CN109913457A - Application and product of the dsRNA in colorado potato bug prevents and treats - Google Patents
Application and product of the dsRNA in colorado potato bug prevents and treats Download PDFInfo
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Abstract
Application and product of the dsRNA in colorado potato bug prevents and treats are related to application and derived product of the gene prod in colorado potato bug prevention and treatment.DsRNA product stability of the present invention is strong, production technology is mature, at low cost, is strong effectively inexpensive specificity, green, environmentally friendly " gene pesticide " product.Application of the dsRNA product of the present invention in colorado potato bug prevents and treats has great and profound significance for the prevention and treatment of colorado potato bug.It can continue insect prevention 28 days or more after potato beetle insecticidal agent low dosage of the present invention sprays potato plant blade, on potting potato plant, 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 still has stronger toxic action to colorado potato bug, and the field prevention and control lasting period was at 3 weeks or so, and preventive effect gradually decreases down 10% or so from 50%.
Description
Technical 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 compares
The features such as economic benefit is obvious constantly increases in China's cultivated area, has become China at present after rice, wheat, corn
4th big staple food grain crop.Colorado potato bug Leptinotarsa decemlineata also known as colorado potato beetles, English name
ColoradoPotato Beetle (CPB) is subordinate to coleoptera, and Chrysomelidae is acknowledged as the destructiveness of potato in world wide
External one of the great quarantine object in pest and China.Its host range relative narrower, main harm potato, eggplant, cultivation
More 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's
Since 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 present
Prominent, most important Plant Protection, and in China Gansu etc. the sound development of the main growing area correlation planting industry of potato
Constitute grave danger.Due to it with high reproductive rate, binge, facultative diapause, migrate and the characteristics such as generation overlap, environment is suitable
Should 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 grind
Study 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 miRNA
transcriptional Gene Silencing,PTGS).It is generally believed that the process is primarily involved in virus immunity, gene expression
The 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 nothing
Place not nuclease degradation, therefore it is unstable；2. production and the dsRNA technology saved are not yet mature, high production cost.
Summary of the invention
DsRNA product stability of the present invention is strong, production technology is mature, at low cost, is that effectively inexpensive specificity is strong, green
Color, environmentally friendly " gene pesticide " product.Application of the dsRNA product of the present invention in colorado potato bug prevents and treats is for colorado potato bug
Prevention and treatment have great and profound significance.
Application of the dsLdRpt3 of the present invention in colorado potato bug prevents and treats, the nucleotide sequence of dsLdRpt3 such as SEQ ID
Shown in NO:1.
Further, active constituent of the dsLdRpt3 as potato beetle insecticidal agent.
Preferably, use the bacterium solution for containing the transgenic engineered bacteria of expression dsLdRpt3 as potato beetle insecticidal agent.
Further, with the expression vector of Prokaryotic expression vector construction expression dsLdRpt3.
Potato beetle insecticidal agent effective component of the present invention is dsLdRpt3, the nucleotide sequence of dsLdRpt3 such as SEQ
Shown in IDNO:1.
Further, potato beetle insecticidal agent is the bacterium solution of the transgenic engineered bacteria containing expression dsLdRpt3.
DsLdRpt3 expression vector of the present invention encodes the DNA sequence dna of dsLdRpt3 as shown in SEQ ID NO:2, and carrier is
Prokaryotic expression carrier.
Further, the prokaryotic expression carrier is pET-2p.
The nucleotide sequence of colorado potato bug lethal gene dsLdRpt3 of the present invention is as shown in SEQ ID NO:1.
The present invention expresses the transgenic engineered bacteria of dsLdRpt3, with Escherichia coli Escherichia coli HTl15
It (DE3) is host, using dsLdRpt3 expression vector as transcriptional expression carrier.
Potato beetle insecticidal agent of the present invention has specificity, lethal efficiency height, mechanism to colorado potato bug larva
Fast advantage.
It, can on potting potato plant after potato beetle insecticidal agent low dosage of the present invention sprays potato plant blade
Continue 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 Ling
Potato 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 dsRNA
Width 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 egg
White 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 L9 be feed
DsLdRpt3 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 L9 be feed
DsLdRpt3 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 L9 be feed
DsLdRpt3 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 L9 be feed
DsLdRpt3 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 L9 is to feed
DsLdRpt3 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 L9 is feeding dsLdRpt3
Bacterium 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 embodiment
Figure, wherein L9 is feeding dsLdRpt3 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 dsLdRpt3 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 embodiment
Statistical chart, wherein L9 is feeding dsLdRpt3 bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium
Liquid.
Figure 10 is that embodiment 1 sprays dsRNA bacterium solution field potato plant to 2 instar larvae mortality statistics of colorado potato bug
Figure, wherein L9 is feeding dsLdRpt3 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 embodiment
Any combination.
Specific embodiment 1: application of the present embodiment dsLdRpt3 in colorado potato bug prevents and treats, wherein
The nucleotide sequence of dsLdRpt3 is as shown in SEQ ID NO:1.
Specific embodiment 2: the difference of present embodiment and specific embodiment one is: dsLdRpt3 is as Ma Ling
The 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 containing
The bacterium solution of the transgenic engineered bacteria of dsLdRpt3 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 expression
The transgenic engineered bacteria of dsLdRpt3 is with Escherichia coli Escherichia coli HTl15 (DE3) for host, with
DsLdRpt3 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 dsLdRpt3 expression
Carrier 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 carrier
For 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 dsLdRpt3
DNA 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 dsLdRpt3, dsLdRpt3
Nucleotide sequence as shown in SEQ ID NO:1.
Specific embodiment 9: the difference of present embodiment and specific embodiment eight is: potato beetle insecticidal agent
For the bacterium solution of the transgenic engineered bacteria containing expression dsLdRpt3.It is other identical as embodiment eight.
Specific embodiment 10: the difference of present embodiment and specific embodiment nine is: described containing expression
The transgenic engineered bacteria of dsLdRpt3 is with Escherichia coli Escherichia coli HTl15 (DE3) for host, with
DsLdRpt3 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 dsLdRpt3 table
It 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 expression
Carrier is pET-2p.It is other identical as embodiment 11.
Specific embodiment 13: present embodiment dsLdRpt3 expression vector, wherein the DNA sequence dna of coding dsLdRpt3
As 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 table
It is pET-2p up to carrier.It is other identical as embodiment 13.
Specific embodiment 15: present embodiment expresses the transgenic engineered bacteria of dsLdRpt3, with Escherichia coli
Escherichiacoli HTl15 (DE3) is host, using dsLdRpt3 expression vector as transcriptional expression carrier.
Specific embodiment 16: the difference of present embodiment and specific embodiment 15 is: the dsLdRpt3
Expression 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 expression
Carrier 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 compiling
The DNA sequence dna of code dsLdRpt3 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 bug
For 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 choosing
It takes, obtains sequence SEQ ID NO:2 (the cDNA segment of dsLdRpt3).
(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, obtains
Positive colony by sequencing and isopropylthio galactolipin (isopropyl-D-hiogalactoside, IPTG) induction fermentation
DsRNA verification result.Expand culture again to OD in bacterium solution600IPTG to final concentration of 0.1mmol/L, fermentation are added when=1.0
Expression 6h can be obtained the dsLdRpt3 for stablizing that the concentration of expression is about 0.05 μ g/ μ L.It is designed using Premier Primer5.0
The primer of dsRNA segment, upstream primer 5 '-GGTCCACTACAGGTTCCA-3 ', downstream primer 5 '-
GTCCATACATCAACACCC-3 ', dsLdRpt3 fragment length are 309bp.
(4) indoor feeding experiment
Through feeding the dsLdRpt3 of colorado potato bug second instar larvae various concentration, with ultrapure water and dsEGFP, LdATPaseE
With LdATPaseA (" the prevention and control corn that LdATPaseE and LdATPaseA were delivered in 2007 in Nature Biotechnology
Disclosed 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 point
Analyse 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 dsLdRpt3, dsATPaseE, dsATPaseA, colorado potato bug larva table
The 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 μ
Larval 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 obviously
The feeding rate of larva on processing group blade.
Processing for 24 hours when after, dsLdRpt3, dsATPaseE, dsATPaseA group blade feeding rate of 50 μ g of dosage and
Feeding rate difference is not significant (P > 0.05) on dsEGFP processing group blade, and feeding rate is 25% hereinafter, hazard rating is I；Agent
DsLdRpt3, dsATPaseE, dsATPaseA group and the dsEGFP group feeding rate difference for measuring 0.05 μ g is not significant (P > 0.05),
But it is variant (P < 0.05) with the feeding rate of other treatment dosage groups.After handling 72h and 96h, dsLdRpt3, 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 super
Pure 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 knot
Fruit 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.With
SPSS20.0 carries out regression analysis to 3 days anti-food rates of larva.DsLdRpt3 anti-food rate regression equation is Y=7.403x-
3.835,20% dosage (AD of feeding20) it is 1.78 ± 0.07 μ g, extension rate is 28 ± 1.5 times；DsATPaseE anti-food rate returns
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, dilution
Multiple is 9.5 ± 3.2 times.
Larva reduces feeding and even stops feeding after the interference of dsLdRpt3, dsATPaseE, dsATPaseA processing group, forces
Its growth and development is inhibited, and 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.DsLdRpt3 all has certain toxic action to potato larva before burying,
Concentration is to show high killing activity to 2 instar larvae of colorado potato bug under 50 μ g/mL, and lethality is up to 90%~93%.
DsLdRpt3 processing group lethality in the case where dsRNA concentration is 5 μ g/mL is more than 50%, and killing activity increases with concentration and increased, table
Reveal 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 pupating
Between be about treated the 7th day, dsLdRpt3 death rate regression equation be Y=3.814x-2.079, the lethal dose of 50 (LD50) be
1.93 ± 0.07 μ g, extension rate are 25.9 ± 3.4 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,
DsLdRpt3 processing group concentration is to colorado potato bug mature larva percentage of pupation under 50 μ g/mL lower than 50%, dsLdRpt3 processing
Group percentage of pupation in the case where dosage is 5 μ g is less than 50%, even if dosage down to 0.05 μ g, also reduces larva 20~30% than control
Percentage 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 about
It is 7 days, dsLdRpt3 percentage of pupation regression equation is Y=3.697x-2.119, middle amount (PD of pupating50) it is 1.24 ± 0.24 μ g, it is dilute
Releasing multiple is 40.5 ± 6.8 times；DsATPaseE percentage of pupation regression equation is Y=3.21x-2.261, middle amount (PD of pupating50) be
0.46 ± 0.02 μ g, extension rate are 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: dsLdRpt3, which has colorado potato bug larva, to be inhibited feeding, inhibits to pupate and killing activity.Its is lethal
Activity increases with concentration and is increased, and shows dose-dependent effect, and compares that there are significant differences.DsLdRpt3 is with higher
Bioactivity, and the virulence of dsLdRpt3 is higher than dsATPaseE and dsATPaseA.
(5) potting feeding is tested
The dilution of dsLdRpt3, dsATPaseE, dsATPaseA, dsEGFP will be expressed on potting potato respectively indoors
10 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 larvaes
Free feeding, 6 repeating groups, each processing amount to 30 larvas.To recycle larva after a week, weighs and count the death rate, weight
5 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 into
Row 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.dsLdRpt3
The processing group larva rate of recovery < 5%, there are extremely significant difference (P < 0.05) with control group.14th day ultrapure water and expression dsEGFP
It dilutes 10 times of bacterium solution control group rate of recovery and is up to 72.5%, 57.5% respectively；The dsLdRpt3 processing group larva rate of recovery is 40%.
It is respectively 60%, 50% that 21st day ultrapure water and expression dsEGFP, which dilute 10 times of bacterium solution control group rate of recovery,；DsLdRpt3 processing
The group larva rate of recovery is less than 30%.28th day ultrapure water dilutes 10 times of bacterium solution control group rate of recovery with expression dsEGFP
62.5%, 65%；The dsLdRpt3 processing group larva rate of recovery is less than 20%.2 age of dsLdRpt3 processing group colorado potato bug children
Worm rate of recovery trend is below control group larva, as shown in Figure 7.Especially experiment the last fortnight tables of data reveals to the test worm rate of recovery
Significant inhibiting effect 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 7d
It 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 larvas
Average weight is respectively 0.149g, 0.135g.DsLdRpt3 processing group recycling larva average weight is less than 0.05g and control group
There 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 body
It is again respectively 0.129g, 0.122g；DsLdRpt3 processing group recycling larva average weight is less than 0.1g.21st day ultrapure water with
It is respectively 0.128g, 0.124g that expression dsEGFP, which dilutes 10 times of bacterium solution group recycling larva average weights, and dsLdRpt3 processing group is returned
Receiving larva average weight is that there are significant difference (P < 0.05) with control group by 0.095g.28th day ultrapure water and expression dsEGFP
Diluting 10 times of bacterium solution group recycling larva average weights is respectively 0.132g, 0.153g, and it is average that dsLdRpt3 processing group recycles larva
Weight is less than 0.1g.2 instar larvae body weight increase trend of dsLdRpt3 processing group colorado potato bug is below control group larva body
Weight growth 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,
With preferable lethal effect.
The result shows that the lethal effect of dsLdRpt3 is 28 days or more, preventive effect can reach as high as 80% or more, then
Decay to 50%.
(6) field plot trial
The dilution of dsLdRpt3, dsATPaseE, dsATPaseA, dsEGFP will be expressed respectively on the potato of field plot
10 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, each
5 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 daily
Pick 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 SPSS
Proceeding 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 is
8%, dsLdRpt3 processing group larval mortality more a height of 5.2%.Ultrapure water control in 14th day dilutes 10 times with expression dsEGFP
Bacterium solution processing group larval mortality is divided into 6%, 10%, and dsLdRpt3, dsATPaseE, dsATPaseA processing group death rate are higher than
Control group；Ultrapure water control in 28th day dilutes 10 times of bacterium solution processing group larval mortalities with expression dsEGFP and is divided into 2%, 6%,
The dsLdRpt3 processing group death rate is higher than control group, 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 hair
Made 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 model
It 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> dsLdRpt3
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 309
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gguccacuac agguuccaau uacuuuguuc guauucucuc cacaaucgau cgggaacuuu 60
ugaaaccuuc agcaaguguu gcucuucaca agcacaguaa ugcacuuguc gaugugcuuc 120
cuccagaagc agauuccucc auaaguaugc ugcaagcuga ugaaaaacca gauguacagu 180
auagugauau uggaggaaug gauaugcaga aacaagaaau uagagaggca guugaauuac 240
cacuuacuca uuuugagcua uacaaacaaa uugguauuga uccaccuaga gggguguuga 300
uguauggac 309
<210> 2
<211> 309
<212> DNA
<213>colorado potato bug (Leptinotarsa decemlineata)
<400> 2
ggtccactac aggttccaat tactttgttc gtattctctc cacaatcgat cgggaacttt 60
tgaaaccttc agcaagtgtt gctcttcaca agcacagtaa tgcacttgtc gatgtgcttc 120
ctccagaagc agattcctcc ataagtatgc tgcaagctga tgaaaaacca gatgtacagt 180
atagtgatat tggaggaatg gatatgcaga aacaagaaat tagagaggca gttgaattac 240
cacttactca ttttgagcta tacaaacaaa ttggtattga tccacctaga ggggtgttga 300
tgtatggac 309
Claims (10)
- Application of the 1.dsLdRpt3 in colorado potato bug prevents and treats, which is characterized in that the nucleotide sequence of dsLdRpt3 such as SEQ Shown in ID NO:1.
- 2. application according to claim 1, which is characterized in that dsLdRpt3 as potato beetle insecticidal agent activity at Point.
- 3. application according to claim 1, which is characterized in that with the bacterium of the transgenic engineered bacteria containing expression dsLdRpt3 Liquid is as potato beetle insecticidal agent.
- 4. application according to claim 3, which is characterized in that with the expression of Prokaryotic expression vector construction expression dsLdRpt3 Carrier.
- 5. potato beetle insecticidal agent, which is characterized in that potato beetle insecticidal agent effective component is dsLdRpt3, dsLdRpt3 Nucleotide sequence as shown in SEQ ID NO:1.
- 6. potato beetle insecticidal agent according to claim 5, which is characterized in that potato beetle insecticidal agent is to contain table Up to the bacterium solution of the transgenic engineered bacteria of dsLdRpt3.
- 7.dsLdRpt3 expression vector, which is characterized in that encode the DNA sequence dna of dsLdRpt3 as shown in SEQ ID NO:2, carrier For prokaryotic expression carrier.
- 8. dsLdRpt3 expression vector according to claim 7, which is characterized in that the prokaryotic expression carrier is pET- 2p。
- 9. expressing the transgenic engineered bacteria of dsLdRpt3, which is characterized in that with Escherichia coli Escherichia coli HTl15 It (DE3) is host, using dsLdRpt3 expression vector as transcriptional expression carrier.
- 10. colorado potato bug lethal gene dsLdRpt3, which is characterized in that the nucleotide sequence of dsLdRpt3 such as SEQ ID NO: Shown in 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274101A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of leptinotarsa decemlineata, method for constructing RNAi expression vector and application thereof |
| CN105980567A (en) * | 2013-07-19 | 2016-09-28 | 孟山都技术有限公司 | Compositions and methods for controlling leptinotarsa |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105980567A (en) * | 2013-07-19 | 2016-09-28 | 孟山都技术有限公司 | Compositions and methods for controlling leptinotarsa |
| CN105274101A (en) * | 2015-11-04 | 2016-01-27 | 重庆大学 | RNAi (ribonucleic acid interference) expression vector for TOR (target of rapamycin) genes of leptinotarsa decemlineata, method for constructing RNAi expression vector and application thereof |
Non-Patent Citations (3)
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| FU,K.: "KY285060.1", 《GENBANK》 * |
| JULIA ULRICH ET AL.: "Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target", 《BMC GENOMICS》 * |
| 周立涛: "马铃薯甲虫保幼激素合成与信号转导相关基因的克隆和功能分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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