CN104404048B - The method for effectively preventing Agricultural Mites with RNAi - Google Patents
The method for effectively preventing Agricultural Mites with RNAi Download PDFInfo
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- CN104404048B CN104404048B CN201210501540.9A CN201210501540A CN104404048B CN 104404048 B CN104404048 B CN 104404048B CN 201210501540 A CN201210501540 A CN 201210501540A CN 104404048 B CN104404048 B CN 104404048B
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Abstract
The present invention relates to field of biotechnology, and in particular to the method for one energetic supersession gene prevention and treatment tetranychid of silencing.According to the method for the present invention the following steps are included: choosing tetranychid lethal gene AK is RNAi target gene；It is connected in plasmid vector L4440, conversion to Escherichia coli HT115 obtains the bacterial strain with expression AK gene dsRNA.The present invention is the important supplement to the Control of Pest Mites method based on insecticide.The present invention provides technical support using the expression of the RNAi technology interference intracorporal energy metabolism related genes of tetranychid for prevention and treatment tetranychid.
Description
Technical field
The invention belongs to agricultural biological technical fields；More particularly it relates to based on RNAi technology prevention and treatment agricultural evil
The target gene and its dsRNA of mite describe that target gene is synthesized double-stranded RNA preparation and directly applies to Agricultural Mites and prevent
The method controlled.
Technical background
In China, method is effectively prevented not yet to Agricultural Mites at present, spraying insecticide is still main means of prevention.
The lavishment of highly toxic and high-persistent pesticide does not only result in crop product quantity decline, the increase of harmful mite drug resistance, and has jeopardized people
Life security and health.Therefore, there is an urgent need to develop strategy safely, effectively, novel to control harmful mite population in production
Harm.
RNAi phenomenon has rapidly developed since discovery in 1991, studies have shown that by the RNAi of specific gene, Ke Yida
To the target gene of orientation interference species, there are certain physiological phenomenons, achievees the purpose that study gene function, meanwhile, this phenomenon
There is a high specificity, i.e. the interference effect that is played different plant species of the specific fragment of homologous gene is not identical, therefore
It is a kind of countermeasure system that ideal pest species are special.
It has been widely used for the function of research insect genes so far by the RNAi that dsRNA is induced, but has been applied to
The research of mite class it is few (Khila, 2007；Campbell, 2010), this is primarily due to mite class individual small (body is mostly in 1 milli
Meter or less), it is difficult using RNAi molecule technical operation.Khila etc. (2007) injects abdomen, success silencing two using dsRNA
The Distal-less gene of class tetranychid (Tetranychus urticae) leads to tetranychid pedipalp truncation deformity and attached foot fusion.
The dsRNA molecule that green fluorescent protein GFP is marked is injected into the abdomen of female adult mite, and also can be detected in the ovum that it is produced,
Show that RNAi system is also likely to be present in mite class to spread through sex intercourse.Campbell etc. (2011) uses 0.9%NaCl solution infusion method, at
Function silencing-Di Shi watts of the mites of parasitics mite (Varroa destructor) of apis mellifera (Apis mellifera)
GlutathioneS-transferase gene, specific silence efficiency reach 87% on transcriptional level.Invention
It is exactly that can result in tetranychid death after interfering from filtering out in Agricultural Mites using the improved dsRNA interference method in laboratory
Important gene, for establish using RNA perturbation technique control harmful mite new strategy sequence and data basis are provided.
Summary of the invention
The purpose of the present invention is the harm of needle Agricultural Mites, the pest-resistant preparation based on RNAi technology and pest-resistant method are provided.
The first aspect of the present invention is to provide the dsRNA and its synthetic method of lethal gene segment AK.
The first aspect of the present invention can be achieved through the following technical solutions:
Agricultural Mites lethal gene segment AK, sequence is as shown in SEQ ID NO.1.
The cloning process of the tetranychid lethal gene segment AK, includes the following steps:
(1) tetranychid is taken, total serum IgE is extracted, first chain of cDNA is synthesized with the tetranychid total serum IgE of extraction；
(2) using first chain of tetranychid cDNA of step (1) synthesis as template, drawn with the upstream that sequence is SEQ ID NO.2
Object P1, the downstream primer P2 that sequence is SEQ ID NO.3 carry out RT-PCR amplification；
(3) PCR product is separated through agarose gel electrophoresis, recycles target DNA fragments；
(4) target DNA fragments of recycling are entered in T3 connection enzyme effect underthrust into PMD18-T carrier, is transformed into large intestine
Bacillus Top-10, is applied to the LB culture medium containing Amp, and 37 DEG C of cultures are stayed overnight；
(5) bacterium solution PCR is identified, screens positive recombinant Fig. 1；
(6) recon is expanded with LB culture solution with ampicillin, extracts cloned plasmids；
(7) full-automatic sequence instrument is sequenced, and obtains AK genetic fragment shown in SEQ ID NO.1.
Wherein, the RT-PCR amplification system are as follows: 10 × Ex PCR Buffer, 2.5 μ L, 2.5mM dNTP
Mixture 2.0 μ L, 10 μM of upstream primer P1 1.0 μ L, 10 μM of 1.0 μ L, cDNA template of downstream primer P2,3 μ L, 5U/ μ L Ex
0.25 μ L, ddH2O, 15.25 μ L of Taq, totally 25 μ L；PCR response procedures are as follows: 94 DEG C of denaturation 5min, 94 DEG C of 1min, 56 DEG C of 1min,
72 DEG C of 1min, 32 circulations, 72 DEG C of extensions.
The dsRNA of tetranychid lethal gene segment AK.
Based on the target gene functional area that analysis clone obtains, design special primer expands the positive double-strand of acquisition respectively
DNA fragmentation and reversed double chain DNA fragment；Use external efficient transcription kit (HiScribe RNAi Transcription
Kit) to two double-stranded DNA target segments of acquisition are transcribed in vitro, annealing synthesizes dsRNA, special dsRNA Fig. 1 is constructed.
Another aspect of the present invention is to provide the building of recombinant plasmid vector and its expression of dsRNA.
The building of the recombinant plasmid vector is realized by following methods:
(1) the recombinant plasmid PMD18-T-AK cloned in first aspect present invention is extracted, is carried out with HindIII and Sac II
Double digestion obtains two cohesive terminus,cohesive terminis.
(2) digestion products are separated through agarose gel electrophoresis, recycle target DNA fragments.
(3) same that double digestion is carried out to L4440 plasmid with HindIII and Sac II, obtain the identical glutinous type end with (1)
End.
(4) digestion products are separated through agarose gel electrophoresis, target DNA fragments are recycled, as carrier.
(5) (2) resulting target fragment is connect with (4) resulting L4440 plasmid vector segment with T4DNA ligase,
It is transformed into Escherichia coli HT115, is applied to the dual anti-LB plate containing (Amp+Tet), 37 DEG C of cultures are stayed overnight；
(6) picking single colonie is inoculated in 2ml LB culture medium (Amp+Tet), and 37 DEG C are incubated overnight, and extracts plasmid and carries out
Digestion identification.
Selection identifies correct bacterium solution 0.75ml and 80% glycerol of 0.25ml is added, and -80 DEG C save.
Another aspect of the present invention is the expression for inducing Escherichia coli HT115 to carry out dsRNA.
It is double that the HT115 bacterium solution containing L4440-AK plasmid of above-mentioned preservation with 1: 100 volume is inoculated in (Amp+Tet)
In anti-2 × YT culture medium, 37 DEG C, 250rpm cultivates to OD595 ≈ 0.4.
Sterile IPTG to final concentration of 0.4mM is added in above-mentioned bacterium solution, induces 4h in 37 DEG C, 250rpm.
Identify the dsRNA of inducing expression.
Another aspect of the present invention is to provide the dsRNA interference method of several tetranychids,
It is slide drop method that another aspect of the present invention tetranychid dsRNA, which interferes first method, and specific steps include:
(1) double faced adhesive tape is cut into 2cm long, is attached to glass slide one end.
(2) the female adult mite of selection health, is gently provoked with small size writing brush, its back is sticked on adhesive tape, be careful not to glue
Firmly foot, pedipalp and mouthpart.
(3) every is glued 30, lines up three rows, and then slide is placed in clean nontoxic and wet pallet, is placed in 28 ± 1
Incubator at DEG C.
(4) torpescence and dead individuals are chosen after 0.5h under binocular anatomical lens and supply 30 it is spare.
(5) appropriate dsRNA is added dropwise toward every cephalont body back side under stereomicroscope with 2.5 μ L liquid-transfering gun of specification.
Death standard: mite body is touched gently with small size writing brush, the motionless person of appendage is death.
It is dsRNA bilateral tube method that another aspect of the present invention tetranychid dsRNA, which interferes second method, and specific steps include:
(1) first add one piece of Parafilm sealed membrane in glass two-way pipe one end, be then added in man-made feeds thereon, then past
Another piece of new Parafilm sealed membrane is covered thereon.
(2) tetranychid to be tested then is put into double-way pipe.
(3) it is finally operated again by method described in (1) step toward the glass two-way pipe other end again.
(4) addition finishes, and it is a micro- (depending on concrete condition) respectively to pierce 20 on the double-deck Parafilm sealed membrane at both ends with needle point
Hole, so as to air and water flow；
(5) tetranychid is placed on growth cabinet raising, toward one block of black cloth of glass tube central cover, so that tetranychid can go to both ends to inhale
Man-made feeds are eaten, per the feed changed for 24 hours once containing dsRNA.
Wherein, the dsRNA is the dsRNA of the tetranychid lethal gene segment AK.
The utility model has the advantages that
Using RNAi technology silencing AK gene, there is apparent lethal effect to tetranychid, illustrate that the gene can be used as utilization
The Effective target site of RNA perturbation technique control pest.
The AK gene dsRNA energy effective reticence AK gene that the present invention synthesizes closes simultaneously better against the degradation of RNA enzyme
At cost is relatively low, used convenient for many experiments.
The present invention carries out RNAi interference experiment using slide drop method and double-way pipe, and relative injection method reduces polypide
Mechanical damage also greatly reduces the dosage of dsRNA, in addition, this experimental construction vivoexpression carrier of dsRNA Escherichia coli,
To reduce experimental expenses, experimental implementation is simplified, solves the experiment insufficient key of dsRNA amount.It is tested eventually by drop
The RNAi for demonstrating AK gene has lethal effect to tetranychid, establishes and is provided using the new strategy of RNA perturbation technique control pest
New laboratory facilities.
Detailed description of the invention
Fig. 1 dsRNA synthesizes electrophoretogram, swimming lane 1:Marker, the dsRNA of swimming lane 2,3:AK；The dsRNA of Fig. 2 various concentration
The corrected mortality of polypide after interference.
Fig. 3, survival rate of the O.turkestanicumvar. tuberculata in different disposal different time.
Specific embodiment
Embodiment 1
The cloning process of 1.AK genetic fragment:
(1) tetranychid 100-150 head is taken, extracts total serum IgE with TRIzol method；
(2) first chain of cDNA is synthesized；
(3) gene fragment order is obtained from tetranychid transcript profile, is carried out in http://www.ncbi.nlm.nih.gov/
After sequence analysis, it is predicted as AK gene, using Primer premier 5.0 software design P1 and P2, with RT-PCR method
It is expanded；
Upstream primer (P1): 5 ' GCGACTTGTGAACCTG 3 ' (SEQ ID NO.2),
Downstream primer (P2): 5 ' TCTGCTGACGCTGAAA 3 ' (SEQ ID NO.3)；
PCR response procedures are as follows: 94 DEG C of denaturation 5min, 94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min, 32 circulations, 72 DEG C are prolonged
It stretches.
PCR reaction system (25 μ L):
(4) PCR product is separated through agarose gel electrophoresis, recycles target DNA fragments；
(5) target fragment of recycling is entered in T3 connection enzyme effect underthrust into pMD18-T carrier, is transformed into Escherichia coli
HD115 (DE), is applied to the LB culture medium of the 100 μ g/ml containing ampicillin, and 37 DEG C of cultures are stayed overnight；
(6) by bacterium solution PCR, positive recombinant is screened；
(7) recon is expanded with LB culture solution with ampicillin, extracts cloned plasmids；
(8) full-automatic sequence instrument is sequenced and (is completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd)
Obtain the AK genetic fragment of nucleotide sequence shown in SEQ IDNO.1.
The building of 2. recombinant plasmid vector of embodiment and its expression of dsRNA
(1) the recombinant plasmid PMD18-T-AK cloned in first aspect present invention is extracted, is carried out with HindIII and SacII
Double digestion obtains two cohesive terminus,cohesive terminis.
(2) digestion products are separated through agarose gel electrophoresis, recycle target DNA fragments.
(3) same that double digestion is carried out to L4440 plasmid with HindIII and SacII, it obtains and (1) identical glutinous type end.
(4) digestion products are separated through agarose gel electrophoresis, target DNA fragments are recycled, as carrier.
(5) (2) resulting target fragment is connect with (4) resulting L4440 plasmid vector segment with T4DNA ligase,
It is transformed into Escherichia coli HT115, is applied to the dual anti-LB plate containing (Amp+Tet), 37 DEG C of cultures are stayed overnight；
(6) picking single colonie is inoculated in 2ml LB culture medium (Amp+Tet), and 37 DEG C are incubated overnight, and extracts plasmid and carries out
Digestion identification.
Selection identifies correct bacterium solution 0.75ml and 80% glycerol of 0.25ml is added, and -80 DEG C save.
It is double that the HT115 bacterium solution containing L4440-AK plasmid of above-mentioned preservation with 1: 100 volume is inoculated in (Amp+Tet)
In anti-2 × YT culture medium, 37 DEG C, 250rpm cultivates to OD595 ≈ 0.4.
Sterile IPTG to final concentration of 0.4mM is added in above-mentioned bacterium solution, induces 4h in 37 DEG C, 250rpm.
Identify the dsRNA of inducing expression.
Embodiment 3.dsRNA drop method
(1) double faced adhesive tape is cut into 2cm long, is attached to glass slide one end.
(2) the female adult mite of selection health, is gently provoked with small size writing brush, its back is sticked on adhesive tape, be careful not to glue
Firmly foot, pedipalp and mouthpart.
Every 30 viscous, lines up three rows, and then slide is placed in clean nontoxic and wet pallet, is placed in 28 ± 1 DEG C
Lower incubator.
(3) torpescence and dead individuals are chosen after 0.5h under binocular anatomical lens and supply 30 it is spare；
(4) appropriate dsRNA is added dropwise toward every cephalont body back side under stereomicroscope with 2.5 μ L liquid-transfering gun of specification.
(5) death standard: mite body is touched gently with small size writing brush, the motionless person of appendage is death.
(6) continuous feeding 48h the results are shown in Table 1 and Fig. 2, by the feeding visible with Fig. 2 of table 1 per a death rate is counted for 24 hours
The dsRNA of lethal gene Tubulin can reach preferable lethal effect.
Table 1
5% level difference conspicuousness (Duncan ' s duncan's new multiple range method) is indicated with lowercase characters different after column of figure in table.
Embodiment 4.dsRNA bilateral tube method
(1) first add one piece of Parafilm sealed membrane in glass two-way pipe one end, be then added in different man-made feeds thereon,
It is past again to cover another piece of new Parafilm sealed membrane thereon.
(2) tetranychid to be tested then is put into double-way pipe.
(3) it is finally operated again by method described in (1) step toward the glass two-way pipe other end again.
(4) addition finishes, and it is a micro- (depending on concrete condition) respectively to pierce 20 on the double-deck Parafilm sealed membrane at both ends with needle point
Hole, so as to air and water flow；
(5) tetranychid is placed on growth cabinet raising, toward one block of black cloth of glass tube central cover, so that tetranychid can go to both ends to inhale
Man-made feeds are eaten, per the feed changed for 24 hours once containing dsRNA.
(1) man-made feeds described in are formula are as follows:
Bacterial concentration remarks
500 × AK 10ul 1000 × express AK-dsRNA HT115 and 10ul cowpea tissue extract mix
50 × AK 10ul 1000 × express AK-dsRNA HT115 and 10ul cowpea tissue extract mix
500 × HT 10ul 1000 × HT115 containing empty carrier is mixed with 10ul cowpea tissue extract
50 × HT 10ul 1000 × HT115 containing empty carrier is mixed with 10ul cowpea tissue extract
CK 10ul ddH2O and 10ul cowpea tissue extract mix
Claims (1)
1. a kind of dsRNA double-way pipe interference method of tetranychid, is characterized mainly in that, includes the following steps:
(1) first add one piece of Parafilm sealed membrane in glass two-way pipe one end, be then added in man-made feeds thereon, then toward thereon
Cover another piece of new Parafilm sealed membrane；
(2) tetranychid to be tested then is put into double-way pipe；
(3) it is finally operated again by method described in (1) step toward the glass two-way pipe other end again；
(4) addition finishes, and 20 micropores is respectively pierced on the double-deck Parafilm sealed membrane at both ends with needle point, so as to air and moisture
Flowing；
(5) tetranychid is placed on growth cabinet raising, toward one block of black cloth of glass tube central cover, so that tetranychid can go to both ends to suck people
Work feed, per the feed changed for 24 hours once containing dsRNA；
The feed containing dsRNA be 10 μ L 1000 × the HT115 for expressing AK-dsRNA and 10 μ L cowpea tissue extracts
It mixes；The bacterium solution of the HT115 for expressing AK-dsRNA the preparation method is as follows:
1) cloning process of tetranychid lethal gene segment AK:
A. tetranychid is taken, total serum IgE is extracted, first chain of cDNA is synthesized with the tetranychid total serum IgE of extraction；
B. upstream primer P1, sequence using first chain of tetranychid cDNA of step A synthesis as template, with sequence for SEQ ID NO.2
The downstream primer P2 for being classified as SEQ ID NO.3 carries out RT-PCR amplification；
C.PCR product is separated through agarose gel electrophoresis, recycles target DNA fragments；
D. the target DNA fragments of recycling are entered in T3 connection enzyme effect underthrust into pMD18-T carrier, is transformed into Escherichia coli
HT115, is applied to the LB culture medium containing ampicillin, and 37 DEG C of cultures are stayed overnight；
E. by bacterium solution PCR, positive recombinant is screened；
F. recon is expanded with LB culture solution with ampicillin, extracts cloned plasmids；
G. full-automatic sequence instrument is sequenced, and obtains AK genetic fragment shown in SEQ ID NO.1；
2) building of recombinant plasmid vector and its expression of dsRNA:
A. recombinant plasmid PMD18-T-AK carries out double digestion with HindIII and SacII, obtains two cohesive terminus,cohesive terminis；
B. digestion products are separated through agarose gel electrophoresis, recycle target DNA fragments；
C. same that double digestion is carried out to L4440 plasmid with HindIII and Sac II, obtain cohesive terminus,cohesive termini identical with step a；
D. digestion products are separated through agarose gel electrophoresis, target DNA fragments are recycled, as carrier；
E. the resulting target fragment of step b is connect with the resulting L4440 plasmid vector segment of step d with T4DNA ligase, is turned
Change into Escherichia coli HT115, be applied to the dual anti-LB plate containing Amp+Tet, 37 DEG C of cultures are stayed overnight；
F. picking single colonie is inoculated in LB culture medium of the 2ml containing Amp+Tet, and 37 DEG C are incubated overnight, and extracts plasmid and carries out digestion
Identification, selection identify correct bacterium solution 0.75ml and 80% glycerol of 0.25ml are added, and -80 DEG C save；
G. it is dual anti-the HT115 bacterium solution containing L4440-AK plasmid that step f is saved to be inoculated in Amp+Tet with 1: 100 volume
2 × YT culture medium in, 37 DEG C, 250rpm cultivates to OD595 ≈ 0.4；
H. sterile IPTG to final concentration of 0.4mM is added in above-mentioned bacterium solution, induces 4h in 37 DEG C, 250rpm；
I. the dsRNA of inducing expression is identified.
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| CN109929871B (en) * | 2019-03-11 | 2023-10-03 | 中国林业科学研究院高原林业研究所 | Method for mediating double-stranded RNA to enter Chinese purple beetle body |
| CN116814641A (en) * | 2023-02-28 | 2023-09-29 | 山西农业大学 | Belle target gene for preventing and controlling spider mites of hawkthorn, application thereof, dsRNA synthesized by Belle target gene and application of dsRNA |
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| CN101084760A (en) * | 2006-06-07 | 2007-12-12 | 北京农学院 | Walnut leaf plant miticide and preparation method thereof |
| WO2011045333A1 (en) * | 2009-10-14 | 2011-04-21 | Vib Vzw | Method to control spider mites |
| CN102220340A (en) * | 2011-05-12 | 2011-10-19 | 南京农业大学 | Gene silencing technique based Laodelphax striatellus lethal gene fragment Tubulin and dsRNA thereof |
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