CN108165582A - A kind of method that dsRNA interference insect gene expressions are permeated by insect body wall - Google Patents
A kind of method that dsRNA interference insect gene expressions are permeated by insect body wall Download PDFInfo
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Abstract
The present invention relates to nano materials and gene jamming field, specifically disclose a kind of method that dsRNA interference insect gene expressions are permeated by insect body wall, by nano-carrier and dsRNA function macromolecular complexes, by the effect of surfactant, external source dsRNA molecules are realized in the attachment of insect body surface, spread, infiltration body wall and enter internal various organ-tissues, efficiently interfere gene expression.The present invention realizes that external source dsRNA molecules are efficient, noninvasive importing polypide is intracellular, final that dsRNA molecules is promoted to be correctly targeted, promotion RNA jamming effectiveness by the dual synergistic effect of nano-carrier and surfactant.The method of the invention is simple, efficient, error is small, and scientific research and highly effective and safe, the application of targeting type RNA pesticidal preparations is interfered insect genes to have important practical significance.
Description
Technical field
The present invention relates to nano material and gene jamming field, permeated specifically, being related to one kind by insect body wall
The method of dsRNA interference insect gene expressions.
Background technology
RNA interference (RNA interference, RNA interference) refer to it is being highly conserved during evolution, by double-stranded RNA
(double-stranded RNA, dsRNA molecule) induce, homologous mRNA efficient selective degradation the phenomenon that.These are special to set
The external source dsRNA molecules of meter can specifically, efficiently, enduringly reduce the expression of specific gene, for studying unknown gene work(
Can, the development of regulation and control insect and behavior, it protect the plants from virus infraction and be of great significance.RNA interference has been used as a kind of heavy
Means are wanted, are widely used in all kinds of Functional identification of genes, functional gene expression regulation field.Simultaneously as dsRNA molecules are high
The characteristics such as degradable after the specificity of degree, release become a kind of special targeting pest, something lost to non-target organism safety
It passes and learns control strategy.
Due to the defence of Insect immunity system and the barrier of organ basilar memebrane and cell membrane, external source dsRNA molecules are difficult to height
Effect enters insect bodies inner cell；Along with caste is more, gene groups are more, injection and feeding method application dsRNA are usually imitated
Fruit is bad.Wherein, it is one of an important factor for insect genes RNA interference effects are bad that the cell absorptivity of dsRNA molecules is low.Note
It is the method that dsRNA molecules are imported to cell most common at present to penetrate method and feeding method, by microinjector directly by dsRNA
Molecule is injected into appointed part in insect bodies, but for the insect that volume is small, body fluid is more, the mechanical trauma of polypide and postoperative
Infection is inevitable, and more demanding to injection device and operating technology, and in being only limitted to, the laboratory science of large-scale insect grinds
Study carefully, operation difficulty is high, is unfavorable for the experimental study work of a large amount of insects, and it is even more impossible to be promoted the use of in farmland.Feeding method is opposite
Have many advantages, such as in injection easy to operate, wound is small and time-consuming few, but the dsRNA molecules in feed penetrate insect gut and enclose food
Film and gut cell membrane barrier efficiency are low, and a large amount of external source dsRNA molecules are discharged with excrement, cause the jamming effectiveness of target gene it is low,
The shortcomings of dsRNA dosages are big, of high cost；And some insects lack effective man-made feeds, feeding method be easy to cause insect nutrition
It is bad and dead, increase error, influence the accuracy of experimental result.
In recent years, nanosecond science and technology are grown rapidly, and are rapidly developed in agriculture field application, one of them is exactly to be carried using nanometer
The targeting delivery and control-release function of body deliver in nucleic acid drug (DNA or RNA) to insect bodies, break the organ base in insect bodies
Counterdie and cell membrane barrier and enteron aisle funnel barrier promote cell to absorb exogenous nucleic acid molecule, improve exogenous nucleic acid molecule
Functioning efficiency.The current successful application in injection and feeding method, nano-carrier delivering dsRNA molecules can be substantially
Degree promotes the jamming effectiveness to insect genes.
But nonetheless, however it remains it is even dead that microinjection mode easily causes insect interior tissue injury, body fluid to be lost in
It dies, and feed mode is difficult to exclude insect because of the error caused by malnutrition the problem of.
Therefore, develop it is a kind of without injection and feeding can efficient interference insect gene expression method, it is unknown to research
Gene function, the development for regulating and controlling insect and behavior protect the plants from virus infraction and are of great significance.
Invention content
In order to solve the problems in the existing technology, the defects of injection and feeding method are made up, the present invention seeks to
A kind of method that dsRNA interference insect gene expressions are permeated by insect body wall is provided.
In order to realize the object of the invention, technical scheme is as follows：
In a first aspect, the present invention provides a kind of method that dsRNA interference insect gene expressions are permeated by insect body wall, it will
Nano-carrier and dsRNA function macromolecular complexes by the effect of surfactant, realize external source dsRNA molecules in insect bodies
Table attachment, spread, infiltration body wall simultaneously enter internal various organ-tissues, efficiently interfere gene expression.
The method specifically comprises the following steps：
1) nano-carrier solution, dsRNA solution with surfactant are mixed, obtains complex liquid；
2) the complex liquid is placed in insect body surface.
Further, in the step 1), in the nano-carrier and the dsRNA solution in the nano-carrier solution
DsRNA according to charge ratio be 1:8~8:1 ratio is mixed to get mixed liquor, and the surfactant is mixed with described
Liquid is mixed according to 0.01%~10.0% volume ratio.
In this mixed process, the nano-carrier and the dsRNA by electrostatic interaction, and/or hydrogen bond, and/or
The effect of Van der Waals force is combined into stable carrier/dsRNA molecular complexes.By the infiltration of surfactant performance, remove
Fat acts on, and dissolves insect body surface wax coat, reduces resisting property of epidermis, and the complex liquid drop of gained is made to be easy to be adsorbed to insect
Body surface simultaneously penetrates insect body wall and enters body cavity, tissue and intracellular, so as to the expression of efficient disturb target gene.
More preferably, in order to improve mixing efficiency, preferably described nano-carrier is with the dsRNA according to charge than 1:1 ratio
Example is mixed to get mixed liquor, and the surfactant is mixed with the mixed liquor according to 1% volume ratio.
Further, dsRNA of the present invention is the double-stranded RNA for being capable of selective degradation homologous mRNA (mRNA)
Molecule is not limited to its clip size and secondary structure type.
Nano-carrier of the present invention has cavity structure, and surface carries the nano-carrier of positive charge to be internal, excellent
Being selected as periphery has the nano-carrier of high density hydrophilic functional group (such as amino, carboxyl).
The internal cavity structures and high density structure of functional groups of the nano-carrier are conducive to the loading shape of dsRNA molecules
Into stable complex, while protect dsRNA molecular biosciences macromolecular from the drop of acid, the damage of alkali environment and enzyme in organism
Solution.
The nano-carrier delivering effect, promote the uniform spread of insect body surface dsRNA molecules penetrate insect body wall into
Enter body cavity, tissue and cell.
The nano-carrier pass through hydrophilic functional group (including amino, carboxyl etc.) functionalization, easily with insect cuticle, cell
The hydrophilic protein matter component " similar to mix " of film improves contained substance and carrier and insect cuticle and the affinity of cell membrane,
DsRNA molecules is promoted to penetrate insect cuticle barrier, into body cavity and intracellular, the target base in interference cell of each organ-tissue
The expression of cause.
Surfactant of the present invention may be selected from all kinds of natural surfactants, organic synthesis surfactant and its
Derivative such as detergent, liquid detergent etc..
On the one hand the surfactant can destroy insect defence wax coat, increase medicinal liquid osmosis power, on the other hand may be used
To reduce water phase medical liquid surface tension, absorption, the spread of insect body wall are easier in sprinkling, dropwise addition, expands and is accumulated by powder.
In the specific embodiment of the present invention, using commercially available goldfish detergent as surfactant, but this
The invention surfactant is not limited to this, as long as with wetting, emulsification and the active any property of grease removal, can drop
The surface tension of low carrier/dsRNA molecular complex liquids promotes infiltration of the drop in insect body wall oily interface (shield wax layer)
With sprawl, dissolve the lipid composition in epidermis, destroy wax coat, that destroys insect body surface resists function, promotes compound suitable
Profit penetrates body wall and enters body cavity.
Further, the step 2) described in the method for the present invention can specifically pass through the dispensers sides such as dripping method, spray-on process, infusion method
The complex liquid is added to insect body surface by method, including with appropriately sized utensil (such as liquid-transfering gun, injection needle, small-sized
Sprayer etc.) it is added drop-wise to insect body surface or directly impregnates insect.
The method of the invention is directly permeated from insect body surface imports dsRNA molecules, easy to operate, quick, avoids biography
The shortcomings of gene jamming effectiveness caused by the injection of system, feeding method import dsRNA molecules is low, unstable, the death rate is high；Together
When, the carrier of special designing can load dsRNA molecules and penetrate insect body wall barrier, into the various organ-tissues of insect bodies intracavitary
Into the cell, the target of dsRNA molecules and gene interference effect are efficiently played, regulates and controls the life such as the growth, development and reproduction of insect
Activity.This method can greatly improve gene jamming effectiveness, significantly reduce the loss of dsRNA molecules, without operation wound, operation letter
Single, safety, the insect genes function to be interfered based on RNA parse experimental study and field application dsRNA preparations spraying prevention evil
Worm provides good technical support.
Second aspect, the present invention provide a kind of complex liquid prepared according to preceding method and the complex medicine
Application of the liquid in gene expression is interfered.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party
Formula.
The beneficial effects of the present invention are：
The present invention by nano-carrier and surfactant dual synergistic effect, realize external source dsRNA molecules efficiently,
Noninvasive importing polypide is intracellular.The internal cavity structures and exterior positive electrode lotus group of nano-carrier can pass through electrostatic with nucleic acid molecules
Effect, and/or hydrogen bond, and/or Van der Waals force and attract each other, the compound of formation stabilization freely be complexed, carrier periphery is highly dense
Degree amido functional group can enhance compound to insect body wall epidermis, the isostructural affinity of internal cell membrane, in addition surface work
Property agent infiltration and peptizaiton, be conducive to complex solution and form liquid drop stable, uniform, that surface tension is small, promote
Drop penetrates insect body wall and is permeated to body cavity and enter polypide in the attachment of insect body surface, spread and delay, promotion dsRNA molecules
With into the cell, final promotion dsRNA molecules are correctly targeted interior various tissues, promote RNA jamming effectiveness.The method of the invention letter
List, efficient, error is small, and scientific research and highly effective and safe, the application of targeting type RNA pesticidal preparations is interfered insect genes to have
There is important practical significance.
The advantage of the method for the invention includes at least：
It is 1. applied widely.Compared to traditional feeding method and injection, insect body wall osmosis of the invention, not by elder brother
The how many limitation of insect population device type, food source, body size, body fluid, can obtain good application.
It is 2. easy to operate, quick.Compared to traditional injection, the present invention omits the arts such as anesthesia, fixed point, injection, disinfection
Preceding preparation and postoperative aftermath only relate to prepare mixed liquor and insect body surface and are added dropwise, impregnate or spraying simple operations, 1 hour
Inside it can be completed, it is high to laboratory apparatus, operating technology tolerance, it is easy to spread and universal.
3. zero death, error are small.Compared to traditional injection, we's noninvasive operation owned by France is evaded because of injection or feeding
The adverse reactions such as malnutrition, mechanical damage, postoperative infection caused by method imports dsRNA molecules, it is ensured that control group 100% is deposited
Motility rate is greatly decreased the influence of systematic error and personal operating error to experiment result accuracy, ensures behaviour every time as much as possible
The validity of work improves working efficiency.
4. jamming effectiveness is high.The dual synergistic effect of nano-carrier and detergent, it is ensured that dsRNA molecules can in liquid
Quick penetration insect body wall, which enters in target cell, to be correctly targeted, and high efficiency regulatory gene expression makes insect growth, developmental deformity,
Oviposition declines even death, either the insect functional gene parsing based on RNA interference or the Genetic control for pest
All there is good application value.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It is it will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention in the case of without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present embodiment is used for the preparation method for illustrating complex liquid of the present invention, specific as follows：
1st, raw material：
(1) nano-carrier：Fluorescence nano carrier molecule disclosed in the present embodiment using Patent No. 201310119837.3 as
Example, the fluorescent core of carrier inside have high-fluorescence quantum yield, and good light, thermally and chemically stability are conducive in organism
Interior tracer forms good cavity structure between each side chain and is conducive to dsRNA molecules with a large amount of specific functional groups
Combination and assembling and form stabilizing dsrna molecule/carrier complex, entire complex has good insect cell compatibility,
Be conducive to dsRNA molecules and rapidly enter cell-targeting target gene.
(2)dsRNA：The dsRNA interfered for the gene synthesized in fat-body.
The external synthesis dsRNA standard methods provided according to Promega, the T7RiboMAX provided using the said firm
Express RNAi System kits synthesize dsRNA, and the nucleotide sequence of dsRNA is as shown in SEQ ID NO.1.
(3) sterile water, general commercial product.
(4) surfactant：Using commercially available goldfish board detergent.
2nd, method：
(1) nano-carrier is configured to the nano-carrier solution of a concentration of 1.0mg/mL using sterile water.
(2) dsRNA is configured to the dsRNA solution of a concentration of 600.0ng/ μ L.
(3) the nano-carrier solution of 1.5 μ g is taken to be mixed in centrifuge tube with the dsRNA solution of 1.0 μ g, passes through vortex oscillation
Come into full contact with two kinds of substances, two kinds of molecules under electrostatic interaction, hydrogen bond and van der Waals interaction by being combined into stable load
Body/dsRNA molecular complex liquids.
(4) surfactant of 1% volume is added in, is mixed with pipette tips piping and druming to uniform, to reduce follow-up liquid drop
Surface tension, the liquid drop for forming small size are added dropwise to insect body surface.
Embodiment 2
The present embodiment illustrates how to enter small-sized piercing and sucking using complex fluid delivery dsRNA described in embodiment 1
In formula mouthpart insect soybean aphid, interference key gene expresses and soybean aphid is controlled to lay eggs.
The present embodiment uses soybean aphid to be tested insect for representativeness.Its abdomen is full of body fluid, and microinjection dsRNA molecules draw
Play that injured, the most of body fluid of soybean aphid interior tissue is lost in and dead；Phytophagous soybean aphid lacks the comprehensive man-made feeds of nutrition,
Fed soy aphid dsRNA molecules are difficult to exclude examination aphid error caused by malnutrition；Its volume is small, and body is about 2 μm,
During dispenser, easily it is atomized insufficient large volume liquid and is drowned.The present embodiment is with RNA interference operation more difficulty
Soybean aphid is representative subject insect, it is intended to illustrate the key technology of the present invention.
Concrete operations are as follows：
1st, it blows and beats the complex liquid that embodiment 1 is prepared repeatedly using liquid-transfering gun, promotes emulsification, with appropriate range
Microinjector forms the droplet of 10nL~100nL and sticks, is added drop-wise to soybean aphid body surface, the surfactant in detergent
Molecule is adsorbed in droplet surface, replaces the hydrone of drop-Air Interface, and the phenomenon that making drop molecule unbalance stress weakens, and is dropped
Low Surface Tension of Liquid Drops, drop are distributed in the surfactant of insect body surface in the rapid spread in grease interface of soybean aphid body surface
Hydrophobic part is inwardly inserted into body wall lipid composition, and hydrophilic segment outwardly, changes the hydrophobicity of insect body surface, further promotes it
Absorption and absorption to water-solubility carrier drop.In order to more preferably embody the effect of detergent in the present invention, removal detergent is introduced
Liquid control treatment 1：Carrier/sterile water, processing 2：Carrier/sterile water/detergent carries out at surface penetration soybean aphid
Reason, 1 Chinese medicine liquid of control treatment are difficult to adsorb at oily epidermis interface due to surface tension effects, it is difficult to absorb；Processing 2 in due to
The immersional wetting liquid of detergent is quickly absorbed in the uniform spread of aphid, liquid by soybean aphid.
In order to show that carrier infiltration soybean aphid body wall enters internal osmotic effect in this method, after 1 hour, shows in light
Micro mirror detects aphid body surface and the positioning of internal carrier, before this first with sterile water washing entire soybean aphid polypide 3-4 times, with removing
The residuals on polypide surface.
The fluorescence nano carrier in processing 2 (carrier/sterile water/detergent) liquid is clearly observable under microscope whole
A aphid polypide is distributed, and carrier fluorescence is not detected in entire polypide body surface in control treatment 1 (carrier/sterile water).
Aphid dissection carrier in fluorescence microscopy Microscopic observation in-vivo tissue in two kinds of processing is positioned：In control treatment 1
Simultaneously carrier fluorescence signal is not detected in (carrier/sterile water) tissue, handles and detects that load is fixed in 2 (carriers/sterile water/detergent)
Each vital tissue of soybean aphid is arrived in position：To the related ooecium of reproduction oviposition, embryo, relevant fat is transported with energetic supersession, storage
Fat body and with digesting and assimilating relevant enteron aisle etc..Illustrate that the surfactant in the method for the invention can promote carrier
Aqueous solution is in the absorption at insect body wall grease interface, spread and infiltration.
2nd, the expression of the delivering of insect body wall osmosis dsRNA molecule, efficient interference insect gene：In order to preferably embody this
Detergent and the dual synergy of carrier promote that external source dsRNA molecules are efficient, noninvasive importing polypide interferes gene table into the cell in invention
The effect reached.It introduces dsRNA molecules and carries out gene interference experiment, control treatment 1：Carrier/sterile water/dsRNA, control treatment 2：
Detergent/sterile water/dsRNA, processing 3：Carrier/detergent/sterile water/dsRNA.Using the gene synthesized in fat-body as target
The dsRNA molecules of gene design, will be in the fluid delivery to soybean aphid body of 3 kinds of processing as described above by body wall osmosis
Fat-body detects gene jamming effectiveness and observes soybean aphid phenotype after 48 hours.Complex is added dropwise using the method for the invention
The target gene of the soybean aphid of liquid processing is suppressed in transcriptional level, and target gene is suppressed in transcriptional level in processing 3, phase
95% is decrease beyond to expression quantity, and the target gene expression quantity being disturbed in control treatment 1, control treatment 2 does not occur significantly
Property variation.
Meanwhile compared to control treatment 1, control treatment 2, soybean aphid normal growth and development is obstructed in processing 3, and production aphid amount declines
Or stopping production aphid, soybean aphid insect density is remarkably decreased in big bean seedlings.This shows the dual synergy in detergent and nano-carrier
Under effect, external source dsRNA molecules can penetrate the body wall barrier of insect, organ substrate envelope barrier, cell membrane barrier, rapidly enter
It is intracellular to play gene interference effect, regulate and control the vital movement of insect, make insect growth impaired development, fecundity declines.
It should be understood that after the dosage of above-described embodiment agents useful for same or raw material is carried out equal proportion expansion or is reduced
Technical solution, it is substantially identical with above-described embodiment.
Nano material described in the embodiment of the present invention 1 is only for example, and can be replaced Patent No. 201310095194.3
Disclosed in water soluble fluorescence dendritic macromole disclosed in Chinese patent or the Chinese patent of Patent No. 201310456288.9
Fluorescence dendroid nanometer disclosed in water-soluble ultrabranching fluorescent polymer or the Chinese patent of Patent No. 201410258179.0
Macromolecular etc..
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>A kind of method that dsRNA interference insect gene expressions are permeated by insect body wall
<141> 2017-12-15
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 493
<212> DNA/RNA
<213> Artificial Sequence
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acagtgcaac aacacgggaa tcattaaaac agaggaccat agtctttgcg ctccagagag 180
cttacccata aatcaaacag tcggtctagt aacagaagac aatccattcc atgatgcgtg 240
cacgaatatc gaaggcatag ttgggttcac ggagtgtcgg ggtctttgcg attcatacac 300
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tttcgataaa ttgtctgtgg tgttacagtg caaagatggt tatcagtata acaagtctgt 420
tccagtgccg tcggcttgtt cttgtacggc gtgtggcgga gagagctttt cgataaaaaa 480
atccagaaaa ctt 493
Claims (9)
1. it is a kind of by insect body wall permeate dsRNA interference insect gene expressions method, which is characterized in that by nano-carrier with
DsRNA function macromolecular complexes by the effect of surfactant, realize external source dsRNA molecules in the attachment of insect body surface, exhibition
Cloth, infiltration body wall simultaneously enter internal various organ-tissues, efficiently interfere gene expression.
2. according to the method described in claim 1, it is characterized in that, described method includes following steps：
1) nano-carrier solution, dsRNA solution with surfactant are mixed, obtains complex liquid；
2) the complex liquid is placed in insect body surface.
3. according to the method described in claim 2, it is characterized in that, in step 1), the nanometer in the nano-carrier solution
Carrier is 1 according to charge ratio with the dsRNA in the dsRNA solution:8~8:1 ratio is mixed to get mixed liquor, by the table
Face activating agent is mixed with the mixed liquor according to 0.01%~10.0% volume ratio.
4. according to the method described in claim 3, it is characterized in that, the nano-carrier and the dsRNA according to charge than 1:1
Ratio be mixed to get mixed liquor, the surfactant is mixed with the mixed liquor according to 1% volume ratio.
5. according to Claims 1 to 4 any one of them method, which is characterized in that the nano-carrier has cavity to be internal
Structure, and surface carries the nano-carrier of positive charge.
6. according to the method described in claim 5, it is characterized in that, the nano-carrier is with high density hydrophilic functional group
Nano-carrier.
7. according to claim 2~4 any one of them method, which is characterized in that the step 2) specifically can by dripping method,
The complex liquid is added to insect body surface by spray-on process or infusion method.
8. a kind of complex liquid for interference insect gene expression, which is characterized in that molten by nano-carrier solution, dsRNA
Liquid is mixed with surfactant.
9. complex liquid according to any one of claims 8 is permeating answering in dsRNA interference insect gene expressions by insect body wall
With.
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| CN110269014A (en) * | 2019-07-23 | 2019-09-24 | 中国海洋大学 | A kind of RNA interference method of Urechis uniconctus larva |
| CN110583581A (en) * | 2019-07-17 | 2019-12-20 | 中国农业科学院蔬菜花卉研究所 | Construction method of novel Bemisia tabaci nymph RNA interference method |
| CN114167047A (en) * | 2021-11-20 | 2022-03-11 | 青岛农业大学 | Insect atomization RNAi device and method |
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