CN103421099A - Vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of vegetative insecticidal protein Vip3AfAa and application - Google Patents
Vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of vegetative insecticidal protein Vip3AfAa and application Download PDFInfo
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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 protein (delta-endotoxin)
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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
- A01N47/44—Guanidine; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- 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
Abstract
The invention discloses a vegetative insecticidal protein Vip3AfAa for bacillus thuringiensis, coding genes of the vegetative insecticidal protein Vip3AfAa and application. An amino acid sequence of the vegetative insecticidal protein for the bacillus thuringiensis is shown as SEQ ID (sequence identifier) No.1, and a base sequence of the coding genes is shown as SEQ ID No.2. The vegetative insecticidal protein for the bacillus thuringiensis is applied to preparing insecticides, and the coding genes are applied to cultivating insect-resistant transgenic plants. Compared with the prior art, the vegetative insecticidal proteinVip3AfAa, the coding genes and the application have the advantages that chimeric genes Vip3AfAa are designed and synthesized on the basis of genes Vip3Af2 and genes Vip3Aa1, and the protein Vip3AfAa obtained by means of expressing the chimeric genes Vip3AfAa is high in lepidopterous insecticidal activity; the chimeric genes Vip3AfAa can be efficiently expressed in cells of plants such as cotton, sweet corn, oilseed rape and soybean and can be used for cultivating the insect-resistant transgenic plants.
Description
Technical field
The present invention relates to the biological prevention field, be specifically related to bacillus thuringiensis vegetative insecticidal protein Vip3A fAa 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 characteristics of causing disaster, and its occurrence scope and severity often cause heavy losses to Chinese national economy, particularly agriculture production.According to statistics, China every year 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.Past is in order to prevent and treat the various insect pests of crop, chemical pesticide is widely used, yet, on the one hand due to long-term a large amount of agricultural chemicals that use, certain resistance or resistance that caused Some Insects to produce, 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 mainly being 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, much has been widely used in the insect-resistant transgenic breeding.Yet, because the genetically modified crops of most of commercialization utilization are the insecticidal crystal protein class, along with the expansion of these genetically modified crops cultivated areas, insect produces to single insecticidal proteins the problem that resistance has become a sternness.Therefore finding new anti insect gene seems particularly important.
Scientist is through unremitting effort, separate the non-parasporal crystal insecticidal proteins that obtains having desinsection toxicity from the Bt bacterial strain of some vegetative growth stages, 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 mainly is 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.
Yet 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, the Zhejiang University Library) by the N of Vip3Ab2 end or C end is chimeric with C end or the N end of Vip3Aa1, 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 the 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 protein Vip3A fAa, this bacillus thuringiensis Vegetative Insecticidal Proteins can be in vegetable cell high efficient expression, the insects such as lepidopteran, Coleoptera and Homoptera are had to higher insecticidal activity.
A kind of bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A aBb, 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 protein Vip3A fAa, and its base sequence is as shown in SEQ ID No.2.This encoding gene designs and synthesizes by following thinking:
(1) obtain 400 amino acid whose 1200 base sequences of coding Vip3Af2 albumen n end in Vip3Af2 gene (GenBank Accession No:DD319822);
(2) obtain 389 amino acid whose 1167 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 restriction endonuclease sites commonly used, carry out the codon displacement under the prerequisite that does not change aminoacid sequence, obtain the base sequence after improvement;
(5) normal chain of the base sequence after improving and corresponding minus strand are carried out to the BLAST2 analysis, carry out the codon displacement under the prerequisite that does not change aminoacid sequence, get rid of the inverted repeats existed in sequence; Finally obtain the base sequence as shown in SEQ ID No.2.
The displacement of codon in step (4) and (5), be adopt the main preference codon described original base sequence of displacement of Plant Genome or correct after base sequence in corresponding codon.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 impact on genetic expression of the codon usage of paddy rice and initial sum terminator codon flanking sequence, 2005, the doctorate paper, the Zhejiang University Library) and dicotyledons intend south (the Duret L.and Mouchiroud D.Expression pattern and that is situated between, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, the codon of the equal preference of nuclear gene group and Arabidopsis.1999.PNAS.96:4482-4487.).
Base sequence shown in the SEQ ID No.2 of acquisition is recombinated in the host cell such as intestinal bacteria and express, obtain the bacillus thuringiensis vegetative insecticidal protein Vip3A fAa with aminoacid sequence shown in SEQID No.1.
The G+C content of described encoding gene is 59.37%, with the highest homology of existing Vip3 gene be 88.56%; And the G+C content of described original base sequence is only 31.05%, both homologys are only 63.05%.
The highest homology that described bacillus thuringiensis vegetative insecticidal protein Vip3A fAa and existing Vip3 albumen are Vip3Aa1 albumen is 98.2%, with the homology of Vip3Af2 albumen be 90.2%.
The present invention also provides expression unit, expression vector or the transformant that contains 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, Vip3AfAa 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 the application of described encoding gene in cultivating insect-resistant transgenic plants.Specifically comprise:
(1) build the plant expression vector that contains described encoding gene;
(2) described plant expression vector is passed through to the 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 to when designing described encoding gene, that the codon that adopts unifacial leaf model plant paddy rice and dicotyledonous model plant to intend the common preference of southern Jie's nuclear gene group carries out the codon displacement, therefore this gene is applicable to unifacial leaf and dicotyledons, for cultivating corresponding insect-resistant transgenic plants.As preferably, described insect-resistant transgenic plants is insect-resistant transgenic cotton, corn, rape and soybean.
The present invention also provides the application of described bacillus thuringiensis vegetative insecticidal protein Vip3A fAa in preparing 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.
Compared with prior art, beneficial effect of the present invention is:
It is source that Vip3Bb1 gene and Vip3Aa1 gene are take in the present invention, designs and synthesizes mosaic gene Vip3AaBb, and mosaic gene Vip3AaBb expresses the Vip3AfAa albumen obtained lepidopterous insects is had to higher insecticidal activity; Mosaic gene Vip3AaBb 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.
The accompanying drawing explanation
The aminoacid sequence comparison chart that Fig. 1 is Vip3Aa1, Vip3Af2 and Vip3AfAa protein.
Embodiment
The molecular biology that following examples are used and biochemical method are known technology, the Molecular Cloning:A Laboratory Mannual published by Cold Spring Harbor Laboratory Press (2001) that the Current Pro tocols in Molecular Biology published by John WTle y and Sons company write at Ausubel and J.Sambrook etc. write, 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
Take Vip3Af2 gene and Vip3Aa1 gene is source, design mosaic gene Vip3AfAa, and concrete steps are as follows:
(1) obtain 400 amino acid whose 1200 base sequences of coding Vip3Af2 albumen n end in Vip3Af2 gene (GenBank Accession No:DD319822);
(2) obtain 389 amino acid whose 1167 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 only has 31.05%;
(4) under the prerequisite that does not change aminoacid sequence, the sequence and the restriction endonuclease sites commonly used that typically cause this unsettled AT of being rich in of plant gene transcription that in described original base sequence, exist are replaced into to monocotyledon rice (Liu Qingpo, the impact on genetic expression of the codon usage of paddy rice and initial sum terminator codon flanking sequence, 2005, the doctorate paper, the Zhejiang University Library) and dicotyledons intend south (the Duret L.and Mouchiroud D.Expression pattern and that is situated between, 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 the BLAST2 analysis, under the prerequisite that does not change aminoacid sequence, the inverted repeats existed in sequence is replaced into to the codon that monocotyledon rice and dicotyledons are intended the equal preference of southern Jie's nuclear gene group, obtain final chimeric gene sequence Vip3AfAa, as shown in SEQ ID No.1, the sequence homology shown in this sequence and SEQ ID No.3 only has 63.05%.Sequence shown in SEQ ID No.1 and the plasmid PUC-Vip3AfAa that contains this DNA fragmentation all entrust Sangon Biotech (Shanghai) Co., Ltd. to complete.
The expression of embodiment 2 mosaic genes
The Vip3AfAa mosaic gene that utilizes embodiment 1 to obtain is expressed cloud gold genus bacillus vegetative insecticidal protein Vip3A fAa, specifically comprises:
The Vip3AfAa 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 ℃ of overnight incubation, then dilute and be cultured to OD in the 1:100 ratio
600For 0.4-0.6, then add the IPTG that final concentration is 1mM to carry out abduction delivering, induction time is 4-6 hour; Centrifugal collection thalline, add the 20mL sterilized water resuspended, liquid nitrogen multigelation 6 times, and the centrifugal thalline that goes, obtain supernatant liquor.
The aminoacid sequence of Vip3AfAa albumen is as shown in SEQ ID No.1.The aminoacid sequence of Vip3AfAa albumen is carried out to Blast2 with Vip3Aa1 albumen and Vip3Af2 albumen respectively and compares, the homology of protein level be respectively 98.2% and 90.2%(see Fig. 1).
The insecticidal activity of embodiment 3Vip3AaBb albumen
The supernatant liquor that embodiment 2 is obtained is fed the lepidopterous insects prodenia litura, and take clear water, to transform the colibacillary fermented supernatant fluid that pET28a (+) empty carrier is arranged be contrast, the insecticidal activity of detection Vip3AfAa albumen to prodenia litura, the results are shown in Table 1.
Three kinds of samples of table 1 compare the insecticidal activity of prodenia litura
As can be seen from Table 1, Vip3AfAa 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 the insect-resistant transgenic cotton
(1) structure of carrier
According to the sequence of SEQ ID NO.1, synthesize respectively two primers (primer sequence entrusts extra large Sani's bio tech ltd to synthesize), from plasmid PUC-Vip3AfAa, pcr amplification goes out the Vip3AfAa gene, and primer sequence is as follows:
Upstream primer:
5’-CACGGGGGACTCTAGAACAATGAACAAGAACAACACTAAG-3’（SEQ?ID?NO:4）；
Downstream primer:
5’-CGGGGGATCCTCTAGTCACTTGATAGAAACGTCGTA-3’（SEQ?ID?NO:5）；
The PCR reaction system is:
The PCR reaction parameter:
98 ℃, 10 seconds, 55 ℃, 15 seconds, 72 ℃, 2 minutes 30 seconds, 35 circulations; 72 ℃ are extended 5 minutes.
The PCR product is after PCR product purification test kit purifying, with XbaI enzyme cutting double T-DNA carrier pLM-B001, with Clontech's
HD Cloning Kit is cloned into pLM-B001 by Vip3AfAa, identifies the positive colony of acquisition check order (PE company, 377 sequenators with XbaI enzyme cutting again; Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) checking, the plasmid called after pLM-Vip3AfAa that checks order correct.
(2) Agrobacterium preparation
By electrization, carrier pLM-Vip3AfAa is imported to Agrobacterium LBA4404; Get containing the Agrobacterium of carrier pLM-Vip3AfAa and draw plate, choose single bacterium colony and cultivate in the 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, with scalper, be cut into the 0.5-0.6cm segment, be inoculated in (MS+B on inducing culture
5Organism+2,4-D0.1mg/L+KT0.1mg/L+ glucose 30g/L+phytagel2.5g/L) evoked callus;
Callus is at subculture medium (MS (saltpetre doubles, and ammonium nitrate reduces by half)+B
5Organism+2,4-D0.05mg/L+KT0.1mg/L glucose 30g/L+phytagel2.5g/L) subculture several times after, select grain of rice shape Granulous callus, it is proceeded to division culture medium (MS+B
5Organism+glucose 30g/L+phytagel2.5g/L+KT0.15mg/L+IBA0.5mg/L), in, further be divided into embryoid;
2) take out the glycerine pipe of the agrobacterium strains of preserving in Ultralow Temperature Freezer in thawing on ice, on the LB plate, rule, 26.5 ℃ dark 36-48hr that cultivates, treat in ware to grow single bacterium colony clearly, picking list bacterium colony, in other LB plate line, is secretly cultivated 36-48hr for 26.5 ℃, treating in ware to grow enough bacterium colonies finishes to cultivate, the media surface bacterium colony is scraped in the MGL substratum in triangular flask, and 27 ℃, 200r/min shake 2hr, and the OD value can be used for infecting between 0.5-1.5;
The callus that 3) will be divided into embryoid proceeds to sterile petri dish from triangular flask, removes rataria, turns white and the dead callus waited out of order, blows and within 5 minutes, makes the micro-a little drying in surface; Activated Agrobacterium bacterium liquid is poured into wherein, Agrobacterium bacterium liquid was advisable just to cover the embryoid surface, stir evenly, standing 5-10 minute, outwell bacterium liquid, with filter paper, blots remaining bacterium liquid, 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 ℃ of dark 38-42 hour that cultivates treats that small part callus surface occurs that distant bacterium colony finishes to cultivate altogether;
4) will take out and immerse containing in the sterilized water of 500mg/L Cef together with filter paper through the callus of cultivating altogether, 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, remove rataria more, turns white and the dead callus waited out of order; Outwell washing lotion, use sterile water wash three times, the filter paper suck dry moisture, rickle divides to intersperse among to be selected on substratum one, blows and within 10 minutes, makes 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, on 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 the kanamycin-resistant callus tissue mono-clonal on substratum three again subculture once to increase callus quantity;
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 the difference clone in the process of differentiation and seedling;
6) kanamycin-resistant callus tissue obtained after screening is transferred on pre-division culture medium and (first secretly cultivated 5-7 days, illumination in then 16 hours differentiation is germinateed) 4-6 week, transfer on root media and take root after the resistance seedling grows up to, 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, identify marker gene and the goal gene in transgenic progeny with the special primer of marker gene and goal gene, obtain the insect-resistant transgenic cotton.
Claims (10)
1. bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A fAa, is characterized in that, aminoacid sequence is as shown in SEQ ID No.1.
2. the encoding gene of bacillus thuringiensis vegetative insecticidal protein Vip3A fAa 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 1 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 cultivating 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.
9. the application of bacillus thuringiensis vegetative insecticidal protein Vip3A fAa in preparing sterilant as claimed in claim 1.
10. application as claimed in claim 9, is characterized in that, the object of killing of described sterilant is lepidopterous insects.
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| CN104151410A (en) | 2014-11-19 |
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