CN111004803A - Application of Nilaparvata lugens NlStit gene as target in preparation of Nilaparvata lugens prevention and treatment medicines - Google Patents
Application of Nilaparvata lugens NlStit gene as target in preparation of Nilaparvata lugens prevention and treatment medicines Download PDFInfo
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- CN111004803A CN111004803A CN201911416589.2A CN201911416589A CN111004803A CN 111004803 A CN111004803 A CN 111004803A CN 201911416589 A CN201911416589 A CN 201911416589A CN 111004803 A CN111004803 A CN 111004803A
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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Abstract
The invention provides application of an NlStit gene of brown planthopper as a target spot in preparation of a medicament for preventing and treating the brown planthopper, wherein the nucleotide sequence of the NlStit gene is shown as SEQ ID NO. 1. The invention also provides dsRNA of the Nilaparvata lugens NlStit gene, which consists of two complementary nucleotide 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 shown as SEQ ID NO. 4. The dsRNA segment of the Nilaparvata lugens NlStit gene provided by the invention can effectively prevent and control Nilaparvata lugens.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of an NlStit gene of brown planthopper as a target spot in preparation of a medicament for preventing and treating the brown planthopper.
Background
Brown plant hoppers (Nilaparvata lugens) belong to Hemiptera (Hemiptera) plant hoppers (Delphacidae), have the characteristics of propagation and migration, are usually damaged by directly puncturing and absorbing rice stems and leaves, and are important pests in rice planting in China at present.
In insects, functional genes encoding important proteins can be used as target genes for intervention, and introduction of these genes can interfere with the functional genes, thereby causing growth and development of insects to be hindered and even insects to die.
The receptor tyrosine kinase Stitcher (Stit), also known as Cad96ca, encodes a Ret family Receptor Tyrosine Kinase (RTK) with a cadherin domain and a TK domain (Tepass et al, 2000). Stit is expressed in all ectodermal epithelial cells in the middle and late embryonic stages, but is subsequently restricted to the epithelial optic nerve (Fung et al, 2007). Stit regulates the transcriptional activation of epithelial regeneration and injury repair genes and the rearrangement of cytoskeleton by stimulating actin-cable assembly in the epithelialization process, induces the repair of extracellular barriers, thereby causing the closure of epidermal wounds, and discovers the phenomenon of death of homozygous deletion mutants in research (Wang et al, 2009). Furthermore, Stit activates ERK, an extracellular signal-regulated kinase, which induces ERK phosphorylation at insect injury sites, and is essential for activation of the transcription factor Grh (Wang et al, 2009; Mace et al, 2009). Thus, it is critical for effective healing of epidermal lesions that the sting is activated.
RNA interference (RNAi), a post-transcriptional silencing phenomenon resulting from the induction of endogenous target mRNA sequence-specific degradation by double-stranded RNA (dsrna), is a novel control strategy that has emerged in recent years in the field of pest control (Zhang et al, 2017).
Disclosure of Invention
The invention aims to provide a technical scheme capable of effectively preventing and treating brown planthopper aiming at the technical problems to be solved.
In order to realize the purpose, the invention provides application of an NlStit gene of the Nilaparvata lugens as a target spot in preparing a medicament for preventing and treating the Nilaparvata lugens, wherein the nucleotide sequence of the NlStit gene is shown as SEQ ID NO. 1.
The invention also provides dsRNA of the Nilaparvata lugens NlStit gene, which consists of two complementary nucleotide 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 shown as SEQ ID NO. 4.
The invention also provides a coding gene of the dsRNA, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The invention also provides an expression cassette, a recombinant vector, a recombinant bacterium, a transgenic cell line or a transgenic plant containing the coding gene.
The invention also provides application of the expression cassette, the recombinant vector, the recombinant bacterium, the transgenic cell line or the transgenic plant in preparation of a medicament for preventing and treating brown planthopper.
The invention also provides a medicament for preventing and treating brown planthopper, which comprises the dsRNA as an active ingredient.
The invention also provides a method for preventing and treating brown planthopper, which comprises the step of introducing the dsRNA into the body of the brown planthopper.
Preferably, in the method, the brown planthopper is a nymph.
Preferably, in the method, the mode of introduction is feeding or injection.
Experimental results show that the dsRNA segment of the Nilaparvata lugens NlStit gene provided by the invention can effectively prevent Nilaparvata lugens, and is expected to become an important direction of a pest control agent.
Drawings
FIG. 1 shows the relative expression of mRNA after injection of dsNlStit.
Figure 2 shows the corrected mortality rate of injected RNAi interfering nlstut.
Figure 3 shows the corrected mortality of feeding RNAi interfering with nlstut.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention.
It is noted that although some conventional technical operation steps are not described in detail in the examples of the present invention, those skilled in the art know these techniques.
Examples
The experiment was performed according to the following procedure.
1. Extracting total RNA of brown planthopper: taking one adult and nymph, grinding in liquid nitrogen, and extracting the brown planthopper total RNA by using TRIzol (conventional operation).
2. First strand cDNA Synthesis: using Beijing-all-gold Biotech Co
One-Step gDNAremoval and cDNA Sythesis SuperMix kit reverse transcribes l. mu.g of total RNA into cDNA.
3. And amplifying primer sequences of dsNlStit genes of the brown planthoppers, and preparing plasmids containing the NlStit genes.
1) And (3) amplifying a target fragment:
the polymerase chain reaction system is as follows:
| name of reagent | Volume/. mu. |
| cDNA template | |
| 2 | |
| ddH2O | 19 |
| 2×SuperStar PCR Mix | 25 |
| Forward primer (10 μm) | 2 |
| Reverse primer (10 μm) | 2 |
| Total volume | 50 |
Reaction procedure: a) pre-denaturation at 98 ℃ for 3 min; b) denaturation at 98 ℃ for 15 s; c) annealing at 60 ℃ for 15s, d) extension at 72 ℃ for 30s, steps b) to d) being set to 35 cycles; e) extending for 7min at 72 ℃; samples were stored at 4 ℃.
The primer sequences are as follows:
2) and (3) purifying an amplification product: and purifying the PCR amplified fragment by using a magenta gel recovery kit to obtain a purified product.
3) Obtaining an intermediate vector: cloning the purified product to a pEASY-Blunt Zero vector (Beijing all-purpose gold Biotechnology company) according to a ligation reaction system, transforming the product to a Transl-T1 competent cell (Beijing all-purpose gold Biotechnology company) according to a conventional operation, and culturing the cell on an LB plate containing I00 mu g/mL of ampicillin overnight (conventional culture conditions) to obtain a colony;
the connection reaction system is as follows: the purified product was 4. mu.L, pEASY-Blunt Zero 1. mu.L, in a total volume of 5. mu.L.
4) And (3) plasmid purification: and (3) inoculating the bacterial colony to an LB liquid culture medium, carrying out shaking overnight culture at 37 ℃, collecting bacterial liquid, carrying out PCR identification and sequencing to obtain a plasmid (pEASY-NlStit plasmid) containing NlStit genes, which is verified by sequencing.
Synthesis of dsRNA
The specific operation steps are as follows:
(1) cross PCR
Reaction system a was synthesized by adding the following reagents to a 0.2mL centrifuge tube without RNase:
| reagent | Volume/. mu.L |
| pEASY- |
1 |
| dsNlStit-F(10μm) | 1 |
| T7-dsNlStit-R(10μm) | 1 |
| 2×HiFi Taq Mix | 12.5 |
| ddH2O | 9.5 |
| Total volume | 25 |
Reaction system B was synthesized by adding the following reagents to a 0.2mL centrifuge tube without RNase:
| reagent | Volume/. mu.L |
| pEASY- |
1 |
| dsNlStit-R(10μm) | 1 |
| T7-dsNlStit-F(10μm) | 1 |
| 2x HiFi Taq Mix | 12.5 |
| ddH2O | 9.5 |
| Total volume | 25 |
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 40s for 30 cycles. Further extension at 72 deg.C for 7min, and storage at 4 deg.C.
T7 RiboMAX from Promega corporation was usedTMThe Express RNAi System kit synthesizes dsRNA of NlStit, named dsNlStit.
5. Injection of brown planthopper dsRNA (dsNlStit)
Brown planthopper was injected with dsNlStit at final concentrations of 2.5, 25, 250 and 250 ng/. mu.L (i.e. injection amounts of 0.1, 1, 10, 100 ng/head, respectively) at a dose of 40nL per head, with green fluorescent protein GFP as control.
Samples of 24h, 48h and 72h after the injection of the short-wing eclosion female adult brown planthopper are collected, and the relative expression quantity detection experiment of mRNA of NlStit is carried out through a fluorescent quantitative PCR experiment, and the result is shown in figure 1. The relative expression of mRNA after intervention with dsNlStit was found to be significantly down-regulated compared to control dsGFP.
In addition, after four low dose (0.1, 1, 10, 100 ng/head) dsNlStits were injected to the four-instar nymphs of brown planthoppers, the death numbers were counted and the corrected mortality was calculated by observing the phenomena for 11 consecutive days, and the results are shown in FIG. 2, in which the corrected mortality continued to rise and the corrected mortality was 100% on day 11 of the injection of the low dose 1 ng/head dsNlStit, indicating that it affects the normal development of the brown planthoppers.
6. Feeding of brown planthopper dsRNA
The synthesized dsRNA (dsNlStit) and the existing artificial feed for the common brown planthopper are respectively prepared into mixed feed of 0.1, 1, 10 and 100 ng/mu L.
The brown planthopper artificial feed is prepared by methods such as Fu Qiang (Fu et al, 2001) and the like.
Feeding short-wing type 4-instar female brown planthopper nymphs with the same size in a glass double-way pipe (9cm multiplied by 2cm), wherein the feeding operation is as follows:
(1) one side of the glass double-way pipe is covered with a Parafilm sealing film, 15 mu L of mixed artificial feed is added, and then another new Parafilm sealing film is covered;
(2) adding 15 brown planthopper nymphs which are grown consistently at 4 instars into a double-way pipe with one sealed end;
(3) the other side of the double-way pipe is sealed in the step (1);
(4) after the feed is added, putting the brown planthopper into an artificial climate box, and covering a piece of black cloth in the center of a glass tube so that the brown planthopper can feed towards the artificial feed with two bright ends;
(5) the artificial feed is replaced every 24h within 10 days, and the death number of the brown planthopper and other data are recorded.
Notably, climatic chambers require external humidifiers to ensure adequate humidity in the incubator.
As shown in figure 3, the results indicate that the corrected mortality was highest, more than 75%, at day 9 after feeding a 100ng/μ L dose of dsnlsting, and as the feeding dose was reduced, the two gene dsRNA corrected mortality showed a decreasing trend, with a clear dose effect.
Sequence listing
<110> Zhongshan university
Application of <120> Nilaparvata lugens NlStit gene as target in preparation of drugs for preventing and treating Nilaparvata lugens
<160>12
<170>SIPOSequenceListing 1.0
<210>1
<211>2623
<212>DNA
<213> Brown planthopper (Nilaparvata lugens)
<400>1
tacgtctgca aggttgtagc cgcgttcatt ttcttacttt taaaatgtgt tacggtctcc 60
aaaacacgag aactccacag agaacgttcc tctaaatcaa gtattatctt tcgcgattcg 120
aaatttattg actagtgatc agaaactcga gttctcgatt gataacagat tgtgtgagat 180
tgttccagct gtgttgttgt cttctttcga acgccaagaa gacaaccaac aacattcttg 240
aatggagttc tcacgaagtt cctctcaaaa acttctacag caaacacttt tcaacaacaa 300
ctatttcggc aaaatgttac gacatcatcg ataagaatgt cgaatcactt gcaccgatta 360
acaaggtgtg acattagtta gttattggag ttttcgtgag ttattcggaa gtgttgaagt 420
gaaagaagcc aacgtgttgg gtgtcagtgg tggaatttca gtgatttcag cagtgttcct 480
attatggatc gattggattt catctcgatt ctgctgttga acttctgctt gctgctggga 540
ggtgtgagca gccagggcga caacactcct cccgtggtat ggatggaacgcaaatgggta 600
gtgtccgaga cagagccagt aggcagtgtg gtgacacgcg tgcgggggtc ggacgccgag 660
ggagatccac tcgagtttgg actcgaacac catcttgggt tcaatgtagc cgcacccgat 720
ccaaatccga gtccgctccc gtttcgcatc gacaaccgga ccggtgttgt ctacactaat 780
gattcgttgg ttggcaaggg cggaaaagac atgttcctgt acgtgacggt aacggatggc 840
aagctgcttc acaagagcga ggtgtgggtg aacgtcattg gtggtggaga cgaccaatca 900
gccgccccca ggaacaaacc caccgacctg cccaacaagc tgctcgccca atacccgagc 960
ccaccacccc cgggaggcct aggcagctac ctgccaggag gcggcttccc acccccggtg 1020
ttcccaggat cacagccaag ggcgcctccc accccaccgc ctccaaagcc gcctccaccg 1080
catcctgatc cgcccccaca tcccaaaccg cccactcctc ccggtgaagt tactgtggcc 1140
actgaggagt cgacaagaag ttcaacgtcc agagcagaag tggaagttgc gacgtccacc 1200
cagccaacag tgacgtcacc aattgccgtg actgatgccg atctgatcat cgtcactgta 1260
gttatggtgg cagtcattgt ctccatctcg tcagtcgtgg cctgcctctt ccggaggagg 1320
atatgcaaga gagccaagtc gaaaaaggat gaaatgagga aggagccagc aagcagcgag 1380
gagccaatgg caatgcagca ttggcaaggt cccagagcgt acgtcaacag atatgagtca 1440
tgggacctgg ataacgtaca gggacagaca gttgagcaaa gcaagctatc agaccgttgg 1500
gaattcccaa gacaccacct caaagtctac agcatcctgg gtgaaggatg tttcggccaa 1560
gtatggcgat gtgaagcagt cggcattgat ggaagagcag gacaatcagt ggtagctgtt 1620
aagacactca aagagaacgc cggagaaaga gagagactgg acttactgca agagctacaa 1680
gtcatgaaat ctctggagcc tcatcctcac gttgtcaggt tacttggctg ttgcactgaa 1740
aaagatccgc tgttcgttat aatggagttc atagcaaatg gcaaactaca gagcttcctt 1800
aggaactcga gggcacagcg ctgctacgat aacatgcacg gcaagagcgc aacactcacc 1860
tcgcgcgatc taacctcgtt ctgctaccag gtagcccgcg gcatggactt tctctcgtcg 1920
aatgggataa ttcaccgtga cttggcggct cgaaatattc tggtcgacga gaaccatatc 1980
tgcaaagtgg ctgactttgg attcgctcgt gacctcatgg ccaactatgt ctatgaacgc 2040
aagtcagagg gtcgactgcc tattagatgg atggcccccg aatcgctcta cgacaacatc 2100
ttctcggtga agtcggacat ctggagcttc ggcgtgctga tctgggaaat agtgaccctg 2160
ggctcgacgc cttaccccgg catggcagct gctgaggtca tgcgcaaagt gcgcgacgga 2220
taccgactcg acaaacccga gcactgccga cgcgagctct acaacatcat gttctactgc 2280
tgggacaacg actccaagga gaggcccgac ttcaaggagc tcgtgaagct gctcgaacag 2340
ctgctgttga ctgagactga gtacattgaa ctggatcggt ttcccgatca ttcctactac 2400
aacatgtcgt cgttgagtgg agaaaagcta tgaaaagagt cagggagtgg gaatccatga 2460
tcatcaaagg ttcatggatc acgcttactc caaacatgtc ttctccaagt ggggagaagt 2520
tgtgattttg tcaaaagtgt tcaatgttac gtacaacata accgtaatgc tagttacaca 2580
cacatcgatt tttggacgta cgatcttttg ccgttcttta tat 2623
<210>2
<211>364
<212>DNA
<213> Brown planthopper (Nilaparvata lugens)
<400>2
ggaggaggat atgcaagaga gccaagtcga aaaaggatga aatgaggaag gagccagcaa 60
gcagcgagga gccaatggca atgcagcatt ggcaaggtcc cagagcgtac gtcaacagat 120
atgagtcatg ggacctggat aacgtacagg gacagacagt tgagcaaagc aagctatcag 180
accgttggga attcccaaga caccacctca aagtctacag catcctgggt gaaggatgtt 240
tcggccaagt atggcgatgt gaagcagtcg gcattgatgg aagagcagga caatcagtgg 300
tagctgttaa gacactcaaa gagaacgccg gagaaagaga gagactggac ttactgcaag 360
agct 364
<210>3
<211>364
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ggaggaggau augcaagaga gccaagucga aaaaggauga aaugaggaag gagccagcaa 60
gcagcgagga gccaauggca augcagcauu ggcaaggucc cagagcguac gucaacagau 120
augagucaug ggaccuggau aacguacagg gacagacagu ugagcaaagc aagcuaucag 180
accguuggga auucccaaga caccaccuca aagucuacag cauccugggu gaaggauguu 240
ucggccaagu auggcgaugu gaagcagucg gcauugaugg aagagcagga caaucagugg 300
uagcuguuaa gacacucaaa gagaacgccg gagaaagaga gagacuggac uuacugcaag 360
agcu 364
<210>4
<211>364
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
agcucuugca guaaguccag ucucucucuu ucuccggcgu ucucuuugag ugucuuaaca 60
gcuaccacug auuguccugc ucuuccauca augccgacug cuucacaucg ccauacuugg 120
ccgaaacauc cuucacccag gaugcuguag acuuugaggu ggugucuugg gaauucccaa 180
cggucugaua gcuugcuuug cucaacuguc ugucccugua cguuauccag gucccaugac 240
ucauaucugu ugacguacgc ucugggaccu ugccaaugcu gcauugccau uggcuccucg 300
cugcuugcug gcuccuuccu cauuucaucc uuuuucgacu uggcucucuu gcauauccuc 360
cucc 364
<210>5
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
ggaggaggat atgcaagaga gc 22
<210>6
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
agctcttgca gtaagtccag tc 22
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<213> Artificial Sequence (Artificial Sequence)
<400>7
ggatcctaat acgactcact ataggggagg aggatatgca agagagc 47
<210>8
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
ggatcctaat acgactcact ataggagctc ttgcagtaag tccagtc 47
<210>9
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
aagggcgagg agctgttcac cg 22
<210>10
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
cagcaggacc atgtgatcgc gc 22
<210>11
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
ggatcctaat acgactcact ataggaaggg cgaggagctg ttcaccg 47
<210>12
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
ggatcctaat acgactcact ataggcagca ggaccatgtg atcgcgc 47
Claims (9)
1. The application of the Nilaparvata lugens NlStit gene as a target spot in preparing a medicament for preventing and treating Nilaparvata lugens is characterized in that the nucleotide sequence of the NlStit gene is shown as SEQ ID No. 1.
2. The dsRNA of the Nilaparvata lugens NlStit gene is characterized by consisting of two complementary nucleotide 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 shown as SEQ ID NO. 4.
3. The dsRNA-encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.
4. An expression cassette, recombinant vector, recombinant bacterium, transgenic cell line or transgenic plant comprising the encoding gene of claim 3.
5. The use of the expression cassette, recombinant vector, recombinant bacterium, transgenic cell line or transgenic plant of claim 4 in the preparation of a medicament for the control of Nilaparvata lugens.
6. A drug for controlling brown planthopper, comprising the dsRNA according to claim 2 as an active ingredient.
7. A method for controlling brown planthopper, comprising introducing the dsRNA of claim 2 into a body of brown planthopper.
8. The method of claim 7, wherein the brown planthopper is a nymph.
9. The method of claim 7 or 8, wherein the introducing is by feeding or injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113337503A (en) * | 2021-04-09 | 2021-09-03 | 广东省农业科学院植物保护研究所 | Application of brown planthopper NLSP7 as target spot in prevention and treatment of brown planthopper |
| CN114807156A (en) * | 2022-04-14 | 2022-07-29 | 浙江大学 | Application of brown planthopper NCS2 gene or encoded protein as target point in preparation of drugs for preventing and treating brown planthopper |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113337503A (en) * | 2021-04-09 | 2021-09-03 | 广东省农业科学院植物保护研究所 | Application of brown planthopper NLSP7 as target spot in prevention and treatment of brown planthopper |
| CN113337503B (en) * | 2021-04-09 | 2022-11-22 | 广东省农业科学院植物保护研究所 | Application of brown planthopper NLSP7 as target spot in prevention and treatment of brown planthopper |
| CN114807156A (en) * | 2022-04-14 | 2022-07-29 | 浙江大学 | Application of brown planthopper NCS2 gene or encoded protein as target point in preparation of drugs for preventing and treating brown planthopper |
| CN114807156B (en) * | 2022-04-14 | 2023-12-22 | 浙江大学 | Application of brown planthopper NCS2 gene or coded protein as target spot in preparation of brown planthopper prevention and treatment drugs |
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