CN102094030B - Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof - Google Patents
Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof Download PDFInfo
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Abstract
The invention provides an insect-resistant gene Cry1Ab-Material. The synthesis of the gene is as follows: according to the amino acid sequence of the N-terminal of the original Bacillus thuringiensis (Bt) protein (Cry1Ab), the preferred codons of monocotyledon (corn) is used to perform human reformation and synthetize a new Cry1Ab DNA sequence; and the prokaryotic expression vector and plant expression vector are constructed, and transformation is performed in the host cells. Vitro tests prove that the transformed and synthetized Bt gene toxic protein has obvious insecticidal effect on Ostrinia nubilalis. The expression of the insect-resistant gene Cry1Ab-Ma in monocotyledon is stable and efficient, thus the gene can be used to produce insect-resistant transgenic plant.
Description
Technical field
The present invention relates to genetically engineered and biological control field, specifically, relate to anti insect gene Cry1Ab-Ma, its expression vector and application.
Background technology
Bt genes encoding insecticidal crystal protein is from bacillus thuringiensis (BacillusthuringHansis).It produces the desinsection parasporal crystal albumen of delta-endotoxin in the gemma forming process, these albumen have very high insecticidal activity.Its action principle is that this pest-resistant albumen can be hydrolyzed to littler active toxin fragment-core fragment (Hofte and Whiteley, 1989) by alkaline intestinal juice dissolving.The further hydrolysis of this active fragments ability protease inhibitor, the protein bound that is activated are on the brush shape vesicle on the insect gut, thereby causing to bore a hole influences osmotic equilibrium, cell expansion and dissolving, and it is also dead at last that target organisms stops to get food.Research shows that the intestinal epithelial cell of many target pests all has the binding site of Bt albumen high-affinity (Hofte and Whiteley, 1989).In decades in the past, confirmed the insecticidal crystal protein of their codings of tens of kinds of bacillus thuringiensis fungus strains and kind more than 130.
1986, first batch of transgenic plant (pest-resistant, antiweed) went through to get into field test.1987, people such as Belgian Vaeck obtained to change the pest-resistant tobacco of Bt insecticidal proteins first, but can only detect faint insect-resistance, its expressing protein almost detect less than, only account for 0.001% of soluble proteins.Report (Barton etc., 1987 that twice acquisition Bt transfer-gen plant arranged again the same year; Fischhoff etc., 1987).Mammals heavy chain of antibody in 1989 and light chain gene successful expression and correctly be assembled into the antibody of function in tobacco.To nineteen ninety, have 7 research groups acquisition Bt transgenic plant at least and be applied to field experiment (Estruch J etc., 1996).Adang etc. (1993) have transformed the Cry3A gene, and this gene efficiently expresses in yam.1993, people such as Michael transformed the Cry1Ab gene, obtained the Bt transfer-gen plant, and Pyrausta nubilalis (Hubern). is had good resistance.KozHal (1993) etc. has cultivated the insect-resistant transgenic corn, and transfer-gen plant can high level expression Cry1A (b) anti insect gene.1996, people such as Joachim W carried out brachymemma with Cry1Ab and modify, rice transformation, and obtaining changes the Bt paddy rice, and the target pest mortality ratio reaches 100%.1998, the codon that people such as Cheng have a preference for according to plant was transformed Cry1Ab and Cry1Ac gene and has been obtained transfer-gen plant with Agrobacterium-mediated Transformation method rice transformation, and R2 tries for worm, and 100% lethality rate was arranged in 5 days.The artificial reconstructed synthetic Cry1B gene of Bohotova etc. (2001) transforms tropical corn and obtains effectively control of maize snout moth's larva of transgenic corns.
China to the research of Bt insecticidal protein gene mainly is: 1992, people such as Guo's three heaps adopted the plant optimizing codon method GFMCry1A killing gene of synthetic total length 1824bp at first at home, have obtained Chinese first-generation unit price Insect Resistant Cotton.(1995) such as fourth groups of stars etc. (1993) and kingdom's English change the pB48.415 plasmid over to ovary injection and particle bombardment respectively and obtain transfer-gen plant in the maize calli, and insect-resistance is measured and shown that it has certain insect-resistance.1994, China Agricultural University at home and abroad changed anti insect gene Bt corn over to and obtains transfer-gen plant with the method for ovary injection first, was that the pest-resistant corn of representative has been showed the excellent development prospect with independent intellectual property right gene Bt.1998, the Chinese Academy of Agricultural Sciences was building up to the GFM killing gene on the pMG6 plasmid, imported to (Zhou Fengyong etc., 1998) in the corn, and the offspring detects and shows that the Bt gene arrives (Liu YJ etc., 2003) of future generation with mendelian inheritance pattern heredity.China has set up the corn gene technical system of comparative maturity aspect transgenic technology research.
Cry1Ab albumen is one type of parasporal crystal that lepidoptera pest is had toxic action that is produced by bacillus thuringiensis, and it has important application prospects in the biological control field.Bacillus thuringiensis delta-endotoxin genes Cry1Ab derives from mikrobe, but its transgenic plant exist problems such as expression amount is low, expression product is unstable, insect-resistance weak effect.Can improve its expression amount in transgenic plant through transforming base sequence raising GC content, reach desinsection purpose (Koziel, 1993).Through comparing the codon service condition of bacillus thuringiensis and corn; Can find that there is very big-difference in both on codon preference; Utilize the preference codon of plant; Again design and transform Cry1Ab and obtain novel pest-resistant gene C ry1Ab-Ma, to realize that changeing new gene crops has insect-resistance preferably.
Corn is an important crops; Be again important feed and industrial raw material; Current corn insect pest (is main with Pyrausta nubilalis (Hubern).) is serious, causes a large amount of underproduction of corn, therefore adopt an effective measure its harm of control to improve corn yield, increasing farmers' income has great importance.Because lack suitable pest-resistant cultivar, the main method that solves insect pest at present is in process of growth, to spray chemical insecticide; But chemical insecticide kills off the insect pests and natural enemy simultaneously, causes ecological imbalance and environmental pollution.Through transgenic technology, can anti insect gene be imported in the corn variety, and then improve the insect-resistance of transgenic corns, reduce the usage quantity of agricultural chemicals, save human and material resources and social resources.Therefore, use new anti insect gene, to improve the expression amount of insecticidal proteins and cultivate novel pest-resistant transgenic corns be one of effective way that addresses the above problem.
Summary of the invention
The purpose of this invention is to provide a kind of can be in plant stability and high efficiency the anti insect gene Cry1Ab-Ma and the expression vector thereof of expressing.
Another object of the present invention provides the application of anti insect gene Cry1Ab-Ma in improving the transgenic plant insect-resistance.
In order to realize the object of the invention; A kind of anti insect gene Cry1Ab-Ma of the present invention; It has the nucleotide sequence shown in the SEQ ID No.1, or this sequence is proteic by SEQ ID No.1 deutero-nucleotide sequence through the coding same function that replaces, lacks and/or add one or several Nucleotide and form.
The aminoacid sequence of above-mentioned anti insect gene Cry1Ab-Ma proteins encoded is shown in SEQ ID No.2.
The carrier that contains anti insect gene Cry1Ab-Ma, preferred two valency carriers.
The host cell that contains above-mentioned carrier, preferred EHA105.
The transformed plant cells that contains anti insect gene Cry1Ab-Ma.
The application of anti insect gene Cry1Ab-Ma in improving the transgenic plant insect-resistance.Preferably, said plant is farm crop, fruit tree or vegetables, like corn, paddy rice, yam, cotton etc.
The object of the invention can also adopt following technical measures further to realize:
Remove one section base sequence of original Bt gene C ry1Ab 3 ' end 1623bp, only stay one section base sequence of 5 ' end 1845bp of excalation; Amino acid keeping sequence C ry1Ab N end protein is formed under the overall constant situation, carries out base substitution with the codon of plant-preference, tentatively obtains the dna sequence dna of a transformation; Get rid of exist in the dna sequence dna cause the unsettled AT of being rich in sequence of plant transcription and restriction enzyme site commonly used (SacI), the method through permutation cipher corrects elimination then; And add terminator codon TAG at 3 ' end; Confirm the Bt gene that also chemosynthesis is transformed, its nucleotide sequence is shown in SEQ ID No.1; Add restriction enzyme enzyme recognition site sequence at the sequence two ends and be building up on the corresponding expression vector according to clone's needs.
Gene C ry1Ab-Ma of the present invention is operably connected with prokaryotic expression carrier, and Preliminary detection synthetic Bt of the present invention gene expression product is to the toxicity of Pyrausta nubilalis (Hubern). fast.Gene C ry1Ab-Ma of the present invention is operably connected with plant expression vector, and expression vector is imported in the corresponding Agrobacterium, and then carry out genetic transformation, cultivate the insect-resistant transgenic corn through agrobacterium-mediated transformation.Also can carry out genetic transformation, make it possess anti-insect activity other farm crop or fruit tree etc.
Those skilled in the art can also produce Bt albumen through large scale fermentation with gene transformation bacterium of the present invention or fungi, and it is prepared into sterilant, are used for the control of crop pests.
The present invention has advantage and beneficial effect at least:
The artificial synthetic anti insect gene of the present invention Cry1Ab-Ma sequence is compared with original Cry1Ab sequence, has strengthened its expression in plant greatly.Use plant-preference property codon; The inverted repeats that is rich in AT sequence and existence and indefinite eukaryotic DNA intron sequences in the original DNA sequence have been reduced; Improved Cry1Ab-Ma gene G+C content is 63.04%, with the homology of original DNA sequence be 66%.Anti insect gene Cry1Ab-Ma of the present invention can be in vegetable cell the expression of efficient stable.
Behind anti insect gene Cry1Ab-Ma importing corn, can obtain the Cry1Ab-Ma transformant of genetic stability.In addition, farm crop such as this gene also can converting cotton, paddy rice, vegetables make it possess corresponding anti-insect activity, thereby reduce the usage quantity of agricultural chemicals, to reduce environmental pollution, have important economic value and wide application prospect.
Description of drawings
Figure 1A～Fig. 1 D is Cry1Ab gene of the present invention and synthetic Cry1Ab-Ma gene coded sequence analysis.
Fig. 2 is CPB (pCAMBIA1300-35S-MCS-Bar) plasmid map.
The commentaries on classics Cry1Ab gene corn of Fig. 3 for adopting agrobacterium-mediated transformation to obtain; Wherein, the screening of callus (left side), regeneration (in) and transfer-gen plant be transplanted to land for growing field crops (right side).
Fig. 4 is T
0For the PCR detected result of goal gene Cry1Ab-Ma in the transformant, wherein, M is M:DL2000plus; The positive plasmid contrast of CK1; CK2 is transgenic negative control not; Blank is that distilled water contrasts; 1-8 is that Ab-Ma1 is to Ab-Ma8.
Fig. 5 is T
0For the PCR detected result of transformant selectable marker gene Bar, wherein, M is M:DL2000plus; The positive plasmid contrast of CK1; CK2 is transgenic negative control not; Blank is that distilled water contrasts; 1-8 is that Ab-Ma1 is to Ab-Ma8.
Fig. 6 is T
0For the immunology detection of transformant target protein Cry1Ab-Ma, wherein CK1 is transgenic seedling negative control not; 1-8 is that Ab-Ma1 is to Ab-Ma8.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
TP in following examples if no special instructions, is ordinary method.Used test materials and reagent in following examples, if no special instructions, all available from routine biochemistry reagent company.
Synthesizing of embodiment 1Cry1Ab-Ma gene
According to the parent nucleotide sequence of Bt gene C ry1Ab, remove one section base sequence of 3 ' end 1623bp, only stay one section base sequence of 5 ' end 1845bp of excalation; Amino acid keeping sequence C ry1Ab N end protein is formed under the overall constant situation, carries out base substitution with the codon of plant-preference, tentatively obtains the dna sequence dna of a transformation; Get rid of exist in the dna sequence dna cause the unsettled AT of being rich in sequence of plant transcription (like ATTTA, AATGAA etc.) and restriction enzyme site commonly used (SacI), the method through permutation cipher corrects elimination then; And add terminator codon TAG at 3 ' end; Confirm the Bt gene that also chemosynthesis is transformed, its nucleotide sequence is shown in SEQ ID No.1; Add restriction enzyme enzyme recognition site sequence at the sequence two ends and be building up on the corresponding expression vector according to clone's needs.
Original Bt gene C ry1Ab and synthetic Cry1Ab-Ma gene coded sequence are compared (Fig. 1), and the homology of two sequences is 66%.The statistics of based composition is: the G+C% of original gene is 37.34%, and the G+C% of new synthetic gene is 63.04%.The compare of analysis of amino acid sequence coded shows, both amino acid sequence coded (its aminoacid sequence is shown in SEQ ID No.2) unanimity.
The synthetic work of new Bt gene C ry1Ab-Ma is accomplished by Sangon Biotech (Shanghai) Co., Ltd..
The expression of embodiment 2Cry1Ab-Ma gene in prokaryotic system and the toxicity detection of product
For the Cry1Ab-Ma gene vivoexpression that detects transformation reaches the toxicity situation to Pyrausta nubilalis (Hubern)., we have made up the Bt prokaryotic expression carrier.According to clone Bt gene needs, at 5 ' end interpolation NdeI restriction endonuclease recognition site sequence C ATATG of primer sequence, 3 ' end adds HindIII restriction endonuclease recognition site sequence A AGCTT.The design primer sequence (upstream primer F1:5 " CATATGGACAACAACCCGAACATC-3 '; Downstream primer R1:5 '-AAGCTTCTAGTACTCCGCCTCG-3 ').
With the pUCAb-Ma plasmid is template, is primer with F1 and R1, and amplification Cry1Ab-Ma gene carries out enzyme with restriction enzyme NdeI and HindIII and cuts, and gel reclaims test kit and reclaims purifying Cry1Ab-Ma gene fragment.Cut pET28b+ with restriction enzyme NdeI and HindIII enzyme, gel reclaims test kit and reclaims purifying 5.3kb fragment.Two fragments are carried out ligation, make up gained prokaryotic expression plasmid called after pETAb-Ma.Carry out enzyme with different restriction enzymes and cut evaluation, show that vector construction is correct.Transform BL21 (DE3) competent cell with pETAb-Ma.
After IPTG induces, extracting Bt albumen with the e. coli bl21 bacterium liquid that contains pETAb-Ma, is contrast with clear water with containing empty carrier pET28b+ e. coli bl21 bacterium liquid, carries out the worm examination with Pyrausta nubilalis (Hubern)..Concrete steps are following:
1. inoculate e. coli bl21 list bacterium colony in 5ml LB (containing kantlex) liquid nutrient medium, 37 ℃ of incubated overnight.
2. bacterium liquid is inoculated in 500ml LB (the containing kantlex) liquid nutrient medium, is cultured to OD ≈ 0.5.
3. adding IPTG continues to shake to final concentration 0.5mM and induces 4 hours.
4. the centrifugal 10min of 4000rpm collects thalline.
5. add 36ml lysis buffer (2mM Tris-HCl; 0.2mM CaCl
2PH=8.0), the thalline that suspends again adds N,O-Diacetylmuramidase to final concentration 1mg/ml, places 30min on ice.
6. ultrasonic disruption thalline (broken parameter: work 10s, 5s at interval, 40 times, ice bath is broken), the centrifugal 10min of 4000rpm collects supernatant.
7. the supernatant of collecting is joined in the feed of raising Pyrausta nubilalis (Hubern)..To contain the negative contrast of empty carrier pET28b e. coli bl21 bacterium liquid.
8. in each test tube, put into a feed, and incubate the Pyrausta nubilalis (Hubern). of not taking food at the beginning of inserting 10, respectively connect 10 test tubes.Put into temperature 26～28 degree; Cultivate in the environment of relative humidity about 70%; Carrying out toxicity after 8 days identifies: the result shows; The Bt albumen of the Cry1Ab-Ma genes encoding of secreting, expressing has insecticidal effect preferably, and the mortality ratio of Pyrausta nubilalis (Hubern). reaches 90.67%, and the growth of Pyrausta nubilalis (Hubern). is had obvious suppression effect (table 1).
The toxicity detected result of table 1Cry1Ab-Ma gene prokaryotic product
The expression of embodiment 3Cry1Ab-Ma gene in transgenic plant and the toxicity detection of expression product
With XbaI and SacI double digestion T vector plasmid pUCCry1Ab-Ma, reclaim purification kit with gel and reclaim the Cry1Ab-Ma fragment.Use XbaI and SacI double digestion plant expression vector CPB simultaneously, reclaim purification kit with gel and reclaim the CPB fragment after enzyme is cut.Two fragments are carried out ligation, make up to obtain eukaryon expression plasmid pCAMBIA1300-Cry1Ab-Ma-Bar (promotor is CaMV35S, and marker gene is the anti-herbicide gene Bar of streptomyces hygroscopicus).Carry out enzyme and cut evaluation, show that vector construction is correct.
Wherein, CPB (pCAMBIA1300-35S-MCS-Bar; Fig. 2) plasmid is the plasmid that on pCAMBIA1300 plasmid basis, makes up, and contains goal gene and inserts box (35S-polyclone restriction enzyme site-Tnos) and the anti-herbicide gene Bar from streptomyces hygroscopicus.This gene order is positioned between cauliflower mosaic virus 35S promoter (pCaMV) and the 3 ' afterbody (TCaMV).
Plasmid pCAMBIA1300 is available from Australian CAMBIA (Center for theApplication of Molecular Biology to International Agriculture), with reference to network address http://www.cambia.org/daisy/cambia/585.html.This carrier can be used for the genetic transformation of corn acceptor material.
Adopt agrobacterium mediation converted method (bacterial classification EHA105) that insertion sequence is imported the callus of acceptor plant, through weedicide bialaphos screening back acquisition transfer-gen plant (Fig. 3).When the transformed plant of transplant survival grows the 7-8 leaf, get blade and extract DNA, adopt round pcr to detect foreign gene.Fig. 4 has shown T
0For the PCR detected result of transformant goal gene Cry1Ab-Ma, wherein M:M:DL2000plus; CK1: positive plasmid contrast; CK2: transgenic negative control not; Blank: the distilled water contrast; 1-8:Ab-Ma1 is to Ab-Ma8.
Primer sequence:
Cry1Ab-Ma：5′-AACAACCCGAACATCAACGAG-3′
Cry1Ab-Ma：5′-CGCGTCCGTGTAGATCGTGA-3′
Purpose clip size: 921bp, 58 ℃ of annealing temperatures, 35 circulations.
Fig. 5 has shown T
0For the PCR detected result of transformant selectable marker gene Bar, wherein M:M:DL2000plus; CK1: positive plasmid contrast; CK2: transgenic negative control not; Blank: the distilled water contrast; 1-8:Ab-Ma1 is to Ab-Ma8.
Primer sequence:
F-Bar：5′-ATGAGCCCAGAACGACGCC-3′
R-Bar：5′-TCAAATCTCGGTGACGGGC-3′
Purpose clip size: 552bp, 60 ℃ of annealing temperatures, 35 circulations.
The detection of target protein Cry1Ab-Ma (Bt-Cry1Ab/1Ac immunology detection):
1, gets about 1cm
2The fresh young leaflet tablet in the left and right sides is placed in the Eppendorf pipe of 1.5ml.The pipe that will be placed with leaf then is inserted in the ice chest, to keep freshness.
2, get liquid nitrogen,, material is pulverized, in pipe, add 500 μ L-1ml SEB4 sample extraction damping fluids rapidly with drill bit with the material quick-frozen.
3, take out detector bar (true Nice, Beijing Bioisystech Co., Ltd; Article No.: STX06200/0050), certification mark is carried out on hand-held detector bar top.Do not remove protective membrane.Keep detector bar vertical, the end of mark is inserted in centrifuge tube or the bag for extracting.Insertion portion does not surpass 0.5cm.In testing process, remain the insertion state.
4, nature controlling line occurs in 3-5 minute, maximum response time is 30 minutes, and this moment, detector bar can take out.Nature controlling line is to be used for guaranteeing accuracy of experimental results.If nature controlling line does not occur, it is invalid to detect.Because sample is mobile different, the time that produces signal is also different.If sample is a male, detection line will occur.If sample is negative, detection line will not occur.If think the prolonged preservation detected result, can cut sample pad, paper towel blots, and this will stop remaining liquid to disturb the result.The depth of detection line has been reacted proteic content to be detected (Fig. 6).
Fig. 6 is T
0For the immunology detection of transformant target protein Cry1Ab-Ma, wherein CK is transgenic seedling negative control not; 1-8 is that Ab-Ma1 is to Ab-Ma8.Last band is a nature controlling line, and following band is that detection line is indicated albumen to be detected.This result shows that the target protein that anti insect gene Cry1Ab-Ma expresses efficiently expresses in transgenic corns.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (5)
1. anti insect gene Cry1Ab-Ma, its nucleotide sequence is shown in SEQ ID No.1.
2. the carrier that contains the said gene of claim 1.
3. carrier as claimed in claim 2, it is two valency carriers.
4. the host cell that contains the said carrier of claim 3, it is EHA105.
5. the application of the described gene of claim 1 in improving the transgenic plant insect-resistance,
Wherein, said plant is a corn, and said insect-resistance is the insect-resistance to Pyrausta nubilalis (Hubern)..
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| CN102533793B (en) * | 2011-12-23 | 2014-09-17 | 北京大北农科技集团股份有限公司 | Insecticide gene and application thereof |
| CN102584961B (en) * | 2012-02-28 | 2013-07-31 | 中国农业科学院作物科学研究所 | Anti-insect protein Cry1A.401 and expression vector and application thereof |
| CN104311648B (en) * | 2014-10-29 | 2017-02-15 | 中国农业科学院作物科学研究所 | Insecticidal protein as well as coded gene and application of insecticidal protein |
| CN109536490B (en) * | 2018-11-09 | 2020-11-17 | 中国农业科学院作物科学研究所 | Transgenic insect-resistant herbicide-resistant corn CM8101 exogenous insert flanking sequence and application thereof |
| CN110055263B (en) * | 2019-03-13 | 2023-07-18 | 河南省农业科学院 | Gene Cry1Ab-MR encoding Bt insecticidal protein, expression vector and application thereof |
| CN110273021A (en) * | 2019-07-09 | 2019-09-24 | 重庆市农业科学院 | The specificity of transformant detection primer of transgenic pest-resistant herbicide-resistant corn C M8101 and its application in backcross transformation |
| CN110981948A (en) * | 2019-12-23 | 2020-04-10 | 隆平生物技术(海南)有限公司 | Plant insect-resistant gene and vector and application thereof |
| CN110951727A (en) * | 2020-02-24 | 2020-04-03 | 中国农业科学院生物技术研究所 | Flanking sequence of exogenous insert of transgenic corn BFL4-2 and application thereof |
| CN111440814A (en) * | 2020-02-26 | 2020-07-24 | 中国农业科学院作物科学研究所 | Insect-resistant fusion gene mCry1AbVip3A, expression vector and application thereof |
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| CN1417330A (en) * | 2001-11-09 | 2003-05-14 | 复旦大学 | Meltiple antigenic compound Bt gene and its application in plant |
| CN1480533A (en) * | 2002-09-04 | 2004-03-10 | 华中农业大学 | Reforming composite insecticidal crystalline gene Cry2A of bacillus thuringiensis |
| CN101580843A (en) * | 2009-04-23 | 2009-11-18 | 中国农业大学 | Artificial synthesized Bt insecticidal gene for transgenic anti-insect plants |
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| CN1417330A (en) * | 2001-11-09 | 2003-05-14 | 复旦大学 | Meltiple antigenic compound Bt gene and its application in plant |
| CN1480533A (en) * | 2002-09-04 | 2004-03-10 | 华中农业大学 | Reforming composite insecticidal crystalline gene Cry2A of bacillus thuringiensis |
| CN101580843A (en) * | 2009-04-23 | 2009-11-18 | 中国农业大学 | Artificial synthesized Bt insecticidal gene for transgenic anti-insect plants |
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