Application and product of the dsRNA in colorado potato bug prevents and treatsTechnical field
The present invention relates to a kind of gene prod colorado potato bug prevention and treatment in application and derived product.
Background technique
Potato is grain important in the world, dish, raises dual-purpose crop and insutrial crop, in recent years, since it comparesThe features such as economic benefit is obvious constantly increases in China's cultivated area, has become China at present after rice, wheat, corn4th big staple food grain crop.Colorado potato bug Leptinotarsa decemlineata also known as colorado potato beetles, English nameColorado Potato Beetle (CPB) is subordinate to coleoptera, and Chrysomelidae is acknowledged as the destructiveness of potato in world wideExternal one of the great quarantine object in pest and China.Its host range relative narrower, main harm potato, eggplant, cultivationMore than 20 plant of Solanaceae such as tomato, henbane seed, Yellow calla.Ma Ling at the beginning of the great Exotic pests from the last century 90'sSince potato beetle is passed to the Xinjiang region in China for the first time, constantly diffusion sprawling has become Xinjiang potato planting industry most at presentProminent, most important Plant Protection, and in China Gansu etc. the sound development of the main growing area correlation planting industry of potatoConstitute grave danger.Due to it with high reproductive rate, binge, facultative diapause, migrate and the characteristics such as generation overlap, environment is suitableShould be able to power and ecological plasticity it is extremely strong, easily generate strong drug resistance, explore and carry out the correlations such as its novel biocontrol Prevention Technique and grindStudy carefully and has become a hot spot.
RNAi (RNA interference, RNA interference) be it is a kind of it is ancient, present in the multi-celled eukaryotes,(the Post- the posttranscriptional gene silencing the phenomenon that caused by dsRNA, siRNA of external source or endogenous miRNAtranscriptional Gene Silencing,PTGS).It is generally believed that the process is primarily involved in virus immunity, gene expressionThe processes such as regulation.
But current RNAi technology still has obvious deficiency: 1. causing the dsRNA or siRNA of RNAi effect in the environment easily by nothingPlace not nuclease degradation, therefore it is unstable；2. production and the dsRNA technology saved are not yet mature, high production cost, shelf lifeIt is short.
Summary of the invention
DsRNA product stability of the present invention is strong, production technology is mature, at low cost, shelf life is long, is effectively inexpensive single-mindedProperty strong, green, environmental protection " gene pesticide " product.Application of the dsRNA product of the present invention in colorado potato bug prevents and treats is for horseThe prevention and treatment of bell potato beetle has great and profound significance.
Application of the dsLdRpn6 of the present invention in colorado potato bug prevents and treats, the nucleotide sequence of dsLdRpn6 such as SEQ IDShown in NO:1.
Further, active constituent of the dsLdRpn6 as potato beetle insecticidal agent.
Preferably, use the bacterium solution for containing the transgenic engineered bacteria of expression dsLdRpn6 as potato beetle insecticidal agent.
Further, with the expression vector of Prokaryotic expression vector construction expression dsLdRpn6.
Potato beetle insecticidal agent effective component of the present invention is dsLdRpn6, the nucleotide sequence of dsLdRpn6 such as SEQ IDShown in NO:1.
Further, potato beetle insecticidal agent is the bacterium solution of the transgenic engineered bacteria containing expression dsLdRpn6.
DsLdRpn6 expression vector of the present invention encodes the DNA sequence dna of dsLdRpn6 as shown in SEQ ID NO:2, and carrier isProkaryotic expression carrier.
Further, the prokaryotic expression carrier is pET-2p.
The nucleotide sequence of colorado potato bug lethal gene dsLdRpn6 of the present invention is as shown in SEQ ID NO:1.
The present invention expresses the transgenic engineered bacteria of dsLdRpn6, with Escherichia coli Escherichia coli HTl15It (DE3) is host, using dsLdRpn6 expression vector as transcriptional expression carrier.
Potato beetle insecticidal agent of the present invention has specificity, lethal efficiency height, mechanism to colorado potato bug larvaFast advantage.
It, can on potting potato plant after potato beetle insecticidal agent low dosage of the present invention sprays potato plant bladeContinue insect prevention 28 days or more, preventive effect is up to 80% or more；It is outdoor strong it is ultraviolet under the conditions of, potato beetle insecticidal agent of the present invention is to Ma LingPotato beetle still has extremely strong toxic action, and the field prevention and control lasting period was at 3 weeks or so, and preventive effect gradually decreases down 10% from 50%Left and right.
Technical solution of the present invention constructs the dsRNA of prokaryotic expression for the first time in the world.It can be big with prokaryotic expression dsRNAWidth reduces the production cost of dsRNA, and every milliliter of bacterium solution amount of fermentation of the present invention is up to 50 μ g, even if selecting expensive import eggWhite peptone, yeast extract etc. make culture medium, and the production cost of 1g is also less than 100 U.S. dollars.
Detailed description of the invention
Fig. 1 be 1 feeding concentration gradient of embodiment dsRNA after first day larval feeding hazard rating, wherein L5 be feedDsLdRpn6 bacterium solution is eaten, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 2 be 1 feeding concentration gradient of embodiment dsRNA after second day larval feeding hazard rating, wherein L5 be feedDsLdRpn6 bacterium solution is eaten, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 3 be 1 feeding concentration gradient of embodiment dsRNA after third day larval feeding hazard rating, wherein L5 be feedDsLdRpn6 bacterium solution is eaten, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 4 be 1 feeding concentration gradient of embodiment dsRNA after the 4th day larval feeding hazard rating, wherein L5 be feedDsLdRpn6 bacterium solution is eaten, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 5 is the 7th day after 1 feeding colorado potato bug of embodiment, 2 instar larvae dsRNA bacterium solution survival rate, and wherein L5 is to feedDsLdRpn6 bacterium solution is eaten, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 6 is the colorado potato bug larvae pupation rate of 1 feeding dsRNA bacterium solution of embodiment, and wherein L5 is feeding dsLdRpn6Bacterium solution, L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 7 is that the rate of recovery counts after potting one month of 2 instar larvae of colorado potato bug of 1 feeding dsRNA bacterium solution of embodimentFigure, wherein L5 is feeding dsLdRpn6 bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Fig. 8 is growing way figure after potting one month of 2 instar larvae of colorado potato bug of 1 feeding dsLdRpn6 bacterium solution of embodiment.
Fig. 9 is that larval weight is recycled weekly in the potting of 2 instar larvae of colorado potato bug of 1 feeding dsRNA bacterium solution of embodimentStatistical chart, wherein L5 is feeding dsLdRpn6 bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacteriumLiquid.
Figure 10 is that embodiment 1 sprays dsRNA bacterium solution field potato plant to 2 instar larvae mortality statistics of colorado potato bugFigure, wherein L5 is feeding dsLdRpn6 bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium solution.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodimentAny combination.
Specific embodiment 1: application of the present embodiment dsLdRpn6 in colorado potato bug prevents and treats, whereinThe nucleotide sequence of dsLdRpn6 is as shown in SEQ ID NO:1.
Specific embodiment 2: the difference of present embodiment and specific embodiment one is: dsLdRpn6 is as Ma LingThe active constituent of potato beetle insecticidal agent.It is other identical as embodiment one.
Specific embodiment 3: the difference of present embodiment and specific embodiment one or two is: being expressed with containingThe bacterium solution of the transgenic engineered bacteria of dsLdRpn6 is as potato beetle insecticidal agent.It is other identical as embodiment one or two.
Specific embodiment 4: the difference of present embodiment and specific embodiment three is: described containing expressionThe transgenic engineered bacteria of dsLdRpn6 is with Escherichia coli Escherichia coli HTl15 (DE3) for host, withDsLdRpn6 expression vector is transcriptional expression carrier.It is other identical as embodiment three.
Escherichia coli HTl15 (DE3) is III deficient strain of RNA enzyme.
Specific embodiment 5: the difference of present embodiment and specific embodiment four is: the dsLdRpn6 expressionCarrier is prokaryotic expression carrier.It is other identical as embodiment four.
Specific embodiment 6: the difference of present embodiment and specific embodiment five is: the prokaryotic expression carrierFor pET-2p.It is other identical as embodiment five.
Specific embodiment 7: the difference of present embodiment and specific embodiment one to six is: coding dsLdRpn6DNA sequence dna as shown in SEQ ID NO:2.It is other identical as embodiment one to six.
Specific embodiment 8: present embodiment potato beetle insecticidal agent, effective component dsLdRpn6, dsLdRpn6Nucleotide sequence as shown in SEQ ID NO:1.
Specific embodiment 9: the difference of present embodiment and specific embodiment eight is: potato beetle insecticidal agentFor the bacterium solution of the transgenic engineered bacteria containing expression dsLdRpn6.It is other identical as embodiment eight.
Specific embodiment 10: the difference of present embodiment and specific embodiment nine is: described containing expressionThe transgenic engineered bacteria of dsLdRpn6 is with Escherichia coli Escherichia coli HTl15 (DE3) for host, withDsLdRpn6 expression vector is transcriptional expression carrier.It is other identical as embodiment nine.
Specific embodiment 11: the difference of present embodiment and specific embodiment ten is: the dsLdRpn6 tableIt is prokaryotic expression carrier up to carrier.It is other identical as embodiment ten.
Specific embodiment 12: the difference of present embodiment and specific embodiment 11 is: the prokaryotic expressionCarrier is pET-2p.It is other identical as embodiment 11.
Specific embodiment 13: present embodiment dsLdRpn6 expression vector, wherein the DNA sequence dna of coding dsLdRpn6As shown in SEQ ID NO:2, carrier is prokaryotic expression carrier.
Specific embodiment 14: the difference of present embodiment and specific embodiment 13 is: the protokaryon tableIt is pET-2p up to carrier.It is other identical as embodiment 13.
Specific embodiment 15: present embodiment expresses the transgenic engineered bacteria of dsLdRpn6, with Escherichia coliEscherichia coli HTl15 (DE3) is host, using dsLdRpn6 expression vector as transcriptional expression carrier.
Specific embodiment 16: the difference of present embodiment and specific embodiment 15 is: the dsLdRpn6Expression vector is prokaryotic expression carrier.It is other identical as embodiment 15.
Specific embodiment 17: the difference of present embodiment and specific embodiment 16 is: the prokaryotic expressionCarrier is pET-2p.It is other identical as embodiment 16.
Specific embodiment 18: the difference of present embodiment and specific embodiment 15 to 17 is: wherein compilingThe DNA sequence dna of code dsLdRpn6 is as shown in SEQ ID NO:2.It is other identical as embodiment 15 to 17.
The experiment of 1 lethal gene lethal efficiency of embodiment
(1) worm sources are tested
The equal random acquisition of colorado potato bug pieces of an egg in Xinjiang academy of agricultural sciences Plant Protection Institute peacefulness canal experimental field.(N:43.9128 E:87.4918).Incubator raising temperature: 26 (± 1) DEG C；Photoperiod: 14L:10D；Relative humidity 50%~60%.It is fed with fresh potato blade, the larva for taking the same time to cast off a skin at the beginning of to 2 ages makees experiment and uses worm.
(2) acquisition of the alternative lethal gene of colorado potato bug
(transcript profile data are mentioned by Agricultural University Of Nanjing teacher Li Guoqing in the transcript profile and genome of colorado potato bugFor being downloaded from U.S. Baylor College of Medicine Human's Genome Sequencing Center website under the genomic data of colorado potato bughttps://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-Project.) in search choosingIt takes, obtains sequence SEQ ID NO:2 (the cDNA segment of dsLdRpn6).
(3) building of dsRNA prokaryotic expression carrier
The Escherichia coli Escherichia coli HTl15 (DE3) that this experiment is given with Agricultural University Of Nanjing be host,The pET-2p dsRNA being given using Agricultural University Of Nanjing is expression vector.
Selecting for sequence SEQ ID NO:2PCR, the recycling of PCR product and purifying, connection and conversion and monoclonal, obtainsPositive colony by sequencing and isopropylthio galactolipin (isopropyl-D-hiogalactoside, IPTG) induction fermentationDsRNA verification result.Expand culture again to OD in bacterium solution600IPTG to final concentration of 0.1mmol/L, fermentation are added when=1.0Expression 6h can be obtained the dsLdRpn6 for stablizing that the concentration of expression is about 0.05 μ g/ μ L.It is designed using Premier Primer5.0The primer of dsRNA segment, upstream primer 5 '-GATAAAGATAATGCCGTGAG-3 ', downstream primer 5 '-GTGTAGCGGACAAATGCT-3 ', dsLdRpn6 fragment length are 323bp.
(4) indoor feeding experiment
Through feeding the dsLdRpn6 of colorado potato bug second instar larvae various concentration, with ultrapure water and dsEGFP, LdATPaseEWith LdATPaseA (" the prevention and control corn that LdATPaseE and LdATPaseA were delivered in 2007 in Nature BiotechnologyDisclosed in the potato of lustrous and transparent chrysomelid and colorado potato bug the transgene expression dsRNA of root and the gene of corn ") to compare, and pointAnalyse the 20% feeding dosage AD to larva20, middle amount PD of pupating50With 7 days lethal dose of 50 LD of larva50。
After the bacterium solution of continuous feeding one week expression dsLdRpn6, dsATPaseE, dsATPaseA, colorado potato bug larva tableThe case where revealing food refusal, hypoevolutism.4 50 μ g of dosage, 5 μ g, 0.5 μ g, 0.05 μ g processing in, in dosage 50 μ g and 5 μLarval feeding rate on g processing blade is extremely significant to be lower than ultrapure water group, dsEGFP processing group and low concentration in the trend that is decreased obviouslyThe feeding rate of larva on processing group blade.
Processing for 24 hours when after, dsLdRpn6, dsATPaseE, dsATPaseA group blade feeding rate of 50 μ g of dosage andFeeding rate difference is not significant (P > 0.05) on dsEGFP processing group blade, and feeding rate is 25% hereinafter, hazard rating is I；AgentDsLdRpn6, dsATPaseE, dsATPaseA group and the dsEGFP group feeding rate difference for measuring 0.05 μ g is not significant (P > 0.05), butIt is variant (P < 0.05) with the feeding rate of other treatment dosage groups.After handling 72h and 96h, dsLdRpn6, dsATPaseE,DsATPaseA processing group larval feeding amount increases with number of days in the trend that is decreased obviously, and concentration is higher, and inhibition reaction is stronger, and superPure water group and dsEGFP processing group larval feeding rate are then in continue to rise, and are caused harm close to even up to IV grades of feeding, experiment knotFruit is as shown in figures 1-4.
In view of larva is after feeding expresses dsRNA bacterium solution, there is significant feeding inhibition phenotype in third day after processing,Each processing group increasing concentrations, anti-food rate increase, i.e., using third day after handling as the analysis moment for inhibiting feeding phenotype.WithSPSS20.0 carries out regression analysis to 3 days anti-food rates of larva.DsLdRpn6 anti-food rate regression equation is Y=4.523x-2.302,20% dosage (AD of feeding20) it is 1.57 ± 0.03 μ g, extension rate is 31.9 ± 0.3 times；DsATPaseE anti-food rate returnsReturning equation is Y=4.076x-1.71,20% dosage (AD of feeding20) it is 3.47 ± 0.06 μ g, extension rate is 14.4 ± 0.8 times；DsATPaseA anti-food rate regression equation is Y=3.46x-1.187,20% dosage (AD of feeding20) it is 5.29 ± 0.27 μ g, dilutionMultiple is 9.5 ± 3.2 times.
DsLdalpha snap, dsATPaseE, dsATPaseA processing group interference after larva reduce feeding even stop intoFood, forces its growth and development to be inhibited, finally results in death.It is daily to count dead head number, it is buried before pupating to mature larva,I.e. dsRNA feeding counts the death rate after handling the 7th day.DsLdalpha snap all has centainly potato larva before buryingToxic action, concentration be 50 μ g/mL under high killing activity is shown to 2 instar larvae of colorado potato bug, lethality is up to90%~93%.DsLdRpn6 processing group lethality in the case where dsRNA concentration is 5 μ g/mL is more than 50%, and killing activity increases with concentrationIt is high and increase, show dose-dependent effect.There are significant difference (P < 0.05) with control group, as shown in Figure 5.
Significance analysis and regression analysis were carried out to 7 days after the processing death rates with SPSS20.0.When burying before pupatingBetween be about treated the 7th day, dsLdRpn6 death rate regression equation be Y=4.087x-2.462, the lethal dose of 50 (LD50) be1.18 ± 0.10 μ g, extension rate are 42.4 ± 3.1 times；DsATPaseE death rate regression equation is Y=4.681x-2.245,The lethal dose of 50 (LD50) it is 4.54 ± 0.54 μ g, extension rate is 11 ± 0.3 times；DsATPaseA death rate regression equation is Y=4.308x-2.183, the lethal dose of 50 (LD50) it is 3.08 ± 0.23 μ g, extension rate is 16.2 ± 0.2 times.
Mature larva buries pupate after the 7th day statistics percentage of pupation, and percentage of pupation reduces with the increase of concentration for the treatment of,DsLdRpn6 processing group concentration is to colorado potato bug mature larva percentage of pupation under 50 μ g/mL down to 0%, dsLdRpn6 processing groupIn the case where dosage is 5 μ g, percentage of pupation is less than 50%, even if dosage down to 0.05 μ g, also reduces larva 20~30% than controlPercentage of pupation, as shown in Figure 6.
Significance analysis and regression analysis are carried out to the mature larva percentage of pupation that buries with SPSS21.0.The time bury aboutIt is 7 days, dsLdRpn6 percentage of pupation regression equation is Y=5.581x-4.518, middle amount (PD of pupating50) it is 0.07 ± 0.00 μ g, it is diluteReleasing multiple is 758.6 ± 130.0 times；DsATPaseE percentage of pupation regression equation is Y=3.21x-2.261, middle amount (PD of pupating50)For 0.46 ± 0.02 μ g, extension rate is 109.8 ± 15.9 times；DsATPaseA percentage of pupation regression equation is Y=3.551x-2.428, middle amount (PD of pupating50) it is 0.50 ± 0.09 μ g, extension rate is 100.8 ± 11.7 times.
The result shows that: dsLdRpn6, which has colorado potato bug larva, to be inhibited feeding, inhibits to pupate and killing activity.Its is lethalActivity increases with concentration and is increased, and shows dose-dependent effect, and compares that there are significant differences.DsLdRpn6 is with higherBioactivity, and the virulence of dsLdRpn6 is higher than dsATPaseE and dsATPaseA.
(5) potting feeding is tested
The dilution of dsLdRpn6, dsATPaseE, dsATPaseA, dsEGFP will be expressed on potting potato respectively indoors10 times of bacterium solutions (concentration is 5 μ g/mL) are sprayed on potting, and after air drying, every plant of potato places 5 colorado potato bug second instar larvaesFree feeding, 6 repeating groups, each processing amount to 30 larvas.To recycle larva after a week, weighs and count the death rate, weight5 second instar larvaes of new placement are repeated to test weekly, be taken pictures to potato plant and larva weekly.
The induction of resistance experiment of potting potato is continuous to carry out surrounding, recycles larva quantity weekly and is counted.Test intoRow dilutes 10 times of bacterium solution control group rate of recovery to the 7th day ultrapure water and expression dsEGFP and is up to 55%, 60% respectively.dsLdRpn6Processing group larva rate of recovery < 5%, there are extremely significant difference (P < 0.05) with control group.14th day ultrapure water and expressionDsEGFP dilutes 10 times of bacterium solution control group rate of recovery and is up to 72.5%, 57.5% respectively；The dsLdRpn6 processing group larva rate of recoveryBeing less than 20%, there are significant difference (P < 0.05) with control group.21st day ultrapure water and expression dsEGFP dilute 10 times of bacterium solutionsThe control group rate of recovery is respectively 60%, 50%；The dsLdRpn6 processing group larva rate of recovery is less than 30%.28th day ultrapure water withExpressing dsEGFP to dilute 10 times of bacterium solution control group rate of recovery is respectively 62.5%, 65%；The dsLdRpn6 processing group larva rate of recoveryIt is less than 20%.2 instar larvae rate of recovery trend of dsLdRpn6 processing group colorado potato bug is below control group larva, such as Fig. 7 instituteShow.Especially experiment the last fortnight tables of data reveals to the significant inhibiting effect of the test worm rate of recovery and preferable lethal effect.
The induction of resistance experiment of potting potato is continuous to carry out surrounding.In test, body is carried out to test worm every 7dIt weighs again, observes its body weight increase rate.Experiment proceeds to the 7th day ultrapure water and expression dsEGFP dilutes 10 times of bacterium solution group larvasAverage weight is respectively 0.149g, 0.135g.DsLdRpn6 processing group recycling larva average weight is less than 0.05g and control groupThere are extremely significant difference (P < 0.05).14th day ultrapure water and expression dsEGFP dilute 10 times of bacterium solution groups recycling larvas and are averaged bodyIt is again respectively 0.129g, 0.122g；DsLdRpn6 processing group recycling larva average weight is less than 0.1g.21st day ultrapure water withIt is respectively 0.128g, 0.124g that expression dsEGFP, which dilutes 10 times of bacterium solution group recycling larva average weights, and dsLdRpn6 processing group is returnedReceiving larva average weight is that there are significant difference (P < 0.05) with control group by 0.093g.28th day ultrapure water and expression dsEGFPDiluting 10 times of bacterium solution group recycling larva average weights is respectively 0.132g, 0.153g, and it is average that dsLdRpn6 processing group recycles larvaWeight is 0.109g.2 instar larvae body weight increase trend of dsLdRpn6 processing group colorado potato bug is below control group larval weightGrowth trend, as shown in Figure 9.Especially experiment the last fortnight tables of data reveals to the significant inhibiting effect of test worm body weight increase, withPreferable lethal effect.
The result shows that the lethal effect of dsLdRpn6 is 28 days or more, preventive effect can reach as high as 80% or more, thenDecay to 50%.
(6) field plot trial
The dilution of dsLdRpn6, dsATPaseE, dsATPaseA, dsEGFP will be expressed respectively on the potato of field plot10 times of bacterium solutions carry out bacterium solution to field and spray.Colorado potato bug second instar larvae is placed in 9cm normal glass ware weekly, each5 larvas are handled, 6 groups of biology repeat.It is fed with the processed blade in crop field is picked up from.The blade of replacement feeding is equal dailyPick up from the processed blade in crop field.The statistics death rate daily, experiment are carried out one month.
Variance analysis, experiment are carried out to 4 weeks death rates of each 2 instar larvae of processing dsRNA colorado potato bug are fed with SPSSProceeding to the 7th day ultrapure water control group larval mortality is 0, and expression dsEGFP dilutes 10 times of bacterium solution processing group larval mortalities and is8%, dsLdRpn6 processing group larval mortality more a height of 9%, as shown in Figure 10.
Although above having made description to the present invention with a general description of the specific embodiments, if in this hairMade on the basis of bright it is some modify or improve, and it is without departing from the spirit of the present invention, belongs to claimed modelIt encloses.
Sequence table
<110>Institute of Plant Protection, Xinjiang Academy of Agricultural Science
<120>application and product of the dsRNA in colorado potato bug prevention and treatment
<130> dsLdRpn6
<160> 2
<170> SIPOSequenceListing 1.0
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<213>artificial sequence (Artificial Sequence)
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<213>colorado potato bug (Leptinotarsa decemlineata)
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