CN104178490B - For the cereal cyst nematode RNAi site sequences and its carrier of biological prevention and control and application - Google Patents

Abstract

It is used for cereal cyst nematode RNAi site sequences and its carrier and the application of biological prevention and control and nematode research the invention discloses a kind of, the present invention is by RNA seq sequencing technologies and combines bioinformatic analysis, gene expression profile when more complete cereal cyst nematode infection resistance plant is obtained for the first time, and three RNAi specific site sequences with lethal effect are have found in Relational database, its sequence is：RCCNY1：SEQ ID NO.1 or RCCNY2：SEQ ID NO.2 or RCCNY3：SEQ ID NO.3.

Description

For the cereal cyst nematode RNAi site sequences and its carrier of biological prevention and control and application

Technical field

It is more particularly to a kind of to be used for the cereal cyst nematode RNAi sites of biological prevention and control the present invention relates to bioengineering field Sequence and its carrier and application.

Background technology

Cereal cyst nematodeAlso known as Heterodera avenae, cereal cyst nematode, to wheat, barley, black Wheat, oat and a variety of graminous pastures have serious harm property, are the pathogenic nematodes being widely present in the world, especially to wheat Production constitutes serious threat.China from 1987 since Hubei Tianmen county finds the disease, so far North China, northwest, 13 provinces, autonomous regions and municipalities such as Central China and East China find to be distributed, and have the gesture gradually spread.Current main prevention and controls include agriculture Industry preventing and treating, chemical pesticide control and biological control.For a long time China peasant mainly take crop rotation, irrigate, increase organic manure, Plough deeply and Exposure to Sunlight soil, plantation trap plants and regulation date of seeding etc. agrotechnical measure preventing and treating nematode, but the work of daily plantation Thing is essentially all the host of cereal cyst nematode, thus prevention effect is not notable.Chemical pesticide prevention and control nematode is effective One of method, but the pollution that it is caused to environment and agricultural product is so that party's law limitation is highlighted.And biological control is reported at present Most is Nematophagous fungi and nemic natural enemy bacterium, but because of its DeGrain, commercialization is difficult, so far still without a kind of raw Anti- preparation is widely popularized.Therefore, find safe and effective control of nematode approach very necessary.

RNAi interference (RNA interference, RNAi) refers to by double-stranded RNA (double-strand RNA, dsRNA) Specifically induce the mRNA of homologous complementary therewith to degrade in the cell, close the expression of corresponding gene, so that triggers turns Record or the gene silencing phenomenon (post-transcriptional gene silencing, PTGS) of post-transcriptional level.It is this existing As if it is biological to protect autogene group from the invasion and attack of external source (such as virus) and endogenous (such as transposable element) sequence, it is specific Ground adjusts or disturbed a kind of self-defense " immune " response phenomenon of gene expression.

It is micro in nematode research, it has been found that there is the mechanism that RNAi signals can be amplified and be transmitted in nematode body DsRNA with regard to body can be guided to produce strong RNAi effects, and filial generation can be delivered to.DsRNA can pass through microinjection Into C.elegans adults, C.elegans adults can also be immersed in the solution containing dsRNA or be fed with to contain DsRNA Escherichia coli.The dsRNA that injection enters is easy to the diffusion in stem cell and causes RNAi, and can be by this effect It is delivered to filial generation.The phenotype genepenetrance that this method is produced is high, is especially suitable for the functional study of embryonic gene.But injection is needed To synthesize dsRNA and manual injection operation in vitro, it is time-consuming, spend that high and technical difficulty is big, it is very difficult to for large-scale function Analysis.Infusion method is the dsRNA solution that nematode is soaked in high concentration.Compared to injection, infusion method can be simultaneously to substantial amounts of Nematode material is handled.But, infusion method needs also exist for synthesizing substantial amounts of dsRNA (generally 1-5mg/ml), cost in vitro It is very high.Compared with injection and infusion method, feeding method has its unique advantage.Feeding method is by building special carrier, bacterial body Interior induced expression dsRNA, then be fed with nematode, can effective suppression target gene expression, save trouble, and can enter on a large scale Row experiment.Thus, if setting up a RNAi bacteriums feeding library, this method can efficiently study the function of large quantities of genes, So as to find out the prevention and controls available for nematode in agricultural production.

Cereal cyst nematode belongs to anchorage endoparasitism nematode with root-knot nematode, and female adult after parasitism is set up with host Feeding site is anchored at no longer to move.Due to can not be cultivated on synthetic medium, so directly being applied in parasitic nematode RNAi technology is relatively difficult.The root-knot nematode bodily form is small, and microinjection infection means become no longer practical.In addition, they are posted in infection Abnormal absorption liquid also causes research not carry out before main plant.Thus, an important breakthrough in recent years is to find The method that RNAi technology is carried out on plant nematode.Successfully inject dsRNA to plant nematode first is that Urwin etc. should Soybean cyst nematode Heterodera glycines (Heterodea glycines) and G.pallida (Globodera pallida) are stimulated with octopamine Oral cavity, make its inhale cysteine proteinase, hgctl, the dsRNA of the main albumen of sperm (MSP) three genes.Treated nematode Infect plant to separate after 14 days, it is found that the expression of three target genes is substantially suppressed, corresponding epigenetic character is also sent out Change is given birth to.After optimized, the method is also used for the research of root-knot nematode.Bakhetia etc. soaks M.incognita larvas As a result bubble detects, total spawning in coding peroxidase (dual oxidase) dsRNA after nematode invades soybean 35 days Amount reduces 70%.The application RNAi technology such as Shingles knocks out Mi-cpl-1 genes, reduces its Transcript abundance, The cysteine protease activity of M.incognita J2s larvas is also reduced.Chitin synthetase is wild cabbage root-knot nematode (Meloidogyne artiellia) generates a kind of important enzyme of chorion chitin.Fanelli etc. proves pieces of an egg to be immersed in It in chitin synthetase dsRNA solution, can reduce the expression of chitin synthetase gene, egg hatching delay.In addition, The research such as Rosso finds that resorcinol can stimulate absorptions of the M.incognita to dsRNA, and observed obvious RANi Phenomenon.In addition to interference nematode in vitro, some scholars reach reduction root knot line by construction expression dsRNA genetically modified plants The parasitic purpose of worm.RNAi is successfully disturbed growing for M.incognita, table by Yadav etc. for transgene tobacco The bright dsRNA that expressed in host plant is a kind of possible strategy of control parasitic nematodiasis.Huang etc. is overexpressed in arabidopsis Root-knot nematode parasitism gene 16D10, as a result root knot reduce 63-90%, and volume very little, the yield of pieces of an egg is decreased 69-93%, successfully reduces the parasitism of 4 kinds of root-knot nematodes.Fairbairn etc. builds transgene tobacco (Nicotiana Tabacum dsRNA hairpin structures, silence javanese root knot nematode (Melododogyne javanica) transcription factor) are expressed MjTis11.Although as a result not significantly reducing the breeding amount of nematode and the incubation rate of ovum, prove that plant can be as one kind The gene silencing of transmission system induction root-knot nematode RNAi mediations.As seen from the above, prevented and treated using RNAi genetically modified plants Parasitic nematode is a new control strategy, will bring new prospect for Plant disease control work.

But foregoing research is the correlative study for concentrating on model animal C. Elegans Automatic Screening and phytotrophy root nematode, due to standing grain The genome background of paddy cyst roundworm is not yet illustrated, so without correlative study report.With transcript profile sequencing technologies in recent years Development, be combined and come into vogue to study the method for plant parasitic nematodes using genome and transcript profile technology.Using turn Record group technology can further identify correlation function gene with the high-throughout candidate gene for obtaining related nematode.We utilize There is the variable mountain of the material of dual anti-effect to cereal cyst nematode (H.avenae) and root-knot nematode (Meloidogyne naasi) The root of sheep's hay has carried out the transcript profile sequencing of depth.By analyzing sequencing result, plant is being eliminated, microorganism, After bacterium and carrier sequence, the related gene 846 that cereal cyst nematode is expressed in infection resistance plant has been obtained first.Enter After one step and existing nematode database are compared, remove and plant (plant), insect (insect) and the mankind (human) homologous gene, finally obtain three and C. Elegans Automatic Screening (C.elegans) it is homologous and with lethal effect gene. DsRNA interference carriers are constructed on this basis, dsRNA immersion interference experiments are carried out, and finally find that three gene locis have There is lethal effect, two of which gene has notable lethal effectiveness.

The content of the invention

To solve the problem of above-mentioned prior art is present, it is used for the standing grain of biological prevention and control it is an object of the invention to provide a kind of Paddy cyst roundworm RNAi site sequences and its carrier and application.

To reach above-mentioned purpose, the technical scheme is that：

A kind of to be used for the cereal cyst nematode RNAi site sequences of 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 method of the interference sequence is：From the variable goat with CCN resistances and RKN resistances Material is sequenced as transcript profile in grass 1, has obtained first cereal cyst nematode by RNA-seq sequencing technologies afterwards and has infected plant The transcript profile of thing, carries out transcript profile sequencing, and by sequence alignment, 36 cereal cyst nematodes are have found based on nematode database RNAi interference site sequence, by further screening, eliminate and plant (Plant), insect (Insect) and the mankind (Human) homologous sequence, finally obtained three cereal cyst nematode RNAi site sequences with lethal effect.

A kind of rna interference vector, it is characterised in that the rna interference vector contain above-mentioned RCCNY1 or RCCNY2 or RCCNY3 cereal cyst nematode RNAi site sequences.

A kind of cereal cyst nematode RNAi site sequences for biological prevention and control and nematode research are ground in cereal cyst nematode Study carefully and biological prevention and control in terms of application.

Relative to prior art, beneficial effects of the present invention are：The present invention is respectively synthesized cereal sporangiocyst by in-vitro transcription Target gene fragment RCCNY1 (Unigene38116), RCCNY2 (Unigene102492) and the RCCNY3 of nematode (CCN) (Unigene38007) dsRNA, then carries out RNAi experiments by infusion method to the J2 larvas of cereal cyst nematode (CCN). This experiment finds that nematode assembles easily dead for a long time, using shaking table culture suspension nematode solution, substantially improves this situation. As a result find that dsRNA significantly reduces the activity of larva, illustrate that target lethal effect gene has in the growing of nematode Important function.

Thus prove that the method that the present invention finds correlation function gene studies gene function using transcript profile technology conscientiously may be used OK, new thinking is provided for Large scale identification species full-length genome function, while the also more identification and screening of system first Cereal cyst nematode and the related gene expressed in resistance plant opponent process.

Brief description of the drawings

Fig. 1 are using the survival rate of nematode after the RNA interfering feeding nematode 24h of gene specific, and dsRNA concentration is 25ng/ μ L, compare (the p that there were significant differences with control<0.05) mark that (it is poor to there is conspicuousness between tri- groups of data of a, b, c with letter It is different).

Fig. 2 is the dsRNA results of 3 cereal cyst nematode target gene of the invention, wherein, A：Water, B:Gene RCCNY1(Unigene38116)、C:Gene RCCNY2 (Unigene102492), D:Gene RCCNY3 (Unigene38007).

Fig. 3 is that Unigene sequence alignment route maps are sequenced in transcript profile.

Fig. 4 is gene RCCNY1 (Unigene38116) sequencer map, as a result shows sequencing sequence and PCR sequences complete one Cause.

Fig. 5 is gene RCCNY2 (Unigene102492), and sequencer map result shows sequencing sequence and PCR sequences complete one Cause.

Fig. 6 is gene RCCNY3 (Unigene38007) sequencer map, as a result shows sequencing sequence and PCR sequences complete one Cause.

Fig. 7 is the PCR electrophoretograms based on candidate's Unigene genetic fragments in transcript profile, wherein, M：DNA marker, 1：Base Because of RCCNY1 (Unigene38116), 2：Gene RCCNY2 (Unigene102492), 3：Gene RCCNY3 (Unigene38007)、4：Control.

Embodiment

Below in conjunction with the accompanying drawings and embodiment is described in further detail to the present invention program：

Test example：First, experiment material and reagent

1. for examination nematode material

Cereal cyst nematode Shandong Feicheng colony (H.avenae, pathological form pathotype Ha43) is by Shandong Agricultural University Professor Liu Feng provides.With aggrieved wheat root and rhizosphere soil is gathered, take 200ml soil to be twisted into pieces in plastic tub, use strong current Rinse, agitation, in precipitation a moment, the aqueous solution is crossed to the mesh screen of 20 and 80 mesh, repeat above step 3 times, then it is gently clear with weep 80 sieve residues are washed, are filtered with filter paper, the available sporangiocyst of picking health under anatomical lens.

2. main agents and enzyme

DNase I and RNaseH is purchased from Quan Shi King Companies；High-purity plasmid extraction kit is purchased from Promega；RNase Inhibitor, LITMUS281 carrier, t7 rna polymerase NTPmix are purchased from NEB companies；Shuttle sodium carboxymethylcellulose pyce is purchased from Sigma Company；Taq enzyme, DNA Marker, pGMTeasy connection kit are Quan Shi King Companies product；Resorcinol is purchased from Beijing Learn chemical reagent work.

2nd, analysis and experiment flow：

RNA-seq is sequenced：

1. vegetable material：Material is sequenced as transcript profile from the Aegilops varibilis with CCN resistances and RKN resistances, with Determine the expression of its all gene in root in the case of inoculation CCN is handled and is not inoculated with two kinds of CCN.First by the kind of material Son carries out surface sterilizing processing and (used again after soaking 5min in the sodium hypochlorite of 3% concentration and 0.01% Tween20 mixed liquors Aqua sterilisa soaks 3 times, then seed is uniformly elaborated is placed on the filter paper of constant moisture in the culture dish of 5cm diameters, at 20 DEG C The room temperature of left and right and 16h/8h periodicity of illumination environment issue seedling.Seedling is divided into two groups after ten days：One group is used to be inoculated with CCN J2 larvas (every plant be inoculated with 1000), another group do not connect worm and as first group of control, while CCN time point will be inoculated with It is defined as 0h/dpi (hours or days post inoculation, hpi or dpi)., will after 30 hours (30hpi) All plant are gone under no nematode and sterile environment to avoid the CCN of unimpinged root from persistently infecting, so that plasomidum (syncytia) development synchronization, while ensuring room temperature that surrounding environment is 20 DEG C or so and 16h/8h periodicity of illumination to promote Develop plant continued growth.

2. transcript profile is sequenced：Mixed in equal amounts is inoculated with cereal cyst nematode and does not connect two kinds of processing of worm 30 hours after worm is connect, 3 The root RNA of it and 9 days carries out Illumina HiSeq^TMThe transcript profile de novo sequencing of 2000 microarray dataset 4G depth, with two Individual assembly program splices to sequencing data.118,064 separate genes are obtained by Trinity method (unigene), its average length is that 500bp, N50 are that 599bp, average sequencing depth are 33.25 times.Further to these independences Gene is explained.

Nematode sequence screening：

1. the transcript profile databases for exceeding 200bp by explaining the whole length of acquisition exist

Swiss-prot/trEMBL albumen databases are compared, with score more than 50 (bit score>50) it is screening Score critical value；

2. the whole nematodes expression that can be downloaded in pair public database (NCBI dbEST)

Sequence label (Nematode EST) is divided into three classes based on life cycle：Free nematode (FLN), animal parasitic nematodes And plant nematode (PPN) (APN).Albumen database comparison postorder is listed in nematode expression database and locally compared (tblastX) nematode related gene is further determined that, simultaneously also by nucleotide sequence comparison (blastN) and protein sequence Row compare (blastX) and (genome project are compared in M.incognita and M.hapla genome database websites(http://www.inra.fr/meloidogyne_incognita,http://www.hapla.org), the above Comparison result selects optimal comparison value (Top hits).

Transcript profile sequencing Unigene sequence alignment routes are as shown in Figure 4.

The discovery in RNAi sites：

1. the nematode gene sequence that a prediction is obtained is using local comparison, based on C. Elegans Automatic Screening radix

According to storehouse WormBase (http://www.wormbase.org, release WS227), by gene the most similar Sequence is remained as cereal cyst nematode candidate gene；

2. the gene that foregoing comparison is obtained compares with the RNAi sites database of C. Elegans Automatic Screening

(WormMart section of WormBase (release WS220)), lethal effect can be corresponded to by selecting Site gene (compare score critical value>40, bit score>40)；

3. by foregoing obtained RNAi site sequences to compare score critical value>40(bit score>40) it is standard, enters One step compared with the non-redundant proteins database of plant (plant nonredundant protein database, Embryophyta, NCBI txid3193), the gene compared less than (No hits) plant non-redundant proteins database is entered one Step and non-redundant proteins database (the nonredundant insect protein database (NCBI of insect (insect) Txid6960)) and the mankind (human) non-redundant proteins database (nonredundant human protein database (NCBI txid9606)) (score critical value is compared>40, bit score>40) it is, last only by lethal type RNAi (lethal RNAi) sequence, while comparing the base less than (No hits) plant, insect and mankind's non-redundant proteins database again Because of RNAi site of the sequence as candidate.

3. the PCR amplifications of three cereal cyst nematode Gene Partial fragments

A. design of primers：

As shown in table 1, have in candidate's cereal cyst nematode transcript profile same in lethal effectiveness gene order and C. Elegans Automatic Screening Source gene

Based on the candidate Unigene genetic fragments (table 1) in transcript profile, the total length of correspondence gene is downloaded from WormBase, Conserved region based on gene, primer is designed using Premier5.0：

Unigene_38116_AS,

CAACCTGCACCGAATACTTCACTACAAA

Unigene38116_S,

ACCCTAAATAATGGAGACCTCACTAACG；

Unigene102492_AS,

ACAAGATGACGGAAATGGAAGAAGAGTT

Unigene102492_siRNA_S,

CGTTAGTGAGGTCTCCATTATTTAGGGT；

Unigene38007_siRNA_AS,

AAGAGCCAACAATCTCCGAGTTCTCCCT

Unigene38007_siRNA_S,

CACCAAGACCAACTACCGAACCACAAGA；

B.PCR is expanded

PCR amplification system

PCR amplification programs are as follows：

PCR electrophoresis results are as shown in Figure 7.

Recovery, clone, the sequencing of 4 amplified fragments

A. the recovery of amplified fragments

Use the QIAquick Gel Extraction Kit of Tiangeng:

(1) target DNA band is scaled off from agarose gel with clean blade, be placed in 1.5ml centrifuge tube, claimed Take weight；

(2) 300 μ l sol solutionses are added per 100mg glue, 50 DEG C of water-bath 10min constantly gently spin upside down centrifuge tube, until glue It is completely dissolved；

(3) previous step resulting solution is added into (adsorption column is put into collecting pipe), 12000r/min in an adsorption column CB 30sec is centrifuged, the waste liquid in collecting pipe is outwelled；

(4) add 700 μ l rinsing liquids PW, 12000r/min centrifugation 30sec, discard waste liquid；

(5) add 500 μ l rinsing liquids PW, 12000r/min centrifugation 30sec, discard waste liquid；

(6) centrifugal adsorbing column is put back in collecting pipe, 12000r/min centrifugation 2min remove rinsing liquid as far as possible；

(7) adsorption column is put into a clean centrifuge tube, appropriate 65-70 DEG C of preheating is added in adsorbed film centre position Elution buffer EB, room temperature place 2min.10⁴R/min centrifuges lmin.The DNA of recovery be stored in -20 DEG C it is standby.

B. connect

Linked system：

C. convert

(1) competent cell (100 μ l) is taken, is placed in and dissolves on ice；

(2) connection product (10 μ l) is added in competent cell with the sterile pipette tip of precooling, gently rotation mixes interior It is tolerant, 30min on ice is placed in, is mixed once every 10min；

(3) centrifuge tube should not be shaken in 42 DEG C of water-bath 60-90sec, 2-3min on ice is placed in immediately after；

(4) the SOC culture mediums of 500 μ l 37 DEG C of preheatings are added, being placed in 37 DEG C of shaking table 180r/min cultures lh makes bacteria resuscitation, And the note gene of antibiotic resistance mark one of expression plasmid coding；

(5) oneself competent cells of conversion of 100 μ l-300 μ l are taken to be transferred on LB flat boards (Amp containing 50mg/L, 16 μ l IPTG (50mg/L), 40 μ l X-gal (20mg/ml)), with a sterile elbow glass rod with gentle cell is uniformly spreadable；

(6) flat board is placed in room temperature until liquid is absorbed, 37 DEG C are inverted culture 12-16h；

(7) blue hickie is screened, picking hickie carries out bacterium solution PCR, digestion identification.

D. plasmid extraction

(l) LBs of the 5ml containing corresponding antibiotic is added in the test tube for the good 15ml that ventilates, single bacterium colony is accessed, in 37 DEG C shaking table 220r/min overnight incubations；

(2) 1.5ml cultures are taken to be added in 1.5ml centrifuge tubes, 12000r/min centrifugation 30sec outwell supernatant, collected Thalline.Collect 2 times, nutrient solution is sucked for the last time, bacterial precipitation is dried as far as possible；

(3) solution I of 100 μ l precoolings is added, vibration makes thalline be suspended in solution I, and room temperature places 10min；

(4) solution II of 200 μ l Fresh is added, overturns and mixes, ice bath 5min；

(5) solution III of 150 μ l precoolings is added, overturns and mixes, ice bath 5min；

(6) 12000r/min, 4 DEG C of centrifugation 10min；

(7) supernatant is shifted, isometric chloroform is added, mixed, room temperature places 5min, 4 DEG C of 12000r/min centrifuge 5min；

(8) supernatant is shifted, the isopropanol of 0.7 times of volume is added, room temperature places 5min, 4 DEG C of 12000r/min centrifugations 15min；

(9) supernatant is abandoned, precipitation is washed with 70% ethanol, room temperature air-dries precipitation；

E. it is sequenced

Handsome company is entrusted to carry out

As a result as Figure 4-Figure 6.

5. vector construction

Carrier (LITMUS281) SacI and Ncol that correct genetic fragment carrier and expression dsRNA is sequenced will be contained Enzyme carries out double digestion, obtains fragment and LJTMUS281 carriers with identical restriction enzyme site, is easy to fragment to be building up to On LITMUS281 carriers.

With T4DNA Ligase16 DEG C connection 12-16h, connection product is converted into TOP10, is screened, selected by blue hickie Hickie carries out double digestion and identifies positive colony, so far successfully introduces T7 sequences at two sections of target gene.

6. high-purity plasmid extraction

To obtain high-purity plasmid, using the QIAquick Gel Extraction Kit of Promega companies, comprise the following steps that:

(1) the 3ml bacterium solutions for staying overnight shaking table culture are poured into 1.5ml centrifuge tubes by several times, 104r/min centrifugations 2min；

(2) supernatant is outwelled, 300 μ l cell resuspension solution is added, mixes thalline；

(3) 300 μ l cell lysis solution are added, are overturned 4 times, are mixed；

(4) 300 μ l neutralization solution are added, are overturned 4 times, are mixed；

(5) 104r/min centrifuges 5min；

(6) piston of 3-5ml syringes is removed, then empty syringe is inserted on micro-column, 1ml is added into syringe resuspend resin；

(7) supernatant obtained by the 5th step is gently transferred to and added in resin syringe；

(8) piston is loaded onto, solution is gently pushed away into centrifugal mini column chromatography；

(9) syringe is removed, piston is extracted out, then syringe is attached on pillar；

(10) 2ml column wash solution are added, piston is plugged, pushes away slowly；

(11) syringe is removed, pillar is transferred in l.5ml centrifuge tube, 10⁴R/min centrifuges 2min；

(12) pillar is transferred in new centrifuge tube, adds 50 μ l nuelease-free water (65 DEG C of water-baths), Place lmin；

(13)10⁴R/min centrifuges 20sec；

(14) resulting solution is stored in -20 DEG C.

Using foregoing obtained plasmid as template, with T7 primers, (sequence is:5’-TAATACGACTCACTATAGG-3') Enter performing PCR amplification.

PCR amplification system

PCR amplification programs are as follows:

PCR primer is purified, purified product as in-vitro transcription template

(1) PCR primer is added to isometric phenol:Chloroform, tip-tap is mixed.4 DEG C, 12000r/min centrifugations 15min；

(2) transfer supernatant adds the absolute ethyl alcohol of 2 times of volumes of supernatant, 4 DEG C of 12000r/min in another centrifuge tube Centrifuge 10min；

(3) ethanol is gently outwelled, then adds the washing of 200 μ l75% ethanol.4 DEG C of 7500r/min centrifuge 5min, outwell supernatant, It is drying precipitated；

(4) DEPC processing water dissolving precipitations DNA is added.

7. in-vitro transcription synthesizes dsRNA

Using foregoing obtained DNA as template, transcribed with t7 rna polymerase

37 DEG C of reaction 2h.Reaction is finished, and in after 70 DEG C of water-bath 15min, is cooled to room temperature.Finally, by transcription product dsRNA Purified:

(l) 1 μ l DNase I and 1 μ l RNaseH, 37 DEG C of incubation 30min are added in the reaction product；

(2) 95% ethanol of 0.1 times of volume sodium acetate (3M, pH5.2) and 2.5 times of volumes is added, ice bath 10min, 4 DEG C 12000r/min centrifuges 10min；

(3) supernatant is gently outwelled, then adds the washing of 500 μ l75% ethanol.4 DEG C of 7500r/min centrifuge 10mins, then outwell Clearly, it is drying precipitated；

(4) DEPC processing water dissolving dsRNA are added, synthetic dsRNA is saved backup in -80 DEG C.

(5) ultraviolet specrophotometer determines the absorbance at 260nm, reaction product concentration is calculated, in 1% Ago-Gel Upper electrophoresis detection dsRNA integrality.

8.dsRNA processing nematode infection experiments

As shown in Figure 2,3, the dsRNA built is fed with cereal cyst nematode (J2s) 24h under 25ng/ μ L concentration, led to The number of disecting microscope microscopy statistics living nematode and dead nematode is crossed to calculate nematode survival rate, each two samples of Setup Experiments This, the statistical comparison between sample uses the Mann-Whitney U-test methods in statistics software SPSS20, significant difference It is worth for (P<0.05).It was found that three sequences have more than 40% fatal rate compared to control, two of which sequence is even more to reach Notable lethal (80%).(Fig. 1)

The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, any The change or replacement expected without creative work, should all be included within the scope of the present invention.Therefore, it is of the invention Protection domain should be determined by the scope of protection defined in the claims.

Claims (4)

1. a kind of be used for the cereal cyst nematode RNAi site sequences of biological prevention and control and nematode research, it is characterised in that its sequence For：RCCNY 1：SEQ ID NO.1.

2. a kind of cereal cyst nematode RNAi sites sequence for nematode research and biological prevention and control according to claim 1 Row, it is characterised in that the construction method of the sequence is：From No. 1 work of Aegilops varibilis with CCN resistances and RKN resistances Material is sequenced for transcript profile, having obtained first cereal cyst nematode by RNA-seq sequencing technologies afterwards infects turning for plant Record group, carries out transcript profile sequencing, and by sequence alignment, the RNAi of 36 cereal cyst nematodes is have found based on nematode database Site sequence is disturbed, by further screening, the sequence with plant, insect and human homology is eliminated, finally obtained tool There are the cereal cyst nematode RNAi site sequences of lethal effect.

3. a kind of rna interference vector, it is characterised in that the rna interference vector contains the standing grain of RCCNY 1 described in claim 1 Paddy cyst roundworm RNAi site sequences.

4. a kind of cereal cyst nematode RNAi site sequences for being used for biological prevention and control and nematode research described in claim 1 are in standing grain Application in terms of the research of paddy cyst roundworm and biological prevention and control.