CN105753951A - Bt insect-resistant gene, protein coded by Bt insect-resistant gene and application of Bt insect-resistant gene - Google Patents
Bt insect-resistant gene, protein coded by Bt insect-resistant gene and application of Bt insect-resistant gene Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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Abstract
The invention discloses a Bt insect-resistant protein belonging to the field of genetic engineering. The insect-resistant protein consists of an amino acid sequence as shown in SEQ ID No: 1 and derived amino acid sequences with insect-resistant specific proteins. The invention also discloses a gene for coding the insect-resistance protein, and the gene consists of a nucleotide sequence as shown in SEQ ID No: 2 and derived nucleotide sequences for coding the insect-resistant specific proteins. The invention further discloses an expression vector containing the gene. The insect-resistant gene is good in cotton bollworm killing effect and the killing efficiency can be up to 98%; secondly, the Bt insect-resistant gene provides a new way for the insect control; moreover, the gene can be efficiently expressed in plants and the original genetic sequence before the transformation cannot be expressed in the plants. The invention further provides an effective method for solving the insect resistance problem.
Description
Technical field
The invention belongs to genetic engineering field, be specifically related to a kind of Bt anti insect gene and the protein of this gene code, further relate to the purposes of this gene.
Background technology
Bollworm (HelicoverpaarmigeraHubner) belongs to Lepidoptera (Lepidoptera) Noctuidae (Noctuidae) Helicoverpa bollworm kind, and Cotton Production harm is very big.Host plant has section more than 20 more than 200 to plant, and except Cotton Gossypii, also includes the important grain such as Semen Maydis, Fructus Lycopersici esculenti and vegetable crop.Past to the preventing and treating of bollworm mainly by chemical pesticide, but chemical pesticide not only endangers human health and environment, but also there is the problem that insect develops immunity to drugs.Utilize anti insect gene carry out Biological control because of pointed strong, be not likely to produce Drug resistance, lasting medicine and advantages of environment protection and obtain favor gradually.
Bacillus thuringiensis (Bacillusthuringiensis is called for short Bt) is a kind of gram positive bacteria being distributed widely in soil.Insecticidal crystal protein (InsecticitalCrystalProteins, it is called for short ICPs) it is produce at spore growth period, ICPs is also referred to as delta-endotoxin (delta-endotoxin) (Bravo.A etc., ComprehensiveMolecularInsectScienceElsevier, 2005,175-206.), by two kinds of gene codes of Cry and Cyt.This insecticidal crystal proteins the various agricultural insects such as Lepidoptera, Diptera, coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga are had special insecticidal effect (explain sub-cattle. Su Yun gold stalk bacterium .1990；Cui Hongzhi etc. Scientia Agricultura Sinica, 1996,29 (1): 93).ICPs, after insect's food-taking, is dissolved release activity toxalbumin molecule (BravoA etc., Toxicon, 2007,49 (4): 423-435) in insect midgut；Then be combined with insect midgut epithelial stricture of vagina velum (BBMV) specific receptors and then insert and be aggregated in cell membrane formation hole or ion selector channel, the permeability destroying cell makes cellular metabolism unbalance, ultimately result in insect death (KnowlesBH etc., AdvancesinInsectPhysiology, 1994,24:275-308；SchwartzJL etc., FEBSLetters, 1997,4l0 (2): 397-402；GazitE etc., ProceedingsoftheNationalAcademyofSciences, 1998,95 (211): 12289-12294), but for harmless (DeMaagdR.A etc., TrendsGenet such as non-target pests, birds and people and animals, 2001,17:193-199；KimEH etc., Planta, 2009,230 (2): 397-405), (SchwemberAR etc., CienciaeInvestigaci ó nAgraria, 2008,35 (3): 231-250) free from environmental pollution.Thus Bt become in the Biological control of insect most widely used microbial insecticide (Wang Jilei etc., biology magazine, 2010,27 (4): 75-78；Chen Jiejun etc., Journal of Agricultural Biotechnology, 201l, 19 (2): 369-374).
Cotton Gossypii can produce the Target crops of effective insect resistance capacity after being introduced into Bt insecticidal protein gene.First business-like pest-resistant crops are Insect Resistant Cotton kind Bollgard and the Ingard of U.S. Meng Shan all artificial reconstructed Cry1Ac genes of (Monsanto) company；NorthrupKing company and Monsanto Company turn pest-resistant corn variety Agrisure and the YieldGard of Cry1Ab.At home, the CrylAc insecticidal proteins of synthetic is imported in Cotton Gossypii by Guo three heaps in 1992 etc., it is thus achieved that China's first generation unit price Insect Resistant Cotton.Along with the commercialization of first generation Cry1A series anti insect gene, create the resistant insect (Tabashnik etc., 2008) of genetically modified crops.As Bt is produced resistance (BETabashnik etc. by field, Hawaii diamondback moth, JournalofEconomicEntomology, 1990,83 (5): 1671-1676), bollworm produces resistance (Tabashnik, B.E etc., NatureBiotechnol.2008 to turning Cry1Ac Insect Resistant Cotton, 26,199-202；Bagla, P.Science, 2010,327,1439) etc..Cry2A albuminoid has different binding site (LiangY etc. from Cry1A albumen, Mol.Microbiol, 1994, 13 (4): 569-575), different Cry2Aa and Cry2Ab receptoroid albumen is not had resistance by the resistant insects of Cry1Ac, both albumen is not easy to produce cross resistance by insect, can be widely applied to the improvement (Liao of pest resistance, C etc., Inwertebr.Pathol.2002, 80, 55-63), therefore, Cry2A class insecticidal proteins starts to be paid much attention to, Meng Shan in 2002 are proposed the Mature Cotton New Variety BollgardII simultaneously expressing two anti insect genes of Cry1Ac and Cry2Ab.Therefore separation screening new strains, clone new Cry2A class killing gene be solve pest resistance problem effective ways.(application number is: 2013101509654) disclose Cry2Ah-like in patent application " to lepidoptera pest high virulence killing gene cry2Ah-like and application ", Cry2Ah-likesp, Cry2Ah-likevp gene pairs bollworm, the Lepidopteras such as Pyrausta nubilalis (Hubern). have a significantly high poisoning ability, but all unconverted do further checking in plant.
By Bt gene transformation to Nicotiana tabacum L. (Vaeck etc., Nature, 1987.328:33-37) and tomato plant (Fischhoff etc., BioTechnology.1987.5:807-813) find that these transfer-gen plants are not all expressed afterwards, illustrate that the expression in plant of the natural Bt gene is too low, be difficult to obtain the plant of insect resistace.Reason is in that lower organisms antibacterial and higher organisms plant exist very big-difference (Willbur etc. in the use of codon, PlantPhysiol.1990.92:1-11), such as codon last wobble bases G/C utilization rate 45%-75% in plant, and only have 24% in Cry2Ah gene；Secondly, the ATTTA sequence and AATAA, AATAAT sequence that exist in natural B t gene can cause that the mRNA transcribed out is imperfect, affect the translation of insecticidal proteins；Additionally, the tRNA in plant is not provided that the translation in plant cell of the lower organisms bacterial gene.According to dicotyledon optimizing codon, obtaining the synthetic gene of Cry1Ac, proceed to and obtain pest-resistant cotton plants in cotton gene group, its expressing quantity is about 50-100 times of (Perlak etc. of natural B t protein gene expression amount, Bio/Technology.8:939-942,1990).Therefore, natural B t gene being transformed so that it is high efficient expression in plant, the insect resistant effect to acquisition transgenic plant is very necessary.
Summary of the invention
The present invention be find in new bacillus thuringiensis lepidoptera pest is had on the insecticidal crystal protein basis of special insecticidal activity, according to Cotton Gossypii codon preference, the original gene sequence of insecticidal crystal protein is carried out gene optimization transformation, can express in Plant Genome, thus completing the present invention.The albumen of improved gene order coding is constant, and compared with original gene sequence (as shown in SEQIDNo:3), after optimization, the G/C content of gene order (as shown in SEQIDNo:2) has brought up to 43.46% from 34.22%.Unnamed gene after optimization is cry2AhM by the present invention.
Present invention aim at providing a kind of Bt insect resistance protein.
Another object of the present invention is in that to provide the gene encoding above-mentioned Bt insect resistance protein.
The present invention the 3rd purpose is in that to provide the expression vector containing said gene.
The present invention the 4th purpose is in that to provide the purposes of said gene.
The present invention the 5th purpose is in that to provide a kind of insecticide containing above-mentioned Bt insect resistance protein.
The present invention the 6th purpose is in that to provide the method utilizing said gene to cultivate transgenic anti-insect plants.
The present invention the 7th purpose is in that to provide for identifying the primer pair containing said gene plant.
The present invention the 8th purpose is in that to provide identifies the method containing said gene plant.
Realize technical scheme as follows:
The present invention provides a kind of Bt insect resistance protein, called after Cry2AhM, and described protein is following (a) or (b):
A () aminoacid sequence shown in SEQIDNo:1 forms；
B aminoacid sequence shown in (a) is passed through replacement and/or the disappearance of one or several amino acid residue and/or adds formation by (), and have the protein of pest-resistant characteristic.
Encoding the gene of above-mentioned Bt insect resistance protein, called after cry2AhM, described gene is following (c), (d) or (e):
C () nucleotide sequence shown in SEQIDNo:2 forms；
D () and the nucleotide sequence shown in SEQIDNo:2 have the homology of more than 90%, and coding has the nucleotide sequence of pest-resistant characteristic protein；
(e) under strict conditions, the nucleotide sequence hybridization limited with (c) or (d), and coding has the nucleotide sequence of pest-resistant characteristic protein.
Expression vector containing said gene.
Described carrier refers to plasmid, cell or plant part etc..
Described plant part includes seed, flower pesticide, pollen, ovule, leaf, plumule, root, the tip of a root, flower, fruit, protoplast or callus etc..But the invention is not restricted to this.
The application on preventing and treating Lepidoptera, coleoptera or Diptera pest of the above-mentioned Bt insect resistance protein.
Described lepidoptera pest refers to bollworm etc..
A kind of insecticide, containing above-mentioned Bt insect resistance protein.
Said gene application on preventing and treating Lepidoptera, coleoptera or Diptera pest
Described lepidoptera pest refers to bollworm etc..
Described application refers to expresses the Bt insect resistance protein obtained as insecticide using said gene.
Described application refers to and is transformed in plant by said gene, it is thus achieved that pest-resistant transgenic plant.Wherein said plant refers to dicotyledon or monocotyledon.
Described plant includes Cotton Gossypii, Semen Maydis, Oryza sativa L., Nicotiana tabacum L., Semen Tritici aestivi or Brassica campestris L etc., but the invention is not restricted to this.
Utilize the method that said gene cultivates transgenic anti-insect plants, import plant including by said gene or expression vector, it is thus achieved that pest-resistant transgenic plant.
Plant described in said method refers to dicotyledon or monocotyledon.
Plant described in said method includes Cotton Gossypii, Semen Maydis, Oryza sativa L., Nicotiana tabacum L., Semen Tritici aestivi or Brassica campestris L etc., but the invention is not restricted to this.
For identifying the PCR primer containing said gene plant, the nucleotide sequence shown in SEQIDNo:6 and SEQIDNo:7 forms.
For detecting the test kit of above-mentioned anti insect gene, containing above-mentioned primer；Described primer nucleotide sequence shown in SEQIDNo:6 and SEQIDNo:7 forms.
For the method identifying the plant containing said gene, including extracting testing sample genomic DNA, then with testing sample genomic DNA for template, arrange with the nucleotides sequence shown in SEQIDNo:6 and SEQIDNo:7 and carry out pcr amplification for primer, acquisition size is about the PCR primer of 1kb, is transgenic plant.
Compared with prior art, the present invention has the advantage that and beneficial effect: (1) the anti insect gene of the present invention good disinsection effect to bollworm, insecticide efficiency is up to 98%.(2) Bt anti insect gene of the present invention is novel gene, provides a kind of new approach for Pest control.(3) anti insect gene of the present invention in plant can high efficient expression, and its transformation before original gene sequence can not express in plant.(4) the present invention solves that pest resistance problem provides a kind of effective ways.
Accompanying drawing explanation
Fig. 1 is expression vector pGBI121-cry2AhM schematic diagram.
Fig. 2 identifies electrophoresis pattern for turning cry2AhM genetic tobacco PCR；Wherein M is Maker；1-12 is transfer-gen plant；13 is blank negative control；14 is pGBI121-cry2AhM plasmid positive control.
Fig. 3 is for turning cry2AhM genetic tobacco RT-PCR electrophoresis pattern；Wherein WT is nontransgenic plants；GO-1, GO-2, GO-3, GO-6, GO-15 are transfer-gen plant.
Fig. 4 is cry2AhM gene relative expression quantity bar diagram in fluorescence quantitative PCR detection transgene tobacco；Wherein WT is nontransgenic plants；GO-1, GO-2, GO-3, GO-6, GO-15 are transfer-gen plant.
Fig. 5 detects electrophoresis pattern for turning cry2AhM genetic tobacco Westernblot；Wherein WT is nontransgenic plants；GO-1, GO-2, GO-3, GO-6, GO-15 are transfer-gen plant.
Fig. 6 is the insect resistace bioassay blade photo turning cry2AhM genetic tobacco；Wherein WT is nontransgenic plants；H-6 is transfer-gen plant GO-6.
Fig. 7 turns the impact contrast photo that larval growth is grown by cry2AhM genetic tobacco, and wherein A is for taking food nontransgenic plants larva；B is for taking food transfer-gen plant larva.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but is not limited to protection scope of the present invention.The experimental technique of unreceipted actual conditions in following example, generally conventionally experiment condition, such as Sambrook etc. " Molecular Cloning: A Laboratory the guide " (third edition, Science Press) described in condition, or the experimental technique provided according to instrument and equipment and reagent manufacturer it is proposed that condition.Reagent involved in experiment and consumptive material are commercially available customary commercial.
The acquisition of embodiment 1cry2AhM gene and the structure of expression vector
(1), the acquisition of cry2AhM gene of the present invention
The new cry2Ah genoid basis that the present invention finds in a new bacillus thuringiensis is realized by the transformation to gene order, a kind of novel B. thuringiensis insecticidal crystal protein of this cry2Ah genoid coding, this albumen is to lepidoptera pest has special insecticidal activity, but its original gene sequence (as shown in SEQIDNo:3) can not be expressed in plant.Its natural original series, according to Cotton Gossypii codon preference, is carried out gene optimization transformation by the present inventor, when the aminoacid sequence ensureing encoding proteins is constant, part antibacterial preferred codons replaces to Cotton Gossypii preference type codon.Compared with original series (as shown in SEQIDNo:3), after optimization, the G/C content of gene order (as shown in SEQIDNo:2) has brought up to 43.46% from 34.22%.Unnamed gene after optimization is cry2AhM by the present inventor.
(2), the structure of plant expression vector
(1). in order to enable cry2AhM gene reading frame to be correctly connected in expression cassette, it is necessary to add Pst I and Xho I restriction enzyme site respectively at gene 5' and 3'.With the cry2AhM gene order (see SEQIDNo:2) of synthetic for template, P1, P2 primer is utilized to carry out pcr amplification, it is thus achieved that pcr amplification product；Wherein said primer is as follows:
P1:5'CCCctgcagGATGAACAAAAACAACACAAAACTCAG3'(SEQIDNo:4) (Italic capitals letter is protection base, and lower case is PstI restriction enzyme site, and capitalization is identical with the 1-27 position of SEQIDNo:2).
P2:5'CCCctcgagTCACTTAATGGACACATCGTAAAAGTGAAC3'(SEQIDNo:5) (Italic capitals letter is protection base; lower case is XhoI restriction enzyme site, and capitalization is identical with the 1866-1896 position of SEQIDNo:2).
Wherein PCR reaction condition: 94 DEG C of denaturation 5min；94 DEG C of degeneration 30s, 59 DEG C of annealing 30s, 72 DEG C extend 30s, 40 circulations；72 DEG C extend 10min.PCR reaction system: template (cry2AhM gene plasmid) 1 μ L, primer P11 μ L, primer P21 μ L, Tag enzyme 1 μ L, dNTP (2.5mmol) 3 μ L, Buffer5 μ L, ddH2O28μL。
(2) the pcr amplification product Pst I step (1) obtained and Xho I double digestion, reclaim the genes of interest fragment of 1896bp after agarose gel electrophoresis.It is connected into the pGUC19 intermediate carrier containing Enhanced expressing sequences such as Cozk, Ω and ployA with PstI and XhoI enzyme action, is built into pGUC19-cry2AhM (35S::Atcry2AhM::Nos) intermediate carrier.
(3) the pGUC19-cry2AhM intermediate carrier obtained with EcoR I and Hind III enzyme action step (2), reclaim about 3.5kb destination gene expression box, it is connected to EcoR I and Hind III double digestion pGBI121 plasmid DNA, it is thus achieved that pGBI121-cry2AhM plant expression vector (see Fig. 1).
The acquisition of 2 turns of cry2AhM genetic tobaccos of embodiment and qualification
(1) plant expression vector converts Agrobacterium
Carry out in accordance with the following steps:
(1) take the Agrobacterium GV3101 competent cell 100 μ L prepared, add the pGBI121-cry2AhM plant expression vector 2-5 μ g of embodiment 1 preparation, mixing, be placed in 5min on ice.
(2) competence mixed liquor adds 2mm electric shock cup (added-time to be avoided producing bubble as far as possible), and 2500V shocks by electricity；Be rapidly added YEB fluid medium 800 μ L after electric shock, 28 DEG C, 180rpm when inducing culture 5h.
(3) take out the bacterium solution induced and be coated on the YEB flat board containing kanamycin and rifampicin (concentration is 50 μ g/mL), cultivate 48h for 28 DEG C.
(4) picking monoclonal is inoculated in the YEB fluid medium containing kanamycin and rifampicin (concentration is 50 μ g/mL) of 5mL, and 180rpm shakes bacterium, 36h, extracts plasmid enzyme restriction in a small amount and PCR identifies.
(5) will verify that correct monoclonal is inoculated in 50mL and contains kanamycin and the YEB fluid medium of rifampicin (concentration is 50 μ g/mL), incubated overnight is until OD600=1.0, add 50% glycerol, save backup in-80 DEG C, obtain the Agrobacterium tumefaciems GV3101 of the pGBI121-mcry2Ab4M containing plant expression vector, by its called after: GV3101/pGBI121-mcry2Ab4M.
(2) acquisition of cry2AhM genetic tobacco is turned
(1) the Agrobacterium GV3101/pGBI121-mcry2Ab4M bacterium solution of the expression vector obtained in () is taken, transferring in the YEB fluid medium of 100mL kanamycin and rifampicin (concentration is 50mg/L), about 12h to OD is cultivated in concussion600=0.6～0.8.
(2) bacterium solution is poured into 50mL sterile centrifugation tube, 4 DEG C, 4500rpm when centrifugal 5min, abandon supernatant, take 50mL sterilizing MS0Fluid medium piping and druming suspension thalline.
(3) take the aseptic tobacco leaf of 28d seedling age, remove leaf explant edge and main lobe arteries and veins, be cut into 0.5cm2Square.
(4) agrobacterium suspension is transferred in glass culture dish, the blade cut is soaked wherein 10min, aseptic filter paper blots bacterium solution, it is placed in and co-cultures culture medium (MS+2mg/L, 6-BA+0.5mg/L, IAA+200mg/L) on, blade respectively covers lid layer aseptic filter paper up and down, 25 DEG C of light culture 3d.
(5) will co-culture in the MS2 screening culture medium that outer implant forwards to containing kanamycin and Carbenicillin, lure bud screening culture medium MS2Composition: MS+2mg/L, 6-BA+0.5mg/L, IAA+200mg/L, Kan+500mg/L, Cep+500mg/L.
(6) switching in every two weeks once, is cultivated 4-8wk, is treated that the regrowth that callus surface grows grows about 4 leaves, cut from wound healing surface, and cuttage carries out root culture in the MS culture medium of 1/2.
(7) seedling rooting to be regenerated cultivates 2-3wk, when root growth is more healthy and stronger, bottle cap is opened in incubator half, adds a small amount of aquesterilisa seedling exercising, is moved in big alms bowl or the big Tanaka in greenhouse cultivates, it is thus achieved that turn cry2AhM genetic tobacco regrowth.
(3) qualification of cry2AhM genetic tobacco is turned
(1) PCR turning cry2AhM genetic tobacco identifies
The regenerated transgenic seedling PhirePlantDirectPCRKit (two) obtained detects, detection method: drill through the small pieces leaf tissue of prototype with the 1mL blue electron gun head of sterilizing at blade to be measured, extracting leaves genomic DNA and carry out PCR qualification as pcr template, primer is as follows:
Cry2Ah-F:5'-GTATCAATACCTACCAGTCTGCTTTC-3'(SEQIDNo:6),
Cry2Ah-R:5'-TATCTCCCTGGTTACCGAACTTTT-3'(SEQIDNo:7).
PCR reaction condition is: 98 DEG C of denaturation 5min；98 DEG C of degeneration 5s, 57 DEG C of annealing 5s, 72 DEG C extend 40s, 40 circulations；72 DEG C of ends extend 1min.
By the 34 strain regeneration of transgenic Nicotiana tabacum L.s obtained are carried out blade PCR qualification, result has 30 strain regrowths to amplify the band (part PCR primer electrophoresis result is shown in Fig. 2) of about 1kb.Randomly drawing 5 strains from 30 strain positive transgenic plant and carry out following experiment as research material, it numbers respectively GO-1, GO-2, GO-3, GO-6, GO-15.
(2) qRT-PCR detection
The positive that (1) is obtained turns 5 the different strains (being numbered: GO-1, GO-2, GO-3, GO-6, GO-15) in cry2AhM genetic material and wild-type tobacco extracts mRNA respectively, and reverse transcription becomes cDNA, carries out qRT-PCR；Primer is as follows:
Cry2Ah-qF:5'-CCCCTGCAGGATGAACTCCGTGCTCAACTCAGGTA-3'(SEQI DNo:8),
Cry2Ah-qR:5'-AAAGTGATTGAAAGTGGCA-3'；(SEQIDNo:9)；
Nicotiana tabacum L. reference gene primer is as follows:
Actin9-F:5'-CTATTCTCCGCTTTGGACTTGGCA-3'；(SEQIDNo:10),
Actin9-R:5'-AGGACCTCAGGACAACGGAAACG-3'(SEQIDNo:11).
PCR reaction condition is: 95 DEG C of denaturation 1min；95 DEG C of degeneration 15s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, 40 circulations；72 DEG C extend 10min.Each material does 3 hole duplicate detection, according to 2-△△CtMethod calculates the relative expression quantity of genes of interest.5 transgenic lines obtained are carried out qRT-PCR analysis, and result shows (see Fig. 3 and Fig. 4) 5 transgenic line equal normal transcription of GO-1, GO-2, GO-3, GO-6, GO-15 anti insect gene, and wherein GO-3 expression is the highest, does not express in wild type.Prove that anti insect gene cry2AhM can high efficient expression in Nicotiana tabacum L. further.
(4) Western hybridization check
5 strains turning cry2AhM genetic tobacco in (2) (are numbered: GO-1, GO-2, GO-3, GO-6, GO-15) tobacco leaf albumen (vegetable protein of KWBIO company extracts test kit) is extracted, PAGE gel electrophoresis is carried out after boiling water treating 5min, transfer to pvdf membrane, then Western hybridization check is carried out, primary antibodie is Cry2Ah rabbit multi-resistance (1: 1000) of Hua Da genome company immunity, the IgG (1:10000) of the horseradish peroxidase-labeled that two anti-employings are ordered by Beijing EMAR company, develop the color through the eECLWesternBlotKit of KWBIO company.Result ((see Fig. 5)) transformed plant all has special immuning hybridization band, Cry2AhM albumen size is about 70kDa, and unconverted Nicotiana tabacum L. is without purpose band, the hybridising band of the positive plant turning cry2AhM is basically identical with positive control size, illustrates that cry2AhM gene has proceeded in Nicotiana tabacum L. and obtained normal expression.
(5) insect resistace is identified
5 strains turning cry2AhM genetic tobacco in (2) (are numbered: GO-1, GO-2, GO-3, GO-6, GO-15) plant carries out biological activity assay, when tobacco plant grows into 8-10 leaf phase, just incubates cotton boll larva and be connected on tobacco leaf after feeding 1d, grow WT lines as negative control using the same period, connect worm 4d " Invest, Then Investigate " mortality rate and blade damage situation.The bollworm corrected mortality of result (see table 1) feeding transgenic tobacco leaf, more than 86.59%, can visually see (see Fig. 6) from blade face, and non-transgenic tobacco blade damage situation is serious, and worm channel number is more.And transgene tobacco blade face only has a small amount of worm channel, substantially intact.By comparing the upgrowth situation (see Fig. 7) of bollworm it can be seen that the bollworm taking food transgenic tobacco leaves is substantially inhibited than the growth of the bollworm taking food non-transgenic Nicotiana tabacum L..
The insect resistace detection statistics result of 1 turn of cry2AhM genetic tobacco of table.
| Transformation event | Mortality rate (%) | Corrected mortality (%) |
| GO-1 | 93.33 | 90.20±9.67 |
| GO-2 | 91.67 | 86.59±9.10 |
| GO-3 | 93.33 | 89.36±6.30 |
| GO-6 | 93.33 | 89.72±6.18 |
| GO-15 | 91.67 | 88.61±13.62 |
Claims (10)
1. a Bt insect resistance protein, it is characterised in that described protein is following (a) or (b):
A () aminoacid sequence shown in SEQIDNo:1 forms；
B aminoacid sequence shown in (a) is passed through replacement and/or the disappearance of one or several amino acid residue and/or adds formation by (), and have the protein of pest-resistant characteristic.
2. the gene of Bt insect resistance protein described in coding claim 1, it is characterised in that described gene is following (c), (d) or (e):
C () nucleotide sequence shown in SEQIDNo:2 forms；
D () and the nucleotide sequence shown in SEQIDNo:2 have the homology of more than 90%, and coding has the nucleotide sequence of pest-resistant characteristic protein；
(e) under strict conditions, the nucleotide sequence hybridization limited with (c) or (d), and coding has the nucleotide sequence of pest-resistant characteristic protein.
3. contain the expression vector of gene described in claim 2.
4. expression vector according to claim 3, it is characterised in that described carrier refers to plasmid, cell or plant part.
5. the application on preventing and treating Lepidoptera, coleoptera or Diptera pest of the Bt insect resistance protein described in claim 1.
6. the application on preventing and treating Lepidoptera, coleoptera or Diptera pest of the gene described in claim 2.
7. application according to claim 6, it is characterised in that described application refers in the gene transformation described in claim 2 to plant, it is thus achieved that pest-resistant transgenic plant.
8. utilize the method that the gene described in claim 2 cultivates transgenic anti-insect plants, it is characterised in that the gene described in claim 2 or the expression vector described in claim 3 are imported plant, it is thus achieved that pest-resistant transgenic plant.
9. for identifying the PCR primer of the plant containing gene described in claim 2, it is characterised in that the nucleotide sequence shown in SEQIDNo:6 and SEQIDNo:7 forms.
10. the method for identifying the plant containing gene described in claim 2, it is characterized in that extracting testing sample genomic DNA, then with testing sample genomic DNA for template, arrange with the nucleotides sequence shown in SEQIDNo:6 and SEQIDNo:7 and carry out pcr amplification for primer, acquisition size is about the PCR primer of 1kb, is transgenic plant.
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| CN109776659A (en) * | 2019-03-14 | 2019-05-21 | 中国农业科学院生物技术研究所 | Application of the cry2Ah-vp gene in anti-armyworm |
| CN110144360A (en) * | 2019-06-06 | 2019-08-20 | 华中农业大学 | The primer pair and application of the protein and application of a kind of striped rice borer SDR gene and its coding, dsRNA and its amplification |
| CN110283833A (en) * | 2019-06-27 | 2019-09-27 | 华侨大学 | Cry2Ab12 gene is in the purposes for preparing aphids resistance genetically modified plants |
| CN116497034A (en) * | 2023-01-12 | 2023-07-28 | 深圳市仙湖植物园(深圳市园林研究中心) | Insect-resistant protein and application thereof |
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