CN101531711B - Bt protein Cry52Bal as well as encoding gene thereof and application thereof - Google Patents

Bt protein Cry52Bal as well as encoding gene thereof and application thereof Download PDF

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CN101531711B
CN101531711B CN2009100815957A CN200910081595A CN101531711B CN 101531711 B CN101531711 B CN 101531711B CN 2009100815957 A CN2009100815957 A CN 2009100815957A CN 200910081595 A CN200910081595 A CN 200910081595A CN 101531711 B CN101531711 B CN 101531711B
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郑爱萍
李平
朱军
王玲霞
王世全
邓其明
李双成
刘怀年
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Sichuan Agricultural University
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Abstract

The invention provides a new Bt protein Cry52Bal as well as an encoding gene thereof; the protein is provided with an aminoacid sequence as shown by SEQ ID No.2 or the protein which has the same activity and is obtained by substituting, deleting and/or adding one or plural aminoacid to an amino acid sequence as shown by SEQ ID No.2. The protein of the invention can be used for preparing Bt insecticides, the gene can be used for converting cotton, maize, rice, vegetables and other crops to ensure that the crops have corresponding anti-insect activity, thereby reducing the use of agricultural chemical, alleviating environment pollution, and having significant economic value and application prospect.

Description

Bt PROTEIN C ry52Ba1, its encoding gene and application
Technical field
The present invention relates to biological technical field, be specifically related to a kind of new Bt albumen and encoding gene and application.
Background technology
In the human being's production process, insect pest is the important factor that causes agriculture production loss and influence human health, adds up according to FAO, and the financial loss that whole world agriculture production every year causes because of insect pest is up to 14%, and disease is with a toll of 12%, and crop smothering is with a toll of 11%.The amount of loss is equivalent to half of the Chinese agriculture gross output value up to 1,260 hundred million dollars, more than 4 times of Britain.In addition, mosquito matchmaker disease is occupied critical positions in preventive medicine, wherein the sick transmissibility of mosquito such as singapore hemorrhagic fever and yellow jack matchmaker strong, popular wide, sickness rate is high, hazardness is big.According to the WHO statistics, the annual singapore hemorrhagic fever number that infects in the whole world reached 8,000 ten thousand, and Hainan Province of China once broke out twice singapore hemorrhagic fever in 1980 and 1986, and morbidity reaches 437469 example and 113589 examples respectively.Singapore hemorrhagic fever and yellow jack are mainly propagated by Aedes aegypti.
In order to reduce these losses, for many years, generally adopt the chemical prevention means to prevent and treat to crop pests and mosquito, but because the long-term, a large amount of of chemical pesticide use, caused the pollution to environment, pesticide residue increases in the agricultural byproducts, has brought harm for human existence and health.In addition, chemical pesticide has has also killed and wounded natural enemy and other useful thing in kill pests, destroyed the eubiosis.Compare safe, effective, persistent characteristics that biological control has with chemical prevention.And a series of problems of having avoided chemical prevention to bring.Therefore, biological control technology has become the focus of people's researchs.In biotic pesticide, bacillus thuringiensis is the quasi-microorganism sterilant that purposes is the widest in the world, output is maximum at present.
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be a kind of gram positive bacterium, its distribution is very extensive, when forming, gemma can form the parasporal crystal of forming by protein with insecticidal activity, have another name called insecticidal crystal protein (Insectididal crystalproteins is called for short ICPs), ICPs is by the cry genes encoding, sensitive insect there is strong toxicity, and to higher animal and people's nontoxicity.In recent decades, Bt has been widely used in controlling insects such as multiple lepidopteran, Diptera, Coleoptera.In addition, Bt also has the effect of control evil to various pests such as Hymenoptera, Homoptera, Orthoptera, Mallophaga and plant pathogeny line insect, mite class, protozoon.At present Bt has become the strong substitute of chemical synthetic pesticide in the control of agricultural pests, injurious forest-insect and sanitary insect pest, and Bt still be that transgenic pest-resistant engineered plant important function of gene is originated.
(Adang M.J et al from Schnepf in 1981 has cloned first gene that can express insecticidal activity from strain HD-1Dipel since, Characterized full-length andtruncated plasmid clones of the crystal protein of Bacillus thuringiensissubsp.kurstaki HD-73 and their toxicity to Manduca sexta, Gene, 1985,36 (3): 289～300.), people separating clone the gene of more than 390 kind of coded insect-killing crystallin, they are defined as different groups respectively according to the amino acid sequence coded homology, subgroup, class and subclass (Crickmore N, Zeigler D R, Feitelson J, et al.Revision of the nomenclature for the Bacillus thuringiensis pesticidalcrystal proteins.Microbiol Mol Biol Rev, 1998,62:807-813; Http:// www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/).Generally speaking, Cry1, toxalbumin such as Cry2 and Cry9 are effective to lepidoptera pest; Wherein the maximum of research are Cry1 and Cry9 proteinoid, the insecticidal crystal protein molecular weight of their codings is 130-140kD, many genes have been widely used in the control (Kozie of the lepidoptera pest of plant at present, 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 crystalprotein with high activity against the spruce bud worm (Choristoneurafnniferana) .Appl.Environ, Microbviol.63:4132-4134; Wang Fei, 2001, the research of bacillus thuringiensis specific strain biological characteristics and the new gene of cry9, Master's thesis, Nankai University).Tribactur Israel subclass (B.thuringiensis subsp.israelensis, abbreviation Bti) toxin protein that produces has fine insecticidal activity to mosquito, extensively applied to control (the Goldberg L J of mosquito, and Margalit J, 1977.A bacterialspore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, and Culexpipiens.Mosqito News, 37:355-358; ).Simultaneously, Cyt albumen has cytolytic, some Cry albumen is had synergism and delays the resistance (Wu of insect, D., Johnson, J.J., and Federici, B.A.1994.Synergism of mosquitocidal toxicity betweenCytA 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)
Find the history in existing so far more than 100 year of Tribactur from the beginning of this century, aspect the preventing and treating of farm crop and gardening plant insect, injurious forest-insect and sanitary insect pest, be widely used, also play good effect.But owing to use Tribactur on a large scale and repeatedly, many insect populations are producing resistance to insecticidal crystal protein in succession in varying degrees.The history in existing more than 50 year of Utilization of pesticides based on the Bt insecticidal crystal protein, the initial resistance of insect that never detect to Bt, but, begin mid-term 80 year last century, resistance problem (the M cGaughey that constantly in laboratory and field test, is confirmed, W.H.1985.Insect resistance to the biological insecticide Bacillus thuringiensis.Science.229:193-195), reason mainly is continue to use single variety and inferiorly cause the Bt of dosage and the application of Bt transgenic anti-insect plants causes insect population to be subjected to the selective pressure of sterilant for a long time.1985, McGaughey report warehouse grain pest Indian meal moth (Plodia interpunctella) under the selective pressure of Dipel (the commodity preparation of Bt subsp.kurstaik HD-1), bred for 15 generations after, resistance increases by 97 times; Under the high dosage selective pressure, resistance can increase by 250 times.Nineteen ninety, the small cabbage moth that confirms big Tanaka in Hawaii has first produced tangible resistance (Tabashnik to the Bt sterilant, 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 nineties in last century, use long Shenzhen of Bt sterilant time in China, Guangzhou, ground such as Shanghai, find that the Bt sterilant obviously descends to the small cabbage moth prevention effect, mean resistance form (Feng Xia .1996. Guangdong small cabbage moth is to the resistance research of Bacillus thuringiensis. insect journal, 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 ofBacillus thuringiensis.Appl.Environ.Microbiol.54:2010-2017).Find at present in the laboratory and the field has at least tens kinds of insects that Bt and insecticidal crystal protein thereof have been produced resistance, arrive with the selective pressure mathematical model prediction, under the condition of Bt transgenic anti-insect plants selective pressure, insect 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, there are some researches prove that Bti does not find resistance problem (Regis L as yet in the use in land for growing field crops, et al., 2000.The useof bacterial larvicides in mosquito and black fly control programsinBrazil.Mem.Instituto Oswaldo Cruz, 95:207-210.), but mosquito constantly is confirmed in the laboratory to its resistance problem, this situation also may (Georghiou G P occur big Tanaka, and Wirth M C, 1997.Influence of exposure to singleversus multiple toxins of Bacillus thuringiensis subsp.israelensis ondevelopment of resistance in the mosquito Culex quinquefasciatus (Diptera:Culicidae) .Applied and Environmental Microbiology, 63:1095-1101.).
Be the loss of avoiding resistant insects to cause, seeking new high virulence gene resource is the effective way that addresses this problem, and this biological control to China has crucial meaning.
Summary of the invention
First purpose of the present invention is to provide a kind of new BT virulence protein resource at above-mentioned deficiency.
Second purpose of the present invention is to provide the gene of encoding said proteins.
The present invention also aims to provide the application of above-mentioned albumen and gene.
The present invention separates the new bacterial strain BM59-2 of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from Sichuan Province's Chengdu Plain soil.By the virulence test shows to BM59-2, BM59-2 all has high virulence to lepidoptera pest, Diptera pest or the like.According to 1 pair of special primer of cry52 genoid conserved sequence design, its genomic dna increases, the result shows that there is the cry52 genoid in this bacterial strain, further its full-length gene primer of design is cloned and is obtained the cry52Ba gene, and its nucleotide sequence is shown in sequence table SEQ ID No.1, this sequence total length 2112bp, analysis revealed, GC content are 36.9%, the albumen that 703 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in SEQ ID No.2.Adopt bacterial sigma7.0 promoter program that complete sequence is predicted in the softberry website and show, contain the sequence in RNA polymerase activation site in the gene coding region upstream, its called after cry52Ba1.The present invention has further analyzed the proteic amino acid of Cry52Ba1 and has formed (seeing Table 1).
The proteic amino acid of table 1 Cry52Ba1 is formed
Amino acid Per-cent % Amino acid Per-cent %
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
Should be appreciated that those skilled in the art can not influence under its active prerequisite according to aminoacid sequence disclosed by the invention, replace, lack and/or increase one or several amino acid, obtain described proteic mutant nucleotide sequence.For example, the 75th Ala is replaced with Met at nonactive section.Therefore, Bt albumen of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.2 is substituted, replaces and/or increases one or several amino acid, has the equal active protein of being derived and being obtained by Cry52Ba1 of Cry52Ba1 albumen.Gene of the present invention comprises the nucleic acids encoding said proteins sequence.
In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Gene of the present invention can be cloned or separate from bacterial strain BM59-2 with protein and be obtained, and perhaps obtains by DNA or peptide synthetic method.
Gene of the present invention can be operably connected with expression vector, obtain to express the proteic recombinant expression vector of the present invention, and then can pass through such as transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage methods, described expression vector is imported the host, obtain changeing the transformant of cry52Ba1 gene, for example plant such as farm crop or fruit tree makes it possess anti-insect activity.
In addition, can also obtain containing the proteic fermented liquid of Cry52Ba1, it is prepared into sterilant, be used for the control of crop pests by fermentation bacterial strain BM59-2 of the present invention.Those skilled in the art can also be with said gene transform bacteria or fungi, by large scale fermentation production Bt albumen of the present invention.
Those skilled in the art can also with farm crop such as its converting cotton, corn, paddy rice, vegetables, make it possess corresponding anti-insect activity according to gene disclosed by the invention.Thereby reduce the usage quantity of agricultural chemicals, reduce environmental pollution, have important economic value and application prospect.
Description of drawings
That Fig. 1 shows is cry52Ba1 full-length gene clone, M wherein, marker; 1, the cry52Ba1 gene.
Fig. 2 shows is that the enzyme of recombinant plasmid pET-52Ba is cut the evaluation collection of illustrative plates, wherein 1 recombinant plasmid pET-52Ba; 2, with Nde I+EcoR I double digestion pET-30a; 3, Nde I+EcoR I double digestion pET-52Ba; 4, the DNA of insertion; M1, M2 are Marker.
What Fig. 3 showed is that the SDS-PAGE that expresses Cry52Ba1 in E.coli BL21 (DE3) detects, and wherein M is albumen marker; 1. negative control (E.coiiBL21 (DE3) (pET-30a)); 2. cracking supernatant; 3.Cry52Ba1 inclusion body.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The clone of embodiment 1cry52Ba1 gene
The present invention separates the new bacterial strain of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from the soil of Muchuan, Sichuan Province virgin forest area, this bacterial strain on January 12nd, 2009 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after bacillus thuringiensis (Bacillus thuringiensis), preserving number is CGMCC No.2871.
This example is cloned the full length sequence that obtains the cry52Ba1 gene by the following method.
Total DNA of BM59-2.The design primer sequence is as follows:
P1：5’ATGAATTCATATCAAAATAAAAATG3’
P2：5’TTACCTCCTACTTCAGTACATACTTT3’
The PCR reaction system:
10×buffer 2.5μl
MgCl 2(25mM) 1.5μl
Taq enzyme 0.2 μ l
dNTPs(2.5mM) 2μl
Primer 2 μ l
Template 5 μ l
End reaction volume 25 μ l
Thermal cycle reaction: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 52 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 5min; 4 ℃ of stopped reaction.The amplified reaction product is electrophoresis on 1% sepharose, puts and observes the pcr amplification result in the gel imaging system.The result has obtained being about the sequence of 2000bp by amplification as shown in Figure 1, and this sequence is checked order, and its nucleotide sequence is shown in SEQ ID No.1, and is consistent with aim sequence.
Embodiment 2 cry52Ba1 expression of gene and insecticidal activity assays
According to cry52Ba1 gene open reading frame two terminal sequences, design and synthesize a pair of Auele Specific Primer cry53F:5 '-GCG CATATG(NdeI) ATGAATTCATATCAAAATAAAAATG-3, cry53R:5 '-CG GAATTC(EcoR I) TTACCTCCTACTTCAGTACATACTTT-3 ' is respectively at 5 ' end primer Nde I and EcoR I restriction enzyme site.With the BM59-2 plasmid is that template increases, the product of amplification adopts Nde I and EcoR I to carry out double digestion, carrier pET-30a (+) after enzyme is cut product and carried out double digestion equally is connected, Transformed E .coli DH5 α competent cell, extract its plasmid enzyme restriction electrophoresis and verified that insertion segment size meets (Fig. 2) after the intended purposes, changes recipient bacterium E.coli.BL21 (DE3) again over to.With recombinant plasmid called after pET-52Ba, contain the recon called after E.coli.BL21 (52Ba) of recombinant plasmid.In the precipitation of SDS-PAGE analysis revealed cry52Ba1 expression of gene product after the thalline ultrasonication (Fig. 3), molecular weight is about about 76kDa, conforms to the molecular weight of albumen of prediction.The Cry52Ba1 gene expression product is respectively to beet armyworm, and the survey result that gives birth to of bollworm and yellow-fever mosquito shows: expression product all has insecticidal activity preferably to these three kinds of worms.The highest to the beet armyworm insecticidal activity, LC 50Be 25.2 μ g/mL; LC to yellow-fever mosquito 50Be 34.63 μ g/mL; Minimum to the bollworm insecticidal activity, LC 50Be 58.19 μ g/mL.Albumen to the lepidopteran insecticidal activity measuring method referring to (SongFP, Zhang J, Gu AX, et al., 2003.Identification of cry1I-type genes fromBacillus thuringiensis strains and characterization of a novel cry1I-typegene.Appl.Environ.Microbiol 69:5207-5211), albumen to the Diptera insecticidal activity measuring method referring to (Ibarra JE, del Rinc ó n MC, Sergio Ord ú z, et al., 2003.Diversity of Bacillus thuringienisis Strains from Latin Americawith Insecticidal Activity against Different Mosquito Species.ApplEnviron Microbiol 69:5269-5274).
Sequence table
＜110〉Sichuan Agricultural University
＜120〉Tribactur BM59-2 and application thereof
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<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?gtc?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.
CN2009100815957A 2009-04-13 2009-04-13 Bt protein Cry52Bal as well as encoding gene thereof and application thereof Active CN101531711B (en)

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PCT/CN2010/000482 WO2010118630A1 (en) 2009-04-13 2010-04-13 Insecticidal crystal protein gene cry52ba1, its encoded protein and uses

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Publication number Priority date Publication date Assignee Title
CN101531711B (en) * 2009-04-13 2011-01-12 四川农业大学 Bt protein Cry52Bal as well as encoding gene thereof and application thereof
AU2014323834A1 (en) * 2013-09-18 2016-03-10 Sichuan Agricultural University Compositions and methods for improving insect resistance

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101531711B (en) * 2009-04-13 2011-01-12 四川农业大学 Bt protein Cry52Bal as well as encoding gene thereof and application thereof
CN101531981B (en) * 2009-04-13 2010-11-10 四川农业大学 Bacillus thuringiensis BM59-2 and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
谭芙蓉 等.苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达.微生物学报48 5.2008,48(5),684-687.
谭芙蓉 等.苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达.微生物学报48 5.2008,48(5),684-687. *

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