CN107474120A - The artificial synthesized Bt killing genes mcry1F for transgenic anti-insect plants - Google Patents

The artificial synthesized Bt killing genes mcry1F for transgenic anti-insect plants Download PDF

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CN107474120A
CN107474120A CN201710701955.3A CN201710701955A CN107474120A CN 107474120 A CN107474120 A CN 107474120A CN 201710701955 A CN201710701955 A CN 201710701955A CN 107474120 A CN107474120 A CN 107474120A
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赖锦盛
赵海铭
宋伟彬
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China Agricultural University
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    • C12N15/8271—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
    • C12N15/8279—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
    • 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 the artificial synthesized Bt killing genes mcry1F for transgenic anti-insect plants.Bt killing genes mcry1F provided by the present invention for transgenic anti-insect plants encodes mCry1F albumen.MCry1F albumen is b1) or b2) or b3)：B1) amino acid sequence is the protein shown in sequence 2 in sequence table；B2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label obtain；B3) the protein related to plant anti-insect for obtaining the amino acid sequence shown in sequence in sequence table 2 by the substitution of one or several amino acid residues and/or missing and/or addition.Experiment shows that mcry1F genes are easier to obtain the transformant of high expression quantity compared with cry1F genes, and compared with cry1F albumen, mcry1F albumen significantly improves to the insecticidal activity of corn borer, armyworm, bollworm and dichocrocis punctiferalis, has important promotional value.

Description

The artificial synthesized Bt killing genes mcry1F for transgenic anti-insect plants
Technical field
The invention belongs to technical field of biological control, and in particular to the Bt mcry1F genes for plant expression.
Background technology
Bt gene code insecticidal crystal proteins, should from bacillus thuringiensis (Bacillus thuringiensis) Bacterium is Gram-positive agrobacterium, belongs to bacillus, and its thalline is rod-short, raw flagellum, single raw or formation short chain.It The desinsection insecticidal crystal proteins that referred to as delta-endotoxin is produced during sporulation (control the gene for synthesizing this protein to exist On plasmid), these albumen have specific insecticidal activity to various insects such as Lepidoptera, Diptera, coleoptera, Hymenopteras.
Schenpf and Whiteley in 1981 clones from bacillus thuringiensis (Bacillus thuringiensis) The cry gene Cs ry1Aa1 of first coding delta-endotoxin.Adang in 1985, M.J etc. are from bacillus thuringiensis
Cry1Ac genes are cloned in (Bacillus thuringiensis), Wabiko, H. et al. in 1986 is from Su Yun Cry1Ab genes are cloned in golden bacillus, 1155 amino acid of the gene code, are that current commercial application is relatively broad Anti insect gene.Chambers in 1991, J have cloned cry1Fa genes.By the end of current, it has been determined that over thousands of kind of Su Yun gold gemma Bacillus fungus strain and more than ten thousand insecticidal crystal proteins planted.
Nineteen eighty-three, Washington, DC university announced that kalamycin resistance gene successfully is imported into tobacco cell, and the same year 4 Month Univ Wisconsin-Madison USA announces that soybean gene successfully is transferred into the birth that sunflower indicates plant transgenic technology jointly. 1986, first batch of genetically modified plants (pest-resistant, antiweed) went through to enter field test.Mammalian antibody heavy in 1989 With light chain gene successful expression and functional antibody is correctly assembled into tobacco.Nineteen ninety, Gordon-Kamm was reported first With via Particle Bombardment Transformation Corn suspension cells, system obtains fertile transformant.Then, corn gene technology starts fast development, A large amount of genetically modified plants develop success successively.Koziel (1993) etc. has cultivated insect-resistant transgenic corn, and transgenosis is planted Strain energy horizontal expression Cry1Ab anti insect genes, lift the insect resistace of corn.Zhang Xiujun (1999) etc. is contained two with particle bombardment The embryo callus of lysine-rich protein matter channel genes corn.Liu great Wen (2000) etc. is by Zm13-Barnase genetic transformation Corn embryo callus, positive plant is obtained by herbicide screening.Ishida (1996) etc. utilizes super binary vector first Establish the rataria of Agrobacterium tumefaciens transformation corn inbred line, Frame (2002) etc. and be successfully realized Agrobacterium tumefaciems using general Logical binary vector conversion rataria.Bt killing genes are imported superior corn by Zhang Yanzhen (2002) etc. to agrobacterium tumefaciens-mediated transformation Self-mating system has carried out more systematic research.Huang and Wei (2005) utilizes Agrobacterium tumefaciems successful conversion ordinary maize certainly Hand over system.Frame (2006) etc. and Lee (2007) etc. are utilized respectively Agrobacterium successful conversion ordinary maize self-mating system.
China has been set up the corn gene technical system of comparative maturity, in state in terms of transgenic technology research Interior, China Agricultural University has carried out the work of maize genetic conversion earlier, and 1994 at home and abroad use the side of Ovule injection first Anti insect gene Bt is transferred to corn and obtains transfer-gen plant by method, using independent intellectual property right gene Bt as representative pest-resistant corn Illustrate good DEVELOPMENT PROSPECT.Meanwhile dropped in marker-free, selectable marker gene deletion and desired gene product timing There is stronger innovation ability in terms of the Core-technologies such as solution, plant tissue specificity predominant expression.It is separated at present Anti insect gene is a lot, and main anti insect gene has：1. the anti insect gene of bacterial origin, mainly Bt toxoprotein genes (Cry1Ab, Cry1Ac, Cry2A, Cry3C etc.)；2. the protease inhibitor gene of plant origin, feature be pest-resistant spectrum it is wide, from plant Thing is easy to be accepted by the public in itself；3. the nutritive insecticidal albumen (Vip1, Vip2, Vip3 etc.) of bacterial origin, the anti-Lepidoptera of wide spectrum, Particularly there is strong effect to black cutworm, mythimna separata and beet tops moth.Due to gene pest-resistant ability and the shadow of insect tolerance Ring, transgenic pest-resistant product is mainly by obtaining the ways such as more more effectively anti insect genes, transformation existing gene, dual anti-plant Footpath obtains.
The U.S. has turned into plants the maximum country of transgenic corns in the world, and transgenic corns popularizing area accounts for american corn The ratio of the gross area by 2000 25% rise to 2015 92%.Data show, due to the use of Bt corns, agricultural chemicals and The dosage of insecticide is decreased obviously, and greatly reduces the pollution to environment.Both reduce agriculture production cost, labor can be saved again Power.The pest-resistant merit of corn brings huge economy and environmental benefit for society, and in China's transgenic corns product Kind is cultivated also has larger gap compared with the U.S., is started for this country in 2008《Genetically modified organism rearing new variety science and technology is great specially 》To improve As-Is.Though China's some reports in terms of transgenic insect-resistant corn breed of variety, but can not be most Whole commercialization, key factor is in the insect resistant effect of Bt genes and the stability of transformed plant.
The content of the invention
The technical problems to be solved by the invention are how to improve plant resistance to insect.
In order to solve the above technical problems, present invention firstly provides mCry1F albumen.
MCry1F albumen provided by the present invention, can be b1) or b2) or b3)：
B1) amino acid sequence is the protein shown in sequence 2 in sequence table；
B2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label obtain；
B3) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or The protein related to plant anti-insect that missing and/or addition obtain.
Wherein, sequence 2 is made up of 600 amino acid residues in sequence table.
In order that b1) in protein be easy to purify, the amino terminal of protein that can be in sequence table shown in sequence 2 or The upper label as shown in table 1 of carboxyl terminal connection.
The sequence of the label of table 1.
Label Residue Sequence
Poly-Arg 5-6 (being usually 5) RRRRR
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
Above-mentioned b3) in protein, the substitution of one or several amino acid residues and/or missing and/or be added to No more than the substitution and/or missing and/or addition of 10 amino acid residues.
Above-mentioned b3) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned b3) in the encoding gene of protein can be by the way that one will be lacked in the DNA sequence dna shown in sequence in sequence table 1 The codon of individual or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.
The nucleic acid molecules for encoding the mCry1F albumen fall within protection scope of the present invention.
The nucleic acid molecules for encoding the mCry1F albumen can be DNA points shown in following (a1) or (a2) or (a3) or (a4) Son：
(a1) DNA molecular of the code area as shown in sequence 1 in sequence table；
(a2) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；
(a3) there is 75% or more than 75% homogeneity with (a1) or (a2) nucleotide sequence limited, and described in coding The DNA molecular of mCry1F albumen；
(a4) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and encode the mCry1F albumen DNA molecular.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made up of 1803 nucleotides in sequence table, the nucleotide coding sequence table of sequence 1 in sequence table Amino acid sequence shown in middle sequence 2.
Those of ordinary skill in the art can be easily using known method, such as the side of orthogenesis and point mutation Method, the nucleotide sequence of the coding mCry1F albumen of the present invention is mutated.Those by manually modified, have with The present invention isolated nucleotide sequence 75% of the mCry1F albumen or the nucleotides of higher homogeneity, as long as coding The mCry1F albumen, it is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair The nucleotide sequence of the mCry1F albumen of amino acid sequence composition shown in the sequence 2 of bright polynucleotide has 75% or more Height, or 80% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity.It is same Property can with the naked eye or computer software is evaluated.Using computer software, the homogeneity between two or more sequences can To be represented with percentage (%), it can be used for evaluating the homogeneity between correlated series.
Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecules fall within this hair Bright protection domain.
The nucleotide sequence of the expression cassette is as shown in sequence 5 in sequence table.In sequence table in sequence 5, from 5 ' ends 1st to 583 nucleotide sequence for corn Gly promoters, the 589th to 2392 nucleotide sequence for mcry1F genes, 2399th to 2652 nucleotide sequence (being used to terminate transcription) for no terminators.
The recombinant vector can be that will encode the nucleic acid molecules of the mCry1F albumen (i.e. in sequence table shown in sequence 1 DNA molecular) insert the recombinant plasmid that plasmid obtains that sets out.
The recombinant vector concretely recombinant plasmid pCAMBIA3301+mCry1F.The recombinant plasmid PCAMBIA3301+mCry1F is by between carrier pCAMBIA3301 restriction enzyme HindIII and BstEII recognition sequence Small fragment replace with double chain DNA molecule of the sequence 5 from 5 ' ends shown in the 1st to 2392 in sequence table.Recombinant plasmid MCry1F albumen in pCAMBIA3301+mCry1F expressed sequence tables shown in sequence 2.
The recombinant microorganism can be obtained by the way that the recombinant vector is imported into the microorganism that sets out.
The microorganism that sets out can be yeast, bacterium, algae or fungi.The bacterium can be gram-positive bacterium or leather Gram-negative bacteria.The gramnegative bacterium can be Agrobacterium tumefaciems (Agrobacteriumtumefaciens).It is described Agrobacterium tumefaciems (Agrobacterium tumefaciens) concretely Agrobacterium tumefaciems EHA105.
The recombinant microorganism concretely EHA105/pCAMBIA3301+mCry1F.EHA105/pCAMBIA3301+ MCry1F is the recombinational agrobacterium for obtaining recombinant plasmid pCAMBIA3301+mCry1F conversions Agrobacterium tumefaciems EHA105.
The transgenic plant cells system does not include propagating materials.The genetically modified plants are interpreted as not only including institute The first generation genetically modified plants that mCry1F genes (i.e. the encoding gene of mCry1F albumen) transformation receptor plant obtains are stated, are also included Its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to which traditional breeding method is by the gene transfer Into other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, callus, complete Whole plant and cell.
The mCry1F albumen, or, the nucleic acid molecules, or, the expression cassette containing the nucleic acid molecules, recombinant vector, The application of recombinant microorganism or transgenic cell line falls within protection scope of the present invention；The mCry1F albumen, or, the core Acid molecule, or, the expression cassette containing the nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic cell line application can For e1) or e2) or e3)：E1 plant resistance to insect) is improved；E2 the product with insect resistant effect) is prepared；E3) insect resistace is cultivated to improve Genetically modified plants.
In above-mentioned application, the product can be medicine.
In order to solve the above technical problems, present invention also offers a kind of method for cultivating genetically modified plants.
The method provided by the present invention for cultivating genetically modified plants, it may include following steps：The mCry1F eggs will be encoded White nucleic acid molecules are imported in recipient plant, obtain genetically modified plants；Compared with the recipient plant, the genetically modified plants Insect resistace strengthens.
In the above method, the nucleic acid molecules for encoding the mCry1F albumen can be following (a1) or (a2) or (a3) or (a4) Shown DNA molecular：
(a1) DNA molecular of the code area as shown in sequence 1 in sequence table；
(a2) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；
(a3) there is 75% or more than 75% homogeneity with (a1) or (a2) nucleotide sequence limited, and described in coding The DNA molecular of mCry1F albumen；
(a4) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and encode the mCry1F albumen DNA molecular.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made up of 1803 nucleotides in sequence table, the nucleotide coding sequence table of sequence 1 in sequence table Amino acid sequence shown in middle sequence 2.
In the above method, it is described " by the nucleic acid molecules for encoding the mCry1F albumen import recipient plant in " can pass through to Recombinant vector is imported in recipient plant to realize；The recombinant vector can be to encode the mCry1F albumen to expression vector insertion The recombinant plasmid that nucleic acid molecules obtain.
The recombinant vector concretely recombinant plasmid pCAMBIA3301+mCry1F.
In order to solve the above technical problems, present invention also offers a kind of plant breeding method.
Plant breeding method provided by the present invention, it may include following steps：Increase mCry1F albumen described in plant Content and/or activity, so as to increase insect resistace.
In above-mentioned plant breeding method, " content and/or activity of mCry1F albumen described in increase plant " can lead to Cross multicopy, change the methods well known in the art such as promoter, regulatory factor, transgenosis, reach described in increase plant The content of mCry1F albumen and/or the effect of activity.
Any of any of the above-described plant can be c1) to c5)：C1) monocotyledon；C2) dicotyledon； C3) grass；C4) corn；C5) corn variety X178.
Any of the above-described insect resistace can be anti-lepidopterous insects.The lepidopterous insects can be d1) to d5) in any Kind：D1) corn borer；D2) east armyworm；D3) bollworm；D4) dichocrocis punctiferalis；D5) Ostrinia furnacalis.
Cry1F gene pairs lepidopterous insects (such as corn borer, armyworm and bollworm) have very strong toxicity, but original Gene can not in plant high efficient expression, do not reach the requirement of plant protection.Activity of the present inventor to Cry1F albumen Region has carried out careful analysis, on the premise of ensureing further to improve its insecticidal activity, is encoded according to monocotyledon special Sign has carried out gene code frame and codon transformation to Cry1F albumen.Improved Cry1F albumen is named as mcry1F albumen. The amino acid sequence of mcry1F albumen is as shown in sequence 2 in sequence table.The gene for encoding mcry1F albumen is (i.e. improved Cry1F genes, hereinafter referred to as mcry1F genes) nucleotide sequence as shown in sequence 1 in sequence table.The present inventor Improved mcry1F genes are connected on the expression vector that corn Gly is promoter.By the method for Agrobacterium-mediated Transformation, Mcry1F genes go to the corn inbred line with Efficient Conversion efficiency.Experiment shows, the mcry1F bases of codon optimization Because compared with the transformant for being easier to obtain high expression quantity before codon optimization, and compared with cry1F albumen, mcry1F albumen is to jade Rice snout moth's larva, armyworm, bollworm and the insecticidal activity of dichocrocis punctiferalis significantly improve, and have important promotional value.
Brief description of the drawings
Fig. 1 is the experimental result of the step 5 of embodiment 3.
Fig. 2 is the experimental result of the step 6 of embodiment 3.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The solute and its concentration of N6E culture mediums be 4g/L N6 salt, 5mL/L N6vitamin Stock (200 ×), The 2 of 2mg/L, 4-D, 0.1g/L inositol, 2.76g/L proline, 30g/L sucrose, 0.1g/L casein hydrolysate, 2.8g/L plant gel and 3.4mg/L silver nitrate；Solvent is distilled water；PH value is 5.8.
N6vitamin Stock(200×)：0.4g/L containing glycine, nicotinic acid 0.1g/L, VB10.2g/L and VB6 0.1g/ The L aqueous solution.
N6E solid plates：About 55 DEG C of N6E culture mediums are poured into culture dish, N6E solid plates are obtained after cooling.
Contaminate culture medium：By micro (100 ×) 10mL, N6 molysite (100 of a large amount of (20 ×) 50mL, the B5 of sucrose 68.4g, N6 ×) organic (200 ×) the 5mL and 100 μm of ol of 10mL, RTV acetosyringone (Acetosyringone, AS) is dissolved in 1L distillations Water, regulation pH value to 5.2.
N6 is a large amount of (20 ×)：Containing (NH4)2SO4 9.26g/L、KNO3 56.60g/L、KH2PO4 8.00g/L、MgSO4· 7H2O 3.70g/L and CaCl2·2H2The O 3.32g/L aqueous solution.
B5 micro (100 ×)：Containing MnSO4·H2O 0.7600g/L、ZnSO4·7H2O 0.2000g/L、H3BO3 0.3000g/L、KI 0.0750g/L、Na2MoO4·2H2O 0.0250g/L、CuSO4·5H2O 0.0025g/L and CoCl2· 6H2The O 0.0025g/L aqueous solution.
N6 molysite (100 ×)：The 1.8300g/L of salt containing the iron edetate aqueous solution.
RTV organic (200 ×)：By Choline Chloride 0.0196g, VB20.0098g, Bio 0.0200g, nicotinic acid 0.0400g, folic acid 0.0097g, VB10.0944g, D-VB5 calcium 0.0200g, VB60.0400g, p-aminobenzoic acid The 0.0098g and VB that 400 μ L concentration are 0.75mg/100mL12The aqueous solution is dissolved in 1L distilled water.
Co-culture culture medium：By MS salt 4.33g, MS Vitamins (500 ×) 2mL, thiamine hydrochloride 0.5mg, sucrose 30.0g, L-PROLINE 1.38g, 2,4-D 0.5mg, 6-BA 0.01mg, plant gel 3.5g and 100 μm of ol AS are dissolved in 1L steamings Distilled water, regulation pH value to 5.7.
MS Vitamins(500×)：1g/L containing glycine, nicotinic acid 0.25g/L, VB10.05g/L and VB60.25g/L's The aqueous solution.
Recovery media：By MS salt 4.33g, MS Vitamins (500 ×) 2mL, thiamine hydrochloride 0.5mg, sucrose 30.0g, L-PROLINE 1.38g, 2,4-D 0.5mg, 6-BA 0.01mg, plant gel 3.5g, Tim 100mg, bialaphos 3.0mg and AgNO33.4mg is dissolved in 1L distilled water, regulation pH value to 5.7.
Selective agar medium：The MS solid mediums of double third ammonia phosphorus containing 1.5mg/L.
Second selecting culture medium：The MS solid mediums of double third ammonia phosphorus containing 3.0mg/L.
Regeneration culture medium I：By MS salt 4.33g, MS Vitamins (500 ×) 2mL, thiamine hydrochloride 0.5mg, sucrose 10.0g, glucose 20g, L-PROLINE 0.7g, plant gel 3.5g, casein hydrolysate 0.2g, glycine 0.04g, inositol 0.1g and bialaphos 3.0mg is dissolved in 1L distilled water, regulation pH value to 5.7.
Regeneration culture medium II：MS salt 2.165g, sucrose 30.0g, plant gel 3.5g and bialaphos 3.0mg are dissolved in 1L Distilled water, regulation pH value to 5.7.
Prokaryotic expression carrier pEASY-E1 is the product of Beijing Quanshijin Biotechnology Co., Ltd.
Embodiment 1, the transformation of Cry1F genes and the acquisition of mcry1F genes
The amino acid sequence of Cry1F albumen is as shown in sequence 4 in sequence table.Encode the gene of Cry1F albumen (hereinafter referred to as For Cry1F genes) nucleotide sequence as shown in sequence 3 in sequence table.
The present inventor has carried out careful analysis to the active region of Cry1F albumen, is ensureing further to improve On the premise of its expression, gene code frame and codon have been carried out to Cry1F albumen according to monocotyledon coding characteristic Transformation.Improved Cry1F albumen is named as mcry1F albumen.Sequence 2 in the amino acid sequence of mcry1F albumen such as sequence table It is shown.Encode the nucleotides sequence of the gene (i.e. improved Cry1F genes, hereinafter referred to as mcry1F genes) of mcry1F albumen Row are as shown in sequence 1 in sequence table.
The sequence homology of mcry1F genes and Cry1F genes only has 66%, and homology region is only 51%, encoder block length It is changed into 600 amino acid from 1174 original amino acid, and G+C contents are also changed into 66.6% from original 38.9%, transformation Front and rear codon utilization rate refers to table 2.
Table 2
Embodiment 2, mcry1F albumen are surveyed to the raw of lepidopterous insects
First, the vivoexpression of mcry1F albumen and purifying
The step of vivoexpression of mcry1F albumen and purifying, is specific as follows：
1st, the double chain DNA molecule in artificial synthesized sequence table shown in sequence 1.
2nd, the double chain DNA molecule that step 1 synthesizes is connected with prokaryotic expression carrier pEASY-E1, obtains recombinant plasmid pEASY-mcry1F。
Recombinant plasmid pEASY-mcry1F is sequenced.Sequencing result shows, contains in recombinant plasmid pEASY-mcry1F DNA molecular in ordered list shown in sequence 1, the mcry1F albumen in expressed sequence table shown in sequence 2.
3rd, recombinant plasmid pEASY-mcry1F is imported into Escherichia coli transetta, obtains recombinant bacterium, the recombinant bacterium is ordered Entitled transetta-mcry1F.
4th, transetta-mcry1F monoclonal is taken, 100mL LB fluid nutrient mediums is seeded to and (contains 50 μ g/mL ammonia benzyls Mycin), 37 DEG C, 200rpm shaken cultivation 12h, obtain cultivating bacterium solution.
5th, culture bacterium solution is taken, is by volume 1:100, which are seeded to 50mL LB fluid nutrient mediums, (it is mould to contain 50 μ g/mL ammonia benzyls Element), 37 DEG C, 200rpm shaken cultivations to OD600nmIt is worth for 0.6, then adds IPTG and make its concentration be 1mM, 28 DEG C, 220rpm Shaken cultivation 4h, 4 DEG C, 10000rpm centrifugation 10min, collects bacterial sediment.
6th, bacterial sediment is taken, adds 100mL pH 8.0,100mM Tris-HCl buffer solutions, ultrasonication is (super after resuspension Acoustic power 600W, cyclic program are：Broken 4s, stops 6s, common 20min), then 4 DEG C, 10000rpm centrifuge 10min, in collection Clear liquid first.
7th, supernatant first is taken, 4 DEG C, 12000rpm centrifugation 10min, collects supernatant second.
8th, the nickel post produced using GE companies is purified (explanation of the specific steps of purifying with reference to nickel post to supernatant second Book), then mcry1F albumen is quantified using the protein quantification kit of match Mo Feishier companies production.
According to the method described above, " double chain DNA molecule in artificial synthesized sequence table shown in sequence 1 " in step 1 is replaced with " double chain DNA molecule in artificial synthesized sequence table shown in sequence 3 ", other steps are constant, obtain cry1F albumen.
2nd, mcry1F albumen is surveyed to the raw of lepidoptera pest
Expression activitiy is carried out to mcry1F albumen and cry1F albumen according to the sod cultivation that Jing Xue (2008) are reported, Respectively determine two kinds of albumen (mcry1F albumen and cry1F albumen) to four Lepidopterous insects (Ostrinia furnacalis, east armyworm, Bollworm and dichocrocis punctiferalis) insecticidal activity.
Part of test results is shown in Table 3.As a result show, mcry1F albumen is substantially good to the insecticidal activity of four Lepidopterous insects In cry1F albumen, cry1F gene pairs Ostrinia furnacalis, bollworm, the anti-insect activity of armyworm and dichocrocis punctiferalis significantly improve.
Table 3
Embodiment 3, the acquisition for turning mcry1F gene corns and insect resistace identification
First, the structure of recombinant vector
Construction recombination plasmid pCAMBIA3301+mcry1F is simultaneously sequenced.According to sequencing result, to recombinant plasmid PCAMBIA3301+mcry1F carries out structure and is described as follows：By carrier pCAMBIA3301 restriction enzyme HindIII and Small fragment between BstEII recognition sequences replaces with double-stranded DNA of the sequence 5 from 5 ' ends shown in the 1st to 2392 in sequence table Molecule.Mcry1F albumen in recombinant plasmid pCAMBIA3301+mcry1F expressed sequence tables shown in sequence 2.
Contain expression cassette in recombinant plasmid pCAMBIA3301+mcry1F, in the nucleotide sequence of the expression cassette such as sequence table Shown in sequence 5.In sequence table in sequence 5, the 1st to 583 nucleotide sequence for corn Gly promoters from 5 ' ends, the 589 to 2392 nucleotide sequences for mcry1F genes, the 2399th to 2652 nucleotide sequence for no terminators (are used Transcribed in terminating).
The nucleotide sequence of mcry1F genes in recombinant plasmid pCAMBIA3301+mcry1F is replaced with into Cry1F genes Nucleotide sequence, other sequences are constant, obtain recombinant plasmid pCAMBIA3301+Cry1F.Recombinant plasmid pCAMBIA3301+ Cry1F albumen in mcry1F expressed sequence tables shown in sequence 4.
2nd, the acquisition of recombinational agrobacterium
Recombinant plasmid pCAMBIA3301+mcry1F is imported in Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, is ordered Entitled EHA105/pCAMBIA3301+mcry1F.
Recombinant plasmid pCAMBIA3301 is imported in Agrobacterium tumefaciems EHA105, recombinational agrobacterium is obtained, is named as EHA105/pCAMBIA3301。
Recombinant plasmid pCAMBIA3301+Cry1F is imported in Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, is named For EHA105/pCAMBIA3301+Cry1F.
3rd, the acquisition of mcry1F gene corns is turned
1st, the acquisition and culture of rataria
(a) in field planting corn variety X178, after self-pollination 9-11d, remove the bract of pollination fruit ear, then Fruit ear, which is put into, to be filled thimerosal ((effective chlorine is for the bleaching agent aqueous solution or aqueous sodium hypochlorite solution to 700mL 50% (v/v) 5.25% (v/v)) in add a drop Tween 20 and obtain) beaker in soak 20min, then with sterile water washing 3 times.Immersion During, need rotation fruit ear frequently gently to pat beaker simultaneously to drive away the bubble on seed surface, disappear so as to reach optimal Toxic effect fruit.
(b) after completing step (a), fruit ear is taken, the point of a knife for shelling embryo knife is inserted between embryo and endosperm, is then gently prized upwards Go out rataria, rataria is gently held up with small operation point of a knife, it is ensured that rataria by any damage, is not close to the plumular axis face of rataria The N6E solid plates of filter paper are placed with, the density of rataria is about 2cm × 2cm (30/ware).
(c) after completing step (b), the N6E solid plates are taken, with ParafilmTM, 28 DEG C of light culture 2-3d.
2nd, the acquisition of During Agrobacterium liquid
(a) EHA105/pCAMBIA3301+mcry1F is inoculated in kanamycins containing 33mg/L (Kanamycin, Kana) On the YEP solid mediums of 50mg/L streptomysins (streptomycin, str), 19 DEG C are cultivated 3 days, are activated.
(b) EHA105/pCAMBIA3301+mcry1F for obtaining step (a), which is inoculated in, contaminates in culture medium, 25 DEG C, 75rpm shaken cultivations, obtain OD550nmFor 0.3-0.4 During Agrobacterium liquid.
3rd, the acquisition of mcry1F gene corns is turned
The condition of alternation of light and darkness culture (i.e. illumination cultivation and light culture alternating) is：25℃.Illumination during illumination cultivation is strong Spend for 15000Lx.The cycle of alternation of light and darkness culture is specially：16h illumination cultivations/8h dark culturings.
(a) rataria of step 1 is taken into, is placed in centrifuge tube, first washs 2 times (every time using dip-dye with dip-dye culture medium Culture medium 2mL), During Agrobacterium liquid is then added, gently overturns centrifuge tube 20 times, then is uprightly statically placed in camera bellows 5min (to ensure Rataria is all immersed in During Agrobacterium liquid).
(b) after completing step (a), the rataria is transferred into co-cultivation culture medium (contacts the plumular axis of rataria to co-culture Media surface, while remove and co-culture the unnecessary Agrobacterium of media surface), then 20 DEG C of light culture 3d.
(c) after completing step (b), the rataria is transferred to recovery media, then 28 DEG C of light culture 7d.
(d) after completing step (c), the rataria is transferred to a Selective agar medium, then 28 DEG C of alternation of light and darkness cultures Two weeks.
(e) after completing step (d), the rataria is transferred to second selecting culture medium, then 28 DEG C of alternation of light and darkness cultures Two weeks, obtain kanamycin-resistant callus tissue.
(f) after completing step (e), kanamycin-resistant callus tissue is transferred to regeneration culture medium I, then 28 DEG C of alternation of light and darkness cultures three Week.
(g) after completing step (f), kanamycin-resistant callus tissue is transferred to regeneration culture medium II, then 28 DEG C of alternation of light and darkness cultures three In week, obtain regrowth.Seedling to be regenerated is transferred to greenhouse when growing to 3-4 piece leaves, normal culture, obtains turning mcry1F genes jade Rice.Will wherein 5 turn mcry1F gene corns and be named as mcry1F-1 successively to mcry1F-5.
According to the method described above, EHA105/pCAMBIA3301+mcry1F is replaced with into EHA105/pCAMBIA3301, it is other Step is constant, obtains turning empty carrier corn.
According to the method described above, EHA105/pCAMBIA3301+mcry1F is replaced with into EHA105/pCAMBIA3301+ Cry1F, other steps are constant, obtain turning Cry1F gene corns.Will wherein 5 turn Cry1F gene corns and be named as successively Cry1F-1 to Cry1F-5.
4th, Molecular Identification
The genomic DNA of mcry1F-1 to mcry1F-5 blade is extracted respectively and using it as template, using primer 1FF：5'-ACAACCACTACAACCGCCTCATC-3' and primer 1FR：5'-TGTTGATGGTGGCGTAGGAGAAG-3' is formed Primer pair enter performing PCR amplification, obtain pcr amplification product first.The genome of Cry1F-1 to Cry1F-5 blade is extracted respectively DNA and using it as template, using primer cry1FF：5'-TATCGTTTGGGCAGGGTTGGG-3' and primer cry1FR：5'- The primer of CGCCACCAGGATTGAAGACCC-3' compositions enters performing PCR amplification, obtains pcr amplification product second.
According to the method described above, the genomic DNA of mcry1F-1 blade is replaced with into water, other step all sames, as Negative control.
According to the method described above, the genomic DNA of mcry1F-1 blade is replaced with to the base for the blade for turning empty carrier corn Because of a group DNA, other step all sames, as control 1.
According to the method described above, the genomic DNA of mcry1F-1 blade is replaced with to the base of corn variety X178 blade Because of a group DNA, other step all sames, as control 2.
According to the method described above, the genomic DNA of mcry1F-1 blade is replaced with into recombinant plasmid pCAMBIA3301+ Mcry1F, other step all sames, as positive control 1.
According to the method described above, the genomic DNA of mcry1F-1 blade is replaced with into recombinant plasmid pCAMBIA3301+ Cry1F, other step all sames, as positive control 2.
Pcr amplification product is entered into row agarose gel electrophoresis.As a result show, with mcry1F-1 to mcry1F-5 blade Genomic DNA or recombinant plasmid pCAMBIA3301+mcry1F are that equal can expand of template obtains the band that size is 1134bp, with The genomic DNA or recombinant plasmid pCAMBIA3301+Cry1F of Cry1F-1 to Cry1F-5 blade are that template can expand To the band that size is 510bp, with the blade of water, the genomic DNA for the blade for turning empty carrier corn or corn variety X178 Genomic DNA is template, can not expand to obtain the band that size is 1134bp or the band that size is 510bp.
Through Molecular Identification, mcry1F-1 to mcry1F-5 is to turn mcry1F gene corns, and Cry1F-1 to Cry1F-5 is equal To turn Cry1F gene corns.
5th, detection turns the content of Cry1F albumen in mcry1F gene corns
Testing sample is mcry1F-1 blade, mcry1F-2 blade, mcry1F-3 blade, mcry1F-4 leaf Piece, mcry1F-5 blade, Cry1F-1 blade, Cry1F-2 blade, Cry1F-3 blade, Cry1F-4 blade, The blade of Cry1F-5 blade, the blade for turning empty carrier corn or corn variety X178.
Bt Cry1F ELISA kits are the product of Agdia companies of the U.S..
Experiment is averaged in triplicate.Comprise the following steps that：
(1) sample and positive control are prepared：Liquid nitrogen grinding testing sample, weigh the ground samples of 0.1g and add 1mL samples Extraction buffer (kit carries), while the positive control carried with 2mL sample extractions buffer solution dilution kit, are fully mixed Low-speed centrifugal 30s afterwards, prepare sample-adding.
(2) with enzyme-linked buffer solution (kit carries) according to 100:1 dilution proportion antibody (kit carries), will dilute Antibody be added in ELISA Plate, per hole add 100 μ l.10 μ L are respectively taken to be added to corresponding sample well in different samples respectively In, while take 0 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 50 μ L, 100 μ L positive controls to add in corresponding sample well respectively, Various kinds sample wells is mended to 200 μ L with board-washing buffer solution (kit carries), 1h or 4 DEG C is incubated under room temperature wet environment overnight.
(3) board-washing：With 1 × PBST (kit carries) board-washing 7 times, every time plus the μ L of board-washing buffer solution 300, after filling it up with a plate Outwell, be inverted ELISA Plate after washing, fully remove internal residual liquid.
(4) develop the color：100 μ L TMB colorbuffers are added per hole, 20min is incubated under room temperature wet environment.
The light absorption value of different samples is analyzed under 650nm wavelength by Thermo MK3 ELIASAs, utilizes painting for positive control Standard curve processed carries out Cry1F protein quantifications, then counts the ratio that Cry1F albumen accounts for testing sample fresh weight, that is, obtains Cry1F The content of albumen.
Light absorption value of the sample segment under 650nm wavelength is shown in Fig. 1.As a result show, mcry1F-1 to mcry1F-5 blade With detectable Cry1F albumen in Cry1F-1 to Cry1F-5 blade, turn the blade and corn variety of empty carrier corn X178 blade can not detect Cry1F albumen.Meanwhile compared with Cry1F-1 to Cry1F-5, mcry1F-1 to mcry1F- The content of Cry1F albumen has different degrees of raising in 5 blade.
6th, the insect resistace identification of mcry1F gene corns is turned
Corn to be measured is mcry1F-1, mcry1F-2, mcry1F-3, mcry1F-4, mcry1F-5, Cry1F-1, Cry1F- 2nd, Cry1F-3, Cry1F-4, Cry1F-5, turn empty carrier corn or corn variety X178.
Experiment setting at least 2 secondary pollutants repeat, and every kind of corn to be measured repeats to set 2 parallel laboratory tests, and specific steps are such as Under：
1st, gather the blade of corn to be measured and be put into culture dish.
2nd, after completing step 1, corn borer larvae is incubated at the beginning of accessing 3 into each culture dish, is subsequently placed in 28 DEG C, photoperiod 16h：8h(L:D), cultivated in relative humidity 70-80% artificial climate incubator.After 144h, the phenotype of blade is observed.
Experimental result is shown in Fig. 2.As a result show, compared with turning cry1F gene corns, the blade for turning mcry1F gene corns resists Worm property significantly improves.
<110>China Agricultural University
<120>The artificial synthesized Bt killing genes mcry1F for transgenic anti-insect plants
<160> 5
<170> PatentInversion3.5
<210> 1
<211> 1803
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
atggagaaca acatccagaa ccagtgcgtc ccctacaact gcctcaacaa ccccgaggtc 60
gagatcctca acgaggagcg ctccaccggc cgcctccccc tcgacatctc cctctccctc 120
acccgcttcc tcctctccga gttcgtcccc ggcgtcggcg tcgccttcgg cctcttcgac 180
ctcatctggg gcttcatcac cccctccgac tggtccctct tcctcctcca gatcgagcag 240
ctcatcgagc agcgcatcga gaccctcgag cgcaaccgcg ccatcaccac cctccgcggc 300
ctcgccgact cctacgagat ttacatcgag gccctccgcg agtgggaggc caaccccaac 360
aacgcccagc tccgcgagga cgtccgcatc cgcttcgcca acaccgacga cgccctcatc 420
accgccatca acaacttcac cctcacctcc ttcgagatcc ccctcctctc cgtctacgtc 480
caggccgcca acctccacct ctccctcctc cgcgacgccg tctccttcgg ccagggctgg 540
ggcctcgaca tcgccaccgt caacaaccac tacaaccgcc tcatcaacct catccaccgc 600
tacaccaagc actgcctcga cacctacaac cagggcctcg agaacctccg cggcaccaac 660
acccgccagt gggcccgctt caaccagttc cgccgcgacc tcaccctcac cgtcctcgac 720
atcgtcgccc tcttccccaa ctacgacgtc cgcacctacc ccatccagac ctcctcccag 780
ctcacccgcg agatttacac ctcctccgtc atcgaggact cccccgtctc cgccaacatc 840
cccaacggct tcaaccgcgc cgagttcggc gtccgccccc cccacctcat ggacttcatg 900
aactccctct tcgtcaccgc cgagaccgtc cgctcccaga ccgtctgggg cggccacctc 960
gtctcctccc gcaacaccgc cggcaaccgc atcaacttcc cctcctacgg cgtcttcaac 1020
cccggcggcg ccatctggat cgccgacgag gacccccgcc ccttctaccg caccctctcc 1080
gaccccgtct tcgtccgcgg cggcttcggc aacccccact acgtcctcgg cctccgcggc 1140
gtcgccttcc agcagaccgg caccaaccac acccgcacct tccgcaactc cggcaccatc 1200
gactccctcg acgagatccc cccccaggac aactccggcg ccccctggaa cgactactcc 1260
cacgtcctca accacgtcac cttcgtccgc tggcccggcg agatttccgg ctccgactcc 1320
tggcgcgccc ccatgttctc ctggacccac cgctccgcca cccccaccaa caccatcgac 1380
cccgagcgca tcacccagat ccccctcgtc aaggcccaca ccctccagtc cggcaccacc 1440
gtcgtccgcg gccccggctt caccggcggc gacatcctcc gccgcacctc cggcggcccc 1500
ttcgcctaca ccatcgtcaa catcaacggc cagctccccc agcgctaccg cgcccgcatc 1560
cgctacgcct ccaccaccaa cctccgcatc tacgtcaccg tcgccggcga gcgcatcttc 1620
gccggccagt tcaacaagac tatggacacc ggcgaccccc tcaccttcca gtccttctcc 1680
tacgccacca tcaacaccgc cttcaccttc cccatgtccc agtcctcctt caccgtcggc 1740
gccgacacct tctcctccgg caacgaggtc tacatcgacc gcttcgagct gatccccgtc 1800
tga 1803
<210>2
<211>600
<212>PRT
<213>Artificial sequence
<220>
<223>
<400>2
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn
1 5 10 15
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30
Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45
Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60
Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln
65 70 75 80
Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95
Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110
Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125
Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn
130 135 140
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val
145 150 155 160
Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175
Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn
180 185 190
Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp
210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp
225 230 235 240
Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255
Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270
Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu
275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe
290 295 300
Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu
305 310 315 320
Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335
Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350
Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365
Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380
Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile
385 390 395 400
Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415
Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro
420 425 430
Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile
450 455 460
Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr
465 470 475 480
Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495
Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe
530 535 540
Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser
545 550 555 560
Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575
Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590
Asp Arg Phe Glu Leu Ile Pro Val
595 600
<210> 3
<211> 3525
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
atggagaata atattcaaaa tcaatgcgta ccttacaatt gtttaaataa tcctgaagta 60
gaaatattaa atgaagaaag aagtactggc agattaccgt tagatatatc cttatcgctt 120
acacgtttcc ttttgagtga atttgttcca ggtgtgggag ttgcgtttgg attatttgat 180
ttaatatggg gttttataac tccttctgat tggagcttat ttcttttaca gattgaacaa 240
ttgattgagc aaagaataga aacattggaa aggaaccggg caattactac attacgaggg 300
ttagcagata gctatgaaat ttatattgaa gcactaagag agtgggaagc aaatcctaat 360
aatgcacaat taagggaaga tgtgcgtatt cgatttgcta atacagacga cgctttaata 420
acagcaataa ataattttac acttacaagt tttgaaatcc ctcttttatc ggtctatgtt 480
caagcggcga atttacattt atcactatta agagacgctg tatcgtttgg gcagggttgg 540
ggactggata tagctactgt taataatcat tataatagat taataaatct tattcataga 600
tatacgaaac attgtttgga cacatacaat caaggattag aaaacttaag aggtactaat 660
actcgacaat gggcaagatt caatcagttt aggagagatt taacacttac tgtattagat 720
atcgttgctc tttttccgaa ctacgatgtt agaacatatc caattcaaac gtcatcccaa 780
ttaacaaggg aaatttatac aagttcagta attgaggatt ctccagtttc tgctaatata 840
cctaatggtt ttaatagggc ggaatttgga gttagaccgc cccatcttat ggactttatg 900
aattctttgt ttgtaactgc agagactgtt agaagtcaaa ctgtgtgggg aggacactta 960
gttagttcac gaaatacggc tggtaaccgt ataaatttcc ctagttacgg ggtcttcaat 1020
cctggtggcg ccatttggat tgcagatgag gatccacgtc ctttttatcg gacattatca 1080
gatcctgttt ttgtccgagg aggatttggg aatcctcatt atgtactggg gcttagggga 1140
gtagcatttc aacaaactgg tacgaaccac acccgaacat ttagaaatag tgggaccata 1200
gattctctag atgaaatccc acctcaggat aatagtgggg caccttggaa tgattatagt 1260
catgtattaa atcatgttac atttgtacga tggccaggtg agatttcagg aagtgattca 1320
tggagagctc caatgttttc ttggacgcac cgtagtgcaa cccctacaaa tacaattgat 1380
ccggagagga ttactcaaat accattggta aaagcacata cacttcagtc aggtactact 1440
gttgtaagag ggcccgggtt tacgggagga gatattcttc gacgaacaag tggaggacca 1500
tttgcttata ctattgttaa tataaatggg caattacccc aaaggtatcg tgcaagaata 1560
cgctatgcct ctactacaaa tctaagaatt tacgtaacgg ttgcaggtga acggattttt 1620
gctggtcaat ttaacaaaac aatggatacc ggtgacccat taacattcca atcttttagt 1680
tacgcaacta ttaatacagc ttttacattc ccaatgagcc agagtagttt cacagtaggt 1740
gctgatactt ttagttcagg gaatgaagtt tatatagaca gatttgaatt gattccagtt 1800
actgcaacat ttgaagcaga atatgattta gaaagagcac aaaaggcggt gaatgcgctg 1860
tttacttcta taaaccaaat agggataaaa acagatgtga cggattatca tattgatcaa 1920
gtatccaatt tagtggattg tttatcagat gaattttgtc tggatgaaaa gcgagaattg 1980
tccgagaaag tcaaacatgc gaagcgactc agtgatgagc ggaatttact tcaagatcca 2040
aacttcaaag gcatcaatag gcaactagac cgtggttgga gaggaagtac ggatattacc 2100
atccaaagag gagatgacgt attcaaagaa aattatgtca cactaccagg tacctttgat 2160
gagtgctatc caacgtattt atatcaaaaa atagatgagt cgaaattaaa accctatact 2220
cgttatcaat taagagggta tatcgaggat agtcaagact tagaaatcta tttgatccgc 2280
tataatgcaa aacacgaaac agtaaatgtg ctaggtacgg gttctttatg gccgctttca 2340
gtccaaagtc caatcagaaa gtgtggagaa ccgaatcgat gcgcgccaca ccttgaatgg 2400
aatcctgatc tagattgttc ctgcagagac ggggaaaaat gtgcacatca ttcgcatcat 2460
ttctccttgg acattgatgt tggatgtaca gacttaaatg aggacttaga tgtatgggtg 2520
atattcaaga ttaagacgca agatggccat gcaagactag gaaatctaga gtttctcgaa 2580
gagaaaccat tagtcgggga agcactagct cgtgtgaaaa gagcagagaa aaaatggaga 2640
gataaacgtg aaaaattgga attggaaaca aatattgttt ataaagaggc aaaagaatct 2700
gtagatgctt tatttgtaaa ctctcaatat gatcaattac aagcggatac gaatattgcc 2760
atgattcatg cggcagataa acgtgttcat agaattcggg aagcgtatct tccagagtta 2820
tctgtgattc cgggtgtaaa tgtagacatt ttcgaagaat taaaagggcg tattttcact 2880
gcattcttcc tatatgatgc gagaaatgtc attaaaaacg gtgatttcaa taatggctta 2940
tcatgctgga acgtgaaagg gcatgtagat gtagaagaac aaaacaacca ccgttcggtc 3000
cttgttgttc cggaatggga agcagaagtg tcacaagaag ttcgtgtctg tccgggtcgt 3060
ggctatatcc ttcgtgtcac agcgtacaag gagggatatg gagaaggttg cgtaaccatt 3120
catgagatcg agaacaatac agacgaactg aagtttagca actgcgtaga agaggaagtc 3180
tatccaaaca acacggtaac gtgtaatgat tatactgcaa atcaagaaga atacgggggt 3240
gcgtacactt cccgtaatcg tggatatgac gaaacttatg gaagcaattc ttctgtacca 3300
gctgattatg cgtcagtcta tgaagaaaaa tcgtatacag atggacgaag agacaatcct 3360
tgtgaatcta acagaggata tggggattac acaccactac cagctggcta tgtgacaaaa 3420
gaattagagt acttcccaga aaccgataag gtatggattg agatcggaga aacggaagga 3480
acattcatcg tggacagcgt ggaattactc cttatggagg aatag 3525
<210> 4
<211> 1174
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 4
Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn
1 5 10 15
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30
Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45
Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60
Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln
65 70 75 80
Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95
Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110
Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125
Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn
130 135 140
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val
145 150 155 160
Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175
Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn
180 185 190
Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp
210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp
225 230 235 240
Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255
Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270
Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu
275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe
290 295 300
Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu
305 310 315 320
Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335
Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350
Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365
Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380
Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile
385 390 395 400
Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415
Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro
420 425 430
Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile
450 455 460
Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr
465 470 475 480
Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495
Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe
530 535 540
Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser
545 550 555 560
Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575
Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr
595 600 605
Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile
610 615 620
Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln
625 630 635 640
Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu
645 650 655
Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp
660 665 670
Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln
675 680 685
Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly
690 695 700
Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp
705 710 715 720
Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu
725 730 735
Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln
740 745 750
Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val
755 760 765
Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro
770 775 780
Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp
785 790 795 800
Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His
805 810 815
His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu
820 825 830
Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp
835 840 845
Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu
850 855 860
Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg
865 870 875 880
Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu
885 890 895
Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln
900 905 910
Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg
915 920 925
Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro
930 935 940
Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr
945 950 955 960
Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe
965 970 975
Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu
980 985 990
Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala
995 1000 1005
Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile
1010 1015 1020
Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val
1025 1030 1035
Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser
1040 1045 1050
Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys
1055 1060 1065
Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr
1070 1075 1080
Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser
1085 1090 1095
Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr
1100 1105 1110
Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly
1115 1120 1125
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu
1130 1135 1140
Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr
1145 1150 1155
Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu
1160 1165 1170
Glu
<210> 5
<211> 2652
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
agattacaag gtagtgaatt gtgacatgta ttcgttccta tccgatccgt cgtttttgag 60
cactaggtgc ggtcactgtg acgcgtggac ttggcttcgc ccactgccat cgtggaccca 120
cgtcatcagc aagtgtccat atccaccacc cgacccgacg accgcttgcc gtccgatccg 180
tgtgctcccg agggcaagga tggcatttcg ccacgcgaga tatttttcgg tggcctgcac 240
aggccggcag tgcagcggcc aaaacgaggt caggtcagtc acgctgggcc ccgcctcacg 300
ctcccgtcct gctccgggtc ccaacaaagc cgtccccggg aggtgctcgt gtgctcgtag 360
cgcggtggcg accccgatgc cccgcatatt ccactgggcg tccgcgccgt cggatgggat 420
caggacggcc gcggcggccc cgcgctcggc tataaagacg ctgcggggga cgcattccct 480
ctccgtgctt tcttagaggt gggttggctt ctcctccccc tccggttcgg gttcgggttc 540
gtgaggttct ccggggttcg ggttcgtggg tgagcggatc gagactagta tggagaacaa 600
catccagaac cagtgcgtcc cctacaactg cctcaacaac cccgaggtcg agatcctcaa 660
cgaggagcgc tccaccggcc gcctccccct cgacatctcc ctctccctca cccgcttcct 720
cctctccgag ttcgtccccg gcgtcggcgt cgccttcggc ctcttcgacc tcatctgggg 780
cttcatcacc ccctccgact ggtccctctt cctcctccag atcgagcagc tcatcgagca 840
gcgcatcgag accctcgagc gcaaccgcgc catcaccacc ctccgcggcc tcgccgactc 900
ctacgagatt tacatcgagg ccctccgcga gtgggaggcc aaccccaaca acgcccagct 960
ccgcgaggac gtccgcatcc gcttcgccaa caccgacgac gccctcatca ccgccatcaa 1020
caacttcacc ctcacctcct tcgagatccc cctcctctcc gtctacgtcc aggccgccaa 1080
cctccacctc tccctcctcc gcgacgccgt ctccttcggc cagggctggg gcctcgacat 1140
cgccaccgtc aacaaccact acaaccgcct catcaacctc atccaccgct acaccaagca 1200
ctgcctcgac acctacaacc agggcctcga gaacctccgc ggcaccaaca cccgccagtg 1260
ggcccgcttc aaccagttcc gccgcgacct caccctcacc gtcctcgaca tcgtcgccct 1320
cttccccaac tacgacgtcc gcacctaccc catccagacc tcctcccagc tcacccgcga 1380
gatttacacc tcctccgtca tcgaggactc ccccgtctcc gccaacatcc ccaacggctt 1440
caaccgcgcc gagttcggcg tccgcccccc ccacctcatg gacttcatga actccctctt 1500
cgtcaccgcc gagaccgtcc gctcccagac cgtctggggc ggccacctcg tctcctcccg 1560
caacaccgcc ggcaaccgca tcaacttccc ctcctacggc gtcttcaacc ccggcggcgc 1620
catctggatc gccgacgagg acccccgccc cttctaccgc accctctccg accccgtctt 1680
cgtccgcggc ggcttcggca acccccacta cgtcctcggc ctccgcggcg tcgccttcca 1740
gcagaccggc accaaccaca cccgcacctt ccgcaactcc ggcaccatcg actccctcga 1800
cgagatcccc ccccaggaca actccggcgc cccctggaac gactactccc acgtcctcaa 1860
ccacgtcacc ttcgtccgct ggcccggcga gatttccggc tccgactcct ggcgcgcccc 1920
catgttctcc tggacccacc gctccgccac ccccaccaac accatcgacc ccgagcgcat 1980
cacccagatc cccctcgtca aggcccacac cctccagtcc ggcaccaccg tcgtccgcgg 2040
ccccggcttc accggcggcg acatcctccg ccgcacctcc ggcggcccct tcgcctacac 2100
catcgtcaac atcaacggcc agctccccca gcgctaccgc gcccgcatcc gctacgcctc 2160
caccaccaac ctccgcatct acgtcaccgt cgccggcgag cgcatcttcg ccggccagtt 2220
caacaagact atggacaccg gcgaccccct caccttccag tccttctcct acgccaccat 2280
caacaccgcc ttcaccttcc ccatgtccca gtcctccttc accgtcggcg ccgacacctt 2340
ctcctccggc aacgaggtct acatcgaccg cttcgagctg atccccgtct gaggtcaccc 2400
gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 2460
ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 2520
cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 2580
tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 2640
tactagatcg gg 2652

Claims (10)

1.mCry1F albumen, be b1) or b2) or b3)：
B1) amino acid sequence is the protein shown in sequence 2 in sequence table；
B2) the fused protein that the N-terminal of the protein in sequence table shown in sequence 2 or/and C-terminal connection label obtain；
B3) by substitution of the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing And/or the protein related to plant anti-insect that addition obtains.
2. encode the nucleic acid molecules of mCry1F albumen described in claim 1.
3. nucleic acid molecules as claimed in claim 2, it is characterised in that：The nucleic acid molecules are following (a1) or (a2) or (a3) Or the DNA molecular shown in (a4)：
(a1) DNA molecular of the code area as shown in sequence 1 in sequence table；
(a2) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table；
(a3) there is 75% or more than 75% homogeneity with (a1) or (a2) nucleotide sequence limited, and encodes claim 1 The DNA molecular of the mCry1F albumen；
(a4) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and encode described in claim 1 The DNA molecular of mCry1F albumen.
4. expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing nucleic acid molecules described in Claims 2 or 3.
5. mCry1F albumen described in claim 1, or, nucleic acid molecules described in Claims 2 or 3, or, contain Claims 2 or 3 The application of the expression cassettes of the nucleic acid molecules, recombinant vector, recombinant microorganism or transgenic cell line, is e1) or e2) or e3)：E1 plant resistance to insect) is improved；E2 the product with insect resistant effect) is prepared；E3 the transgenosis that insect resistace improves) is cultivated to plant Thing.
6. application as claimed in claim 5, it is characterised in that：The product is medicine.
7. a kind of method for cultivating genetically modified plants, comprises the following steps：The core of mCry1F albumen described in claim 1 will be encoded Acid molecule is imported in recipient plant, obtains genetically modified plants；Compared with the recipient plant, the insect resistace of the genetically modified plants Enhancing.
8. a kind of plant breeding method, comprises the following steps：Increase the content of mCry1F albumen described in claim 1 in plant And/or activity, so as to increase insect resistace.
9. mCry1F albumen as claimed in claim 1, or, the application of claim 5 or 6, or, described in claim 7 or 8 Method, it is characterised in that：Any of the plant is c1) to c5)：C1) monocotyledon；C2) dicotyledon；c3) Grass；C4) corn；C5) corn variety X178.
10. mCry1F albumen as claimed in claim 1, or, the application of claim 5 or 6, or, described in claim 7 or 8 Method, it is characterised in that：The insect resistace is anti-lepidopterous insects.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111606984A (en) * 2020-05-19 2020-09-01 隆平生物技术(海南)有限公司 Plant insect-resistant protein and coding gene and application thereof
CN111995690A (en) * 2020-01-16 2020-11-27 黑龙江大鹏农业有限公司 Artificially synthesized insect-resistant protein mCry1Ia2, and preparation method and application thereof
CN114316015A (en) * 2021-12-03 2022-04-12 河北大学 Insect-resistant protein hRI and coding gene and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011075586A1 (en) * 2009-12-16 2011-06-23 Dow Agrosciences Llc Insecticidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance managements
CN102250923A (en) * 2010-05-19 2011-11-23 复旦大学 cryIAc gene having high lepidopteran pest resistance and application thereof in sugarcane
CN102660560A (en) * 2012-04-26 2012-09-12 河南省农业科学院 Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011075586A1 (en) * 2009-12-16 2011-06-23 Dow Agrosciences Llc Insecticidal protein combinations for controlling fall armyworm and european corn borer, and methods for insect resistance managements
CN102250923A (en) * 2010-05-19 2011-11-23 复旦大学 cryIAc gene having high lepidopteran pest resistance and application thereof in sugarcane
CN102660560A (en) * 2012-04-26 2012-09-12 河南省农业科学院 Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111995690A (en) * 2020-01-16 2020-11-27 黑龙江大鹏农业有限公司 Artificially synthesized insect-resistant protein mCry1Ia2, and preparation method and application thereof
CN111995690B (en) * 2020-01-16 2022-10-11 黑龙江大鹏农业有限公司 Artificially synthesized insect-resistant protein mCry1Ia2 and preparation method and application thereof
CN111606984A (en) * 2020-05-19 2020-09-01 隆平生物技术(海南)有限公司 Plant insect-resistant protein and coding gene and application thereof
CN111606984B (en) * 2020-05-19 2021-08-06 隆平生物技术(海南)有限公司 Plant insect-resistant protein and coding gene and application thereof
CN114316015A (en) * 2021-12-03 2022-04-12 河北大学 Insect-resistant protein hRI and coding gene and application thereof

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