CN104151409B - Bacillus thuringiensis vegetative insecticidal protein Vip3A aBb and encoding gene thereof and application - Google Patents
Bacillus thuringiensis vegetative insecticidal protein Vip3A aBb and encoding gene thereof and application Download PDFInfo
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- CN104151409B CN104151409B CN201410391994.4A CN201410391994A CN104151409B CN 104151409 B CN104151409 B CN 104151409B CN 201410391994 A CN201410391994 A CN 201410391994A CN 104151409 B CN104151409 B CN 104151409B
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- 108700031685 Bacillus thuringiensis Vip3A Proteins 0.000 title claims abstract description 14
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- 230000000749 insecticidal effect Effects 0.000 claims abstract description 23
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- 230000014509 gene expression Effects 0.000 claims abstract description 14
- 241001124076 Aphididae Species 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
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Classifications
-
- 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
-
- 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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- C—CHEMISTRY; METALLURGY
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- General Health & Medical Sciences (AREA)
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- Biomedical Technology (AREA)
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- Biophysics (AREA)
- Pest Control & Pesticides (AREA)
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- Physics & Mathematics (AREA)
- Insects & Arthropods (AREA)
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Abstract
The invention discloses a kind of bacillus thuringiensis vegetative insecticidal protein Vip3A aBb and encoding gene thereof and application.The aminoacid sequence of this bacillus thuringiensis Vegetative Insecticidal Proteins is as shown in SEQ ID No.1, and the base sequence of encoding gene is as shown in SEQ ID No.2.Described application is the application in preparing insecticide of the bacillus thuringiensis Vegetative Insecticidal Proteins, and the application that encoding gene is in cultivating insect-resistant transgenic plants.Compared with prior art, the present invention, with Vip3Bb1 gene and Vip3Aa1 gene as source, designs and synthesizes mosaic gene Vip3AaBb, mosaic gene Vip3AaBb and expresses the Vip3AaBb albumen obtained lepidopteran insects, aphid are had higher insecticidal activity；Mosaic gene Vip3AaBb can in the plant cells such as Cotton Gossypii, Semen Maydis, Brassica campestris L and Semen sojae atricolor high efficient expression, can be used for cultivate insect-resistant transgenic plants.
Description
Technical field
The present invention relates to technical field of biological control, be specifically related to bacillus thuringiensis Vegetative Insecticidal Proteins
Vip3AaBb 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 big and often breaks out into
The feature of calamity, its occurrence scope and the order of severity often result in heavy losses to Chinese national economy, particularly agricultural production.According to system
Meter, nearly 50,000,000,000 jin of China's the most due to illness pest injurious loss grain, all kinds of industrial crops 18,000,000 tons.Past is in order to prevent and treat crop
Various insect pests, chemical pesticide is widely used, but, on the one hand owing to using pesticide in a large number for a long time, cause Some Insects to produce
Certain drug resistance or resistance, cause the using dosage of pesticide to increase year by year or using brand-new pesticide；On the other hand, by
Excessively using chemical pesticide in for a long time, the environment causing the mankind to depend on for existence is polluted in various degree.It is, thus, sought for more
The good various pest and disease damage of approach prevention and control.Along with the development of biotechnology, utilizing anti insect gene to cultivate insect-resistant transgenic plants is one
The effective approach of bar.
Bacillus thuringiensis (Bacillus thuringiensis, Bt) is the important donor of Plant Extrinsic Anti-insect Genes
Bacterium, its toxic activity is derived mainly from produced parasporal crystal toxin, i.e. insecticidal crystal protein during sporulation
(insecticidal crystalline protein, ICP), including crystal toxin (crystalline toxin, Cry) and
Cell cracking toxin (cytolytic toxin, Cyt).Sensitive insect, by Cry gene and Cyt gene code, is had by ICP
Strong toxicity, and to higher mammal and people's avirulence.
The insecticidal crystalline gene reported has 748 kinds, is much widely used in insect-resistant transgenic breeding.So
And, the genetically modified crops utilized due to major part commercialization are insecticidal crystal protein class, along with these genetically modified crops are planted
The expansion of area, insect produces resistance to single insecticidal proteins has become a severe problem.Therefore find new pest-resistant
Gene is particularly important.
Scientist is through unremitting effort, and from the Bt bacterial strain of some vegetative growth stages, isolated has parasite killing toxicity
Non-parasporal crystal insecticidal proteins, be i.e. secreted into outside born of the same parents after synthesis, here it is be referred to as the Su Yunjin of second filial generation insect resistance protein
The Vegetative Insecticidal Proteins (vegetativeinsecticidal protein, Vip) of bacillus.Vips is broadly divided into
Vip1, Vip2, Vip3 and Vip4 tetra-type totally 108 insecticidal proteins, wherein 77 albumen belong to Vip3.The insecticidal spectrum of Vip3
Different from ICPs with insecticidal activity, the former has toxic action to insects such as Lepidoptera, coleoptera and Homopteras, and pest-resistant spectrum is more
Extensively.At present, these genes are widely used in pest-resistant transgenic rice, Semen Maydis and cotton breeding.
But, the resource of Vip3 gene is limited after all, and different intergenic resistances exists bigger difference
Different, the gene of antibacterial is enriched in AT simultaneously, and this certainly will affect gene expression in plant, manually changes corresponding gene
Make, the utilization ratio of gene can be improved further.
Document (Fang et al., Characterization of chimeric Bacillus thuringiensis
Vip3toxins,Applied and Environmental Microbiology,2007,73(3):956-961；Fang Jun, Soviet Union
Cloud gold bacillus Vegetative Insecticidal Proteins Vip3 gene and the application in transgenic paddy rice thereof, 2008, thesis for the doctorate, Zhejiang University
Library) by by chimeric with the C end of Vip3Aa1 or N end to N end or the C end of Vip3Ab2, synthetic Vip3AbAa and
Vip3AaAb, result shows, the insecticidal spectrum of mosaic gene is wider, and higher to the insecticidal activity of Some Insects.Therefore, pass through
Synthetic mosaic gene is the effective way of transformation Vip3 gene.
Summary of the invention
The invention provides a kind of bacillus thuringiensis vegetative insecticidal protein Vip3A aBb, this bacillus thuringiensis
Vegetative Insecticidal Proteins can in plant cell high efficient expression, the insecticides such as Lepidoptera, coleoptera and Homoptera are had higher
Insecticidal activity.
A kind of bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A aBb, its ammonia
Base acid sequence is as shown in SEQ ID No.1.
Present invention also offers the encoding gene of described bacillus thuringiensis vegetative insecticidal protein Vip3A aBb, its alkali
Basic sequence is as shown in SEQ ID No.2.This encoding gene is designed and synthesized by following thinking:
(1) coding Vip3Aa1 albumen n end in Vip3Aa1 gene (GenBank Accession No:L48811) is obtained
451 amino acid whose 1353 base sequences；
(2) coding Vip3Bb1 PROTEIN C end in Vip3Bb1 gene (GenBank Accession No:DD319826) is obtained
350 amino acid whose 1050 base sequences；
(3) two base sequences that chimeric step (1) and (2) obtain, it is thus achieved that the original base sequence of a preliminary transformation；
(4) get rid of typically cause present in described original base sequence this instability of plant gene transcription rich in AT
Sequence and conventional restriction endonuclease sites, on the premise of not changing aminoacid sequence, carry out codon substitutions, obtain
Take the base sequence after correction；
(5) normal chain and the corresponding minus strand of the base sequence after improving carry out BLAST2 analysis, are not changing aminoacid
Carry out codon substitutions on the premise of sequence, get rid of inverted repeat present in sequence；Finally obtain such as SEQ ID No.2
Shown base sequence.
Codon substitutions in step (4) and (5), is that the main preference codon using Plant Genome is replaced described former
Codon corresponding in base sequence after beginning base sequence or correction, to improve described encoding gene table in target plant
Reach efficiency.
In the present invention, the main preference codon of described Plant Genome be monocotyledon rice (Liu Qingpo, Oryza sativa L.
Codon usage and the initial and termination codon flanking sequence impact on gene expression, 2005, Ph.D. Dissertation, Zhejiang is big
Learn library) and dicotyledon plan south Jie (Duret L.and Mouchiroud D.Expression pattern and,
surprisingly,gene length shape codon usage in Caenorhabditis,Drosophila,and
The codon of the equal preference of Matrix attachment region Arabidopsis.1999.PNAS.96:4482-4487.).
Base sequence shown in the SEQ ID No.2 of acquisition is recombinated to the host cells such as escherichia coli are expressed, i.e. obtain
Must have the bacillus thuringiensis vegetative insecticidal protein Vip3A aBb of aminoacid sequence shown in SEQID No.1.
The G+C content of described encoding gene is 58.44%, is 79.09% with the highest homology of existing Vip3 gene；
And the G+C content of described original base sequence only has 30.80%；Both sequence homologies only have 70.13%.
Described bacillus thuringiensis vegetative insecticidal protein Vip3A aBb and existing Vip3A_N albumen (GenBank
Accession No:ADI48120) highest homology be 87.6%, be 75.4% with the homology of Vip3Aa1 albumen, with
The homology of Vip3Bb1 albumen is 84.3%.
Present invention also offers expression unit, expression vector or the transformant containing described encoding gene.As preferably, institute
The promoter stating expression unit is T7 promoter, lac promoter or araBAD promoter.Under the effect of these promoteres,
Vip3AaBb albumen directly can realize intracellular soluble-expression in e. coli host cell.The original load of described expression vector
Body can be selected for pET28a (+).
Present invention also offers the application in cultivating insect-resistant transgenic plants of the described encoding gene.Specifically include:
(1) plant expression vector containing described encoding gene is built；
(2) by described plant expression vector by agrobacterium mediation converted plant callus；
(3) transfer to plant callus continue on selective medium to cultivate, after seedling differentiation, transplant to greatly
Field, screening obtains insect-resistant transgenic plants.
Owing to when designing described encoding gene, being to use unifacial leaf model plant Oryza sativa L. and dicotyledonous model plant to intend south
The codon of the common preference of Jie's Matrix attachment region carries out codon substitutions, and therefore this gene is suitable for unifacial leaf and dicotyledon,
For cultivating corresponding insect-resistant transgenic plants.As preferably, described insect-resistant transgenic plants is insect-resistant trans-genie cotton plants, jade
Rice, Brassica campestris L and Semen sojae atricolor.
Present invention also offers described bacillus thuringiensis vegetative insecticidal protein Vip3A aBb in preparing insecticide
Application.The object of killing of described insecticide is preferably lepidopteran insects.Described lepidopteran insects such as Prodenia litura, Pieris rapae,
Bollworm, black cutworm and Pyrausta nubilalis (Hubern). etc., more preferably Prodenia litura.The object of killing of described insecticide is more preferably aphid.
Compared with prior art, the invention have the benefit that
The present invention, with Vip3Bb1 gene and Vip3Aa1 gene as source, designs and synthesizes mosaic gene Vip3AaBb, embedding
The Vip3AaBb albumen closing gene Vip3AaBb expression acquisition has higher insecticidal activity to lepidopteran insects, aphid；Chimeric
Gene Vip3AaBb can in the plant cells such as Oryza sativa L. high efficient expression, can be used for cultivating corresponding insect-resistant transgenic plants.
Accompanying drawing explanation
Fig. 1 is the amino acid alignment figure of Vip3Aa1, Vip3Bb1 and Vip3AaBb albumen；
Fig. 2 A is the transgene cotton resistance result figure to glyphosate；
Fig. 2 B is the non-transgenic Cotton Gossypii resistance result figure to glyphosate；
Fig. 3 A is the non-transgenic Cotton Gossypii resistance result figure to aphid；
Fig. 3 B is the transgene cotton resistance result figure to aphid.
Detailed description of the invention
Molecular biology and biochemical method that following example are used are known technology, compile at Ausubel
The Current Pro tocols inMolecular Biology published by John WTle y and Sons company that writes and
The Molecular published by Cold Spring Harbor Laboratory Press (2001) that J.Sambrook etc. write
The documents such as Cloning:A Laboratory Mannual, 3rd ED. are all discussed in detail.Reality used in following example
Test material and be commercially available purchase product if no special instructions.
The design of embodiment 1 mosaic gene and synthesis
With Vip3Bb1 gene and Vip3Aa1 gene as source, design mosaic gene Vip3AaBb, specifically comprise the following steps that
(1) coding Vip3Aa1 albumen n end in Vip3Aa1 gene (GenBank Accession No:L48811) is obtained
451 amino acid whose 1353 base sequences；
(2) coding Vip3Bb1 PROTEIN C end in Vip3Bb1 gene (GenBank Accession No:DD319826) is obtained
350 amino acid whose 1050 base sequences；
(3) two base sequences that chimeric step (1) and (2) obtain, it is thus achieved that the original base sequence of a preliminary transformation,
As shown in SEQ ID No.3, the G+C content of this sequence only has 30.80%；
(4) on the premise of not changing aminoacid sequence, plant typically causing present in described original base sequence
The sequence rich in AT and conventional restriction endonuclease sites that thing gene transcripts is unstable are replaced into monocotyledon water
Rice (Liu Qingpo, the codon usage of Oryza sativa L. and the initial and termination codon flanking sequence impact on gene expression, 2005, rich
Bachelorship paper, Zhejiang University's library) and dicotyledon 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.) Matrix attachment region is equal
The codon of preference, it is thus achieved that the base sequence after improvement；
(5) normal chain and the corresponding minus strand of the base sequence after improving carry out BLAST2 analysis, are not changing aminoacid
On the premise of sequence, inverted repeat present in sequence is replaced into monocotyledon rice and dicotyledon is intended south and is situated between
The codon of the equal preference of genome, it is thus achieved that final mosaic gene Vip3AaBb, as shown in SEQ ID No.1, this sequence and SEQ
Sequence homology shown in ID No.3 only has 70.13%.Sequence shown in SEQ ID No.1 and the plasmid containing this DNA fragmentation
PUC-Vip3AaBb all entrusts Sangon Biotech (Shanghai) Co., Ltd. to complete.
The expression of embodiment 2 mosaic gene
The Vip3AaBb mosaic gene utilizing embodiment 1 to obtain expresses cloud gold bacillus cereus Vegetative Insecticidal Proteins
Vip3AaBb, specifically includes:
Vip3AaBb mosaic gene is building up to escherichia coli plasmid expression vector pET28a (+) on, and convert large intestine bar
Bacterium expressive host BL21 (DE3)；The single bacterium colony of inoculation in 5 milliliters of LB culture medium, 37 DEG C of overnight incubation, then enter in 1:100 ratio
It is 0.4-0.6 that row dilution is cultivated to OD600, and the IPTG then adding final concentration of 1mM carries out abduction delivering, and induction time is 4-
6 hours；Centrifugal thalline of collecting, addition 20mL sterilized water is resuspended, liquid nitrogen multigelation 6 times, the centrifugal thalline that goes, acquisition supernatant.
The aminoacid sequence of Vip3AaBb albumen is as shown in SEQ ID No.1.Aminoacid sequence by Vip3AaBb albumen
Carrying out Blast2 comparison with Vip3Aa1 albumen and Vip3Bb1 albumen respectively, the homology at protein level is respectively 75.4%
With 84.3% (see Fig. 1).
The insecticidal activity of embodiment 3 Vip3AaBb albumen
The supernatant that embodiment 2 obtained feeds lepidopteran insects Prodenia litura, and with clear water, convert have pET28a (+)
The colibacillary fermented supernatant fluid of empty carrier is comparison, detects the Vip3AaBb albumen insecticidal activity to Prodenia litura, result
It is shown in Table 1.
The insecticidal activity of Prodenia litura is compared by 1 three kinds of samples of table
As can be seen from Table 1, Vip3AaBb albumen has significant insecticidal activity to Prodenia litura, twill night after feeding 24h
The average mortality of moth reaches 86.7%, and after feeding 48h, the average mortality of Prodenia litura reaches 100%.And other two kinds of samples of feeding
The Prodenia litura of product, mortality rate is relatively low.
Embodiment 4: be used for cultivating insect-resistant trans-genie cotton plants
(1) structure of carrier
According to the sequence of SEQ ID NO.1, (primer sequence entrusts sea Sani's biotechnology limited to be respectively synthesized two primers
Company synthesizes), from plasmid PUC-Vip3AaBb, PCR amplifies Vip3AaBb gene, and primer sequence is as follows:
Forward primer:
5’-CACGGGGGACTCTAGAACAATGAACATGAACAACACTAAG-3’(SEQ ID NO:4)；
Downstream primer:
5’-CGGGGGATCCTCTAGTCACTCCTTAACAAGGGAAAC-3’(SEQ ID NO:5)；
PCR reaction system is:
PCR response parameter:
98 DEG C, 10 seconds, 55 DEG C, 15 seconds, 72 DEG C, 2 points 30 seconds, 35 circulations；72 DEG C extend 5 minutes.
PCR primer is after PCR primer Purification Kit, with XbaI enzyme cutting double T-DNA carrier pLM-B001, uses
Clontech'sVip3AaBb is cloned into pLM-B001 by HD Cloning Kit, identifies with XbaI enzyme cutting, obtains
Positive colony check order again (PE company, 377 sequenators；Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) checking, survey
The named pLM-Vip3AaBb of plasmid that sequence is correct.
(2) prepared by Agrobacterium
By electrization, T-DNA carrier pLM-Vip3AaBb is imported Agrobacterium LBA4404.Take containing T-DNA carrier pLM-
The Agrobacterium of Vip3AaBb draws plate, chooses single bacterium colony and cultivates in LB culture medium, prepares Agrobacterium for Cotton Transformation.
(3) acquisition of transgene cotton
1) choose the hypocotyl of Upland Cotton Ke's word 312 aseptic seedling, be cut into 0.5-0.6cm segment with dissecting knife, inoculation
(MS+B on inducing culture5Organic substance+2,4-D0.1mg/L+KT0.1mg/L+ glucose 30g/L+phytagel2.5g/L)
Callus induction；
Callus is at subculture medium (MS (potassium nitrate doubles, and ammonium nitrate halves)+B5Organic substance+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, by its turn
Enter division culture medium (MS+B5Organic substance+glucose 30g/L+phytagel2.5g/L+KT0.15mg/L+IBA0.5mg/L) in,
It is further differentiated into embryoid；
2) the glycerol pipe taking out the agrobacterium strains preserved in ultra cold storage freezer is rule on thawed on ice, LB plate,
26.5 DEG C of light culture 36-48hr, grow single bacterium colony clearly in treating ware, picking list bacterium colony is rule at other LB plate, and 26.5
DEG C light culture 36-48hr, grows enough bacterium colonies and terminates to cultivate, media surface bacterium colony is scraped in triangular flask in treating ware
In MGL culture medium, 27 DEG C, 200r/min shakes 2hr, OD value and i.e. can be used for infecting between 0.5-1.5；
3) callus being divided into embryoid is proceeded to sterile petri dish from triangular flask, remove rataria, turn white and dead
Deng callus out of order, blow 5 minutes and make surface the most micro-dry；Activated Agrobacterium bacterium solution is poured into wherein, agriculture
Bacillus bacterium solution is advisable just to cover embryoid surface, stirs evenly, and stands 5-10 minute, outwells bacterium solution, blots remaining bacterium solution with filter paper,
Blowing 5 minutes makes surface the driest, thin layer be scattered in be lined with filter paper co-culture in culture medium, 19-21 DEG C of light culture 38-42 is little
Time, treat that small part callus surface occurs that distant bacterium colony terminates to co-culture；
4) callus through co-culturing is taken out in the sterilized water immersed containing 500mg/L Cef together with filter paper,
Callus is cleaned up, outwells washing liquid, be placed in the sterilized water containing 500mg/L Cef and soak 15-20min；During immersion
Many agitations, remove rataria, turn white and the dead callus waited out of order；Outwell washing liquid, with sterile water wash three times, filter paper
Suck dry moisture, rickle dispersion is distributed on Selective agar medium one, blows 10 minutes and makes surface the driest；The low light level is cultivated and is continued for about 20 days
In generation, proceeds to Selective agar medium two normal illumination and cultivates, and cultivates about 20 days subcultures and proceeds to Selective agar medium three normal illumination cultivation,
Cultivate 20-30 days, black death wound healing occurs the light yellow granule normal wound healing of less growth is kanamycin-resistant callus tissue.Typically may be used
With kanamycin-resistant callus tissue monoclonal on Selective agar medium three followed by generation once to increase wound healing quantity；
5) from Selective agar medium, picking monoclonal kanamycin-resistant callus tissue is inoculated on division culture medium respectively, about 20 days subcultures
Once, break up and try one's best clear distinguishing between difference clone during seedling；
6) the kanamycin-resistant callus tissue obtained after screening transfer on pre-division culture medium (first light culture 5-7 days, then 16 hours
Illumination differentiation is germinateed) 4-6 week, transfer to take root on root media after resistance seedling grows up to, finally regeneration plant is washed away
Cultivate and cultivate based on greenhouse or field, until results T1 seed；
7) by T1 for seed in land for growing field crops, identify in transgenic progeny with the special primer of marker gene and genes of interest
Marker gene and genes of interest, it is thus achieved that insect-resistant trans-genie cotton plants.
(4) Resistance Identification of transgene cotton
By (3rd) part steps 7) the insect-resistant trans-genie cotton plants seed that obtains is in land for growing field crops, with glyphosate (above-mentioned labelling base
The expression product of cause has resistance to glyphosate) smear transgenic progeny, it is thus achieved that stable express transgenic pure lines (such as Fig. 2 A and
Fig. 2 B).From Fig. 2 A and Fig. 2 B, transgenic progeny has resistance to glyphosate.
Meanwhile, the field test transgenic progeny resistance (see Fig. 3 A and Fig. 3 B) to aphid.From Fig. 3 A and Fig. 3 B, turn
Progeny, apparently without aphid damage, has significant resistance to aphid.
Claims (10)
1. bacillus thuringiensis (Bacillus thuringiensis) vegetative insecticidal protein Vip3A aBb, its feature exists
In, aminoacid sequence is as shown in SEQ ID No.1.
2. the encoding gene of bacillus thuringiensis vegetative insecticidal protein Vip3A aBb as claimed in claim 1, its feature exists
In, base sequence is as shown in SEQ ID No.2.
3. contain the expression unit of encoding gene as claimed in claim 2.
Express unit the most as claimed in claim 3, it is characterised in that promoter is T7 promoter, lac promoter or araBAD
Promoter.
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. encoding gene application in cultivating insect-resistant transgenic plants as claimed in claim 2.
Apply the most as claimed in claim 7, it is characterised in that described insect-resistant transgenic plants is insect-resistant trans-genie cotton plants, jade
Rice, Brassica campestris L or Semen sojae atricolor.
9. bacillus thuringiensis vegetative insecticidal protein Vip3A aBb as claimed in claim 1 answering in preparing insecticide
With.
Apply the most as claimed in claim 9, it is characterised in that the object of killing of described insecticide is aphid.
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