CN102776189A - Cytochrome P450 dsRNA (double-stranded ribonucleic acid) and application to aphid growth inhibition - Google Patents
Cytochrome P450 dsRNA (double-stranded ribonucleic acid) and application to aphid growth inhibition Download PDFInfo
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Abstract
The invention discloses dsRNA (double-stranded ribonucleic acid) and application to aphid growth inhibition. The dsRNA provided by the invention is dsRNA as expressed by 1) or 2) as follows: 1) dsRNA consisting of ribonucleic acid as shown by a sequence 4 in a sequence table and ribonucleic acid as shown by a reverse complementary sequence of the sequence 4; and 2) dsRNA consisting of ribonucleic acid as shown by a sequence 5 in the sequence table and ribonucleic acid as shown by a reverse complementary sequence of the sequence 5. According to experiments, the obtained dsRNA of a conserved sequence of cytochrome P450cDNA (complementary deoxyribonucleic acid) of English grain aphids and green peach aphids can inhibit growth and development of the English grain aphids and the green peach aphids and can cause a lethal effect by adopting a method of feeding the dsRNA in vitro and using an RNAi (ribonucleic acid interfere) technology to silence the in-vivo cytochrome p450 of the English grain aphids and the green peach aphids.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of cytochrome P450 gene dsRNA and the application in suppressing the aphid growth thereof.
Background technology
Wheat aphid (especially grain aphid) is one of primary pest of the Chinese Wheat Production of harm, and according to statistics, Chinese annual wheat aphid hazard area can account for 62% of the total cultivated area of wheat up to 0.17 hundred million hectare, causes the underproduction 15%-30%, can be up to 50% when serious.Black peach aphid is the euryphagy insect, and the host has kind more than 350 approximately, and vegetables such as fruit tree such as main harm tobacco, peach, Lee, plum, pears and Chinese cabbage, radish, capsicum, spinach have been caused very big financial loss.In recent years, because factors such as global warming, cropping system variation significantly strengthen prolificacy of aphid and flexibility, its harm is on the rise.At present, control of aphids is main to spray insecticide, but uses agricultural chemicals in a large number, and is not only harmful to people and animals, and caused the serious environmental pollution.Cultivate anti-aphid wheat and vegetable variety and be the effective way that prevents aphid damage, but owing to lack effective aphid-resistant gene in the existing germ plasm resource, resistance mechanism is still indeterminate, conventional breeding is difficult to prove effective.Excavate and utilize novel aphid-resistant gene and significant through genetically engineered cultivation wheat and the anti-aphid new germ plasm of vegetables.
The RNAi technology of plant mediation has become one of focus of farm crop anti insect gene engineering, expresses the dsRNA of corresponding insect specific gene through host plant, thereby reticent its corresponding gene reaches the purpose of Pest Control harm behind the insect's food-taking plant.Its mechanism of RNAi phenomenon is that double-stranded RNA (dsRNA) gets in the organism; Cut into the siRNA of 21-23nt by the Dicer enzyme, siRNA induces reticent mixture to combine with RNA, combines with the said target mrna of complementary sequence; By Dicer identification, cause the decline of expression of target gene amount.In recent years, utilize that dsRNA is external to feed or inject and screen the RNA target gene, cause target gene to be expressed and reticent, be widely used in evaluation and functional analysis that insect growth is grown key gene.The insect gut specific gene RNAi technology that Monsanto Company mediates through plant has successfully obtained the transgenic corns of anti-Zea mays root snout moth's larva, has effectively alleviated prolonged application Bt transgenic corns and has brought out problems such as insect generation resistance, has accomplished industrial experimentation at present.Special P450 gene can improve the tolerance of cotton bollworm larvae to cotton secondary metabolites and gossypol in the bollworm enteron aisle; Test shows; Transgene tobacco and the cotton leaf of utilize the expressing bollworm P450 gene dsRNA cotton bollworm larvae of feeding; Can cause P450 expression of gene amount decline in the larva body, larvae development is obstructed simultaneously, shows the bollworm resisting performance; Zha etc. have cloned 3 genes of highly expressing from the brown paddy plant hopper midgut: NlHT1, Nlcar and Nltry; Carrier construction; Rice transformation; After brown paddy plant hopper was got the food transgenic paddy rice, the mRNA expression level of 3 kinds of genes had descended 40% to 70% in the body, and has found the existence of dsRNA and siRNA at the phloem of paddy rice.Pitino etc. import MpC002 (mainly in sialisterium, expressing) and 2 genes of Rack-1 (mainly in enteron aisle, expressing) of black peach aphid respectively in tobacco and the Arabidopis thaliana; With the transgenic plant black peach aphid of feeding; Cause the expression amount of intravital MPC002 of black peach aphid or Rack-1 to reduce, the farrowing reduced number of aphid up to 60%.
The anti-aphid germ plasm resource of wheat and vegetables lacks, and resistance mechanism is still indeterminate, and conventional breeding is difficult to prove effective, and cause tremendous economic loss to agriculture prodn every year.Press for and excavate and identify novel aphid-resistant gene and improve wheat and the anti-aphid characteristic of vegetables through genetic engineering breeding.
Summary of the invention
An object of the present invention is to provide a kind of dsRNA.
DsRNA provided by the invention is following 1) or 2):
1) double-stranded RNA of forming by Nucleotide shown in the sequence in the sequence table 4 and the Nucleotide shown in its reverse complementary sequence;
2) double-stranded RNA of forming by Nucleotide shown in the sequence in the sequence table 5 and the Nucleotide shown in its reverse complementary sequence.
The encoding sox of above-mentioned dsRNA also is the scope that the present invention protects; Said encoding sox is specially following 1) or 2): 1) be the Nucleotide shown in the sequence 1 in the sequence table; 2) be the Nucleotide shown in the sequence 2 in the sequence table.
Above-mentioned dsRNA or above-mentioned encoding sox also are the scopes that the present invention protects in the anti-application that eliminates aphis and/or prepare in the anti-product that eliminates aphis.
In the above-mentioned application, said anti-eliminating aphis is embodied in the dead and/or inhibition aphid growth of promotion aphid.
Above-mentioned being applied as imports aphid with above-mentioned dsRNA, realizes promoting that aphid is dead and/or suppresses the aphid growth; Said importing is specially feeds;
Said aphid is specially grain aphid or black peach aphid;
Said promotion aphid is dead and/or the growth of inhibition aphid is concrete through suppressing the expression realization of aphid cells in vivo cytochrome p 450.
Above-mentioned dsRNA or above-mentioned encoding sox also are the scopes that the present invention protects in the application that promotes that aphid is dead and/or suppress in the aphid growth; Said aphid is specially grain aphid or black peach aphid.
Above-mentioned dsRNA or the above-mentioned application of encoding sox in the expression that suppresses aphid cells in vivo cytochrome p 450 also are the scopes that the present invention protects; Said aphid is specially grain aphid or black peach aphid.
The recombinant expression vector, transgenic cell line, reorganization bacterium or the expression cassette that contain above-mentioned dsRNA or above-mentioned encoding sox also are the scopes that the present invention protects.
Above-mentioned recombinant expression vector, transgenic cell line, reorganization bacterium or expression cassette are following 1)-5) in application at least a also be the scope that the present invention protects: 1) anti-eliminating aphis; 2) the anti-product that eliminates aphis of preparation; 3) promote aphid dead; 4) suppress the aphid growth; 5) expression of inhibition aphid cells in vivo cytochrome p 450;
Said aphid is specially grain aphid or black peach aphid.
Another object of the present invention provides a kind of anti-product that eliminates aphis or a kind of application that suppresses the material or the material that said inhibition aphid cells in vivo cytochrome p 450 is expressed of the expression of aphid cells in vivo cytochrome p 450.
Product provided by the invention, its activeconstituents are following A-C:
A, above-mentioned dsRNA;
B, above-mentioned encoding sox;
C, above-mentioned recombinant expression vector, transgenic cell line, reorganization bacterium or expression cassette.
A kind of material that suppresses the expression of aphid cells in vivo cytochrome p 450 provided by the invention is above-mentioned A-C;
The material that said inhibition aphid cells in vivo cytochrome p 450 provided by the invention is expressed is following 1)-5) in application at least a: 1) anti-eliminating aphis; 2) the anti-product that eliminates aphis of preparation; 3) promote aphid dead; 4) suppress the aphid growth; 5) expression of inhibition aphid cells in vivo cytochrome p 450;
Said aphid is specially grain aphid or black peach aphid.
Experiment of the present invention proves; The present invention obtains the dsRNA of conserved sequence of the Cytochrome P450 cDNA of grain aphid and black peach aphid; Adopt external feeding dsRNA method, carried out utilizing RNAi reticent grain aphid of technology and black peach aphid cells in vivo cytochrome p 450, cause grain aphid and black peach aphid to produce lethal effect; And along with dsRNA concentration increases and the feeding time prolongation, the mortality ratio of grain aphid and black peach aphid all increases gradually.Real-time fluorescence quantitative PCR research to P450 in the back survival aphid body of feeding shows that the expression of Cytochrome P450 obviously is suppressed.The conserved sequence that shows the aphid Cytochrome P450 can be applicable to through the RNAi technology raising wheat of plant mediation and the research of vegetables aphid resistance.
Description of drawings
Fig. 1 is the pcr amplification of Cytochrome P450 and GFP
Fig. 2 is the external synthetic dsRNA of Cytochrome P450 and GFP
Fig. 3 is the situation of growing of grain aphid and black peach aphid after feeding 8 days
Fig. 4 is fluorescent quantitative PCR result figure
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Grain aphid among the following embodiment (Qian Youting, Zhou Guanghe, Zhang Shuxiang, Zhang Xiangcai. the research of grain aphid sexual generation. plant protection; 1982,01,14-15.; The public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences) provide by Plant Protection institute, Chinese Academy of Agricultral Sciences, the wheat breed of raising aphid is section's farming 199, and aphid is inoculated on the wheat seedling; Put into growth cabinet (temperature (20 ± 2) ℃, humidity 60%-80%, photoperiod L: D=16: 8) breed.
Black peach aphid among the following embodiment (Jiang Yuwen. melon aphid and black peach aphid. new agricultural, 2001,11,40-41., the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences) pick up from the tobacco of Institute of Crop Science, Chinese Academy of Agricultural Science's chamber planting.
Plasmid extraction kit is available from Biomega company; Restriction endonuclease BamH I, EcoR V and Hiscribe T7 in-vitro transcription test kit are available from NEB company; Coli strain DH5 α, reverse transcription test kit are available from the full formula in Beijing King Company; The rTaq archaeal dna polymerase is available from TaKaRa company, and MinElute PCR Cleaning Kit, MinElute Gel Extraction Kit, RNACleaningKit are available from Qiagen company, and each seed amino acid and other reagent are all available from Beijing Baeyer enlightening company.
The acquisition of embodiment 1, Cytochrome P450 dsRNA
1, the extraction of the total RNA of grain aphid and cDNA's is synthetic
Get about 20 grain aphids and black peach aphid respectively, extract total RNA according to the Trizol test kit specification sheets of the full formula in Beijing King Company, carry out purifying with RNACleaningKit, cDNA first chain, the equal reference reagent box of operation steps specification sheets are synthesized in reverse transcription.
2, design of primers and gene clone
Sequence (the GenBank accession number is respectively NM_001163211.2 and JQ246416.1) according to acyrthosiphum pisim and cotten aphid Cytochrome P450; Compare with DNAman5.0; Select conserved sequence; Utilize Primer Primer5.0 software design primer P1 (table 1), synthetic by the big genome company of Beijing China.The GFP plasmid that green fluorescence protein gene (GFP) fragment improves centralab's preservation from national wheat increases, and utilizes Primer Primer5.0 software design primer P4 (table 1).
The PCR reaction system is 10 * PCR Buffer, 5 μ L, dNTP (2.5mmolL
-1) 4 μ L, rTaq 0.5 μ L, Forward primer (20 μ molL
-1) 1 μ L, Reverse primer (20 μ molL
-1) 1 μ L, cDNA/GFP plasmid 1 μ L, use ddH
2O complements to 50 μ L.The reaction conditions of PCR is 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 39 circulations; 72 ℃ of 10min; 4 ℃ of preservations.
CDNA with grain aphid is a template, increases as primer with P1, obtains 412bp PCR product (the T7 promoter sequence that contains 40bp), and through order-checking, this 412bp PCR product has the Nucleotide shown in the sequence 1 (but synthetic) in the sequence table; 122 amino acid of nucleotide coding shown in the sequence 1 in the sequence table.
CDNA with black peach aphid is a template, increases as primer with P1, obtains 422bp PCR product (the T7 promoter sequence that contains 40bp), and through order-checking, this 422bp PCR product has the Nucleotide shown in the sequence 2 (but synthetic) in the sequence table; 126 amino acid of nucleotide coding shown in the sequence 2 in the sequence table.
With the GFP plasmid is template, increases as primer with P4, obtains 324bp PCR product (the T7 promoter sequence that contains 40bp), and it has the Nucleotide shown in the sequence 3 (but synthetic) in the sequence table.
Above-mentioned PCR electrophoresis result is as shown in Figure 1, M:Trans2K DNA marker; 1: grain aphid Sitobion avenae; 2: black peach aphid Myzus persicae; 3:GFP can find out to obtain the purpose fragment.
Above-mentioned 412bp PCR product (grain aphid) amino acid sequence coded and 422bp PCR product (black peach aphid) amino acid sequence coded are submitted to ExPASy DB (http://www.uniprot.org/uniprot/) carry out the BLAST compare of analysis; ExPASy DB BLAST obtains 250 sequence informations; Be the partial amino-acid series of Cytochrome P450, and nucleotide homology is 90.1%; Therefore think that the amino acid of the nucleotide coding shown in the sequence 2 is the conserved sequence in the Cytochrome P450 in amino acid and the sequence table of the nucleotide coding shown in the sequence 1 in the sequence table.
Table 1 is the pcr amplification primer of Cytochrome P450 and GFP
The underscore place is the t7 rna polymerase promotor.
3, the preparation of the dsRNA of Cytochrome P450 and GFP
Reclaim three kinds of PCR products that obtain by above-mentioned 2 respectively, measure concentration, as the template of in-vitro transcription dsRNA.The in-vitro transcription system of dsRNA is 10 * Transcription Buffer, 4 μ L, 20 * Ribonucleotide Solution Mix, 2 μ L, template (1 μ g) X μ L, 20 * HMW Mix, 2 μ L, T7 RNA Polymerase (500units μ L
-1) 2 μ L, RNase-Free ddH
2O complements to 40 μ L.42 ℃ of night incubation.
After reaction finishes, get 0.5 μ L reaction product agarose gel electrophoresis and detect, add residual template DNA and the single stranded RNA of DNaseI and RNaseA digestion; With MinElute PCR Cleaning Kit purification reaction product; Operating process reference reagent box specification sheets, with no RNase water dissolution dsRNA, spectrophotometer (wavelength 260nm) is quantitative; Place-20 ℃ of refrigerators to preserve, obtain grain aphid P450 dsRNA, black peach aphid P450 dsRNA and GFPdsRNA respectively.The result is as shown in Figure 2, M:Trans2K DNA marker; 1: grain aphid in-vitro transcription product dsRNA; 2: black peach aphid in-vitro transcription product dsRNA; 3:GFP in-vitro transcription product dsRNA; Can see, obtain the purpose transcription product.
Grain aphid P450 dsRNA, black peach aphid P450 dsRNA and GFP dsRNA are checked order respectively, and the result is following:
Grain aphid P450 dsRNA is a double-stranded RNA, is made up of positive-sense strand and antisense strand, and the nucleotides sequence of its positive-sense strand is classified the sequence 4 in the sequence table as, and the nucleotides sequence of its antisense strand is classified the reverse complementary sequence of the sequence 4 in the sequence table as;
Black peach aphid P450 dsRNA is a double-stranded RNA, is made up of positive-sense strand and antisense strand, and the nucleotides sequence of its positive-sense strand is classified the sequence 5 in the sequence table as, and the nucleotides sequence of its antisense strand is classified the reverse complementary sequence of the sequence 5 in the sequence table as.
GFPdsRNA is a double-stranded RNA, is made up of positive-sense strand and antisense strand, and the nucleotides sequence of its positive-sense strand is classified the sequence 6 in the sequence table as, and the nucleotides sequence of its antisense strand is classified the reverse complementary sequence of the sequence 6 in the sequence table as.
Also can synthetic grain aphid P450 dsRNA, black peach aphid P450 dsRNA and GFPdsRNA.
1, the preparation of aphid artificial diet and raise the preparation of device
Set-up procedure reference (Li Caixia, Koryo cutting edge of a knife or a sword, the high tinkling of pieces of jades of device prepared and raised to artificial diet; Li Runzhi. hjolomorphism artificial nutrient liquid is raised the research of aphid. Agricultural University Of Shanxi's journal, 1997,17 (3): 225-228.Li C X; Gao L F, Gao L L, Li R Z.Study on the rearing of aphids on a artificially holidic diets.Journal of Shanxi Agricultural University; 1997,17 (3): 225-228. (in Chinese)) carry out, with the biofilter filtration artificial diet of 0.2 μ m; Divide the centrifuge tube install to the 2.0mL sterilization to be stored in-20 ℃ the refrigerator, avoid multigelation.
2, the application of dsRNA (P450dsRNA) in suppressing the aphid growth
The aphid feeding method is with reference to putting down in writing in the following document: entangle quick; Liu Shusheng. utilize artificial diet to raise the technology of aphid. East China insect journal; 2004,13 (2): 102-109.Jiu M, Liu S S.Aphid rearing with artificial diets.Entomological Journal of East China; 2004,13 (2): 102-109.
Grain aphid P450 dsRNA enteral feeding group (dsP450): each aphid raise put into respectively in the device 15 3 age grain aphid if aphid, according to adding 0,300,500 and the grain aphid P450 dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet successively respectively;
Grain aphid dsGFP group: each aphid raise put into respectively in the device 15 3 age grain aphid if aphid, according to adding 0,300,500 and the GFP dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet successively respectively;
Black peach aphid P450 dsRNA enteral feeding group (dsP450): each aphid raise put into respectively in the device 15 3 age black peach aphid if aphid, according to adding 0,300,500 and the black peach aphid P450 dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet successively respectively;
Black peach aphid dsGFP group: each aphid raise put into respectively in the device 15 3 age black peach aphid if aphid, according to adding 0,300,500 and the GFP dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet successively respectively;
Above-mentioned each group is provided with 3 repetitions, and the survival number that per two days statistics are raised aphid in the device, and feed that more renews and corresponding dsRNA place growth cabinet (temperature (20 ± 2) ℃, humidity 60%-80%, photoperiod L: D=16: 8).Use Excel2003 software that the aphid mortality ratio is carried out statistical analysis, calculate the MV and the variance of every kind of processing, and carry out significant difference analysis (t-test, n=3, P 0.01 or 0.05).
Statistics is respectively organized each number of raising survival aphid in the device, calculates mortality ratio, and the result is shown in table 2 and table 3:
Table 2 is the mortality ratio of grain aphid P450 dsRNA enteral feeding group and grain aphid dsGFP group
*Expression is compared with control group (0ng/ μ L), and the test group result difference is (t-test, n=3, P significantly<0.05),
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P extremely significantly<0.01).
Table 3 is the mortality ratio of black peach aphid P450 dsRNA enteral feeding group and black peach aphid dsGFP group
*Expression is compared with control group (0ng/ μ L), and the test group result difference is (t-test, n=3, P significantly<0.05),
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P extremely significantly<0.01).
Can find out that from table 2 and table 3 mortality ratio of grain aphid and black peach aphid all constantly raises along with time of the P450 dsRNA that feeds and dosage; 3ng μ L wherein
-1After P450 dsRNA fed 6 days and 8 days, the average mortality of grain aphid was 37.78% and 44.44%, and the average mortality of black peach aphid is 24.44% and 37.78%; 5ng μ L
-1After P450 dsRNA fed 2 days, 4 days, 6 days and 8 days, the average mortality of grain aphid was 22.22%, 35.56%, 48.89% and 62.22%, and the average mortality of black peach aphid is 15.55%, 26.67%, 44.44% and 53.33%; 7.5ng μ L
-1After P450dsRNA fed 2 days, 4 days, 6 days and 8 days; The average mortality of grain aphid is 31.11%, 48.89%, 62.22% and 75.56%; The average mortality of black peach aphid is 15.56%, 33.33%, 51.11% and 64.44%; These processing are compared with contrast and are all had significant difference, and the test group mortality ratio of the GFPdsRNA that feeds is compared the difference that does not then have significance with contrast.The Cytochrome P450 dsRNA of 2 kinds of aphids is more or less the same on the aphid lethality rate, and grain aphid P450dsRNA lethal effect is a little better.
Respectively organize the situation of growing of aphid after using microscopic examination to feed 8 days, the result shown in Fig. 3 and table 4, A: grain aphid; B: black peach aphid; CK: the aphid group (0ng/ μ l) that does not have feeding dsRNA; DsGFP: 7.5 ng μ L feed
-1The aphid group of concentration GFP dsRNA; DsP450: 7.5ng μ L feeds
-1The aphid group of concentration P450dsRNA; Can find out; The aphid of the feeding dsRNA and the GFPdsRNA that feeds does not grow normally, the aphid impaired development of the P450dsRNA test group of feeding, and build is little than control group CK; Can not grow and be adult; Do not observe other phenotype as yet and change, explain that the P450dsRNA that feeds can cause the effect of RNAi in the physical efficiency of aphid, causes aphid dead.
Table 4 is grown quantity for aphid
3, dsRNA (P450dsRNA) suppresses the Cytochrome P450 expression
Utilize Primer Primer5.0 software design Cytochrome P450 primer P3 (table 1), the amplified fragments size is 120bp, synthetic internal control gene primer P2 (table 1).
Collect and respectively organize the 7.5ng/ μ ldsRNAs grain aphid (2,4,6 and 8d) of back survival of feeding in above-mentioned 2, extract the RNA of aphid, reverse transcription becomes cDNA dilution 10
0, 10
1, 10
2, 10
3, 10
4, 10
5Doubly, as the template of quantitative fluorescent PCR, carry out relative quantification RT-PCR with P3 as primer and analyze.Confidential reference items (ACTIN) primer is P2.
The real-time fluorescence quantitative PCR system is Forward Primer (10 μ molL
-1) 0.5 μ L, Reverse Primer (10 μ molL
-1) 0.5 μ L, 2 * TransStart
TMGreen qPCR SuperMix 12.5 μ L, Passive Reference Dye 0.5 μ L, template cDNA 1 μ L use ddH
2O to 25 μ L.
The PCR cycling program is 95 ℃ of 30s, 95 ℃ of 5s, 57 ℃ of 15s, 72 ℃ of 10s, 40 circulations, 3 repetitions of each sample.The calculating of net result adopts 2-△ △ Ct method (Ct representes cycle number) to calculate; It is △ △ Ct=(Ct (P450)-Ct (actin)) test group-(Ct (P450)-Ct (actin)) control group; Use Excel2003 software to carry out statistical analysis, calculate the MV and the variance of every kind of processing, and carry out the analysis (t-test of significant difference; N=3, P 0.01 or 0.05).
The result of quantitative fluorescent PCR is as shown in Figure 4, and A is that canonical plotting, B are that amplification curve diagram, C are melt curve analysis figure; Find out that from Fig. 4 A the specificity of goal gene primer and confidential reference items primer is good, and amplification efficiency is consistent relatively; According to the amplification curve and the melt curve analysis (Fig. 4 B and Fig. 4 C) that produce, explain that the result who produces can be used for next step analysis.
Statistics the P450 dsRNA2,4 that feeds, 6 and 8d after, each organizes aphid cells in vivo cytochrome p 450 expression amount, the result is as shown in table 5:
Table 5 for feed P450 dsRNA and GFP dsRNA after grain aphid and black peach aphid Cytochrome P450 relative expression quantity
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P significantly<0.05),
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P extremely significantly<0.01).
Can find out; Grain aphid cells in vivo cytochrome p 450 expression amount has reduced by 41.14%, 31.46%, 47.87% and 63.71% successively; Black peach aphid cells in vivo cytochrome p 450 expression amount has reduced by 37.88%, 77.47%, 82.61% and 93.43% successively; Compare with contrast (0 day expression amount), on statistics, show as significant difference, it is obvious not as the black peach aphid Cytochrome P450 is suppressed effect that the grain aphid Cytochrome P450 is suppressed effect; Its reason possibly be that dsRNA in artificial diet or in the aphid body degraded has taken place, or the aphid body in the mRNA compensation mechanism play a role the institute cause.The intravital Cytochrome P450 expression level of aphid of GFPdsRNA of feeding does not then have the variation of significance; Further specify the RNAi effect that can in grain aphid and black peach aphid body, cause Cytochrome P450 through feeding dsRNA; Cause the expression amount of Cytochrome P450 obviously to reduce, dead until aphid.
Claims (10)
1. a dsRNA is following 1) or 2):
1) double-stranded RNA of forming by Nucleotide shown in the sequence in the sequence table 4 and the Nucleotide shown in its reverse complementary sequence;
2) double-stranded RNA of forming by Nucleotide shown in the sequence in the sequence table 5 and the Nucleotide shown in its reverse complementary sequence.
2. the encoding sox of the said dsRNA of claim 1; Said encoding sox is specially following 1) or 2): 1) be the Nucleotide shown in the sequence 1 in the sequence table; 2) be the Nucleotide shown in the sequence 2 in the sequence table.
3. said dsRNA of claim 1 or the described encoding sox of claim 2 eliminate aphis and/or prepare the application in the anti-product that eliminates aphis anti-.
4. application according to claim 3 is characterized in that: said anti-eliminating aphis is embodied in the dead and/or inhibition aphid growth of promotion aphid.
5. according to claim 3 or 4 described application, it is characterized in that: said being applied as imports aphid with the said dsRNA of claim 1, realizes promoting that aphid is dead and/or suppresses the aphid growth; Said importing is specially feeds;
Said aphid is specially grain aphid or black peach aphid;
Said promotion aphid is dead and/or the growth of inhibition aphid is concrete through suppressing the expression realization of aphid cells in vivo cytochrome p 450.
6. said dsRNA of claim 1 or the described encoding sox of claim 2 are promoting that aphid is dead and/or are suppressing the application in the aphid growth; Said aphid is specially grain aphid or black peach aphid.
7. said dsRNA of claim 1 or the described encoding sox of claim 2 application in the expression that suppresses aphid cells in vivo cytochrome p 450; Said aphid is specially grain aphid or black peach aphid.
8. the recombinant expression vector, transgenic cell line, reorganization bacterium or the expression cassette that contain said dsRNA of claim 1 or the described encoding sox of claim 2.
9. claim 8 recombinant expression vector, transgenic cell line, reorganization bacterium or expression cassette are following 1)-5) in application at least a: 1) anti-eliminating aphis; 2) the anti-product that eliminates aphis of preparation; 3) promote aphid dead; 4) suppress the aphid growth; 5) expression of inhibition aphid cells in vivo cytochrome p 450;
Said aphid is specially grain aphid or black peach aphid.
10. anti-product that eliminates aphis, its activeconstituents is following A-C:
A, the said dsRNA of claim 1;
B, the described encoding sox of claim 2;
C, claim 8 recombinant expression vector, transgenic cell line, reorganization bacterium or expression cassette.
Or a kind of material that suppresses the expression of aphid cells in vivo cytochrome p 450 is said A-C;
Or the material that said inhibition aphid cells in vivo cytochrome p 450 is expressed is following 1)-5) in application at least a: 1) anti-eliminating aphis; 2) the anti-product that eliminates aphis of preparation; 3) promote aphid dead; 4) suppress the aphid growth; 5) expression of inhibition aphid cells in vivo cytochrome p 450;
Said aphid is specially grain aphid or black peach aphid.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103088024A (en) * | 2013-01-09 | 2013-05-08 | 中国农业科学院作物科学研究所 | Application of two dsRNAs (double-stranded ribonucleic acids) and combination thereof in controlling aphid damage |
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