CN1119027A - Method of controlling insects in plants - Google Patents

Abstract

Potatos storage protein 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

The method of control insect

Invention field

The present invention relates to by protein that can be directly used in plant or the protein that is produced thereon by microorganism are provided, perhaps by with the genetic engineering method modified plant to produce the method that described protein comes controlling plant insect infection, the invention still further relates to microorganism useful in described method and plant.Background of the present invention

Use comprises that the natural product of protein is the method for the many insect pests of control known.For example, with bacillus thuringiensis (B.t.) intracellular toxin control lepidopteran and coleopteron insect.Produce that these endotoxic genes have been imported into and by each kind of plant, comprise that cotton, tobacco, tomato express.Yet, also exist some to the insensitive important economically insect pest of B.t. intracellular toxin.The example of these important pests has boll weevil (BWV), Anthonomus grandis and pumpkin 12 asterophyllite first (CRW) (the chrysomelid genus of bar).In addition, other are used to control the different genes product to the insect of B.t. intracellular toxin sensitivity, and if not lethal, it also is important then handling for resistance.

In plant, find other several known insect-killing proteins, comprised phytohemagglutinin, amylase inhibitor and proteinase inhibitor.When using with high dosage, they can influence g and D [Boulter et al., 1989 of insect; Broadway and Duffey, 1986; Czapla and Lang, 1990:Gatehouse et al., 1986; Heusing et al., 1991; Ishimoto and K.Kitamura, 1989; Nurdock et al., 1990; Shukle and Murdock, 1983], but can not provide the acute fatal that is given by B.t. protein.

An object of the present invention is to provide the protein that to control BWV, CRW or other insect pests and produce the used gene of described protein.Another object of the present invention provides genetic constructs and described genetic material is inserted the method for microorganism and vegetable cell.Another object of the present invention provides conversion microorganism and the plant that contains described genetic material.The present invention's general introduction

Have been found that potato storage protein (patatin) (the main storage protein of potato tuber) can control various insects, comprise corn young shoot root leaf Che (WCRW), Diabrotica virgifera, southern corn rootworm (SCRW), Diabrotica undecimpunctata and boll weevil (BWV), Anthonomusgrandis.The potato storage protein is lethal for some larvas, and can hinder survivor's growth so that prevent or seriously delayed the maturation of larva, thereby it can't be bred.These knownly can be had the active protein of esterase (lipid acyl hydrolase enzyme) and be directly used in plant or transduce with other modes, for example grow microorganism, perhaps use the plant itself after the similar conversion by the plant-Ding that uses the described enzyme of production that has transformed.

The potato storage protein is in potato [Gaillaird, 1971; Racusen1984; Andrews et al., 1988] and other plant, especially eggplant shape (solanaceous) plant [Ganal et al., 1991; Vancanneyt et al., 1989] the middle protein families of finding.In potato, the potato storage protein is mainly found in stem tuber, the level in the other plant organ very low [Hofgen and Willmitzer, 1990].Esterase substrate specificity [Hofgen andWillmitzer, 1990 of several potato storage protein isozymes have been detected; Racusen, 1986].

The gene that had separated coding potato storage protein in the past by people (1988) such as people (1988), Stiekema such as people such as Mignery (1984), Mignery with other people.People such as Rosahl (1987) transfer to it in tobacco plant, and have observed the expression of potato storage protein.This shows can be by plant heterogenous expression potato storage protein.

Similarly; can separate potato storage protein gene; be inserted into then in the suitable conversion carrier box; (1) necessarily grows microorganism with described box conversion plant then; when being used for plant, described microbial expression is produced the gene of potato storage protein, controls insect thus; perhaps mix described box in the Plant Genome (2), then by expressing gene and produce the potato storage protein and can protect the attack of himself avoiding insect.In addition, can transform or cultivate plants with the proteinic B.t. gene of one or more coding control insects of coexpression.This just make plant (1) resist more kind insect and/or or (2) have two kinds of binding modes to some insects, this is a kind of important means during resistance is handled.Transform the back and express the plant example of B.t. gene and be disclosed in the open No.0 of European patent, in 385,962, be equivalent to U.S.'s series application number 476,661,1990,2,12 applications people such as [] Fischhoff (document is mixed this paper as a reference).In addition, owing to show that proteinase inhibitor improves the activity of other insecticidal proteins, so can transform or cultivate plants so that the coexpression protease inhibitor gene, as the potato antipain of encoding [Rodis and Hoff, 1984] or those genes of Trypsin inhibitor SBTI [referring to the relevant summary of Ryan, 1990].

In conjunction with content above, according to one of purpose of the present invention, provide the method for controlling plant insect infection, comprise providing the desinsection of significant quantity potato storage protein to ingest for insect.Can finish this method by providing plant-Ding to grow microorganism, described microorganism has been converted expresses potato storage protein gene, imports plant then, expressing said gene, and the potato storage protein of insecticidal effective dose is provided.Also can transform plant to be protected through genetic engineering method with dna molecular and realize this method, described dna molecular contains: the promotor that (i) in vegetable cell, works, to cause the generation of RNA sequence; (ii) the encode structured coding sequence of potato storage protein; 3 ' the non-district that translates of (iii) working in described vegetable cell is to impel polyadenylic acid

Nucleotide is added to 3 ' end of RNA sequence.Wherein said promotor is allogenic for described structured coding sequence, and wherein said promotor links to each other through operating with described structured coding sequence, and described structured coding sequence can be operated and links to each other with the described non-district that translates successively.Preferably plant will be with the gross protein horizontal expression potato storage protein of about 0.1-0.5%.

The present invention also provides insect-resistant corn, cotton, tomato and the potato plants that transforms with genetic engineering method.

Term used herein " control insect infection " refer to or by lethality, delay the growth (dysplasia) of larva, perhaps reduce reproductive efficiency and reduce the insect quantity that causes that useful productive rate reduces.Term used herein " parasiticidal " refers to or by lethality, delays the growth (dysplasia) of larva, perhaps reduces reproductive efficiency and reduces the insect quantity that causes useful productive rate to reduce.

Term used herein " structured coding sequence " refers to the dna sequence dna of coded polypeptide, and described polypeptide is being transcribed into mRNA by cell with DNA, is translated into required polypeptide then and obtains.

Term used herein " potato storage protein " refers to a kind of plant protein, with SEQID NO:31) (hereinafter) encoded protein matter has 75% or higher homology, perhaps at least 80% homology more preferably, or even at least 85% homology more preferably.This term comprises by the protein that the synthetic dna sequence dna produces through being designed for the expression that improves in the monocotyledons.Detailed description of the present invention

The potato storage protein is in potato [Gaillaird, 1971; Racusen, 1984; Andrews et al., 1988] and other plant, particularly eggplant shape plant [Ganal etal., 1991; Vancanneyt et al., 1989] the middle esterase family that finds.In potato, mainly in stem tuber, found the potato storage protein, but it is present in other the plant organ [Hofgen and Willmitzer, 1990] with very low level also.After testing esterase substrate specificity [Hofgen andWillmitzer, 1990 of several potato storage protein isozymes; Racusen, 1986], finding has substrate specificity widely, shows that these enzymes are restricted to substrate.Use potato storage protein and its equivalent (both are all open in detail at this paper) and the homologous protein of all plant derivations, no matter derive from natural DNA sequence or synthetic DNA sequence, so long as for the purpose of controlling plant insect infection all within the scope of the present invention.

Thick potato storage protein goods from potato can be bought from the market.For example, Sigma Chemical Company, St, Louis, MO provide the potato protein goods that are designated as acid phosphatase (P-1146 and P-3752) or apyrase (A-9149) by Sigma.Also need method (Racusen and Foote, 1980 described in potato tuber and the available document; Park et al., 1983) the preparation protein extract.Biopotency detects artificial food's biological detection

At the similar agar food of describing to people such as Marrone 1985 that is used for SCRW, finish the activity detection of anti-SCRW, BWV, colorado potato bug (CPB) and European corn borer (ECB) larva by covering test sample.Protein is dissolved in 4-5ml 10mM HEPES, among the pH7.5, uses in this identical damping fluid, dialysing of 3500 molecular weight to prepare test sample then by test tube.Feed newborn larvae at 26 ℃ with the food of handling, assessed mortality ratio and retardation of growth then at 5 or 6 days.Table 1 has been listed the detected result of P-3752 (Sigma).Show that this thick potato preparation has broad-spectrum insecticidal activity.

Table 1 speed mortality ratio/dysplasia %*

SCRW BWV CPB ECB0.01X 0 11 0 00.03X 0 ^* 20 ^* 0 130.10X 19 ^** 20 ^** 6 0 ^*0.30X 6 ^*****?46 ^** 13 ^* 6 ^*1.00X 6 ^*** 73 ^** 13 ^** 6 ^**

The slight dysplasia of a*=(size reduces about 30-40%)

*=moderate dysplasia (size reduces about 50-80%)

* *=serious dysplasia (size reduces＞90%)

The SCRW (table 2) that contacts after 5 days is carried out the weight measurement of accurate quantification with the ECB (table 3) that contacts after 6 days, the results are shown in hereinafter.When absorption contained the food of P-3752, developmental SCRW larva lost weight 92% compared with the control, and the ECB larva loses weight 62% compared with the control.

Table 2 is handled average survival weight (modified value) weight saving % mortality ratio % Tris 4.00mg (0.60) ^a--control level--P-3752 0.30mg (0.03) ^a92 6

A is on average survived weight at 95% significantly different (ANOVA of a factor).

Table 3 is handled average survival weight (modified value) weight saving % mortality ratio % Tris 5.39mg (0.49) ^a--control level--P-3752 2.05mg (0.27) ^a62 7

A is on average survived and is reset at 95% significantly different (ANOVA of a factor).

By thermolability, ammonium sulfate precipitation, molecular size separate and proteolytic enzyme susceptibility is tested the protein properties of determining to have anti-Southern corn rootworm (SCRW) and the active P-3752 insect disinfestation component of boll weevil (BWV).

React the indirect effect that causes for the effect that confirms P-3752 owing to the direct effect rather than anti-nursing of picked-up potato storage protein, finish food selection research with ECB and SCRW.This selects the result of research to show the food of handling with P-3752 and Tris serves as that food does not have tangible preference.With regard to the food that Tris handles, as if can't abolish the food that P-3752 handles.

Long-term (25 days) test an of-anti-SCRW of P-3752 has utilized the 2nd instar larvae, and several survival insects are transferred on the food of fresh processing.When this research finished, all control larvae were all pupated.Increase by 16% (2.48mg is to 2.14mg) when on the contrary, dead and other 50% its body weight of 50% processing larva is only than beginning.This shows and has suppressed larvae development, and is not only to delay.Because larva can't grow adult, from the angle of insect control, this has important result.Therefore the quantity of offspring SCRW will reduce.

Only in laboratory experiment, used corn young shoot root leaf Che (WCRW) larva in the 2nd instar larvae stage.In order to detect the effect of the anti-WCRW of P-3752, with the 2nd age the SCRW larva designed parallel test.The processing of P-3752 causes SCRW and WCRW the 2nd instar larvae only to increase by 13% and 11% weight respectively.In 7 days, contrast SCRW weightening finish 474% and WCRW increase by 200%.This shows that the anti-WCRW activity of potato storage protein is substantially equal to the activity of its anti-SCRW.

P-3752 has lower activity to oriental tobacco budworm (TBW), Heliothis virescens, beet armyworm (BAW), corn earworm, pink bollworm and maduca sexta, is 1-1.5 in its dysplasia rate of identical concentration, and is 3 to the dysplasia speed of SCRW.P-3752 is 2.5 (above having defined dysplasia speed in the table 1) to the dysplasia speed of black cutworm.In the concentration of being tested, black peach aphid there is not activity.Plant tissue biological detection (1) potato: the thick P-3752 of 1g is dissolved in 4ml 25mM Tris, in the pH7.5 damping fluid, then by dialysis of 0.2 μ m film and filtration.Add Triton ^X-100 is to obtain 0.1% solution.Leaf of potato is immersed in the zymin, is placed on the filter paper that bedews in the culture dish.Put into the CPB larva, flat board was cultivated 3 days in 27 ℃.The leaf of potato that P-3752 handles causes CPB larvae development obstacle and feed to reduce.Test-results is, compares with the leaf that P-3752 handles, and the leaf texture of leaf obviously reduces in contrast.(2) corn and cotton: from agar plate, take out black Mexico's sweet corn callus (BMS) or cotton healing tissue, transfer to then in the 50ml centrifuge tube.Make callus become whirlpool and centrifugal 5 minutes with 8 grades with IEC Clinical whizzer.Slowly pour out supernatant liquor, the liquid agar of 30ml2% is added in the 50ml test tube that contains the 15ml callus.After mixing fully, food is moved on to-detect and carry out the insect biological detection among the arena.The P-3752 that dialysed is added (with 20% volume), finishes detection then as mentioned above as-kind of food coverture.

With thick P-3752 or 25mM Tris, pH7.5 damping fluid vacuum is soaked into Zea mays root and the stem that [Inflt] cuts, and control sample is all organized be immersed in the Iris damping fluid.About 10-15 sheet root or 3 stem tissues are placed in the hole of 24 hole tissue culture plate, repeat 4 times.Every hole adds 4 newborn SCRW larvas.Trier was cultivated 4 days at 26 ℃, at this moment mortality ratio and average larva weight are observed, the results are shown in table 4.

Table 4 organizes insect mortality % weight to reduce %

The BWV of cotton healing tissue 60 that the BMS callus ECB 0 33 that the BMS callus WCRW 0 23 that the BMS callus SCRW 24 51 that the corn stem SCRW 51 52 that Zea mays root SCRW 90 44 vacuum that vacuum is soaked into are soaked into handles handles handles handles does not have data

Therefore, when P-3752 and plant tissue are absorbed jointly, kept insecticidal activity to all four kinds of insects (SCRW, BWV, CPB and ECB).These foods studies show that when the food of only being made up of plant tissue (root, stem, callus or leaf) with its nutrition detected, the potato storage protein had insecticidal activity.Binding mode research

The following potato storage protein (insecticidal active ingredient of P-3752) that studies show that has direct effect to insect itself, and the activity that shows in above-mentioned experiment can not be the effect of active ingredient to insect food before absorbing.Eating effect research

1gP-3752 is dissolved in the damping fluid of 10ml 25mM Tris pH7.6,, in 000 test tube same damping fluid is dialysed then at MWCO 12-14.0.2 after μ m filters,, 50 μ l sample aliquot are added in two insect foods on the flat board adding insect preceding 4 days.Two flat boards were all cultivated 4 days at 27 ℃.After the cultivation, general-individual flat board was heated to 80 ℃, made enzyme deactivation through 1 hour.50 μ l sample aliquot are added on the 3rd flat board.Therefore, carry out the SCRW biological detection with flat board that cultivated, cultivation+heating and that do not cultivate.

In the pre-culture studies of food, the SCRW activity is as follows:

The P-3752 that did not cultivate---6 ^{* *}

The P-3752 that cultivated---0 ^*

The P-3752 that cultivated, heated---0 ^*

Between the food incubation period, lost a part of activity, and heat treated causes active loss fully.Such data and direct picked-up back binding mode-cause, and when detecting with plant tissue-rise when considering show that to the mutability of different insects protein is not the effect of diet to the activity of SCRW and other insect.Identification of proteins

Purified, partial ordering has also been described feature from the insecticidal active ingredient of P-3752.Having identified this promoting agent is the potato storage protein, from the lipid acyl hydrolase enzyme family of potato.Protein separation

SCRW biologically active components with four kinds of method purifying P-3752.

, follow with Q-Sepharose (Pharmacia) anion-exchange chromatography with anion-exchange chromatography purifying SCRW activity-elder generation through MONO-Q (HR5/5, Pharmacia) the SCRW-active ingredient of anion-exchange chromatography purifying P-3752.It is that the food of 31PPm can reach observed that protein level in the SCRW active part shows with protein concn ^*Retardation of growth.SDS-PAGE show in active part exist three kinds of main protein belts (Mr42,000 ,～26,000 and～16,000).

The active 5 step purifying of SCRW-with 5 successive purification steps purifying SCRW active ingredient from P-3752.Film size separation, ammonium sulfate precipitation, Q-Sepharose IEC, S-Sepharose IEC and P-200IEC are arranged.The SDS-PAGE of the purest SCRW active part shows protein belt at Mr42,000 ,～26,000 and～16,000.

Through isoelectrofocusing purifying biological activity-by the explanation of manufacturers, continuous race is 2 times on RF3 Protein Skimmer (Rainin), purifying SCRW biological activity potato protein.The SDS-PAGE figure of SCRW active part is similar to observed figure from the active part of 5 step purifying and anionresin purifying.The IEF gel shows that protein separates from pH4.6 to 5.1, with pH scope (Racusen and Foote, the 1980) unanimity of the potato storage protein of reporting.

By on RF3 a narrow pH range (pH4-5) continuously isoelectrofocusing to separate potato storage protein isozyme.By what expect, when proteinic pH4.6-5.1, see a biological active peak.These parts have the different isozyme patterns and the biological activity of different levels.Bioactive scope is from 0 mortality ratio of 80ppm dosage in these parts, ^*- ^*Dysplasia is to 512ppm.Some biologically-active moiety only has 2 kinds of main isozymes, shows the isozyme that does not need complex patterns for biological activity.

With the electrophoresis P-3752 of natural PAGE purifying-under field conditions (factors), isolate (is substrate with α-Yi Suannaizhi) three bands of esterase activity.The esterase positive material of gel-purified has anti-SCRW activity, produces 1.5 ^*Dysplasia.The SDS-PAGE of this material discloses master tape at Mr42,000 ,～26,000 and～16,000, observed figure is identical with other purification process of former usefulness.This confirms that further the potato storage protein is the insect disinfestation component from potato.Aminoacid sequence

In the active chromatography part of the SCRW of anionresin purifying and 5 step purifying methods, obtained all proteins band (Mr42,000 ,～26,000 and～16,000) NH ₂-terminal amino acid sequence.In a word, obtained the sequence data of all bands in the active part.Most of band and 15-aminoacid sequence are at NH ₂The internal sequence (SEQ ID NO:7) of-terminal (SEQ ID NO:1) or a kind of potato storage protein isozyme.(Stiekemaet al., 1988) have＞85% homology.A 17KD band and the potato storage protein NH that reported ₂8 amino acid of the beginning of-end sequence have 75% homology.In addition-17KD band and the potato storage protein NH that disclosed ₂8 amino acid of the beginning of-end sequence have＞85% homology.The protein hydrolyzate of these band expression potato storage proteins.NH ₂-mutability terminal and internal sequence position 1 and 3 upper amino acids has clearly illustrated that the existence of isozyme.

The-terminal amino acid sequence

The sequence that disclosed: KLEEMVTVLSIDGGG (SEQ ID NO:1) is with 1 (42kD): XLGEMVTVLSIDGGG (SEQ ID NO:2) to be with 2 (28kD): TLGEMVTVLSIDGGG (SEQ ID NO:3) to be with 3 (26kD): TLGEMVTVLSIDGGG (SEQ ID NO:4) to be with 4 (24kD): KLXEMVTVLSIDGGG (SEQ ID NO:5) band 5a (17kD): XXEEMVTV (SEQ ID NO:6)

Internal sequence (amino acid position 224) The sequence that disclosed: SLDYKQMLLLSLGTG (SEQ ID NO:7) band 5b (17kD): KLDVKQML (SEQ ID NO:8) is with 6 (16kD): SLXYKQMLLLSLGTG (SEQ ID NO:9) to be with 7 (15kD): SLNYKQMLLLSLGTG (SEQ ID NO:10) esterase activity

Carry out several experiments to detect the esterase activity in the SCRW active part.

α-Yi Suannaizhi substrate:, just determine with SDS-PAGE (10-20%) if SCRW active part (from 5 step method of purification) shows esterase activity.On the two halves of gel, the negative SCRW active part that heats and do not heat of cutting out.Observe an esterase These positive bands in the sample of not heating, Mr is 55,000.The sample of heating has original Mr42,000 band, but do not have 55,000 bands.This result consistent with the reported literature of the electrophoretic migration of potato storage protein esterase activity [Racusen, 1984].Do not observe Mr55 in the sample of heating, 000 band shows that heat treated has reduced esterase activity in SDS.Do not have Mr55 in heated sample, under the situation of 000 band, observe the Mr42 that saw originally, 000 band with the dyeing of Cowes horse.

Right-nitrophenyl substrate specificity Journal of Sex Research-detection a series of right-nitro phenyl ester (C-2, C-4, C-6, C-8, C-10, C-12, C-14 and C-16) to be to determine substrate specificity.Comparing with other ester, for the esterase activity of most of tested potato storage proteins, is best substrate concerning-nitro C-8 and C-10 ester all the time.

The ability of active purification part (from 5 step purifying methods) several fat of hydrolysis of fat ester substrate-detection SCRW.Every kind of liposoluble is separated, cultivate with the active purification part of a SCRW then.By TLC (Pernes et al., the 1980) analytic sample that uses three kinds of solvent Color Appearance Systems.Four kinds of fat show the modification vestige of being done by TLC.These comprise oleoyl lysolecithin, two oleoyl L-α-Yelkin TTS, the single flax acyl-racemize of 1--glycerine and diolein (Sigma).Compare with inferior oil and fatty acid oil standard, in the organic extraction of these fat/active part reaction mixture, identify at R _f0.37 new TLC point be free fatty acids.Therefore, the SCRW active substance has esterase activity to these four kinds of fat esters.

From the 3rd instar larvae of feeding, take out the midgut of WCRW with Zea mays root.Tripe tallow in the extraction, dissolving is cultivated with the active purification part of SCRW at midgut pH (pH6.55) then.Come analytic sample by the TLC that uses aforesaid method.The SCRW active part that shows purifying has esterase activity to WCRW midgut phosphatide when midgut pH.This has illustrated the possible binding mode of relevant potato storage protein insecticidal activity.Other source of potato storage protein

Because all preliminary experiment all is with P-3752 (a kind of commercially available zymin from MinnesotaRusset var.Kranz potato tuber (Sigma), therefore, be necessary to illustrate also can find again the potato of insecticidal activity storage protein is arranged from fresh potato tuber, basic document (Racusen and Foote, 1980 of pressing; Park et al., 1983) description in prepares tuber extract.Analyze 3 kinds of commercially available potato cultivar (Russet, Desiree and LaChipper) and 7 kinds of wild kind (S.kurtzianum, S.berthaultii, S.tarijense, S.acaule, S.demissum, S.cardiophyllum and S.raphanifolium all derive from Inter-RegionalPotato Introduction Station, USDA, ARS, Sturgeon Bay, WI).By SDS-PAGE and Western engram analysis, all extracts all are potato storage protein positives; Detected by the C-10 esterase, all is esterase positives; And all SCRW is had insecticidal activity, promptly the dysplasia rate is 2-3, sees Table 5.This explanation can separate the potato storage protein that insecticidal activity is arranged from several stem tubers, expect that many members of this whole histone matter have insecticidal properties.

Table 5 kind [Prot] Δ O.D./minmL SCRWa

(mg/mL) 1X 0.1XS.acaule 26.6 82,500 2 1S.berthaultii 23.1 29 3 1S.cardiophyllum?14.6 89 2.5 1.5S.demissum 35.3 375,000 3 1.5S.kurtzianum 25.0 2,700 2.5 1S.raphanifolium?33.7 3,725 3 2S.tarijense 27.2 1008 2.5 1

A represents the slight dysplasia of SCRW activity: 1=(size reduces 30-40%) with the larvae development obstacle, 2=moderate dysplasia (size reduces 50-80%), the serious dysplasia of 3=(size reduce＞90%).

Biological detection S.berthaultii, the activity of anti-other two kinds of target insect CPB of S.kurtzianum and S.tarijense extract and ECB.In table 6, summed up biological detection data.Notice that these extracts almost do not have activity to CPB, and, cause ECB larva moderate to serious dysplasia in 1X speed.Yet, by not having activity to show fully to ECB at 0.1X, the ECB larva to the susceptibility of these extracts a little less than the SCRW larva.

Table 6

Kind CPBa ECBa

1X 0.1X 1X 0.1X

S.berthaultii 0 0 3 0

S.kurtzianum 1 0 2.5 0

Starijense 0 0 3 0

A larvae development obstacle

Protein homology with 9 kinds of different plant genome DNAs of Southern analyzing and testing and potato storage protein.With α- ³²The Southern trace of the SEQ ID NO:11 probe in detecting of p mark shows that in several other plant varieties homologous sequence is arranged.In corn, tomato, beet, rice and potato, obtain strong signal.In this experiment, can not differentiate the wall scroll band: but in all these were planted, the smear size was similar with its intensity.In zucchini, soybean and canola, also see weak signal, and show as a small amount of discontinuous band at the DNA of each kind.May be because as what see in the bromo chemical ingot dyeing of gel, the DNA of load measure still less, therefore under the used condition of this experiment, cucumber and Arabidopsis do not have detectable hybridization with potato storage protein probe.

Those of ordinary skills can be easy to obtain the dna sequence dna of these homologous proteins, and insert in plant or other biology with currently known methods.After for example by baculovirus or E.coli heterogenous expression, the described proteinic insecticidal properties of the easiest detection.Therefore, can obtain can be used for other protein of the inventive method through constant experiment, and be used for being equipped with in after this and avoid insect infection to protect it for plant with currently known methods.Genetic identification

There are several researchists to clone potato storage protein gene.Be called GM203 by the disclosed sequence of people such as Mignery.It has an incomplete signal sequence.People such as Mignery (1988) have identified a genomic clone, are called PS20, comprise GM203, and contain a complete signal sequence.Make up SEQID NO:11, hereinafter referred to as PatA+ with the signal sequence of PS20 and the cDNA of GM203 encoding part.And then a NcoI restriction site and an EcoRI site behind rotaring inter-transtating terminating cipher.Potato cultivar Russet Burbank

From the stem tuber of potato cultivar Russet Burbank, separate 20 cDNA, and sequencing.The aminoacid sequence of inferring shows 11 kinds of different potato storage protein isozymes of these cDNA codings.This 11 kinds of protein and PatA+, SEQ ID NO:11 compare 82% to 100% the identity of having an appointment, and its difference is found in many different positionss of full-length cDNA.In table 7, listed the representational cDNA sequence of 11 kinds of differences of the 11 kinds of different potato storage protein isozymes of encoding.Produce cDNA by the PCR method of using primer SEQ ID NO:26 and SEQ ID NO:27 (be equivalent to coded signal sequence respectively and begin 5 ' Nucleotide of several codons and 3 ' end of encoding sequence), to be used for later clone operations, "+" number expression has comprised the natural signals encoding sequence.Some cDNA does not contain complete natural signals encoding sequence, and has only obtained the mature protein encoding sequence from the similar PCR method of using primer SEQ ID NO:32 and SEQ ID NO:27.These are represented with following " m ".

Table 7

The isozyme sequence is differentiated number

PatA+ SEQ?ID?NO：11

PatAm SEQ?ID?NO：14

PatB+ SEQ?ID?NO：16

PatC+ SEQ?ID?NO：17

PatD _m SEQ?ID?NO：18

PatE+ SEQ?ID?NO：19

PatE _m SEQ?ID?NO：2O

PatF _m SEQ?ID?NO：21

PatG+ SEQ?ID?NO：22

PatH _m SEQ?ID?NO：15

PatI _m SEQ?ID?NO：23

PatL+ SEQ?ID?NO：24

PatM+ SEQ?ID?NO：25Solanum?berthaultii

Reverse transcription stem tuber mRNA by using the PCR of primer SEQ ID NO:26 and SEQ ID NO:27 (above-mentioned), separates the potato storage protein cDNA from diploid potato S.berthaultii then.Finish the separation of a plurality of independently PCR reaction with the repeated cloning avoiding the gene-amplification process and cause.

Totally 14 kinds of potato storage protein cDNA partial orderings, as if all 14 kinds of cDNA (claiming that Patl is to Pat14) have a distinctive nucleotide sequence, illustrate and express 14 kinds of different potato storage protein mRNA at least in the S.berthaultii stem tuber.The sequence of Pat3+ is SEQ ID NO:28.The sequence of Pat10+ is SEQ ID NO:29.The aminoacid sequence of inferring shows at least 11 kinds of different protein of 14 kinds of cDNA codings.In a word, very similar from the cDNA sequence of S.berthaultii stem tuber.In totally 367 residues, only 12 amino acid positions have the sequence mutability.Listed the amino acid that on each position of these positions, occurs in the table 8.On 5 positions in these positions, has only a varient clone with a peculiar residue.This change may reflect the actual variance between the mRNA, perhaps may be to be caused by the mistake that produces in the PCR process.In other 7 positions, bigger mutability is arranged; At least two kinds of cDNA have a variable amino acid.Each of these 9 kinds of different aminoacids sequence set all has the residue type of a uniqueness in these 7 positions.In some cases, variation is guarded, as 164 becoming Ser from Thr in the position.Under other situation, bigger difference is arranged, as 148 importing Pro in the position.

The position of table 8cDNA amino acid difference

89 96 106?113?120?123?148?164?187?200PAT3+ GLN?LEU?GLN TYR?GLU?VAL?ALA?ALA?THR?ASP?ASPPAT4+ GLN SER?ASP?HIS?GLU?VAL?ALA?PRO?SER?ASP?VALPAT5+ GLN SER?ASP?HIS?GLU?VAL?ALA?PRO?THR?ASP?ASPPAT7+ GLN?LEU?GLN TYR?GLU?VAL?ALA?ALA?THR?ASN?ASPPAT8+ LYS SER?GLY?TYR?LYS?VAL?ALA?PRO?THR?ASP?ASPPAT9+ LYS SER?ASP?TYR?LYS?VAL?ALA?PRO?THR?ASP?ASPPAT10+?GLN SER?ASP?HIS?GLU?VAL?THR?PRO?THR?ASP?ASPPAT11+?GLN SER?ASP?HIS?GLU?ALA?ALA?ALA?THR?ASP?ASPPAT12+?GLM SER?GLY?HIS?GLU?VAL?ALA?ALA?THR?ASP?ASPPATA+ HIS?---?SER?---?TYR?GLU?VAL?ALA?ALA?THR?GLU?ASPSolanum?cardiophyllum

With obtaining 10 cDNA clones through PCR from the isolating mRNA of above-mentioned Solanum cardiophyllum stem tuber.At least 75% length according to each clone obtains nucleotide sequence.A full length sequence that is called the clone of Pat17+ is that SEQ ID NO:30, SEQ ID NO:31 are the mature form Pat17m after the processing.These S.cardiophyllum clones almost are identical, only have nucleotide sequence at random to change, and may be actual variance or PCR mistake.But in the position 54 and 519, observing several clones has identical variation, illustrates that they are not caused by amplification procedure.4 kinds of different mRNA have been represented in the Nucleotide mode declaration pattern specification of these positions at least.In this cover cDNA clone, separated several times from two groups mRNA, and two groups mRNA has only separated once in addition.

The aminoacid sequence that the S.cardiophyllum clone is inferred is also very similar.8 aminoacid sequence groups are arranged, and each only has a residue different with other sequence.The cDNA clone of the aminoacid sequence identical with the Patl7+ sequence of encoding has been recovered 2 times, and other 7 cDNA (Patl8+, 19+, 20+, 21+, 22+, 23+ and 24+) contain a distinctive residue.Genetic transformation

By what above discussed, can separate potato storage protein gene from various plant-sourceds.Can be with one or more transform bacteria cells in these genes or vegetable cell so that can produce the potato storage protein and finish method of the present invention.To provide the enforcement of how finishing this purpose below with the sequence of various potato storage proteins.The processing of the cDNA of potato storage protein

Be applicable in the carrier of in the heterologous host cell, expressing the potato storage protein for potato storage protein gene is mixed, must near this gene end, import suitable restriction site.The purpose of mutagenesis is in order to obtain comprising the box of protein coding sequence and minimum non-coding lateral order row like this, and the restriction site that is mixed with usefulness is to move these boxes.Design these boxes so that can move the complete encoding sequence that comprises signal peptide or have only ripe encoding sequence.For PatAm, design two kinds of mutagenic primers to produce these boxes.The mutagenesis of SEQ ID NO:12 is the Lys that replaces ripe protein N-end with two amino acid (Met-Ala), and importing-NcoI site, and SEQ ID NO:13 has added second terminator codon and an EcoRI site.

The modification sequence of gained is accredited as PatAm, SEQ ID NO:14.For all other cDNA, carry out similar modification and import restriction site with SEQ ID NO:27 with SEQ ID NO:27 or primer SEQ ID NO:32 with above-mentioned primer SEQ ID NO:26 with PCR.In E.coli, express the potato storage protein

The dna sequence dna of PatAm (SEQ ID NO:14) of will encoding inserts pMON5766, and an E.coli who derives from pBR327 (Soberon et al., 1980) expresses and cuts out body, has recA promotor and G10 leader sequence (Olin et al., 1989).The carrier pMON19714 of gained is transferred among the E.coli bacterial strain JM101, produce PatAm subsequently, confirmed by Western engram analysis and the esterase activity that uses right-oil of mirbane C-10 ester.

The dna encoding sequence of Pat17m and PatAm is inserted respectively in the E.coli expression vector that derives from pMON6235, and it has AraBAD promotor (can induce), G10 leader sequence and ampicillin resistance gene when cell is grown in pectinose.The gained carrier pMON25213 that will contain Pat17m imports E.coli bacterial strain JM101 with the pMON25216 that contains PatAm.

Express the potato storage protein by the E.co1i that transforms.But it is isolated in the refractive body (refractile body RBs).Carrying out SCRW with complete cell and dissolved RB detects.The results are shown in table 9.

Table 9

Sample multiplicity intact cell dissolved RBs

(pMON) SCRW activity ¹The SCRW activity ¹

19714?A _m 1 2.5 nt

2 1.5 1.0

25216?A _m 1 1.0 0

2 0 1.0

25213?17 _m 1 3.0 3.02

2 1.5 0.5

¹Represent that with the larvae development obstacle (size subtracts the slight dysplasia of SCRW activity: 1=

Little 30-40%), 2=moderate dysplasia (size reduces 50-80%),

The serious dysplasia of 3=(size reduce＞90%).

²The mortality ratio of this sample is 81%.Surely grow expression potato storage protein in the bacterium plant

In order to control insect, also need to grow one or more potato storage proteins of expression in the bacterium surely plant, then this bacterium is used for plant.Because insect is food with the plant, therefore, it can absorb the potato storage protein of the toxicity dose of being produced by entophyte.Entophyte can be those bacteriums in plant surface life, as Rhodopseudomonas or Agrobacterium bacterial classification, or lives in endophyte such as Clavibacter kind in the plant vasular structure.For surperficial bacterial parasite, potato storage protein gene can be inserted in the wide host range carrier that can duplicate in these Gram-negatives host.Described carrier example is the not pKT231 of compatibility class (Bagdasarian et al. of IncQ, 1981) or the pVK100 (Knauf of IncP class, 1982) for endophyte, can mix on the suitable transposon that can in these endophytes, carry out the karyomit(e) insertion by homologous recombination or with gene, thereby potato storage protein gene is inserted karyomit(e).In baculovirus, express the potato storage protein

Potato storage protein gene is arrived among the baculovirus donor carrier pMON14327 (referring to unsettled US.Serial Number 07/941,363,1992.9.4 applies for that the document is mixed this paper as a reference) as the NcoI/EcoRI fragment cloning.Donor carrier pMON14327 contain an ampicillin resistance gene, Tn7 transposon a left side and right arm, a gentamicin resistant gene, strong baculovirus polyhedrin body protein promotor and a polylinker are arranged between this two arm.Baculovirus shuttle vectors or shaft-like plasmid (bacmid) are made up of lacZ gene on the AcNPV viral genome of recombinating and the mini-attTn7 site in the kalamycin resistance gene sign indicating number.By tetracyclin resistance helper plasmid pMON7124, by AcNPV virus (Luckowet al., 1993) that potato storage protein or Gus gene and marker gene swivel base can be obtained recombinating in the viral genome.Following gene is inserted among the pNON14327: listed gene and Pat3+, Pat10+ and Pat17+ in the table 7.

Finish US.Serial No.07/941,363 and people's such as Lockow process after, produce a large amount of potato storage proteins from said gene.Esterase activity by Western engram analysis and the right-oil of mirbane C-10 ester of use is determined the existence of potato storage protein.Except that PatAm, in order to carry out the SCRW biological detection, every kind of isozyme enlarges twice at least in proportion.As if PatE+ and PatEm fermented product almost do not have or do not have the potato storage protein to express all the time, and all other isozymes are all at the acceptable horizontal expression of biological detection.But, not determine to compare with potato, potato storage protein protein is translated the character of post-treatment in the baculovirus.Observe biological activity with Pat17+, PatB+, PatDm, PatIm, PatL+, Pat3+ by the anti-SCRW of isozyme of baculovirus expression.

Determined the influence of multiple isozyme (producing) to insect growth and growth by baculovirus.The sample aliquot of 11 kinds of Russet isozymes is combined the biological detection of carrying out anti-SCRW, ECB, black cutworm and TBW with a kind of sample.In conjunction with the isozyme of each Q-Sepharose purifying of 10-15mg, except that only obtaining the PatDm of 1.7mg, this mixture causes 100% mortality ratio in TBW detects, and ECB weight is reduced 93%.Therefore, every kind of isozyme of separate detection is to the effect of TBW and ECB.Comparing with cutting out the body control larvae, is that the TBW and the ECB of food shows tangible dysplasia (〉=75%) and/or death with the food with isozyme PatC+, PatL+ and PatIm processing.PatB+ and PatDm are respectively 69% and 78% to the influence that TBW grows.Plant gene makes up

The expression of the plant gene that exists with the double-stranded DNA form relates to and is transcribed into messenger RNA(mRNA) (mRNA) by RNA polymerase from a DNA chain processes the mRNA primary transcript subsequently in nuclear.This processing relates to one 3 ' the non-district that translates, and polyadenylic acid Nucleotide is added to the 3 ' end of RNA.DNA is transcribed into mRNA and is regulated by a DNA zone that is commonly referred to " promotor ".This promoter region contains the base sequence that links to each other with DNA of indication RNA polymerase, and initiation transcribing to obtain corresponding RNA chain with the DNA chain mRNA that is template.

Described in the literature many in vegetable cell promoters active.Described promotor can be obtained by plant or plant virus, include but not limited to: courage fat alkali synthetic enzyme (NOS) and octopine synthetic enzyme (OCS) promotor (carrying), cauliflower mosaic virus (CaMV) 19S and 35S promoter, from 1 by the edaphic bacillus tl plasmid, the light inducible promoters and ficus virus 1 (FMV) 35S promoter of the little subunit of 5-diphosphoribulose carboxylase (ssRUBISCO, a kind of abundant plant polypeptide).Various types of DNA that obtained in plant, having expressed with all these promotors (referring to as the open WO84/02913 of PCT).People also can be limited in expression the specified plant position to the attack of insect sensitivity.For example, can expression be limited in root or increase the level of activated protein at root with root enhanced promotor with root-specific promoter.Concerning the plant of food root insect sensitivity, this is preferred.

Specific plant promoter is more effective in monocotyledons.For example, the rice actin promotor of describing in WO9/09948 is effective for expressing in corn.Corn ubiqutin (ubiquitin) promotor of describing in EP0342926 also can be used for monocotyledons.

Can be modified at promotor used in the DNA construct of the present invention (being chimeric plant gene) if desired to influence its control characteristic.For example, can with the CaMV35S promotor be connected to obtain in leaf, activity being arranged and in root, do not have active promotor there not being the light time to suppress ssRUBICO Gene Partial that ssRUBISCO expresses.By described herein, the chimeric promoters of gained also can use.

Therefore on the meaning of this description, " CaMV35S " promotor comprises the varient of CaMV35S promotor, as by with handle the subarea and link to each other, at random or control mutagenesis etc. and the promotor that obtains.In addition, can change promotor to comprise a plurality of " enhancer sequence " that improve genetic expression.Reported the example of described enhancer sequence by people such as kay (1987).

Selected specific promotor should be able to cause giving full expression to produce the potato storage protein of significant quantity of enzyme encoding sequence.Preferred promotor is CaMVE35S promotor (enhanced CaMV35S).

The RNA that is produced by DNA construct of the present invention also comprises 5 ' the non-leader sequence of translating.Can obtain this sequence with expressing gene from selected promotor, and can it be modified to improve translating of mRNA specificity.Also can obtain 5 ' the non-leader of translating from suitable eukaryotic gene or synthetic gene order from viral RNA.The invention is not restricted to the wherein non-district that translates and derive from 5 ' the non-construct of translating the district that accompanies with promoter sequence.

As indicated above, a polyadenylic acid signal that works is contained in 3 ' the non-district that translates of chimeric plant gene of the present invention in plant, so that adenylic acid (AMP) Nucleotide is added to the 3 ' end of RNA.The example in preferred 3 ' district is that (1) contains edaphic bacillus tumor inducing (Ti) plasmid gene, the non-plant gene [Fischhoff et al] of translating district and (2) similar soybean 7s storage protein plasmagene and pea ssRUBISCO E9 gene of transcribing as 3 ' of the polyadenylic acid signal of courage fat alkali synthetic enzyme (NOS) gene.The position

The carrier that contains above-mentioned potato storage protein box is expression activity albumen in the tenuigenin of vegetable cell or vacuole.This just needs major part or whole potato storage protein introduced plant Secretory Pathway.In order to reach this purpose, preferably use signal sequence, but the plant gene of expection is preferred from bacterium or plant gene.The example of described signal sequence is those sequences from endoproteinase B gene (Koehler and Ho) and tobacco PRlb gene (Cornelissen et al.).Disclose in 0385962 disclosed pMON10824 at EP and a kind ofly express the proteinic plant conversion carrier of lepidopteran-active B.t.kurstaki through being designed for.In pMON10824,10 amino acid that B.t.k. encoding sequence and PR1b signal sequence add ripe PR1b encoding sequence merge.In order to obtain the wherein carrier of PR1b signal and potato storage protein gene fusion,, separate the little BglII-NcoI fragment that contains the PR1b signal then with BglII and NcoI cutting pMON10824.In ligation, little BglII-NcoI pMON10824 fragment is mixed with pMON19470 (Brown et al.) from the 10kb NcoI-EcoRI fragment of pMON19714 and BamHI-EcoRI digestion.This reaction makes up a plasmid, and wherein potato storage protein encoding sequence merges with secretion signal from the PR1b gene, and is subjected to the CaMV35S promotor and is used for the control of the intron that monocotyledons expresses.For the genetic expression of dicotyledons, can carry out similar reaction.The NotI-NotI fragment of dicotyledons expression vector can be inserted in the dicotyledons conversion carrier hereinafter described, transfer to then among the unshielded edaphic bacillus host, be used to transform dicotyledons.Therefore, can obtain producing the plant that the potato storage protein is secreted into extracellular space.

The NotI-NotI fragment of this monocotyledons plasmid can be inserted corn conversion carrier (as above-mentioned pMON18181) to produce the maize plant of secretion potato storage protein.

Advantageously instruct the potato storage protein to navigate in another cellular regions chloroplast(id).By being included in the N-end that chloroplast(id) transmits peptide (CTP), protein can be moved on to chloroplast(id).The known little subunit gene of RUBISCO that can make a kind of CTP that heterologous protein is positioned chloroplast(id) from Arabidopsis is called ats1A.Made up the varient of this transmission peptide, 23 amino acid that its coding transmits peptide, ripe RUBISCO sequence add a multiple transmission peptidase cleavage site point, successfully to carry out the proteinic chloroplast(id) of B.t.k. location.The pMON19642 that people such as Brown describe contains the Arabidopsis atslA transmission peptide with the GOX gene fusion, and available certain structure is used for the localized carrier of potato storage protein chloroplast(id).PMON19643 with EcoRI fully and NcoI partly digest, separate (4.0kb) fragment greatly.In ligation, mix with the big fragment of pMON19643 from the NcoI-EcoRI fragment of pMON19714.This reaction has made up a plasmid, wherein potato storage protein encoding sequence with transmit peptides with the amino acid whose Arabidopsises of 23 of ripe RUBISCO and merge, and this plasmid is controlled by the CaMVE35S promotor.Can make up another similar plasmid, replace above-mentioned promotor with the FMV35S promotor.Described plasmid is transferred among the edaphic bacillus host of no defence capability, be used to transform dicotyledons.In addition, by above-mentioned, with the NotI-NotI fragment cloning in the corn conversion carrier.Therefore, can obtain producing the potato storage protein that is arranged in chloroplast(id).Plant Transformation and expression

All the chimeric plant gene that contains structured coding sequence of the present invention can be inserted Plant Genome through any appropriate means.Suitable plant conversion carrier comprises those carriers that derive from the edaphic bacillus Ti-plasmids, and for example by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO disclose 0120516 (Schilperoort et al.) those disclosed, except that the plant conversion carrier of Ti that derives from edaphic bacillus or root induction (Ri) plasmid, also can DNA construct of the present invention be inserted vegetable cell with other method.Described method can comprise that for example, the chemical substance of using liposome, electroporation, increase dissociative DNA to absorb discharges and use the conversion of virus or pollen through the dissociative DNA of micro-emission bullet bombardment (microprojectile bombardment).The transient expression of potato storage protein in tobacco cell

For transforming the useful especially plasmid box of dicotyledons carrier is pMON11794.Expression cassette pMON11794 comprises 3 ' the terminal composition of the polyadenylic acid signal of NOS gene by CaMVE35S promotor, the non-leader sequence of translating of the Hsp705 ' that leads a cow.PMON11794 comprises NcoI and the EcoRI site that is used to insert encoding sequence, is positioned at the NotI-NotI site of plant group expression cassette flank.

PatA (SEQ ID NO:11), PatB+ (SEQ ID NO:16), PatC+ (SEQ ID NO:17) and PatG+ (SEQ ID NO:22) are respectively inserted pMON11794 to obtain pMON19745, pMON19742, pMON19743, pMON19744 respectively.Each carrier is all gone in the tobacco protoplast through electroporation.Determine the potato storage protein expression that the transformation of tobacco cell carries out by the Western engram analysis.The stable conversion of dicotyledons (dicots)

Reported with potato storage protein stable gene by people such as Rosahl and transformed dicotyledons.Under the control of leaf and stem specificity promoter, with potato storage protein gene transformation tobacco.The potato storage protein is expressed.

The NotI-NotI fragment of pMON19745 is inserted pMON17227 (by the Ti-plasmids that people such as Barry disclose and describe, the document is mixed this paper as a reference) to obtain pMON22566 in WO92/04449.This carrier contains the Rebel Garden resistant gene of being described by Barry that is used to select the conversion plant.Similarly obtain carrier pMON22563,22564 and 22565 respectively with SEQ ID NO:16, SEQ ID NO:17 and SEQ IDNO:22.

These carriers are imported among the edaphic bacillus ABI of no defence capability, and be used for the tomato explant that transforming tissue is cultivated.Behind screening Rebel Garden resistance and the plant regeneration, reclaim all plant of expressing the potato storage protein.Determine potato storage protein expression of gene by the Western engram analysis, PRELIMINARY RESULTS show expression level gross protein 0.1 to 0.5% between.Use insect larvae to carry out biological detection.The transient expression of potato storage protein in maize cell

The 1kb NcoI-EcoRI fragment of above-mentioned pMON9729 is inserted pMON19433 (at Wo93/19189 and unsettled U.S. Patent application series No.07/855,857 (1992,3,19 applications) describe in people such as () Brown, the document is mixed this paper as a reference).With the plasmid pMON19731 of NotI digestion gained, the gained fragment is inserted also among the pMON10081 that is described by people such as Brown to obtain pMON19740.This plasmid is pressed Sheen, and 1991 described electroporations are in the leaf of Semen Maydis protoplastis.Determine of the expression of the corn protoplastis of conversion with the Western engram analysis to the potato storage protein.

In order to obtain the cytoplasmic expression of potato storage protein, the NcoI-EcoRI fragment of pMON19714 is inserted pMON19433 to obtain pMON19730.The NotI fragment of pMON19730 is inserted pMON10081, the plasmid pMON19739 electroporation of gained in the leaf of Semen Maydis protoplastis, is determined the potato storage protein of producing by the Western engram analysis.

In the corn protoplastis, also express and have or not with the Pat17 of target signal.The 1.1kb NcoI-EcoRI fragment (SEQ ID NO:30) of coded protein Pat17+ (ripe Pat17 protein and its own signal sequence about the vacuole target) is inserted among the pMON19648.Therefore, pMON19761 contains CaMVE35S promotor, Hsp70 intron, Pat17+ gene and is used for the NOS terminator that maize cell is expressed.

In order to obtain being used for the sanction body of cytoplasmic expression, the Pat17+ sequence among the NcoI-EcoRI fragment replacement pMON19761 of the Pat17m protein (SEQ ID NO:31) of usefulness coding pMON25213 is to form construct pMON25223.

Two fragments of 1Kb NcoI-EcoRI that the chloroplast(id) of Arabidopis thaliana (Arabidopsisthaliana) the SSUla gene (Timko et al.) by will containing pMON19643 (Brown et al.) transmits the 0.3kb XbaI-NcoI fragment of peptide (CTP) and pMON25213 Pat17m are inserted pMON19761 (XbaI-EcoRI) and are obtained pMON25224.Therefore, pMON25224 contains CaMVE35S promotor, Hsp7O intron, CTP/Pat17m encoding sequence and NOS terminator.

In order to reach, 5 ' of the internal protein enzyme B cDNA (Koehler and Ho) of encoded secreted protein extracellular signal peptide is terminally linked to each other with the Pat17m gene of pMON25213 in extracellular target.Obtain containing the BglII-EcoRI fragment of mosaic gene with the overlapping elongation technology of splicing (Horton et al.), be inserted into pMON19761 (BamHI-EcoRI) then to obtain pMON25225.

All these construct electroporations are determined the expression of Par17+ in the leaf of Semen Maydis protoplastis with the Western engram analysis.With potato storage protein stable gene maize transformation

Make up corn conversion carrier pMON18181 from pMON19653 and pMON19643 (Brown et al.).This construct contains CaMVE35S promotor, Hsp70 intron, CP4 Rebel Garden selective marker and NOS and stops molecular box: the box that CaMVE35S promotor, Hsp70 intron, GOX Rebel Garden selective marker, NOS terminator and a NotI site (being used to insert the expression casette that contains potato storage protein base) are formed.SEQ ID NO:11 and SEQ ID NO:30 are respectively inserted pMON18181 to obtain pMON19746 and pMON19764 respectively as the NotI-NotI fragment.

It is described to press people such as Brown, inserts these carriers by the embryo generation tissue culture blast technique that uses the biolistic particle gun.With Rebel Garden resistance screening cell transformed, and the complete plant that regenerates.Use the Western engram analysis, esterase activity detects, and/or the insect-resistant detection confirms that the level of insect-resistant plants expressing gene is the 0.1-0.5% of gross protein.Synthetic gene is used to improve monocotyledonous expression

Proved that modifying encoding sequence can improve other insecticidal protein gene such as bacillus thuringiensis delta-endotoxin sequence (Fischhoff and Perlak; WO93/07278, expression Ciba-Geigy).Therefore modify encoding sequence to improve the potato storage protein plant, the particularly expression in the corn.In SEQ ID NO:33, listed the Pat17 sequence of modified.Synthetic contain the dna fragmentation of SEQ ID NO:33 and be inserted into corn expression cassette carrier, as pMON19470 (Brown et al.).Then but corn expression cassette insertion pMON18181 or other are contained in the maize plant conversion carrier of corn conversion selectable marker gene, just can obtain expressing the complete milpa of Pat17+.From as can be seen above-mentioned, the present invention can obtain apparent and its intrinsic advantage of all above-mentioned results and purpose and the present invention fully.Should understand certain feature and secondary combined tool practicality, and also can not use with reference to further feature and secondary combined.This is realized also within the scope of the claims by claim.Under the situation that does not exceed the scope of the invention, can finish many possible embodiments, therefore, the listed all substances of this paper all are illustrative and meaning without limits.

As if concrete every piece of document or the patent of also having illustrated separately mixed this paper as a reference, and all documents in this specification sheets and patent are mixed this paper as a reference in a word.

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Sequence table (1) physical data: (i) applicant:

(A) title: Monsanto Company

(B) street: 800 North Lindbergh Boulevard

(C) city: St.Louis

(D) state: Missouri

(E) country: the United States of America

(F) postcode: 63167

(G) phone: (314) 694-3131

(H) fax: (314) 694-5435 is the invention exercise question (ii): the (iii) sequence number of method of control insect: 33 (iv) computer-reader forms:

(A) media type: Floppy disk

(B) computer: IBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.25 (EPO) (vi) application materials formerly:

(A) application number: US08/031146

(B) data of the applying date: 12-MAR-1993 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: the data of SEQ ID NO:1:Lys Leu Glu Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly 15 10 15 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): the data of peptide (xi) sequence description SEQ ID NO:2:Xaa Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly1 5 10 15 (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: the data of SEQ ID NO:3:Thr Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly1 5 10 15 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: the data of SEQ ID NO:4:Thr Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly1 5 10 15 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: the data of SEQ ID NO:5:Lys Leu Xaa Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly1 5 10 15 (2) SEQ ID NO:6: (i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:6:

Xaa?Xaa?Glu?Glu?Met?Val?Thr?Val

The data of 15 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:7:

Ser?Leu?Asp?Tyr?Lys?Gln?Met?Leu?Leu?Leu?Ser?Leu?Gly?Thr?Gly

The data of 15 10 15 (2) SEQ ID NO:8: (i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:8:

Lys?Leu?Asp?Tyr?Lys?Gln?Met?Leu

The data of 15 (2) SEQ ID NO:9: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:9:

Ser?Leu?Xaa?Tyr?Lys?Gln?Met?Leu?Leu?Leu?Ser?Leu?Gly?Thr?Gly

The data of 15 10 15 (2) SEQ ID NO:10: (i) sequence signature:

(A) length: 15 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide (xi) sequence description: SEQ ID NO:10:

Ser?Leu?Asn?Tyr?Lys?Gln?Met?Leu?Leu?Leu?Ser?Leu?Gly?Thr?Gly

The data of 15 10 15 (2) SEQ ID NO:11: (i) sequence signature:

(A) length: 1171 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：11：CCATGGCAAC TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60CAACATGTGC TAAGTTGGAA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360TTATTGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATTACTTTA 600TTACTCATAC TAGTAATGGT GATATATATG AGTTCAATCT TGTTGATGGT GGTGTTGCTA 660CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCACA AGAGGCAGCT AAATGGGGTC 840CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TGATAGAATT C 1171 ( 2 ) SEQ ID NO：12： ( i ) ：

(A) length: 37 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA (synthetic) is sequence description (xi): SEQ ID NO:12:

The data of CATGTGCTCT AGAAGATCTC CACCATGGCG TTGGAAG (2) SEQ ID NO:13: (i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA (synthetic) is sequence description (xi): SEQ ID NO:13:

The data of GCTTCTTATT GATAGAATTC AAGGTC (2) SEQ ID NO:14: (i) sequence signature:

(A) length: 1105 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：14：CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60TCATTCCAGC TATCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 240TTGTACCCTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTATTG 300GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATTAC TTTATTACTC 540ATACTAGTAA TGGTGATATA TATGAGTTCA ATCTTGTTGA TGGTGGTGTT GCTACTGTTG 600GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720CTAATTCAGA GTTTGATAAA ACATATACAG CACAAGAGGC AGCTAAATGG GGTCCTCTAC 780GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840ACATTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960TAGTACAAGT TGGTGAAACA TTATTGAAGA AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080ACAAAGCTTC TTATTGATAG AATTC 1105 ( 2 ) SEQ ID NO：15： ( i ) ：

(A) length: 1106 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：15：CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60TCATTCCAGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGATA 240TTGTACCCTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTATTG 300GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATTAC TTTATTACTC 540ATACTAGTAA TGGTGATATA TATGAGTTCA ATCTTGTTGA TGGTGGTGTT GCTACTGTTG 600GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720CTAATTCAGA GTTTGATAAA ACATATACAG CACAAGAGGC AGCTAAATGG GGTCCTCATC 780GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840ACATTTCTAC TGTTTTTCAA GCTGGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960TAGTACAAGT TGGTGAAAAA TTATTGAAGG AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020CTGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGAAAGAAA CTCCGAGCAA 1080ACAAAGCTTC TTATTAATGA GAATTC 1106 ( 2 ) SEQ ID NO：16： ( i ) ：

(A) length: 1172 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：16：CCATGGCAAC TACTAAATCT GTTTTAGTTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60CAACATGTGC TACGTTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300AAGATATTGT ACCTTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTCT AGTGGTTCAA 360TTTTTGGCCC AATGTATGAT GGAAAATATT TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540TGAATGACAT ATGCTATTCC ACAGCAGCAG CTCCAACATA TTTTCCTCCA CATTACTTTG 600TTACTCATAC TAGTAATGGA GATAAATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTCGAAC GAAACTTGCA CAAGTGGATC 720CAAAATTTGC TTCAATTAAG TCATTGAATT ACAACGAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840CTCTACGATG GATATTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900ATTATTACCT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GAAAAATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：17： ( i ) ：

(A) length: 1175 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：17：CCATGGCAAC TACTAAATCT TTTTTAATTT TAATTGTTAT GATATTAGCA ACTACTAGTT 60CAACATTTGC TTCGTTGGAA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGGTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG AAAATGGACA 180ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TTCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300ATGAAATTGT ACCTTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTCT AGGTACTGGC 360CAATTTTTTG GCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAACCTTG 420GAGAAACTCG TGTGCATCAA GCTTTGACTG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480CAAATAAGCC AGTAATATTC ACCAAGTCAA ATTTAGCAAA GTCTCCAGAA TTGGATGCTA 540AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600TTACTACTAA TACTATTAAT GGAGATAAAT ATGAGTTCAA TCTTGTTGAT GGTGCTGTTG 660CTACTGTTGC TGATCCGGCG TTATTATCCA TTAGCGTTGC AACGAGACTT GCAGAAAAGG 720ATCCAGCATT TGCTTCAATT AGGTCATTGA ATTACAAAAA AATGTTGTTG CTCTCATTAG 780GCACTGGCAC TACTTCAGAG TTTGATAAAA CATATACAGC AGAAGAGACA GCTAAATGGG 840GTGCTATACA ATGGATGTTG GTTATACAGC GAATGACTGA TGCAGCAAGT TCTTACATGA 900CTGATTATTA CCTTTCTACT GTTTTTCAAG CTCAAAATTC ACAAAAGAAT TACCTCAGGG 960TTCAAGAAAA TGCGTTAACA GGCACAACTA CTGAAATGGA TGATGCTTCT GAGGCTAATA 1020TGGAATCATT AGTACAAGTT GGTGAAAATT TATTGAAGAA ACCAGTTCCC AAAGACAATC 1080CTGAAACCTA TGAGGAAGCT CTAAAGAGGT TTGCAAAATT GCTTTCTGAT AGGAAGAAAC 1140TTCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC 1175 ( 2 ) SEQ ID NO：18： ( i ) ：

(A) length: 1106 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：18：CCATGGCGTT CGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60/ TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATA 120AAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGGTGCT GCCAAAGATA 240TTGTACCTTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTCTAGTGGT TCAATTTTTG 300GCCCAATGTA TGATGGAAAA TATTTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480ACATATGTTA TTCCACAGCA GCAGCTCCAA CATATTTTCC TCCACATTAC TTTGTTACTC 540ATACTAGTAA TGGAGATAAA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GGTACTGTTC 600GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAAACT TGCACAAGTG GATCCAAAAT 660TTGCTTCAAT TAAGTCATTG AATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720CTAATTCAGA GTTTGATAAA ACATATACAG CAGAAGAGGC AGCTAAATGG GGTCCTCTAC 780GATGGATATT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840ACCTTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900ATGCATTAAC AGGCATAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960TAGTACAAGT TGGTGAAAAA TTATTGAAGA AACCAGTTTC CAAAGACAGT CCTGAAACCT 1020ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080ACAAAGCTTC TTATTAATGA GAATTC 1106 ( 2 ) SEQ ID NO：19： ( i ) ：

(A) length: 1172 base pairs

(B) type: nucleic acid

( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：19：CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCTTTT GCTGCTGCTA 300AAGATATTAT ACCTTTTTAC TTCGAACACG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTCTCCAAA GACAGTCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：20： ( i ) ：

( A ) ：1106 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：20：CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAGTG GACAATAATG 120CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAGGTACA GGAGGTTTAT 180TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC TTTTGCTGCT GCTAAAGATA 240TTATACCTTT TTACTTCGAA CACGGCCCTC ATATTTTTAA TTATAGTGGT TCAATTTTAG 300GCCCAATGTA TGATGGAAAA TATCTTCTGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360GTGTGCATCA AGCTTTGACA GAAGTTGCCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420CAGTAATATT CACTAAGTCA AATTTAGCAA AGTCTCCAGA ATTGGATGCT AAGATGTATG 480ACATATGCTA TTCCACAGCA GCAGCTCCAA TATATTTTCC TCCACATCAC TTTGTTACTC 540ATACTAGTAA TGGTGCTAGA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GCTACTGTTG 600GTGATCCGGC GTTATTATCC CTTAGCGTTG CAACGAGACT TGCACAAGAG GATCCAGCAT 660TTTCTTCAAT TAAGTCATTG GATTACAAGC AAATGTTGTT GCTCTCATTA GGCACTGGCA 720CTAATTCAGA GTTTGATAAA ACATATACAG CAGAAGAGGC AGCTAAATGG GGTCCTCTAC 780GATGGATGTT AGCTATACAG CAAATGACTA ATGCAGCAAG TTCTTACATG ACTGATTATT 840ACATTTCTAC TGTTTTTCAA GCTCGTCATT CACAAAACAA TTACCTCAGG GTTCAAGAAA 900ATGCATTAAA TGGCACAACT ACTGAAATGG ATGATGCGTC TGAGGCTAAT ATGGAATTAT 960TAGTACAAGT TGGTGAAACA TTATTGAAGA AACCAGTCTC CAAAGACAGT CCTGAAACCT 1020ATGAGGAAGC TCTAAAGAGA TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080ACAAAGCTTC TTATTAATGA GAATTC 1106 ( 2 ) SEQ ID NO：21： ( i ) ： ( A ) ：1109 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：21：CCATGGCGTT GGAAGAAATG GTGGCTGTTC TTAGTATTGA TGGAGGTGGA ATTAAGGGAA 60TCATTCCGGG TACCATTCTC GAATTTCTTG AAGGACAACT TCAGAAAATG GACAATAATG 120CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180TGACTGCTAT GATAACTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAATGAAA 240TTGTACCTTT TTACTTCGAA CATGGCCCTC ATATTTTTAA TTCTAGGTAC TGGCCAATTT 300TTTGGCCAAA ATATGATGGA AAATATCTTA TGCAAGTTCT TCAAGAAAAA CTTGGAGAAA 360CTCGTGTGCA TCAAGCTTTG ACAGAAGTTG CCATCTCAAG CTTTGACATC AAAACAAATA 420AGCCAGTAAT ATTCACTAAG TCAAATTTGG CAAAGTCTCC AGAATTGGAT GCTAAGACGT 480ATGACATATG TTATTCGACA GCAGCAGCTC CAACATATTT TCCTCCACAT TACTTTGCTA 540CTAATACTAT TAATGGAGAT AAATATGAGT TCAATCTTGT TGATGGTGCT GTTGCTACTG 600TTGCTGATCC GGCGTTATTA TCCGTTAGCG TTGCAACGAG ACGTGCACAA GAGGATCCAG 660CATTTGCTTC AATTAGGTCA TTGAATTACA AAAAAATGTT GTTGCTCTCA TTAGGCACTG 720GCACTACTTC AGAGTTTGAT AAAACACATA CAGCAGAAGA GACAGCTAAA TGGGGTGCTC 780TACAATGGAT GTTGGTTATA CAGCAAATGA CTGAGGCAGC AAGTTCTTAC ATGACTGATT 840ATTACCTTTC TACTGTTTTT CAAGATCTTC ATTCACAAAA CAATTACCTC AGGGTTCAAG 900AAAATGCATT AACAGGCACA ACTACTAAAG CGGATGATGC TTCTGAGGCT AATATGGAAT 960TATTAGCACA AGTTGGTGAA AATTTATTGA AGAAACCAGT TTCCAAAGAC AATCCTGAAA 1020CCTATGAGGA AGCTCTAAAG AGGTTTGCAA AATTGCTTTC TGATAGGAAG AAACTTCGAG 1080CAAACAAAGC TTCTTATTAA TGAGAATTC 1109 ( 2 ) SEQ ID NO：22： ( i ) ： ( A ) ：1172 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：22：CCATGGCAAC TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60CAACATGTGC TAAGTTGGAA GAAATGGTTA CTGTTCTAAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GAAACATTAT TGAAGAAACC AGTTTCCAAA GACAGTCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：23： ( i ) ：

(A) length: 1104 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：23：CCATGGTTGG AAGAAATGGT GACTGTTCTA AGTATTGATG GAGGTGGAAT TAAGGGAATC 60ATTCCAGCTA TCATTCTCGA ATTTCTTGAA GGACAACTTC AGGAAGTGGA CAATAATAAA 120GATGCAAGAC TTGCAGATTA CTTTGATGTA ATTGGAGGAA CAAGTACAGG AGGTTTATTG 180ACTGCTATGA TAACTACTCC AAATGAAAAC AATCGACCCT TTGCTGCTGC CAAAGATATT 240GTACCCTTTT ACTTCGAACA TGGCCCTCAT ATTTTTAATT ATAGTGGTTC AATTTTAGGC 300CCAATGTATG ATGGAAAATA TCTTCTGCAA GTTCTTCAAG AAAAACTTGG AGAAACTCGT 360GTGCATCAAG CTTTGACGGA AGTTGCCATC TCAAGCTTTG ACATCAAAAC AAATAAGCCA 420GTAATATTCA CTAAGTCAAA TTTAGCAAAG TCTCCAGAAT TGGATGCTAA GATGTATGAC 480ATATGCTATT CCACAGCAGC AGCTCCAATA TATTTTCCTC CACATCACTT TGTTACTCAT 540ACTAGTAATG GTGCTAGATA TGAGTTCAAT CTTGTTGATG GTGCTGTTGC TACTGTTGGT 600GATCCGGCGT TATTATCCCT TAGCGTTGCA ACGAGACTTG CACAAGAGGA TCCAGCATTT 660TCTTCAATTA AGTCATTGGA TTACAAGCAA ATGTTGTTGC TCTCATTAGG CACTGGCACT 720AATTCAGAGT TTGATAAAAC ATATACAGCA GAAGAGGCAG CTAAATGGGG TCCTCTACGA 780TGGATGTTAG CTATACAGCA AATGACTAAT GCAGCAAGTT TTTACATGAC TGATTATTAC 840ATTTCTACTG TTTTTCAAGC TCGTCATTCA CAAAACAATT ACCTCAGGGT TCAAGAAAAT 900GCATTAAATG GCACAACTAC TGAAATGGAT GATGCGTCTG AGGCTAATAT GGAATTATTA 960GTACAAGTTG GTGAAACATT ATTGAAGAAA CCAGTTTCCA GAGACAGTCC TGAAACCTAT 1020GAGGAAGCTC TAAAGAGATT TGCAAAATTG CTCTCTGATA GGAAGAAACT CCGAGCAAAC 1080AAAGCTTCTT ATTAATGAGA ATTC 1104 ( 2 ) SEQ ID NO：24： ( i ) ：

(A) length: 1172 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：24：CCATGGCAAC TACTAAATCT TTTTTAATTT TATTTTTTAT GATATTAGCA ACTACTAGTT 60CAACATGTGC TAAGTTGGAA GAAATGGTTA CTGTTCTAAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCAGCTATC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATAAAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300AAGATATTGT ACCCTTTTAC TTCGAACATG GCCCTCATAT TTTTAATTAT AGTGGTTCAA 360TTTTAGGCCC AATGTATGAT GGAAAATATC TTCTGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGCCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAAGTC TCCAGAATTG GATGCTAAGA 540TGTATGACAT ATGCTATTCC ACAGCAGCAG CTCCAATATA TTTTCCTCCA CATCACTTTG 600TTACTCATAC TAGTAATGGT GCTAGATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660CTGTTGGTGA TCCGGCGTTA TTATCCCTTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTTC TTCAATTAAG TCATTGGATT ACAAGCAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GATAAAACAT ATACAGCAGA AGAGGCAGCT AAATGGGGTC 840CTCTACGATG GATGTTAGCT ATACAGCAAA TGACTAATGC AGCAAGTTCT TACATGACTG 900ATTATTACAT TTCTACTGTT TTTCAAGCTC GTCATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAAATGGC ACAACTACTG AAATGGATGA TGCGTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GCAACATTAT TGAAGAAACC AGTCTCCAAA GACAGTCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGATTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：25： ( i ) ：

(A) length: 1175 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：25：CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCTTTT GCTGCTGCTA 300AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTAAGAT TTTTGAACCT AGTGGTTTTC 360ACCTTTTTGA GCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAAACTTG 420GAGAAACTCG TGTGCATCAA GCTTTGACAG GAGTTGCCAT CTCAAGCTTT GACATCAAAA 480CAAATAAGCC AGTAATATTC ACTAAGTCAA GTTTAGCAAA AACTCCAGAA TTGGATGCTA 540AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACCTCAA TCTTGTTGAT GGCGATGTTG 660CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTGC TTCAATTAAG TCATTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAA TTCAGAGTTT GCTAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840TTCTACAATG GGTATTCTCA CCTTTATGGG AAATGAGAAG TGCAGCAAGT TCTTACATGA 900ATGATTATTA CCTTTCTACT GTTTTTCAAG CTCTTGATTC ACAAAACAAT TACCTCAGGG 960TTCAAGAAAA TGCATTAACA GGCACAGCTA CTACATTTGA TGATGCTTCT CTGGCTAATA 1020TGATATTATT AGTACAAGTT GGTGAAAACT TATTGAAGAA ATCAGTTTCC GAAGACAATC 1080ATGAAACCTA TGAGGTAGCT CTAAAGAGGT TTGCAAAATT GCTCTCTGAT AGGAAGAAAC 1140TCCGAGCAAA CAAAGCTTCT TATTAATGAG AATTC 1175 ( 2 ) SEQ ID NO：26： ( i ) ：

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA (synthetic) is sequence description (xi): SEQ ID NO:26:

The data of GTTAGATCTC ACCATGGCAA CTACTAAATC TTT (2) SEQ ID NO:27: (i) sequence signature:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): DNA (synthetic) is sequence description (xi): SEQ ID NO:27:

The data of CCAGAATTCT CATTAATAAG AAGCTTTGTT TGC (2) SEQ ID NO:28: (i) sequence signature:

(A) length: 1172 base pairs

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：28：CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATACAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCTA 300AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTCAGAT TTTTGAACCT AGTGGTCTTC 360AAATTTTTGG CCCAAAATAT GATGGAAAAT ATCTTATGCA AGTTCTTCAA GAAAAACTTG 420GAGAAACTCG TGTGCATCAA GCTTTGACAG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480CAAATAAGCC AGTAATATTC ACTAAGTCAA ATTTAGCAAA AACTCCAGAA TTGGATGCTA 540AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACTTCAA TCTTGTTGAT GGTGATGTTG 660CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTGC TTCAATTAGG TCGTTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAC TTCAGAGTTT TATAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840TTCTACAATG GCTGTTACCT TTACAGGAAA TGAGAAGTGC AGCAAGTTCT TACATGAATG 900ATTATTACCT TTCTACTGTT TTTCAAGCTC TTGATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAACAGGC ACAGCTACTA AATTTGATGA TGCTTCTGTG GCTAATATGA 1020TATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAATC AGTTTCTGAA GACAATCATG 1080AAACCTATGA GGTAGCTCTA AAGAGGTTTG CAAAATTGCT CTCCGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：29： ( i ) ：

(A) length: 1172 base pairs

(B) type: nucleic acid

( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：29：CCATGGCAAC TACTAAATCT TTTACAATTT TAATTTTTAT GATGTTAGCA ACTACTAGTT 60CAACATTTGC TACATTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120AGGGAATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAGTGGACA 180ATAATACAGA TGCAAGACCT GCAGATTACT TTGATGTAAT TGGAGGAACA GGTACAGGAG 240GTTTATTGAC TGCTATGATA ACTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCTA 300AAGATATTAT ACCTTTTTAC TTCGATCATG GCCCTCAGAT TTTTGAACCT AGTGGTTCAA 360TTTTTGATGG CCCAAAATAT GATGGAAAAC ATCTTATGCA AGTTCTTCAA GAAAAACTAG 420GAGAAACTCG TGTGCATCAA ACTTTGACAG AAGTTGCCAT CTCAAGCTTT GACATCAAAA 480CAAATAAGCC AGTAATATTC ACTAAGTCAA ATTTACCAAA AACTCCAGAA TTGGATGCTA 540AGATGTATGA CATATGTTAT TCCACAGCAG CAGCTCCAAC ATATTTTCCT CCACATTACT 600TTGCTACTAA TACTAGTAAT GGAGATCAAT ATGACTTCAA TCTTGTTGAT GGTGATGTTG 660CTGCTGGTGA TCCGTCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAGAGGATC 720CAGCATTTGC TTCAATTAGG TCGTTGAATT ACAAACAAAT GTTGTTGCTC TCATTAGGCA 780CTGGCACTAC TTCAGAGTTT TATAAAAACT ATACAGCAGA AGAGGCAGCT AAATGGGGTA 840TTCTACAATG GCTGTTACCT TTACAGGAAA TGAGAAGTGC AGCAAGTTCT TACATGAATG 900ATTATTACCT TTCTACTGTT TTTCAAGCTC TTGATTCACA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAACAGGC ACAGCTACTA AATTTGATGA TGCTTCTGTG GCTAATATGA 1020TATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAATC AGTTTCTGAA GACAATCATG 1080AAACCTATGA GGTAGCTCTA AAGAGGTTTG CAAAATTGCT CTCCGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：30： ( i ) ：

(A) length: 1172 base pairs

( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( Xi ) ：SEQ ID NO：30：CCATGGCAAC TACTAAATCT TTTTTAATTT TAATATTTAT GATATTAGCA ACTACTAGTT 60CAACATTTGC TCAGTTGGGA GAAATGGTGA CTGTTCTTAG TATTGATGGA GGTGGAATTA 120GAGGGATCAT TCCGGCTACC ATTCTCGAAT TTCTTGAAGG ACAACTTCAG GAAATGGACA 180ATAATGCAGA TGCAAGACTT GCAGATTACT TTGATGTAAT TGGAGGAACA AGTACAGGAG 240GTTTATTGAC TGCTATGATA AGTACTCCAA ATGAAAACAA TCGACCCTTT GCTGCTGCCA 300AAGAAATTGT ACCTTTTTAC TTCGAACATG GCCCTCAGAT TTTTAATCCT AGTGGTCAAA 360TTTTAGGCCC AAAATATGAT GGAAAATATC TTATGCAAGT TCTTCAAGAA AAACTTGGAG 420AAACTCGTGT GCATCAAGCT TTGACAGAAG TTGTCATCTC AAGCTTTGAC ATCAAAACAA 480ATAAGCCAGT AATATTCACT AAGTCAAATT TAGCAAACTC TCCAGAATTG GATGCTAAGA 540TGTATGACAT AAGTTATTCC ACAGCAGCAG CTCCAACATA TTTTCCTCCG CATTACTTTG 600TTACTAATAC TAGTAATGGA GATGAATATG AGTTCAATCT TGTTGATGGT GCTGTTGCTA 660CTGTTGCTGA TCCGGCGTTA TTATCCATTA GCGTTGCAAC GAGACTTGCA CAAAAGGATC 720CAGCATTTGC TTCAATTAGG TCATTGAATT ACAAAAAAAT GCTGTTGCTC TCATTAGGCA 780CTGGCACTAC TTCAGAGTTT GATAAAACAT ATACAGCAAA AGAGGCAGCT ACCTGGACTG 840CTGTACATTG GATGTTAGTT ATACAGAAAA TGACTGATGC AGCAAGTTCT TACATGACTG 900ATTATTACCT TTCTACTGCT TTTCAAGCTC TTGATTCAAA AAACAATTAC CTCAGGGTTC 960AAGAAAATGC ATTAACAGGC ACAACTACTG AAATGGATGA TGCTTCTGAG GCTAATATGG 1020AATTATTAGT ACAAGTTGGT GAAAACTTAT TGAAGAAACC AGTTTCCGAA GACAATCCTG 1080AAACCTATGA GGAAGCTCTA AAGAGGTTTG CAAAATTGCT CTCTGATAGG AAGAAACTCC 1140GAGCAAACAA AGCTTCTTAT TAATGAGAAT TC 1172 ( 2 ) SEQ ID NO：31：

(i) sequence signature:

( A ) ：1106 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：cDNA ( xi ) ：SEQ ID NO：31：CCATGGCGTT GGAAGAAATG GTGACTGTTC TTAGTATTGA TGGAGGTGGA ATTAGAGGGA 60TCATTCCGGC TACCATTCTC GAATTTCTTG AAGGACAACT TCAGGAAATG GACAATAATG 120CAGATGCAAG ACTTGCAGAT TACTTTGATG TAATTGGAGG AACAAGTACA GGAGGTTTAT 180TGACTGCTAT GATAAGTACT CCAAATGAAA ACAATCGACC CTTTGCTGCT GCCAAAGAAA 240TTGTACCTTT TTACTTCGAA CATGGCCCTC AGATTTTTAA TCCTAGTGGT CAAATTTTAG 300GCCCAAAATA TGATGGAAAA TATCTTATGC AAGTTCTTCA AGAAAAACTT GGAGAAACTC 360GTGTGCATCA GGCTTTGAGA GAAGTTGTCA TCTCAAGCTT TGACATCAAA ACAAATAAGC 420CAGTAATATT CACTAAGTCA AATTTAGCAA ACTCTCCAGA ATTGGATGCT AAGATGTATG 480ACATAAGTTA TTCCACAGCA GCAGCTCCAA CATATTTTCC TCCCCATTAC TTTGTTACTA 540ATACTAGTAA TGGAGATGAA TATGAGTTCA ATCTTGTTGA TGGTGCTGTT GCTACTGTTG 600CTGATCCGGC GTTATTATCC ATTAGCGTTG CAACGAGACT TGCACAAAAG GATCCAGCAT 660TTGCTTCAAT TAGGTCATTG AATTACAAAA AAATGCTGTT GCTCTCATTA GGCACTGGCA 720CTACTTCAGA GTTTGATAAA ACATATACAG CAAAAGAGGC AGCTACCTGG ACTGCTGTAC 780ATTGGATGTT AGTTATACAG AAAATGACTG ATGCAGCAAG TTCTTACATG ACTGATTATT 840ACCTTTCTAC TGCTTTTCAA GCTCTTGATT CAAAAAACAA TTACCTCAGG GTTCAAGAAA 900ATGCATTAAC AGGCACAACT ACTGAAATGG ATGATGCTTC TGAGGCTAAT ATGGAATTAT 960TAGTACAAGT TGGTGAAAAC TTATTGAAGA AACCAGTTTC CGAAGACAAT CCTGAAACCT 1020ATGAGGAAGC TCTAAAGAGG TTTGCAAAAT TGCTCTCTGA TAGGAAGAAA CTCCGAGCAA 1080ACAAAGCTTC TTATTAATGA GAATTC 1106 ( 2 ) SEQ ID NO：32： ( i ) ：

(A) length: 45 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (synthetic)

(xi) sequence description: SEQ ID NO:32:

The data of CCATCTAGAA GATCTCCACC ATGGCGTTGG GAGAAATGGT GACTG (2) SEQ ID NO:33:

(i) sequence signature:

(A) length: 1164 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

( xi ) ：SEQ ID NO：33：ATGGCCACCA CCAAGAGCTT CCTCATCCTG ATCTTCATGA TCCTGGCCAC CACCAGCAGC 60ACCTTCGCCC AGCTCGGCGA GATGGTGACC GTGCTCTCCA TCGACGGCGG TGGCATCAGG 120GGCATCATCC CGGCCACCAT CCTGGAGTTC CTGGAGGGCC AACTCCAGGA GATGGACAAC 180AACGCCGACG CCCGCCTGGC CGACTACTTC GACGTGATCG GTGGCACCAG CACCGGCGGT 240CTCCTGACCG CCATGATCTC CACTCCGAAC GAGAACAACC GCCCCTTCGC CGCTGCGAAG 300GAGATCGTCC CGTTCTACTT CGAACACGGC CCTCAGATTT TCAACCCCTC GGGTCAAATC 360CTGGGCCCCA AGTACGACGG CAAGTACCTT ATGCAAGTGC TTCAGGAGAA GCTGGGCGAG 420ACTAGGGTGC ACCAGGCGCT GACCGAGGTC GTCATCTCCA GCTTCGACAT CAAGACCAAC 480AAGCCAGTCA TCTTCACCAA GTCCAACCTG GCCAACAGCC CGGAGCTGGA CGCTAAGATG 540TACGACATCT CCTACTCCAC TGCTGCCGCT CCCACGTACT TCCCTCCGCA CTACTTCGTC 600ACCAACACCA GCAACGGCGA CGAGTACGAG TTCAACCTTG TTGACGGTGC GGTGGCTACG 660GTGGCGGACC CGGCGCTCCT GTCCATCAGC GTCGCCACGC GCCTGGCCCA GAAGGATCCA 720GCCTTCGCTA GCATTAGGAG CCTCAACTAC AAGAAGATGC TGCTGCTCAG CCTGGGCACT 780GGCACGACCT CCGAGTTCGA CAAGACCTAC ACTGCCAAGG AGGCCGCTAC CTGGACCGCC 840GTCCATTGGA TGCTGGTCAT CCAGAAGATG ACGGACGCCG CTTCCAGCTA CATGACCGAC 900TACTACCTCT CCACTGCGTT CCAGGCGCTT GACTCCAAGA ACAACTACCT CCGTGTTCAG 960GAGAATGCCC TCACTGGCAC CACGACCGAG ATGGACGATG CCTCCGAGGC CAACATGGAG 1020CTGCTCGTCC AGGTGGGTGA GAACCTCCTG AAGAAGCCCG TCTCCGAAGA CAATCCCGAG 1080ACCTATGAGG AAGCGCTCAA GCGCTTTGCC AAGCTGCTCT CTGATAGGAA GAAACTCCGC 1140GCTAACAAGG CCAGCTACTA ATGA 1164

Claims are according to the modification of the 19th of treaty

6. the method for claim 5, wherein said structured coding sequence comprises SEQ IDNO:30 or SEQ ID NO:31.

7. the method for claim 5, wherein said plant is a corn, and described structured coding sequence is a synthetic, to improve the expression in monocotyledons.

8. the method for claim 7, wherein said structured coding sequence comprises SEQ ID NO:32.

9. a plant that is produced by the method for claim 5 wherein comprises the endotoxic gene of one or more expression B.t. in genome.

10. a seed that produces by the plant of claim 9 or plant subdivision.

Claims (11)

1. the method for the plant infection that causes because of food plant insect of a control comprises providing the desinsection of significant quantity potato storage protein for described insect picked-up.

2. the process of claim 1 wherein that described potato storage protein is to grow microorganism surely and provide by being applied to the plant that produces described potato storage protein behind the plant.

3. the process of claim 1 wherein that the potato storage protein gene that is incorporated in the described plant by expression provides described potato storage protein, the parental cell of described plant is in advance through genetic transformation.

4. the method for claim 3, described plant wherein is cotton, corn, tomato or potato.

5. method of producing the insect-resistant plants of the potato storage protein that transforms through genetic engineering method, express effective desinsection amount comprises step:

(a) the reorganization double chain DNA molecule that will contain following component inserts the genome of vegetable cell;

(i) in vegetable cell, cause the promotor that the RNA sequence produces;

(ii) the encode structured coding sequence of potato storage protein;

(iii) in described vegetable cell, make polyadenylic acid Nucleotide be added to RNA sequence 3 '

3 ' the terminal non-district that translates,

Wherein said promotor is allogenic for described structured coding sequence, and

And wherein said promotor links to each other through operating with described structured coding sequence, and is described

The structured coding sequence links to each other successively with the described non-district that translates;

(b) obtain the plant transformed cell; With

(c) effectively kill through the expression that genetic engineering method transforms from plant transformed cell regeneration

The plant of worm amount potato storage protein:

Wherein said promotor is allogenic with regard to described structured coding sequence, and institute wherein

State plant and be selected from cotton, corn, tomato and potato.

6. the method for claim 5, wherein said structured coding sequence contains SEQ ID NO:30 or SEQ ID NO:31.

7. the method for claim 5, wherein said plant is a corn, and described structured coding sequence is a synthetic, to improve the expression in monocotyledons.

8. the method for claim 7, wherein said structured coding sequence contains SEQ ID NO:32.

9. plant that produces by the method for claim 5.

10. the method for claim 9 wherein comprises the endotoxic gene of one or more expression B.t. in genome.

A 11. seed or kind subdivision that produces by the plant of claim 9.