CN103109798A

CN103109798A - Chitosan nano particle, biological pesticide preparation for controlling red fire ants and preparation method thereof

Info

Publication number: CN103109798A
Application number: CN2013100607221A
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: CN103109798B

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 red fire ants 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 red fire ant 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 red fire ant control effect of the biological pesticide preparation are better.

Description

Biopesticide preparation of chitosan nano, control red fire ant 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 red fire ant of double stranded RNA.

Technical background

Red fire ant (Solenop sis invicta Buren), belong to Hymenoptera Hymenoptera, Formicidae Formicidae, Myrmicinae Mymicinae, Solenopsis Solenopsis, also claim external red fire ant, invasion red fire ant, belong to polyphagous pest-insect, because its feeding habits are complicated, habit is violent, breeding is rapid, competitiveness is strong, all have serious harmfulness to invading regional health, public safety, agriculture and forestry production and ecotope, therefore be listed in one of 100 kinds of the most dangerous in the world invasion pests.

Red fire ant is former is distributed in South America Parana basin, red fire ant invasion U.S. Alabama Mobile, after this annual VELOCITY DIFFUSION with 198km between 1918 ~ nineteen thirty.Due to its diffusion rapidly, spread to 13 states and Puerto Rico of the U.S. at present, the soil that surpasses 1.28 hundred million hm2 suffers the red fire ant invasion danger.Annual red fire ant is caused the approximately economic loss of 5,000,000,000 dollars.Calendar year 2001 is invaded New Zealand and Australia.China found red fire ant first in Taiwan in 2003, in September, 2004 in Guangdong, the province such as Guangxi, Hunan, Fujian and Hong Kong and Macao find in succession, cause that the Ministry of Agriculture pays much attention to, formally red fire ant was listed in " plant quarantine pest register enters the territory " and " national plant quarantine pest register " on January 17th, 2005, taked quarantine to block control measure to red fire ant in accordance with the law.

China just began in recent years to the related work of Solenopsis invicta.By means such as field investigation, CLIMEX and the predictions of GARP Niche Model method, point out that red fire ant is one of the strongest insect assembly of invasion danger, the pest that belongs to moderate risk in China, red fire ant invasion can be caused agricultural economy loss, ecotope heavy damage, be threatened public safety etc.

At present, chemical control remains the Main Means that puts out red fire ant, and as beta-cypermethrin, Avermectin, phoxim, Le Siben etc., the medicine liquid irrigation nest is processed, bait formulation is processed, pulvis is processed is the main method of red fire ant chemical control.But the chemical control meeting of red fire ant produces a series of negative effect to ecotope and this geobiocenosis, and the impact that native ant is subject to is particularly serious, and the existence of native ant is played certain inhibitory action to the invasion of red fire ant.Under this prevention and control situation, research is efficient, green, environmental protection, sustainable development pesticidal preparations have been very important research topic.

RNA disturbs (RNA interference, RNAi) refer to that external source or endogenous double-stranded RNA cause the phenomenon of gene silencing specifically, be widely used at present the insect genes functional study, also be considered to new method and the New Policy of agriculture and forestry injurious insect biological control simultaneously.The breakthrough that the RNA perturbation technique is obtained aspect control of insect is that theoretical foundation has been established in the control of red fire ant, and a kind of new Solenopsis invicta strategy is provided, and has opened up biological control and the environmental protection new situation.

Although gene silent technology has been opened the new page of harmful insect biological control, at present a large amount of trial 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 biopesticide preparation of the first control red fire ant based on 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 red fire ant, 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.

Preferably, the cosolvent in chitosan nano 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 particle diameter 100600nm.

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

Concrete scheme is as follows:

A kind of biopesticide preparation of preventing and treating red fire ant, 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 red fire ant, described siRNA is more preferably 17-25nt of 15-50nt() double-stranded RNA,

(B) the 19.95-55% solvent,

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

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

Preferably, the efficient lethal target gene of described red fire ant is ATP synzyme e chain gene, glutathione transferase gene, arginine kinase gene, chondriogen.More preferably, be one or more pairs of in 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 for the double stranded RNA of ATP synzyme e chain gene; Be a pair of or how right in 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 for the double stranded RNA of glutathione transferase gene; Be one or more pairs of in 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 for the double stranded RNA of arginine kinase 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 for the double stranded RNA of chondriogen.

Described active ingredient double stranded RNA few nucleotide is that 15-50nt, molecular weight are 8000-40000.3 ' the end of described siRNA is provided with the suspension base, and more preferably, described suspension base is dTdT.

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 red fire ant.

Concrete technical scheme is as follows:

The preparation method of the biopesticide preparation of a kind of above-mentioned control red fire ant comprises the following steps:

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

2) with step 1) gained solution (preferably with 3000-10000rpm) centrifugal (preferred 10-60min), get supernatant, add surfactant, fully stir, mixing gets solution;

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

(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 the described active ingredient in red fire ant biological control pesticidal preparations 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 the nanoparticle of chitosan nano of the present invention and the little molecular chemistry agricultural chemicals of existing embedding exists own job to distinguish.

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 double stranded RNA and cationic compound, keeps higher pest controling effect.

The 3rd, the present invention is the first red fire ant biological control pesticidal preparations 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.

The 4th, 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 red fire ant ATP synzyme e chain gene double stranded RNA sequence as the chitosan nano biologic product of active ingredient especially.

(1) for design and the synthetic GenBank:GU471223.1 of the former medicine composition of red fire ant ATP synzyme e chain gene double stranded RNA

Si-ATP_001：TCATC?CGCGA?GCAAG?AT

Positive-sense strand (5'-3'): 5'UCAUC CGCGA GCAAG AU dTdT 3'SEQ ID NO.1

Antisense strand (3'-5'): 3'dTdT AGUAG GCGCU CGUUC UA 5'SEQ ID NO.2

Si-ATP_002：GTACGTGACG?CCAAG?CT

Positive-sense strand (5'-3'): 5'GUACG UGACG CCAAG CU dTdT 3'SEQ ID NO.3

Antisense strand (3'-5'): 3'dTdT CAUGC ACUGC GGUUC GA 5'SEQ ID NO.4

Si-ATP_003：GTGCT?GTTTC?AACAT?CA

Positive-sense strand (5'-3'): 5'GUGCU GUUUC AACAU CA dTdT 3'SEQ ID NO.5

Antisense strand (3'-5'): 3'dTdT CACGA CAAAG UUGUA GU 5'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 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, rear and step (2) gained solution mixes, and namely gets chitosan nano, and particle diameter is 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) 0.05% double stranded RNA biopesticide preparations

0.05% double stranded RNA biopesticide preparation is diluted to 100mg/L, is immersed in foodstuff surface (it also is unique water source of red fire ant), corresponding active ingredient concentration at foodstuff surface is 6.7 μ g/cm ²Simultaneously do contrast with clear water, processed once every 2 days, add up the red fire ant death toll after 7d and calculate lethality, result is as shown in the table.

Indicator is that the biologic product of active ingredient is to the control efficiency of red fire ant to the synthetic double stranded RNA of red fire ant ATP synzyme e chain gene design

Double stranded RNA	Lethality (%)
		Si-ATP_001	67.62
Si-ATP_002	71.16
		Si-ATP_003	84.17
CK1	70.69
		CK2	68.25
CK3	20.71

Wherein CK1 is 0.05wt%Si-ATP_003 double stranded RNA+cationic polymer shitosan; CK2 is 0.05wt%Si-ATP_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%), be diluted to 100mg/L, process red fire ant by above-mentioned processing mode, observe its control efficiency in 7d afterwards, result is as follows:

Double stranded RNA	Lethality (%)
		Si-ATP_001	66.95
Si-ATP_002	72.38
		Si-ATP_003	83.50
CK1	71.66
		CK2	68.25
CK3	22.00

Wherein CK1 is 15wt%SiATP_003 double stranded RNA+cationic polymer shitosan; CK2 is 15wt%Si-ATP_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 red fire ant glutathione transferase gene especially.

(1) for design and the synthetic GenBank:AY255670.1 of the former medicine composition of red fire ant glutathione transferase gene double stranded RNA

Si-GSTS1_001：TAGAC?TGCAA?GAACA?TG

Positive-sense strand (5'-3'): 5'UAGAC UGCAA GAACA UG dTdT 3'SEQ ID NO.7

Antisense strand (3'-5'): 3'dTdT AUCUG ACGUU CUUGU AC 5'SEQ ID NO.8

Si-GSTS1_002：CGGTG?GTTAC?TTTGT?CG

Positive-sense strand (5'-3'): 5'CGGUG GUUAC UUUGU CG dTdT 3'SEQ ID NO.9

Antisense strand (3'-5'): 3'dTdT GCCAC CAAUG AAACA GC 5'SEQ ID NO.10

Si-GSTS1_003：ATATT?ATTAT?AAGCG?AT

Positive-sense strand (5'-3'): 5'AUAUU AUUAU AAGCG AU dTdT 3'SEQ ID NO.11

Antisense strand (3'-5'): 3'dTdT UAUAA UAAUA UUCGC UA 5'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 (pH=4-5), 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.

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

15% double stranded RNA biopesticide preparation is diluted to 100mg/L, is immersed in foodstuff surface (it also is unique water source of red fire ant), corresponding active ingredient concentration at foodstuff surface is 6.7 μ g/cm ²Simultaneously do contrast with clear water, processed once every 2 days, add up the red fire ant death toll after 7d and calculate lethality, result is as shown in the table.

The control efficiency of the biologic product of table take the synthetic double stranded RNA of red fire ant glutathione transferase gene design as active ingredient to red fire ant

Double stranded RNA	Lethality (%)
		Si-GSTS1_001	69.71
Si-GSTS1_002	70.46
		Si-GSTS1_003	65.80
CK1	64.25
		CK2	62.03
CK3	10.50

Wherein CK1 is 15wt%Si-GSTS1_001 double stranded RNA+cationic polymer shitosan; CK2 is 15wt%Si-GSTS1_002 double stranded RNA+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%), be diluted to 100mg/L, process as stated above red fire ant, rear 7d observes its control efficiency, and result is as follows:

Double stranded RNA	Lethality (%)
		Si-GSTS1_001	67.85
Si-GSTS1_002	70.50
		Si-GSTS1_003	64.52
CK1	63.84
		CK2	62.53
CK3	11.87

Wherein CK1 is 1wt%Si-GSTS1_001 double stranded RNA+cationic polymer shitosan; CK2 is 1wt%Si-GSTS1_002 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 the double-stranded RNA sequence of red fire ant arginine kinase gene especially.

(1) for the design of the former medicine of red fire ant arginine kinase gene double stranded RNA and synthetic

GenBank：EU817514.1

Si-AK_001：TGGTA?TCTAC?GCGCC?CGAC

Positive-sense strand (5'-3'): 5'UGGUA UCUAC GCGCC CGAC dTdT 3'SEQ ID NO.13

Antisense strand (3'-5'): 3'dTdT ACCAU AGAUG CGCGG GCUG 5'SEQ ID NO.14

Si-AK_002：CCGCT?CACTG?GCATG?AGCA

Positive-sense strand (5'-3'): 5'CCGCU CACUG GCAUG AGCA dTdT 3'SEQ ID NO.15

Antisense strand (3'-5'): 3'dTdT GGCGA GUGAC CGUAC UCGU 5'SEQ ID NO.16

Si-AK_003：GCATC?GCTGA?GCTCA?TCAA

Positive-sense strand (5'-3'): 5'GCAUC GCUGA GCUCA UCAA dTdT 3'SEQ ID NO.17

Antisense strand (3'-5'): 3'dTdT CGUAG CGACU CGAGU AGUU 5'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, isopropyl alcohol 4g, Macrogol 200 4g, be dissolved in 160g citrate buffer (pH=4-5), 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.

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

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

5% double stranded RNA biopesticide preparation is diluted to 100mg/L, is immersed in foodstuff surface (it also is unique water source of red fire ant), corresponding active ingredient concentration at foodstuff surface is 6.7 μ g/cm ²Simultaneously do contrast with clear water, processed once every 2 days, add up the red fire ant death toll after 7d and calculate lethality, result is as shown in the table.

The control efficiency of the biologic product of table take the synthetic double stranded RNA of red fire ant arginine kinase gene design as active ingredient to red fire ant

Double stranded RNA	Lethality (%)
		Si-AK_001	68.65
Si-AK_002	63.20
		Si-AK_003	62.40
CK1	59.72
		CK2	57.56
CK3	22.79

Wherein CK1 is 5WT%Si-AK_003 double stranded RNA+cationic polymer shitosan; CK is 5WT%Si-AK_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%), be diluted to 100mg/L, process as stated above red fire ant, rear 7d observes its control efficiency, and result is as follows:

Double stranded RNA	Lethality (%)
		SiAK_001	67.21
SiAK_002	63.28
		SiAK_003	61.50

CK1	57.95
		CK2	55.74
CK3	20.25

Wherein CK1 is 10WT%Si-AK_001 double stranded RNA+cationic polymer shitosan; CK is 10WT%Si-AK_002 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 especially for red fire ant chondriogen double stranded RNA sequence.

(1) for the design of the former medicine of red fire ant chondriogen ribonucleic acid and synthetic:

Si-M_001：CTCTA?ATTAA?TAATG?ATC

Positive-sense strand (5'-3'): 5'CUCUA AUUAA UAAUG AUC dTdT 3'SEQ ID NO.19

Antisense strand (3'-5'): 3'dTdT GAGAU UAAUU AUUAC UAG 5'SEQ ID NO.20

Si-M_002：TACAT?ATCGC?CGGAA?TAT

Positive-sense strand (5'-3'): 5'UACAU AUCGC CGGAA UAU dTdT 3'SEQ ID NO.21

Antisense strand (3'-5'): 3'dTdT AUGUA UAGCG GCCUU AUA 5'SEQ ID NO.22

Si-M_003：TTATT?CACTA?TAGGA?GGA

Positive-sense strand (5'-3'): 5'UUAUU CACUA UAGGA GGA dTdT 3'SEQ ID NO.23

Antisense strand (3'-5'): 3'dTdT AAUAA GUFAU AUCCU CCU 5'SEQ ID NO.24

Si-M_004：TATGG?CAGAT?TAGTG?CAA

Positive-sense strand (5'-3'): 5'UAUGG CAGAU UAGUG CAA dTdT 3'SEQ ID NO.25

Antisense strand (3'-5'): 3'dTdT AUACC GUCUA AUCAC GAA 5'SEQ ID NO.26

Si-M_005：ATCGC?CCAGA?CCTTG?ACC

Positive-sense strand (5'-3'): 5'AUCGC CCAGA CCUUG ACC dTdT 3'SEQ ID NO.27

Antisense strand (3'-5'): 3'dTdT UAGCG GGUCU GGAAC UGG 5'SEQ ID NO.28

Si-M_006：TAGTC?CCAAC?TGACT?TAG

Positive-sense strand (5'-3'): 5'UAGUC CCAAC UGACU UAG dTdT 3'SEQ ID NO.29

Antisense strand (3'-5'): 3'dTdT AUCAG GGUUG ACUGA AUC 5'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 mitochondria Si-M_001 gene siRNA as active ingredient

(1) active ingredient dissolving: take the 50gSi-M_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 difference 1%, 1%, 1%, 0.1%, 0.1%, 40% preparation chitosan nano, and surplus is water.

1) take shitosan 4g, antifreezing agent 4g, Macrogol 200 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 red fire ant

Table contains the biopesticide preparation of different antifreezing agent agent compositions to the control efficiency of red fire ant

Antifreezing agent	Lethality (%)
		Glycerine	46.83

Ethylene glycol	58.14
		Isopropyl alcohol	49.10
Diethylene glycol (DEG)	50.26
		Glycerine+ethylene glycol	56.37
Glycerine+isopropyl alcohol+diethylene glycol (DEG)	48.95
		CK1	41.25
CK2	45.12
		CK3	19.43

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 mitochondria Si-M_002 gene siRNA 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 red fire ant

Table contains the biopesticide preparation of different surfaces bioactive agent composition to the control efficiency of red fire ant

Surfactant	Lethality (%)
		Polysorbas20	78.21
Polysorbate40	77.46
		Polysorbate60	75.13
Tween 80	70.45
		Polysorbas20+polysorbate60	73.76
Polysorbate40+polysorbate60+Tween 80	74.38
		Polysorbas20+polysorbate40+polysorbate60+Tween 80	77.54
CK1	68.22
		CK2	70.33
CK3	26.31

Wherein CK1 is active ingredient double stranded RNA+cationic polymer gelatin, and CK2 is 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 red fire ant mitochondria Si-M_003 gene siRNA 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 difference 2%, 4%, 3%, 0.5%, 0.5%, 45% preparation chitosan nano, and surplus is water.

1) take shitosan 10g, propane diols 20g, 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 namely gets chitosan nano, particle diameter 100-600nm.

Table contains the biopesticide preparation of different emulsifiers composition to the control efficiency of red fire ant

4, configure the double stranded RNA biopesticide preparation that contains different buffer solutions take red fire ant mitochondria Si-M_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 difference 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 480g buffer solution (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 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: active ingredient solution in step (1) is evenly mixed with step (2) gained chitosan nano solution, namely get 0.05% double stranded RNA biopesticide preparation.

The preventive effect of (4) 0.05% double stranded RNA biopesticide preparations to red fire ant

Table contains the biopesticide preparation of different buffer solution compositions to the control efficiency of red fire ant

Buffer solution

Lethality (%)

Acetate buffer	75.06
		Phosphate buffer	73.18
Citrate buffer	74.67
		Acetate buffer+citrate buffer	69.98
Phosphate buffer+citrate buffer	76.01
		Acetate buffer+phosphate buffer+citrate buffer	73.33
DEPC water	28.15

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

This example provides complex biological preparation and the application of red fire ant chondriogen Si-M_005, Si-M_006 and arginine kinase gene Si-AK_001 double stranded RNA especially.

The preparation of (one) 0.05% double stranded RNA biopesticide preparation

(1) active ingredient dissolving: take Si-M_005+Si-M_006, Si-M_005+Si-AK_001, the former medicinal powder of Si-M_006+Si-AK_001 double stranded RNA end 500mg, be dissolved in 199.5g water, 800r/min stirs it is fully dissolved;

(2) chitosan nano preparation

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) 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.

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

Showing composite double stranded RNA is that the biologic product of active ingredient is to the control efficiency of red fire ant

Double stranded RNA	Lethality (%)
		Si-M_005+Si-M_006	71.05
Si-M_005+Si-AK_001	77.61
		Si-M_006+Si-AK_001	74.31
Si-M_005	65.44
		Si-M_006	63.87
Si-AK_001	67.5
		DEPC water	21.47

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 red fire ant, 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 red fire ant, 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 red fire ant according to claim 6, is characterized in that, the efficient lethal target gene of described red fire ant is ATP synzyme e chain gene, glutathione transferase gene, arginine kinase gene, chondriogen.

8. the biopesticide preparation of control according to claim 7 red fire ant, it is characterized in that, be one or more pairs of in 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 for the double stranded RNA of ATP synzyme e chain gene; Be a pair of or how right in 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 for the double stranded RNA of glutathione transferase gene; Be one or more pairs of in 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 for the double stranded RNA of arginine kinase 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 for the double stranded RNA of chondriogen.

9. the biopesticide preparation of according to claim 6-8 described control of any one red fire ants, 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 red fire ant, 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 described double stranded RNA biopesticide preparation.