CN103233021A - Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 - Google Patents
Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 Download PDFInfo
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Abstract
The invention discloses a preparation method of a bacillus thuringiensis crystal fusion protein Bt Cry35Ba2. The method relates to a codon-optimized bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 gene which is specifically represented by sequence 1 in the sequence list. The bacillus thuringiensis crystal fusion protein preparation method provided by the invention comprises the steps that: (1) a recombinant vector comprising a DNA fragment of the 109th-1269th site of the sequence 1 or the entire sequence 1 is introduced into Escherichia coli, such that recombinant Escherichia coli is obtained; (2) the recombinant Escherichia coli is cultured, and induction expression is carried out; and thallus is collected and cracked, such that the bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 (sequence 2) is obtained. According to the invention, protein activity of the protein obtained by expression of the codon-optimized bacillus thuringiensis crystal fusion protein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) is not influenced by condon optimization.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of preparation method of bacillus thuringiensis crystal fusion rotein, particularly the preparation method of a kind of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram-positive microorganism that is distributed widely in the soil, in the forming process of its gemma, can form the crystallin (Cry albumen) with insecticidal activity.This albumen has higher toxicity to various pests such as lepidopteran, Diptera, Coleoptera, Hymenopteras, is the toxalbumin that is most widely used.Because this characteristic, the gene of coding Cry albumen is taken as be the popular alternative gene of genetically modified crops by the biologist, and existing a plurality of genetically modified crops kinds change this gene and commercialization over to, extensively plantation in the world.The Bt fungus strain contains a large amount of different insecticidal crystal protein encoding genes, so far existing nearly 180 different Bt insecticidal crystalline genes are cloned and are checked order, Bt Cry35Ba2 is exactly wherein a kind of, and the coleoptera larvae that is primarily aimed at harm seeding corn and other crops root has toxicity.
The plantation of genetically modified crops has been satisfied human to grain yield, economic benefit demand.Simultaneously, also exist the environmental safety risk, randomness and uncertainty that foreign gene imports, genetic expression is to the change of the Physiology and biochemistry effect of crop, and threat that ecotope is constituted etc. all is difficult to prediction.There is worry in people to the securities of genetically modified crops, and the effective way of eliminating this worry is exactly that the expressed foreign protein of genetically modified crops is carried out safety evaluation.Therefore, obtain the expressed foreign protein of various Bt insecticidal crystalline genes, significant to carrying out safety evaluation, for the follow-up method of inspection is set up, the enforcement of examination criteria provides the basis, thereby can effectively prevent the generation of potential safety hazard, avoid genetically modified crops to the destruction of ecotope, give full play to the huge applications potentiality of transgenic technology in agriculture production.
Summary of the invention
An object of the present invention is to provide the gene of a kind of bacillus thuringiensis crystal protein B t Cry35Ba2 of codon optimization.
The gene of the bacillus thuringiensis crystal protein B t Cry35Ba2 that described codon is optimized, be under the prerequisite that does not change bacillus thuringiensis crystal protein B t Cry35Ba2 aminoacid sequence (the 1-387 amino acids of GenBank:AY536894.1), the codon of its wild-type encoding gene (the 1-1161 position of GenBank:AY536894.1) replaced with the gene of gained behind the codon of intestinal bacteria preference (high frequency use).Be N-Cry35Ba2 with the unnamed gene after optimizing, specifically can be following a)-c) in arbitrary dna molecular:
A) dna molecular shown in the 109-1269 position Nucleotide of sequence 1 in the sequence table;
B) dna molecular shown in the sequence 1 in the sequence table;
C) and a) or b) dna molecular that limits has 98% identity at least, and protein DNA molecule shown in the 37-423 amino acids of encoding sequence 2 or sequence 2.
Wherein, sequence 1 is made up of 1296 Nucleotide altogether, and wherein 109-1269 position Nucleotide replaces with the gene order of gained behind the intestinal bacteria preference codon for the codon with the 1-1161 position of GenBank:AY536894.1.Protein shown in the sequence 2, wherein the 37-423 amino acids of the 109-1269 position nucleotide coding sequence 2 of sequence 1 in the tabulation of sequence 1 code sequence.Sequence 2 is made up of 431 amino acid altogether.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain described dna molecular (N-Cry35Ba2 gene) also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can be used existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised.For the ease of transgenic cell being identified and being screened, can process used expression vector, but as the gene (GFP gene, luciferase genes etc.) that adds luminophor, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance etc.
In the present invention, the promotor that the described dna molecular of startup (N-Cry35Ba2 gene) is transcribed in the described recombinant expression vector is specially the T7 promotor.
Described recombinant expression vector is specially described dna molecular (N-Cry35Ba2 gene) is inserted into the recombinant plasmid that the multiple clone site place of plasmid pET28a obtains.In one embodiment of the invention, described multiple clone site is EcoR I and Xho I.More concrete, the nucleotide sequence of (pET28a-35Ba2) of described recombinant expression vector is shown in sequence in the sequence table 3.
Wherein, sequence 3 is made up of 6502 Nucleotide altogether, and 137-1432 position Nucleotide is the reverse complementary sequence of sequence 1.
The RNA molecule of being transcribed gained by described dna molecular (N-Cry35Ba2 gene) also belongs to protection scope of the present invention.
A further object of the present invention provides a kind of protein.
Protein provided by the present invention has bacillus thuringiensis crystal protein B t Cry35Ba2 activity, is specially the protein shown in the sequence 2 in the sequence table.
The encoding gene of described protein also belongs to protection scope of the present invention.
Described dna molecular, or described recombinant vectors, expression cassette, transgenic cell line or reorganization bacterium, or described RNA molecule, or described protein also belongs to protection scope of the present invention in the application that preparation has in the product of insecticidal activity.
In described application, described worm is corn rootworm (as three initial stage in age corn rootworms); Described product can be test kit.
The application in the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 expression amount in improving intestinal bacteria of described dna molecular also belongs to protection scope of the present invention; Described bacillus thuringiensis crystal fusion rotein BtCry35Ba2 is specially protein shown in the sequence 2 in the sequence table.
Also purpose of the present invention provides the preparation method of a kind of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
The preparation method of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 provided by the present invention specifically comprises the steps:
1) the described recombinant vectors (as recombinant expression vector pET28a-35Ba2) that will express described dna molecular imports intestinal bacteria, obtains recombination bacillus coli;
2) cultivate described recombination bacillus coli, carry out abduction delivering, collect and the cracking thalline, obtain bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2;
Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is specially protein shown in the sequence 2 in the sequence table.
In aforesaid method, the described expression vector in the step 1) can be can the expressed sequence table in other recombinant prokaryotic expression vectors of dna molecular shown in the sequence 1.
In aforesaid method, step 2) in, described abduction delivering is specially: have to cultivation that to add IPTG in the nutrient solution of described recombination bacillus coli be 1mM to final concentration, 22 ℃ jolt and cultivated 6 hours.
It is between the 0.6-0.8 that described cultivation has the OD600 value of the nutrient solution of described recombination bacillus coli.
The rotating speed that described concussion is cultivated is 180rpm, and rotation radius is 1.5 centimetres.
In aforesaid method, step 2) in, described " collecting and the cracking thalline " afterwards, comprise that also collect centrifugal back and the step of dissolving inclusion body.
At 1mM IPTG, induce under 6 hours the condition for 22 ℃, described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 mainly is expressed in the inclusion body.
Using gene engineering method of the present invention is synthesized bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) at home first.Experiment showed, that in intestinal bacteria the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 gene (N-Cry35Ba2 gene) that codon provided by the present invention is optimized is expressed gained albumen not because of codon optimized its protein-active that influences.The present invention lays the foundation for detection method, physiological function and mechanism of action thereof, the degradation mechanism of further inquiring into bacillus thuringiensis crystal protein B t Cry35Ba2.
Description of drawings
Fig. 1 carries out dna molecular electrophorogram behind the double digestion for recombinant plasmid with EcoR I and Xho I.Wherein, swimming lane Marker for the dna molecular amount standard 10Kbp that buys to 1000bp band (sky root biotechnology (Beijing) company limited, catalog number MD100-1Kb Ladder).Swimming lane 1 carries out band behind the double digestion for recombinant expression vector pET28a-35Ba2 with EcoR I and Xho I.
Fig. 2 is prokaryotic expression and the purification result (SDS-PAGE) of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.
Among the A, swimming lane Marker is for buying molecular weight of albumen standard 10KD to 170KD band (English mill development in science and technology company limited is opened up in Beijing, catalog number TF413-01); Swimming lane 1 changes the inclusion body that the control group thalline of pET28a empty carrier makes over to for cracking.Swimming lane 2 changes the inclusion body that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes over to for cracking.Swimming lane 3 is Cry35Ba2 albumen behind the purifying.
Among the B, swimming lane Marker is for buying molecular weight of albumen standard 10KD to 170KD band (English mill development in science and technology company limited is opened up in Beijing, catalog number TF413-01).Swimming lane 1 changes the supernatant that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes over to for cracking.Swimming lane 2 changes the supernatant that the control group thalline of pET28a empty carrier makes over to for cracking; Swimming lane 3 changes the inclusion body that the experimental group thalline of recombinant expression vector pET28a-35Ba2 makes over to for cracking.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
PGOV4 cloning vector: provided by the Gene Oracle Inc. of gene Synesis Company.Be documented in " Kalle Kantola; Mohammadreza Sadeghi; Anne Lahtinen et al.Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples:Implications for respiratory transmission and latency.Journal of Clinical Virology; 45 (2009) 292-295. " literary composition, the public can obtain from China Agricultural University.One of advantage of this carrier is to remove most conventional restriction enzyme site in its sequence, is conducive to add required site when synthetic gene, and can conflict with existing site in the sequence.In being connected of carrier and purpose fragment, not being to adopt enzyme to cut the method for connection yet, but directly connecting with concordant end.
The pET28a carrier: purchase the company in MERKE NOVAGEN, catalog number is 69864-3.
Corn rootworm (D.baberi): 3 initial stages in age, be documented in " G.MALLOCH; B.FENTON.Super-infections of Wolbachia in byturid beetles and evidence for genetic transfer between A and B super-groups of Wollbachia.Molecular Ecology; (2005) 14; 627-637. " literary composition, the public can obtain from China Agricultural University.
Codon optimized and the full gene of embodiment 1, bacillus thuringiensis crystal protein B t Cry35Ba2 gene is synthetic
According to the wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 gene order (the 1-1161 position Nucleotide of GenBank:AY536894.1) in the bacillus thuringiensis (Bacillus thuringiensis), obtain being suitable for the gene order at the optimization type bacillus thuringiensis crystal protein B t of expression in escherichia coli Cry35Ba2 through design with verifying repeatedly, be about to wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 gene order is transformed into intestinal bacteria preference (high frequency use) under the prerequisite that does not change aminoacid sequence (the 1-387 amino acids of GenBank:AY536894.1) codon optimized sequence, thereby improve the expression level of bacillus thuringiensis crystal protein B t Cry35Ba2 in the intestinal bacteria culture environment.
According to aforesaid method, the final optimization type bacillus thuringiensis crystal protein B t Cry35Ba2 gene that obtains by the design of intestinal bacteria preference, called after N-Cry35Ba2 gene, its gene order are the 109-1269 position of sequence 1 in the sequence table, are made up of 1161 Nucleotide altogether.The albumen of described N-Cry35Ba2 genes encoding is the albumen that the 37-423 amino acids of sequence 2 in the sequence table is formed, and is identical with the albumen of the wild-type bacillus thuringiensis crystal protein B t Cry35Ba2 genes encoding shown in the 1-1161 position Nucleotide of GenBank:AY536894.1.
The prokaryotic expression of embodiment 2, bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
One, the structure of protokaryon recombinant expression vector pET28a-35Ba2
Needs for construction of expression vector, the recognition sequence of restriction enzyme site EcoR I and Xho I is added at the two ends of bacillus thuringiensis crystal protein B t Cry35Ba2 gene (N-Cry35Ba2 gene) respectively after embodiment 1 gained optimization, obtains " the 103-1275 position of sequence 1 ".This sequence of synthetic, and dna fragmentation shown in this sequence linked to each other with the pGOV4 cloning vector, recombinant plasmid obtained.To show the recombinant plasmid called after pGOV4-35Ba2 that in the pGOV4 cloning vector, inserts dna fragmentation shown in " the 103-1275 position of sequence 1 " through order-checking.
The recombinant plasmid pGOV4-35Ba2 of above-mentioned structure is carried out double digestion with restriction enzyme EcoR I and Xho I, reclaim target gene fragment; The skeleton big fragment of gained target gene fragment with the pET28a carrier of cutting through same enzyme linked to each other, connect after product and change in the bacillus coli DH 5 alpha competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), the coated plate screening.The picking positive monoclonal shakes bacterium, extracts plasmid, carries out double digestion with EcoR I and Xho I.Enzyme is cut the result as shown in Figure 1.To cut through enzyme and identify and to obtain the recombinant plasmid sample presentation order-checking that size is about 5341bp and two purpose bands of 1161bp, will be through order-checking proof recombinant plasmid called after pET28a-35Ba2 of the 109-1269 position nucleotide sequence of sequence 1 in the insertion sequence table between the multiple clone site EcoR of pET28a carrier I and Xho I.The nucleotide sequence of recombinant expression vector pET28a-35Ba2 is shown in sequence in the sequence table 3, and wherein 137-1432 position Nucleotide is the reverse complementary sequence of sequence 1.In recombinant expression vector pET28a-35Ba2, all be connected with the His label that the pET28a carrier carries at the upstream and downstream of described N-Cry35Ba2 gene (the 109-1269 position of sequence 1), in order to carry out the purifying of target protein when carrying out prokaryotic expression; Particularly, in recombinant expression vector pET28a-35Ba2, described N-Cry35Ba2 gene (the 109-1269 position of sequence 1) is present among the ORF shown in the whole sequence 1, and wherein the 1270-1296 position of the 1-108 position of sequence 1 and sequence 1 is the sequence that carries on the pET28a carrier.Whole sequence 1 expressed albumen is the albumen shown in the sequence 2 in the sequence table (being designated as " bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 ").In recombinant expression vector pET28a-35Ba2, the promotor that starts described N-Cry35Ba2 genetic transcription is the T7 promotor.
Two, prokaryotic expression, purifying and the evaluation of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
1, the prokaryotic expression of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
A) the recombinant expression vector pET28a-35Ba2 that step 1 is obtained changes in e. coli bl21 (DE3) competent cell (TIANGEN Biotech (Beijing) Co., Ltd.) as experimental group, the e. coli bl21 (DE3) that changes empty carrier pET28a over to is set simultaneously organizes in contrast.Respectively at the LB substratum that contains 50 μ g/mL Kan, 37 ℃ of shaking culture 16h obtain culture with experimental group and control group.
B) step a) gained culture is linked in the LB substratum that contains 50 μ g/mL Kan by 1% volume, 37 ℃ of 200rpm shaking culture 2h, to bacterium liquid OD600 value between 0.6-0.8, obtain nutrient solution.
C) adding IPTG in the step b) gained nutrient solution is 1mM to its final concentration, and 22 ℃ jolt and cultivated 6 hours, and its rotating speed is 180rpm, and the shaking table rotation radius is 1.5 centimetres.
D) with 4 ℃ of step c) products therefroms, the centrifugal 20min of 12000g collects thalline.
E) add BugBuster Master Mix(MERCK company in thalline, catalog number is 71456-3), consumption is the 1mg thalline: 5ml BugBuster Master Mix.Beat or gentle vortex makes BugBuster Master Mix and thalline mixing with inhaling under the room temperature, 50-60rpm shaking culture 20min obtains lysate.
F) with step e) gained lysate with 4 ℃, the centrifugal 20min of 16000g.On the one hand, collect supernatant; On the other hand, collecting precipitation (inclusion body), add behind the BugBuster Master Mix fully vortex (1mg precipitates corresponding 5ml lysate) in the precipitation, 4 ℃, 5000g are centrifugal, and supernatant is removed in the 10min hypsokinesis, in precipitation, add the BugBuster Master Mix diluent with 10 times of deionized water dilutions that adds 6 times of BugBuster Master Mix volumes before again, fully behind the vortex 4 ℃, 5000g centrifugal supernatant is removed in the 10min hypsokinesis, 4 ℃, the centrifugal 15min of 12000g after repeating 3 times, the supernatant that inclines obtains the high purity inclusion body; In inclusion body, add solubilization of inclusion bodies liquid (0.05M CAPS damping fluid contains 0.3%(0.3g/100mL) N-dodecyl musculamine acid sodium; The corresponding 1ml solubilization of inclusion bodies of 10mg inclusion body liquid), light and slow resuspended inclusion body; Draw supernatant behind 4 ℃, the centrifugal 20min of 12000g, obtain solubilization of inclusion bodies liquid.
Step f) gained supernatant and solubilization of inclusion bodies liquid are carried out the SDS-PAGE electrophoresis detection respectively; The SDS-PAGE method is with reference to " biochemical test method and technology " second editions of showing such as Zhang Longxiang, Higher Education Publishing House (2006), 100-106 page or leaf.Adopt 5% to concentrate glue, voltage 50V.Separation gel adopts 12% gum concentration, voltage 120V.Applied sample amount is 15 μ l.
The result is shown in B among Fig. 2.As can be seen from the figure, compare with the control group that changes the pET28a empty carrier over to, the experimental group that changes the recombinant expression vector pET28a-35Ba2 that step 1 obtains over to has the purpose band to occur in the position that size is about 48KD, with consistent according to aminoacid sequence computation purpose molecular weight of albumen size in theory.In addition, it can also be seen that from the result that at 1mM IPTG, induce under 6 hours the condition for 22 ℃, bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 mainly is expressed in the inclusion body.
2, the purifying of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
F in the above-mentioned steps 1) collected solubilization of inclusion bodies liquid is the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that slightly carries, use BugBuster Ni-NTA His Bind purification kit (available from Novagen company, catalog number is 70751-3) and BugBuster His*Bind purification kit (available from Novagen company, catalog number is 70899-3) it is carried out purifying, the bacillus thuringiensis crystal fusion rotein BtCry35Ba2 behind the acquisition purifying.
In the purge process, the resin that adopts BugBuster Ni-NTA His Bind test kit to provide, the purifying damping fluid that adopts BugBuster His*Bind purification kit to provide.Operate according to the method that BugBuster His*Bind purification kit provides.
3, the evaluation of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2
(1) sequence verification
Bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 behind the step 2 gained purifying is carried out the order-checking of N end, and the result confirms that its sequence conforms to sequence 2, has confirmed the exactness of sequence.
(2) SDS-PAGE identifies
The purification result of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 behind step 2 gained of the employing SDS-PAGE purifying is verified.Be contrast with the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that slightly carries before the purifying.
The SDS-PAGE method is with reference to " biochemical test method and technology " second editions of showing such as Zhang Longxiang, Higher Education Publishing House (2006), 100-106 page or leaf.Adopt 5% to concentrate glue, voltage 50V.Separation gel adopts 12% gum concentration, voltage 120V.Applied sample amount is 10 μ l.
SDS-PAGE result is shown in A among Fig. 2.As can be seen from the figure, compare with (swimming lane 2 of A among Fig. 2) before the purifying, the albumen behind the purifying (swimming lane 3 of A among Fig. 2) purpose band is single, has effectively removed foreign protein.
4, the output of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 detects
Adopt BCA protein quantification test kit (BCA Protein Assay Kit, available from Novagen company, catalog number is 71285-3) the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 behind the step 2 gained purifying is carried out concentration determination, and then obtain the ultimate production of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2.The concentration determination concrete operations are carried out referring to the test kit specification sheets.3 repetitions are established in the determination of yield experiment of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 altogether, and the result gets the mean value that repeats three times.
The result shows that the output of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is 0.0236mg albumen/mg thalline, and wherein " thalline " is collected bacterial sediment after the IPTG abduction delivering is cultivated.
The determination of activity of the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 3, embodiment 2 obtain
The Cry35Ba2 albumen that uses embodiment 2 purifying to obtain, use the 0.1%(volume ratio) dilution of the Triton-100 aqueous solution, (protein concentration is followed successively by 10mg/L to be made into 5 concentration, 5.0mg/L, 2.5mg/L, 1.25mg/L and the diluent of 0.625mg/L, simultaneously with the 0.1%(volume ratio) the Triton aqueous solution compares, the root immersion diluent of water planting corn seedling is dried after 10 seconds naturally, being paved with the bottom is covered with in the culture dish of water-soaked filter paper, every ware inserts 10 three initial stage in age corn rootworms (D.baberi), places 25 ℃, relative humidity 65%, cultivate in the dark incubator.The death condition of statistics corn rootworm larva (is touched the larva abdomen end with pen after 120 hours, if its head can not be swung, can not creep forward and then be considered as death), and calculate the LC50 value (median lethal concentration, the protein concentration that 50% corn rootworm larva is killed in expression) of the corn rootworm larva of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 2 obtains accordingly.Each concentration arranges 4 replicate treatment groups, results averaged.
The result shows that the LC50 value of the corn rootworm larva of bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 that embodiment 2 obtains is 2.53mg/L.
Claims (10)
1.DNA molecule, for following arbitrary in a)-c):
A) dna molecular shown in the 109-1269 position Nucleotide of sequence 1 in the sequence table;
B) dna molecular shown in the sequence 1 in the sequence table;
C) and a) or b) dna molecular that limits has 98% identity at least, and protein DNA molecule shown in the 37-423 amino acids of encoding sequence 2 or sequence 2.
2. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain the described dna molecular of claim 1.
3. recombinant vectors according to claim 2, it is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
4. recombinant vectors according to claim 3 is characterized in that: starting the promotor that described dna molecular transcribes in the described recombinant expression vector is the T7 promotor;
Described recombinant expression vector is specially described dna molecular is inserted into the recombinant plasmid that the multiple clone site place of plasmid pET28a obtains.
5. transcribed the RNA molecule of gained by the described dna molecular of claim 1.
6. protein is the protein shown in the sequence in the sequence table 2.
7. the described dna molecular of claim 1, or arbitrary described recombinant vectors among the claim 2-4, expression cassette, transgenic cell line or reorganization bacterium, or the described RNA molecule of claim 5, or the described protein of claim 6 has application in the product of insecticidal activity in preparation.
8. the described dna molecular of claim 1 application in the bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 expression amount in improving intestinal bacteria; Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is protein shown in the sequence 2 in the sequence table.
9. the preparation method of a bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 comprises the steps:
1) arbitrary described recombinant vectors among the claim 2-4 is imported intestinal bacteria, obtain recombination bacillus coli;
2) cultivate described recombination bacillus coli, carry out abduction delivering, collect and the cracking thalline, obtain bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2;
Described bacillus thuringiensis crystal fusion rotein Bt Cry35Ba2 is protein shown in the sequence 2 in the sequence table.
10. method according to claim 9 is characterized in that step 2) in, described abduction delivering is: have to cultivation that to add IPTG in the nutrient solution of described recombination bacillus coli be 1mM to final concentration, 22 ℃ jolt and cultivated 6 hours.
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