CN101531711B - Bt protein Cry52Ba1, its coding gene and application - Google Patents

Abstract

The present invention provides a new Bt protein Cry52Ba1 and its coding gene, said protein has the amino acid sequence shown in SEQ ID No.2, or the amino acid sequence shown in SEQ ID No.2 is substituted, deleted and/or added A protein of one or more amino acids with equivalent activity. The protein of the present invention can be used to prepare Bt insecticides, and the gene can transform crops such as cotton, corn, rice, and vegetables to have corresponding insect-resistant activities, thereby reducing the use of pesticides and reducing environmental pollution, which is of great importance. Economic value and application prospect.

Description

Bt protein Cry52Ba1, its coding gene and application

technical field

The invention relates to the field of biotechnology, in particular to a novel Bt protein and its coding gene and application.

Background technique

In the process of human production, insect pests are an important factor that causes agricultural production losses and affects human health. According to FAO statistics, the annual economic losses caused by insect pests in agricultural production worldwide are as high as 14%, disease losses reach 12%, and weed damage losses reach 11%. %. The loss was as high as 126 billion US dollars, equivalent to half of China's total agricultural output value, and more than four times that of the United Kingdom. In addition, mosquito-borne diseases occupy an important position in preventive medicine, among which mosquito-borne diseases such as dengue fever and yellow fever have strong transmissibility, wide prevalence, high incidence and great harm. According to WHO statistics, as many as 80 million people are infected with dengue fever every year in the world. There were two outbreaks of dengue fever in Hainan Province of my country in 1980 and 1986, and the incidences reached 437,469 and 113,589 cases respectively. Dengue and yellow fever are mainly transmitted by the Aedes aegypti mosquito.

In order to reduce these losses, for many years, chemical control methods have been widely used to control crop pests and mosquitoes. However, due to the long-term and large-scale use of chemical pesticides, the pollution to the environment has been caused, and the amount of pesticide residues in agricultural by-products has increased. and health hazards. In addition, while chemical pesticides kill pests, they also kill natural enemies and other beneficials, destroying the ecological balance. Compared with chemical control, biological control is safe, effective and durable. And avoid a series of problems caused by chemical control. Therefore, biological control technology has become a research hotspot. Among biopesticides, Bacillus thuringiensis is currently the most widely used and most productive type of microbial pesticide in the world.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium with a wide distribution. It can form parasporal crystals composed of proteins with insecticidal activity during the formation of spores, also known as insecticidal crystals. Proteins (Insectididal crystal proteins, ICPs for short), ICPs are encoded by the cry gene, are highly toxic to sensitive insects, but non-toxic to higher animals and humans. In recent decades, Bt has been widely used to control a variety of Lepidoptera, Diptera, Coleoptera and other pests. In addition, Bt also has a harmful effect on various pests such as Hymenoptera, Homoptera, Orthoptera, and Trichophaera, as well as plant pathogenic nematodes, mites, and protozoa. At present, Bt has become a powerful substitute for chemically synthesized pesticides in the control of farmland pests, forest pests and sanitary pests. Bt is also an important gene source for transgenic insect-resistant engineering plants.

Since Schnepf cloned the first gene capable of expressing insecticidal activity from strain HD-1Dipel in 1981 (Adang M.J et al, Characterized full-length and truncated plasma clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their Toxicity to Manduca sexta, Gene, 1985, 36(3): 289～300.), people have isolated and cloned more than 390 genes encoding insecticidal crystal proteins, and they were determined to be different according to the amino acid sequence homology of encoding Groups, subgroups, classes and subclasses of (Crickmore N, Zeigler DR, Feitelson J, et al.Revision of the nomenclature for the Bacillus thuringiensis pesticide crystal proteins. Microbiol Mol Biol Rev, 1998, 62:807-813; http: https://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/). Generally speaking, toxic proteins such as Cry1, Cry2, and Cry9 are effective against Lepidoptera pests; among them, Cry1 and Cry9 are the most studied proteins, and the insecticidal crystal proteins encoded by them have a molecular weight of 130-140kD, and many genes have been widely used. Control of Lepidoptera pests applied to plants (Kozie, M.G., Beland, G.L., Bowman, C., et al. Field performance of elite transgenicmaize plants expressing an insecticidal protein derived from Bacillusthuringiensis. Bio/Technology, 1993, 11: 194 -200; Perlak, F.J., Deaton, R.W., Armstrong, T.A., et al. Insect resistant cotton plants.bio/technology, 1990; 8:939-943; Van Frankenhuyzen, K., Gringorten, L., and Gauhier, D .1997.Cry9Ca1 toxin, a Bacillus thuringiensis insecticidal crystal protein with high activity against the spruce bud worm (Choristoneurafnniferana).Appl.Environ, Microbviol.63:4132-4134; Wang Fei, 2001, Biological characteristics of Bacillus thuringiensis strain and cry9 Research on new genes, master's thesis, Nankai University). The toxin protein produced by Bacillus thuringiensis subsp.israelensis (Bti) has good insecticidal activity against mosquitoes and is widely used in the control of mosquitoes (Goldberg L J, and Margalit J, 1977.A bacterialspore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, and Culexpiens. Mosqito News, 37:355-358;). Simultaneously, Cyt protein is cytolytic, has synergistic effect to some Cry proteins and delays insect resistance (Wu, D., Johnson, J.J., and Federici, B.A.1994.Synergism of mosquito cidal toxicity between CytA and CryIVD Proteins using inclusion sproduced from clonedgenes of Bacillus thuringiensis.Mol.Microbiol.13：965-972；Wirth，M.C.，Georghiou，G.P.，and Federeci，B.A.1997.CytA enables CryIVendotoxins of Bacillus thuringiensis to overcome high levels of CryIVresistance in the mosquito，Culex quinquefasciatus. Proc. Natl. Acad. Sci. 94:10536-10540)

It has been more than 100 years since the discovery of Bacillus thuringiensis at the beginning of this century. It has been widely used in the control of crops and horticultural plant pests, forest pests and sanitary pests, and has also achieved good results. However, due to large-scale and repeated use of Bacillus thuringiensis, many insect populations have successively developed resistance to insecticidal crystal proteins to varying degrees. Insecticides based on Bt insecticidal crystal protein have been used for more than 50 years. At first, insect resistance to Bt was not detected. It has been confirmed in field experiments (M cGaughey, W.H.1985. Insect resistance to the biological insecticide Bacillus thuringiensis. Science. 229: 193-195), mainly due to the continuous use of single species and sublethal doses of Bt and Bt transgenic insect-resistant plants The application of insecticides caused insect populations to be under long-term selection pressure of insecticides. In 1985, McGaughey reported that the warehouse grain pest Indian meal moth (Plodia interpunctella) was under the selection pressure of Dipel (the commercial preparation of Bt subsp. kurstaik HD-1), after 15 generations of reproduction, the resistance increased by 97 times; The resistance can be increased by 250 times. In 1990, it was first confirmed in Hawaii that the diamondback moth in the field showed significant resistance to Bt insecticides (Tabashnik, B.E., Finson, N., Groeters, F.R., et al.1994. Reversal of resistance to Bacillus thuringiensisin Plutella xylostella .Proc.Natl.Acad.Sci.USA.91:4120-4124), since the 1990s, in Shenzhen, Guangzhou, Shanghai and other places where Bt insecticides have been used for a long time in China, it has been found that Bt insecticides have a negative effect on side dishes. The moth control effect has obviously declined, which means that resistance has formed (Feng Xia. 1996. Research on the resistance of diamondback moth in Guangdong to Bacillus thuringiensis. Acta Entomology, 39 (3): 238-244; Hofte, H., Van Rie, J ., Jansens, S., Van Houtven, A., Vanderbruggen, H., and Vaeck, M., 1988.Monoclonal antibody analysis and insecticidal spectrum ofthree types of lepidopteran-specific insecticidal crystal proteins of Bacillus thuringiensis.Appl.Environ.Microbi 54:2010-2017). At present, it is found that at least a dozen kinds of insects have developed resistance to Bt and its insecticidal crystal protein in the laboratory and in the field. It is predicted by the mathematical model of selection pressure that under the condition of selection pressure of Bt transgenic insect-resistant plants, insects will produce Resistance (Schnepf, E., Crickmore, N., Van Pie, J., et al. 1998. Bacillus thuringiensis and its pesticidal Crystalproteins. Microbiol. Mol. Biol. Rev. 65(3): 775-806). In addition, studies have proved that Bti has not found resistance problems in the use of field (Regis L, et al., 2000. The use of bacterial larvicides in mosquito and black fly control programs in Brazil. Mem. Instituto Oswaldo Cruz, 95: 207-210. ), but the problem of mosquito resistance to it has been confirmed in the laboratory, and this situation may also appear in the field (Georghiou GP, and Wirth MC, 1997.Influence of exposure to singleversus multiple toxins of Bacillus thuringiensis subsp . israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology, 63: 1095-1101.).

In order to avoid the losses caused by resistant insects, finding new highly virulent gene resources is an effective way to solve this problem, which is of great significance to the biological control of our country.

Contents of the invention

The first purpose of the present invention is to provide a new BT virulence protein resource to address the above-mentioned deficiencies.

The second object of the present invention is to provide a gene encoding the protein.

The purpose of the present invention is also to provide the application of the above protein and gene.

The present invention is a new bacterial strain BM59-2 of Bacillus thuringiensis isolated from the soil of Chengdu Plain in Sichuan Province. The toxicity test of BM59-2 shows that BM59-2 has extremely high toxicity to Lepidoptera pests, Diptera pests and so on. According to the conserved sequence of the cry52 gene, a pair of specific primers were designed to amplify its genomic DNA. The results showed that the strain had the cry52 gene. The full-length gene primers were further designed and the cry52Ba gene was cloned. Its nucleotide sequence is shown in the sequence table SEQ ID As shown in No.1, the full length of the sequence is 2112bp, the analysis shows that the GC content is 36.9%, and it encodes a protein consisting of 703 amino acids. After determination, its amino acid sequence is shown in SEQ ID No.2. Using the bacterial sigma7.0 promoter program to predict the entire sequence on the softberry website, it was shown that the sequence containing the activation site of RNA polymerase in the upstream of the gene coding region was named cry52Ba1. The present invention further analyzes the amino acid composition of the Cry52Ba1 protein (see Table 1).

Table 1 Amino acid composition of Cry52Ba1 protein

amino acid Percentage% amino acid Percentage % Ala(A): 5.26 Met(M): 1.28 Cys(C): 1.71 Asn(N): 7.54 Asp(D): 4.41 Pro(P): 5.12 Glu(E): 5.41 Gln(Q): 2.56 Phe(F): 4.27 Arg(R): 4.41

Gly(G): 6.97 Ser(S): 7.25 His(H): 1.42 Thr(T): 6.83 Ile(I): 5.26 Val(V): 5.97 Lys(K): 5.69 Trp(W): 1.99 Leu(L): 10.10 Tyr(Y): 6.54

It should be understood that those skilled in the art can substitute, delete and/or add one or several amino acids based on the amino acid sequence disclosed in the present invention without affecting its activity to obtain the mutant sequence of the protein. For example, in the inactive segment, replace Ala at position 75 with Met. Therefore, the Bt protein of the present invention also includes a protein derived from Cry52Ba1 that has the same activity as the Cry52Ba1 protein after the amino acid sequence shown in SEQ ID No. 2 is substituted, substituted and/or increased by one or several amino acids. The gene of the present invention includes the nucleic acid sequence encoding the protein.

In addition, it should be understood that, considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for the expression of specific species as needed.

The gene and protein of the present invention can be cloned or isolated from bacterial strain BM59-2, or obtained by DNA or peptide synthesis.

The gene of the present invention can be operably connected with the expression vector to obtain a recombinant expression vector capable of expressing the protein of the present invention, and then the expression vector can be transformed into The host is introduced to obtain a transformant of the cry52Ba1 gene, such as crops or fruit trees, so that it has insect-resistant activity.

In addition, the fermentation liquid containing Cry52Ba1 protein can also be obtained by fermenting the bacterial strain BM59-2 of the present invention, which can be prepared into an insecticide for the control of crop pests. Those skilled in the art can also transform the above-mentioned gene into bacteria or fungi, and produce the Bt protein of the present invention through large-scale fermentation.

Those skilled in the art can also transform crops such as cotton, corn, rice, and vegetables according to the genes disclosed in the present invention, so that they have corresponding insect-resistant activities. Thereby reducing the use of pesticides and reducing environmental pollution, which has important economic value and application prospects.

Description of drawings

Figure 1 shows the full-length cry52Ba1 gene clone, wherein M, marker; 1, cry52Ba1 gene.

Figure 2 shows the enzyme digestion identification map of recombinant plasmid pET-52Ba, in which 1 recombinant plasmid pET-52Ba; 2, pET-30a was digested with Nde I+EcoR I; 3, pET was double digested with Nde I+EcoR I -52Ba; 4, inserted DNA; M1, M2 are markers.

Figure 3 shows the SDS-PAGE detection of Cry52Ba1 expressed in E.coli BL21(DE3), where M is a protein marker; 1. Negative control (E.coiiBL21(DE3)(pET-30a)); 2. Lysis Qing; 3. Cry52Ba1 inclusion body.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Cloning of embodiment 1cry52Ba1 gene

The present invention is isolated from the new bacterial strain of Bacillus thuringiensis (Bacillus thuringiensis) obtained from the soil of the virgin forest area of Muchuan, Sichuan Province. No. 3, Datun Road, Chaoyang District, City, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) is preserved, and the classification is named Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No.2871.

In this example, the full-length sequence of the cry52Ba1 gene was cloned by the following method.

Total DNA of BM59-2. The primer sequences were designed as follows:

P1: 5'ATGAATTCATATCAAAATAAAAAATG3'

P2: 5'TTACCTCCTACTTCAGTACATACTTT3'

PCR reaction system:

10×buffer 2.5μl

MgCl ₂ (25mM) 1.5μl

Taq enzyme 0.2μl

dNTPs(2.5mM) 2μl

Primer 2μl

Template 5 μl

Final reaction volume 25μl

Thermal cycle reaction: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 52°C for 1 minute, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 5 minutes; stop reaction at 4°C. The amplification reaction product was electrophoresed on 1% agarose gel, and the PCR amplification result was observed in a gel imaging system. The results are shown in Figure 1. A sequence of about 2000 bp was obtained through amplification. The sequence was sequenced, and its nucleotide sequence was shown in SEQ ID No.1, which was consistent with the target sequence.

Example 2 Expression and insecticidal activity of cry52Ba1 gene

According to the sequence at both ends of the open reading frame of the cry52Ba1 gene, a pair of specific primers cry53F: 5′-GCG CATATG (NdeI) ATGAATTCATATCAAAATAAAAATG-3, cry53R: 5′-CG GAATTC (EcoR I) TTACCTCCTACTTCAGTACATACTTT-3′ were designed and synthesized. The 5' end primers Nde I and EcoR I restriction sites. The BM59-2 plasmid was used as a template to amplify, and the amplified product was double-digested with Nde I and EcoR I, and the digested product was ligated with the vector pET-30a(+) after the same double-digestion, and transformed into E.coli DH5α-competent cells were extracted from the plasmid and electrophoresis was performed to verify that the size of the inserted fragment met the expected purpose (Figure 2), and then transferred into the recipient strain E.coli.BL21(DE3). The recombinant plasmid was named pET-52Ba, and the recombinant containing the recombinant plasmid was named E.coli.BL21(52Ba). SDS-PAGE analysis showed that the expression product of the cry52Ba1 gene had a molecular weight of about 76 kDa in the precipitate after sonication of the bacteria (Fig. 3), which was consistent with the predicted protein molecular weight. The bioassay results of the expression product of Cry52Ba1 gene against beet armyworm, cotton bollworm and Aedes mosquito showed that the expression product had good insecticidal activity against these three insects. It has the highest insecticidal activity against beet armyworm with LC ₅₀ of 25.2 μg/mL; LC ₅₀ of Aedes mosquito is 34.63 μg/mL; and the lowest insecticidal activity against cotton bollworm with LC ₅₀ of 58.19 μg/mL. For the assay method of protein to Lepidoptera insecticidal activity, see (SongFP, Zhang J, Gu AX, et al., 2003. Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-typegene.Appl.Environ. Microbiol 69:5207-5211), the assay method of protein to Diptera insecticidal activity sees (Ibarra JE, del Rincón MC, Sergio Ordúz, et al., 2003.Diversity of Bacillus thuringienisis Strains from Latin America with Insecticidal Activity against Different Mosquito Species. ApplEnviron Microbiol 69:5269-5274).

sequence listing

<110>Sichuan Agricultural University

<120> Bacillus thuringiensis BM59-2 and its application

<130>KHP09112216.2

<160>6

<170>PatentIn version 3.5

<210>1

<211>2111

<212>DNA

<213>Bacillus thuringiensis BM59-2

<220>

<221> CDS

<222>(1)..(2109)

<400>1

atg aat tca tat caa aat aaa aat gaa tat gaa ata ttg gat gct tca 48

Met Asn Ser Tyr Gln Asn Lys Asn Glu Tyr Glu Ile Leu Asp Ala Ser

1 5 10 15

caa aac aac tct aat atg tct aat cgt tat cca agg tat cca tta gca 96

Gln Asn Asn Ser Asn Met Ser Asn Arg Tyr Pro Arg Tyr Pro Leu Ala

20 25 30

aat gat cca caa gct tct atg cag aat acg aat tat aaa gat tgg ttg 144

Asn Asp Pro Gln Ala Ser Met Gln Asn Thr Asn Tyr Lys Asp Trp Leu

35 40 45

gct acg tgc aac gga acg cct gcc ccg ctt tat aat tct tca caa cta 192

Ala Thr Cys Asn Gly Thr Pro Ala Pro Leu Tyr Asn Ser Ser Gln Leu

50 55 60

ctt aaa att tca gga aat gta gtt tct agg gct ctc gga atg ctt ccc 240

Leu Lys Ile Ser Gly Asn Val Val Ser Arg Ala Leu Gly Met Leu Pro

65 70 75 80

att cct ggg att gct cct ctt tta agt ttt ctc tct act ttg ctt tgg 288

Ile Pro Gly Ile Ala Pro Leu Leu Ser Phe Leu Ser Thr Leu Leu Trp

85 90 95

cca agt gga tca tcc gga aat act att tgg gaa tcg ttg atg aaa gaa 336

Pro Ser Gly Ser Ser Gly Asn Thr Ile Trp Glu Ser Leu Met Lys Glu

100 105 110

gct gcg gat ttg ata gac caa aag tta gag gag aat ata tta cgc caa 384

Ala Ala Asp Leu Ile Asp Gln Lys Leu Glu Glu Asn Ile Leu Arg Gln

115 120 125

gca acg gcc aat tta gcc gga tta caa gga cta ttg gga tca tat aac 432

Ala Thr Ala Asn Leu Ala Gly Leu Gln Gly Leu Leu Gly Ser Tyr Asn

130 135 140

agc gct ttt gct tca tgg gaa gca ggc ggc aat gca act cct gat ctg 480

Ser Ala Phe Ala Ser Trp Glu Ala Gly Gly Asn Ala Thr Pro Asp Leu

145 150 155 160

gta aaa ggt tat atg gag agc ctt cat cgt acg ttt gtg cag gat att 528

Val Lys Gly Tyr Met Glu Ser Leu His Arg Thr Phe Val Gln Asp Ile

165 170 175

ata ggc agc ttc acg ata cca ggt tat gaa aaa ata tta tta cct acc 576

Ile Gly Ser Phe Thr Ile Pro Gly Tyr Glu Lys Ile Leu Leu Pro Thr

180 185 190

tat gcg att acc gcc aat ttt cat ttg atg tta tta cgt gac att gaa 624

Tyr Ala Ile Thr Ala Asn Phe His Leu Met Leu Leu Arg Asp Ile Glu

195 200 205

att tat gga ggt aaa aaa acc cca gaa ggt aaa gat ggc ctg aat ttt 672

Ile Tyr Gly Gly Lys Lys Thr Pro Glu Gly Lys Asp Gly Leu Asn Phe

210 215 220

gat cca aaa gac cta aat ttt tat aat tgt gaa cta aag aaa tat aag 720

Asp Pro Lys Asp Leu Asn Phe Tyr Asn Cys Glu Leu Lys Lys Tyr Lys

225 230 235 240

gaa ctg tat acg aat cat tgc tta aat act tac aat aaa ggt ttg gcc 768

Glu Leu Tyr Thr Asn His Cys Leu Asn Thr Tyr Asn Lys Gly Leu Ala

245 250 255

tca gaa aaa gaa aaa ggt tgg gtg cct ttc cat cga tat cgt aga gaa 816

Ser Glu Lys Glu Lys Gly Trp Val Pro Phe His Arg Tyr Arg Arg Glu

260 265 270

atg act ttg gct gta tta gat ata att gca tta ttc cca ctc tat gat 864

Met Thr Leu Ala Val Leu Asp Ile Ile Ala Leu Phe Pro Leu Tyr Asp

275 280 285

gca aga ctc tat cct gct aag aat aat aaa gaa atg cca gtt aaa tcc 912

Ala Arg Leu Tyr Pro Ala Lys Asn Asn Lys Glu Met Pro Val Lys Ser

290 295 300

gaa ttg act cga gaa att tat tcg gat gtc att aat agc gat agg ttc 960

Glu Leu Thr Arg Glu Ile Tyr Ser Asp Val Ile Asn Ser Asp Arg Phe

305 310 315 320

gga gtg gta ccc cct tat aat tat gct caa aac gaa gaa cgt tat aca 1008

Gly Val Val Pro Pro Tyr Asn Tyr Ala Gln Asn Glu Glu Arg Tyr Thr

325 330 335

cga cca cct cat ctc ttc act tgg tta cga ggg ctt gac ttt gta acc 1056

Arg Pro Pro His Leu Phe Thr Trp Leu Arg Gly Leu Asp Phe Val Thr

340 345 350

aat gtt ctg act agc gga act tgg gtt tat aga tgg agc gtt tta act 1104

Asn Val Leu Thr Ser Gly Thr Trp Val Tyr Arg Trp Ser Val Leu Thr

355 360 365

ggg ttg tca aaa gaa ata ttc tta tac aaa agg gaa tgg tac tat aac 1152

Gly Leu Ser Lys Glu Ile Phe Leu Tyr Lys Arg Glu Trp Tyr Tyr Asn

370 375 380

tgg tcc ttt tcg ggg tta tcc tgt aga gtc tgg tgg aag aac ttc caa 1200

Trp Ser Phe Ser Gly Leu Ser Cys Arg Val Trp Trp Lys Asn Phe Gln

385 390 395 400

cat tac tat tgc aga agg ttc cta tat tta gaa ctt gtt gcc aag aag 1248

His Tyr Tyr Cys Arg Arg Phe Leu Tyr Leu Glu Leu Val Ala Lys Lys

405 410 415

ctt tca ata tat ttc ccc ttg gta ttt tac gac aaa tat cgc act gat 1296

Leu Ser Ile Tyr Phe Pro Leu Val Phe Tyr Asp Lys Tyr Arg Thr Asp

420 425 430

tac ttt ctt act aac aaa aat aat agt tca aca gaa aaa gtt tat ggt 1344

Tyr Phe Leu Thr Asn Lys Asn Asn Ser Ser Thr Glu Lys Val Tyr Gly

435 440 445

tat gta gcc ggc aac gct aat tta cct act gtt caa aca gat ttt gat 1392

Tyr Val Ala Gly Asn Ala Asn Leu Pro Thr Val Gln Thr Asp Phe Asp

450 455 460

ttt ctt aca aat aaa gaa gta act ggc cct cca aca tac aat aac tat 1440

Phe Leu Thr Asn Lys Glu Val Thr Gly Pro Pro Thr Tyr Asn Asn Tyr

465 470 475 480

aat cat att ttg tca tac ttg ttg cta ggt tat gat tgg aat cag acg 1488

Asn His Ile Leu Ser Tyr Leu Leu Leu Gly Tyr Asp Trp Asn Gln Thr

485 490 495

ggt gga ata ggc aca cac gga tat tca ttt gca ttt aca cat agt agc 1536

Gly Gly Ile Gly Thr His Gly Tyr Ser Phe Ala Phe Thr His Ser Ser

500 505 510

gtt gat cct tat aac acc att gcc cca gat aaa att acg caa att cct 1584

Val Asp Pro Tyr Asn Thr Ile Ala Pro Asp Lys Ile Thr Gln Ile Pro

515 520 525

gca gtg aag gct ttt gaa ata tca gat gca gga cca agt caa gtcata ata 1632

Ala Val Lys Ala Phe Glu Ile Ser Asp Ala Gly Pro Ser Gln Val Ile

530 535 540

gct gga cct ggt cat aca gga gga gat gta gta agg tta tac ctt tca 1680

Ala Gly Pro Gly His Thr Gly Gly Asp Val Val Arg Leu Tyr Leu Ser

545 550 555 560

ggc cgt tta aaa ata cgt tta act cct gca tcc acg aat aaa aat tac 1728

Gly Arg Leu Lys Ile Arg Leu Thr Pro Ala Ser Thr Asn Lys Asn Tyr

565 570 575

ctt gtt aga gtt cgc tat gca agt ccg gta tct ggt acg tta cga gta 1776

Leu Val Arg Val Arg Tyr Ala Ser Pro Val Ser Gly Thr Leu Arg Val

580 585 590

gaa aga tgg tcg cct agt tct gtt aca aat cgt gat ttt act cgt ttg 1824

Glu Arg Trp Ser Pro Ser Ser Val Thr Asn Arg Asp Phe Thr Arg Leu

595 600 605

gct acg ggt ggt ttt aat tca ttt ggc tat gtg gac acc tta gtt act 1872

Ala Thr Gly Gly Phe Asn Ser Phe Gly Tyr Val Asp Thr Leu Val Thr

610 615 620

aca tgt aat caa tca ggt gtt gaa ata att ata caa aat cta ggt gct 1920

Thr Cys Asn Gln Ser Gly Val Glu Ile Ile Ile Gln Asn Leu Gly Ala

625 630 635 640

tct gac gtt atc att gac aaa gtt gaa ttt atc cct tat gac atc cca 1968

Ser Asp Val Ile Ile Asp Lys Val Glu Phe Ile Pro Tyr Asp Ile Pro

645 650 655

att gat aaa tgt acg aaa tgt gaa ttc gaa gga aac gta tgt aca tgt 2016

Ile Asp Lys Cys Thr Lys Cys Glu Phe Glu Gly Asn Val Cys Thr Cys

660 665 670

aga tgt gaa gga gta caa tcc tta gaa aaa gaa aaa gag att gta aat 2064

Arg Cys Glu Gly Val Gln Ser Leu Glu Lys Glu Lys Glu Ile Val Asn

675 680 685

agt tta ttt gtc aaa gaa aac aaa gta tgt act gaa gta gga ggt aa 2111

Ser Leu Phe Val Lys Glu Asn Lys Val Cys Thr Glu Val Gly Gly

690 695 700

<210>2

<211>703

<212>PRT

<213>Bacillus thuringiensis BM59-2

<400>2

Met Asn Ser Tyr Gln Asn Lys Asn Glu Tyr Glu Ile Leu Asp Ala Ser

1 5 10 15

Gln Asn Asn Ser Asn Met Ser Asn Arg Tyr Pro Arg Tyr Pro Leu Ala

20 25 30

Asn Asp Pro Gln Ala Ser Met Gln Asn Thr Asn Tyr Lys Asp Trp Leu

35 40 45

Ala Thr Cys Asn Gly Thr Pro Ala Pro Leu Tyr Asn Ser Ser Gln Leu

50 55 60

Leu Lys Ile Ser Gly Asn Val Val Ser Arg Ala Leu Gly Met Leu Pro

65 70 75 80

Ile Pro Gly Ile Ala Pro Leu Leu Ser Phe Leu Ser Thr Leu Leu Trp

85 90 95

Pro Ser Gly Ser Ser Gly Asn Thr Ile Trp Glu Ser Leu Met Lys Glu

100 105 110

Ala Ala Asp Leu Ile Asp Gln Lys Leu Glu Glu Asn Ile Leu Arg Gln

115 120 125

Ala Thr Ala Asn Leu Ala Gly Leu Gln Gly Leu Leu Gly Ser Tyr Asn

130 135 140

Ser Ala Phe Ala Ser Trp Glu Ala Gly Gly Asn Ala Thr Pro Asp Leu

145 150 155 160

Val Lys Gly Tyr Met Glu Ser Leu His Arg Thr Phe Val Gln Asp Ile

165 170 175

Ile Gly Ser Phe Thr Ile Pro Gly Tyr Glu Lys Ile Leu Leu Pro Thr

180 185 190

Tyr Ala Ile Thr Ala Asn Phe His Leu Met Leu Leu Arg Asp Ile Glu

195 200 205

Ile Tyr Gly Gly Lys Lys Thr Pro Glu Gly Lys Asp Gly Leu Asn Phe

210 215 220

Asp Pro Lys Asp Leu Asn Phe Tyr Asn Cys Glu Leu Lys Lys Tyr Lys

225 230 235 240

Glu Leu Tyr Thr Asn His Cys Leu Asn Thr Tyr Asn Lys Gly Leu Ala

245 250 255

Ser Glu Lys Glu Lys Gly Trp Val Pro Phe His Arg Tyr Arg Arg Glu

260 265 270

Met Thr Leu Ala Val Leu Asp Ile Ile Ala Leu Phe Pro Leu Tyr Asp

275 280 285

Ala Arg Leu Tyr Pro Ala Lys Asn Asn Lys Glu Met Pro Val Lys Ser

290 295 300

Glu Leu Thr Arg Glu Ile Tyr Ser Asp Val Ile Asn Ser Asp Arg Phe

305 310 315 320

Gly Val Val Pro Pro Tyr Asn Tyr Ala Gln Asn Glu Glu Arg Tyr Thr

325 330 335

Arg Pro Pro His Leu Phe Thr Trp Leu Arg Gly Leu Asp Phe Val Thr

340 345 350

Asn Val Leu Thr Ser Gly Thr Trp Val Tyr Arg Trp Ser Val Leu Thr

355 360 365

Gly Leu Ser Lys Glu Ile Phe Leu Tyr Lys Arg Glu Trp Tyr Tyr Asn

370 375 380

Trp Ser Phe Ser Gly Leu Ser Cys Arg Val Trp Trp Lys Asn Phe Gln

385 390 395 400

His Tyr Tyr Cys Arg Arg Phe Leu Tyr Leu Glu Leu Val Ala Lys Lys

405 410 415

Leu Ser Ile Tyr Phe Pro Leu Val Phe Tyr Asp Lys Tyr Arg Thr Asp

420 425 430

Tyr Phe Leu Thr Asn Lys Asn Asn Ser Ser Thr Glu Lys Val Tyr Gly

435 440 445

Tyr Val Ala Gly Asn Ala Asn Leu Pro Thr Val Gln Thr Asp Phe Asp

450 455 460

Phe Leu Thr Asn Lys Glu Val Thr Gly Pro Pro Thr Tyr Asn Asn Tyr

465 470 475 480

Asn His Ile Leu Ser Tyr Leu Leu Leu Gly Tyr Asp Trp Asn Gln Thr

485 490 495

Gly Gly Ile Gly Thr His Gly Tyr Ser Phe Ala Phe Thr His Ser Ser

500 505 510

Val Asp Pro Tyr Asn Thr Ile Ala Pro Asp Lys Ile Thr Gln Ile Pro

515 520 525

Ala Val Lys Ala Phe Glu Ile Ser Asp Ala Gly Pro Ser Gln Val Ile

530 535 540

Ala Gly Pro Gly His Thr Gly Gly Asp Val Val Arg Leu Tyr Leu Ser

545 550 555 560

Gly Arg Leu Lys Ile Arg Leu Thr Pro Ala Ser Thr Asn Lys Asn Tyr

565 570 575

Leu Val Arg Val Arg Tyr Ala Ser Pro Val Ser Gly Thr Leu Arg Val

580 585 590

Glu Arg Trp Ser Pro Ser Ser Val Thr Asn Arg Asp Phe Thr Arg Leu

595 600 605

Ala Thr Gly Gly Phe Asn Ser Phe Gly Tyr Val Asp Thr Leu Val Thr

610 615 620

Thr Cys Asn Gln Ser Gly Val Glu Ile Ile Ile Gln Asn Leu Gly Ala

625 630 635 640

Ser Asp Val Ile Ile Asp Lys Val Glu Phe Ile Pro Tyr Asp Ile Pro

645 650 655

Ile Asp Lys Cys Thr Lys Cys Glu Phe Glu Gly Asn Val Cys Thr Cys

660 665 670

Arg Cys Glu Gly Val Gln Ser Leu Glu Lys Glu Lys Glu Ile Val Asn

675 680 685

Ser Leu Phe Val Lys Glu Asn Lys Val Cys Thr Glu Val Gly Gly

690 695 700

<210>3

<211>25

<212>DNA

<213> Artificial sequence

<400>3

atgaattcat atcaaaataa aaatg 25

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<400>4

ttacctccta cttcagtaca tacttt 26

<210>5

<211>34

<212>DNA

<213> Artificial sequence

<400>5

gcgcatatga tgaattcata tcaaaataaa aatg 34

<210>6

<211>34

<212>DNA

<213> Artificial sequence

<400>6

cggaattctt acctcctact tcagtacata cttt 34

Claims (9)

1. Bt PROTEIN C ry52Ba1, its aminoacid sequence is shown in SEQ ID No.2.

2. coding claim 1 described proteic gene.

3. gene as claimed in claim 2, its nucleotide sequence is shown in SEQ ID No.1.

4. contain claim 2 or 3 described expression carrier.

5. by the described expression vector transformed host cells of claim 4.

6. host cell as claimed in claim 5, it is a plant host cell.

7. contain the described proteic sterilant of claim 1.

8. claim 2 or 3 described genes or the described expression vector of claim 4 application in the preparation transgenic plant.

9. claim 2 or 3 described genes or the described expression vector of claim 4 application in improving plant resistance to insect.

Images