CN104178490A - Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and vector and application thereof - Google Patents
Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and vector and application thereof Download PDFInfo
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Abstract
The invention discloses a Cereal cyst nematode RNAi (ribonucleic acid interference) site sequence for biological control, and a vector and application thereof. A RNA-seq sequencing technique and bioinformation science are combined to obtain the complete gene expression profile for Cereal cyst nematode infection resistant plants for the first time; and the three RNAi specific site sequences with lethal effect are detected in the relational data base: RCCNY1: SEQ ID NO.1 or RCCNY2: SEQ ID NO.2 or RCCNY3: SEQ ID NO.3.
Description
Technical field
The present invention relates to bioengineering field, particularly a kind of cereal cyst nematode RNAi site sequence for biological prevention and control and carrier and application.
Background technology
Cereal cyst nematode
claiming again Heterodera avenae, cereal cyst nematode, wheat, barley, rye, oat and multiple graminous pasture are had to serious harm, is the pathogenic nematode extensively existing in the world, especially the production of wheat is formed to serious threat.China from 1987 since Tianmen county, Hubei finds this disease, so far in North China, 13 provinces, autonomous regions and municipalities such as northwest, Central China and East China find that there is distribution, and have the gesture spreading gradually.The current main method of preventing and treating comprises cultural control, chemical pesticide control and biological control.China peasant mainly takes crop rotation, irrigates, enriches organic manure, ploughs deeply with Exposure to Sunlight soil, plantation trap plants and regulate the agrotechnical measure control nematodes such as date of seeding for a long time, but the crop of daily plantation is all the host of cereal cyst nematode substantially, thereby prevention effect is not remarkable.Chemical pesticide prevention and control nematode is one of effective means, but the pollution that it causes environment and agricultural-food highlights the method defect.And biological control at present report maximum be Nematophagous fungi and nemic natural enemy bacterium, but because of its DeGrain, commercialization difficulty, does not still have a kind of biological prevention and control agent extensively to be promoted so far.Therefore, searching safety and effective control of nematode approach are very necessary.
RNAi disturbs (RNA interference, RNAi) refer to by double-stranded RNA (double-strand RNA, dsRNA) cell internal specific induce the mRNA degraded of homologous complementary with it, the expression of corresponding gene is closed, thereby that causes transcribes or the gene silencing phenomenon (post-transcriptional gene silencing, PTGS) of post-transcriptional level.This phenomenon is biological for protecting autogene group to avoid the invasion and attack of external source (as virus) and endogenous (as transposable element) sequence, regulates specifically or disturb a kind of self-defense " immunity " of genetic expression to reply phenomenon.
In nematode research, find to exist the mechanism that RNAi signal can be amplified and transmits in nematode body, the dsRNA of trace just can guide body to produce strong RNAi effect, and can be delivered to filial generation.DsRNA can enter C.elegans adult by microinjection, also C.elegans adult can be immersed in the solution that contains dsRNA or feed and raise the intestinal bacteria that contain dsRNA.The dsRNA that injection enters is easy to diffusion in stem cell and causes RNAi, and this effect can be delivered to filial generation.The phenotype penetrance that this method produces is high, is applicable to very much the functional study of embryonic gene.But injection needs the operation of external synthetic dsRNA and manual injection, time-consuming, spend high and technical difficulty is large, be difficult to large-scale functional analysis.Infusion method is the dsRNA solution that nematode is soaked in to high density.Compare injection, infusion method can be processed a large amount of nematode materials simultaneously.But infusion method needs external synthetic a large amount of dsRNA (being generally 1-5mg/ml) equally, cost is very high.With injection and infusion method comparison, feeding feeding has its unique advantage.Feed feeding by building special carrier, abduction delivering dsRNA in bacterial body, then feed and raise nematode, can effectively suppress the expression of target gene, save trouble, and can test on a large scale.Thereby, if set up a RNAi bacterium feed raise library, the method just can be studied the function of large quantities of genes efficiently, thereby finds out the method for preventing and treating that can be used for nematode in agriculture production.
Cereal cyst nematode and root knot nematode belong to anchorage endoparasitism nematode, set up after parasitism female worm anchor at feeding site and no longer move with host.Owing to not cultivating on artificial medium, so it is more difficult directly to apply RNAi technology in parasitic nematode.The root knot nematode bodily form is little, and it is no longer practical that microinjection infection means become.In addition, their undesired absorption liquid before infection host plant also makes research to carry out.Thereby an important breakthrough is in recent years to find the method for carrying out RNAi technology on plant nematode.What successfully inject dsRNA to plant nematode first is the oral cavity that the application octopamines such as Urwin stimulate soybean cyst nematode Heterodera glycines (Heterodea glycines) and G.pallida (Globodera pallida), makes it inhale the dsRNA of L-Cysteine HCL Anhydrous, hgctl, three genes of the main albumen of sperm (MSP).The nematode infection plant of processing separates for 14 days afterwards, finds that the expression of three target genes is obviously suppressed, and change has also occurred corresponding epigenetic proterties.After optimizing, this method is also used to the research of root knot nematode.Bakhetia etc. are immersed in M.incognita larva in the dsRNA of coding peroxidase (dual oxidase), and result is invaded soybean nematode and detected afterwards for 35 days, and total egg laying amount has reduced 70%.The application RNAi technology such as Shingles knock out Mi-cpl-1 gene, have reduced its transcript abundance, and the cysteine protease activity of the J2s larva of M.incognita has also reduced.Chitin synthetase is that wild cabbage root knot nematode (Meloidogyne artiellia) generates the chitinous a kind of important enzyme of chorion.The proofs such as Fanelli are immersed in pieces of an egg in chitin synthetase dsRNA solution, can make the expression of chitin synthetase gene reduce, and ovum hatching postpones.In addition, Rosso etc. study discovery, and Resorcinol can stimulate the absorption of M.incognita to dsRNA, and have observed obvious RANi phenomenon.Except in vitro interference nematode, some scholar reaches the object that reduces root knot nematode parasitism by the transgenic plant of construction expression dsRNA.Yadav etc. have successfully disturbed growing of M.incognita RNAi for transgene tobacco, and showing to express dsRNA in host plant is a kind of possible strategy of controlling parasitic nematode disease.Huang etc. cross the parasitic gene 16D10 of expression root knot nematode in Arabidopis thaliana, and result root knot has reduced 63-90%, and volume is very little, and the output of pieces of an egg has also reduced 69-93%, has successfully reduced the parasitism of 4 kinds of root knot nematodes.Fairbairn etc. build transgene tobacco (Nicotiana tabacum) and express dsRNA hairpin structure, reticent javanese root knot nematode (Melododogyne javanica) transcription factor MjTis11.Although result does not have obviously to reduce the breeding amount of nematode and the hatching rate of ovum, prove that plant can be used as the gene silencing of a kind of transmission system induction root knot nematode RNAi mediation.As seen from the above, utilizing RNAi transgenic plant to prevent and treat parasitic nematode is a new control strategy, will bring new prospect for planting disease preventing and controlling.
But aforementioned research is all the correlative studys that concentrate on model animal C. Elegans Automatic Screening and phytotrophy root nematode, because the genome background of cereal cyst nematode is not yet illustrated, so there is no correlative study report.Along with transcribing in recent years the development of group sequencing technologies, utilize genome and transcribe the method that group technology combines to study plant parasitic nematodes to come into vogue.Thereby utilization transcribe group technology can high-throughout acquisition the candidate gene of relevant nematode further identify correlation function gene.The group of transcribing that we utilize the root that cereal cyst nematode (H.avenae) and root knot nematode (Meloidogyne naasi) are had to a variable goatweed of material of dual anti-effect to carry out the degree of depth checks order.By sequencing result is analyzed, removing plant, microorganism, after bacterium and carrier sequence, has obtained 846 of the genes involveds that cereal cyst nematode expresses in the time of infection resistance plant first.After further comparing with existing nematode database, remove and plant (plant), the gene of insect (insect) and the mankind (human) homology, finally obtains three and C. Elegans Automatic Screening (C.elegans) homology and has again the gene of lethal effect.Built on this basis dsRNA interference carrier, carried out dsRNA and soak interference experiment, finally found that three gene locuss all have lethal effect, wherein two genes have remarkable lethal effectiveness.
Summary of the invention
The problem existing for solving above-mentioned prior art, the object of the present invention is to provide a kind of cereal cyst nematode RNAi site sequence for biological prevention and control and carrier and application.
For achieving the above object, technical scheme of the present invention is:
For a cereal cyst nematode RNAi site sequence for biological prevention and control and nematode research, its sequence is: RCCNY1:SEQ ID NO.1 or RCCNY2:SEQ ID NO.2 or RCCNY3:SEQ ID NO.3.
Further, the construction process of described interference sequence is: select No. 1 conduct of variable goatweed with CCN resistance and RKN resistance to transcribe group order-checking material, obtain first cereal cyst nematode by RNA-seq sequencing technologies afterwards and infected the group of transcribing of plant, transcribe group order-checking, and pass through sequence alignment, find the RNAi of 36 cereal cyst nematodes to disturb site sequence based on nematode database, by further screening, get rid of and plant (Plant), the sequence of insect (Insect) and the mankind (Human) homology, three cereal cyst nematode RNAi site sequences with lethal effect are finally obtained.
A kind of rna interference vector, is characterized in that the cereal cyst nematode RNAi site sequence that described rna interference vector contains above-mentioned RCCNY1 or RCCNY2 or RCCNY3.
The application of a kind of cereal cyst nematode RNAi site sequence for biological prevention and control and nematode research aspect cereal cyst nematode research and biological prevention and control.
With respect to prior art, beneficial effect of the present invention is: the present invention synthesizes respectively target gene fragment RCCNY1 (Unigene38116), the RCCNY2 (Unigene102492) of cereal cyst nematode (CCN) and the dsRNA of RCCNY3 (Unigene38007) by in-vitro transcription, then by infusion method, the J2 larva of cereal cyst nematode (CCN) is carried out to RNAi experiment.This experiment finds that the long-time gathering of nematode is easily dead, adopts shaking table to cultivate suspension nematode solution, has greatly improved this situation.Found that dsRNA has obviously reduced the activity of larva, illustrate that target lethal effect gene has vital role in the growing of nematode.
Proving thus that the present invention adopts transcribes group technology and finds the method for correlation function gene studies gene function practical, for the full genome functions of extensive qualification species provides new thinking, simultaneously also first comparatively the qualification of system and screened cereal cyst nematode and resistance plant antagonism process in the genes involved of expressing.
Brief description of the drawings
Fig. 1. utilize the RNA interfering of gene specific to feed the survival rate of raising nematode after nematode 24h, the concentration of dsRNA is 25ng/ μ L, mark (a with letter with contrast (p<0.05) that there were significant differences that compare,, there is significant difference between tri-groups of data of c in b).
Fig. 2 is the dsRNA result of 3 cereal cyst nematode goal gene of the present invention, wherein, A: water, B: gene RCCNY1 (Unigene38116), C: gene RCCNY2 (Unigene102492), D: gene RCCNY3 (Unigene38007).
Fig. 3 is for transcribing group order-checking Unigene sequence alignment route map.
Fig. 4 is gene RCCNY1 (Unigene38116) sequencer map, and result demonstration sequencing sequence and PCR sequence are in full accord.
Fig. 5 is gene RCCNY2 (Unigene102492), and sequencer map result demonstration sequencing sequence and PCR sequence are in full accord.
Fig. 6 is gene RCCNY3 (Unigene38007) sequencer map, and result demonstration sequencing sequence and PCR sequence are in full accord.
Fig. 7 is the PCR electrophorogram based on transcribing the candidate Unigene gene fragment in group, wherein, M:DNA mark, 1: gene RCCNY1 (Unigene38116), 2: gene RCCNY2 (Unigene102492), 3: gene RCCNY3 (Unigene38007), 4: contrast.
Embodiment
Below in conjunction with the drawings and the specific embodiments, the present invention program is described in further detail:
Test example: one, experiment material and reagent
1. for examination nematode material
Cereal cyst nematode Shandong Feicheng colony (H.avenae, pathotype pathotype Ha43) is provided by professor Liu Feng of Shandong Agricultural University.The wheat root of being injured with collection and rhizosphere soil, get 200ml soil twists into pieces in plastic tub, with strong current flushing, stir, precipitation for a moment, is crossed 20 and 80 object mesh screens by the aqueous solution, repeat above step 3 time, clean gently 80 sieve residue with weep again, with filter paper filtering, the healthy available sporangiocyst of picking under anatomical lens.
2. main agents and enzyme
DNase I and RNaseH are purchased from Quan Shi King Company; High purity plasmid extraction kit is purchased from Promega; RNase Inhibitor, LITMUS281 carrier, t7 rna polymerase NTPmix are purchased from NEB company; Shuttle sodium carboxymethylcellulose pyce is purchased from Sigma company; Taq enzyme, DNA Marker, pGMTeasy connect test kit and are Quan Shi King Company product; Resorcinol is purchased from Beijing chemical reagent factory.
Two, analysis and experiment flow:
RNA-seq order-checking:
1. vegetable material: select the variable goatweed with CCN resistance and RKN resistance as transcribing group order-checking material, process and the expression of not inoculating all genes of root in two kinds of situations of CCN at inoculation CCN to measure it.First the seed of material being carried out to surface sterilization processing (soaks 3 times with aqua sterilisa soak 5min in the Tween20 mixed solution of the clorox of 3% concentration and 0.01% after again, again seed is evenly elaborated at the culture dish that continues to be placed on moistening filter paper 5cm diameter, issue seedling in the room temperature of 20 DEG C of left and right and the periodicity of illumination environment of 16h/8h.After ten days, seedling is divided into two groups: one group for inoculating the J2 larva (1000 of every strain inoculations) of CCN, another group does not connect worm and as the contrast of first group, the time point of inoculation CCN is defined as to 0h/dpi (hours or days post inoculation, hpi or dpi) simultaneously.After 30 hours (30hpi), by all plant forward to without under nematode and sterile environment to avoid the CCN that does not invade root to continue to infect, thereby make the development synchronization of synplasm (syncytia), guarantee that surrounding environment is that the room temperature of 20 DEG C of left and right and the periodicity of illumination of 16h/8h are to impel plant continued growth to grow simultaneously.
2. transcribe group order-checking: balanced mix inoculation cereal cyst nematode and do not connect two kinds of processing of worm and carry out Illumina HiSeq at the root RNA that connects after worm 30 hours, 3 days and 9 days
tMthe 2000 order-checking platform 4G degree of depth transcribe group de novo sequencing, use two package programs to splice sequencing data.Obtained 118,064 separate gene (unigene) by Trinity method, its mean length is that 500bp, N50 are 599bp, on average the degree of depth that checks order is 33.25 times.Further these separate gene are explained.
Nematode sequence screening:
By note obtain whole length exceed 200bp transcribe group database exist
Swiss-prot/trEMBL albumen database is compared, and exceedes 50 (bit score>50) as screening score threshold value taking score;
2. the whole nematodes that can download in pair public database (NCBI dbEST) are expressed
Sequence label (Nematode EST) was divided three classes based on life cycle: free nematode (FLN), animal parasitic nematodes (APN) and plant nematode (PPN).Albumen database is compared to postorder to be listed in and in nematode expression database, to carry out this locality comparison (tblastX) and further determine nematode genes involved, simultaneously also compare (blastN) by nucleotide sequence and sequence alignment of protein (blastX) (genome project websites (the http://www.inra.fr/meloidogyne_incognita that compares in the genome database of M.incognita and M.hapla, http://www.hapla.org), above comparison result is all selected best comparison value (Top hits).
Transcribe group order-checking Unigene sequence alignment route as shown in Figure 4.
The discovery in RNAi site:
1. nematode gene sequence prediction being obtained adopts local comparison, based on C. Elegans Automatic Screening radix
According to storehouse WormBase (http://www.wormbase.org, release WS227), the most similar gene order is remained as cereal cyst nematode candidate gene;
2. the RNAi site database of the gene aforementioned comparison being obtained and C. Elegans Automatic Screening is compared
(WormMart section of WormBase (release WS220)), select the gene (comparison score threshold value >40, bit score>40) in the site that can correspond to lethal effect;
By the aforementioned RNAi site sequence obtaining taking comparison score threshold value >40 (bit score>40) as standard, (plant nonredundant protein database further compares with the nonredundancy albumen database of plant, Embryophyta, NCBI txid3193), by compare less than the gene of (No hits) plant nonredundancy albumen database further and the nonredundancy albumen database of insect (insect) (nonredundant insect protein database (NCBI txid6960)) and the mankind's (human) nonredundancy albumen database (nonredundant human protein database (NCBI txid9606)) (score threshold value >40 compares, bit score>40), last only by lethal type RNAi (lethal RNAi) sequence, compare again the plant less than (No hits) simultaneously, the gene order of insect and mankind's nonredundancy albumen database is as candidate's RNAi site.
3. the pcr amplification of three cereal cyst nematode Gene Partial fragments
A. design of primers:
As shown in table 1, candidate's cereal cyst nematode is transcribed has homologous gene in lethal effectiveness gene order and C. Elegans Automatic Screening in group
Based on the candidate Unigene gene fragment (table 1) of transcribing in group, download the total length of corresponding gene from WormBase, based on the conserved regions of gene, utilize Premier5.0 design primer:
Unigene_38116_AS,
CAACCTGCACCGAATACTTCACTACAAA
Unigene38116_S,
ACCCTAAATAATGGAGACCTCACTAACG；
Unigene102492_AS,
ACAAGATGACGGAAATGGAAGAAGAGTT
Unigene102492_siRNA_S,
CGTTAGTGAGGTCTCCATTATTTAGGGT；
Unigene38007_siRNA_AS,
AAGAGCCAACAATCTCCGAGTTCTCCCT
Unigene38007_siRNA_S,
CACCAAGACCAACTACCGAACCACAAGA；
B.PCR amplification
Pcr amplification system
Pcr amplification program is as follows:
PCR electrophoresis result as shown in Figure 7.
The recovery of 4 amplified fragments, clone, order-checking
A. the recovery of amplified fragments
Use the recovery test kit of day root:
(1) with clean blade, target DNA band is scaled off from agarose gel, be placed in the centrifuge tube of 1.5ml, take weight;
(2) every 100mg glue adds 300 μ l sol solutionses, 50 DEG C of water-bath 10min, and constantly gentleness spins upside down centrifuge tube, until glue dissolves completely;
(3) previous step gained solution is added in an adsorption column CB (adsorption column is put into collection tube), the centrifugal 30sec of 12000r/min, outwells the waste liquid in collection tube;
(4) add 700 μ l rinsing liquid PW, the centrifugal 30sec of 12000r/min, discards waste liquid;
(5) add 500 μ l rinsing liquid PW, the centrifugal 30sec of 12000r/min, discards waste liquid;
(6) centrifugal adsorbing column is put back in collection tube, the centrifugal 2min of 12000r/min removes rinsing liquid as far as possible;
(7) adsorption column is put into a clean centrifuge tube, added the elution buffer EB of appropriate 65-70 DEG C preheating in adsorption film mid-way, room temperature is placed 2min.10
4the centrifugal lmin of r/min.Reclaim DNA be kept at-20 DEG C for subsequent use.
B. connect
Linked system:
C. transform
(1) (100 μ l), are placed on ice and dissolve to get competent cell;
(2) (10 μ l) join in competent cell, and rotation mixes content gently, is placed in 30min on ice, mixes once every 10min will to connect product with the aseptic suction nozzle of precooling;
(3) by centrifuge tube at 42 DEG C of water-bath 60-90sec, not shake, be then placed in immediately 2-3min on ice;
(4) add the SOC substratum of 37 DEG C of preheatings of 500 μ l, be placed in 37 DEG C of shaking table 180r/min and cultivate lh and make bacteria resuscitation, and the antibiotics resistance mark one of expression plasmid coding is remembered gene;
(5) competent cell of getting oneself conversion of 100 μ l-300 μ l is transferred on LB flat board (containing 50mg/L Amp, 16 μ l IPTG (50mg/L), 40 μ l X-gal (20mg/ml)), lightly cell is evenly coated with and is opened with an aseptic elbow glass rod;
(6) flat board is placed in to room temperature until liquid is absorbed, is inverted for 37 DEG C and cultivates 12-16h;
(7) screen blue hickie, picking hickie carries out bacterium liquid PCR, enzyme is cut qualification.
D. plasmid extraction
(l) 5ml is joined in the test tube of the good 15ml of ventilation containing corresponding antibiotic LB, access single bacterium colony, in 37 DEG C of shaking table 220r/min overnight incubation;
(2) get 1.5ml culture and join in 1.5ml centrifuge tube, the centrifugal 30sec of 12000r/min, outwells supernatant, collects thalline.Collect 2 times, suck for the last time nutrient solution, bacterial precipitation is dry as far as possible;
(3) add the solution I of 100 μ l precoolings, vibration, is suspended in solution I thalline, and room temperature is placed 10min;
(4) add the freshly prepared solution II of 200 μ l, put upside down and mix, ice bath 5min;
(5) add the solution III of 150 μ l precoolings, put upside down and mix, ice bath 5min;
(6) 12000r/min, 4 DEG C of centrifugal 10min;
(7) shift supernatant, add isopyknic chloroform, mix, room temperature is placed 5min, 4 DEG C of centrifugal 5min of 12000r/min;
(8) shift supernatant, add the Virahol of 0.7 times of volume, room temperature is placed 5min, 4 DEG C of centrifugal 15min of 12000r/min;
(9) abandon supernatant, by 70% washing with alcohol precipitation, the air-dry precipitation of room temperature;
E. order-checking
Entrust handsome company to carry out
Result as Figure 4-Figure 6.
5. vector construction
Carry out double digestion by containing the gene fragment carrier that order-checking is correct and carrier (LITMUS281) SacI and the Ncol enzyme of expressing dsRNA, acquisition has fragment and the LJTMUS281 carrier of identical restriction enzyme site, is convenient to fragment to be building up on LITMUS281 carrier.
Connect 12-16h with T4DNA Ligase16 DEG C, will connect product conversion TOP10, screen by blue hickie, select hickie and carry out double digestion and identify positive colony, so far successfully introduced T7 sequence two sections of goal gene.
6. high purity plasmid extraction
For obtaining high purity plasmid, adopt the recovery test kit of Promega company, concrete steps are as follows:
(1) the 3ml bacterium liquid gradation of shaking table overnight incubation is poured in 1.5ml centrifuge tube into the centrifugal 2min of 104r/min;
(2) outwell supernatant, add 300 μ l cell resuspension solution, thalline is mixed;
(3) add 300 μ l cell lysis solution, put upside down 4 times, mix;
(4) add 300 μ l neutralization solution, put upside down 4 times, mix;
(5) the centrifugal 5min of 104r/min;
(6) piston of 3-5ml syringe is taken off, more empty syringe is inserted on micro-column, in syringe, add 1ml resuspend resin;
(7) supernatant of the 5th step gained is transferred in the syringe that adds resin gently;
(8) load onto piston, push away gently solution in centrifugal mini column chromatography;
(9) take off syringe, extract piston out, then syringe is installed on pillar;
(10) add 2ml column wash solution, plug piston, push away slowly;
(11) take off syringe, l.5ml pillar is transferred in centrifuge tube to 10
4the centrifugal 2min of r/min;
(12) pillar is transferred in new centrifuge tube, added 50 μ l nuelease-free water (65 DEG C of water-baths), place lmin;
(13) 10
4the centrifugal 20sec of r/min;
(14) gained solution is kept at-20 DEG C.
Using the aforementioned plasmid being obtained as template, carry out pcr amplification with T7 primer (sequence is: 5 '-TAATACGACTCACTATAGG-3').
Pcr amplification system
Pcr amplification program is as follows:
PCR product is carried out to purifying, and purified product is as the template of in-vitro transcription
(1) PCR product is added to isopyknic phenol: chloroform, tip-tap mixes.4 DEG C, the centrifugal 15min of 12000r/min;
(2) shift supernatant in another centrifuge tube, add the dehydrated alcohol of 2 times of volumes of supernatant, 4 DEG C of centrifugal 10min of 12000r/min;
(3) outwell gently ethanol, then add 200 μ l75% washing with alcohol.4 DEG C of centrifugal 5min of 7500r/min, outwell supernatant, drying precipitated;
(4) add DEPC to process water dissolution precipitation DNA.
7. the synthetic dsRNA of in-vitro transcription
Using the aforementioned DNA being obtained as template, transcribe with t7 rna polymerase
37 DEG C of reaction 2h.React complete, after 70 DEG C of water-bath 15min, be cooled to room temperature.Finally, transcription product dsRNA is carried out to purifying:
(l) in reaction product, add 1 μ l DNase I and 1 μ l RNaseH, 37 DEG C of incubation 30min;
(2) add 95% ethanol of 0.1 times of volume sodium-acetate (3M, pH5.2) and 2.5 times of volumes, ice bath 10min, 4 DEG C of centrifugal 10min of 12000r/min;
(3) outwell gently supernatant, then add 500 μ l75% washing with alcohol.4 DEG C of centrifugal 10mins of 7500r/min, then outwell supernatant, drying precipitated;
(4) add DEPC to process water dissolution dsRNA, synthetic dsRNA is saved backup in-80 DEG C.
(5) ultraviolet spectrophotometer is measured the absorbancy at 260nm place, calculates reaction product concentration, the integrity of electrophoresis detection dsRNA on 1% sepharose.
8.dsRNA processes nematode infection experiment
As shown in Figure 2,3, the dsRNA building is fed and raises cereal cyst nematode (J2s) 24h under 25ng/ μ L concentration, number by dissecting microscope microscopy statistics living nematode and dead nematode calculates nematode survival rate, each experiment arranges two samples, statistical between sample adopts the Mann – Whitney U-test method in statistics software SPSS20, and significant difference value is (P<0.05).Find that three sequences compare according to all having more than 40% lethality rate, wherein two sequences has reached remarkable lethal (80%) especially.(Fig. 1)
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (4)
1. for a cereal cyst nematode RNAi site sequence for biological prevention and control and nematode research, it is characterized in that, its sequence is: RCCNY1:SEQ ID NO.1 or RCCNY2:SEQ ID NO.2 or RCCNY3:SEQ ID NO.3.
2. a kind of cereal cyst nematode RNAi site sequence for nematode research and biological prevention and control according to claim 1, it is characterized in that, the construction process of described interference sequence is: select No. 1 conduct of variable goatweed with CCN resistance and RKN resistance to transcribe group order-checking material, obtain first cereal cyst nematode by RNA-seq sequencing technologies afterwards and infected the group of transcribing of plant, transcribe group order-checking, and pass through sequence alignment, find the RNAi of 36 cereal cyst nematodes to disturb site sequence based on nematode database, by further screening, get rid of and plant, insect and human homology's sequence, three cereal cyst nematode RNAi site sequences with lethal effect are finally obtained.
3. a rna interference vector, is characterized in that, the cereal cyst nematode RNAi site sequence that described rna interference vector contains RCCNY1 claimed in claim 1 or RCCNY2 or RCCNY3.
4. the application of a kind of cereal cyst nematode RNAi site sequence for biological prevention and control and nematode research claimed in claim 1 aspect cereal cyst nematode research and biological prevention and control.
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| CN106939311A (en) * | 2017-05-16 | 2017-07-11 | 中国农业科学院植物保护研究所 | The genes of cereal cyst nematode Ha 56573 and its application |
| CN106957358A (en) * | 2017-05-16 | 2017-07-18 | 中国农业科学院植物保护研究所 | Cereal cyst nematode Ha34609 albumen, encoding gene and its application |
| CN106967164A (en) * | 2017-05-16 | 2017-07-21 | 中国农业科学院植物保护研究所 | The albumen of cereal cyst nematode Ha 63744, encoding gene and its application |
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| CN106518996B (en) * | 2017-01-13 | 2019-05-28 | 中国农业大学 | From the Ha-18764 albumen and its encoding gene of cereal cyst nematode and application |
| CN106518996A (en) * | 2017-01-13 | 2017-03-22 | 中国农业大学 | Heterodera avenae derived Ha-18764 protein and coding gene and application thereof |
| CN106939311B (en) * | 2017-05-16 | 2019-09-06 | 中国农业科学院植物保护研究所 | Cereal cyst nematode Ha-56573 gene and its application |
| CN106967164A (en) * | 2017-05-16 | 2017-07-21 | 中国农业科学院植物保护研究所 | The albumen of cereal cyst nematode Ha 63744, encoding gene and its application |
| CN107022017A (en) * | 2017-05-16 | 2017-08-08 | 中国农业科学院植物保护研究所 | The albumen of cereal cyst nematode Ha 16674, encoding gene and its application |
| CN107188941A (en) * | 2017-05-16 | 2017-09-22 | 中国农业科学院植物保护研究所 | The albumen of Ha 62292, encoding gene and its application of cereal cyst nematode |
| CN106957358A (en) * | 2017-05-16 | 2017-07-18 | 中国农业科学院植物保护研究所 | Cereal cyst nematode Ha34609 albumen, encoding gene and its application |
| CN106939311A (en) * | 2017-05-16 | 2017-07-11 | 中国农业科学院植物保护研究所 | The genes of cereal cyst nematode Ha 56573 and its application |
| CN106967164B (en) * | 2017-05-16 | 2019-12-06 | 中国农业科学院植物保护研究所 | Ha-63744 protein of heterodera avenae wollenweber, coding gene and application thereof |
| CN106957358B (en) * | 2017-05-16 | 2020-03-10 | 中国农业科学院植物保护研究所 | Ha34609 protein of heterodera avenae wollenweber, coding gene and application thereof |
| CN108727483A (en) * | 2018-05-25 | 2018-11-02 | 中国农业大学 | The HaGLAND5 albumen and its encoding gene of cereal cyst nematode and application |
| CN109762833A (en) * | 2019-02-24 | 2019-05-17 | 中国科学院成都生物研究所 | A kind of Aegilops varibilis Phenylalanine Ammonia-Lyase Gene and its application |
| CN112921013A (en) * | 2021-04-15 | 2021-06-08 | 中国农业科学院植物保护研究所 | Soybean cyst nematode chitin synthetase gene and application thereof |
| CN112921013B (en) * | 2021-04-15 | 2022-06-17 | 中国农业科学院植物保护研究所 | Soybean cyst nematode chitin synthetase gene and application thereof |
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