CN114807156B - Application of brown planthopper NCS2 gene or coded protein as target spot in preparation of brown planthopper prevention and treatment drugs - Google Patents

Application of brown planthopper NCS2 gene or coded protein as target spot in preparation of brown planthopper prevention and treatment drugs Download PDF

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CN114807156B
CN114807156B CN202210393423.9A CN202210393423A CN114807156B CN 114807156 B CN114807156 B CN 114807156B CN 202210393423 A CN202210393423 A CN 202210393423A CN 114807156 B CN114807156 B CN 114807156B
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鲍艳原
张璐
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Zhejiang University ZJU
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Abstract

The invention discloses application of brown planthopper NCS2 genes or coded proteins serving as targets in preparation of brown planthopper prevention and treatment medicines, and relates to the technical field of plant genetic engineering. The invention discovers the role of the brown planthopper NCS2 gene of rice pests in preventing and controlling brown planthopper, and can utilize the advantage to interfere the expression of the brown planthopper NCS2 gene in the bodies of the rice pests because the brown planthopper has a high-efficiency RNAi effect. dsRNA of NCS2 gene is obtained through RNAi technology, and after NCS2 gene is silenced through microinjection, the transcriptional level of the NCS2 gene is reduced, the individual development and the ovarian development of brown planthoppers can be effectively inhibited, the capability of inhibiting the growth and the reproduction of pests is achieved, the propagation of brown planthoppers is inhibited, and the control of brown planthoppers is realized. The invention discovers that RNAi technology is used for interfering the expression of the gene in the 2-5 instar nymphs of brown planthoppers when the function research of NCS2 genes is carried out.

Description

Application of brown planthopper NCS2 gene or coded protein as target spot in preparation of brown planthopper prevention and treatment drugs
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of brown planthopper NCS2 genes or coded proteins serving as targets in preparation of medicines for preventing and treating brown planthopper.
Background
Intracellular calcium ions are important second messengers in eukaryotic cells. Calcium ion binding proteins mediate intracellular calcium signaling. Most calcium ion binding proteins contain one or more pairs of EF-hand domains. The EF-hand domain has a very high affinity for calcium ions. The EF-hand domain is typically 29 amino acids and has a helix-loop-helix structure (Tsujimoto T, jeroman A, saitoh N, roder JC, takahashi T.2002.neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals.science,295 (5563): 2276-2279.). The members of the calcium Neuroreceptor (NCS) family belong to calcium binding proteins that contain an EF-hand domain. The NCS gene is often highly expressed in the nervous system specificity, with the main functions of regulating neurotransmitter release, cell surface receptors and ion channels, gene transcription, neuronal growth, development and survival (McCue HV, haynes LP, burgoyne rd.2010.the diversity of calcium sensor proteins in the regulation of neuronal function.cold Spring Harbor perspectives in biology,2 (8): a 004085.). NCS2 is a newly discovered member of the NCS family, NCS2 being present to date only in nematodes and insects. The function of NCS2 has also been studied only in nematodes, which have an important role in the balance of neuronal synaptic excitation and inhibition in nematodes. However, unlike the NCS2 protein structure of nematodes, the NCS2 protein of insects contains only three EF-hand domains. Since the function of the NCS2 protein is unclear, we analyzed the physiological function of the NCS2 protein with model insect brown planthoppers as subjects. Brown planthoppers (academic name Nilaparvata lugens, english name brown planthopper) only take rice juice to cause plant withering, and are important pests in China and even Asian rice production at present. Brown planthoppers often present intermittent rampant situations and the number of fulminant insects increases. At present, the occurrence rule of the brown planthopper outbreak disaster and the disaster mechanism are not well understood. In order to prevent brown planthoppers from damaging rice crops, chemical control means are generally adopted at the present stage, but the work is often half-done. The pesticide is applied to kill natural enemies of brown planthoppers and simultaneously stimulate pests to reproduce, so that a rampant situation is formed, and meanwhile, the environmental pollution is aggravated, and the quality and safety risks of agricultural products are brought. In view of brown planthoppers becoming the most serious biological disaster endangering the grain safety and ecological safety of rice in China, finding out effective pest target genes, and reducing the economic loss of target pests to rice crops by a biological control method has important scientific significance. Brown planthoppers have a systematic RNAi phenomenon, which can efficiently and for a long time sink expression of host genes, and thus have become new modes of insects that use RNAi to study gene function. Because the NCS2 gene only exists in insects and nematodes, the transgenic plants produced by using the gene cannot produce toxic or side effects on other animals including human beings, and can play a great role in guaranteeing food safety.
Disclosure of Invention
The invention provides application of brown planthopper NCS2 genes as targets in preparing medicines for preventing and treating brown planthopper, dsRNA of brown planthopper NCS2 genes and application of the genes in preparing medicines for preventing and treating brown planthopper, the genes can realize environment-friendly green pest prevention and control strategies, improve insect resistance of transgenic rice crops and provide new gene resources for cultivating new varieties resistant to rice pests.
The specific technical scheme is as follows:
the invention provides application of brown planthopper NCS2 gene or coded protein as a target in preparing a brown planthopper prevention and treatment drug.
The invention provides application of brown planthopper NCS2 gene or coded protein as a target in preparing a medicament for reducing the spawning quantity of brown planthoppers.
The nucleotide sequence of the NCS2 gene is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the NCS2 gene is shown as SEQ ID NO. 2.
Preferably, the brown planthopper is a nymph of 2-5 instar.
The invention provides a dsRNA of brown planthopper NCS2 gene, which consists of two complementary sequences, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is a reverse complementary sequence shown as SEQ ID NO. 3.
The nucleotide sequence which can still achieve the basically same inhibition effect of the sequence shown in the invention after the sequence is transformed by simple deletion, modification and the like also belongs to the protection scope of the invention.
The invention also provides a medicine for preventing brown planthoppers, which comprises the following active ingredients (1) or (2):
(1) dsRNA or siRNA for reducing the expression of the brown planthopper NCS2 gene;
(2) A coding gene for siRNA in (1).
The invention also provides a method for preventing brown planthoppers, and the medicine is used.
The brown planthoppers are nymphs of 2-5 ages.
Only about 20% of nymphs develop into adults, both with low-age (2-age) nymphs and with high-age (5-age) nymphs.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention discovers the role of the brown planthopper NCS2 gene of rice pests in preventing and controlling brown planthopper, and can utilize the advantage to interfere the expression of the brown planthopper NCS2 gene in the bodies of the rice pests because the brown planthopper has a high-efficiency RNA interference (RNAi) effect. dsRNA of NCS2 gene is obtained through RNAi technology, and after NCS2 gene is silenced through microinjection, the transcriptional level of the NCS2 gene is reduced, the individual development and the ovarian development of brown planthoppers can be effectively inhibited, the capability of inhibiting the growth and the reproduction of pests is achieved, the propagation of brown planthoppers is inhibited, and the control of brown planthoppers is realized.
(2) The invention discovers that RNAi technology is used for interfering the expression of the gene in the 2-5 instar nymphs of brown planthoppers when the function research of NCS2 genes is carried out.
Drawings
FIG. 1 is a PCR identification chart of NCS2 gene of the present invention.
FIG. 2 is an identification of double-stranded dsNCS2 synthesized according to the present invention.
FIG. 3 is a graph showing mortality of 2-5-year-old nymphs interfering with the NCS2 gene of the present invention.
FIG. 4 is a diagram showing oviposition of female adults of the present invention interfering with the NCS2 gene; * Represents p < 0.01.
FIG. 5 is a diagram showing the formation of an oocyte in the ovary, which interferes with the NCS2 gene, according to the present invention.
Detailed Description
Example 1
The rice pest brown planthopper NCS2 gene.
Step 1: extraction of total RNA of brown planthoppers and NCS2 gene cloning
Placing brown planthopper adults (100 mg) in a grinding tube, adding appropriate amount of silica grinding beads and 1mL Trizol reagent, and grinding with tissueThe mill was ground 3 times with shaking at 60Hz for 30 seconds each and allowed to stand on ice for 3 minutes. The samples were centrifuged at 12000rpm at 4℃for 10 minutes and 800. Mu.L of the supernatant was pipetted into a fresh 1.5mL RNase free EP tube; 500. Mu.L of chloroform was added thereto, and the mixture was vigorously shaken for 30 seconds and allowed to stand at room temperature for 3 minutes. Centrifuge at 12000rpm,4℃for 10min, aspirate supernatant to a new 1.5mL RNase free EP tube, add equal volume of isopropyl alcohol, gently mix upside down for 30 seconds and stand at room temperature for 20 min. Centrifuge at 12000rpm,4℃for 20 min, pellet was visible at the bottom of the centrifuge tube, and the supernatant was discarded. 1mL of 75% RNase-free ethanol (v/v) was slowly added along the vessel wall and washed. 7500rpm,4 ℃, centrifuging for 5min, discarding supernatant, standing in a ventilated kitchen with a cover, precipitating at the bottom of the tube to be semitransparent, adding 20 μl RNase free ddH 2 O was dissolved sufficiently, and the concentration and quality of RNA were measured by using NanoDrop 2000.
Step 2: RNA reverse transcription
1000ng of RNA prepared in step 1 was pipetted into RNase free PCR tube using RNase free ddH 2 O was made up to 12. Mu.L. mu.L of 4 XgDNA wind Mix was added, mixed well and placed in a PCR apparatus and heated at 42℃for 2 minutes. Add 4. Mu.L of 5X HiScript II qRT SuperMix II, mix well, place in PCR instrument, reaction program: 50 ℃ for 15 minutes; 85℃for 1 minute. 180. Mu.L ddH was added to the product 2 Diluting with O, and storing at-20deg.C.
Step 3: NCS2 Gene fragment cloning
First, the following specific PCR primers containing the T7 promoter were synthesized:
Forward：
Reverse：
the underlined sequence is the T7 promoter sequence.
PCR reaction system
Reagent(s) Volume of
cDNA 1μL
PCR Forward Primer(10μM) 1μL
PCR Reverse Primer(10μM) 1μL
Green Taq Mix 25μL
ddH 2 O 22μL
PCR reaction parameters: pre-denaturation for 3 min at 95 ℃; denaturation at 95℃for 30 sec; annealing at 60 ℃ for 1 minute, annealing at 72 ℃ for 1 minute, and extending for 40 cycles; total extension at 72℃for 10 min. And (3) confirming the PCR product through agarose gel electrophoresis, recovering the corresponding fragment, recovering the fragment by using a DNA gel recovery kit, and constructing the target gene obtained by gel recovery on a pMD19-T vector by using a TA cloning kit.
Step 4: transformation of E.coli
E.coli competent cells were taken out from the-80℃refrigerator, thawed on ice for 5min, and the recombinant pMD19-T vector was added to the competence, mixed with gentle shaking, and ice-bathed for 30min. The sample is put into a water bath kettle with the temperature of 42 ℃ for 90 seconds in a water bath, and immediately subjected to ice bath for 2 minutes. 1mL of the antibiotic-free LB liquid medium was added, and the mixture was cultured on a shaker at 220rpm at 37℃for 2 hours. Cultivation methodCentrifuging the cultured bacterial liquid at 6000rpm for 10min, discarding part of the liquid, suspending the rest 100 μl of liquid, precipitating, and coating on a medium containing Amp + Resistant LB plates; the cells were cultured in an inverted state at 37℃for 12 hours in a bacterial incubator. Single colonies were picked to 1mL containing Amp + In the resistant LB liquid medium, the culture was carried out for 3 hours at 37℃with a shaking table at 220 rpm. The 500. Mu.L turbid bacterial liquid is sucked and sent to a biological company sequencing (ABI 3730XL full-automatic sequencer; zhejiang Shangya biotechnology Co., ltd.) to obtain a 6421bp gene NCS2 (the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence of the coded protein is shown as SEQ ID NO. 2), and the extracted plasmid is named pMD-NCS2.
Example 2
1. Synthesis of dsRNA
Specific primers containing the T7 promoter were used as follows:
Forward：
Reverse：
the underlined sequence is the T7 promoter sequence.
The dsGFP primer sequences are as follows
dsGFP-F：
dsGFP-R：
The recombinant plasmid pMD-NCS2 extracted in example 1 is used as a template, a section of high-concentration DNA fragment with about 550bp (the nucleotide sequence is shown as SEQ ID NO. 4) is amplified by the primer PCR, dsNCS2 (the nucleotide sequence of the sense strand is shown as SEQ ID NO.3, the nucleotide sequence of the antisense strand is the reverse complement of the sequence shown as SEQ ID NO. 3) is synthesized by using the high-concentration DNA fragment as the template, and the template synthesized by dsGFP is a known sequence.
Preparing a double-stranded RNA synthesis reaction system:
ATP solution, UTP solution, CTP solution, and GTP solution each 2. Mu.L, 10X Reaction buffer 2. Mu.L, T7 RNA Polimerase mix. Mu.L, and DNA template 8. Mu.L. The reaction system was mixed well and incubated in a 37℃incubator for 14h. 1. Mu.L DNase I was added and incubated in a metal bath at 37℃for 15 minutes to remove the DNA template. Taking 1 mu L of dsRNA product, detecting the quality of the dsRNA by agarose gel electrophoresis, and preserving at-80 ℃.
2. dsRNA microinjection
And (5) preparing a microinjection needle. The micro-injection needle is manufactured by a micro-electrode needle drawing instrument (P-97 Micropipette Puller) with the caliber of 0.6mm, and the needle drawing instrument comprises the following procedures: heat=750, pull=300, velocity=150, time=150. Microinjection of dsRNA. CO from brown planthopper 2 And (3) anaesthetizing for 15-30 seconds, picking brown planthoppers by using a writing brush, and placing the brown planthoppers on a pre-cooled 2% agarose gel shallow groove, wherein the ventral surface of the insect body faces upwards. Adding the synthesized dsRNA into a microinjection needle, and fixing the needle on a mobile mechanical arm; according to the brown planthopper age, the brown planthopper is pinched into a proper size by using an anatomic forceps, and the microinjection instrument (FemtoJet 4×) is set as follows: injection pressure 900pah, injection time 0.3 seconds, and compensation pressure 10pah; the mechanical arm is operated to inject dsRNA into the insect body from the middle soft part of the chest of the brown planthopper, the second pair and the third pair of foot bases. Placing the brown planthoppers after injection into a transparent pot with fresh rice seedlings, sealing with gauze, and culturing and observing in a climatic chamber.
3. Effect on brown planthopper growth after RNAi
2,3,4 and 5-year-old nymphs raised on rice seedlings were selected and injected with approximately 100ng dsNCS2 per head. After injection, the plant is placed on a rice seedling for 24 hours, and then the plant is transferred to a new rice seedling for observing the growth and development of brown planthoppers and counting the death rate. The results showed that 2-5-year old nymphs injected with dsNCS2 had a survival rate of over 76% the next day after the intervention, and declined shortly thereafter to a survival rate of only 16-24% on day 14. Whereas the survival rate of the 2-5-year-old nymphs of the dsGFP injected control group exceeds 88% the next day after the interference, the survival rate still reaches 67-86% by the 14 th day. The differences in survival rates of brown planthoppers injected with dsNCS2 and dsGFP were shown to be very significant.
4. Effect on brown planthopper reproduction after RNAi
4-year-old nymphs raised on rice seedlings were selected and approximately 100ng dsNCS2 was injected per head. Placing the mixture in rice seedlings after injection, and mating male and female insects after emergence for 4 days. And then removing male and female adults from the rice seedlings, observing the hatching condition of nymphs on the rice seedlings and counting the hatching rate. The results show that male and female adults interfering with dsNCS2 did not spawn after mating, i.e., the spawning rate was 0; after mating of dsNCS 2-injected female adults with dsGFP-injected male adults, more than 80% of females did not lay eggs, with only three females producing an average of 18 eggs. And after mating of male and female adults injected with dsGFP, 84 eggs are produced per female on average, with 77% of the eggs successfully hatched to nymphs. The differences in spawning rates of females from brown planthoppers injected with dsNCS2 and dsGFP were shown to be extremely significant. Eggs laid by female adults injected with dsGFP most have typical eyepoints, while eggs laid by female adults injected with dsNCS2 have no typical eyepoints or eyepoint inversion. Dissections of ovaries on day 5 after emergence of female adults injected with dsNCS2 revealed that there were no mature oocytes in the ovaries. Whereas ovaries on day 5 after emergence of female adults injected with dsGFP are filled with typical banana-type mature eggs. It was demonstrated that interference with the NCS2 gene inhibited the developmental maturation of oocytes in females ovaries.
Only about 20% of nymphs develop into adults, both with low-age (2-age) nymphs and with high-age (5-age) nymphs. In addition, the NCS2 gene is interfered to express in the body of the initial eclosion female adult, and the male adult which interferes with the NCS2 gene cannot lay eggs after mating, so that the reproduction fails. The result shows that the NCS2 gene plays an important role in the growth and development and propagation of brown planthoppers. Therefore, the NCS2 gene can be applied to the field of rice crop breeding, and has important significance for screening and cultivating rice planthopper resistant crops. The transgenic rice strain capable of expressing the double-stranded RNA of the brown planthopper NCS2 gene is cultivated, and the brown planthopper nymphs and adults can generate RNAi effect when eating the rice so as to achieve the aim of inhibiting the development, mutation and spawning of the brown planthopper.
Sequence listing
<110> university of Zhejiang
Application of <120> brown planthopper NCS2 gene or coded protein as target spot in preparation of brown planthopper prevention and treatment medicine
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6457
<212> DNA
<213> brown planthopper (Nilaparvata lugens)
<400> 1
cccggctgtc cagagcagcg gagtagcgga gtagcgggcg gagagcggcg cagttgacag 60
cgagcacata accctctagc acacgcacac gccaggctct tagcacagtt gcgtctcgtg 120
agtgagtcat cccatcatct acgaacatgt atcaccggcc aaacagttct actagttgtg 180
tgcgactcga tcaactctag atattgcatt agtttgtgac tcgaatcgac tgatttcatt 240
caaatcgaac ggctactggc tattgtgtgt gctccaattc gacccgcttt gtgtcaagtt 300
gcatagtttc tgttatcaag tggagtagct gcccccaacc ggcgaccaca ttcaatcggg 360
atggggtgtt tcgggagcaa agacaaatta tccaaagagg atatggattt cctcaaatca 420
caaacacgat acgacgaagc tacaattaaa gaatggtaca aaggttttaa gcaagactgt 480
ccaaacggta gactaacgcc ggccaagttc gtagatatgt acaagatgtt ctttcccagt 540
ggtaatgccg aagagttctg cgaccacgta tttaggacat tcgacatgga caagaacgga 600
tatatcgatt tcaaagagtt cttgcttgct attgatgtga catcgtcggg caccccagaa 660
gaaaaactaa aatgggcgtt taggatgtac gacgttgacg gcaatggtgt catcgacatc 720
caggaaatga caaagattgt tcaggcgatc tacgacatgc tgggcgcatg ctcgtcgaac 780
agaccggccg actcggccga ggagcgcgcc aagaacatct ttgccaagat ggacgagaac 840
aacgacggtc agctcacgca ggaggagttc ctgaagggtt gtctccagga cgaggagctg 900
tccaaaatgc tggcgccata atggaccaat tgcagcagcc actggaaacg agaacaagtg 960
atgttattga agtggtggtc acattgccac cgcctgcttc aggcgtgcaa acatcttgtt 1020
caacaacacc attgccaatt agtctctctc atcgatttta aacggttttc attttatatc 1080
gttgtggtat gattaaacaa attttatatg tgtcacccct attgaagacg ggacattcct 1140
atgtggccgt gtgtgtgtgc gcttgtgcga gtgaccgatc caagtgtgag tgaacatctc 1200
tatctttctc tttctatatt tgtctcaaaa caaactaagt atatactctt ctgtcctgca 1260
tagctttatt acttattgct ggtatcattg tttccttctc ggcaaggctt tgtgcaaatt 1320
ttctccaaga aggaaactcc aatcgaatga caaacacttt tctcactatc atcgtgtaca 1380
acttcctcct ccctctacta atatttttat agtgagtaaa aacaactgat gataacgcat 1440
acaaaaatac gcaccttgaa agctagtaat attaggataa tcaatctagt atcgtcaata 1500
gttgtgtcat tgatcattgt ataagctttt tctattataa ttaacaagtc aaacgtttga 1560
accgtatttg tgtattaatt ccgaaaattg atgacttaaa tttgaattga aaaactcctt 1620
ccccggttaa aaactaaaag taattttcta gcaaactaca atctatttta ttctctttca 1680
aaaagttacc tataaaaatg caattatatt tatcaattag tttaactgat tttcaccttt 1740
tttcatccta ctcctatcta caaaaatgat aacatgttta ttgttatcct ttcataaata 1800
tgtatttctt gtcatgcatt gagaacagag tgatccaggg aaccaactga tgtttagaat 1860
cacactgatt gaaataagca aagtgataga gacatagttc acaatctaat gtgggtttga 1920
acatcaaata atttgagttg gaacaatgtt tcttgtcaca aagaatatca cttcctctat 1980
tgaagtggaa ttattttaga agagtagatt ggtcttgcac agtacgtgaa tctgtcttga 2040
ggttcaaagg tggaacatga tgaaataata gagagtatag tacacttatt tctttcttta 2100
aacttgaaag ttaaccttcc attcatgagt actgcaaatc tagcaccaaa gttgaatttg 2160
attgaaagtc gtcaaatatg ttaccctgga gtactctgtg gaaatgcatt tcacatacat 2220
aatattctct attaaccatt ccgaatggtg atgataataa tattgatgaa cattcttcaa 2280
cattactaca gtcaattgtt tcacatatct actcttcaat aaagcagcaa agtttcgaag 2340
cattgttcgt gtcgtgttgt atacaatgtt tccgtccata aactcaaaat gtagaggtat 2400
gttcagctag tcggaaatga ccttactgac aatcgagttt gtagaattac tattatgaac 2460
tattgagtta tcatatttct tgttgagaaa gttagctgca ccaactctca actcatcaaa 2520
attattaaat attgtatgtt ctcaagtact agtcaataat ttgtctcatt caaattatca 2580
tcattcgtaa tggcatttga ttttttgtga tttttatcta tatgtcatta tatcgaacat 2640
tgaccaagtg ttcatcggtg tgattgttac caaatttatg tcataaataa ctaaatcctt 2700
ctctcatatt actgtcagcc ctttttttct aaattgaata tgaaaatcaa aactattcac 2760
gcttttgaat cttttcaaaa cctgcaattg tatttttata gatgtgtacc taatagatca 2820
ttcacatacc tatcaaagtt tctccgttct tatcaagaaa tgaatttata ataaatctgt 2880
tgattcgaat tggaaatttc atccaagaat ctttaaatgt ctgcattttt ttcagttatt 2940
acataaatca agtttcttct acgtatctac tgcctaacct ttttgtcatt acaattacgt 3000
gaggacctac cttaactcat tgtacgtgat ctaccttatc aattatgtta ttaagaacag 3060
cctatactat tcaccctaaa cattgtcatg atttataatt tgtaccgttt agtttctcct 3120
tgaattttat ttttttgaaa ttccacatat aacggaattc tagtgataag taatgcattt 3180
gactcatttt accacatcta tcaagtagag aatactggag attattatat gaattgatat 3240
attttgtaca tatttagagt aatttgtatg tctatggatt tgtattatat gattttcaca 3300
attataatga ctactttagg aaatataaga gttatggaat aagtattaat actaccacta 3360
atgctaatac tacgattaaa atgctaaccg catgactctc tcttgaaggt gtgctatgat 3420
taccttatca atgatgttat taagaacaga ctatactatt caccttcaac ctttccatag 3480
atggtgaaca ttactgagga agtatattca ttatgtaaat caacttaaaa agtcatcttt 3540
gtttgacatt aatttgcgat ctcattatta cccatccatc aaagaaaaaa agaagatctt 3600
tactcttcaa tattaatcaa tttctaaaaa tataaattgg aacataatat tgaaactaaa 3660
cacgtaaatt tataataaac atattaaatg gtacctgtat tttcatctat gatttgtaaa 3720
atatttggca taaatacaaa aattccaaaa aaataaaata aaagcaagct gccttaaaac 3780
aatattatta atgcatataa actccaatta attgtcaagt atcaaaataa ttaattgcaa 3840
aaaactacga ctgtagaaat gaattcgagc tgcaacattt tttctaacta attgtgatgg 3900
tgagggagca gattcattca ataattttcc acttgagttt tttcacgaat cagtgataaa 3960
gctatctaac agatgtaact atttaataca acttgtaagt gtcaatgaaa attctctagt 4020
cgacaattca catacttaag aacttctaca ccaatcagtc ctaatgcaac tattgtagta 4080
gttcaattta tttcaatagt ctatcatagt acacagttgt aatgtaaatt atataattac 4140
ggtgtgagtg tctttaaaca gtacgaaatt ttctacctaa actaagatat ttctttctga 4200
actttccaaa aatgtaactt cccttgtata aattgaacaa atattctata cacagttata 4260
ggtttttcgc cattttattc cattattgct tcacttttgg aattgattca tgccgtttgt 4320
gaagtaatca cagttttttg ttgaatgatg aagcgatggt gaatcttaat aattgtttta 4380
tattaactga tgagaaattt tcaactcatt acattgtgtg gaaaatccca ctcacctgca 4440
aattggaata aaccagtaac tttcgaccaa ttacttcatt tgtgagaaaa acaacattaa 4500
gtttaaaatc tataaataat gagaactacg tataaataat catttgctct attatctttt 4560
tcaattgttg tttggcttgt gtaattgtaa tgaattattg ataactatcg ataacttatt 4620
taatcaataa catgttttac atttttattt attattctac cttaaaaatt tcacatgtat 4680
taaacactta agtggcatat tttatgataa aattgtttct acttttttca agtaaaaatg 4740
gagctcaaca tggctactat tatgaaacta atggccgatc gtctgaccaa tattgtagaa 4800
aattctacac tgttcactgt attttatatt ggtataaaat cagatgcatg tccatactta 4860
ttgttggtgt tctattcatt tctatgtatc tggtgtctag atattacatt ccatctcttt 4920
tgtacagtat atttttaact attgaagaat ctatttgaag ttgaaaaatt ctatgatagt 4980
gtttaacatt ttaatgttta ctatactatt tttaagagat tatagttagt ggacatagtg 5040
tcaatatatt aattttatgt ttattatgtg aatgccgttg tgtgataaca gaacacatat 5100
ttaaaaactt atctattgat agtaacttgt ttgatcgttt atcacaatat ttattttatt 5160
gagttctttc ggtacttaaa ctgttttggt accttaaaaa ttgtgtagct tttatatatt 5220
tatgaattat tgttcaataa tttctaacta tatgatatct aagcttgaac cagaataaaa 5280
atcaaaacct aagtgtagct gtgtcaattt aatattaaaa acggagcatt ctggtgagat 5340
gcattgcata attgaaatag atgtatgaca attatcataa caattatgag tttttaagtt 5400
agataagact ctgttgagat cagaatgaat tcaataaaaa atatctctca gaaatataaa 5460
tatgaatata ttctcaaaaa tgaacaagaa ttcacatttg atttttcatc gaaatctgga 5520
tgataacaat caacagtaga ggttcaaaaa ttgattgtat cgaatttcga tttaataata 5580
taattgttat acatataaaa tatattaagg ctcacatttg ttggcaatga attctacatt 5640
tcgtgaaaag tagcggaata actaagttat gaaagatgca ttgcccaaag taagttagga 5700
aattgaattt cattgatttc tcatgtgaat ataacattac ttgactataa aacttcttgg 5760
gtcattcata ttattctgtt tgatatgttc accatttaca ctgtaatttc tgagctgaat 5820
gaaattcttg tttaataatt tcaattcttc tgtgatcagt gggctagtta tttctcaatg 5880
aattatgtgt ctaaaattgt ctatttaaat gaatctactt gtacattatt cttgctcagt 5940
agagtaaaat gtgctgggga acagaatatg tttcaaaaat ggaaatattt gattaccgct 6000
atcaattatc ctttcaaatt ccaccaaaca tcaaaatgtt tttctttttt tactgtatta 6060
attcgacgaa taatacgata ttgaattata ctaatgaatt gaatgaatag tatcactgca 6120
agtggaattg ctcaagtgat tgatcggtaa ttctgcgtgg aaagtgattt tgtaaatttg 6180
ctatagttgt caatctaagt attcatatat gcgactgaca atgcagtgtt ttaagattgt 6240
ctttcagttg atgaatgtcc aatgttattg taaatttgaa tcgaacacta tgtgtttaat 6300
aaaataattc aatctacagt gacttgtgtt catcatttgt cgaatattta ctacagttcc 6360
aataagttac tcatatttta ttttcattct cattgcgttt tatattacct attaatgtat 6420
caatttgaaa tgatgtgaat tacattaatc tgtggaa 6457
<210> 2
<211> 186
<212> PRT
<213> brown planthopper (Nilaparvata lugens)
<400> 2
Met Gly Cys Phe Gly Ser Lys Asp Lys Leu Ser Lys Glu Asp Met Asp
1 5 10 15
Phe Leu Lys Ser Gln Thr Arg Tyr Asp Glu Ala Thr Ile Lys Glu Trp
20 25 30
Tyr Lys Gly Phe Lys Gln Asp Cys Pro Asn Gly Arg Leu Thr Pro Ala
35 40 45
Lys Phe Val Asp Met Tyr Lys Met Phe Phe Pro Ser Gly Asn Ala Glu
50 55 60
Glu Phe Cys Asp His Val Phe Arg Thr Phe Asp Met Asp Lys Asn Gly
65 70 75 80
Tyr Ile Asp Phe Lys Glu Phe Leu Leu Ala Ile Asp Val Thr Ser Ser
85 90 95
Gly Thr Pro Glu Glu Lys Leu Lys Trp Ala Phe Arg Met Tyr Asp Val
100 105 110
Asp Gly Asn Gly Val Ile Asp Ile Gln Glu Met Thr Lys Ile Val Gln
115 120 125
Ala Ile Tyr Asp Met Leu Gly Ala Cys Ser Ser Asn Arg Pro Ala Asp
130 135 140
Ser Ala Glu Glu Arg Ala Lys Asn Ile Phe Ala Lys Met Asp Glu Asn
145 150 155 160
Asn Asp Gly Gln Leu Thr Gln Glu Glu Phe Leu Lys Gly Cys Leu Gln
165 170 175
Asp Glu Glu Leu Ser Lys Met Leu Ala Pro
180 185
<210> 3
<211> 556
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gguguuucgg gagcaaagac aaauuaucca aagaggauau ggauuuccuc aaaucacaaa 60
cacgauacga cgaagcuaca auuaaagaau gguacaaagg uuuuaagcaa gacuguccaa 120
acgguagacu aacgccggcc aaguucguag auauguacaa gauguucuuu cccaguggua 180
augccgaaga guucugcgac cacguauuua ggacauucga cauggacaag aacggauaua 240
ucgauuucaa agaguucuug cuugcuauug augugacauc gucgggcacc ccagaagaaa 300
aacuaaaaug ggcguuuagg auguacgacg uugacggcaa uggugucauc gacauccagg 360
aaaugacaaa gauuguucag gcgaucuacg acaugcuggg cgcaugcucg ucgaacagac 420
cggccgacuc ggccgaggag cgcgccaaga acaucuuugc caagauggac gagaacaacg 480
acggucagcu cacgcaggag gaguuccuga aggguugucu ccaggacgag gagcugucca 540
aaaugcuggc gccaua 556
<210> 4
<211> 556
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ggtgtttcgg gagcaaagac aaattatcca aagaggatat ggatttcctc aaatcacaaa 60
cacgatacga cgaagctaca attaaagaat ggtacaaagg ttttaagcaa gactgtccaa 120
acggtagact aacgccggcc aagttcgtag atatgtacaa gatgttcttt cccagtggta 180
atgccgaaga gttctgcgac cacgtattta ggacattcga catggacaag aacggatata 240
tcgatttcaa agagttcttg cttgctattg atgtgacatc gtcgggcacc ccagaagaaa 300
aactaaaatg ggcgtttagg atgtacgacg ttgacggcaa tggtgtcatc gacatccagg 360
aaatgacaaa gattgttcag gcgatctacg acatgctggg cgcatgctcg tcgaacagac 420
cggccgactc ggccgaggag cgcgccaaga acatctttgc caagatggac gagaacaacg 480
acggtcagct cacgcaggag gagttcctga agggttgtct ccaggacgag gagctgtcca 540
aaatgctggc gccata 556
<210> 5
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
taatacgact cactataggg agaggtgttt cgggagcaaa gac 43
<210> 6
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
taatacgact cactataggg agatatggcg ccagcatttt gga 43
<210> 7
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
taatacgact cactataggg agaatgagta aaggagaaga acttttc 47
<210> 8
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
taatacgact cactataggg agatttgtat agttcatcca tgccatgt 48

Claims (7)

1. The application of dsRNA for inhibiting the expression level of the NCS2 gene or the coded protein of brown planthopper in preparing medicines for preventing and treating brown planthopper is characterized in that the dsRNA consists of two complementary sequences, the nucleotide sequence of a sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is the reverse complementary sequence of the sequence shown as SEQ ID NO. 3.
2. The application of dsRNA for inhibiting the expression level of the NCS2 gene or the coded protein of brown planthopper in preparing medicines for reducing the spawning quantity of brown planthopper is disclosed, wherein the dsRNA consists of two complementary sequences, the nucleotide sequence of a sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is the reverse complementary sequence of the sequence shown as SEQ ID NO. 3.
3. The use according to claim 1 or 2, wherein the NCS2 gene has the nucleotide sequence shown in SEQ ID No. 1; the amino acid sequence of the protein coded by the NCS2 gene is shown as SEQ ID NO. 2.
4. The use according to claim 1 or 2, wherein the brown planthopper is a nymph of 2-5 instar.
5. The medicine for preventing and treating brown planthopper is characterized in that the active ingredient is dsRNA for reducing the NCS2 gene expression of brown planthopper;
the dsRNA consists of two complementary sequences, the nucleotide sequence of the sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of the sequence shown as SEQ ID NO. 3.
6. A method for controlling brown planthoppers, characterized in that the medicament of claim 5 is used.
7. The method of claim 6, wherein the brown planthopper is a 2-5 instar nymph.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018205732A1 (en) * 2017-05-08 2018-11-15 广东省农业科学院植物保护研究所 Gene bgiosga015651 for regulating rice resistance to plant hoppers and use thereof
CN110066798A (en) * 2019-04-16 2019-07-30 杭州师范大学 Application of the brown paddy plant hopper NlInR gene as target spot in preparation prevention and treatment brown paddy plant hopper drug
CN110684095A (en) * 2019-10-25 2020-01-14 中国水稻研究所 Protein phosphate gene NlPPP1-Y specifically expressed by brown planthopper males and application thereof
CN111004803A (en) * 2019-12-31 2020-04-14 中山大学 Application of Nilaparvata lugens NlStit gene as target in preparation of Nilaparvata lugens prevention and treatment medicines

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8906876B2 (en) * 2006-01-12 2014-12-09 Devgen Nv Methods for controlling pests using RNAi

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018205732A1 (en) * 2017-05-08 2018-11-15 广东省农业科学院植物保护研究所 Gene bgiosga015651 for regulating rice resistance to plant hoppers and use thereof
CN110066798A (en) * 2019-04-16 2019-07-30 杭州师范大学 Application of the brown paddy plant hopper NlInR gene as target spot in preparation prevention and treatment brown paddy plant hopper drug
CN110684095A (en) * 2019-10-25 2020-01-14 中国水稻研究所 Protein phosphate gene NlPPP1-Y specifically expressed by brown planthopper males and application thereof
CN111004803A (en) * 2019-12-31 2020-04-14 中山大学 Application of Nilaparvata lugens NlStit gene as target in preparation of Nilaparvata lugens prevention and treatment medicines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NADPH Oxidase 5 Is Essential for Molting and Oviposition in a Rice Planthopper Nilaparvata lugens;Lu-Yao Peng;Insects.;第11卷(第9期);全文 *
褐飞虱安全高效RNAi靶基因的筛选 及Snf7 同源基因的控害效果;黎丹;中山大学学报;第59卷(第6期);全文 *

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