACATATACAG CACAAGAGGC AGCTAAATGG GGTCCTCTAC 780 GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840 ACATTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900 ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960 TAGTACAAGT TGGTGAAACA TTATTGAAGA AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020 ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080

ACAAAGCTTC TTATTGATAG AATTC

1105

The string information of SEQ ID NO: 15

Characteristics of the chain:

i) A) Length: 1105 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 15

CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60

TCATTCCAGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120

AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180

TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGGTGCT GCCAAAGATA 240

TTGTACCCTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTATTG 300

GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360

GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420

CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480

ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATTAC TTTATTACTC 540

ATACTAGTAA TGGTGATATA TATGAGTTCA ATCTTGTTGA TGGTGGTGTT GCTACTGTTG 600

GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660

TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720

CTAATTCAGA GTTTGATAAA ACATATACAG CACAAGAGGC AGCTAAATGG GGTCCTCATC 780

GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840

ACATTTCTAC TGTTTTTCAA GCTGGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900

ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960

TAGTACAAGT TGGTGAAAAA TTATTGAAGG AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020

CTGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGAAAGAAA CTCCGAGCAA 1080

ACAAAGCTTC TTATTAATGA GAATTC

1106

The string information of SEQ ID NO: 16

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 16

CCATGGCAAC TACTAAATCT GTTTTAGTTT TATTTTTTAT GATATTAGCA ACTACTAGIT 60 CAACATGTGC TACGTTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGÁAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 AAGATATTGT ACCTTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTCT AGTGGTTCAA 360 TTTTTGGCCC AATGTATGAT GGAAAATATT TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540 TGAATGACAT ATGCTATTCC ACAGCAGCAG CTCCAACATA TTTTCCTCCA CATTACTTTG 600 TTACTCATAC TAGTAATGGA GATAAATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660 CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTCGAAC GAAACTTGCA CAAGTGGATC 720 CAAAATTTGC TTCAATTAAG TCATTGAATT ACAACGAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATATTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACCT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GAAAAATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

The string information of SEQ ID NO: 17

Characteristics of the chain:

i) A) Length: 1175 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 17

CCATGGCAAC TACTAAATCT TTTTTAATTT TAATTGTTAT GATATTAGCA ACTACTAGTT 60 CAACATTTGC TTCGTTGGAA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGGTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG AAAATGGACA 180 ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 GTTTATTGAC TTCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 ATGAAATTGT ACCTTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTCT AGGTACTGGC 360 CAATTTTTTG GCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAACCTTG 420 GAGAAACTCG TGTGCATCAA GCTTTGACTG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480 CAAATAAGCC AGTAATATTC ACCAAGTCAA ATTTAGCAAA GTCTCCAGAA TTGGATGCTA 540 AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600 TTACTACTAA TACTATTAAT GGAGATAAAT ATGAGTTCAA TCTTGTTGAT GGTGCTGTTG 660 CTACTGTTGC TGATCCGGCG TTATTATCCA TTAGCGTTGC AACGAGACTT GCAGAAAAGG 720 ATCCAGCATT TGCTTCAATT AGGTCATTGA ATTACAAAAA AATGTTGTTG CTCTCATTAG 780 GCACTGGCAC TACTTCAGAG TTTGATAAAA CATATACAGC AGAAGAGACA GCTAAATGGG 840 GTGCTATACA ATGGATGTTG GTTATACAGC GAATGACTGA TGCAGCAAGT TCTTACATGA 900 CTGATTATTA CCTTTCTACT GTTTTTCAAG CTCAAAATTC ACAAAAGAAT TACCTCAGGG 960 TTCAAGAAAA TGCGTTAACA GGCACAACTA CTGAAATGGA TGATGCTTCT GAGGCTAATA 1020 TGGAATCATT AGTACAAGTT GGTGAAAATT TATTGAAGAA ACCAGTTCCC AAAGACAATC 1080 CTGAAACCTA TGAGGAAGCT CTAAAGAGGT TTGCAAAATT GCTTTCTGAT AGGAAGAAAC 1140

TTCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC

1175

SEQ ID NO: 18

Characteristics of the chain:

i) A) Length: 1106 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 18

CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60

TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120

AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180

TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 240

TTGTACCTTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTCTAGTGGT TCAATTTTTG 300

GCCCAATGTA TGATGGAAAA TATTTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC. 360

GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420

CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480

ACATATGTTA TTCCACAGCA GCAGCTCCAA CATATTTTCC TCCACATTAC TTTGTTACTC 540

ATACTAGTAA TGGAGATAAA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GCTACTGTTG 600

GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAAACT TGCACAAGTG GATCCAAAAT 660

TTGCTTCAAT TAAGTCATTG AATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720

CTAATTCAGA GTTTGATAAA ACATATACAG CAGAAGAGGC AGCTAAATGG GGTCCTCTAC 780

GATGGATATT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840

ACCTTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900

ATGCATTAAC AGGCATAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960

TAGTACAAGT TGGTGAAAAA TTATTGAAGA AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020

ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080

ACAAAGCTTC TTATTAATGA GAATTC

1106

SEQ ID NO: 19

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 19

CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 gtttattgac TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCTTTT GCTGCTGCTA 300 AAGATATTAT ACCTTTTTAC TTCGAACACG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360 TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540 TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600 TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660 CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 aattattagt ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTCTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

The string information of SEQ ID NO: 20

Characteristics of the chain:

i) A) Length: 1106 pairs of bases B) Type: nucleic acid (C) Coil: single D) Topology: linear

(ii) Type of molecule: CDNA (xi) String description SEQ ID NO: 20 CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60 TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATG 120 CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAGGTACA GGAGGTTTAT 180 TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC TTTTGCTGCT GCTAAAGATA 240 TTATACCTTT TTACTTCGAA CACGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTTTAG 300 GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360 GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420 CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480 ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATCAC TTTGTTACTC ' 540 ATACTAGTAA TGGTGCTAGA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GCTACTGTTG 600 GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660 TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720 CTAATTCAGA GTTTGATAAA ACATATACAG CAGAAGAGGC AGCTAAATGG GGTCCTCTAC 780 GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840 ACATTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900 ATGCATTAAA TGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960 TAGTACAAGT TGGTGAAACA TTATTGAAGA AACCAGTCTC CAAAGACAGT CCTGAAACCT 1020 ATGAGGAAGC TCTAAAGAGA TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080

ACAAAGCTTC TTATTAATGA GAATTC

1106

The string information of SEQ ID NO: 21

Characteristics of the chain:

i) A) Length: 1109 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 21

CCATGGCGTT GGAAGAAATG GTGGCTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60 TCATTCCGGG TACCATTCTC GAATTTCTTG AAGGACAACT TCAGAAAATG GACAATAATG 120 CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180 TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAATGAAA 240 TTGTACCTTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTCTAGGTAC TGGCCAATTT 300 TTTGGCCAAA ATATGATGGA AAATATCTTA TGCAAGTTCT TCAAGAAAAA CTTGGAGAAA 360 CTCGTGTGCA TCAAGCTTTG ACAGAAGTTG CCATCTCAAG CTTTGACATC AAAACAAATA 420 AGCCAGTAAT ATTCACTAAG TCAAATTTGG CAAAGTCTCC AGAATTGGAT GCTAAGACGT 480 ATGACATATG TTATTCGACA GCAGCAGCTC CAACATATTT TCCTCCACAT TACTTTGCTA 540 CTAATACTAT TAATGGAGAT AAATATGAGT TCAATCTTGT TGATGGTGCT GTTGCTACTG 600 TTGCTGATCČ GGCGTTATTA TCCGTTAGCG TTGCAACGAG ACGTGCACAA GAGGATCCAG - 660 CATTTGCTTC AATTAGGTCA TTGAATTACA AAAAAATGTT GTTGCTCTCA TTAGGCACTG 720 GCACTACTTC AGAGTTTGAT AAAACACATA CAGCAGAAGA GACAGCTAAA TGGGGTGCTC 780 TACAATGGAT GTTGGTTATA CAGCAAATGA CTGAGGCAGC AAGTTCTTAC ATGACTGATT 840 ATTACCTTTC TACTGTTTTT CAAGATCTTC ATTCACAAAA CAATTACCTC AGGGTTCAAG 900 AAAATGCATT AACAGGCACA ACTACTAAAG CGGATGATGC TTCTGAGGCT AATATGGAAT 960 TATTAGCACA AGTTGGTGAA AATTTATTGA AGAAACCAGT TTCCAAAGAC aatcctgaaa 1020 CCTATGAGGA AGCTCTAAAG AGGTTTGCAA AATTGCTTTC TGATAGGAAG AAACTTCGAG 1080

CAAACAAAGC TTCTTATTAA TGAGAATTC

1109

The string information of SEQ ID NO: 22

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) winding: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 22

CCA.TGGCAAC TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60 CAACATGTGC TAAGTTGGAA GAAATGGTTA CTGTTCTAAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360 TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540 TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600 TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660 CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT gttgttgctc TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

SEQ ID NO: 23

Characteristics of the chain:

i) A) Length: 1104 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 23

CCATGGTTGG AAGAAATGGT GACTGTTCTA AGTATTGATG GAGGTGGAAT TAAGGGAATC 60 ATTCCAGCTA TCATTCTCGA atttcttgaa GGACAACTTC AGGAAGTGGA CAATAATAAA 120 GATGCAAGAC TTGCAGATTA CTTTGATGTA ATTGGAGGAA t CAAGTACAGG AGGTTTATTG 180 ACTGCTATGA TAACTACTCC AAATGAAAAC AATCGACCCT TTGCTGCTGC CAAAGATATT 240 GTACCCTTTT ACTTCGAACA TGGCCCTCAT ATTTTTAATT ATAGTGGTTC AATTTTAGGC 300 CCAATGTATG ATGGAAAATA TCTTCTGCAA GTTCTTCAAG AAAAACTTGG AGAAACTCGT 360 GTGCATCAAG CTTTGACGGA AGTTGCCATC TCAAGCTTTG ACATCAAAAC AAATAAGCCA 420 GTAATATTCA CTAAGTCAAA TTTAGCAAAG TCTCCAGAAT TGGATGCTAA GATGTATGAC 480 ATATGCTATT CCACAGCAGC AGCTCCAATA TATTTTCCTC CACATCACTT TGTTACTCAT 540 ACTAGTAATG GTGCTAGATA TGAGTTCAAT CTTGTTGATG GTGCTGTTGC TACTGTTGGT 600 GATCCGGCGT TATTATCCCT TAGCGTTGCA ACGAGACTTG CACAAGAGGA TCCAGCATTT 660 TCTTCAATTA agtcattgga TTACAAGCAA ATGTTGTTGC TCTCATTAGG CACTGGCACT 720 AATTCAGAGT TTGATAAAAC ATATACAGCA GAAGAGGCAG CTAAATGGGG TCCTCTACGA 780 tggatgttag. CTATACAGCA AATGACTAAT GCAGCAAGTT TTTACATGAC TGATTATTAC , 840 ATTTCTACTG TTTTTCAAGC TCGTCATTCA CAAAACAATT ACCTCAGGGT TCAAGAAAAT 900 GCATTAAATG GCACAACTAC TGAAATGGAT GATGCGTCTG AGGCTAATAT GGAATTATTA 960 GTACAAGTTG GTGAAACATT ATTGAAGAAA CCAGTTTCCA GAGACAGTCC TGAAACCTAT 1020 GAGGAAGCTC TAAAGAGATT TGCAAAATTG CTCTCTGATA GGAAGAAACT CCGAGCAAAC 1080

AAAGCTTCTT ATTAATGAGA ATTC

1104

The string information of SEQ ID NO: 24

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 24

CCATGGCAAC TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60 CAACATGTGC TAAGTTGGAA GAAATGGTTA CTGTTCTAAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 gtttattgac TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360 TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540 TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600 TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660 CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840 CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900 ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GCAACATTAT TGAAGAAACC AGTCTCCAAA GACAGTCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1X72

The string information of SEQ ID NO: 25

Characteristics of the chain:

i) A) Length: 1175 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: Linear (ii) Molecule Type: cDNA (xi) Chain Description: SEQ ID NO: 25

CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCTTTT GCTGCTGCTA 300 AAGATATTAT acctttttac TTCGATCATG GCCCTAAGAT TTTTGAACCT agtgcttttc 360 ACCTTTTTGA GCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAAACTTG 420 GAGAAACTCG TGTGCATCAA GCTTTGACAG GAGTTGCCAT CTCAAGCTTT GACATCAAAA 480 CAAATAAGCC AGTAATATTC ACTAAGTCAA GTTTAGCAAA AACTCCAGAA TTGGATGCTA 540 AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600 TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACCTCAA TCTTGTTGAT GGCGATGTTG 660 CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTGC TTCAATTAAG TCATTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAA TTCAGAGTTT GCTAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840 TTCTACAATG GGTATTCTCA CCTTTATGGG AAATGAGAAG TGCAGCAAGT TCTTACATGA 900 ATGATTATTA CCTTTCTACT GTTTTTCAAG CTCTTGATTC ACAAAACAAT TACCTCAGGG 960 TTCAAGAAAA TGCATTAACA GGCACAGCTA CTACATTTGA TGATGCTTCT CTGGCTAATA 1020 TGATATTATT AGTACAAGTT GGTGAAAACT TATTGAAGAA ATCAGTTTCC GAAGACAATC 1080 ATGAAACCTA TGAGGTAGCT CTAAAGAGGT TTGCAAAATT GCTCTCTGAT AGGAAGAAAC 1140

TCCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC

1175

SEQ ID NO: 26

Characteristics of the chain:

i) A) Length: 33 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: DNA (synthetic) (xi) Chain description: SEQ ID NO: 26

GTTAGATCTC ACCATGGCAA CTACTAAATC TTT

The string information of SEQ ID NO: 27

Characteristics of the chain:

i) A) Length: 33 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: DNA (synthetic) (xi) Chain description: SEQ ID NO: 27

CCAGAATTCT CATTAATAAG AAGCTTTGTT TGC

The string information of SEQ ID NO: 28

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA

(xi) Chain Description: SEQ ID NO: 28 CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATACAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCŤA 300 AAGATATTAT acctttttac TTCGATCATG GCCCTCAGAT TTTTGAACCT AGTGGTCTTC 360 _AAATTTTTGG CCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAAACTTG 420 GAGAAACTCG TGTGCATCAA GCTTTGACAG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480 CAAATAAGCC AGTAATATTC ACTAAGTCAA ATTTAGCAAA AACTCCAGAA TTGGATGCTA 540 AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600 TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACTTCAA TCTTGTTGAT GGTGATGTTG 660 CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTGC TTCAATTAGG TCGTTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAC TTCAGAGTTT TATAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840 TTCTACAATG GCTGTTACCT TTACAGGAAA TGAGAAGTGC AGCAAGTTCT TACATGAATG 900 attattacct TTCTACTGTT TTTCAAGCTC TTGATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAGCTACTA AATTTGATGA TGCTTCTGTG GCTAATATGA 1020 TATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAATC AGTTTCTGAA GACAATCATG 1080 AAACCTATGA GGTAGCTCTA AAGAGGTTTG CAAAATTGCT CTCCGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

The string information of SEQ ID NO: 29

Characteristics of the chain:

i) A) Length: 1172 base pairs B) Type: nucleic acid (C) Coil: single D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 29

CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60 CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180 ATAATACAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240 GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCTA 300 AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTCAGAT TTTTGAACCT AGTGGTTCAA 360 TTTTTGATGG CCCAAAATAT GATGGAAAAC ATCTTATGCA AGTTCTTCAA GAAAAACTAG 420 GAGAAACTCG TGTGCATCAA ACTTTGACAG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480 CAAATAAGCC AGTAATATTC ACTAAGTCAA ATTTACCAAA AACTCCAGAA TTGGATGCTA 540 AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600 TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACTTCAA TCTTGTTGAT GGTGATGTTG 660 CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720 CAGCATTTGC TTCAATTAGG TCGTTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780 CTGGCACTAC TTCAGAGTTT TATAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840 TTCTACAATG GCTGTTACCT TTACAGGAAA TGAGAAGTGC AGCAAGTTCT TACATGAATG 900 ATTATTACCT TTCTACTGTT TTTCAAGCTC TTGATTCACA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAGCTACTA AATTTGATGA TGCTTCTGTG GCTAATATGA 1020 TATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAATC AGTTTCTGAA GACAATCATG 1080 AAACCTATGA GGTAGCTCTA AAGAGGTTTG CAAAATTGCT CTCCGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

The string information of SEQ ID NO: 30

Characteristics of the chain:

i) A) Length: 1172 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 30

CCATGGCAAC TACTAAATCT TTTTTAATTT TAATATTTAT GATATTAGCA ACTACTAGTT 60 CAACATTTGC TCAGTTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120 GAGGGATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAATGGACA 180 ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240 GTTTATTGAC TGCTATGATA AGTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300 AAGAAATTGT ACCTTTTTAC TTCGAACATG GCCCTCAGAT TTTTAATCCT AGTGGTCAAA 360 TTTTAGGCCC AAAATATGAT GGAAAATATC TTATGCAAGT TCTTCAAGAA AAACTTGGAG 420 AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGTCATCTC AAGCTTTGAC ATCAAAACAA 480 ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAACTC TCCAGAATTG GATGCTAAGA 540 TGTATGACAT AAGfTATTCC ACAGCAGCAG CTCCAACATA TTTTCCTCCG CATTACTTTG 600 TTACTAATAC TAGTAATGGA GATGAATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660 CTGTTGCTGA TCCGGCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAAAGGATC 720 CAGCATTTGC TTCAATTAGG TCATTGAATT ACAAAAAAAT GCTGTTGCTC TCATTAGGCA 780 CTGGCACTAC TTCAGAGTTT GATAAAACAT ATACAGCAAA AGAGGCAGCT ACCTGGACTG 840 CTGTACATTG GATGTTAGTT ATACAGAAAA TGACTGATGC AGCAAGTTCT TACATGACTG 900 ATTATTACCT TTCTACTGCT TTTCAAGCTC TTGATTCAAA AAACAATTAC CTCAGGGTTC 960 AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCTTCTGAG GCTAATATGG 1020 AATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAACC AGTTTCCGAA GACAATCCTG 1080 AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140

GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC

1172

SEQ ID NO: 31

Characteristics of the chain:

i) A) Length: 1106 pairs of flanks

B) Type: nucleic acid (C) Coil: simple

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 31

CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAGAGGGA 60 TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAATG GACAATAATG 120 CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180 TGACTGCTAT GATAAGTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGAAA 240 TTGTACCTTT TTACTTCGAA CATGGCCCTC AGATTTTTAA TCCTAGTGGT CAAATTTTAG 300 GCCCAAAATA TGATGGAAAA TATCTTATGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360 GTGTGCATCA GGCTTTGACA GAAGTTGTCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420 CAGTAATATT CACTAAGTCA AATTTAGCAA ACTCTCCAGA ATTGGATGCT AAGATGTATG 480 ACATAAGTTA TTCCACAGCA GCAGCTCCAA CATATTTTCC TCCGCATTAC TTTGTTACTA 540 ATACTAGTAA TGGAGATGAA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GCTACTGTTG 600 CTGATCCGGC GTTATTATCC ATTAGCGTTG CAACGAGACT TGCACAAAAG GATCCAGCAT 660 TTGCTTCAAT TAGGTCATTG AATTACAAAA AAATGCTGTT GCTCTCATTA GGCACTGGCA 720 CTACTTCAGA GTTTGATAAA ACATATACAG CAAAAGAGGC AGCTACCTGG ACTGCTGTAC 780 ATTGGATGTT AGTTATACAG AAAATGACTG ATGCAGCAAG TTCTTACATG ACTGATTATT 840 ACCTTTCTAC TGCTTTTCAA GCTCTTGATT CAAAAAACAA TTACCTCAGG GTTCAAGAAA 900 ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCTTC TGAGGCTAAT ATGGAATTAT 960 TAGTACAAGT TGGTGAAAAC TTATTGAAGA AACCAGTTTC CGAAGACÁAT CCTGAAACCT 1020 ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080

ACAAAGCTTC TTATTAATGA GAATTC

1106

The string information of SEQ ID NO: 32

Characteristics of the chain:

i) A) Length: 45 pairs of flanks

B) Type: nucleic acid (C) Coil: single

D) Topology: linear (ii) Molecule type: DNA (synthetic) (xi) Chain description: SEQ ID NO: 32>

CCATCTAGAA GATCTCCACC ATGGCGTTGG GAGAAATGGT GACTG 45 é

The string information of SEQ ID NO: 33

Characteristics of the chain:

i) A) Length: 1164 pairs of flanks

B) Type: nucleic acid (C) Coil: simple

D) Topology: linear (ii) Molecule type: cDNA (xi) Chain description: SEQ ID NO: 33

ATGGCCACCA CCAAGAGCTT CCTCATCCTG ATCTTCATGA TCCTGGCCAC CACCAGCAGC 60 ACCTTCGCCC AGCTCGGCGA GATGGTGACC GTGCTCTCCA TCGACGGCGG TGGCATCAGG 120 GGCATCATCC CGGCCACCAT CCTGGAGTTC CTGGAGGGCC AACTCCAGGA GATGGACAAC 180 AACGCCGACG CCCGCCTGGC CGACTACTTC GACGTGATCG GTGGCACCAG CACCGGCGGT 240 CTCCTGACCG CCATGATCTC CACTCCGAAC GAGAACAACC GCCCCTTCGC CGCTGCGAAG 300 GAGATCGTCC CGTTCTACTT CGAACACGGC CCTCAGATTT TCAACCCCTC GGGTCAAATC 360 CTGGGCCCCA AGTACGACGG CAAGTACCTT ATGCAAGTGC TTCAGGAGAA GCTGGGCGAG 420 ACTAGGGTGC ACCAGGCGCT GACCGAGGTC GTCATCTCCA GCTTCGACAT CAAGACCAAC 480 AAGCCAGTCA TCTTCACCAA GTCCAACCTG GCCAACAGCC CGGAGCTGGA CGCTAAGATG 540 TACGACATCT CCTACTCCAC TGCTGCCGCT CCCACGTACT TCCCTCCGCA CTACTTCGTC 600 ACCAACACCA GCAACGGCGA CGAGTACGAG TTCAACCTTG TTGACGGTGC GGTGGCTACG 660 GTGGCGGACC CGGCGCTCCT GTCCATCAGC GTCGCCACGC GCCTGGCCCA GAAGGATCCA 720 GCCTTCGCTA GCATTAGGAG CCTCAACTAC AAGAAGATGC TGCTGCTCAG CCTGGGCACT 780 GGCACGACCT CCGAGTTCGA CAAGACCTAC ACTGCCAAGG AGGCCGCTAC CTGGACCGCC 840 GTCCATTGGA TGCTGGTCAT CCAGAAGATG ACGGACGCCG CTTCCAGCTA CATGACCGAC 900 TACTACCTCT CCACTGCGTT CCAGGCGCTT GACTCCAAGA ACAACTACCT CCGTGTTCAG 960 GAGAATGCCC TCACTGGCAC CACGACCGAG ATGGACGATG CCTCCGAGGC CAACATGGAG 1020 CTGCTCGTCC AGGTGGGTGA GAACCTCCTG AAGAAGCCCG TCTCCGAAGA CAATCCCGAG 1080 ACCTATGAGG AAGCGCTCAA GCGCTTTGCC AAGCTGCTCT CTGATAGGAA GAAACTCCGC 1140

GCTAACAAGG CCAGCTACTA ATGA

Claims (4)

PATENT CLAIMS What is claimed is: 1. A method for controlling infestation of plants by an ingesting plant, comprising providing an effective amount of insecticidal palatine for insect ingestion. t 2. The method of claim 1, wherein said paletin provides plant-settling microorganisms that produce palatin upon application to the plant. 3. The method of claim 1, wherein said palatin is provided by expressing a palatin gene incorporated in said plant prior to genetic transformation of the parent cell of the plant. 4. The method of claim 3 wherein said plant is cotton, corn, tomato or potato.