CN102776189B - A dsRNA of cytochrome P450 gene and its application in inhibiting the growth of aphids - Google Patents
A dsRNA of cytochrome P450 gene and its application in inhibiting the growth of aphids Download PDFInfo
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Abstract
本发明公开了一种dsRNA及其在抑制蚜虫生长中的应用。本发明提供的dsRNA，为如下1）或2）：1）由序列表中序列4所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA；2）由序列表中序列5所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。本发明的实验证明，本发明得到麦长管蚜和桃蚜的细胞色素P450cDNA的保守序列的dsRNA，采用体外饲喂dsRNA方法，进行了利用RNAi技术沉默麦长管蚜和桃蚜体内细胞色素P450，导致麦长管蚜和桃蚜生长发育受阻并产生致死效应。The invention discloses a dsRNA and its application in inhibiting the growth of aphids. The dsRNA provided by the present invention is the following 1) or 2): 1) a double-stranded RNA composed of the nucleotide shown in sequence 4 in the sequence table and the nucleotide shown in its reverse complementary sequence; 2) composed of the sequence A double-stranded RNA composed of nucleotides shown in sequence 5 in the list and nucleotides shown in its reverse complementary sequence. The experiment of the present invention proves that the present invention obtains the dsRNA of the conserved sequence of cytochrome P450 cDNA of Aphid aphid and Peach Aphid, adopts the method of in vitro feeding dsRNA, and uses RNAi technology to silence the cytochrome P450 in Aphid aphid and Peach Aphid. , leading to growth retardation and lethal effects of the wheat long tube aphid and green peach aphid.
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 harm Wheat in China production, 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 approximately has kind more than 350, and the vegetables such as the fruit tree such as main harm tobacco, peach, Lee, plum, pears and Chinese cabbage, radish, capsicum, spinach have been caused very large financial loss.In recent years, because the factors such as global warming, cropping system variation significantly strengthen the fecundity of aphid and adaptability, its harm is on the rise.At present, control of aphids to be spraying insecticide as main, but uses in a large number agricultural chemicals, and is not only harmful to people and animals, and caused serious environmental pollution.Cultivate anti-aphid wheat and vegetable variety and be the effective way that prevents aphid damage, but owing to lacking effective aphid-resistant gene in the existing germ plasm resource, resistance mechanism is still not clear, conventional breeding is difficult to prove effective.Excavate and utilize novel aphid-resistant gene and significant by 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 by host plant, thereby reticent its corresponding gene reaches the purpose that Control pests endangers behind the insect's food-taking plant.Its mechanism of RNAi phenomenon is that double-stranded RNA (dsRNA) enters in the organism, cut into the siRNA of 21-23nt by the Dicer enzyme, siRNA induces reticent mixture to be combined with RNA, is combined with the said target mrna of complementary sequence, identified by Dicer, cause the decline of expression of target gene amount.In recent years, utilize that dsRNA is external to feed or inject to 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.Monsanto Company has successfully obtained the transgenic corns of anti-Zea mays root snout moth's larva by the insect gut specific gene RNAi technology of plant mediation, effectively alleviated prolonged application Bt transgenic corns and brought out the problems such as insect generation resistance, has finished at present industrial experimentation.Special P450 gene can improve cotton bollworm larvae to the tolerance of cotton secondary metabolites and gossypol in the bollworm enteron aisle, test shows, utilize to express the transgene tobacco of bollworm P450 gene dsRNA and the cotton leaf cotton bollworm larvae of feeding, can cause the expression amount of P450 gene in the larva body to descend, larvae development is obstructed simultaneously, shows the bollworm resisting performance; Zha etc. have cloned 3 genes of highly expressing in the intestines from brown paddy plant hopper: NlHT1, Nlcar and Nltry, carrier construction, rice transformation, after brown paddy plant hopper takes food transgenic paddy rice, the mrna expression level of 3 kinds of genes has descended 40% to 70% in the body, and has found the existence of dsRNA and siRNA at the phloem of paddy rice.Pitino etc. mainly express the MpC002(of black peach aphid in sialisterium) and Rack-1(mainly in enteron aisle, express) 2 genes import respectively in tobacco and the Arabidopis thaliana, with the transgenic plant black peach aphid of feeding, cause the interior MPC002 of black peach aphid body or the expression amount of Rack-1 to reduce up to 60% the farrowing reduced number of aphid.
The anti-aphid germ plasm resource of wheat and vegetables lacks, and resistance mechanism is still not clear, and conventional breeding is difficult to prove effective, and cause tremendous economic loss to agriculture production every year.In the urgent need to excavating and identify novel aphid-resistant gene and improving wheat and the anti-aphid characteristic of vegetables by 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 that is formed by the Nucleotide shown in the Nucleotide shown in the sequence in the sequence table 4 and its reverse complementary sequence;
2) double-stranded RNA that is formed by the Nucleotide shown in the Nucleotide shown in the sequence in the sequence table 5 and its reverse complementary sequence.
The encoding gene of above-mentioned dsRNA also is the scope of protection of the invention; Described encoding gene 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 gene also are the scope of protection of the invention in the anti-application that eliminates aphis and/or prepare in the anti-product that eliminates aphis.
In the above-mentioned application, described anti-eliminating aphis is embodied in the death of promotion aphid and/or suppresses the aphid growth.
Above-mentioned being applied as imports aphid with above-mentioned dsRNA, realizes promoting that aphid is dead and/or suppresses the aphid growth; Described importing is specially feeds;
Described aphid is specially grain aphid or black peach aphid;
Described promotion aphid is dead and/or the growth of inhibition aphid is concrete by suppressing the expression realization of aphid cells in vivo cytochrome p 450.
Above-mentioned dsRNA or above-mentioned encoding gene also are the scope of protection of the invention in the application that promotes that aphid is dead and/or suppress in the aphid growth; Described aphid is specially grain aphid or black peach aphid.
Above-mentioned dsRNA or the above-mentioned application of encoding gene in the expression that suppresses aphid cells in vivo cytochrome p 450 also are the scope of protection of the invention; Described aphid is specially grain aphid or black peach aphid.
The recombinant expression vector, transgenic cell line, recombinant bacterium or the expression cassette that contain above-mentioned dsRNA or above-mentioned encoding gene also are the scope of protection of the invention.
Above-mentioned recombinant expression vector, transgenic cell line, recombinant bacterium or expression cassette are following 1)-5) in application at least a also be the scope of protection of the invention: 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;
Described 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 material or the material that described 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 gene;
C, above-mentioned recombinant expression vector, transgenic cell line, recombinant 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 described 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;
Described aphid is specially grain aphid or black peach aphid.
Of the present invention experimental results show that, 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, the reticent grain aphid of RNAi technology and black peach aphid cells in vivo cytochrome p 450 have been carried out utilizing, 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 rear survival aphid body of feeding shows, the expression of Cytochrome P450 obviously is suppressed.The conserved sequence that shows the aphid Cytochrome P450 can be applicable to by 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 if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels 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 Institute of Crop Science, Chinese Academy of Agricultural Science) provided 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, puts 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 Institute of Crop Science, Chinese Academy of Agricultural Science) 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, 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 respectively approximately 20 grain aphids and black peach aphid, 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 the 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 large 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.
Take the cDNA of grain aphid as template, increase as primer with P1, obtain 412bp PCR product (the T7 promoter sequence that contains 40bp), through order-checking, but this 412bp PCR product has the sequence 1(synthetic in the sequence table) shown in Nucleotide; 122 amino acid of nucleotide coding shown in the sequence 1 in the sequence table.
Take the cDNA of black peach aphid as template, increase as primer with P1, obtain 422bp PCR product (the T7 promoter sequence that contains 40bp), through order-checking, but this 422bp PCR product has the sequence 2(synthetic in the sequence table) shown in Nucleotide; 126 amino acid of nucleotide coding shown in the sequence 2 in the sequence table.
Take the GFP plasmid as template, increase as primer with P4, obtain 324bp PCR product (the T7 promoter sequence that contains 40bp), but it has the sequence 3(synthetic in the sequence table) shown in Nucleotide.
With above-mentioned PCR electrophoresis result 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.
The aminoacid sequence of the aminoacid sequence of above-mentioned 412bp PCR product (grain aphid) coding and 422bp PCR product (black peach aphid) coding is submitted to ExPASy database (http://www.uniprot.org/uniprot/) carries out the BLAST compare of analysis, ExPASy database 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 in the amino acid of the nucleotide coding shown in the sequence 1 in the sequence table and the sequence table is the conserved sequence in the Cytochrome P450.
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 respectively three kinds of PCR products that obtain by above-mentioned 2, 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, getting 0.5 μ L reaction product agarose gel electrophoresis detects, 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 is used without RNase water dissolution dsRNA, and spectrophotometer (wavelength 260nm) is quantitative, place-20 ℃ of refrigerators to preserve, obtain respectively grain aphid P450 dsRNA, black peach aphid P450 dsRNA and GFPdsRNA.The result 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 as follows:
Grain aphid P450 dsRNA is double-stranded RNA, is comprised 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 double-stranded RNA, is comprised 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 double-stranded RNA, is comprised 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 (the Li Caixia of device is prepared and raised to artificial diet, the Koryo cutting edge of a knife or a sword, high tinkling of pieces of jades, 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, filter artificial diet with the biofilter of 0.2 μ m, the centrifuge tube that minute installs to the 2.0mL sterilization is stored in-20 ℃ the refrigerator, avoids multigelation.
2, the dsRNA(P450dsRNA) application in suppressing the aphid growth
The reference of aphid feeding method is as putting down in writing in the Publication about 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 nursing group (dsP450): if each aphid raise put into respectively in the device 15 3 age the grain aphid aphid, according to adding respectively successively 0,300,500 and the grain aphid P450 dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet;
Grain aphid dsGFP group: if each aphid raise put into respectively in the device 15 3 age the grain aphid aphid, according to adding respectively successively 0,300,500 and the GFP dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet;
Black peach aphid P450 dsRNA nursing group (dsP450): if each aphid raise put into respectively in the device 15 3 age the black peach aphid aphid, according to adding respectively successively 0,300,500 and the black peach aphid P450 dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet;
Black peach aphid dsGFP group: if each aphid raise put into respectively in the device 15 3 age the black peach aphid aphid, according to adding respectively successively 0,300,500 and the GFP dsRNA of the 750ng aphid of feeding in per 100 μ L artificial diet;
Above-mentioned each group arranges 3 repetitions, and the survival number that per two days statistics are raised aphid in the device, and the 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 mean value and the variance of every kind of processing, and carry out the analysis (t-test, n=3, P＜0.01 or 0.05) of significant difference.
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 nursing 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＜0.05) significantly,
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P＜0.01) extremely significantly.
Table 3 is the mortality ratio of black peach aphid P450 dsRNA nursing 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＜0.05) significantly,
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P＜0.01) extremely significantly.
Can find out from table 2 and table 3, the 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%, all there is significant difference compared with the control in these processing, and the test group mortality ratio of the GFPdsRNA that feeds does not then have the difference of significance compared with the control.The Cytochrome P450 dsRNA of 2 kinds of aphids is more or less the same at the aphid lethality rate, and grain aphid P450dsRNA lethal effect is a little better.
Use microscopic examination to feed and respectively organized afterwards the situation of growing of aphid in 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: 7.5ng μ L feeds
-1The aphid group of concentration GFP dsRNA; DsP450: 7.5ng μ L feeds
-1The aphid group of concentration P450 dsRNA; Can find out, the aphid of feeding dsRNA and the GFPdsRNA that feeds does not grow normal, the aphid impaired development of the P450dsRNA test group of feeding, build is little than control group CK, can not grow and be adult, not yet observe other phenotype and change, illustrate that the P450dsRNA that feeds can cause in the physical efficiency of aphid the effect of RNAi, causes aphid dead.
Table 4 is grown quantity for aphid
3, dsRNA(P450dsRNA) suppressing Cytochrome P450 expresses
Utilize Primer Primer5.0 software design Cytochrome P450 primer P3(table 1), the amplified fragments size is 120bp, synthetic reference gene primer P2(table 1).
Collect and respectively organize the 7.5ng/ μ l dsRNAs grain aphid (2,4,6 and 8d) of rear 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 Dye0.5 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 represents cycle number) to calculate, be △ △ Ct=(Ct (P450)-Ct (actin)) test group-(Ct (P450)-Ct (actin)) control group, use Excel2003 software to carry out statistical analysis, calculate mean value 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 as shown in Figure 4, A is that canonical plotting, B are that amplification curve diagram, C are melt curve analysis figure; Find out from Fig. 4 A, the specificity of goal gene primer and confidential reference items primer is good, and amplification efficiency is relatively consistent; According to the amplification curve and the melt curve analysis (Fig. 4 B and Fig. 4 C) that produce, illustrate that the result who produces can be used for next step analysis.
Statistics the P450dsRNA2 that feeds, 4,6 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 P450dsRNA and GFPdsRNA 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＜0.05) significantly,
*Expression is compared with control group, and the test group result difference is (t-test, n=3, P＜0.01) extremely significantly.
Can find out, grain aphid cells in vivo cytochrome p 450 expression amount has reduced by 41.14% successively, 31.46%, 47.87% and 63.71%, black peach aphid cells in vivo cytochrome p 450 expression amount has reduced by 37.88% successively, 77.47%, 82.61% and 93.43%, compare with contrast (0 day expression amount), show as statistically significant difference, the suppressed effect of grain aphid Cytochrome P450 is not as the suppressed successful of black peach aphid Cytochrome P450, its reason may be that dsRNA in artificial diet or in the aphid body degraded has occured, or the aphid body in the mRNA compensation mechanism play a role the institute cause.The Cytochrome P450 expression level in the aphid body 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 by feeding dsRNA, cause the expression amount of Cytochrome P450 obviously to reduce, until aphid is dead.
Claims (16)
1. a dsRNA is following 1) or 2):
1) double-stranded RNA that is formed by the Nucleotide shown in the Nucleotide shown in the sequence in the sequence table 4 and its reverse complementary sequence;
2) double-stranded RNA that is formed by the Nucleotide shown in the Nucleotide shown in the sequence in the sequence table 5 and its reverse complementary sequence.
2. the encoding gene of the described dsRNA of claim 1; Described encoding gene 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. the described dsRNA of claim 1 or encoding gene claimed in claim 2 eliminate aphis and/or prepare application in the anti-product that eliminates aphis anti-; Described aphid is grain aphid or black peach aphid.
4. application according to claim 3 is characterized in that: described anti-eliminating aphis is embodied in the death of promotion aphid and/or suppresses the aphid growth.
5. according to claim 3 or 4 described application, it is characterized in that: described being applied as imports aphid with the described dsRNA of claim 1, realizes promoting that aphid is dead and/or suppresses the aphid growth; Described importing is specially feeds;
Described promotion aphid is dead and/or the growth of inhibition aphid is concrete by suppressing the expression realization of aphid cells in vivo cytochrome p 450.
6. the described dsRNA of claim 1 or encoding gene claimed in claim 2 application in promoting aphid death and/or the growth of inhibition aphid; Described aphid is specially grain aphid or black peach aphid.
7. the described dsRNA of claim 1 or encoding gene claimed in claim 2 application in the expression that suppresses aphid cells in vivo cytochrome p 450; Described aphid is specially grain aphid or black peach aphid.
8. the recombinant expression vector, transgenic cell line, recombinant bacterium or the expression cassette that contain the described dsRNA of claim 1 or encoding gene claimed in claim 2.
9. the described recombinant expression vector of claim 8, transgenic cell line, recombinant bacterium or expression cassette eliminate aphis and/or prepare application in the anti-product that eliminates aphis anti-; Described aphid is grain aphid or black peach aphid.
10. the described recombinant expression vector of claim 8, transgenic cell line, recombinant bacterium or the expression cassette application in promoting aphid death and/or the growth of inhibition aphid; Described aphid is grain aphid or black peach aphid.
11. the described recombinant expression vector of claim 8, transgenic cell line, recombinant bacterium or expression cassette must be used in the expression that suppresses aphid cells in vivo cytochrome p 450; Described aphid is grain aphid or black peach aphid.
12. an anti-product that eliminates aphis, its activeconstituents is following A-C:
A, the described dsRNA of claim 1;
B, encoding gene claimed in claim 2;
C, claim 8 recombinant expression vector, transgenic cell line, recombinant bacterium or expression cassette;
Described aphid is grain aphid or black peach aphid.
13. one kind is suppressed the material that aphid cells in vivo cytochrome p 450 is expressed, is A-C described in the claim 12; Described aphid is grain aphid or black peach aphid.
14. the material that the described inhibition of claim 13 aphid cells in vivo cytochrome p 450 is expressed eliminates aphis and/or prepares application in the anti-product that eliminates aphis anti-; Described aphid is grain aphid or black peach aphid.
15. the application of material in promoting aphid death and/or the growth of inhibition aphid that the described inhibition of claim 13 aphid cells in vivo cytochrome p 450 is expressed; Described aphid is grain aphid or black peach aphid.
16. the application of material in the expression that suppresses aphid cells in vivo cytochrome p 450 that the described inhibition of claim 13 aphid cells in vivo cytochrome p 450 is expressed; Described aphid is grain aphid or black peach aphid.
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| CN106636148A (en) * | 2017-03-01 | 2017-05-10 | 山西大学 | Locusta migratoria cytochrome P450 reductase gene dsRNA and application thereof |
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| CN103088024B (en) * | 2013-01-09 | 2014-07-09 | 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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| CN114478735B (en) * | 2022-03-18 | 2023-09-29 | 中国农业大学 | High-mortality dsRNA of aphids and application of high-mortality dsRNA in aphid control |
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| BRPI0706266A2 (en) * | 2006-01-12 | 2009-02-25 | Devgen Nv | Pest control methods using rnai |
| CN101063134A (en) * | 2007-04-26 | 2007-10-31 | 浙江大学 | Rice cytochrome P450 gene and its application |
| CN102154271A (en) * | 2011-01-26 | 2011-08-17 | 中国农业科学院作物科学研究所 | Method for breeding aphid-resistant transgenic wheat and special vector |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636148A (en) * | 2017-03-01 | 2017-05-10 | 山西大学 | Locusta migratoria cytochrome P450 reductase gene dsRNA and application thereof |
| CN106636148B (en) * | 2017-03-01 | 2019-07-09 | 山西大学 | A kind of migratory locusts cytochrome P450 reductase gene dsRNA and its application |
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