CN102417538B - Bt protein Cry68Aa1 and encoding gene and application thereof - Google Patents
Bt protein Cry68Aa1 and encoding gene and application thereof Download PDFInfo
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- CN102417538B CN102417538B CN 201110405127 CN201110405127A CN102417538B CN 102417538 B CN102417538 B CN 102417538B CN 201110405127 CN201110405127 CN 201110405127 CN 201110405127 A CN201110405127 A CN 201110405127A CN 102417538 B CN102417538 B CN 102417538B
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Abstract
The invention provides a novel Bt protein Cry68Aa1 and an encoding gene thereof. The protein has an amino acid sequence shown as SEQ ID No.2 or an amino acid sequence which is obtained by performing substitution and/or deletion and/or addition of one or more amino acids on the amino acid sequence shown as SEQ ID No.2 and has equivalent activity. The protein can be used for preparing a Bt pesticide. The gene for encoding the protein can be used for transforming crops such as cotton, corn, paddy rice, vegetables and the like for realizing corresponding anti-insect activities, so that the dosage of a farm chemical is reduced, and the environmental pollution is lowered; and the protein has important 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 gene and application.
Background technology
In the human being's production process, insect pest is the important factor that causes the agriculture production loss and affect human health.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, the agricultural byproducts Pesticide Residues increases, and has brought harm for the mankind's 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 with chemical prevention, biological control has safe, effective, lasting characteristics.And the series of problems of having avoided chemical prevention to bring.Therefore, biological prevention 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, can form the parasporal crystal that is formed by protein with insecticidal activity in sporulation, 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 the insects such as multiple lepidopteran, Diptera, Coleoptera.In addition, Bt also has the effect of control evil to the 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, Bt or the important gene source of transgenic pest-resistant engineered plant in the control of agricultural pests, injurious forest-insect and sanitary insect pest.
Cloned from strain HD-1 since first can express the gene of insecticidal activity from Schnepf in 1981, people separating clone the gene of 500 Multi-encoding insecticidal crystal proteins, they are defined as respectively different group, subgroup, class and subclass (Crickmore N according to the amino acid sequence homology of encoding, et al.Microbiol Mol Biol Rev, 1998,62:807-813; Http:// www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/).Generally speaking, Cry1, the 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 of their codings is 130-140kD, and many genes have been widely used in the control of the lepidoptera pest of plant at present.The toxin protein that Bacillus thuringiensissubsp.israelensis (B.thuringiensis subsp.israelensis is called for short Bti) produces has fine insecticidal activity to mosquito, is widely used in the control of mosquito.Simultaneously, Cyt albumen has cytolytic, the resistance that some Cry albumen is had synergism and delays insect.The Utilization of pesticides history of existing more than 50 year take the Bt insecticidal crystal protein as the basis, at first insect never detected to the resistance of Bt, but, begin mid-term 80 year last century, resistance problem (the McGaughey that constantly is confirmed in laboratory and field test, 1985.Science.229:193-195), reason is mainly 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 subject to for a long time the selective pressure of sterilant.1985, McGaughey report warehouse grain pest Indian meal moth (Plodia interpunctella) was under the selective pressure of Dipel (the commodity preparation of Bt subsp.kurstaik HD-1), and after breeding for 15 generations, 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 first large Tanaka in Hawaii has produced obvious resistance (Tabashnik B.E. to the Bt sterilant, et al.1994.Proc.Natl.Acad.Sci.USA.91:4120-4124), since the nineties in last century, use the ground such as long Shenzhen and Guangzhou of Bt sterilant time, Shanghai in China, find that the Bt sterilant obviously descends to the small cabbage moth prevention effect, means that resistance forms (Feng Xia .1996. insect journal, 39 (3): 238-244; Hofte H., 1988.Appl.Environ.Microbiol.54:2010-2017).Find in the laboratory at present and the field has at least tens kinds of insects to produce resistance to Bt and insecticidal crystal protein thereof, 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., et al.1998.Mol.Biol.Rev.65 (3): 775-806).Be the loss of avoiding resistant insects to cause, seeking new high virulence Bt genetic resources is the effective way that addresses this problem, and this biological control to China has very important meaning.
Summary of the invention
First purpose of the present invention is to provide a kind of new Bt virulence protein resource for above-mentioned deficiency.
Second purpose of the present invention is to provide the gene of encoding said proteins.
The 3rd purpose of the present invention is to provide the application of above-mentioned albumen and gene.
The present invention separates the new bacterial strain MC28 of bacillus thuringiensis that obtains from the virgin forest Soils In The Region of Muchuan, Sichuan Province, this bacterial strain (is called for short CGMCC on October 21st, 2008 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is bacillus thuringiensis (Bacillus thuringiensis), and preserving number is CGMCCNo.2719.This bacterium is open in Chinese patent CN101503666B.
Show by the virulence test to MC28, MC28 all has high virulence to lepidoptera pest, Diptera pest etc.Find that from MC28 genome and plasmid sequencing result there is a new cry gene in this bacterial strain, further design its full-length gene primer, the clone obtains the cry68Aa1 gene, its nucleotide sequence is as shown in sequence table SEQ ID No.1, total length is 2511bp, the analysis showed that, GC content is 35.09%, the albumen that 836 amino acid of encoding form.After measured, its aminoacid sequence is as shown in SEQ ID No.2.Adopt bacterial sigma7.0promoter 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, with its called after cry68Aa1.The amino acid of Cry68Aa1 albumen forms as table 1.
The amino acid of table 1Cry68Aa1 albumen forms
| Amino acid | Per-cent % | Amino acid | Per-cent % |
| Ala(A)： | 4.31 | Met(M)： | 1.08 |
| Cys(C)： | 0.60 | Asn(N)： | 12.32 |
| Asp(D)： | 6.10 | Pro(P)： | 5.50 |
| Glu(E)： | 3.23 | Gln(Q)： | 5.74 |
| Phe(F)： | 4.19 | Arg(R)： | 5.62 |
| Gly(G)： | 4.31 | Ser(S)： | 10.17 |
| His(H)： | 2.75 | Thr(T)： | 6.46 |
| Ile(I)： | 6.34 | Val(V)： | 5.50 |
| Lys(K)： | 1.44 | Trp(W)： | 0.72 |
| Leu(L)： | 7.18 | Tyr(Y)： | 6.46 |
Be to be understood that, those skilled in the art can be according to the aminoacid sequence (SEQ ID No.2) of PROTEIN C ry68Aa1 disclosed by the invention, do not affecting under its active prerequisite, replacing, lack and/or increase one or several amino acid, obtaining the mutant nucleotide sequence of described albumen.For example at nonactive section, the Leu of the 654th is replaced with Gly, the Gly of the 670th is replaced with Ala, with the Thr disappearance of the 799th, with Ala of the 774th increase or increase Lys, do not affect its activity.Therefore, Bt PROTEIN C ry68Aa1 of the present invention comprises that also shown in SEQ ID No.2, aminoacid sequence is substituted, replaces and/or increases one or several amino acid, have with Bt PROTEIN C ry68Aa1 with isoreactivity by the derivative protein that obtains of Cry68Aa1.
Gene of the present invention comprises the nucleotide sequence of encoding said proteins Cry68Aa1.
The invention provides the gene of the above-mentioned Bt PROTEIN C ry68Aa1 of coding, its nucleotides sequence is classified as:
(1) nucleotide sequence shown in sequence table SEQ ID NO.1, or
(2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or increases one or several Nucleotide.
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 Bt bacterial strain MC28 with protein and be obtained, and perhaps obtains by DNA or the synthetic method of peptide.
Gene of the present invention can be operably connected with expression vector, obtain to express the recombinant expression vector of albumen of the present invention, and then can pass through transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage method, described expression vector is imported the host, obtain turning the transformant of cry68Aa1 gene, plants such as farm crop or fruit tree makes it possess anti-insect activity.
In one embodiment of the invention, the acquisition of Bt PROTEIN C ry68Aa1 recombinant expression vector is to obtain recombinant expression vector pET-68Aa by the cry68Aa1 gene being inserted into upper structure of expression vector pET-28a (+).
In addition, can also obtain containing the fermented liquid of Cry68Aa1 albumen by fermentation bacterial strain MC28 of the present invention, it is prepared into sterilant, be used for the control of crop pests.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.
The invention provides the application of Bt PROTEIN C ry68Aa1 in cultivating transgenic plant.
The present invention also provides the application of Bt PROTEIN C ry68Aa1 in improving plant resistance to insect.
Those skilled in the art can also according to cry68Aa1 gene disclosed by the invention, with farm crop such as its converting cotton, corn, paddy rice, vegetables, make it possess corresponding anti-insect activity.For example: the degeneracy of utilizing codon, the gene order that the cry68Aa1 gene design is had paddy rice preference codon, again synthetic cry68Aa1 gene order is connected with carrier pCAMBIA1300, be transferred in rice genome by agriculture bacillus mediated, thus the Transgenic Rice that obtains having anti-insect activity.
The invention provides Cry68Aa1 albumen is a kind of new Bt albumen, has insecticidal activity preferably, uses it for the preparation transgenic plant, can the specific killing insect, and reduce the usage quantity of agricultural chemicals, reduce costs environmental contamination reduction.Also do not have at present insect or insect this albumen to be produced the report of resistance, therefore, Bt PROTEIN C ry68Aa1 of the present invention has important economic worth and application prospect, is fit to large-scale application in the insect-resistance that improves plant.
Description of drawings
Fig. 1 shows is the gel electrophoresis figure of the cry68Aa1 full-length gene that obtains of clone, M wherein, DNA marker; 1, cry68Aa1 gene;
Fig. 2 shows is that the enzyme of recombinant plasmid pET-68Aa is cut the evaluation collection of illustrative plates, M wherein, DNAmarker; 1, the DNA of insertion; 2, the Sac I+Not I double digestion product of recombinant plasmid pET-68Aa; 3, recombinant plasmid pET-68Aa;
What Fig. 3 showed is that the SDS-PAGE of cry68Aa1 gene in E.coli BL21 (DE3) detects figure; M wherein, albumen marker; 1, contain E.coliBL21 (DE3) expressing protein (negative control) of carrier pET-28a; 2, the E.coliBL21 (DE3) that contains recombinant plasmid pET-68Aa induces cellular lysate liquid without IPTG; 3, contain E.coli BL21 (DE3) cellular lysate liquid after IPTG induces of recombinant plasmid pET-68Aa.
Embodiment
Following examples further illustrate 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, biochemical reagents used in embodiment are the commercial reagent, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1cry68Aa1 gene cloning
The present invention separates the new bacterial strain MC28 of bacillus thuringiensis that obtains from the virgin forest Soils In The Region of Muchuan, Sichuan Province, this bacterial strain on October 21st, 2008 in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is bacillus thuringiensis (Bacillus thuringiensis), and preserving number is CGMCC No.2719.
This example is cloned the full length sequence that obtains the cry68Aa1 gene by the following method.
Adopt genomic dna purification kit (available from matching Parkson company) to extract total DNA of bacterial strain MC28 as the template of amplification cry68Aa1 gene.The design primer sequence is as follows:
P1：5’ATGAATACAGATCAAAATA 3’；
P2：5’TCATTTACAATTACAGTTTT 3’
25 μ l PCR reaction systems:
Thermal cycle reaction: 94 ℃ of denaturation 5min; 94 ℃ of sex change 50s, 54 ℃ of annealing 50s, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min; The amplified reaction product is electrophoresis on 1% sepharose, puts and observes the pcr amplification result in gel imaging system.Result has obtained being about the sequence of 2.5kb as shown in Figure 1 by amplification, this sequence is checked order, and its nucleotide sequence is as shown in SEQ ID No.1, and is consistent with aim sequence.
The acquisition of embodiment 2Cry68Aa1 albumen
According to cry68Aa1 gene open reading frame two terminal sequences, design and synthesize a pair of special primer cry68F:5 '-CG
GAGCTCATGAATACAGATCAA AATA-3 ' cry68R:5 '-ATTT
GCGGCCGCTCATTTACAATTACAGTTTT-3 ', 5 ' end primer underscore part base is respectively Sac I and Not I restriction enzyme site.Increase take the total DNA of MC28 as template, response procedures and amplification condition are with embodiment 1, the product of amplification adopts Sac I and NotI to carry out double digestion, carrier pET-28a (+) after enzyme is cut product and carried out equally double digestion 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 expection purpose fragment, then change recipient bacterium E.coli.BL21 (DE3) (buying in the Beijing Quanshijin Biotechnology Co., Ltd) over to.With recombinant plasmid called after pET-68Aa, contain the recon called after E.coli.BL21 (68Aa) of recombinant plasmid.With positive transformant in the LB substratum, in 200r/min, 37 ℃ of incubated overnight, again the ratio of nutrient solution according to 1: 100 is transferred in the 1L triangular flask that contains 400mL LB nutrient solution, 200r/min, 37 ℃ of cultivations when the OD of nutrient solution value reaches 0.6-0.8, add 0.6mmol/L IPTG to carry out abduction delivering 20h, centrifugation medium is collected thalline, abandon supernatant, add 30mL 10mmol/L Tris-HCl (pH8.0) ultrasonic disruption, with SDS-PAGE, expressing protein is detected.
SDS-PAGE the analysis showed that in the precipitation of expression product after the thalline ultrasonication of cry68Aa1 gene (Fig. 3), and molecular weight is about the 95kDa left and right, conforms to the molecular weight of albumen of prediction.
Embodiment 3Cry68Aa1 albumen insecticidal activity assay
The cry68Aa1 gene expression product carries out insecticidal activity assay to beet armyworm and bollworm respectively.
The Cry68Aa1 albumen that embodiment 2 is obtained is mixed with respectively 1 μ g/ml, 10 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/ml to 6 different concns of 100 μ g/ml, and the old tender moderate Caulis et Folium Brassicae capitatae blade of choosing is cleaned, and dries; Shine 15min under ultraviolet lamp, be cut into 2 * 2cm
2Size divides to be placed in 6 kinds of different concns Cry68Aa1 protein liquids, soaks 5min; Taking-up drains unnecessary liquid, be placed in the culture dish of sterilization to dry, the blade that soaks with the LB liquid nutrient medium in contrast, each culture dish is put 4 blades; 30 of healthy 2-3 bollworms in age are put in each culture dish choosing; Every processing repeats 3 times, puts indoorly, observes the larva death condition after 3d, with SPSS 10.0 computed in software LC
50
The Cry68Aa1 albumen that embodiment 2 obtains is the same to the insecticidal activity assay method of beet armyworm.
Result shows: the Cry68Aa1 albumen that embodiment 2 obtains all has insecticidal activity preferably to these two kinds of insects, and is wherein the highest to the bollworm insecticidal activity, LC
50Be 18.33 μ g/mL; Slightly low to the beet armyworm insecticidal activity, LC
50Be 20.16 μ g/mL.E.coli.BL21 (DE3) is as negative control, and detected result shows, E.coli.BL21 (DE3) is not had an insecticidal activity to above two kinds of insects.
The insecticidal activity of table 1Cry68Aa1 to two kinds of insects
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. Bt PROTEIN C ry68Aa1, its aminoacid sequence is
Aminoacid sequence shown in SEQ ID No.2.
2. the gene of coding claim 1 described albumen.
3. gene as claimed in claim 2, its nucleotide sequence is as shown in SEQ IDNo.1 in sequence table.
4. the recombinant expression vector that contains claim 2 or 3 described genes.
5. the host cell that is transformed by the described expression vector of claim 4.
6. host cell as claimed in claim 5, it is plant host cell.
7. the sterilant that contains the described albumen of claim 1, described sterilant has insecticidal activity for beet armyworm and bollworm.
8. albumen claimed in claim 1 or its encoding gene application in the preparation sterilant, described sterilant has insecticidal activity for beet armyworm and bollworm.
9. albumen claimed in claim 1 or its encoding gene application in cultivating transgenic plant.
10. albumen claimed in claim 1 or its encoding gene application in improving plant resistance to insect, described worm is beet armyworm and bollworm.
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Citations (2)
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|---|---|---|---|---|
| WO2006012366A3 (en) * | 2004-07-20 | 2006-09-28 | Phyllom Llc | Methods for making and using recombinant bacillus thuringiensis spores |
| CN101503666A (en) * | 2009-03-05 | 2009-08-12 | 四川农业大学 | Novel strain of Bacillus thuringiensis bacterial strain and use thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006012366A3 (en) * | 2004-07-20 | 2006-09-28 | Phyllom Llc | Methods for making and using recombinant bacillus thuringiensis spores |
| CN101503666A (en) * | 2009-03-05 | 2009-08-12 | 四川农业大学 | Novel strain of Bacillus thuringiensis bacterial strain and use thereof |
Non-Patent Citations (7)
| Title |
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| Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities;GAO, M. et al.;《Biological control》;20081231;Pages 380-388 * |
| GAO, M. et al..Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities.《Biological control》.2008,Pages 380-388. |
| NCBI.GenBank Accession NO.AFU17323.1（Pesticidal crystal protein cry9Aa [Bacillus thuringiensis MC28]）.《NCBI GenBank》.2012,全文. * |
| 四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆;朱军等;《微生物学报》;20090304;第324-330页 * |
| 朱军等.四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆.《微生物学报》.2009,第324-330页. |
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| 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》.2008,第684－687页. |
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