CN103109799A

CN103109799A - Chitosan nano particle, biological pesticide preparation for controlling tobacco caterpillars and preparation method thereof

Info

Publication number: CN103109799A
Application number: CN2013100609369A
Authority: CN
Inventors: 张必良
Original assignee: GUANGZHOU RIBOBIO CO Ltd
Current assignee: GUANGZHOU RIBOBIO CO Ltd
Priority date: 2013-02-26
Filing date: 2013-02-26
Publication date: 2013-05-22
Anticipated expiration: 2033-02-26
Also published as: CN103109799B

Abstract

The invention discloses a chitosan nano particle which comprises the following raw materials in percentage by weight: 0.5-5% of chitosan, 1-10% of cosolvent, 0-5% of antifreezing agent, 0.1-1% of surfactant, 0.1-0.5% of emulsifier, 40-60% of buffer solution and the balance of water. The invention also provides a biological pesticide preparation for controlling tobacco caterpillars and a preparation method thereof. The biological pesticide preparation mainly comprises the following components in percentage by weight: (A) 0.05-15% of active component: a siRNA (small interfering ribonucleic acid) is designed and synthesized in accordance with a tobacco caterpillar high-efficiency killing target gene and is a 15-50nt double-chain RNA; (B) 19.95-55% of solvent; and (C) 30-80% of chitosan nano particle. The chitosan nano particle ensures that the stability and the control effect of the biological pesticide preparation are better.

Description

Biopesticide preparation of chitosan nano, control prodenia litura and preparation method thereof

Technical field

The invention belongs to the agricultural insecticide technical field, be specifically related to a kind of biopesticide preparation and preparation method thereof, a kind of especially chitosan nano, contain the biopesticide preparation and preparation method thereof of the control prodenia litura of double stranded RNA.

Technical background

The spodoptera Lepidoptera, noctuid, various vegetables and the crops such as the Cruciferae of can causing harm, Solanaceae, pulse family, Curcurbitaceae and green onion garlic, also more common on weeds, edible wild herbs, be the extremely assorted insects of a kind of feeding habits, its host plant reaches kind more than 200.In Middle And Lower Reaches of Changjiang River has almost broken out since the nineties in 20th century year after year and causes disaster, one of main insect of flowers and plants with having become brassicaceous vegetable, urban afforestation.Because the annual generable generation number of prodenia litura is many, generating capacity is large, easily breaks out and causes disaster, so prophylactico-therapeutic measures at present is still to use chemical pesticide as main.But due to a large amount of uses of chemical pesticide, add operational lack of standardizationly, make prodenia litura generally produce pesticide resistance.The research of relevant this respect all has report both at home and abroad, since the sixties, on the ground such as India, Egypt, Japan, Turkey and the area priorities such as the Taiwan of China, Shanghai reported that prodenia litura has all produced resistance to multiple insecticide, comprising organochlorine, organic phosphor, carbamate, pyrethroid and bacillus thuringiensis,Bt (Bt) etc.Under this prevention and control situation, research is efficient, green, environmental protection, sustainable development pesticidal preparations have been very important research topic in vegetables production.

RNA disturbs (RNA interference, RNAi) to refer to that external source or endogenous double-stranded RNA cause the phenomenon of gene silencing specifically, are widely used in the lepidoptera pest gene functional research at present.As: Z.-X.Yang etc. import the dsRNA of cadherin gene with the method for microinjection, cause the lethality of this worm and sex ratio to increase, and the diapause phenomenon appears in larval phase.By the method for feeding, import the dsRNA of spodoptera frugiperda cell cytochrome p 450 (CYP6BG1) gene, cause this worm that the resistance of Dalmatian chrysanthemum lipid agricultural chemicals is reduced.In addition, in dsRNA solution, the initiation gene silencing is also laboratory effective means commonly used by polypide is immersed in.The breakthrough that the RNA perturbation technique is obtained aspect preventing and treating at lepidoptera pest is that theoretical foundation has been established in the control of prodenia litura, and a kind of new prodenia litura control strategy is provided, and has opened up the vegetables protection new situation.

Although gene silent technology has been opened the new page of prodenia litura control, this technological means is proved to be to the killing action of prodenia litura, but at present a large amount of trials only limits to laboratory scope, and also unrealized practical operation application yet there are no report based on the biopesticide preparation of RNAi.The factor that limits this technology extensive use is mainly: (one) does not develop can strengthen the dsRNA stable effective ingredients, keep its insecticidal effect, be fit to the mature and stable advanced pesticidal preparations that scale is used; (2) do not probe into out scale production process simple and convenient, the tool operability.

The sign of advanced pesticidal preparations is efficient, environmental friendliness and controlled release.Efficiently depend on active ingredient itself and its particle size in formulation, size is less, and surface area is larger, and efficient is just higher.Water baseization pesticidal preparations such as aqua, emulsifier, aqueous emulsion, suspending agent, suspension emulsion etc. are considered to the environmental friendliness formulation, and the particle size of its agricultural chemicals is increased to micron successively by nanometer, but does not have controlled-release function.For the size that reduces agricultural chemicals with realize slowly-releasing, document is reported for work agricultural chemicals is made nanocrystal, perhaps apply with synthetic high polymer in the nanoparticle outside, or liquid pesticidal is comprised into microcapsules, and with the multiple entry vortex mixer, Biphenthrin is made nano particle, with sodium alginate and shitosan by layer assembly with the Imidacloprid parcel etc., but these methods are loaded down with trivial details, and remarkable, be difficult for carrying out large-scale production.

Chitosan nano is medically becoming the focus of domestic and international research and development as a kind of new drug carrier.Wu Sihui (2009) etc. makes the nanoparticle load insulin with shitosan, control medicine release, prolong drug curative effect, reduce poisonous side effect of medicine, can improve permeability and the medicine stability of hydrophobic drug cell membrane and change method of administration, can also strengthen the target administration ability of preparation.The preparation of the research chitosan nanos such as Liu Hui (2007) also detects its performance, wishes to obtain a kind of pharmaceutical carrier that can use in the mucosal drug delivery system, thereby realizes the sustained release of antibacterials, reaches the target of long-term efficacy.Cell membrane passes in the Sudan (2007) etc. take chitosan nano as carrier loaded siRNA, enter the RNAi path in cytoplasm, for human diseases genesis mechanism, the discriminating of disease gene treatment target spot and the discovery of new drug development such as micromolecular compound and antibody class medicine provide theoretical foundation, but its research only is fit to medical science and is not used in pesticide research.Much more chitosan nano has report in application medically, but the application report on agricultural is also rare.Lin Chunmei (2009) etc. utilizes the cationic characteristic of shitosan band, makes it and occurs from crosslinked with the sodium phosphate trimer of anion, forms nanoparticle after wrapping portion Avermectin particle.Zhao Shuyan (2009) etc. make the rate of release of urea slow down with chitosan nano embedding urea, strengthen slow release effect.Feng Bohua (2009) etc. wrap up rotenone, nimbin botanical pesticide take sodium ricinoleate and shitosan as raw material prepares nano particle, have avoided the use of a large amount of organic solvents and auxiliary agent.Li Min (2011) etc. prepares oligomer by the hydrogen peroxide degradation shitosan, and take Imidacloprid, chlopyrifos as model drug, physicochemical property and the different preparation method of research nano particle form mechanism.Zhang Ziyong (2012) etc. are take shitosan and CMC as carrier, preparation nimbin nano particle aqueous dispersions.Although above-mentioned report has successfully prepared the agricultural chemicals chitosan nanoparticle preparation, but its research and development all are in exploration stage, the physical and chemical indexs such as prepared nanoparticle size, particle diameter distribution, drug loading rate are all unstable, unrealized standardization, secondly technique is loaded down with trivial details, be difficult for realizing large-scale production, thus be confined to so far laboratory research, and have no and be converted into the product formulation circulation market.In addition, the pesticide active ingredient of the agricultural chemicals chitosan preparations embedding that success is at present reported mostly is existing chemical pesticide, and molecular weight is little, and technical operation is relatively simple.

The present invention is based on above-mentioned present situation; invent out the first based on the biopesticide preparation of the control prodenia litura of gene silent technology; utilize simultaneously a kind of preparation method of simple and direct, scale, develop and be applicable to the macromolecular chitosan nano auxiliary agent of active ingredient dsRNA.Biopesticide preparation of the present invention can not only the high-efficiency prevention and control prodenia litura, and can external maintenances dsRNA stable, strengthens its insecticidal effect.Preparation of the present invention is the advanced pesticidal preparations of a kind of efficient, environmental friendliness and controlled release.

Summary of the invention

One of purpose of the present invention is to provide a kind of macromolecular chitosan nano that is applicable to the active ingredient siRNA of biopesticide preparation.

The technical scheme that realizes above-mentioned purpose is as follows:

A kind of chitosan nano, its raw material by following percentage by weight forms:

Shitosan 0.5-5%, cosolvent 1-10%, antifreezing agent 0-5%, surfactant 0.1-1%, emulsifier 0.1-0.5%, buffer solution 40-60%, surplus are water.

Cosolvent in chitosan nano preferably, is one or several mixtures in Macrogol 200, PEG400, ethanol, propane diols for increasing the dissolubility of shitosan.

Antifreezing agent in chitosan nano avoids for the protection of the preparation composition physical hazard that low temperature causes, and preferably, described antifreezing agent is one or several mixtures in glycerine, ethylene glycol, isopropyl alcohol, diethylene glycol (DEG).

Surfactant in chitosan nano is for reducing the surface tension of solution, strengthened preparation at wetting, spread and the adhesive force of plant or insect body surface, thereby improved drug effect.Preferably, described surfactant is one or several mixtures in polysorbas20, polysorbate40, polysorbate60, Tween 80.

emulsifier in chitosan nano is used for improving peptization, emulsification, disperse, crosslinked, preferably, described emulsifier is sodium phosphate trimer, agriculture breast 400#(benzyl dimethyl phenol polyethenoxy ether), agriculture breast 500#(alkyl benzene calcium sulfonate), agriculture breast 600#(phenethyl phenol polyethenoxy ether), Nongru-700 #(alkylphenol polyoxyethylene formaldehyde condensation products), agriculture breast 1600#(phenethyl phenol polyethenoxy polyethenoxy ether), NP series phosphate (polyoxyethylene nonylphenol ether phosphate), phenethyl polyoxyethylene ether ammonium sulfate salt, one or several mixtures in alkyl biphenyl ether disulfonic acid magnesium salts.

Buffer solution in chitosan nano is used for the preparation acid-base accommodation, increases the auxiliary agent dissolubility.Preferably, described buffer solution is one or several mixtures of acetate buffer, phosphate buffer or citrate buffer.

Chitosan nano be different from existing nanoparticle can coating large molecular agricultural nanoparticle, its particle diameter is 100-600nm.

Another object of the present invention is to provide a kind of biopesticide preparation of preventing and treating the lepidoptera pest prodenia litura, and this biopesticide preparation can effectively be controlled prodenia litura, is the environment friendly agricultural of sustainable development.

Concrete scheme is as follows:

A kind of biopesticide preparation of preventing and treating prodenia litura, it mainly is grouped into by the one-tenth of following percentage by weight:

(A) active ingredient of 0.05-15%: for the synthetic siRNA of the efficient lethal target gene design of prodenia litura, described siRNA is that 15-50nt(is preferably 17-25nt) double-stranded RNA,

(B) 19.95-55% be used for dissolving active ingredient double stranded RNA solvent,

(C) the above-mentioned chitosan nano of 30-80%.

Described active ingredient double stranded RNA is by gene silencing mechanism kill harmful insect.

Preferably, the efficient lethal target gene of described prodenia litura is cytochrome P450 gene, acetylcholinesterasegene gene, gamma-aminobutyric acid receptor α subunit gene, moulting hormone gene.More preferably, described siRNA is that the double stranded RNA that is selected from for acetylcholinesterasegene gene is SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; Double stranded RNA for the moulting hormone gene is 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; Double stranded RNA for gamma-aminobutyric acid receptor α subunit gene is SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18; With double stranded RNA for cytochrome P450 gene be one or more pairs of in SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30.

Described active ingredient double stranded RNA few nucleotide is that 15-50nt, molecular weight are 8000-40000.In embodiment, the 3 ' end of described siRNA is provided with the suspension base therein.

Described solvent is used for dissolving active ingredient double stranded RNA, and preferably, described solvent is one or several mixtures in methyl alcohol, methyl-sulfoxide, water, Tris buffer solution.

Another object of the present invention has been to provide the preparation method of the biopesticide preparation of a kind of above-mentioned control prodenia litura.

Concrete technical scheme is as follows:

The preparation method of the biopesticide preparation of a kind of above-mentioned control prodenia litura comprises the following steps:

(A) dissolving: take described siRNA, (preferably under the rotating speed of 800-3000r/min) is dissolved in solvent, and (preferably stirring 5-60min) fully dissolves double stranded RNA, gets solution

(B) chitosan nano preparation:

1) accurately take shitosan, cosolvent and antifreezing agent, join in buffer solution, (preferred 800-3000r/min) stirs it fully dissolved;

2) with mentioned solution (preferably with 3000-10000rpm) centrifugal (preferred 10-60min), get supernatant, add surfactant, fully stir mixing;

3) (preferably with 10-30ml/min speed) after emulsifier water dissolving being added step 2) in solution, mixing namely gets chitosan nano, particle diameter 100-600nm;

(C) preparation preparation: gained solution in step (A) is evenly mixed with the described chitosan nano of step (B), and the double stranded RNA biopesticide preparation that namely gets stable homogeneous is stirred in rear continuation.

The physical and chemical indexs such as the particle diameter distribution of the chitosan nano of preparation and biopesticide preparation, drug loading rate are all more stable according to the method described above, can carry out standardization production.

Compare with existing technologies, the present invention has following innovative point:

The first, the carrier of pesticidal preparations of the present invention is chitosan nano, be different from existing nanoparticle can coating large molecular agricultural nanoparticle.Because active ingredient is double stranded RNA, mean molecule quantity is all between 8000-40000, molecular weight is large, need higher technology to meet the embedding of large molecule active ingredient in chitosan nano preparation, so there is essential distinction in the nanoparticle of chitosan nano of the present invention and the little molecular chemistry agricultural chemicals of existing embedding.

The second, the present invention is the chitosan nanoparticle preparation of double stranded RNA, more is conducive to improve stable effective ingredients than the simple compatibility thing of related double stranded RNA and cationic compound in previous patent, keeps higher pest controling effect.

Three, the present invention is the first Biological Control of Plutella xylostella agent based on gene silent technology, worldwide belongs to the double stranded RNA agricultural chemicals of the first formulation maturation.Based on the mechanism of its special kill pests, said preparation is the biopesticide preparation of a kind of environmental protection, green, health.

Four, the present invention has a technological process that cover is ripe, easy, industrialization operates, related production stage standard, standard, the indexs such as physicochemical property of the nanoparticle biopesticide preparation of producing are optimisation criteria, easily realize the large-scale industrialization operation, utilize this preparation method can break through the laboratory operation scale, realize the marketization of pesticide nano formulation products.

Embodiment

Below by specific embodiment, method of the present invention is described, but the present invention is not limited to this.

Embodiment one

The present embodiment provides preparation and the effect research thereof take prodenia litura acetylcholinesterasegene gene double stranded RNA sequence as the chitosan nano biologic product of active ingredient especially.

(1) for the design of the former medicine composition of prodenia litura acetylcholinesterasegene gene double stranded RNA and synthetic

GenBank：FJ959384.1

Si-Ace1_001: target gene CAGGA ACTGC TACTC TA

Positive-sense strand (5'-3'): 5'CAGGA ACUGC UACUC UA dTdT3'SEQ ID NO.1

Antisense strand (3'-5'): 3'dTdT GUCCU UGACG AUGAG AU5'SEQ ID NO.2

Si-Ace1_002：TGTCG?GTTTC?ATTGC?AT

Positive-sense strand (5'-3'): 5'UGUCG GUUUC AUUGC AU dTdT3'SEQ ID NO.3

Antisense strand (3'-5'): 3'dTdT ACAGC CAAAG UAACG UA5'SEQ ID NO.4

Si-Ace1_003：GGCGC?ATCGT?TATGC?AG

Positive-sense strand (5'-3'): 5'GGCGC AUCGU UAUGC AG dTdT3'SEQ ID NO.5

Antisense strand (3'-5'): 3'dTdT CCGCG UAGCA AUACG UC5'SEQ ID NO.6

3 ' end of above-mentioned sequence is provided with the suspension base of dTdT.Become former medicinal powder last with synthetic double stranded RNA and freeze drying according to above-mentioned sequences Design.

The preparation of (two) 0.05% double stranded RNA biopesticide preparations

1 active ingredient dissolving: take respectively the above-mentioned double stranded RNA of 500mg, be dissolved in 199.5g water, 800r/min stirs it is fully dissolved;

2 chitosan nano preparations

Doing cosolvent, glycerine with Macrogol 200 does antifreezing agent, polysorbas20 and does that surfactant, sodium phosphate trimer are made emulsifier, phosphate buffer is cooked buffer solution and prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are difference 5%, 10%, 5%, 1%, 0.5%, 60% preparation chitosan nano, and surplus is water.

(1) take shitosan 40g, Macrogol 200 80g, glycerine 40g, be dissolved in 480g phosphate buffer (pH4-5), stir 12h under the 800r/min rotating speed, shitosan is fully dissolved;

(2) with step (1) gained solution centrifugal 10min under 3000r/min, get supernatant, add the 8g polysorbas20, fully mix;

(3) take the 4g sodium phosphate trimer and be dissolved in 156g water, fully stir, then mix with step (2) gained solution, namely get chitosan nano, particle diameter 100-600nm;

3 preparations preparations: active ingredient solution in step 1 is evenly mixed with step 2 gained chitosan nano solution, namely get all more stable biopesticide preparations of the physical and chemical indexs such as particle diameter distribution, drug loading rate.

The control efficiency of (three) 0.05% double stranded RNA biopesticide preparations

0.05% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is observed preventive effect after 72h, and result is as shown in the table.

Indicator is that the biologic product of active ingredient is to the control efficiency of prodenia litura to the synthetic double stranded RNA of prodenia litura acetylcholinesterasegene gene design

Double stranded RNA	Lethality (%)
		Si-Ace1_001	68.72
Si-Ace1_002	70.15
		Si-Ace1_003	86.22
CK1	72.66
		CK2	69.50

CK3

19.70

Wherein CK1 is 0.05%Si-Ace1_003 double stranded RNA+cationic polymer shitosan; CK2 is 0.05%Si-Ace1_003 double stranded RNA+cationic polymer polylysine; CK3 is DEPC water.

Composition and preparation method according to above-mentioned biopesticide preparation, the preparation concentration be 15wt% double stranded RNA biopesticide preparation (wherein, described chitosan nano is 35%, water is 50%), it is diluted to 100mg/L, evenly spray on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, every processing triplicate is observed preventive effect after 72h, result is as follows:

Double stranded RNA	Lethality (%)
		Si-Ace1_001	68.33
Si-Ace1_002	73.82
		Si-Ace1_003	83.97
CK1	72.45
		CK2	68.50
CK3	20.73

Wherein CK1 is 15wt%Si-Ace1_003 double stranded RNA+cationic polymer shitosan; CK2 is 15wt%Si-Ace1_003 double stranded RNA+cationic polymer polylysine; CK3 is DEPC water.

Embodiment two

It is preparation and the effect research thereof of the chitosan nano biologic product of active ingredient that the present embodiment provides for the double-stranded RNA sequence of prodenia litura ecdysone receptor gene especially.

(1) for the design of the former medicine composition of prodenia litura ecdysone receptor gene double stranded RNA and synthetic

GenBank:EU180021.1

Si-EcR_001：GTTGC?TCGGA?TTGTG?TTGT

Positive-sense strand (5'-3'): 5'GUUGC UCGGA UUGUG UUGU dTdT3'SEQ ID NO.7

Antisense strand (3'-5'): 3'dTdT CAACG AGCCU AACAC AACA5'SEQ ID NO.8

Si-EcR_002：GAGTG?GTGCA?TGTCC?ATAG

Positive-sense strand (5'-3'): 5'GAGUG GUGCA UGUCC AUAG dTdT3'SEQ ID NO.9

Antisense strand (3'-5'): 3'dTdT CUCAC CACGA ACAGG UAUG5'SEQ ID NO.10

Si-EcR_003：TCGCA?AAGAT?CTCAC?AATC

Positive-sense strand (5'-3'): 5'UCGCA AAGAU CUCAC AAUC dTdT3'SEQ ID NO.11

Antisense strand (3'-5'): 3'dTdT AGCGU UUCUA GAGUG UUAG5'SEQ ID NO.12

The preparation of (two) 15% double stranded RNA biopesticide preparations

1 active ingredient dissolving: take the above-mentioned double stranded RNA of 150g, be dissolved in 350g methyl alcohol, 2000r/min stirs it is fully dissolved;

2 chitosan nano preparations

Doing cosolvent, ethylene glycol with PEG400 does antifreezing agent, polysorbate40 and does that surfactant, agriculture breast 500# are made emulsifier, acetate buffer is cooked buffer solution and prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are difference 2%, 4%, 3%, 0.5%, 0.5%, 45% preparation chitosan nano, and surplus is water.

(1) take shitosan 10g, PEG400 20g, ethylene glycol 15g, be dissolved in 225g acetate buffer solution (pH4-5), stir 24h under the 1000r/min rotating speed, shitosan is fully dissolved;

(2) with step (1) gained solution centrifugal 5min under 2500r/min, get supernatant, add the 2.5g polysorbate40, fully mix;

(3) take 2.5g agriculture breast 500# and be dissolved in 225g water, fully stir, rear and step (2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm;

3 preparation preparations: active ingredient solution in step 1 is evenly mixed with step 2 gained chitosan nano solution, and get final product.

The control efficiency of (three) 15% double stranded RNA biopesticide preparations

15% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is observed preventive effect after 72h, and result is as shown in the table.

The control efficiency of the biologic product of table take the synthetic double stranded RNA of prodenia litura ecdysone receptor gene design as active ingredient to prodenia litura

Double stranded RNA	Symptom or lethality (%) appear
		Si-EcR_001	68.70
Si-EcR_002	71.66
		Si-EcR_003	64.79
CK1	66.25

CK2	62.37
		CK3	10.00

Wherein CK1 is 15%Si-EcR_001 double stranded RNA+85% cationic polymer shitosan; CK2 is 15%Si-EcR_001 double stranded RNA+85% cationic polymer polylysine; CK3 is DEPC water.

Composition and preparation method according to above-mentioned biopesticide preparation, the preparation concentration be 1wt% double stranded RNA biopesticide preparation (wherein, described chitosan nano is 74%, methyl alcohol is 25%), it is diluted to 100mg/L, evenly spray on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, every processing triplicate is observed control efficiency after 72h, result is as follows:

Double stranded RNA	Symptom or lethality (%) appear
		Si-EcR_001	69.87
Si-EcR_002	72.50
		Si-EcR_003	65.40
CK1	67.82
		CK2	60.33
CK3	10.00

Wherein CK1 is 1%Si-EcR_001 double stranded RNA+cationic polymer shitosan; CK2 is 1%Si-EcR_001 double stranded RNA+cationic polymer polylysine; CK3 is DEPC water.

Embodiment three

It is preparation and the effect research thereof of the chitosan nano biologic product of active ingredient that the present embodiment provides for prodenia litura gamma-aminobutyric acid receptor α subunit gene double stranded RNA sequence especially.

(1) for the design of the former medicine of prodenia litura gamma-aminobutyric acid receptor α subunit gene double stranded RNA and synthetic

GenBank:JN792582.1

Si-GABA_001：CTGAA?GTGAA?AATGG?AT

Positive-sense strand (5'-3'): 5'CUGAA GUGAA AAUGG AU dTdT3'SEQ ID NO.13

Antisense strand (3'-5'): 3'dTdT GACUU CACUU UUACC UA5'SEQ ID NO.14

Si-GABA_002：TATGC?AATAT?TGAAA?TC

Positive-sense strand (5'-3'): 5'UAUGC AAUAU UGAAA UC dTdT3'SEQ ID NO.15

Antisense strand (3'-5'): 3'dTdT AUACG UUAUA ACUUU AG5'SEQ ID NO.16

Si-GABA_003：GCGAC?ACCTG?CTAGG?GT

Positive-sense strand (5'-3'): 5'GCGAC ACCUG CUAGG GU dTdT3'SEQ ID NO.17

Antisense strand (3'-5'): 3'dTdT CGCUG UGGAC GAUCC CA5'SEQ ID NO.18

The preparation of (two) 5% double stranded RNA biopesticide preparations

1 active ingredient dissolving: take the 50g double stranded RNA, be dissolved in the 550g methyl-sulfoxide, 3000r/min stirs it is fully dissolved;

The preparation of 2 chitosan nanos

Doing antifreezing agent, Macrogol 200 with isopropyl alcohol does cosolvent, polysorbate60 and does that surfactant, Nongru-700 # are made emulsifier, citrate buffer is cooked buffer solution and prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are difference 1%, 1%, 1%, 0.1%, 0.1%, 40% preparation chitosan nano, and surplus is water.

(1) take shitosan 4g, Macrogol 200 4g, isopropyl alcohol 4g, be dissolved in 160g citrate buffer (pH4-5), stir 24h under the 1000r/min rotating speed, shitosan is fully dissolved;

(2) with step (1) gained solution centrifugal 5min under 2500r/min, get supernatant, add the 0.4g polysorbate60, fully mix;

(3) take 0.4g Nongru-700 # and be dissolved in 227.2g water, fully stir, rear and step (2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm.

The control efficiency of (three) 5% double stranded RNA biopesticide preparations

5% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is added up lethality after 48h, and result is as shown in the table.

Indicator is that the biologic product of active ingredient is to the control efficiency of prodenia litura to the synthetic double stranded RNA of prodenia litura gamma-aminobutyric acid receptor α subunit gene design

Double stranded RNA	Lethality (%)
		Si-GABA_001	78.64
Si-GABA_002	73.21
		Si-GABA_003	72.39

CK1	69.73
		CK2	67.55
CK3	32.80

Wherein CK1 is 5%Si-GABA_003 double stranded RNA+cationic polymer shitosan; CK2 is 5%Si-GABA_003 double stranded RNA+cationic polymer polylysine; CK3 is DEPC water.

Composition and preparation method according to above-mentioned biopesticide preparation, the preparation concentration be 10wt% double stranded RNA biopesticide preparation (wherein, described chitosan nano is 35%, methyl-sulfoxide is 55%), it is diluted to 100mg/L, evenly spray on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, every processing triplicate is added up control efficiency after 48h, result is as follows:

Double stranded RNA	Lethality (%)
		Si-GABA_001	77.25
Si-GABA_002	73.66
		Si-GABA_003	71.95
CK1	68.74
		CK2	65.38
CK3	23.50

Wherein CK1 is 10%Si-GABA_003 double stranded RNA+cationic polymer shitosan; CK2 is 10%Si-GABA_003 double stranded RNA+cationic polymer polylysine; CK3 is DEPC water.

Embodiment four

It is preparation and the effect research thereof of the chitosan nano biologic product of active ingredient that the present embodiment provides for the double-stranded RNA sequence of spodoptera frugiperda cell cytochrome p 450 gene especially.

(1) for the design of the former medicine of spodoptera frugiperda cell cytochrome p 450 gene ribonucleic acid and synthetic:

GenBank：DQ352137.2

Si-CYP4M14v1_001：GGCTC?GACTG?ATCAG?AA

Positive-sense strand (5'-3'): 5'GGCUC GACUG AUCAG AA dTdT3'SEQ ID NO.19

Antisense strand (3'-5'): 3'dTdT CCGAG CUGAC UAGUC UU5'SEQ ID NO.20

Si-CYP4M14v1_002：GAGAG?GTTTC?TGGAG?GT

Positive-sense strand (5'-3'): 5'GAGAG GUUUC UGGAG GU dTdT3'SEQ ID NO.21

Antisense strand (3'-5'): 3'dTdT CUCUC CAAAG ACCUC CA5'SEQ ID NO.22

Si-CYP4M14v1_003：GATGA?TAACA?GCGCT?GC

Positive-sense strand (5'-3'): 5'GAUGA UAACA GCGCU GC dTdT3'SEQ ID NO.23

Antisense strand (3'-5'): 3'dTdT CUACU AUUGU CGCGA CG5'SEQ ID NO.24

Si-CYP4M14v1_004：GACAC?ATTTA?TGTTT?GA

Positive-sense strand (5'-3'): 5'GACAC AUUUA UGUUU GA dTdT3 ' SEQ ID NO.25

Antisense strand (3'-5'): 3'CUGUG UAAAU ACAAA CU dTdT5'SEQ ID NO.26

Si-CYP4M14v1_005：ACCAA?GAAAT?TGTAT?AG

Positive-sense strand (5'-3'): 5'ACCAA GAAAU UGUAU AG dTdT3'SEQ ID NO.27

Antisense strand (3'-5'): 3'dTdT UGGUU CUUUA ACAUA UC5'SEQ ID NO.28

Si-CYP4M14v1_006：TGACT?TGGTA?CTCAG?GA

Positive-sense strand (5'-3'): 5'UGACU UGGUA CUCAG GA dTdT3'SEQ ID NO.29

Antisense strand (3'-5'): 3'dTdT ACUGA ACCAU GAGUC CU5'SEQ ID NO.30

The preparation of (two) 5% biopesticide preparations

1, configure the double stranded RNA biopesticide preparation that contains different antifreezing agents take the Si-CYP4M14v1_001 gene siRNA as active ingredient

(1) active ingredient dissolving: take 50g Si-ace1_001 double stranded RNA and be dissolved in 550g Tris buffer solution, 3000r/min stirs it is fully dissolved;

(2) chitosan nano preparation

Do the antifreezing agent agent with glycerine, ethylene glycol, isopropyl alcohol, diethylene glycol (DEG), glycerine+ethylene glycol, glycerine+isopropyl alcohol+diethylene glycol (DEG) respectively, do cosolvent, polysorbate40 with Macrogol 200 and do that surfactant, agriculture breast 400# are made emulsifier, citrate buffer is cooked buffer solution and prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are respectively 1%, 1%, 1%, 0.1%, 0.1%, 40% preparation chitosan nano, and surplus is water.

1) take shitosan 4g, Macrogol 200 4g, antifreezing agent 4g, be dissolved in 160g citrate buffer (pH4-5), stir 24h under the 1000r/min rotating speed, shitosan is fully dissolved;

2) with step 1) gained solution centrifugal 5min under 2500r/min, get supernatant, add the 0.4g polysorbate40, fully mix;

3) taking 0.4g agriculture breast 400# and be dissolved in 227.2g water, fully stir, rear and step 2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm;

(3) preparation preparation: active ingredient solution in step (1) is evenly mixed with step (2) gained chitosan nano solution, and get final product.

The preventive effect of (4) 5% double stranded RNA biopesticide preparations to prodenia litura

5% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is added up lethality after 48h.Result is as shown in the table.

Table contains the biopesticide preparation of different antifreezing agent agent compositions to the control efficiency of prodenia litura

Antifreezing agent	Lethality (%)
		Glycerine	48.00
Ethylene glycol	57.69
		Isopropyl alcohol	45.74
Diethylene glycol (DEG)	47.61
		Glycerine+ethylene glycol	49.83
Glycerine+isopropyl alcohol+diethylene glycol (DEG)	47.50
		CK1	43.42
CK2	43.67
		CK3	18.06

Wherein CK1 is 5% active ingredient double stranded RNA+cationic polymer gelatin, and CK2 is 5% active ingredient double stranded RNA+cationic polymer shitosan, and CK3 is DEPC water.

2, configure the double stranded RNA biopesticide preparation that contains the different surfaces activating agent take spodoptera frugiperda cell cytochrome p 450 gene Si-CYP4M14v1_002 gene double stranded RNA as active ingredient

(1) take the 150g double stranded RNA, be dissolved in 350g methyl alcohol, 2000r/min stirs it is fully dissolved;

(2) preparation of chitosan nano

Respectively take polysorbas20, polysorbate40, polysorbate60, Tween 80, polysorbas20+polysorbate60, polysorbate40+polysorbate60+Tween 80, polysorbas20+polysorbate40+polysorbate60+Tween 80 as surfactant, do cosolvent, ethylene glycol with PEG400 and do that antifreezing agent, agriculture breast 500# are made emulsifier, phosphate buffer is cooked buffer solution and prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are difference 2%, 4%, 3%, 0.5%, 0.5%, 45% preparation chitosan nano, and surplus is water.

1) take shitosan 10g, PEG400 20g, ethylene glycol 15g, be dissolved in 225g phosphate buffer (pH4-5), stir 24h under the 1000r/min rotating speed, shitosan is fully dissolved;

2) with step 1) gained solution centrifugal 5min under 2500r/min, get supernatant, add the 2.5g surfactant, fully mix;

3) taking 2.5g agriculture breast 500# and be dissolved in 225g water, fully stir, rear and step 2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm;

(3) preparation preparation: active ingredient solution in step (1) is evenly mixed with step (2) gained chitosan nano solution, namely get 15% double stranded RNA biopesticide preparation.

The preventive effect of (4) 15% double stranded RNA biopesticide preparations to prodenia litura

15% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is observed control efficiency after 48h, and result is as shown in the table.

Table contains the biopesticide preparation of different surfaces bioactive agent composition to the control efficiency of prodenia litura

Surfactant	Lethality (%)
		Polysorbas20	60.00
Polysorbate40	63.12
		Polysorbate60	61.87
Tween 80	58.42
		Polysorbas20+polysorbate60	59.15
Polysorbate40+polysorbate60+Tween 80	60.04
		Polysorbas20+polysorbate40+polysorbate60+Tween 80	60.58
CK1	55.62
		CK2	54.56
CK3	22.34

Wherein CK1 is 15% active ingredient double stranded RNA+cationic polymer gelatin, and CK2 is 15% active ingredient double stranded RNA+cationic polymer shitosan, and CK3 is DEPC water.

3, configure the double stranded RNA biopesticide preparation that contains different emulsifiers take spodoptera frugiperda cell cytochrome p 450 Si-CYP4M14v1_003 gene ribonucleic acid as active ingredient

(2) preparation of chitosan nano

respectively with sodium phosphate trimer, agriculture breast 400#(benzyl dimethyl phenol polyethenoxy ether), agriculture breast 500#(alkyl benzene calcium sulfonate), agriculture breast 600#(phenethyl phenol polyethenoxy ether), Nongru-700 #(alkylphenol polyoxyethylene formaldehyde condensation products), agriculture breast 1600#(phenethyl phenol polyethenoxy polyethenoxy ether), NP series phosphate (polyoxyethylene nonylphenol ether phosphate), phenethyl polyoxyethylene ether ammonium sulfate salt, alkyl biphenyl ether disulfonic acid magnesium salts, sodium phosphate trimer+agriculture breast 400#, agriculture breast 500#+ agriculture breast 600#, agriculture breast 400#+NP series phosphate, NP series phosphate+alkyl biphenyl ether disulfonic acid magnesium salts, sodium phosphate trimer+agriculture breast 1600#+NP series phosphate, sodium phosphate trimer+agriculture breast 1600#+NP series phosphate+phenethyl, polyoxyethylene ether ammonium sulfate salt+alkyl biphenyl ether disulfonic acid magnesium salts is emulsifier, make cosolvent with propane diols, isopropyl alcohol is made antifreezing agent, phosphate buffer is cooked buffer solution and is prepared chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are respectively 2%, 4%, 3%, 0.5%, 0.5%, 45% preparation chitosan nano, and surplus is water.

1) take shitosan 10g, propane diols 40020g, isopropyl alcohol 15g, be dissolved in 225g phosphate buffer (pH4-5), stir 24h under the 1000r/min rotating speed, shitosan is fully dissolved;

2) with step 1) gained solution centrifugal 5min under 2500r/min, get supernatant, add the 2.5g polysorbate40, fully mix;

3) taking the 2.5g emulsifier and be dissolved in 225g water, fully stir, rear and step 2) gained solution mixes, and get final product.

15% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is added up lethality after 48h.Result is as shown in the table.

Table contains the biopesticide preparation of different emulsifiers composition to the control efficiency of prodenia litura

4, configure the double stranded RNA biopesticide preparation that contains different buffer solutions take spodoptera frugiperda cell cytochrome p 450 Si-CYP4M14v1_004 gene siRNA as active ingredient

(1) take the 500mg double stranded RNA, be dissolved in 199.5g water, 800r/min stirs it is fully dissolved;

(2) preparation of chitosan nano

Do respectively the buffer solution of chitosan nano preparation with acetate buffer, phosphate buffer, citrate buffer, acetate buffer+citrate buffer, phosphate buffer+citrate buffer, acetate buffer+phosphate buffer+citrate buffer, do with diethylene glycol (DEG) that antifreezing agent, polysorbate60 are made surfactant, agriculture breast 1600# does emulsifier, preparation chitosan nano.Wherein shitosan, cosolvent, antifreezing agent, surfactant, emulsifier, buffer solution percentage by weight are respectively 5%, 10%, 5%, 1%, 0.5%, 60% preparation chitosan nano, and surplus is water.

1) take shitosan 40g, Macrogol 200 80g, diethylene glycol (DEG) 40g, be dissolved in the 480g buffer solution, stir 12h under the 800r/min rotating speed, shitosan is fully dissolved;

2) with step 1) gained solution centrifugal 10min under 3000r/min, get supernatant, add the 8g polysorbate60, fully mix;

3) taking 4g agriculture breast 1600# and be dissolved in 156g water, fully stir, rear and step 2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm;

(3) preparation preparation: with active ingredient solution and step 2 in step 1)) gained chitosan nano solution evenly mixes, and namely gets 0.05% double stranded RNA biopesticide preparation.

The preventive effect of (4) 0.05% double stranded RNA biopesticide preparations to prodenia litura

0.05% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is added up lethality after 48h.Result is as shown in the table.

Table contains the biopesticide preparation of different buffer solution compositions to the control efficiency of prodenia litura

Buffer solution	Lethality (%)
		Acetate buffer	75.65
Phosphate buffer	78.31
		Citrate buffer	69.18
Acetate buffer+citrate buffer	72.64
		Phosphate buffer+citrate buffer	75.45
Acetate buffer+phosphate buffer+citrate buffer	68.74
		CK1	63.81
CK2	65.92
		CK3	21.35

Wherein CK1 is 0.05% active ingredient double stranded RNA+cationic polymer gelatin, and CK2 is 0.05% active ingredient double stranded RNA+cationic polymer shitosan, and CK3 is DEPC water.

5, preparation and the application of composite ribonucleic acid biopesticide preparation

This example provides complex biological preparation and the application of spodoptera frugiperda cell cytochrome p 450 gene Si-CYP4M14v1_005, Si-CYP4M14v1_006 and acetylcholinesterasegene gene Si-Ace1_001 double stranded RNA especially.

(1) active ingredient dissolving: take respectively Si-CYP4M14v1_005+Si-CYP4M14v1_006, Si-CYP4M14v1_005+Si-Ace1_001, the former medicinal powder of Si-CYP4M14v1_006+Si-Ace1_001 double stranded RNA end 500mg, be dissolved in 199.5g water, 800r/min stirs it is fully dissolved;

(2) chitosan nano preparation

1) take shitosan 40g, Macrogol 200 80g, glycerine 40g, be dissolved in 480g phosphate buffer (pH4-5), stir 12h under the 800r/min rotating speed, shitosan is fully dissolved;

2) with step 1) gained solution centrifugal 10min under 3000r/min, get supernatant, add the 8g polysorbas20, fully mix;

3) taking the 4g sodium phosphate trimer and be dissolved in 156g water, fully stir, then with step 2) gained solution mixes, and namely gets chitosan nano, particle diameter 100-600nm;

All more stable according to physical and chemical indexs such as the particle diameter distribution of the above-mentioned chitosan nano of stating method preparation and biopesticide preparation, drug loading rates, can carry out standardization production.

The control efficiency of (4) 0.05% double stranded RNA biopesticide preparations

0.05% biopesticide preparation is diluted to 100mg/L, evenly sprays on the wild cabbage that is connected to Spodoptera litura larvae is potted plant, every basin sprays 5ml, and every processing triplicate is added up lethality after 72h.Result is as shown in the table.

Showing composite double stranded RNA is that the biologic product of active ingredient is to the control efficiency of prodenia litura

Double stranded RNA	Lethality (%)
		Si-CYP4M14v1_005+Si-CYP4M14v1_006	70.00
Si-CYP4M14v1_005+Si-Ace1_001	78.60
		Si-CYP4M14v1_006+Si-Ace1_001	75.42
Si-CYP4M14v1_005	67.50
		Si-CYP4M14v1_006	67.67
Si-Ace1_001	68.72
		DEPC water	21.33

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims

1. a chitosan nano, is characterized in that, its raw material by following percentage by weight forms:

2. chitosan nano according to claim 1, is characterized in that, described cosolvent is one or several mixtures in Macrogol 200, PEG400, ethanol, propane diols.

3. chitosan nano according to claim 1, is characterized in that, described antifreezing agent is one or several mixtures in glycerine, ethylene glycol, isopropyl alcohol, diethylene glycol (DEG).

4. chitosan nano according to claim 1, is characterized in that, described surfactant is one or several mixtures in polysorbas20, polysorbate40, polysorbate60, Tween 80.

5. according to claim 1-4 described chitosan nanos of any one, it is characterized in that, described emulsifier is one or several mixtures in sodium phosphate trimer, benzyl dimethyl phenol polyethenoxy ether, alkyl benzene calcium sulfonate, phenethyl phenol polyethenoxy ether, alkylphenol polyoxyethylene formaldehyde condensation products, phenethyl phenol polyethenoxy polyethenoxy ether, polyoxyethylene nonylphenol ether phosphate, phenethyl polyoxyethylene ether ammonium sulfate salt, alkyl biphenyl ether disulfonic acid magnesium salts; Described buffer solution is one or several mixtures of acetate buffer, phosphate buffer or citrate buffer.

6. a biopesticide preparation of preventing and treating prodenia litura, is characterized in that, it mainly is grouped into by the one-tenth of following percentage by weight:

(A) active ingredient of 0.05-15%: for the synthetic siRNA of the efficient lethal target gene design of prodenia litura, described siRNA be 15-50nt double stranded RNA,

(B) the 19.95-55% solvent,

(C) the described chitosan nano of 30-80% claim 1-5 any one.

7. the biopesticide preparation of control according to claim 6 prodenia litura, it is characterized in that, the efficient lethal target gene of described prodenia litura is cytochrome P450 gene, acetylcholinesterasegene gene, gamma-aminobutyric acid receptor α subunit gene, moulting hormone gene.

8. the biopesticide preparation of control according to claim 7 prodenia litura, it is characterized in that, described siRNA is that the double stranded RNA that is selected from for acetylcholinesterasegene gene is SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; Double stranded RNA for the moulting hormone gene is 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; Double stranded RNA for gamma-aminobutyric acid receptor α subunit gene is SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18; With double stranded RNA for cytochrome P450 gene be one or more pairs of in SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30.

9. the biopesticide preparation of according to claim 6-8 described control of any one prodenia lituras, is characterized in that, described solvent is one or several mixtures in methyl alcohol, methyl-sulfoxide, water, Tris buffer solution.

10. the preparation method of the biopesticide preparation of the described control of a claim 6-9 any one prodenia litura, is characterized in that, comprises the following steps:

(A) dissolving: take described siRNA, be dissolved in solvent, stir, described siRNA is fully dissolved, get solution;

(B) chitosan nano preparation:

1) take described shitosan, cosolvent and antifreezing agent, join in buffer solution, stirring is fully dissolved it, gets solution 1;

2) solution 1 is centrifugal, get supernatant, add surfactant, fully stir, mixing gets solution 2;

3) will add in solution 2 after the dissolving of emulsifier water, mixing namely gets chitosan nano;

(C) preparation preparation: the chitosan nano of gained solution in step (A) and step (B) gained is mixed, namely get the biopesticide preparation of described control prodenia litura.