CN103898111B - The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone binding-protein gene - Google Patents
The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone binding-protein gene Download PDFInfo
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- CN103898111B CN103898111B CN201210576007.9A CN201210576007A CN103898111B CN 103898111 B CN103898111 B CN 103898111B CN 201210576007 A CN201210576007 A CN 201210576007A CN 103898111 B CN103898111 B CN 103898111B
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Abstract
The invention discloses the recombinant baculovirus of the dsRNA of a kind of expression inhibiting corpus allatum hormone binding-protein gene (HaJHBP gene) and the application in preparing insecticide thereof.The invention provides a kind of RNA molecule, for as follows (a) or (b): the RNA molecule shown in the sequence 7 of (a) sequence table；The RNA molecule of sequence 7 reverse complemental of (b) and sequence table.The DNA molecular encoding described RNA molecule falls within protection scope of the present invention.Recombinant vector, recombinant baculovirus, expression cassette, transgenic cell line or recombinant bacterium containing described DNA molecular belong to protection scope of the present invention.Bollworm as model insects, by suppression JH binding protein gene expression in insect bodies, can be promoted the baculovirus insecticidal power to insecticide, thus reach to control the insect purpose to murrain by the present invention.The present invention has substantial worth for agricultural production.
Description
Technical field
The present invention relates to the dsRNA of a kind of expression inhibiting corpus allatum hormone binding-protein gene recombinant baculovirus and
Its application in preparing insecticide.
Background technology
Insecticide cast off a skin and metamorphosis be complexity, high conservative again by the process of precision control, this process is mainly by 20-
Hydroxyecdysone (20-hydroxyecdysone, 20E) and sesquiterpenoids juvenile hormone (juvenile hormone, JH)
Common regulation and control.Enter after JH synthesis JH binding protein in blood, with blood (JH binding protein,
JHBP) combine, form complex and enter into target cell with blood circulation.
The function of JHBP is as follows: 1, transhipment JH is to target cell；2, JH and other oroteins non-specific binding are stoped,
Keep activity；3, JH is stoped to be decomposed.Insect hormone is as a new generation's insecticide, extremely strong in its selectivity, right
Higher mammal is without teratogenesis, carcinogenic, mutagenic action, fool proof to mammal, birds etc., has boundless
Application prospect.Gene in hormone sensitive lipase gene and metabolic pathway, causes ours as the gene with potential insect resistace
Interest.
RNA interference (RNA interference, RNAi) be a kind of newfound mechanisms of gene regulation, be by with target base
A kind of silencing specific genes phenomenon induced because of the double-stranded RNA (dsRNA) of sequence homology.RNAi is in functional genome
The aspects such as, the screening of drug target point, Cell signal propagation pathways analysis, disease treatment are all widely used potentiality.
Baculovirus (baculovirus) is the double-stranded cyclic DNA virus that a class has cyst membrane parcel, and Genome Size is
80-180kb.The architectural feature of baculovirus be virion be shaft-like, outside is coated one layer of albuminous membranae, outward appearance
There are certain rule, referred to as polyhedrosis virus (NPV).Baculovirus single-minded infection arthropod, its natural reservoir (of bird flu viruses) is main
For Lepidoptera, Hymenoptera and dipteral insect, also infect some shell-fish and demodicid mite class.In nature, baculovirus
Exist sprout virion (budded virus, BV) and embedded virus particle (occlusion-derived virus,
ODV；Also known as polyhedral body inclusion body, it is called for short PIB or OBs) two-strain form.BV is to propagate between insect cell
A kind of virion, with the envelope protein of encoding viral, can combine with the special receptor of cell surface,
Propagate in insect bodies or between the cell of cell line.ODV is baculovirus communication form in the environment, is embedded in
In inclusion body.Inclusion body is made up of polyhedrin protein, is polyhedral body cyst membrane outside it, the most highly stable,
Virion is played a protective role.After swallowing the food containing polyhedral body inclusion body without the larva infected, polyhedral body egg
Degrade under white acidic pH environment in insect midgut, releasing virus particle core capsid, viruses contact infected insect children
The epidermis cell tissue of intestinal in worm, manufactures virion of sprouting, and bud virion enters place by sprouting through cytoplasma membrane
In the haemocoele of main insecticide, causing system infections, in the baculovirus infection later stage, virion is by a large amount of polyhedrins
It is coated, forms polyhedral body inclusion body.Host insect is had higher pathogenic by baculovirus, to natural enemies security, the most dirty
Dye environment, especially it can form epidemic diseases and long-term control insect population and not developing immunity to drugs in pest population, bright
Show and be better than other insecticide.
Summary of the invention
It is an object of the invention to provide a kind of expression inhibiting corpus allatum hormone binding-protein gene (HaJHBP gene)
The recombinant baculovirus of dsRNA and the application in preparing insecticide thereof.
The invention provides a kind of RNA molecule, for as follows (a) or (b):
The RNA molecule (single strand RNA molecule or double stranded rna molecule) shown in sequence 7 of (a) sequence table；
The RNA molecule (single strand RNA molecule or double stranded rna molecule) of sequence 7 reverse complemental of (b) and sequence table.
The DNA molecular encoding described RNA molecule falls within protection scope of the present invention.
Described DNA molecular is following 1) or 2) or 3):
1) DNA molecular being made up of DNA fragmentation I, spacer and DNA fragmentation II successively；Described DNA fragmentation I is such as
The sequence 6 of sequence table is from shown in the 1st to 483 nucleotide of 5 ' end；Described DNA fragmentation II such as the sequence of sequence table
Row 6 are from shown in the 697th to 1179 nucleotide of 5 ' end；
2) DNA molecular shown in sequence 6 of sequence table；
3) sequence 2 of sequence table is from the DNA molecular shown in the 207th to 689 nucleotide of 5 ' end.
Recombinant vector, recombinant baculovirus, expression cassette, transgenic cell line or recombinant bacterium containing described DNA molecular
Belong to protection scope of the present invention.
In described recombinant vector, can be by op166 promoter (baculovirus early promoter) and/or P10 promoter (bar
Shape virus late promoter) start the expression of described DNA molecular.Described op166 promoter is concrete such as the sequence of sequence table
Shown in row 5.Described P10 promoter is specifically as shown in the sequence 8 of sequence table.
Described recombinant vector can have expression cassette first and expression cassette second；Described expression cassette first is for being used for expressing described DNA molecular
Expression cassette；Described expression cassette second is to be used for expressing the encoding gene of baculovirus polyhedrin (Ha polh albumen)
Expression cassette.
Described recombinant vector is concretely by described op166 promoter, described DNA molecular and described polyhedrin
Encoding gene inserts expression vector respectively (such as carrier pFastBacTMDUAL) recombiant plasmid that multiple clone site obtains
M.Described op166 promoter can be inserted into carrier pFastBacTMBetween NcoI and the SmaI restriction enzyme site of DUAL.Institute
State described DNA molecular and can be inserted into pFastBacTMThe PvuII restriction enzyme site of DUAL.The coding base of described polyhedrin
Because can be inserted into carrier pFastBacTMBetween BamHI and the HindIII site of DUAL.
Described expression cassette first and described expression cassette second are concretely inserted in Hz8 egt Bacmid by shown recombinant vector
The recombiant plasmid N that obtains of attB site.
Described recombinant vector concretely will include described expression cassette between Tn7L and Tn7R site in described recombiant plasmid M
The recombiant plasmid that first and described expression cassette second attB site in interior DNA fragmentation inserts Hz8 egt Bacmid obtain
Q。
Described Hz8 egt Bacmid is by the steroidal uridine diphosphate glucose transferring enzyme of casting off a skin in HaBacHZ8Bacmid
Obtain after gene (also known as EcdysteroidUDP-Glucosyltransferase gene, be called for short egt gene) inactivation
Recombiant plasmid.Described Hz8 egt Bacmid is specially and is replaced by the egfp gene shown in the sequence 9 of sequence table
The open reading frame of the egt gene in HaBacHZ8Bacmid is from the DNA shown in 116 to 1470 nucleotide of 5 ' end
The recombiant plasmid that fragment obtains.
Described recombinant baculovirus can be to sprout that (polyhedral body is forgiven for the form of the form of virion or polyhedral body inclusion body
Body, compared with virion of sprouting, has the ability of oral infection).
The virion of sprouting of described recombinant baculovirus specifically can be prepared via a method which to obtain: by described restructuring matter
Grain N or described recombiant plasmid Q imports insect cell (such as bollworm HzAm1 cell) and cultivates, and collects and cultivates
Supernatant, be containing described in sprout the virus liquid of virion.
The polyhedral body inclusion body of described recombinant baculovirus can be prepared via a method which to obtain: by described recombiant plasmid N
Or described recombiant plasmid Q imports insect cell (such as bollworm HzAm1 cell) and cultivates, collect cell precipitation,
Cell precipitation i.e. contains described polyhedral body inclusion body.The polyhedral body inclusion body of described recombinant baculovirus can be by by institute
Stating sprouts virion inoculation insecticide and collect insect bodies obtains.The polyhedral body inclusion body tool of described recombinant baculovirus
Body can be prepared via a method which to obtain: by the described virion inoculation insecticide that sprouts, collects corpse also after insect death
Smash to pieces, by filtered through gauze after adding water and stirring evenly, collect filtrate and 300g is centrifuged 10min, collect supernatant 3000g
Centrifugal 30min, collects precipitation, is described polyhedral body inclusion body.Described insecticide can be bollworm.
Baculovirus polyhedrin described in any of the above is following (c) or (d): (c) is by sequence 3 in sequence table
The protein of shown aminoacid sequence composition；D the aminoacid sequence of sequence 3 is passed through one or several aminoacid by ()
The replacement of residue and/or disappearance and/or interpolation and have polyhedrin function the protein derived by sequence 3.
The encoding gene of baculovirus polyhedrin described in any of the above can be following 4) or 5) or 6) or 7)
DNA molecular: 4) sequence 4 of sequence table is from the DNA molecular shown in 5 ' end 157-897 position nucleotide；5) sequence
DNA molecular shown in sequence 4 in list；6) under strict conditions with 4) or 5) the DNA sequence hybridization that limits and
The DNA molecular of coding polyhedrin；7) with 4) or 5) DNA sequence that limits have more than 90% homology and
The DNA molecular of coding polyhedrin.
The present invention also protects a kind of insecticide, and its active component is that suppression corpus allatum hormone binding-protein gene is expressed
Material.Described insecticide can be bollworm insecticide.The thing that described suppression corpus allatum hormone binding-protein gene is expressed
Matter can be for RNA molecule, arbitrary described DNA molecular, arbitrary described recombinant vector, arbitrary described table described in any of the above
Reach box, arbitrary described transgenic cell line, arbitrary described recombinant bacterium or arbitrary described recombinant baculovirus.
The present invention also protects material the answering in preparing insecticide that suppression corpus allatum hormone binding-protein gene is expressed
With.Described insecticide can be bollworm insecticide.The material that suppression corpus allatum hormone binding-protein gene is expressed can be
RNA molecule.The material that described suppression corpus allatum hormone binding-protein gene is expressed can divide for RNA described in any of the above
Described DNA molecular, arbitrary, arbitrary described recombinant vector, arbitrary described expression cassette, arbitrary described transgenic cell
System, arbitrary described recombinant bacterium or arbitrary described recombinant baculovirus.
Corpus allatum hormone associated proteins described in any of the above is following (e) or (f): (e) is by sequence 1 in sequence table
The protein of shown aminoacid sequence composition；F the aminoacid sequence of sequence 1 is passed through one or several aminoacid by ()
The replacement of residue and/or disappearance and/or interpolation and have identical function the protein derived by sequence 1.
Corpus allatum hormone binding-protein gene described in any of the above is following 8) or 9) or 10) or 11) DNA
Molecule: 8) sequence 2 of sequence table is from the 12nd to 740 shown DNA molecular of 5 ' end；9) sequence of sequence table
DNA molecular shown in row 2；10) under strict conditions with 8) or 9) the DNA sequence hybridization that limits and coding described
Protein DNA molecule；11) with 8) or 9) DNA sequence that limits has more than 90% homology and coding is described
Protein DNA molecule.
The present invention using bollworm as model insects, by by suppression JH binding protein gene in insect bodies
Expression, can promote the baculovirus insecticidal power to insecticide, thus reach to control the insect mesh to murrain
's.The present invention has substantial worth for agricultural production.
Accompanying drawing explanation
Fig. 1 is the structural representation of recombiant plasmid pFBD-HaPH-op166-HaJHBPRNAi.
Fig. 2 is the structural representation of the genome of HaBac △ egt.
Fig. 3 is Hz8 egt Bacmid and the PCR of HaBac △ egt-HaJHBPRNAi bacmid identifies electrophoretogram；
1,3:HaBac △ egt-HaJHBPRNAi bacmid:2,4:Hz8 egt Bacmid.
Fig. 4 is the fluorescence photo after cultivating 3 days in embodiment 4.
Fig. 5 is the electrophoretogram of pcr amplification product in embodiment 6.
Fig. 6 is the median lethal time of polyhedral body inclusion body in embodiment 7.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.The English full name of Bacmid is Baculovirus plasmid, and Chinese implication is shaft-like disease
Toxin grain.Grace ' s culture medium powder: purchased from Invitrogen.Escherichia coli DH10B: purchased from Invitrogen
Company, article No. 18290-015.
Carrier pFastBacTMDUAL: list of references: Bac-to-Bac Baculovirus Expression System
Version D 6 April 2004。
Helper plasmid: list of references: Bac-to-Bac Baculovirus Expression System Version D
6 April 2004。
This plasmid of HaBacHZ8Bacmid(has improved Baculovirus Gene group DNA, and described transformation refers to:
Polyhedron gene inserts the attB site inserted for transposable element, former polyhedrin gene inactive): reference
Document: Han-zhong Wang, Fei Deng, Pijlman, Gorben P, Xin-wen Chen, Xiu-lian
Sun,Vlak,Just M,Zhi-hong Hu.Cloning of biologically active genomes from a
Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by
using a bacterial artificial chromosome.Virus Res.2003Nov;97(2):57-63.；
Yan-Hong SI,Ming-Gang FANG,Yi HUANG,Fang-Liang ZHENG,Ting LI,Zhi-Hong HU,
Han-Zhong WANG.Construction and Characterization of a Helicoverpa armigera
Nucleopolyhedrovirus Bacterial Artificial Chromosome with Deletion of
Ecdysteroid UDP-Glucosyltransferase Gene.Bioscience,Biotechnology,and
Biochemistry Vol.71(2007)No.10P2435-2441。
Bollworm HzAm1 cell: Zhang Youhong;Wang Hualin;Zhu Xiongwei;National muscial instrument;Chen Yan;Lv Zhong;Ma Jie;Zhang Jing;It is suitable for cotton boll
The Screening of Media of worm cell HzAm1 growth and low serum domestication [J];Biological engineering journal;04 phase in 2006.
Bollworm (Helicoverpa armigera): list of references: Zhao X., K.Wu, G.Liang and
Y.Guo.2009.Modified female calling behavior in Cry1Ac-resistant Helicoverpa
armigera(Lepidoptera:Noctuidae).Pest Management Science.65(4):353-357.。
Shown in the sequence 1 of the JH binding protein of bollworm such as sequence table (being made up of 242 amino acid residues).Cotton
The cDNA of the encoding gene (HaJHBP gene) of the JH binding protein of earworm as shown in the sequence 2 of sequence table, its
The sequence 2 of open reading frame such as sequence table is from the 12nd to 740 nucleotide composition of 5 ' end.
LB culture medium (pH7.0): solvent is that water, solute and concentration thereof are as follows: yeast extract 5g/L, peptone 10g/L,
NaCl 10g/L。
Embodiment 1, the structure of interference carrier
1, composition sequence table double chain DNA molecule shown in sequence 4 (polyhedron gene of bollworm, also known as
Ha polh gene, its open reading frame is that the sequence 4 of sequence table is from shown in 5 ' end 157-897 position nucleotide；
The Ha polh albumen shown in sequence 3 of Ha polh gene coded sequence table) and it is inserted into carrier
pFastBacTMBetween BamHI and the HindIII site of DUAL, obtain recombiant plasmid pFBD-HaPH.
2, (baculovirus early promoter, also known as op166 for the double chain DNA molecule shown in sequence 5 of composition sequence table
Promoter) and be inserted between NcoI and the SmaI restriction enzyme site of recombiant plasmid pFBD-HaPH, obtain matter of recombinating
Grain pFBD-HaPH-op166.
3, the double chain DNA molecule shown in sequence 6 of composition sequence table is (by its named palindrome fragment；The sequence of sequence table
In row 6, from 5 ' end 1-483 position nucleotide and 697-1179 position nucleotide reverse complementals, 484-696
Position nucleotide is spacer) and it is inserted into the PvuII restriction enzyme site of recombiant plasmid pFBD-HaPH-op166, obtain weight
Group plasmid pFBD-HaPH-op166-HaJHBPRNAi.The knot of recombiant plasmid pFBD-HaPH-op166-HaJHBPRNAi
Fig. 1 is shown in by structure schematic diagram.
Embodiment 2, the preparation of Hz8 egt Bacmid
Open reading by the egt gene in the egfp gene replacement HaBacHZ8Bacmid shown in the sequence 9 of sequence table
Frame, from the DNA fragmentation shown in 116 to 1470 nucleotide of 5 ' end, obtains Hz8 egt Bacmid.
Embodiment 3, the acquisition of HaBac △ egt-HaJHBPRNAi bacmid
Mechanism description: in escherichia coli, Helper plasmid promotes the generation of swivel base, recombiant plasmid
Palindrome fragment and Ha polh gene is included between Tn7L and the Tn7R site of pFBD-HaPH-op166-HaJHBPRNAi
(inserting the polyhedrin gene inactive of the Hz8 egt Bacmid caused in order to cover exogenous sequences) is interior
DNA fragmentation inserts the attB site of Hz8 egt Bacmid by transposition, obtains recombinant baculovirus plasmid.
1, Helper plasmid is imported the competent cell of escherichia coli DH10B, obtains recombinant bacterium I.
2, the recombinant bacterium I obtained by Hz8 egt Bacmid steps for importing 1, obtains recombinant bacterium II.
3, the recombiant plasmid pFBD-HaPH-op166-HaJHBPRNAi step of converting 2 embodiment 1 obtained obtains
Recombinant bacterium II, then coat LB culture medium flat plate (containing 50 μ g/mL kanamycin, 7 μ g/mL gentamycins,
10 μ g/mL tetracyclines, 100 μ g/mL X-gal and 40 μ g/mL IPTG), cultivate 24-48 hour for 37 DEG C, check
Blue white macula on flat board, white colony is rule again a fresh LB culture medium flat plate (containing 50 μ g/mL cards that
Mycin, 7 μ g/mL gentamycins, 10 μ g/mL tetracyclines, 100 μ g/mL X-gal and 40 μ g/mL IPTG),
37 DEG C cultivate 24-48 hour, extracting waste bacterium colony, be containing recombinant baculovirus plasmid (also known as
HaBac △ egt-HaJHBPRNAi bacmid) bacterium colony.
The PCR of Hz8 egt Bacmid and HaBac △ egt-HaJHBPRNAi bacmid identifies that electrophoretogram is shown in Fig. 3,
Use primer (5 '-CCCAGTCACGACGTTGTAAAACG-3 ') and the correspondence institute of corresponding Hz8 egt Bacmid
State the primer pair that the primer (5 '-GAAATTTTTGAATCTTGCAGTCAGC-3 ') of palindrome fragment forms,
The purpose fragment of HaBac △ egt-HaJHBPRNAi bacmid display about 2.1kb, Hz8 egt Bacmid does not shows
Show described purpose fragment.Use the primer of P2R and P2F composition to (P2R:GGCCTTTTGTAAATCATCT；P2F:
TGGTCGATGAAGGCTTGGGA), HaBac △ egt-HaJHBPRNAi bacmid shows the purpose fragment of about 350bp,
Hz8 egt Bacmid does not show described purpose fragment.
Embodiment 4, the acquisition of recombinant baculovirus (virion of sprouting)
One, the acquisition of recombinant baculovirus (virion of sprouting)
1, inoculation 5 × 10 in the capsule (Grace ' the s culture medium equipped with containing 10%FBS) of 35mm5Individual bollworm
HzAm1 cell, 27 ° of C overnight incubation, inhale in Biohazard Safety Equipment and abandon supernatant, add 1ml Grace ' s culture medium
Room temperature is placed 1 hour.
2, by 5 μ g HaBac △ egt-HaJHBPRNAi bacmid and 10 μ l Lipofectin respectively with Grace '
S culture medium is diluted to 100 μ l, then both is mixed, and mixes once every 7min, after 45min, adds 400 μ l
Grace ' s culture medium, mixing.
3, taking into the capsule of step 1, inhale and abandon supernatant and add the mixed liquor that step 2 obtains, 27 ° of C cultivate 30min
(every 15min shakes up capsule once)；Being subsequently adding 400 μ l Grace ' s culture medium, mixing, 27 ° of C cultivate 6
Hour；Being subsequently adding 1mL Grace ' the s culture medium containing 20%FBS, mixing, 27 ° of C collect after cultivating 6 days
Clear liquid, is the f1 disease venom of recombinant baculovirus (also known as HaBac △ egtHaJHBPRNAi).27 ° of C cultivate
During 6 days, under fluorescence inverted microscope, observe the generation situation of green fluorescence every day, glimmering after cultivating 3 days
Radiograph is shown in Fig. 4 (it is observed that green fluorescence, represent and infect successfully).
4, at 25cm2Batch cultur bottle in press MOI=0.1 dosage f1 disease venom infect bollworm HzAm1
Cell, cultivates for 27 DEG C and collects supernatant after 96 hours, be second filial generation virus liquid.
5, at 25cm2Batch cultur bottle in press MOI=0.1 dosage second filial generation virus liquid infect bollworm HzAm1
Cell, cultivates for 27 DEG C and collects supernatant after 96 hours, be third generation virus liquid.
Two, the acquisition of control baculovirus (virion of sprouting)
Replace HaBac △ egt-HaJHBPRNAi bacmid to carry out step one with Hz8 △ egt Bacmid, obtain shaft-like
F1 disease venom, second filial generation virus liquid and the third generation virus liquid of virus (also known as HaBac △ egt).HaBac△
The structural representation of the genome of egt is shown in Fig. 2.
Three, the acquisition of control baculovirus (virion of sprouting)
Recombiant plasmid pFBD-HaPH-op166 replacement recombiant plasmid pFBD-HaPH-op166-HaJHBPRNAi is carried out reality
Execute example 3, the DNA fragmentation including Ha polh gene between Tn7L and the Tn7R site of carrier pFBD-HaPH
The polyhedron gene site (polyhedrin locus) of Hz8 egt Bacmid is inserted by transposition,
To recombinant baculovirus plasmid (also known as HaBac △ egt-A bacmid).
Replace HaBac △ egt-HaJHBPRNAi bacmid to carry out step one with HaBac △ egt-A bacmid, obtain
F1 disease venom, second filial generation virus liquid and the third generation virus liquid of baculovirus (also known as HaBac △ egt-A).
Embodiment 5, the acquisition of polyhedral body inclusion body
One, the acquisition of HaBac △ egtHaJHBPRNAi polyhedral body inclusion body
1, bollworm was supported to 3 age Mos, put to the most numb with cold before injection, took HaBac △ with the microsyringe of sterilization
The third generation virus liquid of egtHaJHBPRNAi, second from the bottom at bollworm abdominal foot and the 3rd between be injected in hemolymph,
Every bollworm injects 5 μ l, and after 5 days, bollworm liquefaction death, collects worm corpse.
2, add distilled water stirring evenly after being smashed to pieces by the worm corpse of step 1, with 3 layers of filtered through gauze and collect filtrate, will filter
Liquid 300g is centrifuged 10min and collects supernatant, supernatant carries out 3000g and is centrifuged 30min, collects precipitation (HaBac
△ egtHaJHBPRNAi polyhedral body inclusion body).
Two, the acquisition of HaBac △ egt polyhedral body inclusion body
The third generation virus liquid of HaBac △ egtHaJHBPRNAi is replaced with the third generation virus liquid of HaBac △ egt, its
It is with step one, obtains HaBac △ egt polyhedral body inclusion body.
Three, the acquisition of HaBac △ egt-A polyhedral body inclusion body
The third generation virus liquid of HaBac △ egtHaJHBPRNAi is replaced with the third generation virus liquid of HaBac △ egt-A,
Other is with step one, obtains HaBac △ egt-A polyhedral body inclusion body.
Embodiment 6, the half lethal dose of polyhedral body inclusion body
The HaBac △ egtHaJHBPRNAi polyhedral body inclusion body, the HaBac △ egt that embodiment 5 are prepared respectively are polygonal
Body inclusion body and HaBac △ egt-A polyhedral body inclusion body carry out the detection of following half lethal dose:
1, select uniform two age end cotton bollworm larvaes, be placed in 96 aseptic orifice plates, 28 DEG C of constant temperature illumination trainings
Hungry cultivation 16 hours in supporting case, allow they decortications enter in next age.
2, the sterilized water that polyhedral body inclusion body eats light blue with the sucrose containing 4g/100mL and 1mg/mL is diluted to 1
×106Individual polyhedral body inclusion body/mL, 3 × 105Individual polyhedral body inclusion body/mL, 1 × 105Individual polyhedral body inclusion body/mL, 3
×104Individual polyhedral body inclusion body/mL or 1 × 104The suspension of individual polyhedral body inclusion body/mL is (many with blood counting chamber counting
Angle body inclusion body).
3, the cotton bollworm larvae completing step 1 is divided into five groups, often 48 larvas of group, obtains by step 2 respectively
Suspension feeds, and from feeding beginning timing, the larva that in 10min, middle intestinal becomes blueness proceeds to manually raise containing fresh
In 24 orifice plates of material, observe 2 every day and add up mortality rate until all confession is tried dead larvae or pupate.Carry out three
Secondary repetition is tested.The median lethal dense dosage (LC of polygonal inclusion body is calculated with Probit regression analysis50) and standard error,
Relatively two-strain LC50Between the significance of difference.
The half lethal concentration of HaBac △ egtHaJHBPRNAi polyhedral body inclusion body is 2.26 × 104Individual polyhedral body inclusion body
/ ml, standard deviation is 0.81 × 104Individual polyhedral body inclusion body/ml, 95% confidence interval is 1.06-4.21 × 104Individual polygonal
Body inclusion body/ml.The half lethal concentration of HaBac △ egt polyhedral body inclusion body is 8.12 × 104Individual polyhedral body inclusion body
/ ml, standard deviation is 1.16 × 104Individual polyhedral body inclusion body/ml, 95% confidence interval is 6.16-10.7 × 104Individual polygonal
Body inclusion body/ml.The half lethal concentration of two kinds of polyhedral body inclusion bodys has significant difference (z=7.27, p < 0.05).HaBac
The half lethal concentration of △ egt-A polyhedral body inclusion body is consistent with the half lethal concentration of HaBac △ egt polyhedral body inclusion body.
4, take bollworm dead in step 3, extract total serum IgE and reverse transcription is cDNA, with cDNA as template, adopt
It is reference gene with ACTIN gene, is identified the expression of HaJHBP gene by semiquantitive PCR.
As follows for identifying the primer of ACTIN gene:
Forward primer: 5 '-CCTGGTATTGCTGACCGTATGC-3 '；
Downstream primer: 5 '-CTGTTGGAAGGTGGAGAGGGAA-3 '.
As follows for identifying the primer of HaJHBP gene:
Forward primer: 5 '-ATTGCTTGCATTTGCGAGTTGTGT-3 '；
Downstream primer: 5 '-TCCGGGAAACCATTGCTTGT-3 '.
The electrophoretogram of pcr amplification product is shown in Fig. 5.Compared with the bollworm feeding HaBac △ egt polyhedral body inclusion body,
Feed the expression of HaJHBP gene in the Helicoverpa armigera of HaBac △ egtHaJHBPRNAi polyhedral body inclusion body significantly to drop
Low.
Result shows, the dsRNA of described palindrome fragment expression can promote HaJHBP gene expression amount to lower, so that
The half lethal concentration of baculovirus declines, and is greatly improved the baculovirus lethal efficiency to bollworm.
Embodiment 7, the median lethal time of polyhedral body inclusion body
The HaBac △ egtHaJHBPRNAi polyhedral body inclusion body, the HaBac △ egt that embodiment 4 are prepared respectively are polygonal
Body inclusion body and HaBac △ egt-A polyhedral body inclusion body carry out the detection of following median lethal time:
1, select uniform two age end cotton bollworm larvaes, be placed in 96 aseptic orifice plates, 28 DEG C of constant temperature illumination trainings
Hungry cultivation 16 hours in supporting case, allow they decortications enter in next age.
2, it is diluted to hang by the sterilized water that polyhedral body inclusion body eats light blue with the sucrose containing 4g/100mL and 1mg/mL
Liquid.
3, taking into 96 larvas of step 1, the suspension obtained by step 2 carries out feeding that (feeding dosage is 100
Times LC50), from feeding beginning timing, the larva that in 10min, middle intestinal becomes blueness proceeds to 24 containing fresh man-made feeds
In orifice plate, observe 4 times (respectively 7:00,13:00,18:00 and 23:00) every day and add up mortality rate until owning
For trying dead larvae or pupating.Carry out three times repeating experiment.
The infected test dead larvae time is carried out survival analysis (SPSS software, Life-table or
Kaplan-Meier method), and calculate ST50Value, analysis result is shown in Fig. 6.HaBac △ egtHaJHBPRNAi is polygonal
The median lethal time of body inclusion body is 68 hours (meansigma methodss of three tests).HaBac △ egt polyhedral body inclusion body
Median lethal time is 119 hours (meansigma methodss of three tests).The semilethal of HaBac △ egt-A polyhedral body inclusion body
Time is consistent with the median lethal time of HaBac △ egt polyhedral body inclusion body.
Claims (6)
1. the recombinant vector containing target DNA molecule, it is characterised in that: described recombinant vector has expression cassette first and
Expression cassette second；Described expression cassette first is the expression cassette for target DNA molecule；Described expression cassette second is for being used for expressing shaft-like disease
The expression cassette of the encoding gene of poison polyhedrin；
In described expression cassette first, op166 promoter and/or P10 promoter start the expression of target DNA molecule；
Described target DNA molecule is following 1) or 2):
1) DNA molecular being made up of DNA fragmentation I, spacer and DNA fragmentation II successively；Described DNA fragmentation I is such as
The sequence 6 of sequence table is from shown in the 1st to 483 nucleotide of 5 ' end；The sequence 6 of described DNA fragmentation II such as sequence table is from 5 '
Shown in the 697th to 1179 nucleotide of end；
2) DNA molecular shown in sequence 6 of sequence table；
Described recombinant vector is by described expression cassette first and described expression cassette second are inserted the attB position in Hz8 egt Bacmid
Point obtains；
Described Hz8 egt Bacmid is by the steroidal uridine diphosphate glucose transferase gene of casting off a skin in HaBacHZ8Bacmid
The recombiant plasmid obtained after inactivation；
Described baculovirus polyhedrin is made up of the aminoacid sequence shown in sequence in sequence table 3.
Recombinant vector the most according to claim 1, it is characterised in that: described target DNA molecule encoding one RNA
Molecule；
Described RNA molecule, for as follows (a) or (b):
The RNA molecule shown in sequence 7 of (a) sequence table；
The RNA molecule of sequence 7 reverse complemental of (b) and sequence table.
3. one kind contains target DNA molecular recombination baculovirus, it is characterised in that:
Described target DNA molecule is following 1) or 2):
1) DNA molecular being made up of DNA fragmentation I, spacer and DNA fragmentation II successively；Described DNA fragmentation I is such as
The sequence 6 of sequence table is from shown in the 1st to 483 nucleotide of 5 ' end；The sequence 6 of described DNA fragmentation II such as sequence table is from 5 '
Shown in the 697th to 1179 nucleotide of end；
2) DNA molecular shown in sequence 6 of sequence table；
Described recombinant baculovirus is the sprout form of virion or the form of polyhedral body inclusion body；
The virion of sprouting of recombinant baculovirus is prepared via a method which to obtain: by the restructuring matter described in claim 1 or 2
Grain imports insect cell and also cultivates, and collects culture supernatant, be containing described in sprout the virus liquid of virion.
Recombinant baculovirus the most according to claim 3, it is characterised in that: described target DNA molecule encoding one RNA
Molecule；Described RNA molecule, for as follows (a) or (b):
The RNA molecule shown in sequence 7 of (a) sequence table；
The RNA molecule of sequence 7 reverse complemental of (b) and sequence table.
5. an insecticide, its active component is the material that suppression corpus allatum hormone binding-protein gene is expressed；
Described " material that suppression corpus allatum hormone binding-protein gene is expressed " is at least one in following material:
(I) recombinant baculovirus described in claim 3 or 4；
(II) recombinant vector described in claim 1 or 2.
6. the material that suppression corpus allatum hormone binding-protein gene is expressed application in preparing insecticide；Described " suppression insecticide
The material of JH binding protein gene expression " be at least one in following material:
(I) recombinant baculovirus described in claim 3 or 4；
(II) recombinant vector described in claim 1 or 2.
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| CN1429910A (en) * | 2001-12-31 | 2003-07-16 | 中国科学院武汉病毒研究所 | Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method |
| CN102464710A (en) * | 2010-11-09 | 2012-05-23 | 中国科学院遗传与发育生物学研究所 | Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof |
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| CN1429910A (en) * | 2001-12-31 | 2003-07-16 | 中国科学院武汉病毒研究所 | Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method |
| CN102464710A (en) * | 2010-11-09 | 2012-05-23 | 中国科学院遗传与发育生物学研究所 | Helicoverpa armigera juvenile hormone binding protein (Ha-JHBP) and encoding gene and application thereof |
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