CN104630275A - RNAi technology for preventing and controlling pests through irrigation, and applications thereof - Google Patents
RNAi technology for preventing and controlling pests through irrigation, and applications thereof Download PDFInfo
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Abstract
The present invention relates to RNAi for regulating phytophagous pest growth related gene and applications thereof. Particularly the present invention discloses a gene fragment leading poor phytophagous pest growth based on the RNA interference technology, wherein a dsRNA-containing composition is irrigated on the plant root, and the plant uptakes the dsRNA so as to lead the insect eating the plant to die. The present invention relates to the dsRNA preparation and the convenient and fast use method of the dsRNA preparation, wherein the process for insect pest preventing and controlling and requiring the transgenic plant preparing is simplified, and the pest preventing and controlling purpose can be achieved only through the common irrigation method.
Description
Technical field
The invention belongs to biotechnology and agricultural application field, particularly, the present invention relates to for the RNAi technology by irrigating pest control and application thereof.
Background technology
Large quantifier elimination is verified, and utilize RNA to disturb (RNAi) technology can realize the specificity control of pest species, therefore, the application of this technology can not affect other biological, is a kind of eco-friendly pest control New Policy.But this technology also exists some problems in actual applications, wherein topmost problem how accurately to be delivered in target pest body by the double-stranded RNA (dsRNA) of target gene.More feasible method has two kinds at present:
One is the dsRNA utilizing expression of plants target pest target gene, when this transgenic plant of pests, dsRNA can be taken in body, thus reach the object of pest control.But under field conditions (factors), often kind of crop all will in the face of the infringement of various disease, worm, and we can not carry out transgeneic procedure all crops makes it avoid causing harm of multiple biology.
Two is produce double-stranded RNA preparation in a large number, carries out sprinkling and carry out pest control as chemical insecticide.But; this method has the insect (as brown paddy plant hopper) of wax protective layer for some borer pests (as the snout moth's larva of rice etc.), subterranean pest-insect, some body walls; or be hidden in the insect at the places such as the plant leaf back side, gap; be difficult to be processed in the process of spraying, do not reach the effect of control.
In addition, for sucking pest, the especially less but reproduction speed kind (as aphid, aleyrodid etc.) faster of individuality, there is no the screening of good target gene and utilisation technology at present.
In order to solve Problems existing in above-mentioned technology, this area is easy to use in the urgent need to developing one, extensive effective RNAi utilisation technology, especially for the insect of boring moth property and aculeus type, thus reaches the effect of pest control, cover crop.
Summary of the invention
The object of the present invention is to provide a kind of based on RNAi technology, for piercing-sucking mouthparts and bore preparation that moth property harmful insect carries out preventing and treating and use of the new technology.
A first aspect of the present invention, provide a kind of method double-stranded RNA (dsRNA) being imported plant, comprise step: the root described double-stranded RNA being applied to plant, by the root absorption effect of described plant to double-stranded RNA, described double-stranded RNA is made to be sucked by described plant and be transported to the plant part except root.
In another preference, described double-stranded DNA comprises insect-resistance double-stranded DNA.
Second aspect present invention, provide a kind of method improving plant resistance to insect, comprise step: root insect-resistance double-stranded RNA (dsRNA) being applied to plant, described insect-resistance double-stranded RNA is absorbed by described plant root, and the plant part be transported to except root, thus improve the insect-resistance of plant.
In another preference, the described plant part except root comprise stem, branch, leaf, flower, really or its combination.
In another preference, described conveying is carried in plant body.
In another preference, the length of described pest-resistant dsRNA is 50-2000bp, is preferably 100-1000bp.
In another preference, the application concentration of described pest-resistant dsRNA is 0.0001-5mg/ml, is preferably 0.001-1mg/ml.
In another preference, the plant part except root is upper 3/4 part of plant ground or water surface part, is preferably 2/3 part.
In another preference, described pest-resistant dsRNA is the dsRNA for insect growth genes involved.
In another preference, described insect growth genes involved comprises planthopper P450 gene (Cyp18A1), planthopper carboxylesterase gene (carboxylesterase, Ces) or Ostrinia furnacalis K type serpin gene (KPIs).
In another preference, described insect comprises lepidopteran, Hemiptera, dipteral insect.
In another preference, described planthopper P450 (Cyp18A1) gene fragment order is as shown in SEQ ID NO.1.
Described planthopper Procaine esterase (carboxylesterase, Ces) gene fragment order is as shown in SEQ ID NO.2.
K type serpin (KPIs) gene order of described Ostrinia furnacalis is as shown in SEQ ID NO.3.
In another preference, described dsRNA is as shown in SEQ ID NOs.:4-6.
In another preference, described method of application comprises irrigation, immersion, pouring, drip irrigation.
In another preference, described pest-resistantly comprise anti-phytophagy insect.
In another preference, described phytophagy insect comprises sucking pest, chewing type insect, borer pest and subterranean pest-insect.
In another preference, described sucking pest comprises Brown Planthopper, white backed planthopper or small brown rice planthopper.
In another preference, described borer pest comprises Ostrinia furnacalis, European corn borer or dichocrocis punctiferalis.
In another preference, described plant comprises dicotyledons, monocotyledons or gymnosperm.
In another preference, described plant comprises grass, leguminous plants, cress, plant of Solanaceae and cucurbitaceous plant.
In another preference, described grass comprises corn, paddy rice, barley, wheat, oat, Chinese sorghum, rye.
In another preference, described leguminous plants comprises soybean, peanut, broad bean, pea, red bean, mung bean, cowpea, string bean and French beans.
In another preference, described cress comprises green vegetables, Chinese cabbage, cauliflower, radish, wild cabbage.
Third aspect present invention provides a kind of agricultural composition, and described agricultural composition comprises acceptable carrier in the insect-resistance double-stranded RNA of insecticidal effective dose and Pesticide Science.
In another preference, described agricultural composition is applied to plant root.
In another preference, the application concentration of described dsRNA is 0.0001-5mg/g or 0.0001-5mg/ml, is preferably 0.001-1mg/ml or 0.001-1mg/g.
In another preference, in described agricultural composition, the application concentration of dsRNA is 0.01-5mg/g or 0.01-5mg/ml, is preferably 0.1-1mg/ml or 0.1-1mg/g.
In another preference, described agricultural composition is liquid or solid-state.
In another preference, in described pharmacy or Pesticide Science, acceptable carrier comprises water, synergistic agent, tensio-active agent or stablizer.
In another preference, described agricultural composition also comprises RNA stablizer.
In another preference, described RNA stablizer comprises hydrochloric acid cyanoguanidine, RNA protective material, deionized methane amide equal solvent.
Fourth aspect present invention, provides a kind of purposes of insect-resistance double stranded RNA sequences, for the preparation of being used by root thus improving the composition of plant resistance to insect.
Fifth aspect present invention, provides a kind of polynucleotide of separation, and described polynucleotide are selected from lower group:
Sequence a () SEQ ID NO.:1-6 is arbitrary shown in;
The polynucleotide of b complementary that () and (a) limit; Or
C sequence that () and (a) limit has at least 70% ((preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) arbitrary polynucleotide of the sequence identity more than or complementary sequence, wherein, with the dsRNA of the chain of described arbitrary polynucleotide sequence complementation after phytophagy insect absorbs, there is the activity of the growth suppressing described phytophagy insect.
In another preference, by irrigating, soak, water or being absorbed by plant root after drip irrigation plant root, and the activity of the growth suppressing described phytophagy insect can be had after the picked-up of phytophagy insect with the dsRNA of the chain of described arbitrary polynucleotide sequence complementation.
In another preference, described suppression phytophagy insect growth activity comprises induction or causes phytophagy insect dead.
Sixth aspect present invention, provides a kind of dsRNA construction, and the construction of described dsRNA is double-strand, and its normal chain or minus strand contain the structure shown in formula I:
Seq
forward-X-Seq
oppositely
Formula I
In formula,
Seq
forwardfor the nucleotide sequence of phytophagy insect growth related gene or fragment;
Seq
oppositelyfor with Seq
forwardsubstantially complementary nucleotide sequence;
X is for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelyit is not complementary,
Wherein, described phytophagy insect growth related gene is selected from: planthopper P450 gene (Cyp18A1), planthopper carboxylesterase gene (carboxylesterase, Ces) or Ostrinia furnacalis K type serpin gene (KPIs).
In another preference, described phytophagy insect comprises sucking pest or borer pest.
In another preference, described dsRNA construction can form the dsRNA shown in formula II,
Formula II
In formula,
Seq'
forwardfor Seq
forwardthe RNA sequence that sequence pair is answered or sequence fragment;
Seq'
oppositelyfor with Seq'
forwardsubstantially complementary sequence;
X' is nothing; Or for being positioned at Seq'
forwardand Seq'
oppositelybetween intervening sequence, and described intervening sequence and Seq'
forwardand Seq'
oppositelyit is not complementary,
|| represent at Seq
forwardand Seq
oppositelybetween formed hydrogen bond.
In another preference, Seq
forward, Seq
oppositelylength be at least 50bp.
In another preference, the length of described intervening sequence X' is 0-300bp.
Seventh aspect present invention, provides a kind of expression vector, and described expression vector contains the dsRNA construction described in sixth aspect present invention.
Eighth aspect present invention, provides a kind of genetically engineered host cell, containing the DNA sequence dna be integrated with in the expression vector described in seventh aspect present invention or karyomit(e) corresponding to the dsRNA construction described in sixth aspect present invention in described host cell.
In another preference, described host cell is vegetable cell, is preferably maize cell or rice cell.
Ninth aspect present invention, provides a kind of dsRNA sequence, and described dsRNA sequence is insect-resistance dsRNA, and its length is 100-800bp, is preferably 150-600bp.
In another preference, the sequence source of described dsRNA is from the species being selected from lower group: agriculture and forestry injurious insect or vegetables pest.Preferably, described species comprise the Ostrinia furnacalis of lepidopteran Pyralidae and the Brown Planthopper of Homoptera Delphacidae.
In another preference, described dsRNA sequence is synthetic or artificial preparation.
In another preference, described " being derived from " refers to that the homology (homogeny) of the nucleotide sequence of the target gene in described dsRNA and described insect is 95-100%, is preferably 99-100%.
In another preference, described target gene is that described insect is distinctive.
In another preference, the sequence of described dsRNA is selected from lower group: SEQ ID NOs.:4-6; Or the dsRNA of its length >=50bp.
Tenth aspect present invention, provides a kind of method of pest control, and the agricultural composition described in third aspect present invention or the dsRNA described in ninth aspect present invention are applied to the plant needing pest control.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, this is no longer going to repeat them.
Accompanying drawing explanation
Fig. 1 shows rice root and absorbs dsRNA and the expression of lowering genes involved.
Fig. 2 shows the suppressed and lethality rate to brown paddy plant hopper of brown paddy plant hopper related gene expression.
Fig. 3 shows the suppressed and lethality rate to Pyrausta nubilalis (Hubern). of Ostrinia furnacalis related gene expression.
Fig. 4 shows stability and the degradation rate of dsRNA.
Fig. 5 shows by dsRNA drop of the present invention in Rice Leaf surface after 24 hours, and the blade face of distance drop place far-end, can observe fluorescence phenomenon equally, in figure, grey parts is shown fluorescence.
It is the total serum IgE that clear water process contrast is extracted that Fig. 6 shows CK; DS-is that different double-stranded RNA soaks the total serum IgE extracted after 24 hours, and the arrow below it shows there is a specific band being different from CK in its total serum IgE, is the external source double-strand dsRNA of plant absorption, because all CK do not have this specific band in contrasting.
It is material extraction total serum IgE that Fig. 7 shows that three kinds of different dsRNA to soak after 24h with paddy rice stem, and after being reversed to cDNA, the dsRNA fragment of amplification foreign gene, result obtains the fragment consistent with target sizes, but does not then have band in clear water contrasts.
Embodiment
The present inventor is through extensive and deep research, important gene during phytophagy insect grows is screened, be surprised to find that, the dsRNA for planthopper P450 (Cyp18A1) gene as shown in SEQ ID NO:4, as shown in SEQ ID NO:5 for planthopper Procaine esterase (carboxylesterase, Ces) dsRNA of the dsRNA of gene and K type serpin (KPIs) gene for Ostrinia furnacalis as shown in SEQ ID NO:6, described dsRNA can be made into irrigation liquid, through root system of plant, take in body by phytophagy insect after blade absorbs, exercise the interference to insect target gene, suppress the expression of target gene, finally cause the death of boring moth property and sucking pest, reach the object of pest control.DsRNA of the present invention can adopt simple preparation and application process: irrigate and can reach vermins-proof effect.The present invention, without the need to carrying out transgenic culturing to plant, just can utilize dsRNA to carry out pest control, especially cannot prevent and treat spraying insecticide, the insect pest be hidden in Gen Ye and cane effectively prevents and treats.
As used herein, " substantially complementary " refers to that the sequence of Nucleotide is enough complementary, can interact, as formed secondary structure (as loop-stem structure) in the foreseeable mode of one.Usually, the nucleotide sequence of two " substantially complementary " mutually between have at least the Nucleotide of 70% to be complementary; Preferably, the Nucleotide of 80% is had at least to be complementary; Preferred, have at least the Nucleotide of 90% to be complementary; Preferred further, have at least the Nucleotide of 95% to be complementary; As 98%, 99% or 100%.Usually, maximum 7 unmatched Nucleotide can be had between two enough complementary molecules; Preferably, there are maximum 6 unmatched Nucleotide; Preferred, there are maximum 5 unmatched Nucleotide; Preferred further, there are maximum 4 unmatched Nucleotide, as having 0,1,2,3,4 unmatched Nucleotide.
As used herein, the sequence of " complementation " typically refers to the sequence (as the 5'ATCG3' → GCTA) sequence in 5'-3' direction being converted to its 3'-5' direction, and then gets its complementary sequence (as GCTA → 5'CGAT3').
As used herein, " stem ring " structure is also referred to as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form the secondary structure that one comprises double-stranded region (stem), described double-stranded region is formed by two regions (being positioned on same a part) of this nucleic acid molecule, the both sides of two region apportion double stranded section; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. single-stranded regions.Even if two of this nucleic acid molecule regions are not complete complementaries, the double stranded section of nucleic acid also can keep double-stranded state.Such as, insertion, disappearance, replacement etc. can cause the not complementary of a zonule or this zonule self to form the secondary structure of loop-stem structure or other form, but these two regions still can be substantially complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, usually obtain one there is the nucleic acid of the nucleotide sequence of primary structure after, those skilled in the art can determine whether this nucleic acid can form loop-stem structure.
In the present invention, preferred loop-stem structure can such as formula shown in the dsRNA of II, and X' is for being positioned at Seq'
forwardand Seq'
oppositelybetween intervening sequence, and described intervening sequence and Seq'
forwardand Seq'
oppositelynot complementary, when X ' for without time, namely the dsRNA of formula II is by Seq'
forwardand Seq'
oppositelythat formed, complementary duplex-RNA constructs.
As used herein, described " nucleic acid inhibitor " refers to based on of the present invention for preventing and treating general name that the useful target gene of phytophagy insect (fragment) or its fragment or clipped form prepare, that have the class material preventing and treating phytophagy pest activity.Described " nucleic acid inhibitor " is such as some disturbing molecules, comprise dsRNA (being also called double-stranded RNA, double stranded RNA or double-stranded ribonucleotides sequence), antisense nucleic acid, siRNA, Microrna etc., or can express or be formed the construction of described dsRNA, antisense nucleic acid, siRNA or Microrna.
In the present invention, nucleic acid inhibitor is preferably dsRNA.
As used herein, described " connecting with can operating (property) " or " operability connection " refers to functional spatial disposition of two or more nucleic acid region or nucleotide sequence.Such as: promoter region is placed in the specific position relative to goal gene nucleotide sequence, what make nucleotide sequence transcribes the guiding being subject to this promoter region, thus promoter region is " operably connected " on this nucleotide sequence.
As used herein, " the RNA sequence corresponding with DNA sequence dna " refers to a kind of RNA sequence, if DNA sequence dna is " AT ", RNA sequence is " AU ".
" contain ", " having " or " comprising " include " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
RNA disturbs (RNAi)
As used herein, term " RNA disturbs (RNA interference; RNAi) " refers to: some little double-stranded RNAs can block the expression of specific gene in body efficiently, specifically, mRNA is impelled to degrade, entice cell shows the phenotype of specific gene disappearance, and it is intervened also referred to as RNA or RNA interferes.RNA interference is the gene silencing mechanism in mRNA level in-site of high special.
As used herein, term " siRNA (small interfering RNA; siRNA) " or " dsRNA " refer to a kind of short-movie section double stranded rna molecule, can with the mRNA of homologous complementary sequence for target to be degraded specific mRNA, this process is exactly RNA interference channel (RNA interference pathway).
In the present invention, the ultimate principle of described RNA interference is: RNA interfering is prepared as irrigation liquid, routinely after irrigation method irrigating plant, insect is taken containing disturbing the siRNA (siRNA) of its genetic expression or the plant of dsRNA, thus the death of induction insect.
As the preferred mode of one, utilize an intron sequences, two ends connect upper complementary gene order, after transfered cell, " stem-ring " structure can be produced, and " stem " shape part can in insect body the processed tiny RNA into about about 21-25nt, this tiny RNA especially effectively can suppress the expression of goal gene.
In the present invention, preferred RNA interfering is dsRNA.DsRNA is a kind of double stranded RNA sequences formed by normal chain and its complementary strand.Wherein, normal chain and minus strand can be mated or semi-match completely.A kind of preferred dsRNA has such as formula the dsDNA shown in II structural formula.
In the present invention, the length of dsRNA sequence is not particularly limited, and its length is 100-800bp usually, is preferably 150-600bp.
In another preference, the sequence source of described dsRNA is from the species being selected from lower group: agriculture and forestry injurious insect or vegetables pest.Preferably, described species comprise the Ostrinia furnacalis of lepidopteran Pyralidae and the Brown Planthopper of Homoptera Delphacidae.
In another preference, described dsRNA sequence is synthetic or artificial preparation.
In another preference, described " being derived from " refers to that the homology (homogeny) of the nucleotide sequence of the target gene in described dsRNA and described phytophagy insect is 95-100%, is preferably 99-100%.
In another preference, described target gene is that described phytophagy insect is distinctive.
In another preference, the sequence of described dsRNA is selected from lower group: SEQ ID NOs.:4-6; Or its length is the >=dsRNA of 50bp.
Insect growth genes involved
As used herein, term " insect growth genes involved ", " phytophagy insect growth related gene " word are used interchangeably, all refer to the gene of being correlated with at the insect growth being mainly food with plant (especially farm crop), described " growth is relevant " refers to the low expression of described gene or does not express the process such as growth, growth, metabolism, the breeding generation exception that will cause insect, even causes the death of insect.In a preference of the present invention, described phytophagy insect growth related gene comprises K type serpin gene (KPIs) of the P450 gene (Cyp18A1) of planthopper, carboxylesterase gene (carboxylesterase, Ces) or Ostrinia furnacalis.
" insect growth genes involved " refers to those and the closely-related gene of insect growth, and as in P450 gene, have a lot of detoxification participating in food, the expression of this genoid, once suppressed, will cause insect poisoning.Esterase is the Chlorogenic Acid that in insect body, a class is important, and it by the ester bond of ester hydrolysis class toxic chemical, or can be combined with lipophilic class toxic compounds, reduces its effective concentration, reduces the toxicity of toxic compounds.Many sterilants are the ester compounds containing ester bond, and as organophosphorus, carbamate and pyrethroid pesticide, esterase also plays an important role in the resistance of insect, as Procaine esterase.Therefore, the expression of interference insect body lactonase, not only can reduce its detoxification ability, can also prevent its drug-fast raising.And proteinase inhibitor is the material of a class arrestin enzymic activity.Serpin can regulate and control the multiple physiological response in organism, the katalysis of inhibitory enzyme, stops proenzyme to be converted into activated enzyme, therefore, affects insect and grow normally.
Preferably, in brown paddy plant hopper, with for P450 gene (Cyp18A1) fragment and carboxylesterase gene (carboxylesterase, Ces) dsRNA of fragment design, soak brown paddy plant hopper of being fed by the plant organ except immersion position after rice root, the expression of corresponding gene is lowered (Fig. 2 a, 2b); Mortality ratio after brown paddy plant hopper takes food 5 days on corresponding dsRNA process paddy rice is respectively 26.67% and 48.33% (Fig. 2 c).In Ostrinia furnacalis, with the dsRNA designed for K type serpin gene (KPIs) fragment, to feed Ostrinia furnacalis after pouring corn, the expression of this gene is significantly down-regulated that (Fig. 3 a).Mortality ratio after Ostrinia furnacalis takes food 5 days on corresponding dsRNA process corn is 43.33% (Fig. 3 b).
As optimal way of the present invention, the length of the fragment of the present invention preferred phytophagy insect growth related gene is at least 50bp, can be such as 60bp, 80bp, 100bp, 200bp, 500bp, 1000bp.Described gene, for time of the present invention, can be full-length gene or gene fragment, preferably, for the dsRNA of the P450 gene (Cyp18A1) of planthopper as shown in SEQ ID NO:4; For the dsRNA of the carboxylesterase gene (carboxylesterase, Ces) of planthopper as shown in SEQ ID NO:5; For the dsRNA of K type serpin gene (KPIs) of Ostrinia furnacalis as shown in SEQ ID NO:6.
Present invention also offers the dsRNA of the EYFP gene for non-insect origin, the sequence of described EYFP gene is as shown in SEQ ID NO:7.With planthopper P450 gene, carboxylesterase gene is compared with Ostrinia furnacalis K type serpin gene, the poor effect of EYFP gene.
The invention provides the dsRNA for phytophagy insect growth related gene, by dsRNA being mixed with the root of irrigation liquid irrigated crop and absorbing through plant root, insect edible absorb the crop of dsRNA after, its growth is suppressed and occurs mortality.
Illustrated dsRNA construction is such as formula shown in I, dsRNA is such as formula shown in II, the length of intervening sequence X or X' adopted has no particular limits, as long as form construction in itself and forward sequence and reverse sequence and be directed to after in body, can form the dsRNA shown in formula II.As optimal way of the present invention, the length of intervening sequence of the present invention is 80-300bp; Be more preferably 100-250bp.
In a preference of the present invention, the construction of described expression phytophagy insect growth related gene dsRNA is imported in host, described host can be vegetable cell, tissue or organ, described construction can express insect genes dsRNA in plant materials, and dsRNA is processed to siRNA.Usually, the length of siRNA is about about 21-25nt.
Usually, described construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described construction.Described expression vector is usually also containing promotor, replication orgin and/or the marker gene etc. that are connected with described construction operability.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as kalamycin, gentamicin, Totomycin, amicillin resistance.
Comprise the carrier of above-mentioned suitable gene order and suitable promotor or control sequence, may be used for transforming suitable host.In the method for the invention, described host carries the host that described expression vector also can be passed to vegetable cell by described expression vector any being suitable for.Preferably, described host is Agrobacterium.
Although the phytophagy insect of illustrating in example of the present invention is Ostrinia furnacalis and planthopper.But should understand, the present invention has no particular limits for being applicable to insect of the present invention, described insect can be any one can be the phytophagy insect eaten with plant, and such as it can be lepidopterous insects, hemipteran, Homoptera insect or dipteral insect.
The present invention has no particular limits for being applicable to plant of the present invention, and can be various common cash crop or ornamental plant, as plants such as Gramineae, Cruciferae, pulse families, be preferably corn or paddy rice.
The nucleotide sequence of target gene
P450 (Cyp18A1) gene fragment of planthopper is as shown in SEQ ID NO.1; Procaine esterase (carboxylesterase, the Ces) gene fragment of planthopper is as SEQ ID NO.2: shown in; The full length cDNA sequence of K type serpin (KPIs) gene of Ostrinia furnacalis is as shown in SEQ ID NO.3.
The target sequence of dsRNA
In one embodiment of the invention, based on RNAi technology, using the P450 gene (Cyp18A1) of planthopper as target, screen the RNA interfering fragment for P450 gene, preferably, the dsRNA sequence of described P450 gene fragment is as shown in SEQ ID NO:4 (dsRNA1:dsCyp18A1).
In one embodiment of the invention, based on RNAi technology, with planthopper carboxylesterase gene (carboxylesterase, Ces) as target, screen the RNA interfering fragment for carboxylesterase gene, preferably, the dsRNA sequence of described carboxylesterase gene fragment is as shown in SEQ ID NO:5 (dsRNA2:dsCes).
In one embodiment of the invention, based on RNAi technology, using K type serpin (KPIs) gene of Ostrinia furnacalis as target, screen the RNA interfering fragment for K type serpin (KPIs) gene, preferably, the dsRNA sequence of described K type serpin (KPIs) gene fragment is as shown in SEQ ID NO:6.
In one embodiment of the invention, based on RNAi technology, with the enhancement type yellow fluorescent protein gene EYFP of jellyfish, as foreign gene contrast (SEQ ID NO.:7).The dsRNA sequence of described EYFP gene fragment is as shown in SEQ ID NO:41 (dsRNA:dsEYFP).
DsRNA construction and application thereof
Any that prepare based on target gene provided by the invention (fragment) or its fragment or clipped form, there is the material preventing and treating phytophagy pest activity all can be used as nucleic acid inhibitor, for preventing and treating phytophagy insect.Preferably some disturbing molecules such as described nucleic acid inhibitor such as grade, such as, the construction of dsRNA, antisense nucleic acid, siRNA or Microrna, maybe can express or be formed the construction of described dsRNA, antisense nucleic acid, siRNA or Microrna.It is more preferably the construction that dsRNA maybe can express described dsRNA.
According to gene provided by the present invention and sequence thereof, the construction for expressing dsRNA, antisense nucleic acid, siRNA or Microrna can be designed.Therefore, the invention provides a kind of artificial constructed construction.Designing described construction according to gene provided by the invention and sequence thereof is that those skilled in the art are apprehensible, usually this construction can be made to comprise an intron sequences (not complementary with both sides sequence), two ends connect upper complementary gene order, after transfered cell, " stem ring " structure can be produced, and " stem " shape part can form dsRNA, antisense nucleic acid, siRNA or Microrna, and this dsRNA, antisense nucleic acid, siRNA or Microrna especially effectively can suppress the expression of goal gene.
A kind ofly preferably realize method that the present invention prevents and treats phytophagy insect and comprise and described nucleic acid inhibitor directly irrigated or soaks the object (as plant) being used for needing to prevent and treat, to reach the object of preventing and treating phytophagy insect.
The invention provides a kind of dsRNA construction, the construction of described dsRNA is double-strand, and its normal chain or minus strand contain the structure shown in formula I:
Seq
forward-X-Seq
oppositelyformula I
In formula,
Seq
forwardfor the nucleotide sequence of phytophagy insect growth related gene or fragment;
Seq
oppositelyfor with Seq
forwardsubstantially complementary nucleotide sequence;
X is nothing, or for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelyit is not complementary,
Wherein, described phytophagy insect growth related gene is K type serpin gene (KPIs) of the P450 gene (Cyp18A1) of planthopper, carboxylesterase gene (carboxylesterase, Ces) and Ostrinia furnacalis.
In a preference of the present invention, Seq
forward, Seq
oppositelylength be at least 50bp.
In a preference of the present invention, described dsRNA construction absorbs through crop root and after being ingested by phytophagy insect, forms the dsRNA shown in formula II,
formula II
In formula,
Seq'
forwardfor Seq
forwardthe RNA sequence that sequence pair is answered or sequence fragment;
Seq'
oppositelyfor with Seq'
forwardsubstantially complementary sequence;
X' is nothing; Or for being positioned at Seq'
forwardand Seq'
oppositelybetween intervening sequence, and described intervening sequence and Seq'
forwardand Seq'
oppositelyit is not complementary,
|| represent at Seq'
forwardand Seq'
oppositelybetween formed hydrogen bond.
Certainly, described construction also can be positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described construction.Described expression vector is usually also containing promotor, replication orgin and/or the marker gene etc. that are connected with described construction operability.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as kantlex, gentamicin, Totomycin, amicillin resistance.
Comprise the carrier of above-mentioned suitable gene order and suitable promotor or control sequence, may be used for transforming suitable host.In the method for the invention, described host carries the host that described expression vector also can give expression to nucleic acid inhibitor any being suitable for.Such as, described host is intestinal bacteria, fungi, yeast, vegetable cell, zooblast etc.
Transform host with recombinant DNA to carry out with routine techniques well known to those skilled in the art, specifically depending on floristic difference.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.The conversion of fungi and yeast cell, vegetable cell, zooblast is also well known to those skilled in the art.
Carry described construction or expression vector and the host that can give expression to nucleic acid inhibitor directly can be applied to the object (as plant) needing control, to reach the object of preventing and treating phytophagy insect.
Namely " stem " shape part of above-described loop-stem structure is oppositely interacted by Seq forward and Seq and is formed, and can be processed to form nucleic acid inhibitor.The nucleic acid inhibitor formed has following structure:
Wherein, Seq'
forwardbe selected from SEQ ID NO:1-3 arbitrary shown in the sequence pair RNA sequence of answering or sequence fragment; Seq'
oppositelyfor with Seq'
forwardsubstantially complementary sequence.X' is for being positioned at Seq'
forwardand Seq'
oppositelybetween intervening sequence, described intervening sequence and Seq'
forwardand Seq'
oppositelynot complementary.X' sequence can be excised in vitro, also can not excise, and enters after in insect body process excision by the enzyme (as nuclease Dicer) in insect body until dsRNA.
Composition and application thereof
Present invention also offers a kind of composition, described composition comprises dsRNA construction and/or dsRNA, and acceptable carrier in pharmacy or Pesticide Science.In another preference, described composition is the composition for disturbing phytophagy insect to grow, induce or cause the death of phytophagy insect.
In another preference, described dsRNA has following sequence:
DsRNA1: as shown in SEQ ID NO.:4;
DsRNA2: as shown in SEQ ID NO.:5;
DsRNA3: as shown in SEQ ID NO.:6;
In a preference of the present invention, composition is the aqueous solution, and pH is about 5-8 usually, and preferably, pH is about 6-8.
As used herein, term " carrier " comprises various vehicle and thinner.This kind of carrier comprises (but being not limited to): water, salt solution, damping fluid, glucose, glycerine, ethanol and combination thereof.
Composition of the present invention can directly irrigate or water in crop organ, and wherein said organ comprises the root of plant, stem, leaf, flower, really; In addition, also can the organ of plant be soaked in the present composition, to reach the object of the present composition through plant absorption.Certainly, preferred mode is for preparing agricultural composition of the present invention or joining in irrigation liquid, and the root of irrigating in plant, dsRNA of the present invention is made to pass through root absorption, bring to non-irrigation part, thus make insect after the non-irrigation part of feeding plant, there is growth-inhibiting or death, to reach the pest-resistant object of plant.
In composition of the present invention, water and the aqueous solution containing other assistant agents are prepared by ordinary method.Described composition should manufacture under aseptic or condition without RNA enzyme.
Described phytophagy insect growth related gene derives from K type serpin gene (KPIs) of the P450 gene (Cyp18A1) of planthopper, carboxylesterase gene (carboxylesterase, Ces) and Ostrinia furnacalis;
In another preference, described insect is sucking pest or borer pest, preferably from phytophagy insect, best from Brown Planthopper and Ostrinia furnacalis.
Advantage of the present invention
(1) the dsRNA preparation of effective target gene is applied to Field Pests control in the mode of irrigating or soak by the present invention easily, plant can be made effectively to absorb dsRNA of the present invention and composition thereof, thus avoid all crops being carried out complicated transgeneic procedure;
(2) the present invention is directed to pierce-suck type, bore moth property and subterranean pest-insect, carry out the high flux screening of phytophagy insect target gene;
(3) the present invention by target in turn and mixing application solve issuable resistance problem in pest control process;
(4) dsRNA preparation of the present invention has relative stability, ensures the function playing sterilant in crop growth period;
(5) under the illumination condition of field, dsRNA preparation of the present invention by fast degradation after crop growth period, can have good biological safety and ecological security.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Universal method
The plantation of vegetable material is cultivated
Vegetable material used by this research has in rice varieties spends 11 (ZH11, purchased from China Paddy Rice Inst) and corn variety Zheng Dan 958 (ZD958, purchased from Henan Agricultural Sciences research institute).Rice paddy seed sowing is at high 10cm, and diameter is in the cylinder shape plastic cup of 5cm; Corn seed sowing is at 15cm, and diameter is in the cylinder shape plastic cup of 10cm.All plant growings are placed in greenhouse to be cultivated.In greenhouse daytime/night temperature be 28/22 DEG C, daytime/night photoperiod is 14/10 hour, relative humidity 50-60%.
The raising of insect
Brown Planthopper and Ostrinia furnacalis pick up from village, often used in village names farm, Songjiang, Shanghai five.Population of Rice Brown Planthopper is raised on the ZH11 rice varieties of above-mentioned plantation, gets 2-3 nymph in age and tests for this.
Ostrinia furnacalis artificial diet, raise in illumination box.Incubator temperature is 25 DEG C, daytime/night photoperiod is 14/10 hour, relative humidity 75%.
Pyrausta nubilalis (Hubern). raises formula: Semen Maydis powder 120.0 grams, Semen Maydis powder 32.0 grams, soyflour 120.0 grams, vitamins C 4.0 grams, 12.0 grams, agar, yeast powder 72 grams, Sorbic Acid 4.0 grams, glucose 60.0 grams, formaldehyde 1.6ml, 1 liter, water.
Embodiment 1
Screening target gene
1. paddy rice, corn, the extraction of brown paddy plant hopper and Ostrinia furnacalis total serum IgE
Blade got by paddy rice and corn, brown paddy plant hopper gets the nymph in 2-3 age, Ostrinia furnacalis carries out the extraction of total serum IgE respectively with 3 instar larvaes, conventional Trizol method is adopted to extract RNA, ordinary method purifying, DNA enzymatic process, obtains the total serum IgE sample that concentration >=300ng/ul, total amount >=6ug, OD260/280 are 1.8 ~ 2.2.
The separation of 2.mRNA and the synthesis of cDNA
Go out the mRNA with polyA with the Beads enrichment with oligo-dT, then synthesize cDNA first chain with the Superscript II reverse transcriptase test kit of random sexamer and Invitrogen.
3. the amplification of gene and order-checking
Utilize the primer that the target gene shown in table 1 is special, with the cDNA of above-mentioned synthesis for template show in the amplification of listed gene, by the gene fragment purifying obtained, be connected in pMD18-T carrier (Takara company), be transformed into intestinal bacteria Top10 bacterial strain, blue hickie screening, positive strain checks order, and is used as the template of dsRNA synthesis after test is correct.
Table 1, gene chemical synthesis the primer
Embodiment 2
The synthesis of dsRNA
Obtain the target gene sequence of synthesis dsRNA with primer (table 2) amplification with T7 joint and check order, utilizing MEGAscript RNAi kit (Ambion, Huntingdon, UK) to synthesize and purifying dsRNA.
Concrete steps and method
1, Trizol method extracting target species total serum IgE purifying.
(1) by powdered for 100mg tissue liquid nitrogen grinding (using the mortar of DEPC process);
(2) dry powder is poured into be equipped with
centrifuge tube in;
(3) after room temperature places 5min, add 200 μ l chloroforms, be inverted mixing 15s;
(4) after room temperature leaves standstill 5min, 4 DEG C of centrifugal 15min of 12000rpm;
(5) get supernatant liquid in new centrifuge tube, add isopyknic Virahol (~ 500 μ l), be inverted mixing for several times;
(6) after room temperature leaves standstill 10min, 4 DEG C of centrifugal 10min of 12000rpm;
(7) outwell supernatant gently, retain precipitation, add 500 ~ 1000 μ l75% ethanol rinses a little;
After (8) 4 DEG C of centrifugal 5min of 7600rpm, outwell ethanol gently, tip upside down on thieving paper and control dry ethanol, keep 10min;
(9) add appropriate DEPC water, dissolve 10min.
(10) RNA sample is added DEPC water and expand 100 μ l to, add 100 μ l (equivalent) phenol/chloroform/primary isoamyl alcohol (25:24:1), fully mix;
(11) the centrifugal 5min of 13000rpm room temperature, supernatant liquid moves to another centrifuge tube;
(12) add 100 μ l (equivalent) chloroform/primary isoamyl alcohol (24:1), fully mix;
(13) the centrifugal 5min of 13000rpm room temperature, supernatant liquid is transferred to another centrifuge tube;
(14) the 3M NaOAc (pH5.2) of 10 μ l (1/10 amount) is added;
(15) 250 μ l (2.5 times) cold dehydrated alcohol is added ,-70 DEG C of overnight precipitation (or-20 DEG C of precipitation 2h);
(16) 4 DEG C of centrifugal 5min of 13000rpm, go supernatant to stay precipitation, with the cold ethanol rinse of 1ml75%, and dry 5min;
(17) add appropriate DEPC water, dissolve 10min;
(18) dilute 100 times and measure concentration, and electrophoresis detection quality.
2, reverse cDNA.
(1) following reagent is mixed:
(2) Oligo dT Primer (25pmol/ μ l) 1 μ l is added;
(3) template ribonucleic acid (1 below μ g) x μ l is added;
(4) ThermoScript II 1 μ l is added;
(5)30℃10min，42℃20min，99℃5min；
(6) 5min is on ice placed in immediately;
(7) moment centrifugal after, cDNA is in-20 DEG C of preservations.
3, utilize the dsRNA primer amplification dsRNA template in table 2, and product is cut glue recovery.
(1) electrophoresis, rubber tapping;
(2) add isopyknic Binding Buffer (XP2), 55 ~ 60 DEG C of incubation 7min or dissolve completely to glue, every 2 ~ 3min vibration therebetween once;
(3) will
dNA column puts into 2ml collection tube;
(4) on column, add 700 μ l glue melt liquid, 10, the centrifugal 1min of 000 × g room temperature;
(5) abandon filtered solution, column puts into former collection tube;
(6) on column, add 300 μ l Binding Buffer (XP2), the centrifugal 1min of 10,000 × g room temperature, abandon filtered solution, column puts into former pipe;
(7) on column, add 700 μ l SPW Wash Buffer (dehydrated alcohol dilution), the centrifugal 1min of 10,000 × g room temperature, abandon filtered solution, column puts into former pipe;
(8) OPTIONAL: repeating step (7);
(9) abandon filtered solution, empty column is with (>=13,000 × g) centrifugal 2min the most at a high speed;
(10) column is put into clean 1.5ml centrifuge tube, Xiang Zhu center adds 30 ~ 50 μ lElution Buffer, and room temperature places 1min, and (>=13,000 × g) centrifugal 1min is with eluted dna the most at a high speed.
4, dsRNA synthesis
(1)8μL ATP/CTP/UTP/GTP solution
DNA after (2) 1 μ g purifying
(3)2μL Enzyme Mix
(4) mixed solution is placed in 37 DEG C of incubators and hatches 4h, and product is dsRNA.
(5) add 1 μ L TURBO DNase, mix latter 37 DEG C and hatch 15min, product is dsRNA
Show the primer of 2-in-1 one-tenth used by dsRNA
Wherein, EYFP is enhancement type yellow fluorescent protein gene, contrasts as foreign gene.
The absorption of embodiment 3 plant organ exogenous dsRNA
This experiment adopts the dsRNA (SEQ ID NO.:40) of blank (CK), dsRNA of the present invention (SEQ ID NOs.:4-6), paddy rice endogenous gene Actin respectively and contrasts exogenous dsRNA and dsEYFP (SEQ ID NO.:41) by after Root Absorption dsRNA, the dsRNA expression of stem (stem do not contacted with dsRNA soak solution) is observed and measured, the final concentration of all dsRNA is 0.5mg/ml, and it is grouped as follows:
A. blank group: be designated as CK, this contrast is by the plant (paddy rice or corn) of test, same method plantation, growth conditions is consistent, but without dsRNA solution-treated, as in the present invention, paddy rice soaks with clear water, and corn is then irrigate with clear water;
B. dsRNA of the present invention: the dsRNA (dsCyp18A1), the carboxylesterase gene (carboxylesterase that adopt the P450 gene (Cyp18A1) of the endogenous expression of brown paddy plant hopper, after the dsRNA (dsKPI) of dsRNA (dsCES) Ces) or K type serpin gene (KPIs) of the endogenous expression of Ostrinia furnacalis irrigates plant root or soaks, being measured by the dsRNA expression of stem after root absorption dsCES, dsCyp18A1, dsKPI of the present invention;
C. adopt the dsRNA of paddy rice native gene Actin to soak rice root, and measure the expression of dsRNA by stem Actin after root absorption;
D. all non-existent foreign gene in insect and plant materials is adopted: after the dsRNA (dsEYFP) of the enhancement type yellow fluorescence protein (EYFP) of Victoria's multitube jellyfish irrigates plant root or soaks, measure the expression of stem dsRNA.
Result:
After soaking rice root 24h with the fluorescently-labeled dsEYFP of band FAM that final concentration is 0.5mg/ml, obvious green fluorescence can be observed at the leaf sheath place of paddy rice (Fig. 1 a) under fluorescent microscope, obvious green fluorescence (Fig. 1 b) is presented by also seeing in rice stem after rice stem crosscut, extract the Culm of Rice RNA after absorbing dsRNA, as template amplification dsRNA, can obtain and the band of foreign gene dsEYFP formed objects (Fig. 1 c).And after soaking rice root 24h with the dsRNA of paddy rice native gene Actin, the growth of rice root is significantly suppressed (Fig. 1 d, e), and the expression of this gene is also suppressed (Fig. 1 f) by significant.These results illustrate that exogenous dsRNA can be entered in the body of plant by root system, and suppress the expression of genes involved.
As shown in Figure 6, CK is the total serum IgE that clear water process contrast is extracted; DS-is that different double-stranded RNA is irrigated plant root or soaks the total serum IgE that after 24 hours, stem extracts, arrow below it shows there is a specific band being different from CK in its total serum IgE, be the external source double-strand dsRNA of plant absorption, because all CK do not have this specific band in contrasting.Fig. 7 shows with dsCYP18A1; DsCes; After the dsRNA that dsEYFP etc. three kinds are different soaks paddy rice 24h, be material extraction total serum IgE with paddy rice stem, after being reversed to cDNA, the dsRNA fragment of amplification foreign gene, result obtains the fragment consistent with target sizes, but does not then have band in clear water contrast.
In addition, by dsRNA drop of the present invention in Rice Leaf surface, find after 24 hours, the blade face of distance drop place far-end, can observe fluorescence phenomenon equally, illustrate that dsRNA of the present invention can be passed through the Foliage Absorption of plant.(Fig. 5)
Conclusion: can by plant organ especially root absorption to the stem of plant with fluorescently-labeled exogenous dsRNA, the dsRNA of plant gene itself can affect the growth of plant by Root Absorption, and be also all transported to plant stem by plant organ especially root absorption for the dsRNA of insect growth in the present invention, and leaf also can realize the transport to dsRNA.As can be seen here, exogenous dsRNA by the organ of plant, as root, leaf etc. are absorbed into inside plants.
Embodiment 4 is tested the lethal effect of insect by the exogenous dsRNA of root absorption.
This experiment adopts the dsRNA of synthesis to carry out simulation irrigation experiment to paddy rice and corn, and observes its impact on insect, and utilizes qPCR to carry out the detection of expression conditions to sample, and primer is in table 3, and sequence and the gene source information of dsRNA are as shown in table 4.
Table 3. is for the primer of quantitative PCR detection
The title of table 4dsRNA and source-information
Lethal effect to brown paddy plant hopper after 4.1 rice roots absorption dsRNA
Paddy rice ZH11 seed grew to for two leaf phases after vernalization in nutritive medium, by rice transplantation in the dsRNA solution of 0.5mg/ml, rice root was all soaked in the solution, after soaking 24h, obtained the paddy rice through immersion treatment.
Received by 1-2 brown paddy plant hopper in age above-mentioned on the paddy rice of immersion treatment again, every strain 10, plant hopper mainly concentrates on paddy rice stem, and distance root 1-4cm position (non-steeped position) takes food.Plant hopper is got, gene expression detection situation after meeting worm 24h.In whole process of the test, take safeguard procedures, make plant hopper directly not contact dsRNA solution.
Wherein, dsRNA solution is P450 gene (Cyp18A1) and or dsRNA or dsEYFP (concentration is respectively 0.5mg/mL) of carboxylesterase gene (carboxylesterase, Ces) of synthesis brown paddy plant hopper endogenous expression.
Result shows, the expression of these two genes of brown paddy plant hopper is significantly down-regulated (Fig. 2 a, b).With double-stranded RNA process after 24 hours, the mortality ratio after paddy rice brown paddy plant hopper being placed on corresponding dsRNA process takes food 5 days is continuously respectively 26.67% and 48.33% (Fig. 2 c and table 5).
Lethality rate to planthopper after table 5. brown paddy plant hopper gene dsRNA pouring paddy rice
Lethal effect to Ostrinia furnacalis after 4.2 maize root systems absorption dsRNA
Zheng Dan 985 corn seed is planted in nutrition pot, start to irrigate root with the dsRNA solution of 0.5mg/ml when corn grew to for 3 leaf phase, Pyrausta nubilalis (Hubern). is accessed after 24h, every strain maize seedling accesses 2 age corn borer larvae 10, Pyrausta nubilalis (Hubern). mainly takes food the top of leaf of Semen Maydis position and stem, can not pierce in soil and take food.Pyrausta nubilalis (Hubern). is got, gene expression detection situation after 24h.Wherein, dsRNA solution is dsRNA or dsEYFP (concentration is respectively 0.5mg/mL) of K type serpin gene (KPIs) of the endogenous expression of Ostrinia furnacalis of synthesis.
Result shows, the expression of this gene of Pyrausta nubilalis (Hubern). is significantly down-regulated that (Fig. 3 a).With double-stranded RNA process after 24 hours, it is 43.33% (Fig. 3 b and table 6) that corn Ostrinia furnacalis being placed on corresponding dsRNA process takes food the mortality ratio after 5 continuously.
Lethality rate to Pyrausta nubilalis (Hubern). after table 6. Ostrinia furnacalis gene dsRNA pouring corn
Embodiment 5dsRNA stability and degradability test
Synthetic dsRNA is placed in respectively 4 DEG C, under three kinds of conditions such as natural lighting environment or shading, result shows, dsRNA deposits 90 days under 4 DEG C of conditions, and (Fig. 4 a) not find significantly degraded.When room temperature keeps in Dark Place, there is Partial digestion, the dsRNA (Shading in Fig. 4 b and 4c) of 50% can also be detected 90 days time.Under room temperature light conditions, 45 days start obvious degradation, within 90 days, substantially can't detect dsRNA (sunlight in Fig. 4 b and 4c).These results illustrate that double-stranded RNA is a kind of metastable preparation on the one hand, play a role within a certain period of time, on the other hand, under illumination condition after can ensureing the stability that preparation is deposited and application, comparatively fast can be degraded, thus can not environmental pollution be caused.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (17)
1. double-stranded RNA (dsRNA) is imported the method for plant by one kind, it is characterized in that, comprise step: the root described double-stranded RNA being applied to plant, by the root absorption effect of described plant to double-stranded RNA, described double-stranded RNA is made to be sucked by described plant and be transported to the plant part except root.
2. one kind is improved the method for plant resistance to insect, it is characterized in that, comprise step: root insect-resistance double-stranded RNA (dsRNA) being applied to plant, described insect-resistance double-stranded RNA is absorbed by described plant root, and the plant part be transported to except root, thus improve the insect-resistance of plant.
3. method as claimed in claim 1 or 2, it is characterized in that, described dsRNA is the dsRNA for insect growth genes involved.
4. method as claimed in claim 3, it is characterized in that, described dsRNA is as shown in SEQ ID NOs.:4-6.
5. method as claimed in claim 1 or 2, is characterized in that, described method of application comprises irrigation, immersion, pouring or drip irrigation.
6. method as claimed in claim 2, is characterized in that, described pest-resistantly comprises anti-phytophagy insect.
7. method as claimed in claim 1 or 2, it is characterized in that, described plant comprises dicotyledons, monocotyledons or gymnosperm.
8. an agricultural composition, is characterized in that, described agricultural composition comprises acceptable carrier in the insect-resistance double-stranded RNA of insecticidal effective dose and Pesticide Science.
9. agricultural composition as claimed in claim 8, it is characterized in that, the application concentration of described dsRNA is 0.0001-5mg/g or 0.0001-5mg/ml, is preferably 0.001-1mg/ml or 0.001-1mg/g.
10. a purposes for insect-resistance double stranded RNA sequences, is characterized in that, for the preparation of being used by root thus improving the composition of plant resistance to insect.
11. 1 kinds of polynucleotide be separated, it is characterized in that, described polynucleotide are selected from lower group:
Sequence a () SEQ ID NO.:1-6 is arbitrary shown in;
The polynucleotide of b complementary that () and (a) limit; Or
C sequence that () and (a) limit has at least 70% ((preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) arbitrary polynucleotide of the sequence identity more than or complementary sequence, wherein, with the dsRNA of the chain of described arbitrary polynucleotide sequence complementation after phytophagy insect absorbs, there is the activity of the growth suppressing described phytophagy insect.
12. 1 kinds of dsRNA constructions, is characterized in that, the construction of described dsRNA is double-strand, and its normal chain or minus strand contain the structure shown in formula I:
Seq
forward-X-Seq
oppositely
Formula I
In formula,
Seq
forwardfor the nucleotide sequence of phytophagy insect growth related gene or fragment;
Seq
oppositelyfor with Seq
forwardsubstantially complementary nucleotide sequence;
X is for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelyit is not complementary,
Wherein, described phytophagy insect growth related gene is selected from: planthopper P450 gene (Cyp18A1), planthopper carboxylesterase gene (carboxylesterase, Ces) or Ostrinia furnacalis K type serpin gene (KPIs).
13. dsRNA constructions as claimed in claim 12, it is characterized in that, described dsRNA construction can form the dsRNA shown in formula II,
Formula II
In formula,
Seq'
forwardfor Seq
forwardthe RNA sequence that sequence pair is answered or sequence fragment;
Seq'
oppositelyfor with Seq'
forwardsubstantially complementary sequence;
X' is nothing; Or for being positioned at Seq'
forwardand Seq'
oppositelybetween intervening sequence, and described intervening sequence and Seq'
forwardand Seq'
oppositelyit is not complementary,
|| represent at Seq
forwardand Seq
oppositelybetween formed hydrogen bond.
14. 1 kinds of expression vectors, is characterized in that, described expression vector contains dsRNA construction according to claim 12.
15. 1 kinds of genetically engineered host cells, is characterized in that, containing the DNA sequence dna be integrated with in expression vector according to claim 14 or karyomit(e) corresponding to dsRNA construction according to claim 12 in described host cell.
16. 1 kinds of dsRNA sequences, is characterized in that, described dsRNA sequence is insect-resistance dsRNA, and its length is 100-800bp, are preferably 150-600bp.
The method of 17. 1 kinds of pest controls, is characterized in that, agricultural composition according to claim 8 or dsRNA according to claim 16 are applied to the plant needing pest control.
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| PCT/CN2014/090946 WO2015070765A1 (en) | 2013-11-12 | 2014-11-12 | Rnai-based pest control method using irrigation, and application thereof |
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| CN111955486A (en) * | 2020-08-27 | 2020-11-20 | 湖南省植物保护研究所 | Method for preventing and controlling plant pests, nucleic acid pesticide, and preparation method and application thereof |
| CN115124669A (en) * | 2022-05-27 | 2022-09-30 | 北京化工大学 | Preparation method and application of linear block copolymer nano-carrier with double-carrier genes and medicines |
| CN115124669B (en) * | 2022-05-27 | 2024-03-26 | 北京化工大学 | Preparation method and application of linear block copolymer nano-carrier for double-gene and medicine |
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