CN101531712B - Bt protein Cry30Bal as well as encoding gene thereof and application thereof - Google Patents
Bt protein Cry30Bal as well as encoding gene thereof and application thereof Download PDFInfo
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- CN101531712B CN101531712B CN2009100815976A CN200910081597A CN101531712B CN 101531712 B CN101531712 B CN 101531712B CN 2009100815976 A CN2009100815976 A CN 2009100815976A CN 200910081597 A CN200910081597 A CN 200910081597A CN 101531712 B CN101531712 B CN 101531712B
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Abstract
The invention provides a new Bt protein Cry30Bal as well as an encoding gene thereof; the protein is provided with an amino acid 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 amino acid 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
Technical field
The present invention relates to biological technical field, be specifically related to a kind of new Bt albumen and encoding sox and application.
Background technology
In the human being's production process, insect pest is the important factor that causes agriculture prodn loss and influence human health, adds up according to FAO, and the financial loss that whole world agriculture prodn 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 the half the of the Chinese agriculture gross output value, more than 4 times of Britain up to 1,260 hundred million dollars.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 present the widest, the maximum quasi-microorganism sterilant of output of purposes in the world.
Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram positive bacterium, and its distribution is very extensive;, gemma can form the parasporal crystal of forming by protein when forming 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 confirmed as different crowd, subgroup, class and subclass (Crickmore N respectively according to the amino acid sequence coded homology; 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, and the insecticidal crystal protein molecular weight that their are encoded is 130-140kD, and 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.Cry9Cal 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, is extensively applied to control (Goldberg L J, the and Margalit J of mosquito; 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, and some Cry albumen is had synergism and delays resistance (Wu, the D. of insect; 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 the Utilization of pesticides that is the basis with 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 constantly is confirmed in laboratory and field test (M cGaughey, W.H.1985.Insect resistance to the biological insecticide Bacillus thuringiensis.Science.229:193-195), and 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 receive 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, confirm first that in Hawaii big Tanaka's small cabbage moth has produced tangible resistance (Tabashnik, B.E. to the Bt sterilant; 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,, found that the Bt sterilant obviously descends to the small cabbage moth control effect on China Application of B t sterilant time long Shenzhen and Guangzhou, Shanghai and other places; 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.et al. as yet in the use in land for growing field crops; 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 above-mentioned deficiency a kind of new BT virulence protein resource to be provided.
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 HS18-1 of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from Sichuan Province's Chengdu Plain soil.Through the virulence test shows to HS18-1, HS18-1 all has high virulence to lepidoptera pest, Diptera pest or the like.
According to 1 pair of special primer of cry30 genoid conserved sequence design, its genomic dna that increases, the result shows that there is the cry30 genoid in this bacterial strain; Further its full-length gene primer of design is cloned and is obtained the cry30Ba gene, and its nucleotide sequence is shown in sequence table SEQ ID No.1; The total length of sequence SEQ ID NO 1 is 1995bp; Analysis revealed, GC content are 32.53%, the albumen that 664 amino acid of encoding are formed.Through measuring, its aminoacid sequence is shown in SEQ ID NO2.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 at the gene coding region upper reaches, its called after cry30Ga1.The present invention has further analyzed the proteic amino acid of Cry30Ga1 and has formed (seeing table 1).
The proteic amino acid of table 1 Cry30Ga1 is formed
| Amino acid | Per-cent % | Amino acid | Per-cent % |
| Ala(A)： | 6.78 | Met(M)： | 2.11 |
| Cys(C)： | 1.05 | Asn(N)： | 9.34 |
| Asp(D)： | 3.16 | Pro(P)： | 5.72 |
| Glu(E)： | 4.82 | Gln(Q)： | 4.37 |
| Phe(F)： | 3.31 | Arg(R)： | 3.92 |
| Gly(G)： | 6.17 | Ser(S)： | 6.93 |
| His(H)： | 0.90 | Thr(T)： | 7.83 |
| Ile(I)： | 10.09 | Val(V)： | 3.01 |
| Lys(K)： | 4.22 | Trp(W)： | 1.20 |
| Leu(L)： | 9.19 | Tyr(Y)： | 5.57 |
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 said proteic mutant nucleotide sequence.For example, the 390th Tyr is replaced with Pro at nonactive section.Therefore, Bt albumen of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.2 through replacing, replace and/or increasing one or several amino acid, has the equal active protein of being derived and being obtained by Cry30Ga1 of Cry30Ga1 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 HS18-1 with protein and obtained, and perhaps obtains through DNA or peptide synthetic method.
Can gene of the present invention be operably connected with expression vector; Obtain to express the proteic recombinant expression vector of the present invention; And then can said expression vector be imported host, the transformant that obtains changeing the cry30Ga1 gene through such as transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage methods; 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 Cry30Ga1, it is prepared into sterilant, be used for the control of crop pests through fermentation bacterial strain HS18-1 of the present invention.Those skilled in the art can also be with said gene transform bacteria or fungi, through 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 cry30Ga1 full-length gene clone, M wherein, marker; 1, the cry30Ga1 gene.
Fig. 2 shows is that the enzyme of recombinant plasmid pET-30Ga is cut the evaluation collection of illustrative plates, wherein 1 recombinant plasmid pET-30Ga; 2, with Nde I+EcoR I double digestion pET-30a; 3, Nde I+EcoR I double digestion pET-30Ga; 4, the DNA of insertion; M1, M2 are Marker.
What Fig. 3 showed is that the SDS-PAGE that in E.coli BL21 (DE3), expresses Cry30Ga1 detects, and wherein M is albumen marker; 1. negative control (E.coiiBL21 (DE3) (pET-30a)); 2. cracking supernatant; 3.Cry30Ga1 inclusion body.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, 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 1 cry30Ga1 gene
The present invention separates the new bacterial strain of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from Sichuan Province's Chengdu Plain soil; This bacterial strain on October 21st, 2008 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.2817.
This example obtains the full length sequence of cry30Ga1 gene through following method clone.
Adopt genomic dna purification kit (available from match Parkson company) to extract total DNA of bacterial strain HS18-1.The design primer sequence is following:
P1：5’ATGAATTTATATCAAAATGAAAATGA 3’
P2：5’TTAGTTCATTTTACAAGCTTCTACAC 3’
The PCR reaction system:
10×buffer 2.5μl
MgCl
2(25mM) 1.5μl
Taq enzyme 0.2 μ l
dNTPs(2.5mM) 2μl
End reaction volume 25 μ l
Thermal cycle reaction: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 52 ℃ of annealing, 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 is as shown in Figure 1, has obtained being about the sequence of 2000bp through amplification, and this sequence is checked order, and its nucleotide sequence is shown in SEQ ID No.1, and is consistent with aim sequence.
According to cry30Ga gene ORFs two terminal sequences, design and synthesize a pair of Auele Specific Primer cry30F:5 '-GCG
CATATG(NdeI) ATGAAGCCGTATCAAAGTG-3 '; Cry30R:5 '-CG
GAATTC(EcoR I) TTAGTTCATTTTACAAGCTTCTACAC-3 ' is respectively at 5 ' end primer Nde I and EcoR I restriction enzyme site.With the BtMC28 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 extracts its plasmid enzyme restriction electrophoresis and has verified (Fig. 2) after the insertion segment size accord with expectation purpose, changes recipient bacterium E.coli.BL21 (DE3) again over to.With recombinant plasmid called after pET-30Ga, contain the recon called after E.coli.BL21 (30Ga) of recombinant plasmid.In the deposition of SDS-PAGE analysis revealed cry30Ga 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 cry30Ga 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 19.82 μ g/mL; LC to yellow-fever mosquito
50Be 21.27 μ g/mL; Minimum to the bollworm insecticidal activity, LC
50Be 34.03 μ g/mL.Albumen to the lepidopteran insecticidal activity measuring method referring to (Song FP; Zhang J; Gu AX; Et al., 2003.Identification of cry1I-type genes from Bacillusthuringiensis strains and characterization of a novel cry1I-type gene.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 America withInsecticidal Activity against Different Mosquito Species.Appl EnvironMicrobiol 69:5269-5274).
Sequence table
< 110>Sichuan Agricultural University
< 120>Bt PROTEIN C ry30Ga1, its encoding sox and application
<130>KHP09112220.6
<160>6
<170>PatentIn version 3.5
<210>1
<211>1995
<212>DNA
<213>Bacillus thuringiensis HS18-1
<220>
<221>CDS
<222>(1)..(1995)
<400>1
atg aat tta tat caa aat gaa aat gaa tat aaa ata ttg gat gtt tta 48
Met Asn Leu Tyr Gln Ash Glu Asn Glu Tyr Lys Ile Leu Asp Val Leu
1 5 10 15
cca aat tat tcg aac atg gtc aat gct tat tca agt tat cca tta gca 96
Pro Asn Tyr Ser Asn Met Val Asn Ala Tyr Ser Ser Tyr Pro Leu Ala
20 25 30
aat aat cca caa gtt ccc tta caa aat acg agt tat aaa gat tgg ctc 144
Asn Asn Pro Gln Val Pro Leu Gln Asn Thr Ser Tyr Lys Asp Trp Leu
35 40 45
aat atg tgt caa act att act cca ctt tgt acc act ata gac tct gac 192
Ash Met Cys Gln Thr Ile Thr Pro Leu Cys Thr Thr Ile Asp Ser Asp
50 55 60
att aat tca gtc gct gcc gct ata ggg gta ata gct tct ata ata ggt 240
Ile Asn Ser Val Ala Ala Ala Ile Gly Val Ile Ala Ser Ile Ile Gly
65 70 75 80
ctt att cgt ggt cca gga gaa gct ata gga tta att tta gga act ttt 288
Leu Ile Arg Gly Pro Gly Glu Ala Ile Gly Leu Ile Leu Gly Thr Phe
85 90 95
tca tca ata ata cct ttt ctt tgg cca gag aac aaa act att ata tgg 336
Ser Ser Ile Ile Pro Phe Leu Trp Pro Glu Asn Lys Thr Ile Ile Trp
100 105 110
gaa gag ttt aca cat aga ggg tta aac ctt att aga cca gaa ctg aca 384
Glu Glu Phe Thr Hi s Arg Gly Leu Asn Leu Ile Arg Pro Glu Leu Thr
115 120 125
cca gca gaa ata gaa ata ata tta aac cct ctc aaa gga tct tac aat 432
Pro Ala Glu Ile Glu Ile Ile Leu Asn Pro Leu Lys Gly Ser Tyr Asn
130 135 140
gca tta cgt gaa cag ctg gtg aat ttt gag aga gag ttt gca ata tgg 480
Ala Leu Arg Glu Gln Leu Val Asn Phe Glu Arg Glu Phe Ala Ile Trp
115 150 155 160
gcc ggt gca aaa aat caa gct act aca ggg gat tta tta aga aga att 528
Ala Gly Ala Lys Asn Gln Ala Thr Thr Gly Asp Leu Leu Arg Arg Ile
165 170 175
tca gct att gaa ggt gct att ata caa ctt aaa aat caa tta aca gta 576
Ser Ala Ile Glu Gly Ala Ile Ile Gln Leu Lys Asn Gln Leu Thr Val
180 185 190
agc gaa gct aat aag cct gca tta ctc agt ctc tat gca caa acc gca 624
Ser Glu Ala Asn Lys Pro Ala Leu Leu Ser Leu Tyr Ala Gln Thr Ala
195 200 205
aat att gat tta ata tta ttc caa aga ggc gcc aaa tat gga gat gaa 672
Asn Ile Asp Leu Ile Leu Phe Gln Arg Gly Ala Lys Tyr Gly Asp Glu
210 215 220
tgg gca aaa tac gct cgc aat caa ccc ata cct ttt aaa aca tca cga 720
Trp Ala Lys Tyr Ala Arg Asn Gln Pro Ile Pro Phe Lys Thr Ser Arg
225 230 235 240
gaa tat tat gca tca tta ata gaa aaa ata aaa act tat act aat gat 768
Glu Tyr Tyr Ala Ser Leu Ile Glu Lys Ile Lys Thr Tyr Thr Asn Asp
245 250 255
att gca gga aca tat aga aat ggt tta aat aaa atc aaa aat ata caa 816
Ile Ala Gly Thr Tyr Arg Asn Gly Leu Asn Lys Ile Lys Asn Ile Gln
260 265 270
aat atc tca tgg gat act ttc aat gaa tat cgt aga ggg atg act cta 864
Asn Ile Ser Trp Asp Thr Phe Asn Glu Tyr Arg Arg Gly Met Thr Leu
275 280 285
agt gca tta gat tta gtt gca tta ttc cca aat tac gat ata tgt att 912
Ser Ala Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr Asp Ile Cys Ile
290 295 300
tat cca ata caa aca aaa aca gaa ctt act aga aaa att tat atg cca 960
Tyr Pro Ile Gln Thr Lys Thr Glu Leu Thr Arg Lys Ile Tyr Met Pro
305 310 315 320
tca ttc tat tta caa gca ctt caa caa agc gga aat cta gaa tca ttg 1008
Ser Phe Tyr Leu Gln Ala Leu Gln Gln Ser Gly Asn Leu Glu Ser Leu
325 330 335
gaa aac caa ctt aca cat ccc cca tca tta ttt act tgg tta aac gaa 1056
Glu Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu
340 345 350
tta aac ctt tat aca ata agt gaa aat ttc aat ccg gct ata ctt cct 1104
Leu Asn Leu Tyr Thr Ile Ser Glu Asn Phe Asn Pro Ala Ile Leu Pro
355 360 365
aat ccg gct caa gga att aca ggt ggc aca cca ata cca ata ggg tta 1152
Asn Pro Ala Gln Gly Ile Thr Gly Gly Thr Pro Ile Pro Ile Gly Leu
370 375 380
aat aac ttg ttt att tat aaa tta tca atg tca caa tat cat gat cca 1200
Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met Ser Gln Tyr His Asp Pro
385 390 395 400
aat ggt tgt tat cca ata gct gga att tct gat atg acc ttt tat aaa 1248
Asn Gly Cys Tyr Pro Ile Ala Gly Ile Ser Asp Met Thr Phe Tyr Lys
405 410 415
agt gac tat aat ggt aat gcg tcc aca act caa cct tat cat gca ggt 1296
Ser Asp Tyr Asn Gly Asn Ala Ser Thr Thr Gln Pro Tyr His Ala Gly
420 425 430
aga aac tca aat aat gtc ata gat aca ttt atg aat ggc cca caa aat 1344
Arg Asn Ser Asn Asn Val Ile Asp Thr Phe Met Asn Gly Pro Gln Asn
435 440 445
gca tca agc tca aat aat att tct att aaa gaa aca aaa cat ata cta 1392
Ala Ser Ser Ser Asn Asn Ile Ser Ile Lys Glu Thr Lys His Ile Leu
450 455 460
tct gat att aaa atg gta tat tcg cga tct ggc gtc tat agt ctt gga 1440
Ser Asp Ile Lys Met Val Tyr Ser Arg Ser Gly Val Tyr Ser Leu Gly
465 470 475 480
tat tca ttt gcc tgg aca tgt act agt gta aat cct gat aat cta att 1488
Tyr Ser Phe Ala Trp Thr Cys Thr Ser Val Asn Pro Asp Asn Leu Ile
485 490 495
gtt cca aat aga att aca caa att cct gct gtt aaa gct aat ctt ttg 1536
Val Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Ala Asn Leu Leu
500 505 510
aat tcg cca gct aga gta att gcg ggc cct ggt cat aca ggt gga gac 1584
Asn Ser Pro Ala Arg Val Ile Ala Gly Pro Gly His Thr Gly Gly Asp
515 520 525
tta gtt gct ctt ctg aac agt ggt act caa tcc ggt aga atg gaa att 1632
Leu Val Ala Leu Leu Asn Ser Gly Thr Gln Ser Gly Arg Met Glu Ile
530 535 540
aaa tgt aaa aca ggt agc ttt act gaa act tcc aga cgt tat ggt ata 1680
Lys Cys Lys Thr Gly Ser Phe Thr Glu Thr Ser Arg Arg Tyr Gly Ile
545 550 555 560
cgc atg cgt tat gct gca aat aat gca ttt aca gtg agt cta tca tat 1728
Arg Met Arg Tyr Ala Ala Asn Asn Ala Phe Thr Val Ser Leu Ser Tyr
565 570 575
aca tta cag ggt ggg aat cca ata ggt ata aca ttt ggt aca gaa cgt 1776
Thr Leu Gln Gly Gly Asn Pro Ile Gly Ile Thr Phe Gly Thr Glu Arg
580 585 590
aca ttt tta aga act aat aat ata ata cca aca gat tta aaa tac gag 1824
Thr Phe Leu Arg Thr Asn Asn Ile Ile Pro Thr Asp Leu Lys Tyr Glu
595 600 605
gag ttt aaa tat aaa gaa tat aat caa att att aca atg act gca cct 1872
Glu Phe Lys Tyr Lys Glu Tyr Asn Gln Ile Ile Thr Met Thr Ala Pro
610 615 620
caa aat aca ata gta act ata gct gtt tac caa tca act ccg agt tta 1920
Gln Asn Thr Ile Val Thr Ile Ala Val Tyr Gln Ser Thr Pro Ser Leu
625 630 635 640
aat aat caa tta att att gac agg atc gaa ttc tat cca atg gat caa 1968
Asn Asn Gln Leu Ile Ile Asp Arg Ile Glu Phe Tyr Pro Met Asp Gln
645 650 655
ggt gta gaa gct tgt aaa atg aac taa 1995
Gly Val Glu Ala Cys Lys Met Asn
660
<210>2
<211>664
<212>PRT
<213>Bacillus thuringiensis HS18-1
<400>2
Met Asn Leu Tyr Gln Asn Glu Asn Glu Tyr Lys Ile Leu Asp Val Leu
1 5 10 15
Pro Asn Tyr Ser Asn Met Val Asn Ala Tyr Ser Ser Tyr Pro Leu Ala
20 25 30
Asn Asn Pro Gln Val Pro Leu Gln Asn Thr Ser Tyr Lys Asp Trp Leu
35 40 45
Asn Met Cys Gln Thr Ile Thr Pro Leu Cys Thr Thr Ile Asp Ser Asp
50 55 60
Ile Asn Ser Val Ala Ala Ala Ile Gly Val Ile Ala Ser Ile Ile Gly
65 70 75 80
Leu Ile Arg Gly Pro Gly Glu Ala Ile Gly Leu Ile Leu Gly Thr Phe
85 90 95
Scr Ser Ile Ile Pro Phe Lcu Trp Pro Glu Asn Lys Thr Ile Ile Trp
100 105 110
Glu Glu Phe Thr His Arg Gly Leu Asn Leu Ile Arg Pro Glu Leu Thr
115 120 125
Pro Ala Glu Ile Glu Ile Ile Leu Asn Pro Leu Lys Gly Ser Tyr Asn
130 135 140
Ala Leu Arg Glu Gln Leu Val Asn Phe Glu Arg Glu Phe Ala Ile Trp
145 150 155 160
Ala Gly Ala Lys Asn Gln Ala Thr Thr Gly Asp Leu Leu Arg Arg Ile
165 170 175
Ser Ala Ile Glu Gly Ala Ile Ile Gln Leu Lys Asn Gln Leu Thr Val
180 185 190
Ser Glu Ala Asn Lys Pro Ala Leu Leu Ser Leu Tyr Ala Gln Thr Ala
195 200 205
Asn Ile Asp Leu Ile Leu Phe Gln Arg Gly Ala Lys Tyr Gly Asp Glu
210 215 220
Trp Ala Lys Tyr Ala Arg Asn Gln Pro Ile Pro Phe Lys Thr Ser Arg
225 230 235 240
Glu Tyr Tyr Ala Ser Leu Ile Glu Lys Ile Lys Thr Tyr Thr Asn Asp
245 250 255
Ile Ala Gly Thr Tyr Arg Asn Gly Leu Asn Lys Ile Lys Asn Ile Gln
260 265 270
Asn Ile Ser Trp Asp Thr Phe Asn Glu Tyr Arg Arg Gly Met Thr Leu
275 280 285
Ser Ala Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr Asp Ile Cys Ile
290 295 300
Tyr Pro Ile Gln Thr Lys Thr Glu Leu Thr Arg Lys Ile Tyr Met Pro
305 310 315 320
Ser Phe Tyr Leu Gln Ala Leu Gln Gln Ser Gly Asn Leu Glu Ser Leu
325 330 335
Glu Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu
340 345 350
Leu Asn Leu Tyr Thr Ile Ser Glu Asn Phe Asn Pro Ala Ile Leu Pro
355 360 365
Asn Pro Ala Gln Gly Ile Thr Gly Gly Thr Pro Ile Pro Ile Gly Leu
370 375 380
Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met Ser Gln Tyr His Asp Pro
385 390 395 400
Asn Gly Cys Tyr Pro Ile Ala Gly Ile Ser Asp Met Thr Phe Tyr Lys
405 410 415
Ser Asp Tyr Asn Gly Asn Ala Ser Thr Thr Gln Pro Tyr His Ala Gly
420 425 430
Arg Asn Ser Asn Asn Val Ile Asp Thr Phe Met Asn Gly Pro Gln Asn
435 440 445
Ala Ser Ser Ser Asn Asn Ile Ser Ile Lys Glu Thr Lys His Ile Leu
450 455 460
Ser Asp Ile Lys Met Val Tyr Ser Arg Ser Gly Val Tyr Ser Leu Gly
465 470 475 480
Tyr Ser Phe Ala Trp Thr Cys Thr Ser Val Asn Pro Asp Asn Leu Ile
485 490 495
Val Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Ala Asn Leu Leu
500 505 510
Asn Ser Pro Ala Arg Val Ile Ala Gly Pro Gly His Thr Gly Gly Asp
515 520 525
Leu Val Ala Leu Leu Asn Ser Gly Thr Gln Ser Gly Arg Met Glu Ile
530 535 540
Lys Cys Lys Thr Gly Ser Phe Thr Glu Thr Ser Arg Arg Tyr Gly Ile
545 550 555 560
Arg Met Arg Tyr Ala Ala Asn Asn Ala Phe Thr Val Ser Leu Ser Tyr
565 570 575
Thr Leu Gln Gly Gly Asn Pro Ile Gly Ile Thr Phe Gly Thr Glu Arg
580 585 590
Thr Phe Leu Arg Thr Asn Asn Ile Ile Pro Thr Asp Leu Lys Tyr Glu
595 600 605
Glu Phe Lys Tyr Lys Glu Tyr Asn Gln Ile Ile Thr Met Thr Ala Pro
610 615 620
Gln Asn Thr Ile Val Thr Ile Ala Val Tyr Gln Ser Thr Pro Ser Leu
625 630 635 640
Asn Asn Gln Leu Ile Ile Asp Arg Ile Glu Phe Tyr Pro Met Asp Gln
645 650 655
Gly Val Glu Ala Cys Lys Met Asn
660
<210>3
<211>26
<212>DNA
< 213>artificial sequence
<400>3
atgaatttat atcaaaatga aaatga 26
<210>4
<211>26
<212>DNA
< 213>artificial sequence
<400>4
ttagttcatt ttacaagctt ctacac 26
<210>5
<211>28
<212>DNA
< 213>artificial sequence
<400>5
gcgcatatga tgaagccgta tcaaagtg 28
<210>6
<211>34
<212>DNA
< 213>artificial sequence
<400>6
cggaattctt agttcatttt acaagcttct acac 34
Claims (1)
1. contain the application of sterilant in killing yellow-fever mosquito of Bt PROTEIN C ry30Ga1, the aminoacid sequence of said Bt PROTEIN C ry30Ga1 is:
1) aminoacid sequence shown in the SEQ ID No.2; Or
2) aminoacid sequence shown in the SEQ ID No.2 is through replacing, lack and/or increasing one or several amino acid and have equal active aminoacid sequence.
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|---|---|---|---|
| CN2009100815976A CN101531712B (en) | 2009-04-13 | 2009-04-13 | Bt protein Cry30Bal as well as encoding gene thereof and application thereof |
| PCT/CN2010/000486 WO2010118634A1 (en) | 2009-04-13 | 2010-04-13 | Insecticidal crystal protein gene cry30ga1, its encoded protein and uses |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100815976A CN101531712B (en) | 2009-04-13 | 2009-04-13 | Bt protein Cry30Bal as well as encoding gene thereof and application thereof |
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