CN109468336A

CN109468336A - Brown paddy plant hopper protein phosphatase gene NlPP1, albumen and its dsRNA and application

Info

Publication number: CN109468336A
Application number: CN201811493977.6A
Authority: CN
Inventors: 王渭霞; 赖凤香; 朱廷恒; 万品俊; 傅强
Original assignee: China National Rice Research Institute
Current assignee: Zhejiang Fuwo Agricultural Biotechnology Co.,Ltd.
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2019-03-15
Anticipated expiration: 2038-12-07
Also published as: CN109468336B

Abstract

The present invention relates to the albumen (SEQ ID NO.2) that brown paddy plant hopper protein phosphatase gene NlPP1 (SEQ ID NO.1), the gene encode, and dsRNA (SEQ ID NO.3) and its application in prevention and treatment brown paddy plant hopper insect pest according to gene design.The present invention provides a kind of brown paddy plant hopper protein phosphatase gene NlPP1, and the dsRNA according to the gene chemical synthesis.The gene plays critical function in maintaining the normal growth and development of brown paddy plant hopper and reproductive process, and function, which is suppressed, will lead to brown paddy plant hopper nymphal development and be obstructed, and final dead.The present invention provides new control strategy and target site for brown paddy plant hopper prevention and treatment, is provided the foundation to establish using RNA perturbation technique control pest, has preferable application prospect.

Description

Brown paddy plant hopper protein phosphatase gene NlPP1, albumen and its dsRNA and application

(1) technical field

The present invention relates to the albumen that brown paddy plant hopper protein phosphatase gene NlPP1, the gene encode, and are designed according to the gene DsRNA and its prevention and treatment brown paddy plant hopper insect pest in application.

(2) background technique

Rice is the most important cereal crops of one of which now in the world, is accounted in the world by the population of staple food of rice More than half of population.Brown paddy plant hopper (the Nilaparvata l μ gens of Homoptera Delphacidae)(Brown Planthopper) be special feeding rice phloem sap oligophagy pest, have concealment, it is sudden, explosive, ruin Going out property the features such as.They usually reside in rice basal part of stem or fringe portion, and piercing and sucking rice strain juice excretes poison, transmitted virus, sternly Rice clump base portion dark brown when endangering again, gradually complete stool is withered.Often there is " the yellow pool ", " emit and wear ", " lodging " etc. among killed rice field Classical symptom is gradually expanded in flakes, causes full field famine withered when serious.Therefore, rice pest planthopper is efficiently controlled to rice Volume increase is of great significance.

Cultivating resistant rice varieties is generally acknowledged environment amenable brown paddy plant hopper control strategy the most cost-effective, cut-off Though there are 29 resistance gene of brown planthopper to be identified from long-grained nonglutinous rice, japonica rice and wild rice respectively at present, wherein 14 genes by It is pin-pointed in 3,4,6 and No. 12 specific chromosome regions of rice, 5 genes are cloned.But much more research Show that certain resistance gene is solely imported by molecular marking supplementary breeding is equally easy to that new brown paddy plant hopper is caused to cause evil property population Occur and due to lacking effective Resistance resource, rice can not be significantly improved to brown by solely importing existing resistant gene The resistance of plant hopper.It polymerize different resistant genes at present into the developing direction of Rice Resistance plant hopper breeding.And it is actually answering at present Chemical insecticide is still with the primary method of middle control planthopper harm.Chemical insecticide is main point from the point of view of its mechanism of action For nervous system toxic agent and interference metabolism toxic agent two major classes.The prevention and treatment most common chemical insecticide of brown paddy plant hopper mainly has anabasine Insecticide such as imidacloprid, Nitenpyram, Diacloden.Carbamic acid insecticides Bassa, Mobucin and Buprofezin and pyrrole aphid Ketone.Wherein most mainly acts on acetylcholinergic receptor, has nerve block effect to the synaptic receptor of pest.Buprofezin category It is metabolized class toxic agent in interference, blocks pest development by inhibiting insect chitin synthesis.And pymetrozine can produce lancet resistance Filling in effect is that pest is dead because of starvation.Chemical insecticide such as continues largely to use at many-sided reason for a long time, gives brown paddy plant hopper Very big resistance to insecticides is caused to select pressure, the brown paddy plant hopper of many countries and regions produces difference to common chemical insecticide The drug resistance of degree.The decline of target sensibility, the enhancing etc. for enzyme effect of detoxifying is the main reason for brown paddy plant hopper develops drug resistance.Cause This, finding new insecticidal action mode and target site is the key that break through chemical insecticide application bottleneck.

Recent study shows that the double-stranded RNA of some short-movie sections can be special using the mRNA of sequence homology complementation as target It is different, efficiently degrade mRNA, blocks the expression of specific gene, lure into cells show go out specific gene missing phenotype, referred to as RNA It interferes (RNA interferenceRNAi).RNA interference is the skill of the inhibition of gene expression rapidly developed in recent years Art.The function that indispensable gene is developed and bred in insect can be specifically inhibited to lead to pest development and breeding in turn using the technology It is obstructed, due to this pesticidal hybridization pairing for being to rely on RNA sequence that RNAi is mediated, the mutation of a small amount of nucleotide can't A possibility that effect of influence RNAi, insect will overcome this resistance, is also just relatively small, therefore RNAi technology is in pest control It has broad application prospects in system.And a key factor is for RNAi target gene sequence in using RNAi control pest The selection of column.Target gene must be the key gene of insect, while want continuous expression in the entire history of life of insect.It is another A key be target-gene sequence selection must between species conservative it is low, to avoid undershooting-effect.Although in Lepidoptera cotton DsRNA silencing insect genes are generated to reach pest control by transgenic plant in earworm and diabroticavirgifera coleoptera Purpose it has been reported that but due to lack pest functional gene is understood in depth, realize currently with the technology to pest Control action also can not show a candle to the plant of trans Bt gene.Pest is controlled using the genetically modified plants of expression dsRNA, first choice needs It filters out suitably for the target gene for being transferred to plant, this gene reduces at transcriptional level can cause pest development It is obstructed, leads to the high death rate.The sequence of the gene and target plant and other insects especially natural enemy etc. are beneficial to elder brother simultaneously Worm etc. is without the similar sequence of continuous 20bp or more height.

Serine/threonine protein phosphatase (serine/threonine phosphatases, STPs) is eucaryote The important executor of horizontal dephosphorylation after vivo transcription.It can remove the phosphorus being connected on phosphorylation serine threonine residue Acid groups, presence corresponding with protein kinase together constitute phosphorylation and dephosphorylation this important protein active Switching system participates in many important life processes.Wherein serine/threonine protein phosphatase 1 (serine/threonine Protein phosphatase 1, PP1) be the family important member, in eucaryote participate in cell division, protein The adjusting of the various kinds of cell biological processes such as synthesis, cytoskeleton recombination and signal transduction.The present invention passes through in clone brown paddy plant hopper PP1 gene, analyze its sequence signature and by synthesis double-stranded RNA (double RNA dsRNA) import brown paddy plant hopper Immune inducing in vivo The RNAi of the gene observes individual phenotype and survival rate after silencing, prevents and treats for brown paddy plant hopper and provides new control strategy and effectively Target site.

(3) summary of the invention

It is an object of the present invention to provide a kind of serine/threonine protein phosphatase 1 NlPP1 cloned from Brown Planthopper The cDNA sequence of gene, the dsRNA and protein sequence of the sequence and its application in prevention and treatment brown paddy plant hopper insect pest are The research and development of against-plant hopper transgenic paddy rice provide new gene source.

The technical solution adopted by the present invention is that:

A kind of brown paddy plant hopper protein phosphatase gene NlPP1, nucleotide sequence is as shown in SEQ ID NO.1.

SEQ ID NO.1 sequence is as follows:

The protein amino acid sequence of gene coding is as shown in SEQ ID NO.2.

SEQ ID NO.2 sequence is as follows:

MADIEKLNIDSVIARLLEVRGGRPGRNVQLLESEIRGLCLKSREIFLSQPILLELEAPLKICGDIHGQY YDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHECASINRIYGFYDECKRRYNI KLWKTFTDCFNCLPVSAIVDEKIFCCHGGLSPDLHSMEQIRRIMRPTDVPDQGLLCDLLWSDPDKDTMGWGENDRGV SFTFGVEVVAKFLHKLDFDLICRAHQVVEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPAD KRKFTYGGLNVGRPVTPPRGATNKNKKK

Brown paddy plant hopper protein phosphatase gene NlPP1 cloning process of the present invention is as follows:

1) brown paddy plant hopper is taken, total serum IgE is extracted, synthesizes first chain of cDNA；

2) first chain synthesized using step 1 is template, using NlPP1-F as upstream primer, using NlPP1-R as downstream primer Carry out RT-PCR amplification；

NlPP1-F:5 '-CTCACTTCGTTCGTTCGCATT-3 '；

NlPP1-R:5 '-AGACCTACTCCAGGTAGCCTT-3 '；

3) PCR product is separated through agarose gel electrophoresis, recycles the target DNA fragments at 1262bp；

4) recycling segment T3 connection enzyme effect underthrust enter toIn 2.1 carriers, it is transformed into escherichia coli DH5a, is applied In containing X-gal, the LB culture medium of IPTG and kanamycins, 37 DEG C are incubated overnight；

5) white single colonie is selected, is expanded positive colony with the LB culture solution containing kanamycins, extracts cloned plasmids pTOP-NlPP1；

6) plasmid pTOP-NlPP1 is sequenced, and obtains NlPP1 genetic fragment shown in SEQ ID NO.1.

Wherein RT-PCR amplification system are as follows: include the cDNA, upstream and downstream primer each 20pmol, 0.2mM after 3.0 μ L dilution The r-Taq DNApolymerase (Takara) of dNTP, 1 × PCR buffer, 2.5units, totally 25 μ L.PCR amplification program Are as follows: 95 DEG C of denaturation 2min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 90s of 35 circulations；Last 72 DEG C of extensions 10min。

The invention further relates to the dsRNA of brown paddy plant hopper protein phosphatase gene NlPP1, nucleotide sequence such as SEQ ID NO.3 It is shown.

SEQ ID NO.3 sequence is as follows:

The dsRNA synthetic method is as follows:

1) according to the NlPP1 gene fragment order having verified that, simultaneously synthetic primer dsNlPP1-F:TAATACGA is separately designed CTCACTATAGGGTTTCGAGTACGGAGGATTCCC and dsNlPP1-R:TAATACGACTCACTATAGGGGCATTGGCAAC TTCTCTCCAC.Using the plasmid pTOP-NlPP1 of the gene containing NlPP1 as template, using dsNlPP1-F as upstream primer, with DsNlPP1-R is that downstream primer carries out PCR amplification, to obtain the dsDNA segment of the entitled dsNlPP1 containing T7 promoter.

2) PCR product of step 1 acquisition is separately recovered, using this amplified production as template, is transcribed in vitro synthesis NlPP1's dsRNA.Synthetic system are as follows: template about 1.5-2.0 μ g, 2.0 μ L 10 × T7buffer, 2.0 μ L ATP, 2.0 μ L GTP, 2.0 μ L CTP, 2.0 μ L UTP, 2.0 μ L T7 enzyme, totally 20.0 μ L.37 DEG C keep the temperature 5 hours, and 72 DEG C inactivate 5 minutes；Synthesize dsRNA After degradation removal DNA and single stranded RNA, purifying obtains dsRNA.

Wherein plasmid PCR amplification system are as follows: 1.0 μ L Plasmid DNA, upstream and downstream each 20pmol, 0.2mM dNTP, 1 × PCR The r-Taq DNA polymerase (Takara) of buffer, 2.5units, totally 25 μ L.PCR amplification program are as follows: 95 DEG C of denaturation 2min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 40s of 35 circulations；Last 72 DEG C of extensions 10min.

The invention further relates to application of the brown paddy plant hopper protein phosphatase gene NlPP1 in prevention and treatment brown paddy plant hopper insect pest.Experiment It was found that by 3 age brown paddy plant hoppers of the dsRNA microinjection of NlPP1 synthesis or feeding, when dsRNA concentration is higher than 50ng/uL, injection The 4th day brown paddy plant hopper nymph death rate reaches significant difference compared with the control afterwards, and the nymph death rate is up to 80% or more at the 7th day.When When injection dsRNA concentration is down to 0.05ng/uL, the 10th day brown paddy plant hopper survival rate with compare that there were significant differences.Utilize feeding method The silencing gene equally can cause brown paddy plant hopper survival rate to be remarkably decreased, when feeding dsRNA concentration is in 600ng/uL, feeding the Brown paddy plant hopper survival rate is lower than 10% within 6 days.The above experimental result prompts the dsRNA to can be used for preparing the drug for preventing and treating brown paddy plant hopper.

Specifically, the application are as follows: dsRNA is designed according to the brown paddy plant hopper protein phosphatase gene NlPP1, with described The drug of dsRNA preparation prevention and treatment brown paddy plant hopper.

Preferably, the dsRNA nucleotide sequence is as shown in SEQ ID NO.3.

The invention further relates to the brown paddy plant hopper protein phosphatase gene NlPP1 answering in building brown planthopper resistant transgenic paddy rice With.Gene order involved in the gene is cloned into plant RNAi expression vector, by means of turning for the mediations such as Agrobacterium Gene means are transferred in rice, and gained transgenic paddy rice can synthesize the dsRNA of the gene-correlation, the brown paddy plant hopper feeding transgenosis water The mRNA for inducing NlPP1 gene after rice specifically degrades, and expression of the gene in brown paddy plant hopper is blocked, so as to cause brown paddy plant hopper Impaired development is dead.

The beneficial effects are mainly reflected as follows: the present invention provides a kind of brown paddy plant hopper protein phosphatase gene NlPP1, with And the dsRNA according to the gene chemical synthesis.The gene plays important in maintaining the normal growth and development of brown paddy plant hopper and reproductive process Function, function, which is suppressed, will lead to brown paddy plant hopper nymphal development and is obstructed, and final dead.The present invention provides for brown paddy plant hopper prevention and treatment New control strategy and target site controls pest using RNA perturbation technique and provides the foundation, have preferably application to establish Prospect.

(4) Detailed description of the invention

Fig. 1 is the PCR amplification electropherogram of brown paddy plant hopper NlPP1 gene；Left side swimming lane M is 100bpDNA marker, right side swimming Road is the target stripe of the 1262bp of amplification.

Fig. 2 is the dsRNA and its template dsDNA electrophoretogram of external synthesis brown paddy plant hopper NlPP1 gene；M is 100bpDNA marker。

Fig. 3 is expression analysis of the NlPP1 gene in brown paddy plant hopper different tissues and developmental stage；M is 100bpDNA marker。

Fig. 4 is shadow of the dsNlPP1 of injection various concentration to 3 instar of brown paddy plant hopper to survival rate (A) and gene expression amount (B) It rings.Different letters indicate processing, and there were significant differences (p < 0.05) with survival rate after injection or gene expression amount is compareed.DsGFP, For the dsRNA for injecting GFP gene, it is used as control in this experiment；DsNlPP1-1000 indicates injection NlPP1 gene DsRNA concentration is 1000ng/uL, and so on, every worm injection dsRNA amount is 0.1uL.

Fig. 5 is precocious molt and emergence phenotype in injection teleonymph development.

Fig. 6 is influence of the dsRNA of feeding various concentration to 3 instar survival rate (A) and gene expression amount (B) of brown paddy plant hopper.* Indicate that there were significant differences (p < 0.05) for the brown paddy plant hopper survival rate for handling with compareing.Different letters are indicated to handle and be compareed after injection There were significant differences for survival rate or gene expression amount (p < 0.05).For the dsRNA for injecting GFP gene, it is used as control in this experiment； DsNlPP1-100 indicates that the dsRNA concentration of feeding NlPP1 gene is 100ng/uL, and so on.

(5) specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:

The clone of embodiment 1:NlPP1 gene, the synthesis of dsNlPP1

1) clone of NlPP1 gene

The whole worm RNA of 5-10 brown paddy plant hoppers is extracted using the RNeasy Mini kit (74106) of Qiagen company, is used The ReverTra AceqPCR RT Kit kit synthesis cDNA sequence of Toyobo company, 10 μ L synthetic system RNA containing 1ng, 2.0 μ L5xRT buffer, 0.5 μ L primer mix, 0.5 μ L RT enzyme mix.37 DEG C keep the temperature 45 minutes, 98 DEG C 5 points Clock inactivates reverse transcriptase, the template that cDNA is expanded after diluting 10 times as target gene cDNA.

The sequence fragment of brown paddy plant hopper NlPP1 gene, root are obtained from the transcript profile database matching sequence assembling in this laboratory Upstream primer NlPP1-F:CTCACTTCGTTCGTTCGCATT, downstream primer NlPP1-R are designed according to NCBI primer blast: AGACCTACTCCAGGTAGCCTT。

25 μ L of PCR amplification system: include the cDNA, upstream and downstream primer each 20pmol, 0.2mM dNTP, 1 after 3.0 μ L dilution × PCR buffer, 2.5units of r-Taq DNA polymerase (Takara), totally 2 repetitions.PCR amplification program Are as follows: 95 DEG C of denaturation 2min；95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 90s of 35 circulations；Last 72 DEG C of extensions 10min.Amplified production is separated through 2.0% agarose electrophoresis.

PCR product is recycled using the DNA purification and recovery kit (DP214-02) of Tiangeng company.Add in 50 μ L PCR products Enter the mixing of 50 μ L PC solution, mixed liquor is transferred to and has been used in the processed Filter column of equilibrium liquid, 1min is placed at room temperature for； 12000rpm is centrifuged 1min, discards filtrate in collecting pipe, and 700 μ Lwash buffer (dehydrated alcohol has been added) are added, 12000rpm is centrifuged 1min, discards filtrate in collecting pipe, and 500 μ Lwash buffer (dehydrated alcohol has been added) are added, 12000rpm is centrifuged 1min, discards filtrate in collecting pipe；Liquid is not added to be transferred to Filter column after 12000rpm is centrifuged 2min In 1.5mL centrifuge tube, 40 μ L Nuclease-free water are added, are placed at room temperature for 2min, 12000rpm is centrifuged 2min, and collection obtains The PCR product electrophoresis of PCR product, recycling is shown in Fig. 1.

PCR product is connected to by the connection kit for taking 4.0 μ L recovery products to be produced with handsome company2.1 carrier In (being purchased from Invitrogen company), 10 μ L of linked system: contain 1.0 μ 10 × Ligation of L Buffer, 2.0 μ L 2.1vector (25ng/ μ L), 2.0 μ L water and 1.0 μ L T4DNA Ligase.16 DEG C of connection 12h.6.0 μ L connection products are taken to turn Enter in 100 μ L bacillus coli DH 5 а competent cells, ice bath 30min, 42 DEG C of heat shock 45s, after then putting 2min on ice, is added 200 μ L are preheating to 37 DEG C of SOC culture medium, and uniform coated plate is in containing 50mg/mL card, that is mould after 100rpm, 37 DEG C of recovery 1h Element, on the LB solid medium of 0.2g/mL IPTG, 0.02g/mL X-gal (agar powder containing 15g/L), 37 DEG C are incubated overnight. SOC culture medium group becomes (tryptone 20g/L, yeast powder 5g/L, NaCl 0.5g/L, KCl 0.185g/L, MgCl₂ 0.95g/L, MgSO₄1.2g/L, glucose 3.6g/L, solvent are deionized water, and pH is natural).LB culture medium group becomes (pancreas egg White peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, solvent are deionized water, and pH is natural).

Random picking 5 white positive colonies into the LB liquid medium of 3.0mL kanamycins containing 50mg/mL, 200rpm, 37 DEG C are shaken bacterium 10h, positive colony are carried out amplification verifying, according to the plasmid extraction kit of Roche Holding Ag (11754785001) specification extracts plasmid.Bacterium solution is transferred to 12000rpm in 2.0mL centrifuge tube and is centrifuged 1min, discards supernatant liquid, The Suspension buffer of 250 μ L enzymes containing RNase is added in bacterial sediment, 250 μ L Lysis are added after mixing in oscillation Buffer is mixed by inversion 3-6 times.It is placed at room temperature for 5min.The Binding buffer that 350 μ L are pre-chilled on ice is added, is mixed by inversion 3-6 times, solution turbid, puts 12000rpm centrifugation 10min after 5min on ice at this time.Supernatant is transferred in Filter column, at full speed from Heart 1min.Liquid in collecting pipe is outwelled, 700 μ L Wash buffer II are added in Filter column, are centrifuged 1min at full speed.It will Liquid is outwelled in collecting pipe, and repeating washed once, and outwells liquid in collecting pipe, and liquid centrifugation 2min at full speed is not added in Filter column. Filter column is transferred in 1.5mL centrifuge tube, 100 μ L Elution buffer are added, are placed at room temperature for 2min, 12000rpm from Heart 2min, collection obtain plasmid pTOP-NlPP1.

Plasmid pTOP-NlPP1 is sequenced (by Shanghai, Invitrogen Corp. is completed), is compared confirmation and is obtained SEQ ID The NlPP1 genetic fragment of nucleotide sequence shown in NO.1.1262 base of cDNA sequence overall length, the exploitation containing 987 bases are read Discovery only 1 copy is compared in brown paddy plant hopper genome in frame (area ORF), gene order by 7 exons (exon) and 6 introne (intron) compositions.Coding contains the protein sequence of 328 amino acid.The gene cDNA sequence and other insects into Row sequence analysis, discovery nucleotide sequence identity is up to 78.0%, coverage 70%.Protein sequence and other insects one Cause degree is up to 96%.

2) expression pattern analysis of the NlPP1 gene in brown paddy plant hopper

According to the NlPP1 gene fragment order having verified that, qNlPP1-F is designed and synthesized with NCBI primer blast Upstream primer TCTGCTCAAATGGCTGACATAGA, qNlPP1-R downstream primer TCCACGGATTTCCGACTCAA, with brown paddy plant hopper Different tissues include head, chest, abdomen, ovary, salivary gland, fat-body, middle intestines, hemolymph, leg etc. and different development ages The RNA that phase (1-5 age nymph and female male imago) extracts is that reverse transcription is cDNA to template respectively.RNA is extracted and cDNA synthesis is same as above. 25 μ L of PCR amplification system: include the cDNA, upstream and downstream primer each 20pmol, 0.2mM dNTP, 1 × PCR after 3.0 μ L dilution The r-Taq DNA polymerase (Takara) of buffer, 2.5units, totally 3 repetitions.PCR amplification program are as follows: 95 DEG C of changes Property 2min；95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 90s of 40 circulations；Last 72 DEG C of extensions 5min.Amplification produces Object is separated through 2.0% agarose electrophoresis (see Fig. 3).The result shows that the gene has table in brown paddy plant hopper different tissues and developmental stage It reaches, is the gene of constitutive expression.

3) synthesis and purifying, the recycling of dsRNA

According to the NlPP1 gene fragment order having verified that, dsNlPP1- is designed and synthesized with NCBI primer blast F upstream primer, ds NlPP1 downstream primer, and held in upstream and downstream primer 5 ' and T7 promoter sequence is added TAATACGACTCACTATAGGG。

Ds NlPP1-F upstream primer:

TAATACGACTCACTATAGGGTTTCGAGTACGGAGGATTCCC,

Ds NlPP1-R downstream primer:

TAATACGACTCACTATAGGGGCATTGGCAACTTCTCTCCAC。

Using the plasmid pTOP-NlPP1 of the gene containing NlPP1 as template, PCR system are as follows: 1.0 μ L Plasmid DNA, upstream and downstream primer The r-Taq DNA polymerase (Takara) of each 20pmol, 0.2mM dNTP, 1 × PCR buffer, 2.5units, totally 25 μ L, is repeated 10 times.PCR amplification program are as follows: 95 DEG C of denaturation 2min；95 DEG C of denaturation 30s of 35 circulations, 58 DEG C of annealing 30s, 72 DEG C Extend 40s；Last 72 DEG C of extensions 10min.PCR product separates (see Fig. 2) through the agarose gel electrophoresis of 2.0% concentration, sequence See SEQ ID NO.2.DsDNA sequence compares in the library NCBI, and removing has at two with anopheles costalis (Anopheles gambiae) It include natural enemy etc. without the similar sequence of continuous 20bp or more height with other insects continuously outside the similar sequence of 20bp or more height Column, show that the sequence causes the probability of RNAi effect very low in other insects.

10 duplicate PCR products are separately recovered using the DNA purification and recovery kit of Tiangeng company, with the PCR of recycling Product is template, synthesizes dsRNA using the RNAi synthetic agent box of life technology company.Synthetic system are as follows: template is about 1.5-2 μ g, 2.0 μ L10 × T7buffer, 2.0 μ LATP, 2.0 μ L GTP, 2.0 μ L CTP, 2.0 μ L UTP, 2.0 μ L T7enzyme, totally 20.0 μ L.37 DEG C keep the temperature 5 hours, and 72 DEG C inactivate 5 minutes；21.0 μ are added in synthesis 20.0 μ L of dsRNA LNuclease-free water, 5.0 μ 10 × digestion of L buffer, 2.0 μ L DNase, 2.0 μ LRNase.37 DEG C of heat preservations 45min removes DNA and single stranded RNA to degrade.

150.0 μ L Nuclease-free water, 50.0 μ L 10x are added in the product containing dsRNA of upper 50.0 μ L of step Binding buffer, 250.0 μ L100% ethyl alcohol shift in Filter column after mixing, are placed at room temperature for 2min, 12000rpm centrifugation 2min discards filtrate in collecting pipe, and Filter column is added 500 μ Lwashing buffer, 12000rpm and is centrifuged 2min, not liquid feeding Filter column is transferred in 1.5mL centrifuge tube by body after 12000rpm is centrifuged 2min, and 50 μ L are added and are preheating to 95.0 DEG C Elutionbuffer is placed at room temperature for 2min, and 12000rpm is centrifuged 2min, collects the dsRNA purified, and electrophoresis detection is simultaneously surveyed After determining concentration.- 80.0 DEG C save backup.Electrophoresis detection is shown in Fig. 2.

Embodiment 2:dsNlPP1 tests the injection of 3 age brown paddy plant hoppers

It makes the agarose gel groove of fixed brown paddy plant hopper: preparing the agarose gel that mass concentration is 2.0-2.5%, will dissolve Agarose gel afterwards pours into the culture dish of diameter 9cm, and the height of agarose gel is slightly above culture dish, places 4- above culture dish The capillary of 5 diameter 0.5-0.8mm.After agarose gel is cooling, remove capillary to get to the agar that can fix brown paddy plant hopper Carbohydrate gum groove；It chooses 3-4 age brown paddy plant hopper nymph to be blown into teat glass, uses CO₂After 2 points of anesthesia, brown paddy plant hopper is poured into and is filled in advance Have in the culture dish of Ago-Gel, it will be in the neat groove for being placed in agarose gel of brown paddy plant hopper with banister bruss.

Using eppendorf microinjection instrument (model TransferMan NK2, Shanghai Ai Bende company), in brown paddy plant hopper The soft each brown paddy plant hopper in place of chest mesopodium base portion injects 0.1 μ LdsRNA, dsRNA concentration and sets from 1000~0.025ng/ μ L Set 9 concentration.Brown paddy plant hopper is inoculated on rice varieties (the Taichung Native 1) TN1 in seedling stage after injection, interior for 24 hours to reject Because of dead worm caused by injection operation.Worm point living is shrouded into raising, i.e., each rice single plant is covered with shrouding, and each shrouds interior raising 35,4 repetitions.It is placed in shrouding in the big basin with water, control room temperature is at 25 ± 1 DEG C, humidity 80% or so.Often It rejects dead worm, records borer population living.The 4th day after injection, 5 worms living is taken to extract RNA respectively from each repetition, using quantitatively The silence efficiency of round pcr analysis target gene NlPP1.RNA is extracted and cDNA synthesis is same as above.Real-time fluorescence quantitative PCR amplification 20 μ L of system: comprising 3.0 μ L dilution after cDNA, upstream and downstream primer (qNlPP1-F:TCTGCTCAAATGGCTGACATAGA and QNlPP1-R:TCCACGGATTTCCGACTCAA) each 10pmol, 1 × SYBRGreenPCR mix (ABI).PCR amplification program Are as follows: 95 DEG C of denaturation 2min；95 DEG C of denaturation 30s, 60 DEG C of annealing 45s of 40 circulations.It is brown after the dsNlPP1 of injection various concentration to fly Lice nymph survival rate and gene silencing efficiency are shown in Fig. 4.The processing group of dsRNA concentration 50ng/uL or more is injected the 4th day after injection Start it is dead, the death rate up to 60% with compare between there were significant differences, the death rate is up to 90% (see Fig. 4) at the 10th day.After injection Death due to cannot normally complete husking or emergence of brown paddy plant hopper 35~42% (see Fig. 5).

Embodiment 3: influence of the feeding dsNlPP1 to 2-3 age brown paddy plant hopper

Glass tube (15cm × 2.5cm) setting of both ends open is placed on superclean bench table top, upper end is square The sealed membrane of shape uniformly to two laybacks, seals upper open end, draws 50.0 μ L drop of man-made feeds in sealed membrane with pipettor Centre, control is only plus dsRNA (final concentration of 100,300 and 600ng/ μ is added in feed (formula is shown in Table 1), processing group in feed L), with a new sealed membrane, paster is face-down, and Uniform Tension is attached to above the sealed membrane added with feed, and feed and dsRNA are sealed Between two layers of sealed membrane.

It draws 25 2-3 age brown paddy plant hoppers with pest sucking device to be gently blown into glass tube from glass tube open end, with sterilizing rear yarn Cloth seals open end；Each processing is repeated 4 times；Feed will have been added to lie on feeding worm frame with the glass tube for being blown into brown paddy plant hopper, One layer of lid wet black cloth moisturizing above glass tube.Illumination is given at glass tube feed end, 26 DEG C of raising temperature, is made using its phototaxis Its feeding feed, every 48h count a survival rate, change a feed and dsRNA.Continuous feeding 10 days.As a result see Fig. 6, feeding When the concentration of dsNlPP1 is 600ng/uL, after feeding 4 days the death rate up to after 80%, 6 day up to 98%.Gene expression amount decline Up to 50% or so, there were significant differences with control group.When feeding dsRNA concentration is down to 100ng/uL, the expression of target gene and Brown paddy plant hopper survival rate indifference compared with the control.Therefore the dsRNA of feeding 300ng/uL or more NlPP1 has brown paddy plant hopper obvious Lethal effect.

Table 1: Chemically defined diet ingredient

Adjusting pH is 6.8 after preparing feed, filtration sterilization.

Sequence table

<110>China Paddy Rice Inst

<120>brown paddy plant hopper protein phosphatase gene NlPP1, albumen and its dsRNA and application

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1262

<212> DNA

<213> Nilaparvata lugens

<400> 1

ctcacttcgt tcgttcgcat ttactcacaa cactacagat taatgcttat tttatcagcg 60

taaatttgtt ttaaattcaa ttttctctgt catcgaattt cagtttaatt atctgctcaa 120

atggctgaca tagaaaaact taatattgac agtgttatag caagattact tgaagtacgt 180

ggaggtagac ctggaaggaa tgttcagctt cttgagtcgg aaatccgtgg attgtgtcta 240

aaatccagag aaattttctt gtctcagccc attttgttgg aattggaggc accattgaaa 300

atttgcggtg acatccatgg tcaatactat gatctgctgc gacttttcga gtacggagga 360

ttcccgccag aatcgaatta cttgttcctc ggtgactatg tagacagagg taaacagtcg 420

ctcgagacaa tctgcttgct gttggcgtac aaaattaaat atccagaaaa cttcttcctg 480

cttcgaggca atcatgagtg cgccagtatc aataggatat atggattcta cgatgaatgc 540

aagaggcgct acaatatcaa actgtggaag actttcaccg actgcttcaa ttgtttaccg 600

gtgtcagcca ttgtcgatga gaaaatcttc tgctgtcacg ggggtttgag tccggacctg 660

cacagcatgg aacagattcg tcgtatcatg cgacccactg atgttcctga tcagggattg 720

ctctgcgatc tgctctggtc tgatcccgac aaagatacga tgggttgggg tgagaacgac 780

agaggcgttt cattcacgtt tggcgttgaa gtagtggcga aattcttaca taaactcgac 840

ttcgatctca tatgccgcgc tcatcaggtt gttgaagatg ggtacgaatt cttcgccaag 900

cgtcaacttg tgactttgtt ttctgcccct aattactgtg gcgaattcga caatgccggt 960

gctatgatgt cagttgacga gactctcatg tgctcgttcc aaatcctcaa gcctgctgac 1020

aagaggaaat tcacttacgg tggtttgaat gttgggcgtc cagtgactcc tccccgtggc 1080

gctactaata aaaataaaaa gaaataaact tcctctcgag tggagagaag ttgccaatgc 1140

ctcctgccct tccgtctgtt aagaagttgt tcatttttta tctgctcctc ccctttgtag 1200

aaaattattc aaattgcttt aatacgtaca gggagaaaca gaaaggctac ctggagtagg 1260

tc 1262

<210> 2

<211> 328

<212> PRT

<213> Nilaparvata lugens

<400> 2

Met Ala Asp Ile Glu Lys Leu Asn Ile Asp Ser Val Ile Ala Arg Leu

1 5 10 15

Leu Glu Val Arg Gly Gly Arg Pro Gly Arg Asn Val Gln Leu Leu Glu

20 25 30

Ser Glu Ile Arg Gly Leu Cys Leu Lys Ser Arg Glu Ile Phe Leu Ser

35 40 45

Gln Pro Ile Leu Leu Glu Leu Glu Ala Pro Leu Lys Ile Cys Gly Asp

50 55 60

Ile His Gly Gln Tyr Tyr Asp Leu Leu Arg Leu Phe Glu Tyr Gly Gly

65 70 75 80

Phe Pro Pro Glu Ser Asn Tyr Leu Phe Leu Gly Asp Tyr Val Asp Arg

85 90 95

Gly Lys Gln Ser Leu Glu Thr Ile Cys Leu Leu Leu Ala Tyr Lys Ile

100 105 110

Lys Tyr Pro Glu Asn Phe Phe Leu Leu Arg Gly Asn His Glu Cys Ala

115 120 125

Ser Ile Asn Arg Ile Tyr Gly Phe Tyr Asp Glu Cys Lys Arg Arg Tyr

130 135 140

Asn Ile Lys Leu Trp Lys Thr Phe Thr Asp Cys Phe Asn Cys Leu Pro

145 150 155 160

Val Ser Ala Ile Val Asp Glu Lys Ile Phe Cys Cys His Gly Gly Leu

165 170 175

Ser Pro Asp Leu His Ser Met Glu Gln Ile Arg Arg Ile Met Arg Pro

180 185 190

Thr Asp Val Pro Asp Gln Gly Leu Leu Cys Asp Leu Leu Trp Ser Asp

195 200 205

Pro Asp Lys Asp Thr Met Gly Trp Gly Glu Asn Asp Arg Gly Val Ser

210 215 220

Phe Thr Phe Gly Val Glu Val Val Ala Lys Phe Leu His Lys Leu Asp

225 230 235 240

Phe Asp Leu Ile Cys Arg Ala His Gln Val Val Glu Asp Gly Tyr Glu

245 250 255

Phe Phe Ala Lys Arg Gln Leu Val Thr Leu Phe Ser Ala Pro Asn Tyr

260 265 270

Cys Gly Glu Phe Asp Asn Ala Gly Ala Met Met Ser Val Asp Glu Thr

275 280 285

Leu Met Cys Ser Phe Gln Ile Leu Lys Pro Ala Asp Lys Arg Lys Phe

290 295 300

Thr Tyr Gly Gly Leu Asn Val Gly Arg Pro Val Thr Pro Pro Arg Gly

305 310 315 320

Ala Thr Asn Lys Asn Lys Lys Lys

325

<210> 3

<211> 796

<212> DNA

<213>unknown (Unknown)

<400> 3

tttcgagtac ggaggattcc cgccagaatc gaattacttg ttcctcggtg actatgtaga 60

cagaggtaaa cagtcgctcg agacaatctg cttgctgttg gcgtacaaaa ttaaatatcc 120

agaaaacttc ttcctgcttc gaggcaatca tgagtgcgcc agtatcaata ggatatatgg 180

attctacgat gaatgcaaga ggcgctacaa tatcaaactg tggaagactt tcaccgactg 240

cttcaattgt ttaccggtgt cagccattgt cgatgagaaa atcttctgct gtcacggggg 300

tttgagtccg gacctgcaca gcatggaaca gattcgtcgt atcatgcgac ccactgatgt 360

tcctgatcag ggattgctct gcgatctgct ctggtctgat cccgacaaag atacgatggg 420

ttggggtgag aacgacagag gcgtttcatt cacgtttggc gttgaagtag tggcgaaatt 480

cttacataaa ctcgacttcg atctcatatg ccgcgctcat caggttgttg aagatgggta 540

cgaattcttc gccaagcgtc aacttgtgac tttgttttct gcccctaatt actgtggcga 600

attcgacaat gccggtgcta tgatgtcagt tgacgagact ctcatgtgct cgttccaaat 660

cctcaagcct gctgacaaga ggaaattcac ttacggtggt ttgaatgttg ggcgtccagt 720

gactcctccc cgtggcgcta ctaataaaaa taaaaagaaa taaacttcct ctcgagtgga 780

gagaagttgc caatgc 796

<210> 4

<211> 41

<212> DNA

<213>unknown (Unknown)

<400> 4

taatacgact cactataggg tttcgagtac ggaggattcc c 41

<210> 5

<211> 41

<212> DNA

<213>unknown (Unknown)

<400> 5

taatacgact cactataggg gcattggcaa cttctctcca c 41

<210> 6

<211> 21

<212> DNA

<213>unknown (Unknown)

<400> 6

ctcacttcgt tcgttcgcat t 21

<210> 7

<211> 21

<212> DNA

<213>unknown (Unknown)

<400> 7

agacctactc caggtagcct t 21

<210> 8

<211> 23

<212> DNA

<213>unknown (Unknown)

<400> 8

tctgctcaaa tggctgacat aga 23

<210> 9

<211> 20

<212> DNA

<213>unknown (Unknown)

<400> 9

tccacggatt tccgactcaa 20

Claims

1. a kind of brown paddy plant hopper protein phosphatase gene NlPP1, nucleotide sequence is as shown in SEQ ID NO.1.

2. the albumen of brown paddy plant hopper protein phosphatase gene NlPP1 coding, amino acid sequence is as shown in SEQ ID NO.2.

3. the dsRNA of Brown Planthopper protein phosphatase gene NlPP1, nucleotide sequence is as shown in SEQ ID NO.3.

4. application of the brown paddy plant hopper protein phosphatase gene NlPP1 described in claim 1 in prevention and treatment brown paddy plant hopper insect pest.

5. application as claimed in claim 3, it is characterised in that the application are as follows: according to the brown paddy plant hopper protein phosphatase gene NlPP1 designs dsRNA, and the drug of prevention and treatment brown paddy plant hopper is prepared with the dsRNA.

6. application as claimed in claim 4, it is characterised in that the dsRNA nucleotide sequence is as shown in SEQ ID NO.1.

7. application of the brown paddy plant hopper protein phosphatase gene NlPP1 described in claim 1 in building brown planthopper resistant transgenic paddy rice.