CN104845983B - Bacillus thuringiensis vip3A-6 genes, expression albumen and its application - Google Patents
Bacillus thuringiensis vip3A-6 genes, expression albumen and its application Download PDFInfo
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Abstract
6 genes of bacillus thuringiensis vip3A, expression albumen and its application, belong to technological field of biochemistry, 6 gene expression albumen of vip3A carries out primary dcreening operation and secondary screening to beet armyworm biological activity test, investigates number of dead and live insects after 7d respectively, calculates corrected mortality and LC50；As a result known to, the 6 gene expression albumen of vip3A of bacillus thuringiensis is 4.783 μ g/g (2.234 7.784 μ g/g) to beet armyworm half lethal concentration in secondary screening, when expression albumen concentration reaches 81 μ g/g, all beet armyworms are dead, it can be seen that the 6 gene expression albumen of vip3A of bacillus thuringiensis has high virulence to beet armyworm, it is characterised in that the nucleotide sequence such as Seq ID No of 6 genes of bacillus thuringiensis vip3A, expression albumen and its application:Shown in 1,6 genes of vip3A are a new gene.
Description
Technical field
The present invention relates to a kind of bacillus thuringiensis vip3A-6 genes, expression albumen and its applications, belong to biochemistry
Technical field.
Background technology
Beet armyworm, scientific name：Laphygma exigua Hubner, English name：Beet army worm, alias：Greedy night
Moth.Worm state has adult, ovum, larva, pupa, is caused harm plant with larva.Newly hatched larvae cluster blade back, spinning netting, the feeding in leaf
Mesophyll leaves epidermis, at transparent aperture；Blade can be eaten into hole after 3 ages or incised；Larva can also brill moth green pepper,
Tamato fruit.Pests occurrence rule and harmful characteristics:First instar larvae spins in blade back cluster knots, and appetite is small, and after 3 ages, dispersion is caused harm,
Appetite increases, and hides by day and comes out at night, and endangers blade pore-forming and incises, and when serious, can eat up mesophyll, only stays vein, or even stripping food cane skin
Layer.Larva can migrate in groups, slightly spun and landed by shock, there is seemingly-dead property.After 3-4 ages, dives daytime in plant lower part or soil seam, be close to
Evening removes feeding and causes harm.6-98 generations occurring within 1 year, occurs 7-8 months more, high temperature, dry year are more, and normal mixed with prodenia litura is sent out,
Leaf vegetables is threatened very big.The polyphagous pest-insect of the big generation of this intermittence of beet armyworm, in cotton since the nineties in last century
Since area's occurrence injury, the trend gradually aggravated is presented in occurrence injury in recent years, has spread over wild cabbage, Chinese cabbage, tomato, blueness
The various vegetables such as green pepper, potato, celery and soybean etc. more than 170 plant plant.Beet armyworm is caused harm wildness in recent years, population outbreak,
The many frequent medication of peasant still preventive effects are bad, have to capture to field sooner or later daily, field is hoped to heave a sigh.Bacillus thuringiensis
(Bacillus thuringiensis, abbreviation Bt) exists《Primary Jie Shi Bacteria Identifications handbook》The second class the tenth is belonged in 9th edition
Eight groups, Gram-positive, one kind of bacillus.During it forms gemma, in endobacillary one or both ends shape
The parasporal crystal (parasporal crystal) being the same or different at one or more shapes.This parasporal crystal it is main at
Point it is that there is the protein of insecticidal activity, therefore also known as insecticidal crystal protein (ICPs) or delta-endotoxin (δ-endotoxin).It should
Insecticidal crystal protein to various insects such as Lepidoptera, Diptera, coleoptera, Hymenoptera, Homoptera, Orthoptera, Mallophagas, and
Nematode, mite class and protozoan etc. have insecticidal activity (G.M.Faust etc. 1983 of specificity；D.L.Edwards etc. 1988；
F.Baroy etc. is 1998).Bt is free from environmental pollution to person poultry harmless, thus Bt has been obtained most widely in the biological control of pest
Using.In recent years, it has been found that a kind of new type disinsection toxalbumin, that is, Vegetative Insecticidal Proteins (VIPs) that Bt secretes in the trophophase,
It is different from the insecticidal crystal protein of Bt, is that one kind being secreted into extracellular toxin egg the phase in Bt nutrient growth mid-term and production born of the same parents
In vain, reach top (J.J.Estruch etc. 1996 early periods until stablizing；G.W.Warren etc. 1998；J.J.Estruch etc.
2000), it is generally not formed parasporal crystal.With thermal instability (E.Schnepf etc. 1998)；To Lepidoptera, coleoptera elder brother
Worm has the insecticidal activity of a wider spectrum, more even has the insect such as black cutworm of very strong resistance all to Bt insecticidal crystal proteins
Have more strongly active and all active to a series of pests such as Spodopterafrugiperda, beet armyworm, cigarette aphid noctuid, bollworms
(L.L.Loguercio etc. 2002), toxic effect is all in nanogram (ng) level.The discovery of the albumen makes Bt insecticides live in desinsection
Property, obtain very big breakthrough in terms of insecticidal range, overcome many pests to a certain extent to Bt endotoxin muting sensitive senses or unwise
The drawbacks of sense.It extremely enriches as it can be seen that VIPs is one and is the resource with great potential.The field has become Bt researchs
Hot spot.The foundation and application of PCR identification systems, greatly accelerate the separation of Bt bacterial strain screenings, biological activity determination, new gene
The speed of the items such as clone research.Studies have shown that it is to collect trophophase desinsection egg in Bt to be combined by PCR and biological activity determination
White active preferred tactful (L.L.Loguercio etc. 2002).The discovery of vip genes is with identification for the various of research Bt resources
Property, predict its insecticidal activity, quickly screen strain excellent, separation is cloned new vip genes and is of great significance.How to seek
Finding bacillus thuringiensis expression albumen has the gene of high virulence to become urgent need to solve the problem beet armywormSo hair
Bright bacillus thuringiensis vip3A-6 genes, expression albumen and its application, searching out an expression albumen has beet armyworm
High virulence is necessary with the bacillus thuringiensis vip3A-6 genes applied to biological control of insect pests.
Invention content
There is high virulence gene to beet armyworm to overcome the problems, such as to find bacillus thuringiensis expression albumen, the present invention
Provide a bacillus thuringiensis vip3A-6 gene, expression albumen and its application, the bacillus thuringiensis vip3A-6
Its expression albumen of gene, expression albumen and its application carries out primary dcreening operation and secondary screening to beet armyworm biological activity test, distinguishes after 7d
Number of dead and live insects is investigated, corrected mortality and LC are calculated50.As a result it is found that the vip3A-6 gene expression eggs of bacillus thuringiensis
It is in vain 4.783 μ g/g (2.234-7.784 μ g/g) to beet armyworm half lethal concentration in secondary screening, when expression albumen concentration reaches
When 81 μ g/g, all beet armyworms are dead, it is seen that vip3A-6 gene expressions albumen has high virulence to beet armyworm.
The technical solution adopted by the present invention to solve the technical problems is：
The nucleic acid sequence of bacillus thuringiensis vip3A-6 genes, expression albumen and its application is Seq ID No:1, tool
Body is built and verification process is as follows：
1. the PCR amplification of gene：The Bt bacterium genomic DNAs of extraction are subjected to PCR expansions using vip-5/vip-3 primer pairs
Increase, primer sequence is shown in Table 1.With reference to the N-terminal and C-terminal sequence homology of the gene coding regions vip3A announced in GenBank, design
A pair of of specific primer vip-5/vip-3 of synthesis introduces NdeI and SalI restriction enzyme sites (shown in arrow) at 5 ' ends respectively, is used for
Expand full-length gene.
2. 2.PCR reaction systems：50 μ L of KOD polymeric enzyme reactions system；1 μ L of KOD enzymes；1 μ L of primer pair (10mmol/L)；
1 μ L of template；10x PCR Buffer 5μL；MgSO43μL；dNTP 5μL；DdH2O complements to 50 μ L；
3. PCR reaction conditions (table 2)
4. DNA is recycled：(1) gel containing target DNA fragment is cut using sterilizing scalpel, be placed in 1.5mL centrifugations；
(2) the colloidal sol buffer solution A of 3 times of volume colloidal sol is added, is placed in 10min or so in 50 DEG C of water-baths；(3) add after glue is completely dissolved
Enter the B solution of 0.5 A colloidal sol buffer solution volume；(4) recovery column is transferred to after mixing, 12000r/min centrifuges 1min；(5) it removes
Residual liquid is added elution buffer W1 500 μ L, 12000r/min and centrifuges 1min；(6) residual liquid, elution buffer are removed
700 μ L of W2, elution is twice；(7) the TE solution of 30 μ L is added in recovery column, is stored at room temperature 2min；(8) by upper step recovery column
12000r/min centrifuges 1min, spare.It such as needs that 7-8 steps can be repeated primary.
5. connection reaction：By DNA glue recovery product and pEB carriers according to linked system (4 μ L of target fragment DNA, carrier
I 5 μ L of 1 μ L of DNA, connection kit Solution) it is attached reaction.After mixing well, under the conditions of 16 DEG C of 4h or 4 DEG C of connections
Connection is overnight.
6. Escherichia coli heat shock converts：Connection product full dose is added to the competent cell of 100 μ L e. coli jm109s
In, mixing, ice bath 30min or more takes out the accurate thermal shock 90s of 42 DEG C of centrifuge tube, then ice bath 3min, and the training of 900 μ L LB liquid is added
37 DEG C of base culture 1h or so is supported, 200 μ L is taken to be coated in LB solid plates, is added corresponding antibiotic, 40 μ L of X-gal solution,
4 μ L of IPTG solution, 37 DEG C of overnight incubations carry out blue day shift screening, and screening outgoing direction is connected correct positive colony, carried with PEB
The forward primer pEBF of body and the reverse primer of institute's clone gene carry out PCR identifications.The plasmid for extracting positive colony bacterial strain, is transferred to
In Escherichia coli Rosetta bacterial strain competent cells.Again by sequencing after the identifications such as PCR, digestion, what is identified is correct
Recombinant conversion carries out induction and express express target protein.
7. e. coli plasmid dna extracts：(1) picking positive transformant overnight incubation in LB liquid medium, takes 1-
4ml bacterium solutions centrifuge 1min in 12000r/min, discard supernatant；(2) it is outstanding that the S1 solution that 250 μ L contain 50mg/ml RNAase is added
It drifts along shallow lake；(3) 250 μ L bacterial lysates S2 are added and fully slowly leniently spin upside down 4-6 times, until solution transparent clear
Until, the step for should not be more than 5 minutes；(4) 350 μ L S3 neutralizers are added and fully leniently spin upside down 6-8 times,
It is centrifuged 10 minutes under 12000r/min；(5) supernatant is then drawn, is then transferred into and prepares in pipe, under the conditions of 12000r/min
It centrifuges 1 minute and discards filtrate；(6) it then centrifuges 1 minute and discards under the conditions of 500 μ L cleaning solutions W1,12000r/min of addition
Filtrate；(7) 700 μ L, W2 are then added, washes away excess salt, centrifuges 1 minute under the conditions of 12000r/min, be repeated once later；
(8) column will be collected to be transferred on new 1.5ml centrifuge tubes, collect column center be added dropwise 60-80 μ L be preheated to 65 DEG C TE buffer solutions or
Aqua sterilisa static 5 minutes, centrifuges 1 minute under the conditions of 12000r/min, air-dry and be stored in -20 DEG C it is spare.
8. the induced expression of gene：(1) single bacterium colony of Escherichia coli is inoculated under the conditions of 37 DEG C of LB liquid medium
220rpm activates 12-16h；(2) 1% inoculum concentration is used to be inoculated with positive expression bacterial strain in the conical flask of 200mL LB liquid mediums
In, under the conditions of 37 DEG C, 220rpm shaken cultivations about 2h to OD600=0.6 or so；The IPTG200 μ L of 1M are added later；It places
Induce about 4-16h, strain different condition also different under 150rpm low speed culture on shaking table, 16-30 DEG C of cryogenic conditions；(3) will
The culture solution of induction centrifuges 5min under conditions of 6000rpm and collects thalline, with the 10mmol/L TrisCl (pH of precooling
It is adjusted to 8.0 or so) suspension thalline 2-3 times；(4) ultrasonic disruption thalline in mixture of ice and water, super 3s stop 3s, and 50ml suspends
Bacterium solution about ultrasound 10min；(5) centrifugation 10min collects supernatant precipitation, precipitation group respectively under the conditions of 4 DEG C and 12000rpm
Divide and still 10mmol/L TrisCl is used to suspend.It is SDS-PAGE to carry out protein electrophoresis detection respectively later.
9. protein electrophoresis detects：Electrophoresis：Crude protein sample and 3x loadings Buffer are pressed 2:1 mixing, boiling water bath processing
10min takes analyte sample fluid 10L loadings, electrophoresis apparatus that 120V prerunning 10-15min are arranged, then carries out 150V constant pressure electrophoresis
1h.Dyeing：Protein gel is taken out after electrophoresis, is put into 50mLSI solution, and microwave stove heat 30s, 60rpm oscillation is set
5min；It is put into after glue is taken out in the mixed liquor of SII and SIII and (adds 200L SIII per 50mL SII), then microwave stove heat 30s
Shaking table 60rpm is taken out afterwards vibrates, gel imaging system imaging high-visible to protein adhesive band.
10. determination and analysis of sequence:By Beijing six directions lead to company complete sequencing, with Clone Manager, Omiga,
The softwares analytical sequence such as NCBI BLAST, DNAMAN, result are Seq ID No:1 and Fig. 4.
11. expressing biological activity determination of the albumen to beet armyworm:It weighs 30g man-made feeds to set in sterilizes culture dish, add
Enter 3mL testing sample solutions, stirred with spoon, is placed at room temperature for, feed excessive moisture is made to evaporate.By whole feeds point
In the tissue culture plate sterilized loaded on 3.First larva is gently shaken off on a blank sheet of paper, then blank sheet of paper is inverted, it is young at this time
Worm will glide in wire drawing since on blank sheet of paper, and with writing brush, gently larva is connected in culture plate by Pick Wire；After having connect, in culture plate upper cover
One layer of blowing paper, then lid lid, are used in combination rubber band to fix, stringent to seal, and prevent larva from escaping.It is positioned over 25 in illumination box
DEG C culture, the photoperiod is 12h illumination, and 12h is dark.From the 2nd day, humidity<When 30%, need additionally to add basin increase it is wet
Degree.Observation daily, checks whether illumination, humidity, temperature and feed go mouldy, if has Water vapor condensation.It expresses albumen pair
Beet armyworm biological activity test carries out primary dcreening operation and secondary screening, investigates number of dead and live insects after 7d respectively, calculates corrected mortality and LC50。
As a result it is found that the vip3A-6 gene expressions albumen of bacillus thuringiensis is to beet armyworm half lethal concentration in secondary screening
4.783 μ g/g (2.234-7.784 μ g/g), when expression albumen concentration reaches 81 μ g/g, all beet armyworms are dead, it is seen that
Vip3A-6 gene expressions albumen has high virulence to beet armyworm.
Beneficial effects of the present invention are：Bacillus thuringiensis vip3A-6 genes of the present invention, expression albumen and its apply it
It expresses albumen and primary dcreening operation and secondary screening is carried out to beet armyworm biological activity test, investigate number of dead and live insects after 7d respectively, it is dead to calculate correction
Die rate and LC50.As a result it is found that the vip3A-6 gene expressions albumen of bacillus thuringiensis causes beet armyworm half in secondary screening
Dead a concentration of 4.783 μ g/g (2.234-7.784 μ g/g), when expression albumen concentration reaches 81 μ g/g, all beet armyworms are equal
It is dead, it is seen that vip3A-6 gene expressions albumen has high virulence to beet armyworm；Shown with NCBI BLAST comparison results
Vip3A-6 gene orders are all different with existing all vip gene orders, similitude 99%, determine that vip3A-6 genes are one
New gene.
Description of the drawings
The following further describes the present invention with reference to the drawings.
Fig. 1 is nucleic acid sequence (the Seq ID No of bacillus thuringiensis vip3A-6 genes, expression albumen and its application:
1)。
Fig. 2 is the PCR amplification result figure of bacillus thuringiensis vip3A-6 genes, expression albumen and its application.
Fig. 3 is the full-length gene of bacillus thuringiensis vip3A-6 genes, expression albumen and its application in Escherichia coli
Protein expression result figure.
Fig. 4 is the sequence alignment result figure of bacillus thuringiensis vip3A-6 genes, expression albumen and its application.
Specific implementation mode
In the context of the present specification, otherwise any term used in this specification has this field unless specifically stated otherwise
Technical staff's normally understood meaning in the art, and the experimental method that detailed conditions are not specified is according to conventional methods
Or carried out according to the operational manual proposed by supplier.
Embodiment one
1. the PCR amplification of gene：
The Bt bacterium genomic DNAs of extraction are subjected to PCR amplification using vip-5/vip-3 primer pairs, primer sequence is shown in Table 1.
With reference to the N-terminal and C-terminal sequence homology of the gene coding regions vip3A announced in GenBank, a pair of of specific primer of design synthesis
Vip-5/vip-3 introduces NdeI and SalI restriction enzyme sites (shown in arrow), for expanding full-length gene at 5 ' ends respectively.
Table 1：Primer sequence
2.PCR reaction systems
3.PCR reaction conditions
Table 2：PCR reaction conditions
4.DNA is recycled
(1) gel containing target DNA fragment is cut using sterilizing scalpel, be placed in 1.5mL centrifugations；
(2) the colloidal sol buffer solution A of 3 times of volume colloidal sol is added, is placed in 10min or so in 50 DEG C of water-baths；
(3) B solution of 0.5 A colloidal sol buffer solution volume is added after glue is completely dissolved；
(4) recovery column is transferred to after mixing, 12000r/min centrifuges 1min；
(5) residual liquid is removed, elution buffer W1 500 μ L, 12000r/min is added and centrifuges 1min；
(6) residual liquid is removed, 700 μ L of elution buffer W2, elution is twice；
(7) the TE solution of 30 μ L is added in recovery column, is stored at room temperature 2min；
(8) upper step recovery column 12000r/min is centrifuged into 1min, it is spare.It such as needs that 7-8 steps can be repeated primary.
5. connection reaction
DNA glue recovery product is attached with pEB carriers according to following linked system and is reacted.After mixing well, 16 DEG C
It is connected overnight under the conditions of 4h or 4 DEG C of connection.
4 μ L of target fragment DNA
1 μ L of carrier DNA
Connect I 5 μ L of kit Solution
6. Escherichia coli heat shock converts
Connection product full dose is added in the competent cell of 100 μ L e. coli jm109s, mixing, ice bath 30min with
On, the accurate thermal shock 90s of 42 DEG C of centrifuge tube, then ice bath 3min are taken out, 900 μ L LB liquid mediums, 37 DEG C of culture 1h or so are added,
It takes 200 μ L to be coated in LB solid plates, corresponding antibiotic is added, 40 μ L, IPTG solution of X-gal solution, 4 μ L, 37 DEG C were cultivated
Night carries out blue day shift screening, and screening outgoing direction connects correct positive colony, with the forward primer pEBF of PEB carriers and institute gram
The reverse primer of grand gene carries out PCR identifications.The plasmid for extracting positive colony bacterial strain, is transferred to Escherichia coli Rosetta bacterial strain senses
In by state cell.Again by sequencing after the identifications such as PCR, digestion, correct recombinant conversion that identifies carry out induction and
Express express target protein.
7. e. coli plasmid dna extracts
(1) picking positive transformant overnight incubation in LB liquid medium takes 1-4ml bacterium solutions to be centrifuged in 12000r/min
1min is discarded supernatant；
(2) the S1 solution suspensions precipitation that 250 μ L contain 50mg/ml RNAase is added；
(3) 250 μ L bacterial lysates S2 are added and fully slowly leniently spin upside down 4-6 times, until solution is transparent clear
Until bright, the step for should not be more than 5 minutes；
(4) 350 μ L S3 neutralizers are added and fully leniently spin upside down 6-8 times, 10 points are centrifuged under 12000r/min
Clock；
(5) supernatant is then drawn, is then transferred into and prepares in pipe, centrifuged 1 minute under the conditions of 12000r/min and is discarded
Filtrate；
(6) it is then centrifuged 1 minute under the conditions of 500 μ L cleaning solutions W1,12000r/min of addition and discards filtrate；
(7) 700 μ L, W2 are then added, wash away excess salt, centrifuge 1 minute under the conditions of 12000r/min, later repeatedly one
It is secondary；
(8) column will be collected to be transferred on new 1.5ml centrifuge tubes, collects column center and the TE that 60-80 μ L are preheated to 65 DEG C is added dropwise
Buffer solution or aqua sterilisa static 5 minutes, centrifuge 1 minute under the conditions of 12000r/min, air-dry and be stored in -20 DEG C it is spare.
8. the induced expression of gene
(1) 220rpm activates 12-16h under the conditions of the single bacterium colony of Escherichia coli being inoculated in 37 DEG C of LB liquid medium；
(2) 1% inoculum concentration is used to be inoculated with positive expression bacterial strain in the conical flask of 200mL LB liquid mediums, 37 DEG C of items
Under part, 220rpm shaken cultivations about 2h to OD600=0.6 or so；The IPTG200 μ L of 1M are added later；It is positioned on shaking table
150rpm low speed cultures induce about 4-16h, strain different condition also different under 16-30 DEG C of cryogenic conditions；
(3) culture solution of induction is centrifuged to 5min under conditions of 6000rpm and collects thalline, with the 10mmol/L of precooling
TrisCl (pH is adjusted to 8.0 or so) suspension thalline 2-3 times；
(4) ultrasonic disruption thalline in mixture of ice and water, super 3s stop 3s, 50ml suspension bacteria liquids about ultrasound 10min；
(5) centrifugation 10min collects supernatant precipitation respectively under the conditions of 4 DEG C and 12000rpm, and deposited components are still used
10mmol/L TrisCl suspend.It is SDS-PAGE to carry out protein electrophoresis detection respectively later.
9. protein electrophoresis detects
Electrophoresis：Crude protein sample and 3x loadings Buffer are pressed 2:1 mixing, boiling water bath handle 10min, take analyte sample fluid
120V prerunning 10-15min are arranged in 10L loadings, electrophoresis apparatus, then carry out 150V constant pressure electrophoresis 1h.
Dyeing：Protein gel is taken out after electrophoresis, is put into 50mL SI solution, microwave stove heat 30s is set, and 60rpm shakes
Swing 5min；It is put into after glue is taken out in the mixed liquor of SII and SIII and (adds 200L SIII per 50mL SII), then microwave stove heat
Shaking table 60rpm is taken out after 30s vibrates, gel imaging system imaging high-visible to protein adhesive band.
3 SDS polyacrylamide gels of table prepare table
10. determination and analysis of sequence
Company is led to by Beijing six directions and completes sequencing, with Clone Manager, Omiga, NCBI BLAST, DNAMAN
Equal softwares analytical sequence, vip3A-6 gene orders are Seq ID No:1 (Fig. 1).
11. expressing biological activity determination of the albumen to beet armyworm
It weighs 30g man-made feeds to set in sterilizes culture dish, 3mL testing sample solutions is added, are stirred with spoon,
It is placed at room temperature for, feed excessive moisture is made to evaporate.Whole feeds are sub-packed in the tissue culture plate that 3 have sterilized.First by larva
Gently shake off on a blank sheet of paper, then blank sheet of paper is inverted, larva will glide in wire drawing since on blank sheet of paper at this time, gently be chosen with writing brush
Larva is connected in culture plate by silk；After having connect, in one layer of blowing paper of culture plate upper cover, then lid lid, it is used in combination rubber band to fix, sternly
Lattice seal, and prevent larva from escaping.25 DEG C of cultures in illumination box are positioned over, the photoperiod is 12h illumination, and 12h is dark.From the 2nd
It rises, humidity<When 30%, need additionally to add basin increase humidity.Observation daily checks illumination, humidity, temperature and feed
Whether go mouldy, if having Water vapor condensation.It is expressed albumen and carries out primary dcreening operation (table 4) and secondary screening to beet armyworm biological activity test
(table 5) investigates number of dead and live insects after 7d, calculates corrected mortality and LC respectively50.As a result it is found that bacillus thuringiensis
Vip3A-6 gene expressions albumen is 4.783 μ g/g (2.234-7.784 μ g/g) to beet armyworm half lethal concentration in secondary screening,
When expression albumen concentration reaches 81 μ g/g, all beet armyworms are dead, it is seen that vip3A-6 gene expressions albumen is to beet night
Moth has high virulence.
Table 4 expresses primary dcreening operation of the albumen to beet armyworm virulence
Table 5 expresses secondary screening of the albumen to beet armyworm virulence
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it will be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this hairs
Bright principle, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these variations
It all fall within the protetion scope of the claimed invention with improvement, its is equivalent by appended claims for the claimed scope of the invention
Object defines.
Claims (1)
1. the vip3A-6 genes of bacillus thuringiensis, it is characterised in that：Its nucleotide sequence such as Seq ID No:Shown in 1.
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