CN104151408A - Bacillus thuringiensis vegetative-period insecticidal protein Vip3DAa, and coding gene and applications thereof - Google Patents
Bacillus thuringiensis vegetative-period insecticidal protein Vip3DAa, and coding gene and applications thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- 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 a bacillus thuringiensis vegetative-period insecticidal protein Vip3DAa, and a coding gene thereof and applications thereof. The bacillus thuringiensis vegetative insecticidal protein has an amino acid sequence represented by SEQ ID No.1, and a coding gene base sequence is represented by SEQ ID No.2. The applications are the application of the bacillus thuringiensis vegetative insecticidal protein in preparing an insecticide, and the application of the coding gene in cultivating insect-resistant transgenic plants. Compared with prior arts, according to the invention, Vip3D gene and Vip3Aa1 gene are adopted as models, and a chimeric gene Vip3DAa is designed and synthesized. The Vip3DAa protein obtained by chimeric gene Vip3DAa expression has high insecticidal activity against lepidopteran insects and aphids. The chimeric gene Vip3DAa can be highly efficiently expressed in cells of plants such as cotton, corn, canola, soybean, and the like, and can be used for cultivating insect-resistant transgenic plants.
Description
Technical field
The present invention relates to biological prevention field, be specifically related to bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa and encoding gene thereof and application.
Background technology
Diseases and pests of agronomic crop is one of Main Agricultural disaster of China, and it has, and kind is many, impact is large, also often break out the feature of causing disaster, and its occurrence scope and severity often cause heavy losses to Chinese national economy, particularly agriculture production.According to statistics, China due to illness nearly 50,000,000,000 jin of pest injurious loss grain, 1,800 ten thousand tons of all kinds of cash crop every year.Past is in order to prevent and treat the various insect pests of crop, chemical pesticide is widely used, but, on the one hand due to long-term a large amount of agricultural chemicals that use, cause Some Insects to produce certain resistance or resistance, the using dosage that causes agricultural chemicals is increasing year by year or is using brand-new agricultural chemicals; On the other hand, due to the long-term chemical pesticide that excessively uses, cause the environment that the mankind depend on for existence to be polluted in various degree.Therefore, need to find the various disease and pests of better approach prevention and control.Along with the development of biotechnology, utilizing anti insect gene to cultivate insect-resistant transgenic plants is an effective approach.
Bacillus thuringiensis (Bacillus thuringiensis, Bt) be the important donor bacterium of Plant Extrinsic Anti-insect Genes, the parasporal crystal toxin that its toxicity activity produces while being mainly derived from sporulation, be insecticidal crystal protein (insecticidal crystalline protein, ICP), comprise crystal toxin (crystalline toxin, Cry) and lysis toxin (cytolytic toxin, Cyt).ICP, by Cry gene and Cyt genes encoding, has strong toxicity to sensitive insect, and to higher animal and people's nontoxicity.
The insecticidal crystalline gene of having reported has 748 kinds, has much been widely used in insect-resistant transgenic breeding.But because the genetically modified crops of most of commercialization utilization are insecticidal crystal protein class, along with the expansion of these genetically modified crops cultivated areas, insect produces resistance to single insecticidal proteins has become a severe problem.Therefore find new anti insect gene and seem particularly important.
Scientist is through unremitting effort, from the Bt bacterial strain of some vegetative growth stages, separate the non-parasporal crystal insecticidal proteins that obtains having desinsection toxicity, after synthetic, be secreted into outside born of the same parents, Here it is is called as the Vegetative Insecticidal Proteins (vegetative insecticidal protein, Vip) of the Tribactur of the pest-resistant albumen of the s-generation.Vips is mainly divided into Vip1, Vip2, Vip3 and Vip4 Four types totally 108 insecticidal proteins, and wherein 77 albumen belong to Vip3.The insecticidal spectrum of Vip3 is different from ICPs with insecticidal activity, and the former has toxic action to insects such as lepidopteran, Coleoptera and Homopteras, and pest-resistant spectrum is wider.At present, these genes are widely used in pest-resistant transgenic rice, corn and cotton breeding.
But the resource of Vip3 gene is limited after all, and there is larger difference in different intergenic resistances, the gene of bacterium is rich in AT simultaneously, this certainly will affect the expression of gene in plant, corresponding gene is carried out artificial reconstructed, can further improve the utilising efficiency of gene.
Document (Fang et al., Characterization of chimeric Bacillus thuringiensis Vip3toxins, Applied and Environmental Microbiology, 2007,73 (3): 956-961; Fang Jun, Bacillus thuringiensis Vegetative Insecticidal Proteins Vip3 gene and the application in transgenic paddy rice thereof, 2008, Ph D dissertation, Library of Zhejiang University) by by chimeric with C end or the N end of Vip3Aa1 to the N end of Vip3Ab2 or C end, synthetic Vip3AbAa and Vip3AaAb, result shows, the insecticidal spectrum of mosaic gene is wider, and higher to the insecticidal activity of Some Insects.Therefore, by synthetic mosaic gene be the effective way of transformation Vip3 gene.
Summary of the invention
The invention provides a kind of bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa, this bacillus thuringiensis Vegetative Insecticidal Proteins can be in vegetable cell high efficient expression, lepidopterous insects is had to higher insecticidal activity.
A kind of bacillus thuringiensis (Bacillus thuringiensis) Vegetative Insecticidal Proteins Vip3DAa, its aminoacid sequence is as shown in SEQ ID No.1.
The present invention also provides the encoding gene of described bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa, and its base sequence is as shown in SEQ ID No.2.This encoding gene designs and synthesizes by following thinking:
(1) obtain 257 amino acid whose 771 base sequences of coding Vip3D albumen n end in Vip3D gene (GenBank Accession No:DQ054848);
(2) obtain 532 amino acid whose 1596 base sequences of coding Vip3Aa1 PROTEIN C end in Vip3Aa1 gene (GenBank Accession No:L48811);
(3) two base sequences that chimeric step (1) and (2) obtain, obtain a preliminary original base sequence of transforming;
(4) get rid of the sequence that typically causes this unsettled AT of being rich in of plant gene transcription that exists in described original base sequence and conventional restriction endonuclease sites, under the prerequisite that does not change aminoacid sequence, carry out codon displacement, obtain the base sequence after improvement;
(5) normal chain of the base sequence after improving and corresponding minus strand are carried out to BLAST2 analysis, under the prerequisite that does not change aminoacid sequence, carry out codon displacement, get rid of the inverted repeats existing in sequence; Finally obtain the base sequence as shown in SEQ ID No.2.
Codon displacement in step (4) and (5), to adopt the main preference codon of Plant Genome to replace corresponding codon in the base sequence after described original base sequence or correction, to improve the expression efficiency of described encoding gene in target plant.
In the present invention, the main preference codon of described Plant Genome is monocotyledon rice (Liu Qingpo, the codon usage of paddy rice and the impact of initial sum terminator codon flanking sequence on genetic expression, 2005, doctorate paper, Library of Zhejiang University) and dicotyledons plan south Jie (Duret L.and Mouchiroud D.Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis.1999.PNAS.96:4482-4487.) the codon of the equal preference of nuclear gene group.
Base sequence shown in the SEQ ID No.2 of acquisition is recombinated in the host cells such as intestinal bacteria and expressed, obtain the bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa with aminoacid sequence shown in SEQID No.1.
The G+C content of described encoding gene is 59.32%, with the highest homology of existing Vip3 gene be 88.64%; And the G+C content of described original base sequence is only 30.84%, both homologys are only 68.55%.
The highest homology that described bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa and existing Vip3 albumen are Vip3Aa1 albumen is 96.3%, with the homology 92.5% of Vip3D albumen.
The present invention also provides the expression unit, expression vector or the transformant that contain described encoding gene.As preferably, the promotor of described expression unit is T7 promotor, lac promotor or araBAD promotor.Under the effect of these promotors, Vip3Da albumen can directly be realized soluble-expression in born of the same parents in e. coli host cell.The initial carrier of described expression vector can be selected pET28a (+).
The present invention also provides described encoding gene in the application of cultivating in insect-resistant transgenic plants, comprising:
(1) build the plant expression vector that contains described encoding gene;
(2) described plant expression vector is passed through to agrobacterium mediation converted plant callus;
(3) plant callus is transferred on selective medium and continued to cultivate, after seedling differentiation, transplant to land for growing field crops, screening obtains insect-resistant transgenic plants.
Due in the time designing described encoding gene, that the common preference codon that adopts unifacial leaf model plant paddy rice and dicotyledonous model plant to intend southern Jie's nuclear gene group carries out codon displacement, therefore this encoding gene is applicable to monocotyledons and dicotyledons, for cultivating corresponding insect-resistant transgenic plants.As preferably, described insect-resistant transgenic plants is insect-resistant transgenic cotton, corn, rape or soybean.
The present invention also provides described bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa in the application of preparing in sterilant.The object of killing of described sterilant is preferably lepidopterous insects.Described lepidopterous insects is as prodenia litura, cabbage caterpillar, bollworm, black cutworm and Pyrausta nubilalis (Hubern). etc., more preferably prodenia litura.Described sterilant kill more preferably aphid of object.
Compared with prior art, beneficial effect of the present invention is:
The present invention, taking Vip3D gene and Vip3Aa1 gene as source, designs and synthesizes mosaic gene Vip3DAa, and mosaic gene Vip3DAa expresses the Vip3DAa albumen obtaining lepidopterous insects, aphid are had to higher insecticidal activity; Mosaic gene Vip3DAa can be in the vegetable cells such as cotton, corn, rape and soybean high efficient expression, can be used for cultivating corresponding insect-resistant transgenic plants.
Brief description of the drawings
Fig. 1 is the aminoacid sequence comparison chart of Vip3Aa1, Vip3D and Vip3DAa protein;
Fig. 2 A is the resistance result figure of transgene cotton to glyphosate;
Fig. 2 B is the resistance result figure of non-transgenic cotton to glyphosate;
Fig. 3 A is the resistance result figure of non-transgenic cotton to aphid;
Fig. 3 B is the resistance result figure of transgene cotton to aphid.
Embodiment
The molecular biology that following examples are used and biochemical method are known technology, the Molecular Cloning:A Laboratory Mannual being published by Cold Spring Harbor Laboratory Press (2001) that the Current Pro tocols in Molecular Biology and the J.Sambrook etc. that are published by John WTle y and Sons company writing at Ausubel writes, the documents such as 3rd ED. all have detailed explanation.In following examples, experiment material used is commercially available purchase product if no special instructions.
The design of embodiment 1 mosaic gene and synthetic
Taking Vip3D gene and Vip3Aa1 gene as source, design mosaic gene Vip3DAa, concrete steps are as follows:
(1) obtain 257 amino acid whose 771 base sequences of coding Vip3D albumen n end in Vip3D gene (GenBank Accession No:DQ054848);
(2) obtain 532 amino acid whose 1596 base sequences of coding Vip3Aa1 PROTEIN C end in Vip3Aa1 gene (GenBank Accession No:L48811);
(3) two base sequences that chimeric step (1) and (2) obtain, obtain a preliminary original base sequence of transforming, and as shown in SEQ ID No.3, the G+C content of this sequence is only 30.84%;
(4) do not changing under the prerequisite of aminoacid sequence, the sequence and the conventional restriction endonuclease sites that typically cause this unsettled AT of being rich in of plant gene transcription that in described original base sequence, exist are replaced into monocotyledon rice (Liu Qingpo, the codon usage of paddy rice and the impact of initial sum terminator codon flanking sequence on genetic expression, 2005, doctorate paper, Library of Zhejiang University) and dicotyledons plan south Jie (Duret L.and Mouchiroud D.Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis.1999.PNAS.96:4482-4487.) codon of the equal preference of nuclear gene group, obtain the base sequence after improving,
(5) normal chain of the base sequence after improving and corresponding minus strand are carried out to BLAST2 analysis, do not changing under the prerequisite of aminoacid sequence, the inverted repeats existing in sequence is replaced into monocotyledon rice and dicotyledons and intends the codon of the equal preference of southern Jie's nuclear gene group, obtain final mosaic gene Vip3DAa, as shown in SEQ ID No.1, the sequence homology shown in this sequence and SEQ ID No.3 only has 68.55%.Sequence shown in SEQ ID No.1 and the plasmid PUC-Vip3DAa that contains this DNA fragmentation all entrust Sangon Biotech (Shanghai) Co., Ltd. to complete.
The expression of embodiment 2 mosaic genes
Utilize the Vip3DAa mosaic gene that embodiment 1 obtains to express cloud gold genus bacillus Vegetative Insecticidal Proteins Vip3DAa, specifically comprise:
Vip3DAa mosaic gene is building up to escherichia coli plasmid expression vector pET28a (+) upper, and transforms escherichia coli expression host BL21 (DE3); Inoculate single bacterium colony in 5 milliliters of LB substratum, 37 DEG C of overnight incubation, then dilute and be cultured to OD in 1:100 ratio
600for 0.4-0.6, then adding final concentration is that the IPTG of 1mM carries out abduction delivering, and induction time is 4-6 hour; Centrifugal collection thalline, adds 20mL sterilized water resuspended, liquid nitrogen multigelation 6 times, and the centrifugal thalline that goes, obtains supernatant liquor.
The aminoacid sequence of Vip3DAa albumen is as shown in SEQ ID No.1.The aminoacid sequence of Vip3DAa albumen is carried out to Blast2 with Vip3Aa1 albumen and Vip3D albumen respectively and compare, be respectively 96.3% and 92.5% (seeing Fig. 1) in the homology of protein level.
The insecticidal activity of embodiment 3 Vip3DAa albumen
The supernatant liquor that embodiment 2 is obtained is fed lepidopterous insects prodenia litura, and the colibacillary fermented supernatant fluid that has pET28a (+) empty carrier taking clear water, conversion is contrast, detect the insecticidal activity of Vip3DAa albumen to prodenia litura, the results are shown in Table 1.
The insecticidal activity comparison of three kinds of samples of table 1 to prodenia litura
As can be seen from Table 1, Vip3DAa albumen has significant insecticidal activity to prodenia litura, and after feeding 24h, the average mortality of prodenia litura reaches 86.7%, and after feeding 48h, the average mortality of prodenia litura reaches 100%.And the prodenia litura of other two kinds of samples of feeding, mortality ratio is lower.
Embodiment 4: for cultivating insect-resistant transgenic cotton
(1) structure of carrier
According to the sequence of SEQ ID NO.1, synthetic two primers (primer sequence entrusts extra large Sani bio tech ltd to synthesize) respectively, from plasmid PUC-Vip3DAa, pcr amplification goes out Vip3DAa gene, and primer sequence is as follows:
Upstream primer:
5’-CACGGGGGACTCTAGAACAATGAACATGAACAACACTAAG-3’(SEQ?ID?NO:4)；
Downstream primer:
5’-CGGGGGATCCTCTAGTCACTTGATAGAAACGTCGTA-3’(SEQ?ID?NO:5)；
PCR reaction system is:
PCR reaction parameter:
98 DEG C, 10 seconds, 55 DEG C, 15 seconds, 72 DEG C, 2 points 30 seconds, 35 circulations; 72 DEG C are extended 5 minutes.
PCR product is after PCR product purification test kit purifying, with XbaI enzyme cutting double T-DNA carrier pLM-B001, with Clontech's
vip3DAa is cloned into pLM-B001 by HD Cloning Kit, with XbaI enzyme cutting qualification, and the positive colony of acquisition check order again (PE company, 377 sequenators; Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) checking, the plasmid called after pLM-Vip3DAa that checks order correct.
(2) Agrobacterium preparation
By electrization, carrier pLM-Vip3DAa is imported to Agrobacterium LBA4404, get containing the Agrobacterium of carrier pLM-Vip3DAa and draw plate, choose single bacterium colony and cultivate in LB substratum, for Cotton Transformation is prepared Agrobacterium.
(3) acquisition of transgene cotton
1) choose the hypocotyl of Upland Cotton Ke word 312 aseptic seedling, be cut into 0.5-0.6cm segment with scalper, be inoculated in (MS+B on inducing culture
5organism+2,4-D 0.1mg/L+KT 0.1mg/L+ glucose 30g/L+phytagel 2.5g/L) evoked callus;
Callus is at subculture medium (MS (saltpetre doubles, and ammonium nitrate reduces by half)+B
5organism+2,4-D 0.05mg/L+KT 0.1mg/L glucose 30g/L+phytagel 2.5g/L) subculture several times after, select grain of rice shape Granulous callus, proceeded to division culture medium (MS+B
5organism+glucose 30g/L+phytagel 2.5g/L+KT 0.15mg/L+IBA 0.5mg/L) in, be further divided into embryoid;
2) the glycerine pipe that takes out the agrobacterium strains of preserving in Ultralow Temperature Freezer is in thawing on ice, on LB plate, rule, 26.5 DEG C of dark 36-48hr that cultivate, treat to grow single bacterium colony clearly in ware, picking list bacterium colony, in other LB plate line, is secretly cultivated 36-48hr for 26.5 DEG C, treat that in ware, growing enough bacterium colonies finishes to cultivate, media surface bacterium colony is scraped in the MGL substratum in triangular flask, and 27 DEG C, 200r/min shake 2hr, and OD value can be used for infecting between 0.5-1.5;
3) callus that is divided into embryoid is proceeded to sterile petri dish from triangular flask, remove rataria, turn white and the dead callus waiting out of order, blow and within 5 minutes, make surface micro-a little dry; Activated Agrobacterium bacterium liquid is poured into wherein, Agrobacterium bacterium liquid was advisable just to cover embryoid surface, stir evenly, leave standstill 5-10 minute, outwell bacterium liquid, blot remaining bacterium liquid with filter paper, blow and within 5 minutes, make surface dry a little, thin layer is scattered in the common culture medium that is lined with filter paper, and the 19-21 DEG C of dark 38-42 hour that cultivates treats that small part callus surface occurs that distant bacterium colony finishes common cultivation;
4) callus through cultivating is altogether taken out and immersed containing in the sterilized water of 500mg/L Cef together with filter paper, callus is cleaned up, outwell washing lotion, be placed in the sterilized water that contains 500mg/L Cef and soak 15-20min; Between soak period, stir, removes rataria more, turns white and the dead callus waiting out of order; Outwell washing lotion, use sterile water wash three times, filter paper suck dry moisture, rickle divides to intersperse among to be selected, on substratum one, to blow and within 10 minutes, make surface dry a little; The low light level is cultivated 20 days left and right subcultures and is proceeded to the two normal illuminations cultivations of selection substratum, cultivate 20 days left and right subcultures and proceed to the three normal illuminations cultivations of selection substratum, cultivate 20-30 days, in the dead callus of black, occur that the normal callus of the less growth of light yellow particle is kanamycin-resistant callus tissue.Generally can select on substratum three again subculture once to increase callus quantity kanamycin-resistant callus tissue mono-clonal;
5) from selecting substratum, picking mono-clonal resistance callus is inoculated into respectively on division culture medium, and 20 days left and right subcultures once, as far as possible clear distinguishing between difference clone in the process of differentiation and seedling;
6) kanamycin-resistant callus tissue obtaining after screening is transferred to (the first dark 5-7 days of cultivation on pre-division culture medium, illumination in then 16 hours differentiation is germinateed) 4-6 week, after resistance seedling grows up to, transfer on root media and take root, finally regeneration plant is washed away to cultivate based on greenhouse or field and cultivate, until results T1 seed;
7) by T1 for seed in land for growing field crops, with marker gene and goal gene in the special primer qualification transgenic progeny of marker gene and goal gene, obtain insect-resistant transgenic cotton.
(4) Resistance Identification of transgene cotton
By (3) part steps 7) obtain insect-resistant transgenic cotton seeds kind in land for growing field crops, smear transgenic progeny with glyphosate (expression product of above-mentioned marker gene has resistance to glyphosate), obtain the transgenosis pure lines (as Fig. 2 A and Fig. 2 B) of stably express.From Fig. 2 A and Fig. 2 B, transgenic progeny has resistance to glyphosate.
Meanwhile, the resistance (see Fig. 3 A and Fig. 3 B) of field test transgenic progeny to aphid.From Fig. 3 A and Fig. 3 B, transgenic progeny does not obviously have aphid damage, and aphid is had to significant resistance.
Claims (10)
1. bacillus thuringiensis (Bacillus thuringiensis) Vegetative Insecticidal Proteins Vip3DAa, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.
2. the encoding gene of bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa as claimed in claim 1, is characterized in that, base sequence is as shown in SEQ ID No.2.
3. contain the expression unit of encoding gene as claimed in claim 2.
4. expression as claimed in claim 3 unit, is characterized in that, promotor is T7 promotor, lac promotor or araBAD promotor.
5. contain the expression vector of encoding gene as claimed in claim 2.
6. contain the transformant of encoding gene as claimed in claim 2.
7. the application of encoding gene in cultivation insect-resistant transgenic plants as claimed in claim 2.
8. application as claimed in claim 7, is characterized in that, described insect-resistant transgenic plants is insect-resistant transgenic cotton, corn, rape or soybean.
As claimed in claim 1 bacillus thuringiensis Vegetative Insecticidal Proteins Vip3DAa in the application of preparing in sterilant.
10. application as claimed in claim 9, is characterized in that, the object of killing of described sterilant is aphid.
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