CN109913456A - 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
Colorado Potato 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 dsLdalpha snap of the present invention in colorado potato bug prevents and treats, the nucleotides sequence of dsLdalpha snap
Column are as shown in SEQ ID NO:1.
Further, active constituent of the dsLdalpha snap as potato beetle insecticidal agent.
Preferably, use the bacterium solution for containing the transgenic engineered bacteria of expression dsLdalpha snap as potato beetle insecticidal
Agent.
Further, with the expression vector of Prokaryotic expression vector construction expression dsLdalpha snap.
Potato beetle insecticidal agent effective component of the present invention is dsLdalpha snap, the nucleotide of dsLdalpha snap
Sequence is as shown in SEQ ID NO:1.
Further, potato beetle insecticidal agent is the bacterium of the transgenic engineered bacteria containing expression dsLdalpha snap
Liquid.
DsLdalpha snap expression vector of the present invention encodes the DNA sequence dna such as SEQ ID NO:2 of dsLdalpha snap
Shown, carrier is prokaryotic expression carrier.
Further, the prokaryotic expression carrier is pET-2p.
The nucleotide sequence of colorado potato bug lethal gene dsLdalpha snap of the present invention is as shown in SEQ ID NO:1.
The present invention expresses the transgenic engineered bacteria of dsLdalpha snap, with Escherichia coli Escherichia coli
HTl15 (DE3) is host, using dsLdalpha snap 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 L1 be feed
DsLdalpha snap 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 L1 be feed
DsLdalpha snap 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 L1 be feed
DsLdalpha snap 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 L1 be feed
DsLdalpha snap 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 L1 is to feed
DsLdalpha snap 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 L1 is feeding dsLdalpha
Snap 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 L1 is feeding dsLdalpha snap bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium
Liquid.
Fig. 8 is long after potting one month of 2 instar larvae of colorado potato bug of 1 feeding dsLdalpha snap bacterium solution of embodiment
Gesture figure.
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 L1 is feeding dsLdalpha snap bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding
DsATPaseE bacterium solution.
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 L1 is feeding dsLdalpha snap bacterium solution, and L11 is feeding dsATPaseA bacterium solution, L12 is feeding dsATPaseE bacterium
Liquid.
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 dsLdalpha snap in colorado potato bug prevents and treats, wherein
The nucleotide sequence of dsLdalpha snap is as shown in SEQ ID NO:1.
Specific embodiment 2: the difference of present embodiment and specific embodiment one is: dsLdalpha snap makees
For 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 dsLdalpha snap is as potato beetle insecticidal agent.Other and embodiment one or two-phase
Together.
Specific embodiment 4: the difference of present embodiment and specific embodiment three is: described containing expression
The transgenic engineered bacteria of dsLdalpha snap is with Escherichia coli Escherichia coli HTl15 (DE3) for host, with
DsLdalpha snap 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 dsLdalpha
Snap expression vector 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 dsLdalpha
The DNA sequence dna of snap is 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 is dsLdalpha snap,
The nucleotide sequence of dsLdalpha snap is 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 dsLdalpha snap.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 dsLdalpha snap is with Escherichia coli Escherichia coli HTl15 (DE3) for host, with
DsLdalpha snap 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 dsLdalpha
Snap expression vector is prokaryotic expression 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 dsLdalpha snap expression vector, wherein encoding dsLdalpha
For the DNA sequence dna of snap 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 dsLdalpha snap, with large intestine bar
Bacterium Escherichia coli HTl15 (DE3) is host, using dsLdalpha snap expression vector as transcriptional expression carrier.
Specific embodiment 16: the difference of present embodiment and specific embodiment 15 is: the dsLdalpha
Snap 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 dsLdalpha snap 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 https under the genomic data of colorado potato bug: //
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 dsLdalpha snap).
(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.IPTG to final concentration of 0.1mmol/L, hair are added when bacterium solution expands culture to OD600=1.0 again
Ferment expression 6h can be obtained the dsLdalpha snap for stablizing that the concentration of expression is about 0.05 μ g/ μ L.Using Premier
Primer5.0 designs the primer of dsRNA segment, upstream primer 5 '-TTCGGTAGTTCTAGTCGT-3 ', downstream primer 5 '-
AATAGTCTGGTGGTGCTT-3 ', dsLdalpha snap fragment length are 288bp.
(4) indoor feeding experiment
Through feeding the dsLdalpha snap of colorado potato bug second instar larvae various concentration, with ultrapure water and dsEGFP,
(LdATPaseE and LdATPaseA were delivered in 2007 in Nature Biotechnology by LdATPaseE and LdATPaseA
Disclosed in " potato of lustrous and transparent chrysomelid and colorado potato bug the transgene expression dsRNA of prevention and control corn root and the gene of corn ") be
Control, and analyze 20% feeding dosage AD20,7 days lethal dose of 50 LD50 of middle amount PD50 and larva of pupating to larva.
After continuous feeding expresses the bacterium solution of dsLdalpha snap, dsATPaseE, dsATPaseA for one week, colorado potato bug
Larva shows the case where 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
Larval feeding rate on μ g and 5 μ g processing blade in being decreased obviously trend, it is extremely significant lower than ultrapure water group, dsEGFP processing group and
The feeding rate of larva on low concentration processing group blade.
After when handling for 24 hours, dsLdalpha snap of 50 μ g of dosage, dsATPaseE, dsATPaseA group blade feeding rate
Not significant (P > 0.05) with feeding rate difference on dsEGFP processing group blade, feeding rate is 25% hereinafter, hazard rating is I；
DsLdalpha snap, dsATPaseE, dsATPaseA group and the dsEGFP group feeding rate difference of 0.05 μ g of dosage it is not significant (P >
0.05), but variant (P < 0.05) with the feeding rate of other treatment dosage groups.After handling 72h and 96h, dsLdalpha snap,
DsATPaseE, dsATPaseA processing group larval feeding amount increase with number of days in the trend that is decreased obviously, the higher inhibition reaction of concentration
It is stronger, and be then in continue to rise in ultrapure water group and dsEGFP processing group larval feeding rate, close to even up to IV grades of feeding
Cause harm, experimental result 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.DsLdalpha snap anti-food rate regression equation is Y=
5.873x-3.064,20% dosage of feeding (AD20) are 1.51 ± 0.06 μ g, and extension rate is 33.2 ± 0.7 times；dsATPaseE
Anti-food rate regression equation is Y=4.076x-1.71, and 20% dosage of feeding (AD20) is 3.47 ± 0.06 μ g, and extension rate is
14.4 ± 0.8 times；DsATPaseA anti-food rate regression equation is Y=3.46x-1.187, and 20% dosage of feeding (AD20) is 5.29
± 0.27 μ g, extension rate are 9.5 ± 3.2 times.
DsLdalpha snap, dsATPaseE, dsATPaseA processing group interference after larva reduce feeding even stop into
Food, 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 burying
Toxic action, concentration be 50 μ g/mL under high killing activity is shown to 2 instar larvae of colorado potato bug, lethality is up to
90%~93%.DsLdalpha snap processing group dsRNA concentration be 5 μ g/mL under lethality be more than 50%, killing activity with
Concentration increases and increases, and shows 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, dsLdalpha snap death rate regression equation be Y=3.45x-1.593, the lethal dose of 50
It (LD50) is 3.52 ± 0.36 μ g, extension rate is 14.2 ± 0.7 times；DsATPaseE death rate regression equation is Y=
4.681x-2.245, the lethal dose of 50 (LD50) are 4.54 ± 0.54 μ g, and extension rate is 11 ± 0.3 times；The dsATPaseA death rate
Regression equation is Y=4.308x-2.183, and the lethal dose of 50 (LD50) is 3.08 ± 0.23 μ g, and 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,
DsLdalpha snap processing group concentration is to colorado potato bug mature larva percentage of pupation under 50 μ g/mL down to 0%,
DsLdalpha snap processing group percentage of pupation in the case where dosage is 5 μ g is less than 50%, even if dosage is also compared down to 0.05 μ g
According to the percentage of pupation for reducing larva 20~30%, 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
Be 7 days, dsLdalpha snap percentage of pupation regression equation be Y=5.073x-3.443, middle amount of pupating (PD50) be 0.39 ±
0.01 μ g, extension rate are 128.4 ± 5.7 times；DsATPaseE percentage of pupation regression equation is Y=3.21x-2.261, is pupated
Measuring (PD50) is 0.46 ± 0.02 μ g, and extension rate is 109.8 ± 15.9 times；DsATPaseA percentage of pupation regression equation is Y=
3.551x-2.428, middle amount of pupating (PD50) are 0.50 ± 0.09 μ g, and extension rate is 100.8 ± 11.7 times.
The result shows that: dsLdalpha snap, which has colorado potato bug larva, to be inhibited feeding, inhibits to pupate and killing activity.
Its killing activity increases with concentration and is increased, and shows dose-dependent effect, and compares that there are significant differences.dsLdalpha
Snap bioactivity with higher, and the virulence of dsLdalpha snap is higher than dsATPaseE and dsATPaseA.
(5) potting feeding is tested
DsLdalpha snap, dsATPaseE, dsATPaseA, dsEGFP will be expressed on potting potato respectively indoors
10 times of bacterium solutions of dilution (concentration be 5 μ g/mL) be sprayed on potting, after air drying, every plant of potato places 5 colorado potato bugs two
The free feeding of instar larvae, 6 repeating groups, each processing amount to 30 larvas.To recycle larva after a week, weighs and count dead
Rate reapposes 5 second instar larvaes, repeats to test weekly, take 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.
There are extremely significant difference (P < 0.05) with control group down to 0 for the dsLdalpha snap processing group larva rate of recovery.14th day ultrapure
Water and expression dsEGFP dilute 10 times of bacterium solution control group rate of recovery and are up to 72.5%, 57.5% respectively；At dsLdalpha snap
The reason group larva rate of recovery is less than 20%, and there are significant difference (P < 0.05) with control group.21st day ultrapure water and expression
It is respectively 60%, 50% that dsEGFP, which dilutes 10 times of bacterium solution control group rate of recovery,；The dsLdalpha snap processing group larva rate of recovery
It is 52.5%.It is respectively 62.5%, 65% that 28th day ultrapure water and expression dsEGFP, which dilute 10 times of bacterium solution control group rate of recovery,；
The dsLdalpha snap processing group larva rate of recovery is 45%.2 instar larvae of dsLdalpha snap processing group colorado potato bug returns
Yield trend is below control group larva, as shown in Figure 7.Especially experiment the last fortnight tables of data reveals significant to the test worm rate of recovery
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.DsLdalpha snap processing group recycle larva average weight down to 0.02g with it is right
According to group, there are extremely significant difference (P < 0.05).It is flat that 14th day ultrapure water and expression dsEGFP dilute 10 times of bacterium solution group recycling larvas
Equal weight is respectively 0.129g, 0.122g；DsLdalpha snap processing group recycling larva average weight is less than 0.1g.21st
It is respectively 0.128g, 0.124g that its ultrapure water and expression dsEGFP, which dilute 10 times of bacterium solution groups recycling larva average weights,
DsLdalpha snap processing group recycling larva average weight is less than 0.05g, and there are significant difference (P < 0.05) with control group.
It is respectively 0.132g, 0.153g that 28th day ultrapure water and expression dsEGFP, which dilute 10 times of bacterium solution groups recycling larva average weights,
DsLdalpha snap processing group recycling larva average weight is less than 0.1g.DsLdalpha snap processing group colorado potato bug
2 instar larvae body weight increase trend are below control group larval weight growth trend, as shown in Figure 9.Especially test the last fortnight number
According to showing to the significant inhibiting effect of test worm body weight increase, with preferable lethal effect.
The result shows that the lethal effect of dsLdalpha snap is 28 days or more, preventive effect can reach as high as 80% with
On, then decay to 50%.
(6) field plot trial
DsLdalpha snap, dsATPaseE, dsATPaseA, dsEGFP will be expressed respectively on the potato of field plot
10 times of bacterium solutions of dilution, to field carry out bacterium solution spray.Colorado potato bug second instar larvae is placed in 9cm normal glass ware weekly
In, 5 larvas of each processing, 6 groups of biology repeat.It is fed with the processed blade in crop field is picked up from.Replacement feeding daily
Blade 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%, dsLdalpha snap processing group larval mortality more a height of 8%, 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> dsLdalpha snap
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 288
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
uucgguaguu cuagucguau cgaagaugca gucgaauguu aucagagagc ugcuaaucuu 60
uuuaaaaugg ccaaaagcug ggauucugca gggagcgcuu ucugcgaagc agccaaccuu 120
caucuacgaa guggggcacg ucaugaugcu gcuacaaauu auguagaugc ugcaaauugu 180
uacaagaagg cagauaucaa cgaagcuguu aauuguuuau uaaaagcuau agaaauauac 240
acagacaugg guagauuuac uauggcggcc aagcaccacc agacuauu 288
<210> 2
<211> 288
<212> DNA
<213>colorado potato bug (Leptinotarsa decemlineata)
<400> 2
ttcggtagtt ctagtcgtat cgaagatgca gtcgaatgtt atcagagagc tgctaatctt 60
tttaaaatgg ccaaaagctg ggattctgca gggagcgctt tctgcgaagc agccaacctt 120
catctacgaa gtggggcacg tcatgatgct gctacaaatt atgtagatgc tgcaaattgt 180
tacaagaagg cagatatcaa cgaagctgtt aattgtttat taaaagctat agaaatatac 240
acagacatgg gtagatttac tatggcggcc aagcaccacc agactatt 288
Claims (10)
- Application of the 1.dsLdalpha snap in colorado potato bug prevents and treats, which is characterized in that the nucleosides of dsLdalpha snap Acid sequence is as shown in SEQ ID NO:1.
- 2. application according to claim 1, which is characterized in that dsLdalpha snap is as potato beetle insecticidal agent Active constituent.
- 3. application according to claim 1, which is characterized in that with the transgenic engineering containing expression dsLdalpha snap The bacterium solution of bacterium is as potato beetle insecticidal agent.
- 4. application according to claim 3, which is characterized in that express dsLdalpha snap with Prokaryotic expression vector construction Expression vector.
- 5. potato beetle insecticidal agent, which is characterized in that potato beetle insecticidal agent effective component is dsLdalpha snap, The nucleotide sequence of dsLdalpha snap is 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 dsLdalpha snap.
- 7.dsLdalpha snap expression vector, which is characterized in that the DNA sequence dna such as SEQ ID of coding dsLdalpha snap Shown in NO:2, carrier is prokaryotic expression carrier.
- 8. dsLdalpha snap expression vector according to claim 7, which is characterized in that the prokaryotic expression carrier For pET-2p.
- 9. expressing the transgenic engineered bacteria of dsLdalpha snap, which is characterized in that with Escherichia coli Escherichia coli HTl15 (DE3) is host, using dsLdalpha snap expression vector as transcriptional expression carrier.
- 10. colorado potato bug lethal gene dsLdalpha snap, which is characterized in that the nucleotide sequence of dsLdalpha snap As shown in SEQ ID NO:1.
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|---|---|---|---|---|
| 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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2019
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| 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 |
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| FU,K.: "KY285070.1", 《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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