CN103898112A - Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone methyltransgerase gene - Google Patents

Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone methyltransgerase gene Download PDF

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CN103898112A
CN103898112A CN201210576020.4A CN201210576020A CN103898112A CN 103898112 A CN103898112 A CN 103898112A CN 201210576020 A CN201210576020 A CN 201210576020A CN 103898112 A CN103898112 A CN 103898112A
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sequence
recombinant
gene
rna molecule
baculovirus
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CN103898112B (en
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朱祯
王晓芳
戴艳
魏晓丽
张磊
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Institute of Genetics and Developmental Biology of CAS
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Institute of Genetics and Developmental Biology of CAS
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Abstract

The invention discloses a recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone methyl transferase gene (HaJHAMT gene), and its application in the preparation of insecticides. The invention provides an RNA molecule. The RNA molecule is one of RNA molecules comprising an RNA molecule (a) represented by sequence 7 in a sequence table and an RNA molecule (b) reversely complementing with the sequence 7 in the sequence table. DNA molecules coding the RNA molecule also belong to the scope of protection of the invention. Recombinant vectors containing the DNA molecules, recombinant baculoviruses containing the DNA molecules, expression cassettes containing the DNA molecules, transgenic cell lines containing the DNA molecules or recombinant bacteria containing the DNA molecules also belong to the scope of protection of the invention. In the invention, cotton bollworm is used as a model insect, and the expression of the juvenile hormone methyltransgerase gene in the insect is inhibited, so the insect killing effectiveness of baculoviruses can be promoted in order to control the harms of insects to crops. The recombinant baculovirus is valuable to agricultural production.

Description

The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone methyl transferase gene
Technical field
The present invention relates to a kind of expression inhibiting corpus allatum hormone methyl transferase gene dsRNA recombinant baculovirus and in the application of preparing in sterilant.
Background technology
The growing of insect, cast off a skin, participation that physiological process and the behavior reaction etc. such as metamorphosis, diapause, reproduction, polytypism all be unable to do without hormone.Neotonin (juvenile hormone, JH) be the terpene compound of a kind of lower molecular weight, fat-soluble, nonprotein, verified have 7 kinds of natural JH to exist, specific as follows: (1) JH0, (2) JHI, (3) JHII, (4) JHIII, (5) 4-methyl-JHI, (6) JHIII-bisepoxide, (7) mehtyl famesoate.Wherein (1), (2), (3), (5) are present in lepidopteran, and (6) are present in Diptera, and (7) are present in cockroach, and (4) all exist in all insects.JH is synthesized by corpus allatum and discharges, and is the distinctive hormone of arthropods, does not see in other animal.
The biosynthetic pathway of JH is mainly: caproic acid, propionic acid → mevalonic acid (MVA) → isopentenyl pyrophosphate (IPP) → farnesyl pyrophosphate → method Buddhist nun acid (FA) → JH.Acting as of neotonin methyltransgerase (JHAMT) forms methyl method Buddhist nun ester by the Methyl transporters in external source S-adenosine-methionine(Met) molecule to method Buddhist nun acid molecule.The route of synthesis of method acid → JH, because of caste different different.Methyl-esterified and epoxidation are the synthetic peculiar reactions of JH, and corresponding methyltransgerase (JHAMT), JH cyclooxygenase are the synthetic peculiar reaction enzymes of JH.
Insect hormone has become major fields of sterilant research and development, its mechanism of action is different from the past acts on neural conventional pesticides, toxicity is low, pollute less, little on natural enemy and beneficial organism impact, contribute to sustainable agriculture development, be conducive to that pollution-free green food is produced, health is beneficial to man.
It is a kind of newfound mechanisms of gene regulation that RNA disturbs (RNA interference, RNAi), is by the reticent phenomenon of a kind of specific gene of inducing with the double-stranded RNA (dsRNA) of target-gene sequence homology.The RNAi potentiality that are all widely used at aspects such as functional genomics, the screening of drug target point, cell signaling path analysis, disease treatments.
Baculovirus (baculovirus) is the double-stranded cyclic DNA virus that a class has cyst membrane parcel, and Genome Size is 80-180kb.It is shaft-like that the constitutional features of baculovirus is that virus particle is, and outside is being coated with one deck albuminous membranae, and outward appearance has certain rule, is called polyhedrosis virus (NPV).The single-minded infection of baculovirus arthropods, its natural reservoir (of bird flu viruses) is mainly lepidopteran, Hymenoptera and dipteral insect, also infects some crustaceans and mite class.At occurring in nature, there is sprout virus particle (budded virus, BV) and embedding C-type virus C particle (occlusion-derived virus, ODV in baculovirus; Claim again polyhedron inclusion body, be called for short PIB or OBs) two-strain form.BV is a kind of virus particle of propagating between insect cell, with the envelope protein of encoding viral, can combine with the special receptor of cell surface, in insect body or between the cell of clone, propagates.ODV is the communication form of baculovirus in environment, is embedded in inclusion body.Inclusion body is made up of polyhedrin protein, and its outside is polyhedron cyst membrane, highly stable in environment, and virus particle is played a protective role.Swallow after the food containing polyhedron inclusion body without the larva infecting, under the acid pH environment of polyhedrin in insect midgut, degrade, releasing virus particle core capsid, in virus contact infected insect larva, the epidermic cell tissue of intestines, manufactures the virus particle of sprouting, and bud virus particle enters in the haemocoele of host insect through cytoplasmic membrane by sprouting, cause system to infect, in the baculovirus infection later stage, virus particle is coated by a large amount of polyhedrins, forms polyhedron inclusion body.Baculovirus has higher pathogenic to host insect, to natural enemies security, free from environmental pollution, and especially it can form prevailing disease and long-term control insect population and not developing immunity to drugs in pest population, is obviously better than other sterilant.
Summary of the invention
The object of this invention is to provide a kind of expression inhibiting corpus allatum hormone methyl transferase gene (HaJHAMT gene) dsRNA recombinant baculovirus and in the application of preparing in sterilant.
The invention provides a kind of RNA molecule, for following (a) or (b):
(a) the RNA molecule shown in the sequence 7 of sequence table (single stranded RNA molecule or double stranded rna molecule);
(b) with the RNA molecule (single stranded RNA molecule or double stranded rna molecule) of sequence 7 reverse complementals of sequence table.
The DNA molecular of described RNA molecule of encoding also belongs to protection scope of the present invention.
Described DNA molecular is following 1) or 2) or 3):
1) DNA molecular being formed by DNA fragmentation I, transcribed spacer and DNA fragmentation II successively; Described DNA fragmentation I is if the sequence 6 of sequence table is from as shown in 5 ' end 1-405 position Nucleotide; Described DNA fragmentation II is if the sequence 6 of sequence table is from as shown in 5 ' end 630-1034 position Nucleotide;
2) DNA molecular shown in the sequence 6 of sequence table;
3) sequence 2 of sequence table is from the DNA molecular shown in the 206th to 610 Nucleotide of 5 ' end.
The recombinant vectors, recombinant baculovirus, expression cassette, transgenic cell line or the recombinant bacterium that contain described DNA molecular all belong to protection scope of the present invention.
In described recombinant vectors, can be started by op166 promotor (baculovirus early promoter) and/or P10 promotor (baculovirus late promoter) expression of described DNA molecular.Described op166 promotor is specifically as shown in the sequence 5 of sequence table.Described P10 promotor is specifically as shown in the sequence 8 of sequence table.
Described recombinant vectors can have expression cassette first and expression cassette second; Described expression cassette first is the expression cassette for expressing described DNA molecular; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin body protein (Ha polh albumen).
Described recombinant vectors specifically can be the encoding gene of described op166 promotor, described DNA molecular and described polyhedrin is inserted respectively to expression vector (as carrier pFastBac tMdUAL) the recombinant plasmid M that multiple clone site obtains.Described op166 promotor can insertion vector pFastBac tMbetween the NcoI of DUAL and SmaI restriction enzyme site.Described DNA molecular can insert pFastBac tMthe PvuII restriction enzyme site of DUAL.The encoding gene of described polyhedrin can insertion vector pFastBac tMbetween the BamHI of DUAL and HindIII site.
Shown in recombinant vectors specifically can be described expression cassette first and described expression cassette second inserted to the recombinant plasmid N that the attB site in Hz8 ⊿ egt Bacmid obtains.
Described recombinant vectors specifically can be the recombinant plasmid Q that in described recombinant plasmid M, between Tn7L and Tn7R site, the attB site in the insertion of the DNA fragmentation including described expression cassette first and described expression cassette second Hz8 ⊿ egt Bacmid obtains.
Described Hz8 ⊿ egt Bacmid is the recombinant plasmid obtaining after the steroidal uridine diphosphate glucose transferase gene of casting off a skin in HaBacHZ8Bacmid (claim again Ecdysteroid UDP-Glucosyltransferase gene, be called for short egt gene) deactivation.Described Hz8 ⊿ egt Bacmid is specially the recombinant plasmid that the open reading frame of the egt gene in the egfp gene replacement HaBacHZ8Bacmid shown in the sequence of sequence table 9 is obtained from the DNA fragmentation shown in 116 to 1470 Nucleotide of 5 ' end.
Described recombinant baculovirus can be the form of the virus particle of sprouting or the form of polyhedron inclusion body (polyhedron inclusion body, compared with the virus particle of sprouting, has the ability of oral infection).
The virus particle of sprouting of described recombinant baculovirus specifically can prepare by the following method: described recombinant plasmid N or described recombinant plasmid Q are imported to insect cell (as bollworm HzAm1 cell) and cultivate, collect culture supernatant, be the virus liquid of the virus particle of sprouting described in containing.
The polyhedron inclusion body of described recombinant baculovirus can prepare by the following method: described recombinant plasmid N or described recombinant plasmid Q are imported to insect cell (as bollworm HzAm1 cell) and cultivate, collecting cell precipitation, contains described polyhedron inclusion body in cell precipitation.The polyhedron inclusion body of described recombinant baculovirus can be inoculated insect and be collected insect corpse by the virus particle of sprouting described in general and obtain.The polyhedron inclusion body of described recombinant baculovirus specifically can prepare by the following method: the virus particle of sprouting described in inciting somebody to action inoculation insect, after insect death, collect corpse and smash to pieces, add water and stir evenly rear by filtered through gauze, collect filtrate the centrifugal 10min of 300g, collect the centrifugal 30min of supernatant liquor 3000g, collecting precipitation, is described polyhedron inclusion body.Described insect can be bollworm.
Arbitrary described baculovirus polyhedrin body protein is following (c) or (d) above: the protein (c) being made up of the aminoacid sequence shown in sequence in sequence table 3; (d) by the aminoacid sequence of sequence 3 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have polyhedrin function the protein being derived by sequence 3.
The encoding gene of arbitrary described baculovirus polyhedrin body protein can be following 4 above) 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) DNA molecular shown in sequence 4 in sequence table; 6) under stringent condition with 4) or 5) the DNA sequence dna hybridization limiting and the DNA molecular of coding polyhedrin; 7) with 4) or 5) DNA sequence dna that limits has 90% above homology and the DNA molecular of the polyhedrin of encoding.
The present invention also protects a kind of sterilant, and its activeconstituents is to suppress the material that corpus allatum hormone methyl transferase gene is expressed.Described sterilant can be bollworm sterilant.The material of described inhibition HaJHAMT genetic expression can be above arbitrary described RNA molecule, arbitrary described DNA molecular, arbitrary described recombinant vectors, arbitrary described expression cassette, arbitrary described transgenic cell line, arbitrary described recombinant bacterium or arbitrary described recombinant baculovirus.
The present invention also protects the material that suppresses the expression of corpus allatum hormone methyl transferase gene in the application of preparing in sterilant.Described sterilant can be bollworm sterilant.The material that suppresses the expression of corpus allatum hormone methyl transferase gene can be RNA molecule.The material of described inhibition HaJHAMT genetic expression can be above arbitrary described RNA molecule, arbitrary described DNA molecular, arbitrary described recombinant vectors, arbitrary described expression cassette, arbitrary described transgenic cell line, arbitrary described recombinant bacterium or arbitrary described recombinant baculovirus.
Arbitrary described corpus allatum hormone methyltransgerase is following (e) or (f) above: the protein (e) being made up of the aminoacid sequence shown in sequence in sequence table 1; (f) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and have identical function the protein being derived by sequence 1.
Arbitrary described corpus allatum hormone methyl transferase gene is following 8 above) or 9) or 10) or 11) DNA molecular: 8) sequence 2 of sequence table is from the DNA molecular shown in the 83rd to 937 of 5 ' ends; 9) DNA molecular shown in the sequence 2 of sequence table; 10) under stringent condition with 8) or 9) the DNA sequence dna hybridization that limits and the DNA molecular of code for said proteins; 11) with 8) or 9) DNA sequence dna limiting has the DNA molecular of 90% above homology and code for said proteins.
The present invention, using bollworm as model insects, by suppressing the expression of neotonin methyl transferase gene in insect body, can promote the desinsection effect of baculovirus to insect, thereby reach the object of Control pests to murrain.The present invention has great value for agriculture production.
Brief description of the drawings
Fig. 1 is the structural representation of recombinant plasmid pFBD-HaPH-op 166-HaJHAMTRNAi.
Fig. 2 is the genomic structural representation of HaBac △ egt.
Fig. 3 is the PCR qualification electrophorogram of Hz8 ⊿ egt Bacmid and HaBac △ egt-HaJHAMTRNAi bacmid; 1:Hz8 ⊿ egt Bacmid; 2:HaBac △ egt-HaJHAMTRNAi bacmid.
Fig. 4 cultivates the fluorescence photo after 3 days in embodiment 4.
Fig. 5 is the electrophorogram of pcr amplification product in embodiment 6.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.The English full name of Bacmid is Baculovirus plasmid, and Chinese implication is baculovirus plasmid.Grace ' s substratum pulvis: purchased from Invitrogen.Intestinal bacteria DH10B: purchased from Invitrogen company, article No. 18290-015.
Carrier pFastBac tMdUAL: reference: Bac-to-Bac Baculovirus Expression System Version D 6 April 2004.
Helper plasmid: reference: Bac-to-Bac Baculovirus Expression System Version D6 April 2004.
This plasmid of HaBacHZ8 Bacmid(has improved Baculovirus Gene group DNA, described transformation refers to: in polyhedron gene, inserted the attB site of inserting for transposable element, former polyhedron gene inactivation): reference: 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; Be applicable to Screening of Media and the low serum domestication [J] of bolworm cell HzAm1 growth; Biotechnology journal; 04 phase in 2006.
Bollworm (Helicoverpa armigera): reference: 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..
The neotonin methyltransgerase of bollworm (is made up of 284 amino-acid residues) as shown in the sequence 1 of sequence table.The cDNA of the encoding gene (HaJHAMT gene) of the neotonin methyltransgerase of bollworm is as shown in the sequence 2 of sequence table, and its open reading frame is if the sequence 2 of sequence table is from the 83rd to 937 Nucleotide compositions of 5 ' end.
LB substratum (pH7.0): solvent is water, and solute and concentration thereof are as follows: yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L.
The structure of embodiment 1, interference carrier
1, (polyhedron gene of bollworm, claims again Ha polh gene to the double chain DNA molecule shown in the sequence 4 of composition sequence table, and the sequence 4 that its open reading frame is sequence table is from shown in 5 ' end 157-897 position Nucleotide; Ha polh albumen shown in the sequence 3 of Ha polh gene coded sequence table) and be inserted into carrier pFastBac tMbetween the BamHI of DUAL and HindIII site, obtain recombinant plasmid pFBD-HaPH.
2, the double chain DNA molecule shown in the sequence 5 of composition sequence table (baculovirus early promoter claims again op166 promotor) being inserted between the NcoI and SmaI restriction enzyme site of recombinant plasmid pFBD-HaPH, obtains recombinant plasmid pFBD-HaPH-op166.
3, the double chain DNA molecule shown in the sequence 6 of composition sequence table is (by its called after palindrome fragment; In the sequence 6 of sequence table, from 5 ' end 1-405 position Nucleotide and 630-1034 position Nucleotide reverse complemental, 406-629 position Nucleotide is transcribed spacer) and be inserted into the PvuII restriction enzyme site of recombinant plasmid pFBD-HaPH-op166, obtain recombinant plasmid pFBD-HaPH-op166-HaJHAMTRNAi.The structural representation of recombinant plasmid pFBD-HaPH-op166-HaJHAMTRNAi is shown in Fig. 1.
The preparation of embodiment 2, Hz8 ⊿ egt Bacmid
The open reading frame that egfp gene shown in the sequence of sequence table 9 is replaced to the egt gene in HaBacHZ8Bacmid, from the DNA fragmentation shown in 116 to 1470 Nucleotide of 5 ' end, obtains Hz8 ⊿ egt Bacmid.
The acquisition of embodiment 3, HaBac △ egt-HaJHAMTRNAi bacmid
Mechanism description: in intestinal bacteria, Helper plasmid promotes the generation of swivel base, DNA fragmentation between the Tn7L of recombinant plasmid pFBD-HaPH-op166-HaJHAMTRNAi and Tn7R site including palindrome fragment and Ha polh gene (inserting the polyhedron gene inactivation of the Hz8 ⊿ egt Bacmid causing in order to cover external source fragment) inserts Hz8 ⊿ egt Bacmid attB site by transposition, obtains recombinant baculovirus plasmid.
1, the competent cell that Helper plasmid is imported to intestinal bacteria DH10B, obtains recombinant bacterium I.
2, Hz8 ⊿ egt Bacmid is imported to the recombinant bacterium I that step 1 obtains, obtain recombinant bacterium II.
3, the recombinant bacterium II that the recombinant plasmid pFBD-HaPH-op166-HaJHAMTRNAi step of converting 2 that embodiment 1 is obtained obtains, then coat LB culture medium flat plate (containing 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, 100 μ g/mL X-gal and 40 μ g/mL IPTG), cultivate 24-48 hour for 37 DEG C, check the blue hickie on flat board, white colony is rule at a fresh LB culture medium flat plate again (containing 50 μ g/mL kantlex, 7 μ g/mL gentamicins, 10 μ g/mL tsiklomitsins, 100 μ g/mL X-gal and 40 μ g/mL IPTG), cultivate 24-48 hour for 37 DEG C, extracting waste bacterium colony, be and contain the recombinant baculovirus plasmid bacterium colony of (claiming again HaBac △ egt-HaJHAMTRNAi bacmid).
The PCR qualification electrophorogram of Hz8 ⊿ egt Bacmid and HaBac △ egt-HaJHAMTRNAi bacmid is shown in Fig. 3, adopt the primer pair of the primer (5 '-CCCAGTCACGACGTTGTAAAACG-3 ') of corresponding Hz8 ⊿ egt Bacmid and the primer of corresponding described palindrome fragment (5 '-TTCGCGAATGATCATCATTGT-3 ') composition, HaBac △ egt-HaJHAMTRNAi bacmid shows the object fragment of 2.1kb left and right, and Hz8 ⊿ egt Bacmid does not show described object fragment.
The acquisition of embodiment 4, recombinant baculovirus (virus particle of sprouting)
One, the acquisition of recombinant baculovirus (virus particle of sprouting)
1, inoculation 5 × 10 in the capsule (Grace ' the s substratum containing 10%FBS is housed) of 35mm 5individual bollworm HzAm1 cell, 27 ° of C overnight incubation are inhaled and are abandoned supernatant liquor in Biohazard Safety Equipment, add 1ml Grace ' s substratum room temperature to place 1 hour.
2, use respectively Grace ' s substratum to be diluted to 100 μ l 5 μ g HaBac △ egt-HaJHAMTRNAi bacmid and 10 μ l Lipofectin, then both are mixed, mix once every 7min, after 45min, add 400 μ lGrace ' s substratum, mix.
3, get the capsule of completing steps 1, inhale the mixed solution of abandoning supernatant liquor and adding step 2 to obtain, 27 ° of C cultivate the every 15min of 30min(and shake up capsule once); Then add 400 μ l Grace ' s substratum, mix, 27 ° of C cultivate 6 hours; Then add 1mL to contain Grace ' the s substratum of 20%FBS, mix, 27 ° of C cultivate after 6 days and collect supernatant liquor, are the recombinant baculovirus f1 disease venom of (claiming again HaBac △ egtHaJHAMTRNAi).In 27 ° of C cultivations process of 6 days, observe the generation situation of green fluorescence every day under fluorescence inverted microscope, the fluorescence photo of cultivating after 3 days is shown in Fig. 4 (can observe green fluorescence, represent to infect successfully).
4, at 25cm 2batch cultur bottle in press MOI=0.1 dosage f1 disease venom infect bollworm HzAm1 cell, cultivate after 96 hours for 27 DEG C and collect supernatant liquor, be s-generation virus liquid.
5, at 25cm 2batch cultur bottle in press MOI=0.1 dosage s-generation virus liquid infect bollworm HzAm1 cell, cultivate after 96 hours for 27 DEG C and collect supernatant liquor, be third generation virus liquid.
Two, the acquisition of control baculovirus (virus particle of sprouting)
Replace HaBac △ egt-HaJHAMTRNAi bacmid to carry out step 1 with Hz8 △ egt Bacmid, obtain baculovirus f1 disease venom, s-generation virus liquid and the third generation virus liquid of (claiming again HaBac △ egt).The genomic structural representation of HaBac △ egt is shown in Fig. 2.
Three, the acquisition of control baculovirus (virus particle of sprouting)
Replace recombinant plasmid pFBD-HaPH-op166-HaJHAMTRNAi to carry out embodiment 3 recombinant plasmid pFBD-HaPH-op166, DNA fragmentation between the Tn7L of carrier pFBD-HaPH and Tn7R site including Ha polh gene inserts the polyhedron gene site (polyhedrin locus) of Hz8 ⊿ egt Bacmid by transposition, obtain recombinant baculovirus plasmid (claiming again HaBac △ egt-A bacmid).
Replace HaBac △ egt-HaJHAMTRNAi bacmid to carry out step 1 with HaBac △ egt-A bacmid, obtain baculovirus f1 disease venom, s-generation virus liquid and the third generation virus liquid of (claiming again HaBac △ egt-A).
The acquisition of embodiment 5, polyhedron inclusion body
One, the acquisition of HaBac △ egtHaJHAMTRNAi polyhedron inclusion body
1, bollworm was supported to 3 age ends, before injection, put to numb with cold on ice, get the third generation virus liquid of HaBac △ egtHaJHAMTRNAi with the microsyringe of sterilization, second from the bottom and be injected in hemolymph between the 3rd at bollworm abdominal foot, every bollworm is injected 5 μ l, after 5 days, bollworm liquefaction death, collects worm corpse.
2, add distilled water and stir evenly after the worm corpse of step 1 is smashed to pieces, by 3 layers of filtered through gauze and collect filtrate, by centrifugal filtrate 300g 10min and collect supernatant liquor, supernatant liquor is carried out to the centrifugal 30min of 3000g, collecting precipitation (HaBac △ egtHaJHAMTRNAi polyhedron inclusion body).
Two, the acquisition of HaBac △ egt polyhedron inclusion body
The third generation virus liquid that replaces HaBac △ egtHaJHAMTRNAi with the third generation virus liquid of HaBac △ egt, other same step 1, obtains HaBac △ egt polyhedron inclusion body.
Three, the acquisition of HaBac △ egt-A polyhedron inclusion body
The third generation virus liquid that replaces HaBac △ egtHaJHAMTRNAi with the third generation virus liquid of HaBac △ egt-A, other same step 1, obtains HaBac △ egt-A polyhedron inclusion body.
The medial lethal dose of embodiment 6, polyhedron inclusion body
HaBac △ egtHaJHAMTRNAi polyhedron inclusion body, HaBac △ egt polyhedron inclusion body and the HaBac △ egt-A polyhedron inclusion body respectively prepared by embodiment 5 carry out the detection of following medial lethal dose:
1, select uniform two age Mo cotton bollworm larvae, be placed in 96 aseptic orifice plates, hungry cultivation 16 hours in 28 DEG C of constant temperature illumination boxs, allows their decortications enter in next age.
2, be 1 × 10 by polyhedron inclusion body with the sterilized water dilution of the sucrose that contains 4g/100mL and the edible light blue of 1mg/mL 6individual polyhedron inclusion body/mL, 3 × 10 5individual polyhedron inclusion body/mL, 1 × 10 5individual polyhedron inclusion body/mL, 3 × 10 4individual polyhedron inclusion body/mL or 1 × 10 4the suspension (with blood counting chamber counting polyhedron inclusion body) of individual polyhedron inclusion body/mL.
3, the cotton bollworm larvae of completing steps 1 is divided into five groups, every group of 48 larvas, the suspension obtaining by step 2 is respectively fed, from the beginning timing of feeding, the interior middle intestines of 10min become blue larva and proceed in 24 orifice plates that contain fresh artificial diet, observe every day 2 times and add up mortality ratio until all for trying larva death or pupating.Carry out repeating for three times experiment.Calculate the lethal dense dosage (LC of half of polygonal inclusion body with Probit regression analysis 50) and standard error, relatively two-strain LC 50between the significance of difference.
The toxic limit medium dose of HaBac △ egtHaJHAMTRNAi polyhedron inclusion body is 1.24 × 10 4individual polyhedron inclusion body/ml, standard deviation is 0.49 × 10 4individual polyhedron inclusion body/ml, 95% fiducial interval is 0.49-2.42 × 10 4individual polyhedron inclusion body/ml.The toxic limit medium dose of HaBac △ egt polyhedron inclusion body is 8.12 × 10 4individual polyhedron inclusion body/ml, standard deviation is 1.16 × 10 4individual polyhedron inclusion body/ml, 95% fiducial interval is 6.16-10.7 × 10 4individual polyhedron inclusion body/ml.The toxic limit medium dose of two kinds of polyhedron inclusion bodys has significant difference (z=7.27, p<0.05).The toxic limit medium dose of HaBac △ egt-A polyhedron inclusion body is consistent with the toxic limit medium dose of HaBac △ egt polyhedron inclusion body.
4, get bollworm dead in step 3, extracting total RNA reverse transcription is cDNA, and taking cDNA as template, adopting ACTIN gene is reference gene, by the expression amount of sxemiquantitative PCR qualification HaJHAMT gene.
Primer for the identification of ACTIN gene is as follows:
Upstream primer: 5 '-CCTGGTATTGCTGACCGTATGC-3 ';
Downstream primer: 5 '-CTGTTGGAAGGTGGAGAGGGAA-3 '.
Primer for the identification of HaJHAMT gene is as follows:
Upstream primer: 5 '-GGGTACTCGCGCGCAACAACAA-3 ';
Downstream primer: 5 '-ATCCTGCGAGTCAGGCTTCCG-3 '.
The electrophorogram of pcr amplification product is shown in Fig. 5.Compared with the bollworm of the HaBac △ egt polyhedron inclusion body of feeding, in the Helicoverpa armigera of the HaBac △ egtHaJHAMTRNAi polyhedron of feeding inclusion body, the expression amount of HaJHAMT gene significantly reduces.
Result shows, the dsRNA of described palindrome fragment expression can impel HaJHAMT gene expression amount to lower, thereby the toxic limit medium dose of baculovirus is declined, and greatly improves the lethal efficiency of baculovirus to bollworm resisting.

Claims (10)

1. a RNA molecule, for following (a) or (b):
(a) the RNA molecule shown in the sequence 7 of sequence table;
(b) with the RNA molecule of sequence 7 reverse complementals of sequence table.
2. the DNA molecular of RNA molecule described in coding claim 1.
3. DNA molecular as claimed in claim 2, is characterized in that: described DNA molecular is following 1) or 2) or 3):
1) DNA molecular being formed by DNA fragmentation I, transcribed spacer and DNA fragmentation II successively; Described DNA fragmentation I is if the sequence 6 of sequence table is from as shown in 5 ' end 1-405 position Nucleotide; Described DNA fragmentation II is if the sequence 6 of sequence table is from as shown in 5 ' end 630-1034 position Nucleotide;
2) DNA molecular shown in the sequence 6 of sequence table;
3) sequence 2 of sequence table is from the DNA molecular shown in the 206th to 610 Nucleotide of 5 ' end.
4. contain recombinant vectors, recombinant baculovirus, expression cassette, transgenic cell line or the recombinant bacterium of DNA molecular described in claim 2 or 3.
5. recombinant vectors as claimed in claim 4, is characterized in that: in described recombinant vectors, by the expression of DNA molecular described in op166 promotor and/or P10 promotor startup claim 2 or 3.
6. recombinant vectors as claimed in claim 5, is characterized in that: in described recombinant vectors, have expression cassette first and expression cassette second; Described expression cassette first is the expression cassette for expressing DNA molecular described in claim 2 or 3; Described expression cassette second is the expression cassette of the encoding gene for expressing baculovirus polyhedrin body protein.
7. recombinant baculovirus as claimed in claim 4, is characterized in that: described recombinant baculovirus is the sprout form of virus particle or the form of polyhedron inclusion body.
8. a sterilant, its activeconstituents is to suppress the material that corpus allatum hormone methyl transferase gene is expressed.
9. suppress the material of corpus allatum hormone methyl transferase gene expression in the application of preparing in sterilant.
10. sterilant as claimed in claim 8, or application as claimed in claim 9, is characterized in that: described " suppressing the material that corpus allatum hormone methyl transferase gene is expressed " is at least one in following material:
Recombinant baculovirus described in (I) claim 7;
Recombinant vectors, recombinant baculovirus, expression cassette, transgenic cell line or recombinant bacterium described in (II) claim 4;
Recombinant vectors described in (III) claim 5 or 6;
DNA molecular described in (IV) claim 2 or 3;
RNA molecule described in (V) claim 1.
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