CN103103203A

CN103103203A - Bt cry54Ab1 gene, protein encoded by gene and application of gene or protein

Info

Publication number: CN103103203A
Application number: CN2013100254785A
Authority: CN
Inventors: 郑爱萍; 李平; 关鹏; 余宗兰; 王世全; 邓其明; 刘怀年
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2013-01-23
Filing date: 2013-01-23
Publication date: 2013-05-15

Abstract

The invention provides a Bt cry54Ab1 gene, protein encoded by the gene and application of the gene or the protein. A nucleotide sequence of the cry54Ab1 gene is shown in SEQ ID NO.2, or is a nucleotide sequence which expresses the same functional protein and is formed by carrying out substitution, deletion or addition of one or more nucleotides on the nucleotide sequence shown in SEQ ID NO.2. The Bt protein Cry54Ab1 encoded by the cry54Ab1 gene can be used for preparing a Bt pesticide; or the cry54Ab1 gene can convert crops such as cotton, corn, rice and vegetables, so that the Bt cry54Ab1 operon gene has corresponding anti-insect activity, so as to reduce the dosage of the pesticide, and reduce the environmental pollution. The Bt cry54Ab1 gene has important economic value and application prospect.

Description

A kind of Bt cry54Ab1 gene and proteins encoded and application

Technical field

The present invention relates to biological technical field, be specifically related to a kind of Bt cry54Ab1 gene and proteins encoded 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 crystal proteins 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 630 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, etal.Microbiol Mol Biol Rev, 1998,62:807-813; Http:// www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/).Now separating clone the insecticidal proteins of 72 monoids.Generally speaking, Cry albumen is all encoded by single manipulator, as Cry1, and Cry2, Cry3, the toxalbumin such as Cry4 and Cry9, the insecticidal crystal protein molecular weight of these genes encodings is 130-140kD; The insecticidal crystal protein molecular weight of the genes encoding such as Cry54 and Cry56 is 70-80kDa.Along with going deep into the research of Bt killing gene, some have the killing gene albumen of two manipulator codings and are found, in the middle of these cry genes, cry operon gene of the orf2 genomic constitution with same-code direction of some type cry genes and its sequence back is arranged.These cry operon genes are comprised of 3 parts, first part is a cry genes encoding 60-80kDa Cry albumen, second section is an orf2 genes encoding 50-60kDa Orf2 albumen, and is positioned between the two one and is about the noncoding intervening sequence of 30-80bp.In former research, some investigator thinks that orf2 has the effect of stablizing cry mRNA, perhaps the Orf2 albumen of its coding may as a molecular chaperones, help the formation (Rosso ML, 1997.Appl Environ Microbiol63:4449-4455) of cry albumin crystal; Some investigator thinks that Orf2 albumen has important effect (J.Eleazar Barboza-Corona, 2012.Appl Environ Microbiol78 (6): 2005-2012) for the stable crystal configuration.The C end of the macromolecule Cry albumen such as Orf2 albumen and Cry1, Cry4, Cry7, Cry8, Cry9 is similar, therefore some macromolecular cry genes are divided into two portions because the factors such as transgenation cause complete gene order during evolution, formation is by cry genetically manipulated (Thorne L, 1986.JBacteriology.166:801-811) of two genomic constitutions.

Molecular chaperones is a large proteinoid in cell, that a class does not have dependency but the protein of common function is arranged on sequence, they help other structures that contain polypeptide to complete correct assembling in cell, but the component when not consisting of these protein structures and carrying out functions.

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 130-140kDa, 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).In addition, there are some researches prove that Bti not yet finds resistance problem in the use in land for growing field crops, but mosquito constantly is confirmed in the laboratory to its resistance problem, this situation also may (Georghiou G P occur large Tanaka, 1997.Applied and Environmental Microbiology, 63:1095-1101.).

Be the loss of avoiding resistant insects to cause, many methods that are directed to this problem are suggested, for example strengthen the collaborative use of using dosage and polygene, but still seeking of can fundamentally addressing this problem is new, Bt genetic resources high virulence, wide insecticidal spectrum, and this biological control to China has very important meaning.

Summary of the invention

The purpose of this invention is to provide a kind of Bt cry54Ab1 gene and proteins encoded and application.

The purpose of this invention is to provide a kind of Bt cry54Ab1 gene, it has the nucleotide sequence shown in (1) or (2):

(1) nucleotide sequence shown in SEQ ID NO.2;

(2) nucleotide sequence shown in SEQ ID No.2 is substituted, lacks or add the nucleotide sequence of one or several Nucleotide and expression identical function protein.

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.

The present invention also provides a kind of Bt cry54Ab1 operon gene, described cry54Ab1 operon gene comprises a cry54Ab1 gene, orf2 gene and the non-coding transcribed spacer between them, and described cry54Ab1 operon gene has the nucleotide sequence shown in (1) or (2):

(1) nucleotide sequence shown in SEQ ID NO.1;

(2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks or add the nucleotide sequence of one or several Nucleotide and expression identical function protein.

The present invention is by being CGMCCNo.2719 to the new bacterial strain MC28(of bacillus thuringiensis preserving number) genome and plasmid order-checking study, find that there is a new cry operon gene in this bacterial strain, design its full-length gene primer, the clone obtains the complete genome sequence of cry54Ab1 operon gene, its nucleotide sequence is as shown in SEQ ID No.1, total length is 3823bp, comprises the orf2 gene of the cry54Ab1 gene of a 2232bp and a 1524bp and the non-coding transcribed spacer of 67bp between them.The analysis showed that, cry54Ab1 operon gene GC content is 32.28%.

Wherein, the new bacterial strain MC28 of described bacillus thuringiensis separates in 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: 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), preserving number is CGMCC No.2719, has been open in 200910078897.9 application for a patent for invention at application number.Show by the virulence test to MC28, MC28 all has high virulence to lepidoptera pest, Diptera pest etc.

Wherein, the nucleotide sequence of described cry54Ab1 operon gene is as shown in SEQ ID NO.1, and the nucleotide sequence of cry54Ab1 gene is as shown in SEQ ID NO.2.The nucleotide sequence of orf2 gene is as shown in SEQ ID NO.3.

Described cry54Ab1 operon gene comprises the nucleotide sequence of proteins encoded Cry54Ab1 and Orf2.Wherein, the Cry54Ab1 albumen of 743 amino acid compositions of cry54Ab1 genes encoding; The Orf2 albumen that 507 amino acid of orf2 genes encoding form.Adopt bacterial sigma7.0promoter program that the cry gene in this operon 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 cry54Ab1.

The albumen that the purpose of this invention is to provide described cry54Ab1 genes encoding.

The purpose of this invention is to provide a kind of Bt PROTEIN C ry54Ab1, its aminoacid sequence is as shown in SEQ ID No.4.

The present invention also provides a kind of Bt PROTEIN C ry54Ab1+Orf2, it comprises PROTEIN C ry54Ab1 and albumen Orf2, the aminoacid sequence of PROTEIN C ry54Ab1 is as shown in SEQ ID No.4, and the aminoacid sequence of albumen Orf2 is as shown in SEQ ID No.5, and albumen Orf2 is the molecular chaperones of PROTEIN C ry54Ab1.

The amino acid of Cry54Ab1 and Orf2 albumen forms as table 1 and table 2.

The amino acid of table 1Cry54Ab1 albumen forms

Amino acid

Per-cent %

Amino acid

Per-cent %

[0027]?

Ala(A):	5.38	Met(M):	1.35
				Cys(C):	0.94	Asn(N):	10.36
Asp(D):	4.85	Pro(P):	4.71
				Glu(E):	3.90	Gln(Q):	4.31
Phe(F):	4.04	Arg(R):	3.90
				Gly(G):	5.25	Ser(S):	8.08
His(H):	0.67	Thr(T):	6.59
				Ile(I):	8.21	Val(V):	4.31
Lys(K):	5.38	Trp(W):	1.75
				Leu(L):	9.83	Tyr(Y):	6.19

The amino acid of table 2Orf2 albumen forms

Amino acid	Per-cent %	Amino acid	Per-cent %
				Ala(A):	3.94	Met(M):	4.73
Cys(C):	0.99	Asn(N):	7.50
				Asp(D):	6.51	Pro(P):	2.96
Glu(E):	6.71	Gln(Q):	6.31
				Phe(F):	2.76	Arg(R):	2.76
Gly(G):	6.31	Ser(S):	7.10
				His(H):	3.55	Thr(T):	6.90
Ile(I):	5.92	Val(V):	4.73
				Lys(K):	6.11	Trp(W):	1.18
Leu(L):	7.10	Tyr(Y):	5.92

Be to be understood that, those skilled in the art can be according to PROTEIN C ry54Ab1(disclosed by the invention as shown in SEQ ID No.4) and the aminoacid sequence (as shown in SEQ ID No.5) of Orf2, do not affecting under its active prerequisite, replace, lack or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.For example at the nonactive section of Cry54Ab1, the Leu of the 707th is replaced with Gly, the Gly of the 645th is replaced with Ala, with the Leu disappearance of the 740th, with Ala of the 732nd increase or increase Lys, do not affect its activity.Therefore, Bt PROTEIN C ry54Ab1(of the present invention is as shown in SEQ ID No.4) and Orf2 albumen (as shown in SEQ ID No.5) also comprise shown in aminoacid sequence be substituted, lack or add one or several amino acid and the aminoacid sequence with same function that forms, thereby obtain having with Bt PROTEIN C ry54Ab1+Orf2 with isoreactivity or function by the derivative protein that obtains of Cry54Ab1+Orf2.

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.

Another object of the present invention is to provide the recombinant expression vector that contains described gene.

Take the DNA of Bt bacterial strain MC28 as template, take CO-F(as shown in SEQ ID No.6) and C-R(as shown in SEQ ID No.7) as primer increases, obtain the cry54Ab1 gene, wherein, the sequence of primer CO-F and C-R is as follows:

Primer CO-F:5 '-GCC GGATCCGATGTACAGGTGGGTTCTTGTTAAAC-3 ';

Primer C-R:5 '-CGC GTCGACTCAATTAATAAATAGATTATTCAC-3 '.

Take the DNA of Bt bacterial strain MC28 as template, take O-F(as shown in SEQ ID No.8) and CO-R(as shown in SEQ ID No.9) as primer increases, obtain the orf2 gene, wherein, the sequence of primer O-F and CO-R is as follows:

Primer O-F:5 '-GCC GGATCCGATGTTTACAAGTGGGGCGAAAAAC-3 ';

Primer CO-R:5 '-CGC GTCGACCTAACTATAATTGCTATCATAGC-3 '.

Underscore part GGATCC is restriction enzyme site BamH I, and GTC GAC is restriction enzyme site SalI.

Take the DNA of Bt bacterial strain MC28 as template, take above-mentioned CO-F and CO-R as primer increases, obtain described Cry54Ab1 operon gene.

Cry54Ab1 gene, orf2 gene and cry54Ab1 operon gene are inserted into respectively on bacillus-bacillus coli shuttle expression carrier pSTK, build and obtain recombinant expression vector pSTK-cry54Ab1, pSTK-orf2 and pSTK-cry54Ab1-orf2.

Another object of the present invention is to provide the host cell that contains described recombinant expression vector.

Described host cell is intestinal bacteria or fungi.

Preferably, described host cell is plant host cell, as farm crop such as cotton, corn, paddy rice, vegetables.

Another object of the present invention is to provide the sterilant that contains Bt PROTEIN C ry54Ab1.

The host cell that contains the expression vector of cry54Ab1 gene by fermentation obtains containing the fermented liquid of Bt PROTEIN C ry54Ab1, and it is prepared into sterilant, is used for the control of crop pests.Those skilled in the art can also with above-mentioned cry54Ab1 gene transformation bacterium or fungi, produce Bt PROTEIN C ry54Ab1 by large scale fermentation.

The present invention also provides the sterilant that contains Bt PROTEIN C ry54Ab1+Orf2.

The host cell that contains the expression vector of cry54Ab1 operon gene by fermentation obtains containing the fermented liquid of Bt PROTEIN C ry54Ab1+Orf2, and it is prepared into sterilant, is used for the control of crop pests.Those skilled in the art can also with above-mentioned cry54Ab1 operon gene transform bacteria or fungi, produce Bt PROTEIN C ry54Ab1+Orf2 by large scale fermentation.

Another object of the present invention is to provide the application of said gene and albumen.

The present invention also provides cry54Ab1 gene or the application of Bt PROTEIN C ry54Ab1 in the preparation sterilant.

The present invention also provides cry54Ab1 operon gene or the application of Bt PROTEIN C ry54Ab1+Orf2 in the preparation sterilant.

The present invention also provides cry54Ab1 gene or the application of Bt PROTEIN C ry54Ab1 in cultivating transgenic plant.

The present invention also provides cry54Ab1 operon gene or the application of Bt PROTEIN C ry54Ab1+Orf2 in cultivating transgenic plant.

Another object of the present invention is to provide cry54Ab1 gene or the application of Bt PROTEIN C ry54Ab1 in improving the transgenic plant insect-resistance.

Described being applied as is connected the cry54Ab1 gene with expression vector, obtain to express the recombinant expression vector of Bt PROTEIN C ry54Ab1, by transgenic method, described recombinant expression vector is imported plant again, obtain containing the transformant of cry54Ab1 gene, express Bt PROTEIN C ry54Ab1, improve plant resistance to insect.

The cry54Ab1 gene is operably connected with expression vector, obtain to express the recombinant expression vector of Bt PROTEIN C ry54Ab1, and then by transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage method, described recombinant expression vector is imported the host, obtain containing the transformant of cry54Ab1 gene, plants such as farm crop or fruit tree is expressed Bt PROTEIN C ry54Ab1, it is possessed and its anti-insect activity is provided.

Those skilled in the art can also according to cry54Ab1 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 cry54Ab1 gene design is had the codon of paddy rice numbering, again synthetic cry54Ab1 gene 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 present invention also provides cry54Ab1 operon gene or the application of Bt PROTEIN C ry54Ab1+Orf2 in improving the transgenic plant insect-resistance.

Described being applied as is connected the cry54Ab1 operon gene with expression vector, obtain to express the recombinant expression vector of Bt PROTEIN C ry54Ab1+Orf2, by transgenic method, described recombinant expression vector is imported plant again, obtain containing the transformant of cry54Ab1 operon gene, express Bt PROTEIN C ry54Ab1+Orf2, improve plant resistance to insect.

The cry54Ab1 operon gene is operably connected with expression vector, obtain to express the recombinant expression vector of Bt PROTEIN C ry54Ab1+Orf2, and then by transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage method, described recombinant expression vector is imported the host, obtain containing the transformant of cry54Ab1 operon gene, plants such as farm crop or fruit tree is expressed Bt PROTEIN C ry54Ab1+Orf2, it is possessed and its anti-insect activity is provided.

Those skilled in the art can also according to cry54Ab1 operon 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 design of cry54Ab1 operon gene is had the codon of paddy rice numbering, again synthetic cry54Ab1 operon gene is connected with carrier pCAMBIA1300, be transferred in rice genome by agriculture bacillus mediated, thus the Transgenic Rice that obtains having anti-insect activity.

Cry54Ab1 gene of the present invention and proteins encoded Bt PROTEIN C ry54Ab1 thereof have following advantage:

Cry54Ab1 gene provided by the invention is a kind of new gene, its proteins encoded Bt PROTEIN C ry54Ab1 is a kind of new Bt albumen, has insecticidal activity preferably, use it for the preparation transgenic plant, can the specific killing insect, and the usage quantity of reduction 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 ry54Ab1 of the present invention has important economic worth and application prospect, is fit to large-scale application in the insect-resistance that improves plant.

Wherein, the new bacterial strain MC28 of described bacillus thuringiensis separates in 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: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature are bacillus thuringiensis (Bacillus thuringiensis), and preserving number is CGMCC No.2719.Show by the virulence test to MC28, MC28 all has high virulence to lepidoptera pest, Diptera pest etc.

Description of drawings

Fig. 1 is cry54Ab1 operon gene different piece amplification electrophorogram; Wherein, M is DNA marker; 1 is the orf2 gene; 2 is the cry54Ab1 gene; 3 is complete cry54Ab1 operon gene.

Fig. 2 is that recombinant plasmid pSTK-orf2 enzyme is cut the evaluation electrophorogram; Wherein, M is DNAmarker; 1 is the BamH I of pSTK-orf2 and the double digestion product of Sal I; The 2 orf2 genes for insertion; 3 for to cut the recombinant plasmid pSTK-orf2 of processing without enzyme.

Fig. 3 is that recombinant plasmid pSTK-cry54Ab1 enzyme is cut the evaluation electrophorogram; Wherein, M is DNA marker; The 1 cry54Ab1 gene for insertion; 2 is the BamHI of pSTK-cry54Ab1 and the double digestion product of Sal I; 3 for to cut the recombinant plasmid pSTK-cry54Ab1 of processing without enzyme.

Fig. 4 is that recombinant plasmid pSTK-cry54Ab1-orf2 enzyme is cut the evaluation electrophorogram; Wherein, M is DNA marker; 1 is the cry54Ab1 operon gene; 2 is the BamH I of pSTK-cry54Ab1-orf2 and the double digestion product of Sal I; 3 for to cut the recombinant plasmid pSTK-cry54Ab1-orf2 of processing without enzyme.

Fig. 5 is that the expressing protein SDS-PAGE of HD73 △ (cry54Ab1+orf2), HD73 △ (cry54Ab1) and HD73 △ (orf2) transformant detects figure; Wherein, M is albumen marker; 1 is the expressing protein of HD73 △ (cry54Ab1+orf2); 2 is the expressing protein of HD73 △ (cry54Ab1); 3 is the expressing protein of HD73 △ (orf2); 4 is to transform the bacillus thuringiensis of pSTK plasmid without crystal mutant strain HD73 ^-Expressing protein, as negative control; Arrow represents respectively cry54Ab1 and orf2 expressing protein.

Fig. 6 is the microscopic examination figure of HD73 △ (cry54Ab1+orf2), HD73 △ (cry54Ab1) and HD73 △ (orf2) transformant;

Wherein, figure A is observation by light microscope figure; A is transformant HD73 △ (cry54Ab1+orf2); B is transformant HD73 △ (cry54Ab1); C is transformant HD73 △ (orf2);

Figure B is scanning electron microscopic observation figure; In figure B in a, b, c and figure A a, b, c represent identical.

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.

The genomic dna purification kit is available from matching Parkson company;

E.coli Trans110 is available from the Beijing Quanshijin Biotechnology Co., Ltd;

Opticmicroscope BX51 is available from Japanese Olympus company (Olympus);

Scanning electron microscope S-4800 is available from HIT (Hitachi);

If do not specialize, biochemical reagents used in the embodiment of the present invention are the commercial reagent, as do not illustrate, the conventional means that in the embodiment of the present invention, technique means used is well known to the skilled person.

Embodiment 1cry54Ab1 operon gene, cry54Ab1 gene and orf2 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: 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 (Bacillusthuringiensis), and preserving number is CGMCC No.2719.

The present embodiment is cloned full length sequence and cry54Ab1 gene and the orf2 gene that obtains the cry54Ab1 operon gene by the following method.

Adopt the genomic dna purification kit to extract total DNA of bacterial strain MC28 as the template of amplification cry54Ab1 operon gene and cry54Ab1 gene and orf2 gene, the design primer sequence is as follows:

Primer CO-F:5 '-GCC GGATCCGATGTACAGGTGGGTTCTTGTTAAAC-3 ';

Primer C-R:5 '-CGC GTCGACTCAATTAATAAATAGATTATTCAC-3 '.

Primer O-F:5 '-GCC GGATCCGATGTTTACAAGTGGGGCGAAAAAC-3 ';

Primer CO-R:5 '-CGC GTCGACCTAACTATAATTGCTATCATAGC-3 '.

Underscore part GGATCC is restriction enzyme site BamH I, and GTC GAC is restriction enzyme site SalI.Wherein, the sequence of each primer is as shown in table 3.

Table 3cry54Ab1 operon gene different piece amplimer

In table 3, underscore part GGATCC is restriction enzyme site BamH I, and GTC GAC is restriction enzyme site SalI.

The amplified reaction of above-mentioned each gene is as follows:

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 3.5min, 30 circulations; 72 ℃ are extended 10min; The amplified reaction product is electrophoresis on 0.7% sepharose, puts and observes the pcr amplification result in gel imaging system.Result has obtained being about the sequence of 3.8kb 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 cry54Ab1 operon gene sequence; Obtained being about the sequence of 2.2kb by amplification, this sequence is checked order, its nucleotide sequence is as shown in SEQ ID No.2, and is consistent with the cry54Ab1 gene order; Obtained being about the sequence of 1.5kb by amplification, this sequence is checked order, its nucleotide sequence is as shown in SEQID No.3, and is consistent with the orf2 gene order.

The acquisition of embodiment 2Cry54Ab1 albumen and Orf2 albumen

Product orf2 gene, cry54Ab1 gene and the cry54Ab1 operon gene of embodiment 1 amplification all carry out double digestion with BamH I and Sal I, shuttle vectors pSTK 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 checking insertion segment size and meet expection purpose fragment (as shown in Fig. 2,3,4), obtain respectively recombinant plasmid pSTK-orf2, pSTK-cry54Ab1 and pSTK-cry54Ab1-orf2.Three kinds of recombinant plasmids are changed over to respectively make the recombinant plasmid dna demethylation in E.coli Trans110.Then extract plasmid, three kinds of recombinant plasmids after demethylation are transformed by electricity respectively change Bacillus thuringiensis with crystal negative mutant strain HD73 over to ^-Middle expression.The parameter setting that electricity transforms is 2.2kV, 1000 Ω and 25 μ F.Recombinant bacterial strain difference called after HD73 △ (orf2), the HD73 △ (cry54Ab1) and the HD73 △ (cry54Ab1+orf2) that contain three kinds of recombinant plasmids.Simultaneously with pSTK Plasmid Transformation bacillus thuringiensis without crystal mutant strain HD73 ^-As negative control, with positive transformant in the 1/2LB substratum, cultivate 72h under 200r/min, 28 ℃, centrifugation medium is collected thalline, abandon supernatant, wash thalline 3 times with sterilized water, add 30mL10mmol/L Tris-HCl (pH8.0) ultrasonic disruption, extract expressing protein, with SDS-PAGE, expressing protein is detected.

As shown in Figure 5, SDS-PAGE the analysis showed that: transformant HD73 △ (54Ab+orf2) expresses the albumen of two kinds of different molecular weight sizes, and Cry54Ab1 albumen that is about 80kDa and one is the Orf2 albumen of 60kDa approximately; Only express the approximately Cry54Ab1 albumen of 80kDa in transformant HD73 △ (54Ab); And transformant HD73 △ (orf2) only expresses the Orf2 albumen of 60kDa.The molecular weight of these expressing proteins conforms to the molecular weight of albumen of prediction.

Embodiment 3cry54Ab1, orf2 and complete cry54Ab1 operon gene are at HD73 ^-The microscopic examination of middle expression

In embodiment 2, with each positive transformant in the 1/2LB substratum, cultivate under 200r/min, 28 ℃, collect thalline after the sporulation more than 90%, whether film-making can produce crystallin in optical microphotograph Microscopic observation transformant, adopts the scanning electron microscopic observation crystal habit if produce crystallin.

Observe by Optical microscope and SEM, complete cry54Ab1 operon gene is at HD73 ^-Middle expression also forms spherical parasporal crystal, and cry54Ab1 gene or orf2 gene are separately at HD73 ^-During middle expression, can not form parasporal crystal (as shown in Figure 6).

Embodiment 4Cry54Ab1 and Orf2 albumen insecticidal activity assay

HD73 △ (cry54Ab1), the HD73 △ (orf2) of embodiment 2 acquisitions and gemma and the mixed crystal after the cultivation of HD73 △ (cry54Ab1+orf2) transformant are carried out insecticidal activity assay to culex respectively.At first mixture is mixed with 6 different concentration gradients such as 0.1,4,8,16,32,64mg/mL, then the processing of each concentration gradient drops into 20 Ability of Culex Pipiens Larvaes, and each is processed and repeats 3 times, transforms the bacillus thuringiensis of pSTK plasmid without crystal mutant strain HD73 ^-As negative control, clear water is blank; Statistics after 12h, LC ₅₀Use the SPSS10.0 software analysis.

As can be seen from Table 4: HD73 △ (cry54Ab1+orf2) transformant has good insecticidal activity to Ability of Culex Pipiens Larvae, and its LC50 is 13.8 μ g/mL; HD73 △ (cry54Ab1) also has certain insecticidal activity to culex, and its LC50 is 146.3 μ g/mL.HD73 △ (orf2) and negative control all to culex without insecticidal activity.

The insecticidal activity of three kinds of different transformant expressing proteins of table 4 to culex

In table 4, N represents without insecticidal activity.

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

1. a Bt cry54Ab1 gene, is characterized in that, described cry54Ab1 gene has the nucleotide sequence shown in (1) or (2):

(1) nucleotide sequence shown in SEQ ID NO.2;

2. the albumen of the described genes encoding of claim 1.

3. albumen according to claim 2, is characterized in that, described albumen is Bt PROTEIN C ry54Ab1, and its aminoacid sequence is as shown in SEQ ID No.4.

4. the recombinant expression vector that contains the described cry54Ab1 gene of claim 1.

5. recombinant expression vector according to claim 4, is characterized in that, described recombinant expression vector is pSTK-cry54Ab1.

6. recombinant expression vector according to claim 5, is characterized in that, builds the cry54Ab1 gene the primer that increases in described recombinant expression vector process to be:

Primer CO-F:5 '-GCC GGATCCGATGTACAGGTGGGTTCTTGTTAAAC-3 ';

Primer C-R:5 '-CGC GTCGACTCAATTAATAAATAGATTATTCAC-3 '.

7. the host cell that contains the described recombinant expression vector of claim 4-6 any one.

8. the sterilant that contains claim 2 or 3 described albumen.

9. cry54Ab1 gene claimed in claim 1 or Bt PROTEIN C ry54Ab1 claimed in claim 3 application in improving the transgenic plant insect-resistance.

10. application according to claim 9, it is characterized in that, described being applied as is connected the cry54Ab1 gene with expression vector, obtain to express the recombinant expression vector of Bt PROTEIN C ry54Ab1, by transgenic method, described recombinant expression vector is imported plant again, obtain containing the transformant of Cry54Ab1 gene, express Bt PROTEIN C ry54Ab1, improve plant resistance to insect.