CN103571844B - DsRNA and the application be combined in control aphid damage thereof - Google Patents
DsRNA and the application be combined in control aphid damage thereof Download PDFInfo
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Abstract
The invention discloses dsRNA and be combined in the application controlled in aphid damage.DsRNA provided by the invention is following 1), 2) or 3): the double-stranded RNA 1) be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 4 He its reverse complementary sequence; 2) double-stranded RNA be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 5 He its reverse complementary sequence; 3) be made up of following two double-stranded RNAs: the double-stranded RNA 1) and 2) shown in double-stranded RNA.Experiment of the present invention proves, the present invention obtains grain aphid and to grow the dsRNA of genes involved 8273 and 22544cDNA, adopt external feeding dsRNA method, carry out utilizing RNAi technology reticent grain aphid tumor growth development related gene 8273 and 22544, caused grain aphid to grow and be obstructed and produce lethal effect.
Description
this application be to the applying date be on January 9th, 2013, application number is 201310007173.1, denomination of invention is " dsRNA
and be combined in the application controlled in aphid damage " the divisional application of patent application
Technical field
The present invention relates to biological technical field, particularly relate to dsRNA and be combined in the application controlled in aphid damage.
Background technology
Wheat aphid (especially grain aphid) is one of primary pest of harm Wheat in China production, and according to statistics, the annual wheat aphid hazard area of China up to 0.17 hundred million hectare, can account for 62% of the total cultivated area of wheat, cause the underproduction 15%-30%, can up to 50% time serious.In recent years, due to factors such as global warming, cropping system changes, the fecundity of aphid and adaptability are significantly strengthened, and its harm is on the rise.At present, control of aphids to spray insecticide, but uses agricultural chemicals in a large number, is not only harmful to people and animals, and causes serious environmental pollution.Cultivating aphids resistance wheat and vegetable variety is the most effective way preventing aphid damage, but owing to lacking effective aphid-resistant gene in existing germ plasm resource, resistance mechanism is still not clear, and conventional breeding is difficult to prove effective.Excavate and utilize novel aphid-resistant gene and to cultivate wheat anti-aphid new germ plasm by genetically engineered significant.
The RNAi technology of mediated plant has become one of focus of farm crop Insect resistant gene engineer, is expressed the dsRNA of corresponding insect specific gene by host plant, its corresponding gene reticent after insect's food-taking plant thus reach the object of Control pests harm.Its mechanism of RNAi phenomenon is that double-stranded RNA (dsRNA) enters in organism, cut into the siRNA of 21-23nt by Dicer enzyme, siRNA and RNA induces silencing complex to combine, and 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 RNA target gene, cause target gene to be expressed and reticent, be widely used in qualification and functional analysis that insect growth grows key gene.Monsanto Company have successfully been obtained the transgenic corns of anti-Zea mays root snout moth's larva by the insect gut specific gene RNAi technology of mediated plant, effectively alleviates prolonged application Bt transgenic corns and brings out the problems such as insect generation resistance, complete industrial experimentation at present.In bollworm enteron aisle, special P450 gene can improve the tolerance of cotton bollworm larvae to cotton secondary metabolites and gossypol, test shows, the transgene tobacco of expressing bollworm P450 gene dsRNA and cotton leaf is utilized to feed cotton bollworm larvae, the expression amount of P450 gene in larva body can be caused to decline, larvae development is obstructed simultaneously, shows bollworm resisting performance; Zha etc. have cloned 3 genes of highly expressing from brown paddy plant hopper in intestines: NlHT1, Nlcar and Nltry, carrier construction, rice transformation, after brown paddy plant hopper takes food transgenic paddy rice, in body, the mrna expression level of 3 kinds of genes have dropped 40% to 70%, and has found the existence of dsRNA and siRNA at the phloem of paddy rice.The MpC002(of black peach aphid mainly expresses by Pitino etc. in sialisterium) and Rack-1(mainly express in enteron aisle) 2 genes import in tobacco and Arabidopis thaliana respectively, to feed black peach aphid with transgenic plant, the expression amount of MPC002 or Rack-1 in black peach aphid body is caused to reduce up to 60%, the farrowing reduced number of aphid.
Wheat aphids resistance germ plasm resource lacks, and resistance mechanism is still not clear, and conventional breeding is difficult to prove effective, and causes tremendous economic to lose every year to agriculture production.In the urgent need to excavating and identifying novel aphid-resistant gene and improve the anti-aphid characteristic of wheat by genetic engineering breeding.
Summary of the invention
An object of the present invention is to provide dsRNA.
DsRNA provided by the invention is following 1), 2) or 3):
1) double-stranded RNA be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 4 He its reverse complementary sequence;
2) double-stranded RNA be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 5 He its reverse complementary sequence;
3) be made up of following two double-stranded RNAs: the double-stranded RNA 1) and 2) shown in double-stranded RNA.
Above-mentioned 3) double-stranded RNA and 2 in the dsRNA shown in, 1)) shown in the mass ratio of double-stranded RNA be 1:1.
Above-mentioned 1), 2) or 3) shown in the encoding gene of described dsRNA be also the scope of protection of the invention, specific as follows:
1) encoding gene of the dsRNA shown in is specially the Nucleotide shown in sequence 1 in sequence table;
2) encoding gene of the dsRNA shown in is specially the Nucleotide shown in sequence 2 in sequence table;
3) encoding gene of the dsRNA shown in is specially the composition be made up of the DNA molecular shown in the Nucleotide shown in sequence 2 in the DNA molecular shown in the Nucleotide shown in sequence in sequence table 1 and sequence table.
Any one application following of above-mentioned dsRNA or above-mentioned encoding gene is also the scope of protection of the invention:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
Above-mentioned dsRNA is imported aphid by above-mentioned being applied as, and realizes the anti-expression eliminating aphis, promote that aphid is dead, suppress aphid growth, suppress aphid tumor growth development related gene 8273 and/or 22544; Described importing is specially feeds;
Described aphid is specially grain aphid.
The nucleotides sequence of said gene 8273 is classified as the sequence 1 in sequence table, and the nucleotides sequence of gene 22544 is classified as the sequence 2 in sequence table.
The recombinant expression vector of the encoding gene containing above-mentioned dsRNA, transgenic cell line, recombinant bacterium or expression cassette are also the scope of protection of the invention.
Any one application following of above-mentioned recombinant expression vector, transgenic cell line, recombinant bacterium or expression cassette is also the scope of protection of the invention:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
Described aphid is specially grain aphid.
Another object of the present invention is to provide product, and its activeconstituents is at least one in 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;
Described product is following 1)-4) in any one: 1) the anti-product that eliminates aphis; 2) the dead product of aphid is promoted; 3) aphid growth product is suppressed; 4) the expression product of aphid tumor growth development related gene 8273 and/or 22544 is suppressed;
Described aphid is specially grain aphid.
Any one application following of the material suppressing aphid tumor growth development related gene 8273 and/or 22544 to be expressed also is the scope of protection of the invention:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) the described aphid of application in aphid growth product is suppressed to be specially grain aphid in the application suppressed in aphid growth or in preparation.
The material suppressing aphid tumor growth development related gene 8273 and/or 22544 to be expressed in the present invention be in upper A-C any one.
Experiment of the present invention proves, the present invention obtains grain aphid and to grow the dsRNA of sequence of genes involved 8273 and 22544cDNA, adopt external feeding dsRNA method, it is no matter the mixture of a kind of dsRNA or feeding dsRNA of feeding separately, grain aphid is all caused to produce lethal effect, and along with dsRNA concentration increases and feeding time prolongation, the mortality ratio of grain aphid increases all gradually, and research shows to utilize RNAi technology reticent grain aphid tumor growth development related gene 8273 and 22544 to express.Show that the grow conserved sequence of genes involved 8273 and 22544 of aphid can be applicable to be improved by the RNAi technology of mediated plant the research of wheat resistance to aphids.
Accompanying drawing explanation
Fig. 1 is the pcr amplification of gene 8273,22544 and GFP
Fig. 2 is the situation of growing of grain aphid after feeding 8 days
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Grain aphid in following embodiment (Qian Youting, Zhou Guanghe, Zhang Shuxiang, Zhang Xiangcai. the research of grain aphid sexual generation. plant protection, 1982,1:14-15.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 agriculture 199; aphid is inoculated on wheat seedling; put into growth cabinet (temperature (20 ± 2) DEG C; humidity 60%-80%, photoperiod L: D=16: 8) breed.
Plasmid extraction kit is purchased from Biomega company, restriction endonuclease BamH I, EcoR V and Hiscribe T7 in-vitro transcription test kit are purchased from NEB company, coli strain DH5 α, Reverse Transcription box are purchased from Beijing Quan Shi King Company, rTaq archaeal dna polymerase is purchased from TaKaRa company, MinElute PCR Cleaning Kit, MinElute Gel Extraction Kit, RNA Cleaning Kit are purchased from Qiagen company, and each seed amino acid and other reagent are all purchased from Beijing Baeyer enlightening company.
The acquisition of embodiment 1, genes involved 8273dsRNA and 22544dsRNA that grow
1, the extraction of grain aphid total serum IgE and the synthesis of cDNA
Get about 20 grain aphids respectively, extract total serum IgE according to the Trizol test kit specification sheets of Beijing Quan Shi King Company, carry out purifying with RNA Cleaning Kit, reverse transcription synthesis cDNA first chain, operation steps equal reference reagent box specification sheets.
2, design of primers and gene clone
According to this laboratory, genes involved 8273(sequence 1 of growing is obtained for the result that grain aphid transcript profile checks order) and 22544(sequence 2), utilize Primer Primer5.0 software design primer P1 and P2(table 1), synthesized by Beijing Hua Da genome company.The GFP plasmid that green fluorescence protein gene (GFP) fragment is preserved from national wheat flour quality centralab increases, and utilizes Primer Primer5.0 software design primer P3(table 1).
PCR reaction system is 10 × PCR Buffer5 μ L, dNTP(2.5mmolL
-1) 4 μ L, rTaq0.5 μ L, Forward primer(20 μm of olL
-1) 1 μ L, Reverse primer(20 μm olL
-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 DEG C of 4min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 39 circulations; 72 DEG C of 10min; 4 DEG C of preservations.
With the cDNA of grain aphid for template, increase as primer with P1, obtain 279bp PCR primer and be 8273 gene order upper part fragments (the T7 promoter sequence containing 40bp), through order-checking, this 279bp PCR primer has sequence 1(in sequence table can synthetic) shown in Nucleotide.
With the cDNA of grain aphid for template, increase as primer with P2, obtain 263bp PCR primer 22544 gene order upper part fragment (the T7 promoter sequence containing 40bp), through order-checking, this 263bp PCR primer has sequence 2(in sequence table can synthetic) shown in Nucleotide.
With GFP plasmid for template, increase with P3 as primer, obtain 324bp PCR primer (the T7 promoter sequence containing 40bp), it has sequence 3(in sequence table can synthetic) shown in Nucleotide.
By above-mentioned PCR electrophoresis result as shown in Figure 1, M:Trans2K DNA marker; 1:8273 gene; 2:22544 gene; 3:GFP, can find out and obtain object fragment.
Table 1 is pcr amplification primer
Underscore place is t7 rna polymerase promotor.
3, the preparation of the dsRNA of 8273 genes, 22544 genes and GFP
Reclaim the three kinds of PCR primer obtained by above-mentioned 2 respectively, measure concentration, as the template of in-vitro transcription dsRNA.The In vitro transcripts of dsRNA is 10 × Transcription Buffer4 μ L, 20 × Ribonucleotide Solution Mix2 μ L, template (1 μ g) X μ L, 20 × HMW Mix2 μ L, T7RNA Polymerase(500units μ L
-1) 2 μ L, RNase-Free ddH
2o complements to 40 μ L.42 DEG C of night incubation.
After reaction terminates, get 0.5 μ L reaction product agarose gel electrophoresis to detect, add the residual template DNA of DNaseI and RNaseA digestion and single stranded RNA, with MinElute PCR Cleaning Kit purification reaction product, operating process reference reagent box specification sheets, with without RNase water dissolution dsRNA, spectrophotometer (wavelength 260nm) is quantitative, be placed in-20 DEG C of refrigerators to preserve, obtain grain aphid 8273dsRNA, 22544dsRNA and GFPdsRNA respectively.
Checked order respectively by grain aphid 8273dsRNA, 22544dsRNA and GFP dsRNA, result is as follows:
Grain aphid 8273dsRNA is double-stranded RNA, be made up of positive-sense strand and antisense strand, the nucleotides sequence of its positive-sense strand is classified as the sequence 4 in sequence table, the nucleotides sequence of its antisense strand is classified as the reverse complementary sequence of the sequence 4 in sequence table, and the nucleotides sequence of 8273dsRNA encoding gene is classified as sequence 1 in sequence table;
Grain aphid 22544dsRNA is double-stranded RNA, be made up of positive-sense strand and antisense strand, the nucleotides sequence of its positive-sense strand is classified as the sequence 5 in sequence table, the nucleotides sequence of its antisense strand is classified as the reverse complementary sequence of the sequence 5 in sequence table, and the nucleotides sequence of 22544dsRNA encoding gene is classified as sequence 2 in sequence table.
GFPdsRNA is double-stranded RNA, be made up of positive-sense strand and antisense strand, the nucleotides sequence of its positive-sense strand is classified as the sequence 6 in sequence table, and the nucleotides sequence of its antisense strand is classified as the reverse complementary sequence of the sequence 6 in sequence table, and the nucleotides sequence of GFPdsRNA encoding gene is classified as sequence 3 in sequence table.
Also can synthetic grain aphid 8273dsRNA, 22544dsRNA and GFPdsRNA.
Embodiment 2, dsRNA are suppressing the application in aphid growth
1, aphid artificial diet preparation and raise the preparation of device
Artificial diet are prepared and are raised the set-up procedure reference (Li Caixia of device, Koryo cutting edge of a knife or a sword, high tinkling of pieces of jades, Li Runzhi. hjolomorphism artificial nutrient liquid raises the research of aphid. Agricultural University Of Shanxi's journal, 1997, 17 (3): 225-228.Li C X, GaoL F, Gao L L, Li R Z.Study on the rearing of aphids on a artificially holidic diets.Journal ofShanxi Agricultural University, 1997, 17 (3): 225-228. (in Chinese)) carry out, with the bacterial filter artificial diet of 0.2 μm, the centrifuge tube being dispensed into 2.0mL sterilizing is stored in the refrigerator of-20 DEG C, avoid multigelation.
2, dsRNA(22544dsRNA, 8273dsRNA) suppressing the application in aphid growth
The reference of aphid feeding method is as recorded in 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 22544dsRNA nursing group (ds22544): if each aphid raise in device put into respectively 15 3 age grain aphid aphid, to feed aphid according to the grain aphid 22544dsRNA adding 0 and 750ng in every 100 μ L artificial diet successively respectively;
Grain aphid 8273dsRNA nursing group (ds8273): if each aphid raise in device put into respectively 15 3 age grain aphid aphid, to feed aphid according to the grain aphid 8273dsRNA adding 0 and 750ng in every 100 μ L artificial diet successively respectively;
Grain aphid dsGFP group: if each aphid raise in device put into respectively 15 3 age grain aphid aphid, to feed aphid according to the GFPdsRNA adding 0 and 750ng in every 100 μ L artificial diet successively respectively;
Grain aphid 22544dsRNA+8273dsRNA nursing group: if each aphid raise in device put into respectively 15 3 age grain aphid aphid, to feed aphid according to the feed grain aphid 22544dsRNA of aphid and 750ng of the grain aphid 8273dsRNA adding 750ng in every 100 μ L artificial diet successively; Not add any dsRNA for contrast (CK).
Above-mentioned each group arranges 3 repetitions, and within every two days, statistics raises the survival number of aphid in device, and the feed more renewed and corresponding dsRNA, be placed in growth cabinet (temperature (20 ± 2) DEG C, humidity 60%-80%, photoperiod L: D=16: 8).Use Excel2003 software to carry out statistical analysis to aphid mortality ratio, calculate mean value and the variance of often kind of process, and carry out the analysis (t-test, n=3, P<0.01 or 0.05) of significant difference.
Add up each group of each number of raising in device the aphid that survives, calculate mortality ratio, result is as shown in table 2-table 4:
Table 2 is the mortality ratio of grain aphid 8273dsRNA nursing group and grain aphid dsGFP group
*represent compared with control group (0ng/ μ L), test group result difference remarkable (t-test, n=3, P<0.05),
*represent compared with control group, test group result difference extremely significantly (t-test, n=3, P<0.01).
Table 3 is the mortality ratio of grain aphid 22544dsRNA nursing group and grain aphid dsGFP group
*represent compared with control group (0ng/ μ L), test group result difference remarkable (t-test, n=3, P<0.05),
*represent compared with control group, test group result difference extremely significantly (t-test, n=3, P<0.01).
Table 4 is the mortality ratio of grain aphid 22544dsRNA+8273dsRNA nursing group and grain aphid dsGFP group
*expression is compared with control group (CK group, 0ng/ μ L), test group result difference remarkable (t-test, n=3, P<0.05),
*represent compared with control group, test group result difference extremely significantly (t-test, n=3, P<0.01).
As can be seen from table 2, table 3 and table 4, the mortality ratio of grain aphid all constantly raises along with the time of feeding dsRNA; 7.5ng μ L
-1after 8273dsRNA feeds 2 days, 4 days, 6 days and 8 days, the average mortality of grain aphid is 33.33%, 48.89%, 60% and 60%, average mortality after 22544dsRNA feeds 2 days, 4 days, 6 days and 8 days is 16.67%, 43.30%, 56.67% and 63.30%, the average mortality of mixed feeding after 2 days, 4 days, 6 days and 8 days is that 33.33%, 44.44%, 60.00%, 68.89% these process all exist significant difference compared with the control, and the test group mortality ratio of the GFPdsRNA that feeds then does not have the difference of significance compared with the control.
Use microscopic examination to feed and within 8 days, respectively organize the situation of growing of aphid afterwards, (left figure is dsGFP group to result such as Fig. 2, and right figure is ds8273 group; CK group and dsGFP group result are without significant difference; Ds8273+ds22544 group, ds22544 group and ds8273 group result are without significant difference) and table 5 shown in, CK: aphid group (the 0ng μ L not having feeding dsRNA
-1); DsGFP: feed 7.5ng μ L
-1the aphid group of concentration GFP dsRNA; Ds8273: feed 7.5ng μ L
-1the aphid group of concentration 8273dsRNA; Ds22544: feed 7.5ng μ L
-1the aphid group of concentration 22544dsRNA; Ds8273+ds22544: feed 7.5ng μ L
-1concentration 8273dsRNA and 7.5ng μ L
-1the aphid group of 22544dsRNA.Can find out, the aphid of non-feeding dsRNA and the GFPdsRNA that feeds grows normal, the aphid impaired development of 8273dsRNA, 22544dsRNA and mixed feeding 8273dsRNA, 22544dsRNA test group of feeding respectively, build is little compared with control group CK, can not grow for adult, not yet observe other character mutation, illustrate feed 8173 and 22544dsRNA can cause the effect of RNAi in the physical efficiency of aphid, cause aphid dead.
Table 5 is grain aphid growth quantity
3, dsRNA(8273,22544dsRNA) suppress the expression of body internal object gene 8273 and 22544
Remove T7 promoter sequence synthesis fluorescent quantitation 8273 primer P4 and 22544 primer P5(tables 1), synthesis reference gene primer P6(table 1).
To collect in above-mentioned 2 each group 7.5ng/ μ l dsRNAs feed after the grain aphid (2,4,6 and 8d) of survival, extract the RNA of aphid, reverse transcription becomes cDNA to dilute 10
0, 10
1, 10
2, 10
3, 10
4, 10
5doubly, as the template of quantitative fluorescent PCR, relative RT-PCR analysis is carried out using P4 and P5 as primer.Internal reference (ACTIN) primer is P6.
Real-time fluorescence quantitative PCR system is Forward Primer(10 μm olL
-1) 0.5 μ L, Reverse Primer(10 μm olL
-1) 0.5 μ L, 2 × TransStart
tMgreen qPCR SuperMix12.5 μ L, Passive Reference Dye0.5 μ L, template cDNA1 μ L, use ddH
2o to 25 μ L.
PCR cycling program is 95 DEG C of 30s, 95 DEG C of 5s, 57 DEG C of 15s, 72 DEG C of 10s, 40 circulations, the repetition of 3, each sample.The calculating of net result adopts 2-△ △ Ct method (Ct represents cycle number) to calculate, i.e. △ △ Ct=(Ct (dsRNA)-Ct (actin)) test group-(Ct (dsRNA)-Ct (actin)) control group, Excel2003 software is used to carry out statistical analysis, calculate mean value and the variance of often kind of process, and carry out the analysis (t-test of significant difference, n=3, P<0.01 or 0.05).
Statistics after 2,4,6 and the 8d of feed 8273dsRNA, 22544dsRNA, 8273dsRNA+22544dsRNA, 8273(sequence 1 in each group aphid body) and 22544(sequence 2) expression amount, not feed as contrasting (0d), result is as table 6 and table 7:
Table 6 is grain aphid 8273 and 22544 relative expression quantity after the different dsRNA that feeds separately
* represent compared with control group, test group result difference remarkable (t-test, n=3, P<0.05),
*represent compared with control group, test group result difference extremely significantly (t-test, n=3, P<0.01).
Table 7 is grain aphid 8273 and 22544 relative expression quantity after the different dsRNA of mixed feeding
Can find out, 8273(sequence 1 in grain aphid body) expression amount in turn reduces 31%, 51%, 42% and 65%, 22544(sequence 2) expression amount in turn reduces 19%, 38%, 78% and 91%, , compared with contrast (expression amount of 0 day), 8273 and 22544 gene expression doses of feeding in the aphid body of GFP dsRNA then do not have the change of significance, further illustrate the RNAi effect that can be caused corresponding gene by feeding dsRNA in grain aphid body, the expression amount of genes involved 8273 and 22544 of causing growing obviously reduces, aphid death or growth is caused to be suppressed.After mixed feeding result basic with when feeding separately 23028 and coo2 expression amount the change of divergence basically identical.
Claims (15)
1.dsRNA is following 1), 2) or 3):
1) double-stranded RNA be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 4 He its reverse complementary sequence;
2) double-stranded RNA be made up of the Nucleotide shown in the Nucleotide shown in sequence in sequence table 5 He its reverse complementary sequence;
3) be made up of following two double-stranded RNAs: the double-stranded RNA 1) and 2) shown in double-stranded RNA.
2. the double-stranded RNA and 2 dsRNA according to claim 1, is characterized in that: in the dsRNA described 3), 1)) shown in the mass ratio of double-stranded RNA be 1:1.
3. in claim 1 1), 2) or 3) shown in the encoding gene of described dsRNA;
1) encoding gene of the dsRNA shown in is specially the Nucleotide shown in sequence 1 in sequence table;
2) encoding gene of the dsRNA shown in is specially the Nucleotide shown in sequence 2 in sequence table;
3) encoding gene of the dsRNA shown in is specially the composition be made up of the DNA molecular shown in the Nucleotide shown in sequence 2 in the DNA molecular shown in the Nucleotide shown in sequence in sequence table 1 and sequence table.
4. any one application following of dsRNA described in claim 1 or 2 or encoding gene according to claim 3:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
5. application according to claim 4, it is characterized in that: described in be applied as dsRNA described in claim 1 or 2 imported aphid, realize the anti-expression eliminating aphis, promote that aphid is dead, suppress aphid growth, suppress aphid tumor growth development related gene 8273 and/or 22544.
6. application according to claim 5, is characterized in that:
Described importing is for feeding.
7. the recombinant expression vector of the encoding gene containing the dsRNA described in claim 1 or 2.
8. the transgenic cell line of the encoding gene containing the dsRNA described in claim 1 or 2.
9. the recombinant bacterium of the encoding gene containing the dsRNA described in claim 1 or 2.
10. the expression cassette of the encoding gene containing the dsRNA described in claim 1 or 2.
11. recombinant expression vectors according to claim 7 are in following application in any one:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
12. transgenic cells according to claim 8 tie up to following application in any one:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
13. recombinant bacteriums according to claim 9 are in following application in any one:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
14. expression cassettes according to claim 10 are in following application in any one:
1) application in anti-eliminating aphis or in the anti-application eliminated aphis in product of preparation;
2) application in the dead product of aphid is promoted in the application promoted in aphid death or in preparation;
3) application in aphid growth product is suppressed in the application suppressed in aphid growth or in preparation;
4) application in the expression suppressing aphid tumor growth development related gene 8273 and/or 22544 or the application in the expression product of preparation suppression aphid tumor growth development related gene 8273 and/or 22544.
15. products, its activeconstituents is at least one in following A-F:
DsRNA described in A, claim 1 or 2;
B, encoding gene according to claim 3;
C, recombinant expression vector according to claim 7;
D, transgenic cell line according to claim 8;
E, recombinant bacterium according to claim 9;
F, expression cassette according to claim 10;
Described product is following 1)-4) in any one: 1) the anti-product that eliminates aphis; 2) the dead product of aphid is promoted; 3) aphid growth product is suppressed; 4) the expression product of aphid tumor growth development related gene 8273 and/or 22544 is suppressed.
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| CN104109671B (en) * | 2014-07-10 | 2017-01-11 | 中国农业科学院作物科学研究所 | DsRNA of gene of symbiotic bacterium of aphids and application thereof to inhibition of growth of aphids |
| CN104263731B (en) * | 2014-10-09 | 2016-08-03 | 南阳师范学院 | The dsRNA of a kind of aphid fungal component gene and the application in reducing aphid survival rate thereof |
| CN105695467B (en) * | 2016-03-31 | 2018-11-23 | 中国农业科学院植物保护研究所 | The siRNA of conservative olfactory receptor Orco gene design based on grain aphid Sitobion avenae and its application |
| CN105695468B (en) * | 2016-03-31 | 2018-11-23 | 中国农业科学院植物保护研究所 | The siRNA of conserved sequence design based on grain aphid Sitobion avenae olfactory receptor SaveOrco and its application |
| CN105755003B (en) * | 2016-04-05 | 2018-11-23 | 中国农业科学院植物保护研究所 | SiRNA and its application based on the design of grain aphid Sitobion avenae olfactory receptor SaveOrco gene |
| CN107446947A (en) * | 2017-09-26 | 2017-12-08 | 中国农业科学院作物科学研究所 | A kind of methods and techniques system for cultivating anti-aphid tobacco and wheat |
| CN110628771B (en) * | 2019-08-05 | 2021-09-14 | 华南农业大学 | Kit for preventing and treating harmonia axyridis |
| CN111567566B (en) * | 2020-05-28 | 2021-11-30 | 西南大学 | Aphid control agent, RNAi and beauveria bassiana combined application aphid control method |
| CN114540350B (en) * | 2022-01-28 | 2023-08-22 | 南京农业大学 | Screening, preparation and application of dsRNA (double-stranded ribonucleic acid) medicament for targeting genes of aphids AK, SOD and CHS and killing aphids |
| CN114561411A (en) * | 2022-03-14 | 2022-05-31 | 贵州大学 | Dicer1 gene and application of dsRNA thereof in pest control |
| 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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| EP1794303B1 (en) * | 2004-09-29 | 2011-09-07 | Advanced Technologies (Cambridge) Limited | Modification of plant development and morphology |
| CN102206668A (en) * | 2011-04-28 | 2011-10-05 | 上海交通大学 | Method for enhancing aphid resistance of plant |
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| EP1794303B1 (en) * | 2004-09-29 | 2011-09-07 | Advanced Technologies (Cambridge) Limited | Modification of plant development and morphology |
| CN102206668A (en) * | 2011-04-28 | 2011-10-05 | 上海交通大学 | Method for enhancing aphid resistance of plant |
| CN102703392A (en) * | 2012-06-12 | 2012-10-03 | 石河子大学 | Preparation method of transgenically cloned pig integrating O-type foot and mouth disease virus shRNA (Short Hairpin Ribonucleic Acid) |
| CN102776189A (en) * | 2012-06-18 | 2012-11-14 | 中国农业科学院作物科学研究所 | Cytochrome P450 dsRNA (double-stranded ribonucleic acid) and application to aphid growth inhibition |
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