CN109776659A - Application of the cry2Ah-vp gene in anti-armyworm - Google Patents

Abstract

The present invention relates to application of the cry2Ah-vp gene in anti-armyworm.The applicant is inserted into a proline behind the 354th valine of Cry2Ah and therefrom obtains Cry2Ah1-vp mutant as shown in SEQ ID No.1.Cry2Ah-vp gene is obtained after codon optimization, and cry2Ah-vp gene is transferred to corn using agrobacterium-mediated transformation, obtains T₀For transgenic corns.The experimental results showed that cry2Ah-vp gene can be with normal transcription in transgenic corns different tissues, expression quantity highest of the six leaf phase Cry2Ah-vp albumen at each position of corn, the expression quantity of blade is 2.20 μ g/g (fresh weight), and also there is higher expression at tasseling stage and pustulation period each position.Pest-resistant experimental result shows that transgenic corns have good insecticidal activity to armyworm, and armyworm is all dead after connecing worm 3 days；There is apparent growth inhibition effect to corn borer larvae.

Description

Application of the cry2Ah-vp gene in anti-armyworm

Technical field

The present invention relates to field of biotechnology, more particularly to application of the cry2Ah-vp gene in anti-armyworm.

Background technique

Corn is important cereal crops, and is important feed and the raw material of industry, but since the generation of insect pest is beautiful every year The rice underproduction about 7%, up to 20% when serious.Insect-resistant transgenic crops in 1996 start commercial growth, Bt crop it is extensive Effectively preventing pest is planted, reduces the use of insecticide, reduces production cost, to environment, beneficial insect and people and animals Deng [Bates S L, Zhao J Z, Roush R T, Shelton A the M.Insect resistance that has no adverse effects management in GM crops:past,present and future.Nature Biotechnology,2005,23: 57-62.Estruch J J,Carozzi NB,Desai N,Duck N B,Warren G W,Koziel MG.Transgenic plants:an emerging approach to pest control.Nature Biotechnology,1997,15:137- 141.].The gene that insect-resistant transgenic corn mainly converts in the world at present includes Bt cry1Ab gene, such as Mon810 and Bt11； Cry1F gene, such as TC1507；Vip3A gene, such as MIR162；Cry1A.105 (cry1F and cry1Ac heterozygosis) and cry2Ab2 base The MON89034 etc. of cause；The gene for the pest-resistant corn transformation that the country is developing mainly has cry1Ab, cry1Ac, cry1Ab/ [Han Lanlan, Song Fuping, Zhang Jie, Zhao Kuijun thuringiensis insecticidal crystal protein are to bollworm by cry2Aj and cry1Ah etc. Activity analysis Northeast Agricultural University journal, 2008,39 (8): 21-24.Yang F, Kerns D L, Head G P, Leonard B R,Niu Y,Huang F N.Occurrence,distribution,and ear damage of Helicoverpa zea (Lepidoptera:Noctuidae)in mixed plantings of non-Bt and Bt corn containingSmartStaxTM traits.Crop Protection,2014,55:127-132.].Armyworm (Mythimna Separata it is) a kind of common Corn Pests, belongs to Lepidoptera Noctuidae, it is serious to occur in larva period gluttony maize leaf When, blade can be eaten up in a short time, and causing the corn underproduction even to have no harvest, [Jiang Xingfu, Zhang Lei, Cheng Yunxia, Luo Lizhi China armyworm send out Life endangers new feature and trend analysis applied entomology report, 2014,51 (06): 1444-1449.].Existing Bt gene pairs is beautiful Rice snout moth's larva has a good control efficiency, but also need in armyworm prevention and treatment effective anti insect gene [Yang Sujuan, Huang Minzhong, Li Yanqiu, Zhou Zishan, Jiang Xingfu, Gao Jiguo, Cheng Yunxia, Zhang Jie there is the Bt protein screening of insecticidal activity and analysis plant to protect armyworm Shield, 2016,42 (03): 30-35.].Cry2Ah gene be from soil sample mixed collection exempt from culture obtain, Cry2Ah albumen There is weight inhibitory activity to corn borer, has growth inhibitory activity to bollworm, while also having very well to cry1Ac resistant bollworm Growth inhibitory activity [Shu C L, Zhang J T, Chen Gui H, Liang G M, He K L, Crickmore N, Huang D F,Zhang J,Song F P.Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.Journal of Invertebrate Pathology,2013,114:31-33.].It is existing studies have shown that Cry2Ah albumen has weight inhibition to armyworm, but (Liu Chenchen beautiful jade etc., four kinds of Bt albumen are to six kinds of important squama wings without lethal effect The Insecticidal Activity of mesh pest, Chinese biological preventing and treating journal, 2017,33 (6) 774-779).By the 354th figured silk fabrics of Cry2Ah A proline is inserted into behind propylhomoserin and therefrom obtains Cry2Ah1-vp mutant, and there is phase with Cry2Ab on mutated site Same carbon skeleton.After two genes are transferred to tobacco plant respectively, bollworm are carried out and cry1Ac resistant bollworm desinsection is living Property test, the results showed that Cry2Ah-vp compared with Cry2Ah have higher insecticidal activity [Li S Y, Wang Z Y, Zhou Y Y,Li C H,Wang G P,Wang H,Zhang J,Liang G M,Lang Z H.Expression of cry2Ah1and two domain II mutants in transgenic tobacco confers high resistance to susceptible and Cry1Ac-resistant cotton bollworm.Scientific Reports,2018,8(1):508.].The applicant is " artificial synthesized to the high virulence desinsection base of lepidoptera pest in patent application Because and application " to mcry2Ah-vp gene order and its turn the application of tobacco and carried out in detail in (application number 2017101451666) Thin explanation.

Summary of the invention

For the demand in above-mentioned field, the present invention provides application of the cry2Ah-vp gene in anti-armyworm, by Bt Cry2Ah-vp gene carries out codon optimization, is transferred in corn, and experiment shows that cry2Ah-vp gene has fabulous anti-armyworm Effect.

PROTEIN C ry2Ah-vp is applied in anti-armyworm, the amino acid sequence of the PROTEIN C ry2Ah-vp such as SEQ ID Shown in No.1.

The application is that the carrier for the gene for containing coding PROTEIN C ry2Ah-vp is transferred in corn, makes it have anti-armyworm Characteristic.

The gene order of the coding PROTEIN C ry2Ah-vp is as shown in SEQ ID No.2.

The carrier is pC2HBvp, and skeleton carrier is pCAMBIA3300.

The structure of the carrier pC2HBvp is as shown in Figure 2.

The Cry2Ah-vp albumen that the applicant is optimized by early period is shown in SEQ ID No.1, i.e., by Cry2Ah (SEQ ID No.3 a proline is inserted into behind the 354th valine) and therefrom obtains Cry2Ah1-vp mutant (see patent application " artificial synthesized to the high virulence killing gene of lepidoptera pest and application " (application number 2017101451666)), and the phase is according to jade The codon preference of rice gene consider simultaneously tRNA transhipment it is hungry to Bt cry2Ah-vp gene carry out codon optimization (see SEQ ID No.2), the gene constructed plant expression vector pC2HBvp of cry2Ah-vp after optimization is turned using agrobacterium-mediated transformation Enter in corn, obtains T₀For transgenic corns.It is examined in our current research by glufosinate-ammonium screening, PCR identification and destination protein content It surveys, screening has obtained transgenic event VP1-5, and carries out copy number analysis and insect resistace experiment to VP1-5 transgenic corns. Southern blot testing result shows that cry2Ah-vp gene is inserted into Maize genome in the form singly copied；qRT- PCR testing result shows that cry2Ah-vp gene can be with normal transcription in VP1-5 transgenic corns different tissues；Enzyme linked immunological Determining adsorption (ELISA) testing result shows, expression quantity highest of the six leaf phase Cry2Ah-vp albumen at each position of corn, leaf The expression quantity of piece is 2.20 μ g/g FW (fresh weight), and also there is higher expression at tasseling stage and pustulation period each position.Pest-resistant experimental result is aobvious Show that VP1-5 transgenic corns have good insecticidal activity to armyworm, armyworm is all dead after connecing worm 3 days；Have to corn borer larvae Apparent growth inhibition effect.

Armyworm harm seriously affects corn quality and yield, needs new pest-resistant material, Cry2Ah- in its prevention and treatment at present Vp albumen is that have very highly resistance armyworm activated protein after being transformed, and can be expressed at each position of corn, therefore VP1-5 transgenosis is beautiful Rice will be corn breeding for pest resistance and the good germ plasm resource of control of insect, before having commercialization well on corn breeding for pest resistance Scape.

Detailed description of the invention

Fig. 1 plant expression vector pC2HB constructs schematic diagram,

Fig. 2 plant expression vector pC2HB-vp constructs schematic diagram,

Plant growth condition and Bar test strips testing result after Fig. 3 glufosinate-ammonium sprays 7 days,

Fig. 4 pcr amplification product, wherein A be cry2Ah-vp, B bar,

M:marker in figure；CK+: positive control；1-5:vp1-5 material；CK-: Zheng 58 compares；0: blank control；

The Southern blot of Fig. 5 VP1-5 transgenic event is detected,

Wherein M:DNA marker；+: positive control；III digestion Post genome of H:Hind；B:BamHI digestion Post genome；E: EcoRI digestion Post genome；S:SacI digestion Post genome；: negative control；

The rna expression analysis of cry2Ah-vp in Fig. 6 different times and tissue,

The expression quantity of Cry2Ah-vp albumen in Fig. 7 different times and tissue,

Indoor biometrics at Fig. 83 days as a result,

Wherein: being VP1-5 blade in figure A, C；Scheme to be 58 blade of Zheng in B, D；Scheming A, B is armyworm processing group；Scheming C, D is jade Rice snout moth's larva processing group,

Corn borer upgrowth situation at Fig. 9 indoor biometrics 3 days,

Wherein: being vp1-5 blade in figure A；Scheme to be 58 blade of Zheng in B,

Figure 10 Field inoculation armyworm larva is raw survey as a result,

Wherein six leaf phase worm of A:vp1-5 is surveyed；B: 58 6 leaf phase worm of Zheng is surveyed.

Specific embodiment

The present invention will be further described in detail below with reference to the embodiments.

Embodiment 1

1.1 material

Vegetable material turns Bt cry2Ah gene pest-resistant corn material (being saved by this laboratory), corn inbred line Zheng 58.

Selected insect source armyworm, egg stage of Ostrinia furnacalis are purchased from Beijing Mei Yan agricultural science and technology Co., Ltd.

The Zhejiang limited public affairs of agriculture bioscience forever are purchased from for examination pesticide glufosinate-ammonium (aqua, active constituent content: 200 grams per liters) Department.

BAR Rapid detection test strip is purchased from Beijing Ao Chuanjinbiao biotech firm.

1.2 transgenic corns

1.2.1 the building of plant expression vector

In order to which the mcry2Ah (SEQ ID No.4) of transformation and mcry2Ah-vp (SEQ ID No.2) gene are transferred to corn In, construct two plant expression vectors.The skeleton carrier of plant expression vector is commercial vectors pCAMBIA3300, Riddled basins phosphinothricin (PPT) is had existed on pCAMBIA3300 carrier, can be used for glufosinate-ammonium sieve Choosing.The both ends of artificial synthesized mcry2Ah and mcry2Ah-vp gene have increased separately multiple cloning sites, and 5 ' ends are contained KpnI, XhoI and EcoRI are contained in HindIII, XbaI, XmaI, SmaI and BamHI, 3 ' ends, and the gene constructed of synthesis arrives pUC57 Carrier, being referred to as pUm2Ah and pUm2Ah-vp, (plasmid is stored in Biological Technology institute, Chinese Academy of Agricultural Sciences's Lang Zhihong class Topic group can be provided to the public).Two genes will be connected in respectively from the ubiquitin promoter of corn and NOS terminator 5 ' end and 3 ' end, building obtain pUbim2AhN and pUbim2AhvpN (plasmid is stored in Chinese Academy of Agricultural Sciences's biotechnology Lang Zhihong seminar, research institute can provide to the public), with Hind III and EcoR I double digestion pUbim2AhN and PUbim2AhvpN is connected to the pCAMBIA3300 with same digestion, and the carrier for constructing acquisition is known as pC2HB and pC2HBvp (is carried Body building schematic diagram is shown in Fig. 1, Fig. 2).

1.2.2 the acquisition of transgenic corns

With Agrobacterium-mediated Transformation method by pC2HB the and pC2HBvp maize transformation of building, method for transformation is Agrobacterium-mediated Transformation method, Corn Agrobacterium-mediated Transformation method has been that maize immature embryos are placed in by conventional method for transformation first using maize immature embryos as receptor tissue The infecting in culture medium for Agrobacterium EHA105 of pC2HB containing plant expression vector and pC2HBvp is infected, under 26 DEG C of dark conditions It is cultivated on base in co-culturing, then goes to Fiber differentiation on recovery media and go out callus；Again by callus be transferred to containing It is cultivated on the screening and culturing medium of 1mM glufosinate-ammonium；It is then transferred to light-exposed differentiation on regeneration culture medium, green bud point occurs, it will more Hurting block cutting separates green bud point and is transferred on regeneration culture medium to cultivate, and when stem is extended to 3-4cm, is shifted Root induction on to regeneration culture medium；After plant grows sturdy and well developed root system, plantation to flowerpot or greenhouse or Field.By corn transformation, acquisition turns 10 plants of pC2HB carrier plant, turns 31 plants of pC2HBvp carrier plant.Pass through field grass ammonium Phosphine screening, turns pC2HBvp carrier plant VP1-5 resistance.

The culture medium:

Infect culture solution: N6 salt and N6 vitamin (Chu etc., Science Sinica, 1975,18:659-668), 1.5mg/L 2,4-D, 0.7/L g proline, 68.4g/L sucrose, 36g/L glucose (pH 5.2), filtration sterilization are stored up in 4 DEG C It deposits；Using the acetosyringone (AS) of preceding addition filtration sterilization, final concentration of 100 μM；

Co-culture medium: N6 salt and N6 vitamin, 1.5mg/L 2,4-D, 0.7g/L proline, 30g/L sucrose, 3g/L The silver nitrate of the final concentration of 0.85mg/L through filtration sterilization is added after high pressure sterilization for plant gel (pH 5.8), 100 μM The cysteine of AS, 300mg/L；

Recovery media: N6 salt and N6 vitamin, 1.5mg/L 2,4-D, 0.7g/L proline, 30g/L sucrose, 0.5g/L MES, 4g/L plant gel (pH 5.8), be added after high pressure sterilization the final concentration of 0.85mg/L through filtration sterilization silver nitrate and The carbenicillin of 200mg/L；

Screening and culturing medium: selective agent 1mM glufosinate-ammonium is added in recovery media；

Regeneration culture medium: MS salt and MS vitamin, 30g/L sucrose, 100mg/L inositol, 3g/L plant gel (pH5.8), High pressure sterilization.

The maize immature embryos are the rataria of the 1.2mm long of fresh removing.

100 μM of acetosyringones are added in the Agrobacterium bacterium solution.

1.2.3 turn the detection of pC2HBvp carrier plant

1.2.3.1 glufosinate resistance screening and the detection of Bar test strips

Field planting T₁For corn, glufosinate resistance screening, the glufosinate-ammonium aqueous solution that compound concentration is 2 ‰, in jade are carried out Rice four leaf stage is sprayed, and observing effect after 7 days counts number of live vaccine.It is the positive in order to further determine survival plant, then uses BAR Rapid detection test strip is detected, 1 ㎝ of clip²The maize leaf of left and right size is put into the 1.5mL's equipped with 0.5mL water It in EP pipe, is ground using tip glass bar, by test strips insertion EP pipe, waits 2min or so, observe test strips nature controlling line And detection line.

1.2.3.2 the PCR detection of transformed plant

Genomic DNA [Porebski S, Bailey L G, Baum are isolated from maize leaf tissue using CTAB method B R.Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components.Plant Molecular Biology Reporter, 1997,15:8-15.].PCR detection is carried out respectively with the specific primer of Cry2Ah-vp and bar, and primer sequence is shown in Table 1. The detection of Cry2Ah-vp is with the DNA profiling containing 100ng in 20 μ L reaction mixtures, and each 0.1 μM of primer, 10 2 × taq of μ L MasterMix (Dye), ddH2O complement to 20 μ L.Reaction condition are as follows: 94 DEG C of 5min, (94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 60s) 30 circulations, 72 DEG C of 5min.The detection of Bar is with the DNA profiling containing 100ng in 20 μ L reaction mixtures, and each 0.1 μM of primer, 10 μ 2 × taq of L MasterMix (Dye), ddH2O complement to 20 μ L.Reaction condition are as follows: 94 DEG C of 5min, (94 DEG C of 30s, 56 DEG C 30s, 72 DEG C of 20s) 30 circulations, 72 DEG C of 5min.PCR product passes through the agarose gel electrophoresis of 1% (W/V), uses Doc^TM XR+ (Bio-Rad, the U.S.) gel imager is observed and is stored.

1 PCR the primer sequence of table

1.2.3.3Southern blot is analyzed

50 μ g corn gene group DNAs carry out digestion with Hind III, BamHI, EcoRI, SacI respectively, and in 0.7% (W/V) 30V electrophoresed overnight on Ago-Gel, is transferred to HyBord^TM- N+ nylon membrane (GE, Britain).With cry2Ah-vp gene 862bp segment is probe, and probe preparation uses forward primer 5'-GAGTGGATGGAGTGGAAG-3', reverse primer 5'- CGATGTTTGGGAAGGTCT-3' amplifies purpose band, gel extraction.DNA probe PCR DIG Probe Synthesis Kit (Roche, Germany) is labeled as DIG-11-dUTP, according to digoxin DNA marker and detection kit II (Roche, Germany) Scheme carry out.Image analysis is carried out with AI600 (GE, the U.S.) robotics luminescence imaging analysis system.

1.2.3.4 fluorescence quantitative RT-RCR is analyzed

Cry2Ah-vp gene is detected in the expression of transcriptional level using quantitative fluorescent PCR (quantitative RT-PCR) Difference.Tassel, lobus cardiacus, flat leaves, bract, filigree, cob, seed, the stem, root tissue at corn heading stage and pustulation period are taken, is used Trizol (TransGen, China) extracts total serum IgE from 100mg tissue.It is reversed using reverse transcription reagent box (Thermo, the U.S.) Record synthesis cDNA, quantitative RT-PCR use equipment3Real-Time PCR System (ThermoFisher Scientific, the U.S.) carries out.Using SYBR GreenI quantitative PCR kit (Roche, Switzerland), It is the transcription of cry2Ah-vp to be detected with primer vp1-5-qF1/R1 respectively, with primer actin- with reference to gene with actin gene QF1/R1 detects the transcription of actin, and primer sequence is shown in Table 2.In 20 μ L reaction mixtures containing 10 μ L 2 × Top Green qPCR SuperMix (TransGen, China), each 0.2 μM of primer, 1 μ L of cDNA template.Reaction condition is as follows: 95 DEG C of 30s initial denaturations, 95 DEG C of 5s and 60 DEG C of 34s carry out 40 circulations, then carry out solubility curve analysis.

2 qPCR the primer sequence of table

1.2.3.4 enzyme linked immunosorbent assay (ELISA) (ELISA)

Three strains of VP1-5 material different growing stage are acquired, each strain takes 3 plants of positive samples, respectively six leaves Phase, the tassel at heading stage and pustulation period, lobus cardiacus, flat leaves, bract, filigree, cob, seed, stem, root tissue, are exempted from using enzyme-linked The Cry2Ah-vp of epidemic disease determining adsorption (enzyme-linked immunosorbent assay, ELISA) measurement transgenic plant Expressing quantity.According to Cry2A immue quantitative detection reagent box (Envirologix, the U.S.) specification, by 100mg maize leaf group Milling and extracting Cry2Ah-vp albumen is knitted, enzyme linked immunosorbent assay (ELISA) is carried out.Using microplate reader BioTek ELx808 (BioTek, The U.S.) at wavelength 450nm measure OD, repeat read 3 times as a result, calculate mean value, according to Cry2Ah-vp protein standard substance OD value is drawn standard curve, is analyzed using Excel and SPASS software data.

1.2.3.5 bioassay

When corn growth is to six leaf phases, indoor and field bioassay is carried out.Measure transgenic corn plant to armyworm, The insecticidal activity of corn borer larvae.The indoor bioassay of armyworm and corn borer larvae uses 24 hole culture dishes, takes 1 ㎝²Size Maize leaf be placed in 24 holes, a newly hatched larvae is put into each hole, records the quantity of living worm, dead worm, record 7 daily It.Every kind of processing carries out three secondary pollutant repetitions, and the blade of 58 material of inbred Zheng is as control.Ultimate mortality table is shown as dead larvae The ratio (%) of the total larva of Zhan.The raw survey in field will just incubate armyworm larva respectively and just incubate corn borer larvae and use at the six leaf phase of corn Writing brush is inoculated into different plants, and 58 plant of Zheng compares, and can be counted after connecing worm 2 weeks, and observation plant is endangered feelings Condition simultaneously photographs to record.

1.2.4 turn pC2HBvp carrier plant testing result

1.2.4.1 the glufosinate-ammonium screening of transformed plant is detected with test strips

Glufosinate-ammonium screening is carried out to the maize transformation plant of field planting, 2 ‰ glufosinate-ammonium sprays rear 7 days observation plant, It converting positive plant energy normal growth (A in Fig. 3), negative plant then turns to be yellow, it wilts, dead situation (B in Fig. 3).It is right The positive plant of acquisition further uses Bar test strips to detect, and band is nature controlling line in test strips, and lower band is detection line, and two Band some is positive plant (C in Fig. 3).Test strips A-E is the testing result of VP1-5 material in C in Fig. 3, is shown as sun Property, F is Zheng 58, is detected as feminine gender.

1.2.4.2 the PCR of maize transformation plant is identified

CTAB method extract maize leaf genome using PCR detection transgenic plant (Fig. 4), the results show that in Fig. 4 A with Cry2Ah-vp is primer, carries out PCR by template of VP1-5 material genomic DNA, amplified production length is 1353bp.B in Fig. 4 In using bar as primer, using VP1-5 material genomic DNA as template carry out PCR, amplified production length be 495bp.Show Cry2Ah-vp gene and bar gene all normal expression in VP1-5 material.

1.2.4.3 Southern blot hybridizes

By the genomic DNA of transgenic corns material VP1-5 use respectively restriction enzyme Hind III, BamHI, EcoRI, SacI carry out digestion, carry out Southern blot hybridization using digoxin kit, the results showed that, in transgenosis jade In rice plant VP1-5 material, cry2Ah-vp gene is integrated into Maize genome (Fig. 5), adjoining tree in the form singly copied Hybridization signal (Fig. 5) is not detected in 58 material of Zheng.Southern blot results of hybridization confirms foreign gene by successful conversion simultaneously It is integrated into Maize genome.

1.2.4.4 transcription analysis of the cry2Ah-vp gene in transgenic corns

VP1-5 transgenic plant is detected using transcriptional level of the qRT-PCR method to cry2Ah-vp.Have chosen jade The rice different tissues such as tasseling stage and the root of pustulation period, stem, leaf, filigree, cob are with reference to gene, cry2Ah- with actin gene Vp gene is purpose gene, and each sample repeats three times.Using 2^-△△CTMethod analyzes data.QRT-PCR the result shows that, Cry2Ah-vp gene is correctly transcribed in transgenic corn plant is respectively organized, and different times different parts transgenic plant turns Record level existence difference, the transcriptional level highest (Fig. 6) in tasseling stage and the blade and filigree of pustulation period.

1.2.4.5 in transgenic corns Cry2Ah-vp albumen quantitative analysis

ELISA method carries out the quantitative detection of Cry2Ah-vp protein expression to VP1-5 transgenic corns, when difference is grown Three strains of phase, each strain take 3 plants of samples, and sample carries out 3 reprocessings.The hero of six leaf phases, heading stage and pustulation period Fringe, lobus cardiacus, flat leaves, bract, filigree, cob, seed, stem, root tissue quantitative analysis results are shown in the Cry2Ah- of Fig. 7: six leaf phases Expression quantity of the vp albumen in root, stem, leaf is all very high, and expression quantity is up to 2.20 μ gg in blade^-1FW (fresh weight), tasseling stage Expression quantity of the Cry2Ah-vp albumen at each position of corn is higher than pustulation period each position expression quantity, and conventional kind of material Zheng 58 is equal Cry2Ah albumen is not detected.There is the expression of Cry2Ah-vp albumen in each tissue site of different times, it is ensured that beautiful Rice is caused harm in growth course from target pest, and corn yield loss is reduced.

1.2.4.6 bioassay

Turn cry2Ah-vp gene corn to the insecticidal activity of armyworm and corn borer, to VP1-5 transgenic corns to study Twig and shoot pest is carried out with ordinary maize Zheng 58.Indoor biometrics are as the result is shown: compared with the control group, transgenic corn plant is connecing Kind shows apparent resistance (A in Fig. 8) to armyworm after newly hatched larvae three days, shows apparent growth inhibition to corn borer and makees With.58 blade of control group Zheng is impaired serious, most of larvae alives, and polypide is larger flushes (B, D in Fig. 8), opposite institute Have the leaf damage of genetically modified plants slight, only a small number of needle prick shape worm channels, most dead larvaes, the growth of survival larva by Inhibit (A, C in Fig. 8) to obvious.With whole death in armyworm larva three days that VP1-5 plant is food, corrected mortality is 96.82% ± 4.40%；With corrected mortality when VP1-5 plant is food corn borer larvae three days be 25.00% ± 15.59% (table 3).Although the death rate of corn borer is lower than armyworm, corn borer polypide is significantly less than control group, and vigor is low, It hardly feeds, cannot further complete growth and development (Fig. 9).Simultaneously to turn pC2HB carrier plant 2AhL246,2AhL247 and 2AhL248 has carried out insect resistace detection, and the corrected mortality of armyworm is lower than VP1-5 (table 3).Resist to pC2HB carrier plant is turned Worm detection effect is consistent substantially with " Insecticidal Activity of four kinds of Bt albumen to six kinds of important lepidoptera pests ".

The corrected mortality of armyworm and corn borer statisticallys analyze at table 33 days

When corn growth is to the 6-8 leaf phase, inoculation just incubates armyworm larva in transgenic line VP1-5 and conventional kind of material Zheng 58, it connects worm and observes plant strain growth situation, the smooth no hole (Figure 10, A) in VP1-5 material blade surface, 58 material leaf of Zheng after two weeks Piece is incised and worm channel (Figure 10, B).

Experiment shows that cry2Ah-vp is a Bt killing gene to lepidoptera pest armyworm with insecticidal activity；Turn Cry2Ah-vp gene corn VP1-5 has high insecticidal activity to armyworm, can be used for pest-resistant corn breeding, before having good industrialization Scape.

Sequence table

<110>Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>application of the cry2Ah-vp gene in anti-armyworm

<141> 2019-03-14

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<170> SIPOSequenceListing 1.0

<210> 1

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Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr

545 550 555 560

Ile Asn Gly Arg Val Tyr Thr Ala Ser Asn Val Asn Thr Thr Thr Asn

565 570 575

Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile

580 585 590

Gly Asn Val Val Ala Ser Asp Asn Thr Asn Val Pro Leu Asp Ile Asn

595 600 605

Val Thr Leu Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile Met Phe

610 615 620

Val Pro Thr Asn Leu Pro Pro Leu Tyr

625 630

<210> 2

<211> 1902

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

atgaacaacg tcctcaacag cggcagggct acgaacggcg acgcgtacaa cgtggtcgcc 60

cacgacccct tctccttcca gcacaagagc ctcgacacca tccaggagga gtggatggag 120

tggaagaagg acaaccacat cctctacgtg gacccgatcg tgggcaccgt cgcctccttc 180

ctcctgaaga aggtcggcag cctcgtcgag aagcgcatcc tctccgagct gaggaacctc 240

atcttcccgt ccggcagcac gaacctcatg caggacatcc tgcgcgagac cgagaagttc 300

ctgaaccagc gcctcaacac ggacaccctg gctagggtca acgctgagct gaccggcctc 360

caggctaacg tcgaggagtt caaccgccag gtggacaact tcctcaaccc gaacaggaac 420

gccgtccccc tgtccatcac gtccagcgtg aacaccatgc agcagctctt cctgaacagg 480

ctcccccagt tccagatgca gggctaccag ctcctgctcc tgccactgtt cgctcaggct 540

gcgaacctcc acctgtcctt catccgcgac gtgatcctga acgctgacga gtggggcatc 600

agcgctgcta cgctcaggac ctaccagaac cacctgcgca actacacgag ggagtactcc 660

aactactgca tcaccacgta ccagacggcg ttccgcggcc tgaacaccag gctccacgac 720

atgctggagt tccgcaccta catgttcctc aacgtgttcg agtatgtgtc catctggagc 780

ctgttcaagt accagagcct cctggtctcc agcggcgcca acctctacgc ttccggcagc 840

ggcccacagc agacgcagtc cttcaccagc caggactggc cgttcctgta ctccctcttc 900

caggtgaaca gcaactacgt cctcaacggc ttctccggcg ctaggctgac gcagaccttc 960

ccaaacatcg tgggcctgcc aggcaccacg accacgcacg cgctcctggc tgctagggtg 1020

aactactccg gcggcgtctc cagcggcgac atcggcgctg tgcccttcaa ccagaacttc 1080

tcctgcagca cgttcctccc accactcctg accccattcg tccgcagctg gctggactcc 1140

ggcagcgaca ggggcggcat caacacggtg accaactggc agacggagag cttcgagacc 1200

acgctcggcc tgcgctccgg cgccttcacc gcgaggggca acagcaacta cttcccggac 1260

tacttcatcc gcaacatctc cggcgtgccc ctcgtggtca ggaacgagga cctccgcagg 1320

ccgctgcact acaaccagat caggaacatc gagtccccaa gcggcacccc aggcggcctc 1380

agggcgtaca tggtgagcgt ccataacagg aagaacaaca tctacgcggt ccacgagaac 1440

ggcacgatga tccacctggc cccggaggac tacacgggct tcaccatctc ccccatccac 1500

gcgacccagg tgaacaacca gacgcgcacc ttcatcagcg agaagttcgg caaccagggc 1560

gactccctca ggttcgagca gagcaacacc acggctaggt acaccctgag gggcaacggc 1620

aactcctaca acctctacct gcgcgtctcc agcatcggca acagcacgat ccgcgtgacc 1680

atcaacggca gggtctacac cgcctccaac gtgaacacca cgaccaacaa cgacggcgtg 1740

aacgacaacg gcgcgaggtt ctccgacatc aacatcggca acgtggtcgc cagcgacaac 1800

acgaacgtcc ccctcgacat caacgtgacg ctgaacagcg gcacccagtt cgagctgatg 1860

aacatcatgt tcgtgccgac caacctgccg cccctctact ga 1902

<210> 3

<211> 632

<212> PRT

<213>bacillus thuringiensis (Bacillus thuringiensis)

<400> 3

Met Asn Asn Val Leu Asn Ser Gly Arg Ala Thr Asn Gly Asp Ala Tyr

1 5 10 15

Asn Val Val Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu Asp

20 25 30

Thr Ile Gln Glu Glu Trp Met Glu Trp Lys Lys Asp Asn His Ile Leu

35 40 45

Tyr Val Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys Lys

50 55 60

Val Gly Ser Leu Val Glu Lys Arg Ile Leu Ser Glu Leu Arg Asn Leu

65 70 75 80

Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu

85 90 95

Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg

100 105 110

Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe Asn

115 120 125

Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro Leu

130 135 140

Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg

145 150 155 160

Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu

165 170 175

Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val Ile

180 185 190

Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr

195 200 205

Gln Asn His Leu Arg Asn Tyr Thr Arg Glu Tyr Ser Asn Tyr Cys Ile

210 215 220

Thr Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp

225 230 235 240

Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val

245 250 255

Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly

260 265 270

Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe

275 280 285

Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser

290 295 300

Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Thr Gln Thr Phe

305 310 315 320

Pro Asn Ile Val Gly Leu Pro Gly Thr Thr Thr Thr His Ala Leu Leu

325 330 335

Ala Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Asp Ile Gly

340 345 350

Ala Val Phe Asn Gln Asn Phe Ser Cys Ser Thr Phe Leu Pro Pro Leu

355 360 365

Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg Gly

370 375 380

Gly Ile Asn Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Thr Thr

385 390 395 400

Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn Tyr

405 410 415

Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val Val

420 425 430

Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Gln Ile Arg Asn

435 440 445

Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Leu Arg Ala Tyr Met Val

450 455 460

Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Val His Glu Asn Gly

465 470 475 480

Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile Ser

485 490 495

Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile Ser

500 505 510

Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Ser Asn

515 520 525

Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn Leu

530 535 540

Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr Ile

545 550 555 560

Asn Gly Arg Val Tyr Thr Ala Ser Asn Val Asn Thr Thr Thr Asn Asn

565 570 575

Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile Gly

580 585 590

Asn Val Val Ala Ser Asp Asn Thr Asn Val Pro Leu Asp Ile Asn Val

595 600 605

Thr Leu Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile Met Phe Val

610 615 620

Pro Thr Asn Leu Pro Pro Leu Tyr

625 630

<210> 4

<211> 1899

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

atgaacaacg tcctcaacag cggcagggct acgaacggcg acgcgtacaa cgtggtcgcc 60

cacgacccct tctccttcca gcacaagagc ctcgacacca tccaggagga gtggatggag 120

tggaagaagg acaaccacat cctctacgtg gacccgatcg tgggcaccgt cgcctccttc 180

ctcctgaaga aggtcggcag cctcgtcgag aagcgcatcc tctccgagct gaggaacctc 240

atcttcccgt ccggcagcac gaacctcatg caggacatcc tgcgcgagac cgagaagttc 300

ctgaaccagc gcctcaacac ggacaccctg gctagggtca acgctgagct gaccggcctc 360

caggctaacg tcgaggagtt caaccgccag gtggacaact tcctcaaccc gaacaggaac 420

gccgtccccc tgtccatcac gtccagcgtg aacaccatgc agcagctctt cctgaacagg 480

ctcccccagt tccagatgca gggctaccag ctcctgctcc tgccactgtt cgctcaggct 540

gcgaacctcc acctgtcctt catccgcgac gtgatcctga acgctgacga gtggggcatc 600

agcgctgcta cgctcaggac ctaccagaac cacctgcgca actacacgag ggagtactcc 660

aactactgca tcaccacgta ccagacggcg ttccgcggcc tgaacaccag gctccacgac 720

atgctggagt tccgcaccta catgttcctc aacgtgttcg agtatgtgtc catctggagc 780

ctgttcaagt accagagcct cctggtctcc agcggcgcca acctctacgc ttccggcagc 840

ggcccacagc agacgcagtc cttcaccagc caggactggc cgttcctgta ctccctcttc 900

caggtgaaca gcaactacgt cctcaacggc ttctccggcg ctaggctgac gcagaccttc 960

ccaaacatcg tgggcctgcc aggcaccacg accacgcacg cgctcctggc tgctagggtg 1020

aactactccg gcggcgtctc cagcggcgac atcggcgctg tgttcaacca gaacttctcc 1080

tgcagcacgt tcctcccacc actcctgacc ccattcgtcc gcagctggct ggactccggc 1140

agcgacaggg gcggcatcaa cacggtgacc aactggcaga cggagagctt cgagaccacg 1200

ctcggcctgc gctccggcgc cttcaccgcg aggggcaaca gcaactactt cccggactac 1260

ttcatccgca acatctccgg cgtgcccctc gtggtcagga acgaggacct ccgcaggccg 1320

ctgcactaca accagatcag gaacatcgag tccccaagcg gcaccccagg cggcctcagg 1380

gcgtacatgg tgagcgtcca taacaggaag aacaacatct acgcggtcca cgagaacggc 1440

acgatgatcc acctggcccc ggaggactac acgggcttca ccatctcccc catccacgcg 1500

acccaggtga acaaccagac gcgcaccttc atcagcgaga agttcggcaa ccagggcgac 1560

tccctcaggt tcgagcagag caacaccacg gctaggtaca ccctgagggg caacggcaac 1620

tcctacaacc tctacctgcg cgtctccagc atcggcaaca gcacgatccg cgtgaccatc 1680

aacggcaggg tctacaccgc ctccaacgtg aacaccacga ccaacaacga cggcgtgaac 1740

gacaacggcg cgaggttctc cgacatcaac atcggcaacg tggtcgccag cgacaacacg 1800

aacgtccccc tcgacatcaa cgtgacgctg aacagcggca cccagttcga gctgatgaac 1860

atcatgttcg tgccgaccaa cctgccgccc ctctactga 1899

Claims (5)

1. PROTEIN C ry2Ah-vp is applied in anti-armyworm, the amino acid sequence of the PROTEIN C ry2Ah-vp such as SEQ ID No.1 It is shown.

2. applying according to claim 1, it is that the carrier for the gene for containing coding PROTEIN C ry2Ah-vp is transferred in corn, makes Its characteristic with anti-armyworm.

3. applying according to claim 2, the gene order of the coding PROTEIN C ry2Ah-vp is as shown in SEQ ID No.2.

4. applying according to claim 3, the carrier is pC2HBvp, and skeleton carrier is pCAMBIA3300.

5. applying according to claim 4, the structure of the carrier pC2HBvp is as shown in Figure 2.