CN107119052B - The miRNA molecule that engineer synthesizes and its Pest control efficacy to aphid - Google Patents
The miRNA molecule that engineer synthesizes and its Pest control efficacy to aphid Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
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Abstract
The present invention relates to a kind of miRNA molecule, miRNA molecule is duplex structure, contains positive-sense strand and antisense strand；Wherein, the nucleotide sequence of positive-sense strand is as shown in the SEQ ID NO:1 in sequence table, and the nucleotide sequence of antisense strand is as shown in the SEQ ID NO:2 in sequence table；Or miRNA molecule is single-stranded structure, nucleotide sequence is as shown in the SEQ ID NO:3 in sequence table.The miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir that the present invention designs synthesis has lethal to aphid, provides novel effective product for the prevention and control of aphid；And the target gene of its effect can be used as ideal candidate gene resource, for formulating the novel anti-pest crop of RNAi mediation, new Research Thinking is provided for pest control, has very high researching value and wide application prospect.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to the miRNA molecule of a kind of engineer synthesis and its to aphid
Pest control efficacy.
Background technique
Wheat aphid is a kind of global agricultural pests, mainly includes grain aphid Sitobion avenae
(Fabricius), green bugs Schizaphis graminum (Rondani), rhopalosiphum padi Rhopalosiphum padi
(Linnaeus) and Acyrthosiphon dirhodum Acyrthosiphon dirhodum (Walker) etc..Wheat aphid, which has, is distributed wide, quantity
Greatly, the features such as reproductive capacity is strong, harm is serious, and be able to carry out and migrate at a distance, and can in Migrating transmitted virus,
The honeydew of excretion can also multiply a variety of moulds, influence the photosynthesis and respiration of wheat plant, lead to crop failure, give agriculture
Industry production brings about great losses, and thus becomes one of the hot spot of agriculture field prevention and control of plant diseases, pest control research.The country is for aphid at present
Control method mainly use chemical pesticide, excessively spraying chemical pesticide can bring sternly to the life of natural environment and the mankind
The consequence of weight.The many scholars of recent domestic are dedicated to screening and the research on utilization of wheat resistant variety, it is desirable to be able to cultivate
Prevention effect can be played in the integrated control of wheat aphid with using the anti-aphid kind of wheat/tie up to.Currently, due in existing germ plasm resource
Effective aphid-resistant gene is also lacked, therefore urgently researcher develops prevention and control of the new target gene for wheat pest.
Summary of the invention
It is an object of that present invention to provide a kind of miRNA molecules: miRNA molecule is duplex structure or single-stranded structure, miRNA
When molecule is duplex structure, contain positive-sense strand and antisense strand；Wherein, the nucleotide sequence of positive-sense strand such as the SEQ ID in sequence table
Shown in NO:1, and the nucleotide sequence of antisense strand is as shown in the SEQ ID NO:2 in sequence table；Or miRNA molecule is single-stranded knot
When structure, nucleotide sequence is specific as follows as shown in the SEQ ID NO:3 in sequence table:
Positive-sense strand (SEQ ID NO:1): 5 '-AAUGGCACUGAAAGAAUUCACGGG-3 '
Antisense strand (SEQ ID NO:2): 5 '-CGUGAAUUCUUUCAGUGCCAUUUU-3 '
Single-stranded (SEQ ID NO:3): 5 '-CCCGUGAAUUCUUUCAGUGCCAUU-3 '
Preferably, in duplex structure, the chemical modification of antisense strand includes: that 3 ' ends carry out cholesterol modification, and 5 ' ends carry out two
A thio backbone modification, 3 ' ends carry out four thio backbone modifications, obtain: 5 '-CsGsUG
AAUUCUUUCAGUGCCAUsUsUsUs-Chol 3 ', and full chain carries out methoxyl group modification；Or the chemical modification of single-stranded structure
It include: that 3 ' ends carry out cholesterol modification, 5 ' ends carry out two thio backbone modifications, and 3 ' ends carry out four thio backbone modifications, obtain
It arrives: 5 '-CsCsCGUGAAUUCUUUCAGUGCCsAsUs Us-Chol 3 ', and full chain carries out methoxyl group modification.
MiRNA molecule provided by the invention is designed with the mature sequence of the miRNAavi-miR-263a of aphid
Corresponding miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi-miR-263a antagomir
(miRNA antagonist).MiRNA avi-miR-263a agomir therein be duplex structure, antisense strand (SEQ ID NO:2) into
Row modification, 3 ' ends carry out cholesterol modification, 5 ' the two thio backbone modifications in end, 3 ' the four thio backbone modifications in end, full chain methoxy
Base modification；Positive-sense strand (SEQ ID NO:1) is effect chain, and antisense strand is the auxiliary chain filled, when design be staggered preceding 2 bases into
The design of row reverse complemental, and add UU pendency convenient for unwinding in end.MiRNA avi-miR-263a antogimir therein is
Single-stranded structure (SEQ ID NO:3), 3 ' ends carry out cholesterol modification, 5 ' the two thio backbone modifications in end, 3 ' the four thio bones in end
Un-wheeling repair decorations, full chain methoxyl group modification.MiRNA avi-miR-263a agomir and miRNA avi-miR- provided by the invention
263a antagomir is that commission Suzhou GenePharma Co., Ltd. is designed synthesis.
The present invention protects the kit including the miRNA molecule of above-mentioned duplex structure or the miRNA molecule of single-stranded structure.
The present invention also protects a kind of kit, and kit includes miRNA molecule solution, and miRNA molecule solution includes quality
The miRNA molecule or single-stranded structure for the above-mentioned duplex structure that the sucrose solution and concentration that score is 30% are 300~500nM
MiRNA molecule；Wherein, the solvent of sucrose solution is water.
Wherein, it is 30% that the process for preparation of miRNA molecule solution, which can be such that (1) uses ultrapure water configuration quality score,
Then sucrose solution is sterilized using 0.2 μm of micro-pore-film filtration, obtained filtrate is sub-packed in the sterile centrifugation tube of 1.5ml,
- 20 DEG C of refrigerators are set to save backup；(2) the miRNA avi-miR-263a agomir by synthesis for being 30% using mass fraction
Or to be diluted to 300~500nM concentration respectively spare by miRNA avi-miR-263a antagomir.
The present invention also protects application of the mentioned reagent box in preparation pest controlling aphid product.Aphid includes wheat aphid, wheat aphid packet
Include grain aphid Sitobion avenae (Fabricius), green bugs Schizaphis graminum (Rondani), standing grain
Paddy Rhopalosiphum spp Rhopalosiphum padi (Linnaeus) and Acyrthosiphon dirhodum Acyrthosiphon dirhodum
One of (Walker) or it is a variety of.
The present invention also provides a kind of method of mentioned reagent box pest controlling aphid, include the following steps: that access head larvae aphid exists
72h is fed in feeding aphid device containing miRNA molecule solution；Then it is linked into wheat strain and continues to raise, until development is adult aphid.It needs
It is noted that growing it is preferred that being linked into fresh potting wheat strain, the death condition and individual hair of aphid are recorded during raising
Situation is educated, development is adult aphid after about 6-7 days, counts the survival rate of aphid；Larvae aphid is preferably derived from: aphid is continuously single
In head raising N generation, collects the larvae aphid of reproduction in the aptery adult aphid primiparity of N+1 generation for 24 hours, and N generation was preferably 3 generations.
Preferably, the feeding aphid device containing miRNA molecule solution is the semiclosed feeding aphid device containing miRNA molecule solution, is contained
There is the preparation method of the semiclosed feeding aphid device of miRNA molecule solution to include the following steps: to hang down the cylindrical glass tube after sterilizing
It is directly placed on blotting paper, top utilizes seals with a thickness of 0.8~2 μm of olefin film, is added in the upper surface of olefin film
Then miRNA molecule solution is bound with the olefin film with a thickness of 8~15 μm again, obtain half envelope containing miRNA molecule solution
It closes and raises aphid device；Wherein, the diameter of cylindrical glass is 2cm, is highly 3cm, and the volume of miRNA molecule solution is 200~300 μ
L；The condition of feeding include: by access several larvae aphids the semiclosed feeding aphid device containing miRNA molecule solution bottom end and water suction
Then entire feeding system is placed in insectary by paper close contact, the temperature of insectary is 20 ± 1 DEG C, and humidity 60 ±
10%, photoperiod L:D=14h:10h.It should be noted that the cylindrical glass tube of both ends open, is gone out using preceding high temperature and pressure
Bacterium is disposed vertically on blotting paper, and top is sealed using olefin film (Parafilm, the U.S.), is instilled above olefin film
MiRNA molecule solution, then one layer of olefin film is sealed, the sandwich of " olefin film-miRNA molecule solution-olefin film " is made
Structure；After accessing aphid, semiclosed feeding aphid device is placed on the pallet for completing blotting paper, is raised in insectary, guarantees to raise aphid
The bottom end bore and blotting paper close contact of device are aphid escapes in order to prevent.
Preferably, several are 10~20.
Preferably, in the upper growth period of wheat strain, with the covering wheat strain of feeding worm cover, the purpose is to avoid aphid from escaping.
MiRNAs mainly participates in biology in rear transcriptional level by three transcriptional control, Transcription inhibition, Translational repression modes
The life processes such as ontogeny, cell differentiation, cell apoptosis and proliferation.Due to important function of the miRNA in insect growth,
Perhaps, their expression inhibiting and overexpression can all influence the normal development of biology.Because of the limitation of miRNA size, miRNA's is straight
Connecing knockout can not realize well.The research of general miRNA function mainly by enhance or weaken the expression of purpose miRNA come
Identification.Using now at most is exactly to utilize specific miRNAs sequence, chemical synthesis miRNA antagonist (miRNA
Antagomir) and miRNA agonist (miRNA agomir), with common miRNA inhibitor (miRNA inhibitor) and
MiRNA analogies (miRNA minics) are compared, and have stronger cellular affinity, stability and inhibition.miRNA
Antgomir is designed according to the mature sequence of miRNA, is synthesized by a series of chemical modification and special marking single-stranded small
RNA can block the combination of miRNA and target gene by the competitive binding with cylinder mature miRNA, reach and inhibit miRNA hair
The effect of waving.MiRNA agomir is that a kind of double-strand tiny RNA is mutually paired by miRNA in analogue body with target gene, enhances mesh
Gene expression in vivo.By the way that miRNA agomir and miRNA antagomir are imported in organism, enhancing or decrease
The expression in vivo of target gene, to influence the normal growth and development process of organism.Especially those prevention and treatment evils
Worm target gene is with strong points, and has lethal to wheat pest after silencing, these genes can be used as ideal candidate gene money
Source, for formulating the novel anti-pest crop of RNAi mediation.
The present invention designs the miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi- of synthesis
MiR-263a antagomir (miRNA antagonist) has lethal to aphid, provides a kind of effective production for the prevention and control of aphid
Product；Also, its target gene acted on can be used as ideal candidate gene resource, for formulating the novel pest-resistant work of RNAi mediation
Object, the prevention and control for crop especially wheat pest provide new Research Thinking, have very high researching value and wide answer
Use prospect.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is raising schematic diagram of the wheat aphid in semiclosed feeding aphid device in the embodiment of the present invention；
Fig. 2 is wheat aphid in the embodiment of the present invention through miRNA avi-miR-263a agomir, miRNA avi-miR-263a
Antagomir, agomir NC, antagomir NC and NC feeding treated mortality statistics figure.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description.The following examples are only intended to illustrate the technical solution of the present invention more clearly, therefore is intended only as example, without
It can be limited the scope of the invention with this.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.
Aphid used in the embodiment of the present invention is grain aphid Sitobion avenae (Fabricius).
One: miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir's of embodiment sets
Meter and synthesis
It designs to obtain corresponding miRNA avi-miR-263a with the mature sequence of the miRNAavi-miR-263a of aphid
Agomir (miRNA agonist) and miRNA avi-miR-263a antagomir (miRNA antagonist).MiRNA therein
Avi-miR-263a agomir is duplex structure, the nucleotide sequence of two chains respectively as in sequence table SEQ ID NO:1 and
Shown in SEQ ID NO:2 in sequence table；MiRNA avi-miR-263a antogimir therein is single-stranded structure, nucleosides
Acid sequence is as shown in the SEQ ID NO:3 in sequence table.
In order to preferably characterize miRNA avi-miR-263a agomir provided by the invention, miRNA avi-miR-
The effect of 263a antagomir, negative control agomir NC and antagomir NC has also been devised in the present invention, therein
The nucleotide sequence of two chains of agomir NC is respectively such as the SEQ ID in the SEQ ID NO:4 and sequence table in sequence table
Shown in NO:5, antagomir NC single-stranded nucleotide sequence is as shown in the SEQ ID NO:6 in sequence table.
MiRNA avi-miR-263a agomir provided by the invention, miRNA avi-miR-263a antagomir with
And corresponding negative control agomir NC and antagomir NC is that commission Suzhou GenePharma Co., Ltd. is set
Meter synthesis.Particular sequence information is as shown in table 1 below.
The sequence information of table 1 miRNA agomir and antagomir
The antisense strand (SEQ ID NO:2) of miRNA avi-miR-263a agomir is modified, is obtained: 5 '-
CsGsUGAAUUCUUUCAGUGCCAUsUsUsUs-Chol 3 ', modification specifically: it is solid that gallbladder is carried out to 3 ' ends of antisense strand
Alcohol modification, 5 ' ends carry out two thio backbone modifications, and 3 ' ends carry out four thio backbone modifications, and last full chain carries out methoxyl group and repairs
Decorations.Positive-sense strand (SEQ ID NO:1) is effect chain above, below antisense strand be the auxiliary chain filled, when design, is staggered preceding 2 alkali
Base carries out reverse complemental design, and adds UU pendency convenient for unwinding in end.
MiRNA avi-miR-263a antogimir therein be single-stranded structure (SEQ ID NO:3), and to its into
Row modification, obtain: 5 '-CsCsCGUGAAUUCUUUCAGUGCCsAsUsUs-Chol3 ', modification specifically: to 3 ' hold into
Row cholesterol modification, 5 ' ends carry out two thio backbone modifications, and 3 ' ends carry out four thio backbone modifications, and full chain carries out methoxyl group
Modification.
Embodiment two: it is raised using miRNA avi-miR-263a agomir and miRNAavi-miR-263a antagomir
Feed aphid and caused lethal effect
1, miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomi, agomir NC and
Antagomir NC preparation method:
(1) then the sucrose solution for being 30% with ultrapure water configuration quality score is gone out using 0.2 μm of micro-pore-film filtration
Obtained filtrate is sub-packed in 1.5ml sterile centrifugation tube by bacterium, sets -20 DEG C of refrigerators and saves backup；
(2) sucrose solution for being 30% using mass fraction is by the miRNA avi-miR-263a after synthesis and modification
Agomir, miRNA avi-miR-263a antagomir, agomir NC and antagomir NC are diluted to concentration respectively
300nM, obtain miRNA avi-miR-263a agomir (+sucrose solution), miRNA avi-miR-263a antagomir (+
Sucrose solution), agomir NC (+sucrose solution) and antagomir NC (+sucrose solution), it is spare.
2、miRNA avi-miR-263a agomir、miRNA avi-miR-263a antagomi、agomir NC、
Antagomir NC and NC feed aphid
(1) the continuous single head of aphid was raised into for 3 generations, collects the larvae aphid of reproduction in the 4th generation aptery adult aphid primiparity for 24 hours, takes respectively
10 miRNA avi-miR-263a agomir (+sucrose solution), the miRNA avi-miR-263a being put into containing 200 μ L
Antagomir (+sucrose solution), agomir NC (+sucrose solution), antagomir NC (+sucrose solution) and mass fraction are
It is fed in the semiclosed feeding aphid device of 30% sucrose solution.Wherein, it is semiclosed raise aphid device preparation method include the following steps: by
Cylindrical glass tube after sterilizing is disposed vertically on blotting paper, and top is sealed using with a thickness of 1 μm of olefin film, in paraffin
The upper surface of film is separately added into corresponding solution (i.e. miRNA avi-miR-263a agomir (+sucrose solution), miRNA
Avi-miR-263a antagomir (+sucrose solution), agomir NC (+sucrose solution), (+sucrose is molten by antagomir NC
Liquid), mass fraction be 30% sucrose solution), then bound again with the olefin film with a thickness of 10 μm, " olefin film-be made
The sandwich structure of miRNA molecule solution/sucrose solution-olefin film " obtains the semiclosed feeding aphid device containing different solutions；
Also, the diameter of cylindrical glass is 2cm, is highly 3cm, the volume of different solutions is 200 μ L.It, will be partly after accessing aphid
Closing is raised aphid device and is placed on the pallet for completing blotting paper, as shown in Figure 1, raising in insectary, guarantees the low port for raising aphid device
Diameter and blotting paper close contact, prevent aphid from escaping.The condition of feeding includes: that will be respectively connected to the molten containing correspondence of 10 larvae aphids
The semiclosed feeding aphid device of liquid is placed in insectary, and the temperature of insectary is 20 ± 1 DEG C, humidity 60 ± 10%, photoperiod L:D=
14h:10h。
(2) as above after feeding 72h, the larvae aphid in aphid device will be raised and be respectively connected in fresh potting wheat strain, with feeding worm cover
Covering so as not to aphid escape, during raising record aphid death condition and ontogeny situation, after 6-7 days development be at
Aphid counts the survival rate of each processing.
In order to guarantee repeatability, every kind of processing is arranged 10 operations and repeats.
3, miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomir, agomir are fed
The lethal effect of NC, antagomir NC and NC to aphid
Concrete outcome is as shown in Fig. 2 and the following table 2: finding through statistical analysis, feeds miRNA avi-miR-263a
The death rate of wheat aphid is 90.00 ± 3.74% after agomir, wheat aphid after feeding miRNA avi-miR-263a antagomir
The death rate is 69.00 ± 5.38%, and comparing with 3 controls has significant difference；Wheat aphid after feeding antagomir NC control group
The death rate be 30.00 ± 2.83%, the death rate of wheat aphid is 16.00 ± 2.90% after feeding agomir NC control group, feeding
The death rate of wheat aphid is 9.00 ± 2.63% after NC control group.Between processing group and control group having differences property it is significant (P <
0.05)。
Table 2 feeds different material to the lethal effect of aphid
The present invention designs the miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi- of synthesis
MiR-263a antagomir (miRNA antagonist) has lethal to aphid, provides a kind of effective production for the prevention and control of aphid
Product；Also, its target gene acted on can be used as ideal candidate gene resource, for formulating the novel pest-resistant work of RNAi mediation
Object, the prevention and control for crop especially wheat pest provide new Research Thinking, have very high researching value and wide answer
Use prospect.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
SEQUENCE LISTING
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>miRNA molecule of engineer's synthesis and its Pest control efficacy to aphid
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Claims (3)
1. a kind of method using kit pest controlling aphid, it is characterised in that:
The kit includes miRNA molecule solution, and the miRNA molecule is duplex structure, contains positive-sense strand and antisense strand；Its
In, the nucleotide sequence of the positive-sense strand is as shown in the SEQ ID NO:1 in sequence table, and the nucleotide sequence of the antisense strand
As shown in the SEQ ID NO:2 in sequence table；
Or the miRNA molecule is single-stranded structure, nucleotide sequence is as shown in the SEQ ID NO:3 in sequence table；
In the duplex structure, the chemical modification of the antisense strand includes: that 3 ' ends carry out cholesterol modification, and 5 ' ends carry out two sulphur
For backbone modification, 3 ' ends carry out four thio backbone modifications, and full chain carries out methoxyl group modification；
Or the chemical modification of the single-stranded structure includes: that 3 ' ends carry out cholesterol modification, 5 ' ends carry out two thio backbone modifications,
3 ' ends carry out four thio backbone modifications, and full chain carries out methoxyl group modification；
The miRNA molecule solution includes the sucrose solution that mass fraction is 30% and concentration is the described of 300~500nM
MiRNA molecule；Wherein, the solvent of the sucrose solution is water；
The method of the kit pest controlling aphid, includes the following steps:
Access head larvae aphid feeds 72h in the feeding aphid device containing miRNA molecule solution, is then linked into wheat strain and continues to raise；
The feeding aphid device containing miRNA molecule solution is the semiclosed feeding aphid device containing miRNA molecule solution, described to contain
The preparation method of the semiclosed feeding aphid device of miRNA molecule solution includes the following steps: that the cylindrical glass tube after sterilizing is vertical
It is placed on blotting paper, top utilizes seals with a thickness of 0.8~2 μm of olefin film, adds in the upper surface of the olefin film
Enter miRNA molecule solution, then bound again with the olefin film with a thickness of 8~15 μm, obtains described containing miRNA molecule solution
Semiclosed feeding aphid device；Wherein, the diameter of the cylindrical glass is 2cm, is highly 3cm, the body of the miRNA molecule solution
Product is 200~300 μ L；
The condition of the feeding includes: by the bottom end for the semiclosed feeding aphid device containing miRNA molecule solution for accessing several larvae aphids
With blotting paper close contact, then entire feeding system is placed in insectary, the temperature of the insectary is 20 ± 1 DEG C,
Humidity 60 ± 10%, photoperiod L:D=14h:10h.
2. the method for kit pest controlling aphid according to claim 1, it is characterised in that:
Described several are 10~20.
3. the method for pest controlling aphid according to claim 2, it is characterised in that:
In the upper growth period of wheat strain, the wheat strain is covered with feeding worm cover.
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