CN103233022A - Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry9C - Google Patents
Preparation method of bacillus thuringiensis crystal fusion protein Bt Cry9C Download PDFInfo
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Abstract
The invention discloses a preparation method of a bacillus thuringiensis crystal fusion protein Bt Cry9C. The method relates to a codon-optimized bacillus thuringiensis crystal fusion protein Bt Cry9C 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-1548th 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 Cry9C (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 Cry9C gene (N-Cry9C 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 Cry9C.
Background technology
(Bacillus thuringiensis is a kind of gram-positive microorganism that is distributed widely in the soil Bt) to bacillus thuringiensis, 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, and existing nearly 180 different Bt insecticidal crystalline genes are cloned and checked order so far, and cry9C is exactly wherein a kind of.
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 Cry9C of codon optimization.
The gene of the bacillus thuringiensis crystal protein B t Cry9C that described codon is optimized, be under the prerequisite that does not change bacillus thuringiensis crystal protein B t Cry9C aminoacid sequence (the 1-480 amino acids of GenBank:AX100534.1), the codon of its wild-type encoding gene (the 1-1440 position of GenBank:AX100534.1) replaced with the gene of gained behind the codon of intestinal bacteria preference (high frequency use).Be N-Cry9C with the unnamed gene after optimizing, specifically can be following a)-c) in arbitrary dna molecular:
A) dna molecular shown in the 109-1548 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 37-516 amino acids of encoding sequence 2 or sequence 2.
Wherein, sequence 1 is made up of 1575 Nucleotide altogether, and wherein 109-1548 position Nucleotide replaces with the gene order of gained behind the intestinal bacteria preference codon for the codon with the 1-1440 position of GenBank:AX100534.1.Protein shown in the sequence 2, wherein the 37-516 amino acids of the 109-1548 position nucleotide coding sequence 2 of sequence 1 in the tabulation of sequence 1 code sequence.Sequence 2 is made up of 524 amino acid altogether.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain described dna molecular (N-Cry9C 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-Cry9C gene) is transcribed in the described recombinant expression vector is specially the T7 promotor.
Described recombinant expression vector is specially described dna molecular (N-Cry9C 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-9C) of described recombinant expression vector is shown in sequence in the sequence table 3.
Wherein, sequence 3 is made up of 6781 Nucleotide altogether, and 137-1711 position Nucleotide is the reverse complementary sequence of sequence 1.
The RNA molecule of being transcribed gained by described dna molecular (N-Cry9C 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 Cry9C activity, is specially following I) or II):
I) protein that the 37-516 amino acids of sequence 2 is formed in the sequence table;
II) 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 can be various pests such as lepidopteran, Diptera, Coleoptera or Hymenoptera; In one embodiment of the invention, described worm be specially paddy rice yellow rice borer (Scirpophaga incertulas (Walker)) (as first age the paddy rice yellow rice borer).Described product can be test kit.
The application in the bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 Bt Cry9C 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 Cry9C.
The preparation method of bacillus thuringiensis crystal fusion rotein Bt Cry9C provided by the present invention specifically comprises the steps:
1) the described recombinant vectors (as recombinant expression vector pET28a-9C) 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 Cry9C;
Described bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 its final concentration, 25 ℃ jolt and cultivated 9 hours.
Described cultivation has the OD600 value of nutrient solution of described recombination bacillus coli between 0.6-0.8.
The rotating speed that described concussion is cultivated is 120rpm, and rotation radius is 1.5cm.
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 9 hours the condition for 25 ℃, described bacillus thuringiensis crystal fusion rotein Bt Cry9C mainly is expressed in the inclusion body.
Using gene engineering method of the present invention is synthesized bacillus thuringiensis crystal fusion rotein Bt Cry9C gene (N-Cry9C gene) at home first.Experiment showed, that in intestinal bacteria the bacillus thuringiensis crystal fusion rotein Bt Cry9C gene (N-Cry9C 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 Cry9C.
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 M is that dna molecular amount standard 10Kbp is to 1000bp band (day 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-9C with EcoR I and Xho I.
Fig. 2 is bacillus thuringiensis crystal fusion rotein Bt Cry9C prokaryotic expression result (SDS-PAGE).Wherein, swimming lane M is that molecular weight of albumen standard 10KD is to 170KD band (English mill development in science and technology company limited, catalog number TF413-01 are opened up in Beijing).Swimming lane 1 changes the supernatant that the control group thalline of pET28a empty carrier makes over to for cracking; Swimming lane 2 and 3 changes the inclusion body that the experimental group thalline of recombinant expression vector pET28a-9C makes over to for cracking.Swimming lane 4 changes the supernatant that the experimental group thalline of recombinant expression vector pET28a-9C makes over to for cracking.The arrow indication is target protein.
Fig. 3 is bacillus thuringiensis crystal fusion rotein Bt Cry9C prokaryotic expression and purification result (SDS-PAGE).Wherein, swimming lane M is that molecular weight of albumen standard 10KD is to 170KD band (English mill development in science and technology company limited, catalog number TF413-01 are opened up in Beijing); Swimming lane 1 is bacillus thuringiensis crystal fusion rotein Bt Cry9C albumen behind the purifying.Swimming lane 2 changes the supernatant that the experimental group thalline of recombinant expression vector pET28a-9C makes over to for cracking.The bacillus thuringiensis crystal fusion rotein Bt Cry9C of swimming lane 3 for slightly carrying.Swimming lane 4 changes the supernatant that the control group thalline of pET28a empty carrier makes over to for cracking.The arrow indication is target protein.
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.
Paddy rice yellow rice borer (Scirpophaga incertulas (Walker)): first age, Plant Protection institute, Chinese Academy of Agricultral Sciences.Be documented in " Edwin P.Alcantara; Remedios M.Aguda; April Curtiss; et al.Bacillus thuringiensis δ-Endotoxin Binging to Brush Border Membrane Vesicles of Rice Stem Borers.Archiver of Insect Biochemistry and Physiology; 55:169-177 (2004). " 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 Cry9C gene is synthetic
At present, the wild-type bacillus thuringiensis crystal protein B t Cry9C gene in the bacillus thuringiensis (Bacillus thuringiensis) has following two kinds: the short sequence shown in the 194-1354 position Nucleotide of the one, GenBank:AY346129.1; Another is the long sequence shown in the 1-1878 position Nucleotide of GenBank:AX100534.1.Wherein, the 1-1161 position Nucleotide of GenBank:AX100534.1 is consistent with the 194-1354 position Nucleotide of GenBank:AY346129.1.Because the protein by above two kinds of genes encoding gained all has bacillus thuringiensis crystal protein B t Cry9C activity, the present invention selects the 1-1440 position Nucleotide of GenBank:AX100534.1 as carrying out codon optimized wild type gene sequence, be intended to take into account when selecting the protein-active position, the target protein size is controlled a scope that is easy to carry out prokaryotic expression.Specific as follows:
According to the wild-type bacillus thuringiensis crystal protein B t Cry9C gene order (the 1-1440 position Nucleotide of GenBank:AX100534.1) 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 Cry9C through design with verifying repeatedly, be about to wild-type bacillus thuringiensis crystal protein B t Cry9C gene order is transformed into intestinal bacteria preference (high frequency use) under the prerequisite that does not change aminoacid sequence (the 1-480 amino acids of GenBank:AX100534.1) codon optimized sequence, thereby improve the expression level of bacillus thuringiensis crystal protein B t Cry9C in the intestinal bacteria culture environment.
According to aforesaid method, the final optimization type bacillus thuringiensis crystal protein B t Cry9C gene that obtains by the design of intestinal bacteria preference, called after N-Cry9C gene, its gene order are the 109-1548 position of sequence 1 in the sequence table, are made up of 1440 Nucleotide altogether.The albumen of described N-Cry9C genes encoding is the albumen that the 37-516 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 Cry9C genes encoding shown in the 1-1440 position Nucleotide of GenBank:AX100534.1.
The prokaryotic expression of embodiment 2, bacillus thuringiensis crystal fusion rotein Bt Cry9C
One, the structure of protokaryon recombinant expression vector pET28a-9C
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 Cry9C gene (N-Cry9C gene) respectively after embodiment 1 gained optimization, obtains sequence " 103-1554 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 pGOV4-9C that in the pGOV4 cloning vector, inserts dna fragmentation shown in " 103-1554 of sequence 1 " through order-checking.
The recombinant plasmid pGOV4-9C 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 the double digestion restriction enzyme mapping as shown in Figure 1 with EcoR I and Xho I.To cut evaluation through enzyme and obtain the recombinant plasmid sample presentation order-checking that size is about 5341bp and two purpose bands of 1440bp.To show between the multiple clone site EcoR of pET28a carrier I and Xho I the recombinant plasmid called after pET28a-9C of the 109-1548 position nucleotide sequence of sequence 1 in the insertion sequence table through order-checking.The nucleotide sequence of recombinant expression vector pET28a-9C is shown in sequence in the sequence table 3, and wherein 137-1711 position Nucleotide is the reverse complementary sequence of sequence 1.In recombinant expression vector pET28a-9C, all be connected with the His label that the pET28a carrier carries at the upstream and downstream of described N-Cry9C gene (the 109-1548 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-9C, described N-Cry9C gene (the 109-1548 position of sequence 1) is present among the ORF shown in the whole sequence 1, and wherein the 1549-1575 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 Cry9C ").In recombinant expression vector pET28a-9C, the promotor that starts described N-Cry9C genetic transcription is the T7 promotor.
Two, prokaryotic expression, purifying and the evaluation of bacillus thuringiensis crystal fusion rotein Bt Cry9C
1, the prokaryotic expression of bacillus thuringiensis crystal fusion rotein Bt Cry9C
A) the recombinant expression vector pET28a-9C 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 ℃ shaking culture 2-3 hour 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 25 ℃ jolt and cultivated 9 hours, and its rotating speed is 120rpm, and the shaking table rotation radius is 1.5cm.
D) with 4 ℃ of step c) products therefroms, the centrifugal 20min of 10000g 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, 50-100rpm 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 ℃, 12000g are centrifugal, and supernatant is removed in the 20min hypsokinesis, institute adds the BugBuster Master Mix diluent with 10 times of deionized water dilutions of 6 times of BugBuster Master Mix volumes before adding in the precipitation, and 4 ℃, the centrifugal 15min hypsokinesis of 5000g remove supernatant behind the abundant vortex; In precipitation, add the BugBuster Master Mix diluent with 10 times of deionized water dilutions that adds 3 times of BugBuster Master Mix volumes before again, fully behind the vortex 4 ℃, 5000g centrifugal supernatant is removed in the 15min hypsokinesis, 4 ℃, the centrifugal 15min of 16000g after repeating 2 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, 4 ℃ of dissolvings are spent the night; 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 100V.Applied sample amount is 10 μ l.
The result as shown in Figure 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-9C that step 1 obtains over to has the purpose band to occur in the position that size is about 58KD, 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 9 hours the condition for 25 ℃, bacillus thuringiensis crystal fusion rotein Bt Cry9C mainly is expressed in the inclusion body.
2, the purifying of bacillus thuringiensis crystal fusion rotein Bt Cry9C
F in the above-mentioned steps 1) collected solubilization of inclusion bodies liquid is the bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 Bt Cry9C 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.The concrete operations of purifying are undertaken by the specification sheets that the BugBusterHis*Bind purification kit provides.
3, the evaluation of bacillus thuringiensis crystal fusion rotein Bt Cry9C
(1) sequence verification
Bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 Cry9C behind step 2 gained of the employing SDS-PAGE purifying is verified.Be contrast with the bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 100V.Applied sample amount is 6 μ l.
SDS-PAGE result as shown in Figure 3.As can be seen from the figure, and compare before the purifying, the albumen purpose band behind the purifying is single, has effectively removed foreign protein.
4, the output of bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 Cry9C behind the step 2 gained purifying is carried out concentration determination, and then obtain the ultimate production of bacillus thuringiensis crystal fusion rotein Bt Cry9C.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 Cry9C 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 Cry9C is 0.0268mg albumen/g 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 Cry9C that embodiment 3, embodiment 2 obtain
Get the bacillus thuringiensis crystal fusion rotein Bt Cry9C that purifying obtains among the embodiment 2, use the 0.1%(volume ratio) dilution of the Triton-100 aqueous solution, be made into 5 dilution diluents, simultaneously with the 0.1%(volume ratio) the Triton aqueous solution compares, the clean rice stem section that is slightly smaller than the culture dish bottom is immersed diluent dries after 10 seconds naturally, bottom putting into, the section of stem section is covered with the culture dish through water-soaked filter paper, every ware inserts 10 first paddy rice yellow rice borers in age (Scirpophaga incertulas (Walker)), cultivates under the room temperature condition.The death condition of statistics paddy rice yellow rice borer larva (is touched larva with pen after 72 hours, if its head can not be swung, can not creep forward and then be considered as death), and utilize the LC50 value (median lethal concentration, the protein concentration that 50% paddy rice yellow rice borer larva is killed in expression) of the paddy rice yellow rice borer of the bacillus thuringiensis crystal fusion rotein Bt Cry9C larva that POLO computed in software embodiment 2 obtains accordingly.Each concentration arranges 4 replicate treatment groups, results averaged.
The result shows that the LC50 value of the paddy rice yellow rice borer of the bacillus thuringiensis crystal fusion rotein Bt Cry9C larva that embodiment 2 obtains is 83.42mg/L.
Claims (10)
1.DNA molecule, for following arbitrary in a)-c):
A) dna molecular shown in the 109-1548 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 37-516 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 following I) or II):
I) protein that the 37-516 amino acids of sequence 2 is formed in the sequence table;
II) protein shown in the sequence 2 in the sequence table.
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 Cry9C expression amount in improving intestinal bacteria; Described bacillus thuringiensis crystal fusion rotein Bt Cry9C is protein shown in the sequence 2 in the sequence table.
9. the preparation method of a bacillus thuringiensis crystal fusion rotein Bt Cry9C 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 Cry9C;
Described bacillus thuringiensis crystal fusion rotein Bt Cry9C 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, 30 ℃ jolt and cultivated 9 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1375009A (en) * | 1999-09-17 | 2002-10-16 | 阿温提斯作物科学公司 | Insect-resistant rice plants |
| CN102140444A (en) * | 2010-01-28 | 2011-08-03 | 复旦大学 | Low temperature alkaline phosphatase and preparation method thereof |
| CN102586286A (en) * | 2012-03-07 | 2012-07-18 | 复旦大学 | Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a |
Non-Patent Citations (2)
| Title |
|---|
| 练云等: "人工改造的CRY1AC和CRY1IE基因在烟草中共表达对棉铃虫有更好的杀虫活性", 《科学通报》 * |
| 高洁荣等: "苏云金芽孢杆菌Cry2Aa蛋白体外表达及纯化", 《湖北农业科学》 * |
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