CN101919404A - Biopesticide and insect preventing and controlling method - Google Patents
Biopesticide and insect preventing and controlling method Download PDFInfo
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Abstract
The invention provides an insect preventing and controlling method, which is to feed lepidopterous insets with a mixture of siRNA for the specific gene of the lepidopterous insets and a cationic polymer, wherein the siRNA is 17 to 50 nt double-chain RNA chemically synthesized in vitro; and the cationic polymer consists of polyethyleneimine (PEI), chitosan, polylysine and gelatin. The invention also relates to a biopesticide, which uses the siRNA for the specific gene of the lepidopterous insets as an active ingredient. In the invention, the gene expression of the lepidopterous insets is inhibited by feeding the lepidopterous insets with the siRNA which contains the specific gene of the lepidopterous insets, such as an interfering insect mitochondrion composite IIIFe-S subunit gene, a cholinesterase gene, a gamma-amino butyric acid receptor gene and the like, to interfere the normal growth and development of the insects, so the effective control of the insects is realized; and pot culture experiment result shows that the biopesticide is very effective.
Description
Technical field
The present invention relates to the small molecules interference RNA field of chemosynthesis and modification, particularly, relate to a kind of biopesticide and insect control method.
Background technology
RNA disturbs that (RNA interference RNAi) is meant that external source or endogenous double-stranded RNA (dsRNA) cause the phenomenon of gene expression silence specifically, and it is to evolve to go up high conservative and in the ubiquitous a kind of gene regulation mechanism of biosphere.In nematode Caenorhabditis elegans, find this phenomenon first in 1998, and in fungi, plant, insect and animal, also found this phenomenon subsequently.The mechanism of action of RNAi is: long-chain dsRNA external importing or that endogenous is transcribed generation is cut into the siRNA of 21-25nt (base) by the RNase-III of Dicer family, siRNA further forms RISC (RNA-induced silencing complex) with the Argonaute protein combination, combines the specificity degraded that causes homology said target mrna molecule with the said target mrna complementary element by RISC mediation siRNA antisense strand at last.RNAi research has in recent years obtained breakthrough, is chosen as one of ten big sciences progress of calendar year 2001 by " Science " magazine, and ranks first of 2002 the ten big science progress.Reject or close the basic ideas that are expressed as of insect specific gene with specificity, explore the New Policy of control of insect, be expected to hot technology into the novel biopesticide initiative.
Nematode is behind absorption or local injection dsRNA, and its RNAi effect can spread all over whole organism or even be delivered to the offspring, and this is defined as systemic RNAi.Insect must have the mechanism of propagating the RNAi effect in the body, and just can be expected to realize with siRNA is main biopesticide control strategy of imitating the factor.Insect systematicness RNAi effect is found in red flour beetle (Triboliumcastaneum) first, the dsRNA of this worm larval phase injection Tc-ASH gene, show phenotype (the Tomoyasu Y of disappearance hair on the neck in the adult stage, Denell RE.Larval RNAi in Tribolium (Coleoptera) for analyzing adult development.Dev Genes Evol.2004,214:575-578), and the isogenic RNAi effect of this insect Distalless can be passed to filial generation (Bucher G.Parental RNAi in Tribolium (Coleoptera) .Curr Biol from parental generation, 2002,12:R85-R86).At present, systemic RNAi effect has existed in the following insect and must verify: the dipteral insect tsetse fly; The coleopteron red flour beetle; The hymenopteran honeybee; The orthopteran locust; Blattaria insect Groton bug; Lepidopterous insects prodenia litura, diamond-back moth, beet armyworm, shallow brown apple moth; The hemipteran acyrthosiphum pisim.The popularity of systematicness RNAi effect in insect shows that siRNA has application potential widely aspect control of insect.
Second largest order in Lepidoptera (Lepidoptera) Insecta.Complete metamorphosis.Larva is commonly referred to as caterpillar, also Cheng “ Zhan Si ".Pupa is an obtected pupa.Adult claims moth or butterfly, and is close by scale on its wing and the body, so name.Tool absorbs mouthpart, forms microscler and beak that can roll; Compound eye is big; It is many that feeler changes, and is thread, pinniform or comb shape etc.The whole world is known to be had about 140,000 kinds, on the books about 20,000 kinds of China.Most of kinds and national economy have significant relationship, as snout moth's larva, mythimna separata, pine caterpillars and imported cabbageworm, diamond-back moth etc., are the important pests of agriculture and forestry plant; Silkworm, tussah and eri silkworm etc. are famous resource insects.
According to the relevant report of recent years, insect is expressed plant or the bacterial strain of dsRNA by feeding, and can effectively block the expression of insect target gene.Mao Yingbo etc. (2007) allow plant self produce the dsRNA of P450 gene by the transgenosis means.Then, with plant feeding cotton bollworm (Helicoverpa armigera).DsRNA enters cell from esophagus, suppresses P450 expression of gene in the cotton boll polypide, causes cotton bollworm that the resistance of gossypol is reduced.At last, cotton bollworm is caused fatal influence.Corn root firefly chrysomelid (Diabrotica virgifera virgifera Leconte) is fed and contains the isogenic dsRNA plant feed of V-ATPase A, and diapause or death appear in larva, have significantly reduced the harm of this worm to corn root.Tian Honggang etc. (2009) utilization has the carrier L4440 of double T 7 promotors and HT115 (DE3) bacterial strain of RNase III disappearance, made up the engineered strain that abduction delivering can produce seCHSA gene dsRNA, the beet armyworm of feeding causes indivedual polypides deformity or dead to occur.
The RNA perturbation technique has been applied to the research of diamond-back moth functional gene.As: Z.-X.Yang etc. cause lethality of this worm and sex ratio to increase, and the diapause phenomenon appear in larval phase with the dsRNA of the method importing cadherin gene of microinjection.By the method for feeding, import the dsRNA of diamond-back moth Cytochrome P450 (CYP6BG1) gene, cause this worm that the resistance of Dalmatian chrysanthemum lipid agricultural chemicals is reduced (Ma, A.M. B., Tadashi, M., Ken M., Toshiharu, T.RNAinterference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin.Insect.2009.Biochem.Mol.Biol.39:38-46.).In view of the above, we think that diamond-back moth has systemic RNA interference capability, might realize with the RNA perturbation technique being the control strategy of means.
Though new one page that the RNAi technology is used has been opened in present research aspect control of insect, wherein gene specific dsRNA only comes from genetically modified plants or engineered strain, or is that in-vitro transcription generates.Great general character and key technology that restriction at present utilizes the RNAi technology to produce novel biopesticide are: one, existingly utilize transgenic technology to change over to do the technical system and the strategy of objects system pest control to have the genetically modified plants food safety question; Two, the engineered strain of expressing dsRNA takes effect slowly, preventive effect is low, and environmental suitability is difficult to assess; Three, the synthetic cost height of the in-vitro transcription of dsRNA, stability are bad etc.These problems have limited the extensive use of this technology in field of pest control greatly.
Summary of the invention
For overcoming above shortcoming, the invention provides a kind of new insect control method.
Concrete technical scheme is as follows:
A kind of insect control method mainly comprises: will be at the siRNA feeding lepidopterous insects of lepidopterous insects particular target gene, and described siRNA is the double-stranded RNA of external chemosynthesis 17-50nt.
More preferably, above-mentioned siRNA directly is sprayed on the blade of the edible crops of lepidopterous insects, allows insect get food naturally.
Insect control method of the present invention, method by external chemosynthesis 17-50nt double-stranded RNA (siRNA), also can stability and the drug effect of siRNA have been strengthened by chemical modification and with the cationic polymer compatibility, the feed that contains the particular target gene siRNA by feeding naturally or spraying is effectively blocked the expression of insect target gene mRNA, and insect is caused fatal influence.The preparation of this method is main biopesticide of imitating the factor with siRNA, has advantages such as cycle weak point, instant effect, nontoxic, non-environmental-pollution, can use widely.
Another object of the present invention provides a kind of biopesticide.
A kind of biopesticide is an effective ingredient with the siRNA at lepidopterous insects particular target gene, and described siRNA is the double-stranded RNA of external chemosynthesis 17-50nt.
Preferably, it is effective ingredient that described biopesticide also includes cationic polymer, and more preferably, described cationic polymer is polymine, shitosan, polylysine or gelatin.
Preferably, described siRNA also is provided with suspension base with the combination of two deoxyribonucleosides through chemical modification or siRNA3 ' end, and described chemical modification is 2 '-methylates, fluoro, 5 '-PEG, cholesterol, or polypeptide etc.
Preferably, described specific gene is mitochondria composite I IIFe-S subunit gene or acetylcholinesterasegene gene or gamma-aminobutyric acid receptor gene.More preferably, be one or more pairs of among SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and the SEQ ID NO.6 at the siRNA of mitochondria composite I IIFe-S subunit gene; At the siRNA of acetylcholinesterasegene gene is one or more pairs of among SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and the SEQ ID NO.18; At the siRNA of gamma-aminobutyric acid receptor gene is one or more pairs of among SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ IDNO.23 and the SEQ ID NO.24.
Preferably, described lepidopterous insects is a diamond-back moth.
The present invention directly feeds to insect and contains the siRNA of particular target gene, causes the variation of this gene mRNA level of insect or protein level, suppresses by the expression of this method to a certain specific gene of insect correspondence.Owing to adopt the method for chemosynthesis to prepare 17-50nt siRNA, produce fast, and can strengthen stability and the effect of siRNA by chemical modification with the cationic polymer compatibility, method is easy; Because the siRNA that is produced directly is sprayed on the blade, allow insect get food naturally and get final product, simple to operate, realized agricultural chemicals insecticide-applying way truly, can promote widely.Therefore, adopt this method to suppress the expression of insect genes, can provide new method for control of insect.Further, show that this is a class biological insecticides efficiently by potted plant experimental result.
Description of drawings
Fig. 1: for the diamondback moth larvae of the siRNA that feeds among the embodiment 1 shows dead result schematic diagram;
Fig. 2: be the qRT-PCR result schematic diagram among the embodiment 1;
Fig. 3: be western blot result schematic diagram among the embodiment 1;
Fig. 4: be the ATP measurement result schematic diagram in the example 1;
Fig. 5: be the 5th day control efficiency schematic diagram relatively after the dispenser among the embodiment 3;
Diamond-back moth shows the not most symptom schematic diagram of decortication among Fig. 6: the embodiment 5.
Embodiment
The concentration of siRNA in the biopesticide of the present invention is preferably greater than 10ppm usually greater than 1ppm, more preferably 50ppm-500ppm.Those skilled in the art also can be according to general knowledge, and according to actual needs, proportioning becomes suitable concentration, and the blade face concentration when selecting suitable dispenser, and usually, concentration is big more, and effect is good more.The concentration of cationic polymer is the same with siRNA, can be according to actual needs, and proportioning becomes suitable concentration, usually can be greater than 10ppm.Preferably, the ratio of N (cationic polymer): P (siRNA) is 1 in described biopesticide: 1-100: 1.Specifically need not to give unnecessary details.
Below by embodiment the present invention is carried out concrete description, only be used for the present invention is further specified, can not be interpreted as qualification protection domain of the present invention.
Embodiment one
This example provides the siRNA of diamond-back moth mitochondria composite I IIFe-S subunit gene especially, by behind this target gene siRNA that feeds naturally lepidopterous insects such as diamond-back moth having been caused fatal influence.
This example is the method that is used to prevent and treat lepidopterous insects diamond-back moth (Plutella xylostella).Contain the siRNA of reticent mitochondria composite I IIFe-S subunit gene (the GeneBank accession number is EU815629) by feeding, transmit, suppress the formation of insect ATP, and then realized effective improvement insect thereby block insect mitochondria electronics.
Mitochondria composite I II claims ubiquinone cytochrome C-reductase (Ubiquinol-cytochrome c reductase) again, is made up of 9-11 subunit, comprises two cytochrome bs (b562 and b566), a cytochrome C1 and a Fe-S albumen.Wherein the Fe-S protein protomer is responsible for an electronics is passed to cytochrome C1 from reduced coenzyme Q, and a proton is discharged into intermembrane space, produce formation (the Trumpower B L.Cytochrome bc1 complexes ofmicroorganisms.Microbiological reviews that film potential is used for ATP with this, 1990,54:101-29).The Biological Principles of present embodiment is exactly by reticent insect mitochondria composite I IIFe-S subunit gene mRNA, and then the transmission of blocking-up insect mitochondria electronics, suppresses the formation of ATP, thereby reaches the purpose of control of insect.
The siRNA of chemosynthesis is evenly coated the wild cabbage blade face after with the DEPC dilution, feed and scribble cabbage leaves by insect larvae being carried out nature at mitochondria composite I IIFe-S subunit gene siRNA, thereby the expression at blocking-up insect specific gene such as diamond-back moth mitochondria composite I IIFe-S subunit gene realizes the effective improvement to this insect.This interference effect can be determined by the change that detects insect mRNA or protein level, as passing through to detect diamond-back moth mitochondria composite I IIFe-S Subunit mRNA expression and its protein expression situation, or be the synthetic situation of diamondback moth larvae ATP, determine interference effect.Show that as experiment after diamond-back moth was got the siRNA of Shiqi mitochondria composite I IIFe-S subunit, this gene mRNA and protein expression level reduced, show the electronics transmission and be obstructed, finally dead because of can't normally synthesizing ATP.
Described method mainly may further comprise the steps:
(1) selection of insect specific function gene:
Consider the key effect of insect composite I IIFe-S subunit gene in the mitochondria electronics transmits, thus the present invention to select the gene of this specific function be reticent object, its gene order is EU815629 referring to the GeneBank accession number.
(2) design of diamond-back moth mitochondria composite I IIFe-S subunit gene siRNA and synthetic:
Si-UQCR_001：GCAAGTCCGTCACCTTCAA(19nt)
Positive-sense strand (5 '-3 '): 5 ' GCAAGUCCGUCACCUUCAA dTdT, 3 ' SEQ ID NO.1
Antisense strand (3 '-5 '): 3 ' dTdT CGUUCAGGCAGUGGAAGUU, 5 ' SEQ ID NO.2,
Si-UQCR_002：CATCCAGTGTAGTGAGCAA(19nt)
Positive-sense strand (5 '-3 '): 5 ' CAUCCAGUGUAGUGAGCAA dTdT, 3 ' SEQ ID NO.3
Antisense strand (3 '-5 '): 3 ' dTdT GUAGGUCACAUCACUCGUU, 5 ' SEQ ID NO.4
Si-UQCR_003：CAACAACCTCTGAGAAGTT(19nt)
Positive-sense strand (5 '-3 '): 5 ' CAACAACCUCUGAGAAGUU dTdT, 3 ' SEQ ID NO.5
Antisense strand (3 '-5 '): 3 ' dTdT GUUGUUGGAGACUCUUCAA, 5 ' SEQ ID NO.6
3 ' end of the siRNA of above-mentioned SEQ ID NO1-6 is provided with the suspension base of two TT.
As effective ingredient, the dilution of DEPC water is prepared into described biopesticide with above-mentioned every couple of siRNA.
(3) with above-mentioned be that the biopesticide of active ingredient is evenly coated cabbage leaves and feeding diamond-back moth second instar larvae with siRNA: siRNA is diluted to the solution that concentration is 100ppm (biopesticide) with DEPC (diethypyrocarbonate) water, evenly coat then on the cabbage leaves, blade face concentration is 3.0 μ g/cm
2, with this blade feeding diamond-back moth in two ages.Every couple of siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 36h, 48h, 60h, 72h investigate insect respectively, calculate lethality.
(4) detect the reticent level of ISP gene mRNA in the insect bodies with the method for quantitative fluorescent PCR: the death toll of waiting to have investigated insect, just collect the insect that shows the symptom of showing one's color, as: show the individuality of dead proterties, extract total RNA, be inverted to the first chain cDNA, be used for the template of qRT-PCR.Use quantitative real time PCR Instrument supporting soft afterwards
Part calculates relative expression's value.
Fluorescence quantification PCR primer:
EU815629_FP GTTGTGAGGTCAGGGCATTT SEQ?ID?NO.31
EU815629_RP GGAGAGGCTGAGACACCAAC SEQ?ID?NO.32
(5) detect the expression of destination protein in the insect bodies of the siRNA that fed with WB: treat the insect specific siRNA that feeds, as: behind Si-UQCR_003 (SEQ ID the NO 5 and 6) siRNA, at 12h, 24h, 48h, 72h collects the individuality that shows the dominance symptom, extract total protein, measure the protein concentration of each sample with the BCA method, on adjusting, under the situation of sample total protein concentration unanimity, separate the total protein of each sample with the 15%SDS-PAGE gel electrophoresis, afterwards, after one anti-, two anti-hatching, exposure imaging obtains as Fig. 3 result.
(6) the fed detection of ATP amount in the insect bodies of siRNA: treat the insect specific siRNA that feeds, as: behind the Si-UQCR_003 siRNA, at 6h, 12h, 24h, 36h, 48h, 72h collects live body insect sample, and collects the live body insect sample of the DEPC water of having fed simultaneously, afterwards with the green skies production ATP of company detection kit (production code member: S0026) measure the content of its ATP.
Find by this experiment, the process nature is fed behind the siRNA blade that scribbles selected genes, the expression silencing of the mitochondria composite I IIFe-S subunit gene in the insect bodies, genes of interest mRNA expression reduces, protein expression level also is suppressed simultaneously, and then the transmission of blocking-up insect electronics, suppress the formation of ATP, thereby cause insect individual significantly dead.
Table 1: feeding scribbles behind the biopesticide cabbage leaves that contains the ISP gene siRNA contrast statistics to the diamond-back moth lethality
Described chemical modification is that 3 ' and 5 '-terminal each three base are that 2 '-methyl and fluoro are modified.
Fig. 2: be qRT-PCR result.The explanation of this table is fed behind the gene specific siRNA, and the mRNA that shows ISP gene in the diamond-back moth body of dead dominant character significantly reduces.
Fig. 3: be western blot result.The explanation of changing plan is fed behind the ISP gene Si-UQCR_003 siRNA, and at 12 hours, the ISP expressing quantity significantly reduced in the diamond-back moth body, and along with progressively the growing up of diamond-back moth, the expression of this albumen progressively increases afterwards.
Fig. 5: be the ATP measurement result.1-6 represents 6,12,24,36,48,72 hours respectively.
Embodiment two
This example provides the siRNA of diamond-back moth mitochondria composite I IIFe-S subunit gene especially, is lepidopterous insects such as diamond-back moth to have been caused fatal influence behind the compatibilities such as polymine (PEI), shitosan, polylysine and gelatin with cationic polymer respectively by this target gene Si-UQCR_001 that feeds naturally (SEQ ID NO 5 and 6) siRNA.
Si-UQCR_001 (SEQ ID NO 1 and 2) siRNA and above-mentioned cationic polymer are prepared into biopesticide, and evenly coating cabbage leaves and feeding diamond-back moth second instar larvae, among the group 1-4, the N in the biopesticide: P is 1: 1, among the group 5-6, the N in the biopesticide: P is 20: 1.The blade face concentration of siRNA is 3.0 μ g/cm
2Every couple of siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 36h, 48h, 60h, 72h investigate insect respectively, calculate lethality, experimental result such as table 2.
Table 2: the contrast to the diamond-back moth lethality behind the cabbage leaves that scribbles ISP gene siRNA and various cationic polymers of feeding is added up
1-8 siRNA biopesticide in the experiment: 1 and 5 is that polymine (PEI), 2 and 6 is that shitosan, 3 and 7 is that polylysine, 4 and 8 is gelatin.
Embodiment three
This example provides the siRNA of diamond-back moth acetylcholinesterasegene gene especially, by behind this target gene siRNA that feeds naturally lepidopterous insects such as diamond-back moth having been caused fatal influence.
This example is the method that is used to prevent and treat lepidopterous insects diamond-back moth (Plutella xylostella).The siRNA that contains reticent acetylcholinesterasegene gene 1 and 2 (the GeneBank accession number is respectively: AY970293 and AY061975) by feeding, thereby influence the normal nerve impulse transmission of insect, make insect continue to be in excitatory state and death, and then realized effective improvement insect.
Acetylcholinesterase is a kind of serine hydrolase, its major function is to end transmission (the Taylor T of nerve impulse by quick hydrolysis neurotransmitter acetylcholine (ACh) in cholinergic synapses, Radie Z, 1994.Thecholinesterases:from genes to proteins.Annu Rev Pharmacol.Toxicol., 34:281-320).Biological Principles of the present invention is exactly by reticent insect acetylcholinesterasegene gene mRNA, and then influences the transmission of insect nerve impulse, makes insect continue to be in excitatory state and death.
Particularly, the method for this siRNA restraint insect gene expression of feeding is insect directly to be fed contain the siRNA of specific gene, causes growing of insect to be obstructed, and suppresses by the expression of this method to a certain specific gene of insect correspondence.Described method mainly may further comprise the steps:
(1) selection of insect specific function gene:
Consider the key effect of insect acetylcholinesterasegene gene in nerve impulse is transmitted, thus the present invention to select the gene of this specific function be reticent object.Sequence specifically is respectively referring to the GeneBank accession number: AY970293 and AY061975.
(design of 2 diamond-back moth acetylcholinesterasegene gene gene siRNAs and synthetic:
GenBank：AY970293?ace1
Si-ace1_001：CATGCATGGTGATGAAATA
Positive-sense strand (5 '-3 '): 5 '-CAUGCAUGGUGAUGAAAUATT-3 ' SEQ ID NO.7
Antisense strand (3 '-5 '): 3 '-TTGUACGUACCACUACUUUAU-5 ' SEQ ID NO.8
Si-ace1_002(R?S27/A29)：GAATGATGTTGCCAGACAA
Positive-sense strand (5 '-3 '): 5 '-GAAUGAUGUUGCCAGACAATT-3 ' SEQ ID NO.9
Antisense strand (3 '-5 '): 3 '-TTCUUACUACAACGGUCUGUU-5 ' SEQ ID NO.10
Si-ace1_003(R?S27/A29)：CAGAGAGGAGAGTGTGATA
Positive-sense strand (5 '-3 '): 5 '-CAGAGAGGAGAGUGUGAUATT-3 ' SEQ ID NO.11
Antisense strand (3 '-5 '): 3 '-TTGUCUCUCCUCUCACACUAU-5 ' SEQ ID NO.12
GenBank：AY061975?ace2
Si-ace2_001：CGGCGACACTTGATCTATA
Positive-sense strand (5 '-3 '): 5 '-CGGCGACACUUGAUCUAUATT-3 ' SEQ ID NO.13
Antisense strand (3 '-5 '): 3 '-TTGCCGCUGUGAACUAGAUAU-5 ' SEQ ID NO.14
Si-ace2_002：CAGACACGATGATGAAAGA
Positive-sense strand (5 '-3 '): 5 '-CAGACACGAUGAUGAAAGATT-3 ' SEQ ID NO.15
Antisense strand (3 '-5 '): 3 '-TTGUCUGUGCUACUACUUUCU-5 ' SEQ ID NO.16
Si-ace2_003：CTGGCTATTCGTTGGATAA
Positive-sense strand (5 '-3 '): 5 '-CUGGCUAUUCGUUGGAUAATT-3 ' SEQ ID NO.17
Antisense strand (3 '-5 '): 3 '-TTGACCGAUAAGCAACCUAUU-5 ' SEQ ID NO.18
3 ' end of the siRNA of above-mentioned SEQ ID NO7-18 is provided with the suspension base of two TT.
As effective ingredient, the dilution of DEPC water is prepared into described biopesticide respectively with above-mentioned every couple of siRNA.
(3) siRNA is evenly coated cabbage leaves and feeding diamond-back moth second instar larvae: siRNA is diluted to the solution that concentration is 100ppm (biopesticide) with DEPC water, evenly coats then on the cabbage leaves, blade face concentration is 3.0 μ g/cm
2, with this blade feeding diamond-back moth in two ages.Every couple of siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 48h investigate insect respectively, calculate lethality.
(4) calculating of above-mentioned siRNA lethal concentration of 50 LC50 value: siRNA is diluted to variable concentrations gradient: 0ppm, 10ppm, 50ppm, 100ppm, 200ppm with DEPC water, is prepared into described biopesticide, get 400 μ l and evenly be applied to cabbage leaves (8cm
2), each repeats 10 cephalonts, establishes three repetitions, adds up dead insect population number at 48h afterwards, calculates LC50 and LC90 value.
(5) the indoor control effect of Si-ace2_001siRNA: the indoor control effect of determining Si-ace2_001siRNA with the method for potted plant experiment.Place 50 diamond-back moths on each potted plant brassica oleracea plants, what then every potted plant wild cabbage is sprayed 5ml is the biopesticide of effective ingredient with Si-ace2_001 siRNA, and its concentration is 100ppm.If two repetitions.With DEPC water as contrast.
(6) be 10ppm with Si-ace2_001siRNA and above-mentioned cationic polymer as the concentration that effective ingredient is prepared into biopesticide siRNA, N: P is 10: 1, evenly coat cabbage leaves and feeding diamond-back moth second instar larvae, siRNA blade face concentration is 3.0 μ g/cm2.Each siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 48h investigate insect respectively, calculate lethality, experimental result such as table 5.
Find that by this experiment after the process nature was fed and scribbled the blade of described biopesticide, significantly dead (seeing also Fig. 5) appearred in insect.
Table 3: behind the cabbage leaves of the biopesticide that scribbles the acetylcholine esterase gene siRNA of feeding to the contrast statistics of diamond-back moth lethality
The calculating of the lethal concentration of 50 value of table 4:Si-ace2_001siRNA.
| The concentration of 2-1siRNA (ppm) | Total borer population (head) | Dead borer population (head) |
| 0 | 30 | 8 |
| 10 | 30 | 16 |
| 50 | 30 | 18 |
| 100 | 30 | 23 |
| 200 | 30 | 28 |
Table calculates the LC50=8.20ppm of Si-ace2_001siRNA thus; LC95=935.70ppm
Table 5: behind the cabbage leaves of the biopesticide that scribbles acetylcholine esterase gene Si-ace2_001siRNA and cationic polymer of feeding to the contrast statistics of diamond-back moth lethality
1-4 siRNA biopesticide in the experiment: No. 1 is that polymine (PEI), 2 is polylysine, is shitosan for gelatin and 4 No. 3.
Embodiment four
This example provides the Si-ace1_001siRNA of diamond-back moth acetylcholinesterasegene gene and mitochondria composite I IIFe-S subunit gene Si-UQCR_003siRNA to mix use especially, by behind this target gene siRNA mixed biologic agricultural chemicals of feeding naturally lepidopterous insects such as diamond-back moth having been caused fatal influence.
This example is the method that is used to prevent and treat lepidopterous insects diamond-back moth (Plutella xylostella).Contain the biopesticide of the mixing siRNA of reticent acetylcholinesterasegene gene 1 and mitochondria composite I IIFe-S subunit gene by feeding, (1) shows the electronics transmission and is obstructed, and is final because of can't normally synthesizing ATP; (2) influence the normal nerve impulse transmission of insect, make insect continue to be in excitatory state, thereby cause insect death, and then realized effective improvement insect.
The siRNA virulence experiment that is mixed: Si-UQCR_003siRNA siRNA and Si-ace1_001siRNA isoconcentration are mixed, evenly coat then on the cabbage leaves, blade face siRNA concentration is 3 μ g/cm
2, with this blade feeding diamond-back moth in three ages.Handled 10 cephalonts, investigated its lethality at 12h, 24h, 48h respectively and be respectively: 40%, 60%, 75%.
Find that by this example feed behind the mixing siRNA blade that scribbles selected genes through nature, insect death obviously increases than single siRNA.
Embodiment five
This example provides the siRNA of diamond-back moth gamma-aminobutyric acid receptor gene especially, by behind this target gene siRNA that feeds naturally lepidopterous insects such as diamond-back moth having been caused fatal influence.
This example is the method that is used to prevent and treat lepidopterous insects diamond-back moth (Plutella xylostella).Contain the siRNA of reticent gamma-aminobutyric acid receptor (the GeneBank accession number is EU273945) by feeding, thereby influence the normal cynapse transmission of insect, cause nervous function not normal, and then realize effective improvement insect.
γ-An Jidingsuan (GABA) is a kind of main inhibiting nerve mediator in animal (insect) body; GABA is a kind of important amino acid that derives from nonprotein; its the synthetic glutamic acid depickling enzyme that is subjected to is controlled; by causing the inhibition of neurotransmission with the GABA receptors bind; make postsynaptic neuron be in protectiveness holddown (KerrD.IB.; Ong J.GABA agonists andantagonists.Med Res Rev; 2002; 12 (6): 593～636), the GABA acceptor is one of most important target of insecticide.Biological Principles of the present invention is exactly by reticent insect gamma-aminobutyric acid receptor gene mRNA, and then influences the normal cynapse transmission of insect, causes nervous function not normal, thereby reaches the purpose of control of insect.
Particularly, the method of this biopesticide restraint insect gene expression of containing siRNA of feeding, be insect directly to be fed contain the siRNA of specific gene, cause insect significantly dead, suppress by a certain particular target expression of gene of this method to the insect correspondence.Described method mainly may further comprise the steps:
(1) selection of insect specific function gene:
Consider the key effect of insect gamma-aminobutyric acid receptor in nerve impulse is transmitted, thus the present invention to select the gene of this specific function be reticent object.
The design of diamond-back moth gamma-aminobutyric acid receptor gene siRNA and synthetic, sequence is as follows:
Si-GABA_001：GGGTCTATTACCAGAAGTA
Positive-sense strand (5 '-3 '): 5 '-GGGUCUAUUACCAGAAGUATT-3 ' SEQ ID NO.19
Antisense strand (3 '-5 '): 3 ' TTCCCAGAUAAUGGUCUUCAU-5 ' SEQ ID NO.20
Si-GABA_002：CCATGTATGTGCTCTCTAT
Positive-sense strand (5 '-3 '): 5 '-CCAUGUAUGUGCUCUCUAUTT-3 ' SEQ ID NO.21
Antisense strand (3 '-5 '): 3 '-TTGGUACAUACACGAGAGAUA-5 ' SEQ ID NO.22
Si-GABA_003：GTGGAGGAGACGAGGATAA
Positive-sense strand (5 '-3 '): 5 '-GUGGAGGAGACGAGGAUAATT-3 ' SEQ ID NO.23
Antisense strand (3 '-5 '): 3 '-TTCACCUCCUCUGCUCCUAUU-5 ' SEQ ID NO.24
As effective ingredient, the dilution of DEPC water is prepared into described biopesticide respectively with above-mentioned every couple of siRNA.
(3) siRNA is evenly coated cabbage leaves and feeding diamond-back moth second instar larvae: siRNA is diluted to the solution that concentration is 100ppm (biopesticide) with DEPC water, evenly coats then on the cabbage leaves, blade face concentration is 3.0 μ g/cm
2, with this blade feeding diamond-back moth in two ages.Each siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 36h, 48h, 60h, 72h investigate insect respectively, calculate lethality (table six).
(4) Si-GABA_001siRNA and above-mentioned cationic polymer are prepared into biopesticide, and evenly coat cabbage leaves and feeding diamond-back moth second instar larvae, its blade face concentration is 3.0 μ g/cm2.Each siRNA handles 30 cephalonts, establishes 3 repetitions, and with rotenone as positive controls.Afterwards, in the death toll that 24h, 48h, 72h investigate insect respectively, calculate lethality, experimental result such as table 7.
Find by this experiment, feed behind the siRNA blade that scribbles selected genes, cause insect individual significantly dead through nature.
Table 6: the contrast to the diamond-back moth lethality behind the cabbage leaves that scribbles the γ-An Jidingsuan gene siRNA of feeding is added up
Table 7: behind the cabbage leaves of the biopesticide (N: P is 50: 1) that scribbles γ-An Jidingsuan gene Si-GABA_001siRNA (SEQ ID NO.19 and 20) and cationic polymer of feeding to the statistics of diamond-back moth lethality
| Borer population (head) | 24 hours death toll | 48 hours death toll | 72 hours death toll | 72 hours lethality | |
| ?001 | 30 | 9 | 17 | 29 | 97％ |
| ?002 | 30 | 5 | 15 | 25 | 83％ |
| ?003 | 30 | 3 | 11 | 21 | 70％ |
| ?004 | 30 | 6 | 20 | 27 | 90％ |
| ?005 | 30 | 0 | 1 | 11 | 36％ |
001-005 number: No. 001 is shitosan, and No. 002 is polylysine, and No. 003 is gelatin, and No. 004 is polymine (PEI), and No. 005 is rotenone.
Embodiment six
This example provides the siRNA of diamond-back moth moulting hormone acceptor gene especially, by behind this target gene siRNA that feeds naturally lepidopterous insects such as diamond-back moth decortication growth course having been caused harmful effect.
This example is the method that is used to prevent and treat lepidopterous insects diamond-back moth (Plutella xylostella).Contain the siRNA of reticent moulting hormone acceptor (the GeneBank accession number is EF417852) by feeding, thereby influence the insect process of peeling normally, and then realize effective improvement insect.
The casting off a skin of insect, abnormal and breeding are subjected to the strictness regulation and control of moulting hormone. and the moulting hormone action target is by ecdysone receptor (ecdvsteroid receptor, EcR) and super valve albumen (ultraspiracle protein, USP) form, moulting hormone and EcR/USP effect start the cascade reaction process of casting off a skin. and the present invention attempts the expression by blocking-up diamond-back moth moulting hormone acceptor gene, disturb its normal decortication process, and then realize the purpose of pest control.
Particularly, the method for this siRNA restraint insect gene expression of feeding is insect directly to be fed contain the siRNA of specific gene, causes insect depauperation or death, suppresses by the expression of this method to a certain specific gene of insect correspondence.Described method mainly may further comprise the steps:
(1) selection of insect specific function gene:
Consider insect moulting hormone acceptor in the insect developmental key effect of peeling, thus the present invention to select the gene of this specific function be reticent object.
The design of diamond-back moth moulting hormone acceptor gene siRNA and synthetic:
Si-EcR_001 (R S27/A29): CTCACTCAAGCTCAAGAACAAGAAGCTGC (frame is represented the siRNA target spot)
Positive-sense strand (5 '-3 '): 5 '-CACUCAAGCUCAAGAACAAGAAGCUGC 3 ' SEQ ID NO.25
Antisense strand (3 '-5 '): 3 ' GAGUGAGUUCGAGUUCUUGUUCUUCGACG, 5 ' SEQ ID NO.26
Si-EcR_002 (R S27/A29): AGGCACAAAGGGAGAAGGATAAGCTGCCT (frame is represented the siRNA target spot)
Positive-sense strand (5 '-3 '): 5 '-GCACAAAGGGAGAAGGAUATT-3 ' SEQ ID NO.27
Antisense strand (3 '-5 '): 3 '-TTCGUGUUUCCCUCUUCCUAU-5 ' SEQ ID NO.28
Si-EcR_003 (R S27/A29): CTGCATGTACTCGCTGAATATGGACAATA (frame is represented the siRNA target spot)
Positive-sense strand (5 '-3 '): 5 '-GCAUGUACUCGCUGAAUAUTT-3 ' SEQ ID NO.29
Antisense strand (3 '-5 '): 3 '-TTCGUACAUGAGCGACUUAUA-5 ' SEQ ID NO.30
3 ' end of the siRNA of above-mentioned SEQ ID NO27-30 is provided with the suspension base of two TT.As effective ingredient, the dilution of DEPC water is prepared into described biopesticide respectively with above-mentioned every couple of siRNA.
(3) siRNA is evenly coated cabbage leaves and feeding diamond-back moth second instar larvae: siRNA is diluted to the solution that concentration is 100ppm with DEPC water, and (biopesticide) evenly coated on the cabbage leaves then, and blade face concentration is 3.0 μ g/cm
2, with this blade feeding diamond-back moth in two ages.Each siRNA handles 30 cephalonts, establishes 3 repetitions, and with DEPC water as negative control group.Afterwards, in the death toll that 12h, 24h, 36h, 48h, 60h, 72h investigate insect respectively, calculate lethality.Find by this experiment, feed behind the siRNA blade that scribbles selected genes, cause the insect individuality to show the not most symptom of decortication (referring to Fig. 6), but death is not remarkable through nature.
Sequence table
＜110〉the sharp rich bio tech ltd in Guangzhou
＜120〉a kind of biopesticide and insect control method
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Claims (10)
1. a biopesticide is characterized in that, is effective ingredient with the siRNA at the lepidopterous insects target gene, and described siRNA is the double-stranded RNA of external chemosynthesis 17-50nt.
2. biopesticide according to claim 1 is characterized in that, also including cationic polymer is effective ingredient.
3. biopesticide according to claim 2 is characterized in that, described cationic polymer is polymine, shitosan, polylysine or gelatin.
4. according to each described biopesticide of claim 1-3, it is characterized in that described siRNA also is provided with suspension base with the combination of two deoxyribonucleosides through chemical modification or siRNA3 ' end.
5. according to each described biopesticide of claim 1-3, it is characterized in that described target gene can be mitochondria composite I IIFe-S subunit gene, acetylcholinesterasegene gene and/or gamma-aminobutyric acid receptor gene.
6. according to the described biopesticide of claim 5, it is characterized in that, be one or more pairs of among SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ IDNO.5 and the SEQ ID NO.6 at the siRNA of mitochondria composite I IIFe-S subunit gene; At the siRNA of acetylcholinesterasegene gene is one or more pairs of among SEQID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and the SEQ ID NO.18; At the siRNA of gamma-aminobutyric acid receptor gene is one or more pairs of among SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQID NO.23 and the SEQ ID NO.24.
7. according to each described biopesticide of claim 1-3, it is characterized in that described lepidopterous insects is a diamond-back moth.
8. an insect control method is characterized in that, with each described biopesticide feeding lepidopterous insects of claim 1-7.
9. insect control method according to claim 8, it is characterized in that, described feeding mode is: described biopesticide directly is sprayed on the blade of the edible crops of lepidopterous insects, allows insect get food naturally, the blade face concentration of described siRNA is greater than 0.1 μ g/cm2.
10. siRNA who is used to prepare at the biopesticide of lepidopterous insects, it is characterized in that described siRNA is at the siRNA of mitochondria composite I IIFe-S subunit gene being one or more pairs of among SEQ ID NO.1 and SEQ ID NO.2, SEQID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and the SEQ ID NO.6; At the siRNA of acetylcholinesterasegene gene is one or more pairs of among SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ IDNO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and the SEQ ID NO.18; At the siRNA of gamma-aminobutyric acid receptor gene is one or more pairs of among SEQ ID NO.19 and SEQ IDNO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and the SEQ ID NO.24.
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| CN107858405A (en) * | 2017-10-12 | 2018-03-30 | 华南农业大学 | A kind of methods of measure external source dsRNA to ladybug toxic effect |
| CN109439685A (en) * | 2018-12-26 | 2019-03-08 | 菏泽学院 | A kind of construction method of the plant expression vector that can express gypsymoth USP gene dsRNA and its application |
| CN110964734A (en) * | 2019-12-13 | 2020-04-07 | 西北农林科技大学 | Insecticidal gene sequence and application thereof |
| CN112913784A (en) * | 2019-12-06 | 2021-06-08 | 中国林业科学研究院资源昆虫研究所 | Diapause induction method of Kallima inachus |
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| CN103109799A (en) * | 2013-02-26 | 2013-05-22 | 广州市锐博生物科技有限公司 | Chitosan nano particle, biological pesticide preparation for controlling tobacco caterpillars and preparation method thereof |
| CN103109798A (en) * | 2013-02-26 | 2013-05-22 | 广州市锐博生物科技有限公司 | Chitosan nano particle, biological pesticide preparation for controlling red fire ants and preparation method thereof |
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| CN103109798B (en) * | 2013-02-26 | 2015-04-08 | 广州市锐博生物科技有限公司 | Chitosan nano particle, biological pesticide preparation for controlling red fire ants and preparation method thereof |
| CN105613568B (en) * | 2014-10-30 | 2018-08-31 | 江苏命码生物科技有限公司 | It is a kind of using siRNA using plant as the biological pesticide of generator |
| CN105613568A (en) * | 2014-10-30 | 2016-06-01 | 江苏命码生物科技有限公司 | Biological pesticide using siRNA and taking plant as generator |
| CN107858405A (en) * | 2017-10-12 | 2018-03-30 | 华南农业大学 | A kind of methods of measure external source dsRNA to ladybug toxic effect |
| CN109439685A (en) * | 2018-12-26 | 2019-03-08 | 菏泽学院 | A kind of construction method of the plant expression vector that can express gypsymoth USP gene dsRNA and its application |
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| CN110964734A (en) * | 2019-12-13 | 2020-04-07 | 西北农林科技大学 | Insecticidal gene sequence and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102960363A (en) | 2013-03-13 |
| CN101919404B (en) | 2015-01-14 |
| CN102960363B (en) | 2015-08-19 |
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