Brown planthopper nivha-c gene and its dsrna in the prevention and treatment of brown planthopper and method

Abstract

This invention belongs to the field of biotechnology, specifically relating to the brown planthopper. NIVHA-c Application and methods of genes and their dsRNA in the control of brown planthoppers. This was achieved through the preparation of... NIVHA-c RNAi treatment of the brown planthopper with the gene's dsRNA revealed that silencing this gene significantly reduced the planthopper's survival rate, and markedly decreased honeydew secretion and weight gain in adults. L4440- was constructed. NIVHA-c The dsRNA expression vector was transformed into *E. coli*, and dsRNA was produced after IPTG induction. The survival rate of brown planthoppers that fed on artificial feed containing dsRNA also decreased significantly; indicating that... NIVHA-c The gene plays an important role in the growth, development, and feeding process of the brown planthopper, and its dsRNA has a good pest control effect. This invention provides... NIVHA-c NIVHA-c Genes and their dsRNAs can serve as important targets for the development of biopesticides and the biological control of brown planthoppers, which is of great significance for the green control of brown planthoppers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to the biological control of brown planthoppers. Background Technology

[0002] Rice is a crucial food crop for ensuring global food security and alleviating the imbalance between food supply and demand. Increasing rice yield is of great significance for safeguarding national and global food security. However, the brown planthopper (Nilaparvata lugens) is a major pest in rice production, characterized by its short life cycle, high reproductive rate, and strong migratory ability, and has long been one of the main pests affecting rice production. Since the 1970s, my country's rice production has long relied heavily on chemical insecticides such as organophosphates and neonicotinoids. While this has controlled brown planthopper damage to some extent, it has also led to varying degrees of resistance to multiple insecticides, resulting in a decline in natural enemy populations and an imbalance in the paddy field ecosystem. Therefore, developing efficient, highly specific, and environmentally friendly new technologies for brown planthopper control has become a critical issue that urgently needs to be addressed in the field of agricultural pest control.

[0003] RNA interference (RNAi) is a biological process that specifically inhibits the expression of a specific target gene by inducing exogenous or endogenous double-stranded RNA (dsRNA). This technology achieves target gene silencing by triggering the degradation of homologous mRNAs through dsRNA. Compared with traditional chemical control techniques, RNAi technology has advantages such as high species specificity, biodegradability, and low environmental residue risk. Therefore, it is considered one of the important technical approaches to replace traditional chemical control and achieve green control of agricultural pests, providing new ideas for precise, efficient, and environmentally friendly control of brown planthoppers. For example, patent 202310050947.2 discloses the application of the brown planthopper NlA7 gene and its dsRNA in the control of brown planthoppers, silencing brown planthoppers through microinjection of dsRNA. NlA7 Genes that induce brown planthoppers to exhibit a feeding refusal effect.

[0004] Although RNAi technology has shown great potential in pest control, its industrial application still faces technical bottlenecks in areas such as target screening and delivery efficiency. On the one hand, insect genomes contain a vast number of gene families, and some genes have functional redundancy. Therefore, it is necessary to screen for target genes that play a crucial role in pest survival, growth, development, or reproduction, and whose functions are difficult to substitute. On the other hand, the screened target genes should also avoid high homology with non-target organisms as much as possible to reduce potential ecological risks. Therefore, screening key functional genes of the brown planthopper and applying them as precision control targets in the development of biopesticides and the biological control of the brown planthopper has promising application prospects and is of great significance for promoting the development of green control technologies for the brown planthopper. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention proposes the brown planthopper. NlVHA-c Applications and methods of genes and their dsRNAs in the control of brown planthoppers.

[0006] The technical solution of this invention is implemented as follows:

[0007] On the one hand, the present invention provides a brown planthopper NlVHA-c The dsRNA of the gene, wherein the dsRNA comprises a nucleotide sequence as shown in SEQ ID No. 3 and a nucleotide sequence that is inversely complementary to it.

[0008] Secondly, the present invention provides an engineered strain expressing the above-mentioned dsRNA in prokaryotes, and the method for constructing the engineered strain is as follows:

[0009] (1) Using brown planthopper cDNA as a template, primers containing the SacⅠ site cL- NlVHA-c -F and primers containing the KpnⅠ site cL- NlVHA-c -R was used for PCR amplification, and the amplification product was purified and recovered after double enzyme digestion to obtain the target fragment;

[0010] (2) After double enzyme digestion, the prokaryotic expression vector is ligated with the target fragment in step (1), and the ligation product is transformed into RNase III-deficient Escherichia coli. The positive clone strains obtained by screening are the engineered strains that express dsRNA in prokaryotes.

[0011] dsRNA can be extracted from engineered strains after induction with IPTG (isopropyl thiogalactoside).

[0012] Preferably, the above primer cL- NlVHA-c The -F sequence is shown in SEQ ID No. 5, and the primer cL- NlVHA-cThe sequence of -R is shown in SEQ ID No. 6; the prokaryotic expression vector is vector L4440, and the RNase III-deficient Escherichia coli is Escherichia coli strain HT115.

[0013] Thirdly, the present invention provides a brown planthopper as described above. NlVHA-c The in vitro synthesis method of dsRNA of a gene includes the following steps:

[0014] (1) Using brown planthopper cDNA as a template, primers ds with a T7 promoter were used. NlVHA-c -F and primer ds NlVHA-c -R is used for PCR amplification to obtain an amplification product containing the T7 promoter sequence;

[0015] (2) The amplified product was purified and used as a transcription template for in vitro transcription to synthesize dsRNA.

[0016] Preferably, the above in vitro synthesis NlVHA-c The dsRNA nucleotide sequence is shown in SEQ ID No. 3. EGFP The dsRNA nucleotide sequence is shown in SEQ ID No. 4.

[0017] Fourthly, the present invention provides a reagent for controlling brown planthoppers, comprising the aforementioned brown planthoppers. NlVHA-c The dsRNA of the gene or the engineered strain mentioned above.

[0018] Preferably, the dosage of dsRNA in the above reagents is 40-230 ng / head.

[0019] Fifthly, the present invention provides brown planthoppers. NlVHA-c Application of genes, dsRNAs, or reagents mentioned above in the control of brown planthoppers.

[0020] Preferably, the above NlVHA-c Genes can be used as targets to control brown planthoppers. NlVHA-c The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2. NlVHA-c The gene is 3618 bp in length and contains a complete open reading frame of 471 bp that encodes a small molecular weight protein of 157 amino acid residues.

[0021] In a sixth aspect, the present invention provides a method for controlling brown planthoppers, wherein the above-mentioned reagent is injected into the brown planthopper or artificially fed to the brown planthopper to control the brown planthopper.

[0022] Preferably, the above-mentioned artificial feeding method involves adding the bacterial solution of the above-mentioned engineered strain to artificial feed and feeding it to brown planthoppers.

[0023] Preferably, the dosage of dsRNA in the above-mentioned injection reagent is 40-230 ng / head; when artificially feeding, 20 μL of bacterial solution is added per 100 μL of artificial feed.

[0024] The present invention has the following beneficial effects:

[0025] 1. This invention provides brown planthopper NlVHA-c Application of dsRNA in the control of brown planthoppers. Preparation via in vitro transcription. NlVHA-c The dsRNA of the gene was extracted, and RNA interference was performed on brown planthoppers using microinjection. Results showed that silencing... NlVHA-c After gene administration, the survival rate of brown planthoppers decreased significantly, and the amount of honeydew secreted and the weight gain of adults decreased significantly, indicating that this gene plays an important role in the survival and feeding process of brown planthoppers and can be used as an effective target for the control of brown planthoppers.

[0026] 2. This invention constructs L4440- NlVHA-c dsRNA expression vector and transformation Escherichia coli HT115, obtained after IPTG induction NlVHA-c dsRNA. Brown planthoppers, after ingesting artificial feed containing this dsRNA, [their...] NlVHA-c Gene expression levels were significantly downregulated at both 48 h and 72 h, and survival rates were significantly reduced, indicating that this dsRNA has good interference effects and control potential. The present invention provides... NlVHA-c The gene, its encoded protein, and dsRNA can be used for the research and development of biological pesticides and the biological control of brown planthoppers. It has the advantages of strong targeting, clear control effect, and good application prospects, and is of great significance for the green control of brown planthoppers. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Brown planthopper NlVHA-c A diagram illustrating the spatiotemporal expression patterns of genes. Figure A shows... NlVHA-c The relative expression levels of B in brown planthoppers of different instars. NlVHA-c Relative expression levels in different tissues. Different letters indicate significant differences.

[0029] Figure 2 For in vitro synthesis of ds NlVHA-c The results of agarose gel electrophoresis are shown in the figure.

[0030] Figure 3 For microinjection ds NlVHA-c Figure A shows the results of fitness testing of the brown planthopper on rice. NlVHA-c The results of gene silencing efficiency testing, expressed in ds EGFP B represents the control group; C represents the survival rate of brown planthoppers feeding on TN1 rice after RNAi treatment; D represents the amount of honeydew secreted by emerging females 48 h after feeding on rice after RNAi treatment; and the percentage weight gain of emerging females 48 h after feeding on rice after RNAi treatment. An asterisk indicates a significant difference: * indicates a statistically significant difference. P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0031] Figure 4 For recombinant plasmid L4440- NlVHA-c A structural diagram.

[0032] Figure 5 For L4440- NlVHA-c Figure showing the results of agarose gel electrophoresis of dsRNA-expressing products.

[0033] Figure 6 Brown planthoppers feed on ds NlVHA-c Figure A shows the survival test results after artificial feeding. NlVHA-c Figure 1 shows the validation results of gene silencing effect; B represents brown planthoppers feeding on food containing ds NlVHA-c Graph showing changes in mortality rates after artificial feed administration. Asterisks indicate significant differences: * indicates... P <0.05. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] V-ATPase (Vacuolar-type ATPase) is a multi-substrate pump complex composed of a peripheral V1 complex and an intramembrane-embedded V0 complex. It drives H⁺ transmembrane transport via ATP hydrolysis, playing a crucial role in maintaining cellular and organelle pH homeostasis, osmotic pressure regulation, and neurotransmitter release. In insects, V-ATPase is widely distributed in the midgut epithelium and salivary glands, providing essential electrochemical driving forces for feeding, digestion, and ion balance. Studies have shown that insect V-ATPase subunits differ significantly in sequence and structure from those in mammals, and targeting insect-specific V-ATPase subunits holds promise for achieving high selective toxicity. However, there are currently no reports on using the V-ATPase subunit genes of the brown planthopper as RNA interference targets for its control. The function of these genes and their roles in the growth, feeding, and survival of the brown planthopper require further research and development.

[0037] In the preparation and delivery of dsRNA, the RNase III-deficient *E. coli* strain HT115, lacking the ability to degrade dsRNA, can efficiently express exogenous dsRNA, making it a low-cost and simple method for dsRNA production. Furthermore, using engineered bacteria to express dsRNA and inducing RNAi through feeding avoids operational errors caused by frequent injections, thus helping to maintain consistency among treated individuals. Achieving efficient target gene silencing by synthesizing dsRNA in vitro or by expressing dsRNA using engineered bacteria and feeding it to brown planthoppers, thereby inhibiting their growth and survival, is a promising control strategy.

[0038] Brown planthoppers were tested. The brown planthoppers were reared on TN1 rice variety at a temperature of 28℃, a humidity of 75%, and a photoperiod of 16L:8D.

[0039] The main reagents used in this invention are shown in Table 1:

[0040] Table 1. Reagents used in this invention

[0041]

[0042] Table 2 Primers used in this invention

[0043]

[0044] The primers used in this invention are shown in Table 2:

[0045] Example 1: Brown planthopper NlVHA-c Cloning of genes

[0046] 1. Extraction of total RNA

[0047] Four brown planthoppers were collected using a homemade trematode and placed in a 1.5 mL centrifuge tube. Total RNA was extracted from the brown planthoppers using the TRIzol method, as follows:

[0048] (1) Add one grinding bead and 500 μL of RNAiso Plus reagent to an RNasefree centrifuge tube containing brown planthoppers, tighten the cap, grind thoroughly in a grinder, and let stand on ice for 5 min.

[0049] (2) Add 100 μL of chloroform to the centrifuge tube, invert the tube to mix thoroughly, and let it stand on ice to separate the layers. Centrifuge at 12,000 rpm for 15 min at 4℃.

[0050] (3) Take the supernatant into a brand new 1.5 mL RNase-free centrifuge tube, add 200 μL of isopropanol, invert the tube, and let it stand on ice for 1 h to precipitate. Centrifuge at 4℃, 12000 rpm for 10 min.

[0051] (4) Discard the supernatant, add 500 μL of 75% anhydrous ethanol (prepared fresh for use), gently invert and wash the precipitate. Centrifuge at 7500 rpm for 5 min at 4℃.

[0052] (5) Repeat step (4).

[0053] (6) Drying.

[0054] (7) Add 10 μL of RNase-free H2O to dissolve the RNA.

[0055] (8) RNA quality was detected by agarose gel electrophoresis. 1 μL of RNA was taken and its purity and concentration were detected by micro-spectrophotometer.

[0056] Brown planthopper cDNA was synthesized according to the HiScript IV All-in-One Ultra RT SuperMix for qPCR instructions and used as a PCR amplification template.

[0057] 2. NlVHA-c Gene cloning

[0058] Obtained from NCBI database NlVHA-c The nucleotide sequence of the gene was used to design PCR primers using Primer 5, and primers c in Table 2 were used. NlVHA-c -F and c NlVHA-c -R is used for PCR amplification.

[0059] The PCR reaction system consisted of: 25 μL Phanta Flash Master Mix, 1 μL each of the PCR primers, 1 μL of cDNA, and double-distilled water to a final volume of 50 μL.

[0060] The PCR reaction program was as follows: 98℃ pre-denaturation for 30 sec; 98℃ denaturation for 10 s; 60℃ annealing for 5 s; 72℃ extension for 2 s; 35 cycles; and a final extension at 72℃ for 1 min.

[0061] After electrophoresis of the PCR products using 1% agarose gel, the PCR products were ligated into pCE3 Blunt Vector (Vazyme) using the Ultra-Universal TOPOCloning Kit and transformed into *E. coli* DH5α competent cells. The cells were plated on ampicillin-containing plates and cultured overnight. Single colonies were picked for PCR verification. Correct colonies were sent to our company (Qingke Biotechnology Co., Ltd.) for sequencing. Sequencing results were compared with sequences in the NCBI database using Bioedit software. Gene information was obtained from the NCBI database, and primers were designed for gene cloning. Successful cloning was achieved. NlVHA-c The gene CDS sequence is shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 2.

[0062] 3. Brown planthopper NlVHA-c Spatiotemporal expression patterns of genes

[0063] Brown planthoppers were dissected under a stereomicroscope in 1% PBS buffer. Samples were taken from the intestine, fat body, salivary glands, testes, and ovaries, and then rapidly frozen in liquid nitrogen. Simultaneously, samples from different instars of brown planthoppers (nymphal instars and males / females) were collected using a homemade trematode tube, rapidly frozen in liquid nitrogen, and then stored in an ultra-low temperature freezer. Detection was performed using quantitative real-time PCR. NlVHA-c Gene expression in different tissues and at different ages was investigated using Primer Primer 5 for quantitative primer design. Following the instructions for the SupReal QPurple Universal SYBR qPCR MasterMix (U+) (Novizan), a 10 μL PCR reaction system was selected, including 5 μL of SYBR, 2 μL of cDNA template, 0.4 μL of primers (mixed), and 2.6 μL of ddH2O. The amplification program was: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 1 min, for 40 cycles. NlRPS The gene was used as an internal reference gene, and the relative expression level of the target gene was measured using a 2-1 ratio. -△△Ct The method is used for calculation.

[0064] The results showed that NlVHA-c The gene is expressed at all stages of life, with the highest expression in females. [[ID= A), is expressed most highly in the midgut, followed by the salivary glands ( ​ B).

[0065] Example 2: Synthesis, microinjection, and silencing effect verification of dsRNA

[0066] 1. Synthesis of dsRNA

[0067] Example 1 ​ and ​ The target fragment (enhanced green fluorescent protein) was amplified, ligated, transformed, and identified by PCR, followed by sequencing. 100 μL of the correctly sequenced bacterial culture was added to 5 mL of liquid LB medium containing ampicillin and incubated overnight in a shaker.

[0068] Plasmids were extracted from the overnight culture using the M5 plasmid miniprep plus kit. dsRNA primers were designed using Primer primier5, and the T7 promoter sequence was added to the front end of the primers.

[0069] Using dsRNA primer pairs ​ and ​ The gene plasmid was amplified by PCR, and the amplified product was then purified using the FastPure Gel DNA Extraction Mini kit. Once the purified DNA template passed quality checks and concentration tests, it became the template for dsRNA synthesis. ​ Genes and ​ The nucleotide sequences of the gene's dsRNA are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. The dsRNA was synthesized according to the instructions for the MEGAscript™ T7 Transcription Kit. ​ Electrophoresis diagram of gene dsRNA synthesis as shown below ​ As shown. ​ The sequence of the gene's dsRNA is shown in SEQ ID No. 3. ​ The sequence of the gene's dsRNA is shown in SEQ ID No. 4.

[0070] 2. Microinjection of dsRNA

[0071] Third instar nymphs of brown planthoppers were selected, and 40 ng ds was injected into the middle portion of the midcoxel and hindcoxel of their thorax using a microinjector. ​ and ds ​ The specific method is as follows:

[0072] (1) Collect a number of 3rd instar brown planthopper nymphs and anesthetize them with CO2 for 10 s;

[0073] (2) Use a micro-pipette to add the sample. ​ The dsRNA of the gene was injected into a glass capillary;

[0074] (3) Install the glass capillary tube containing the sample into a microinjector and inject dsRNA into the brown planthopper under a stereomicroscope.

[0075] (4) Using the same method, ​ The dsRNA of the gene was injected into brown planthoppers as a negative control;

[0076] (5) After the brown planthopper nymphs recover after injection, transfer them to rice plants.

[0077] 3. Verification of dsRNA silencing effect

[0078] Samples were taken at 24 h, 48 h, and 72 h post-injection and detected using a real-time PCR instrument. ​ Gene expression status is analyzed using the following methods:

[0079] (1) Collect brown planthopper nymphs injected with dsRNA and obtain the injected brown planthopper RNA and cDNA using the total RNA extraction method in Example 1;

[0080] (2) Using brown planthopper cDNA as a template, following the instructions of SupReal QPurple Universal SYBR qPCRMaster Mix (U+) (Novizan), a 10 μL PCR reaction system was selected, including 5 μL of SYBR, 2 μL of cDNA template, 0.4 μL of primers (mixed) and 2.6 μL of ddH2O; the amplification program was: 95 ℃ reaction for 10 min; 95 ℃ reaction for 10 s, 60 ℃ reaction for 1 min, 40 cycles;

[0081] (3) Using 2 -△△Ct Calculating brown planthoppers ​ The expression levels were assessed using an independent samples t-test, which showed significant differences between different treatment groups.

[0082] After confirming silencing, ds will be injected. ​ and ds ​ The brown planthoppers were transferred to rice paddies, and the number of survivors was recorded daily until all of them emerged.

[0083] Samples were taken at 24 h, 48 h and 72 h after microinjection of dsRNA into brown planthoppers for analysis. ​Gene silencing effect. Quantitative results showed that, compared with the control group, ​ Gene expression levels decreased significantly at all time points, with a 68.00% decrease at 24 h, a 42.57% decrease at 48 h, and an 89.90% decrease at 72 h. These results indicate that microinjection of ds ​ Effectively silences brown planthoppers ​ Genes, with good interference effect ( ​ A).

[0084] Example 3: Brown planthopper ​ Survival rate in rice after gene silencing

[0085] The injection will be completed. ​ Brown planthoppers with the dsRNA gene were reared in rice (TN1 variety), with approximately 30 brown planthoppers per group of rice, and three replicates were performed. Mortality was recorded daily. After the brown planthopper nymphs emerged, the honeydew and percentage weight gain of the newly emerged females were measured and calculated 48 hours later.

[0086] Experimental results show that, compared with ds ​ Compared with the control group, injection of ds ​ The survival rate of brown planthoppers generally decreased at all observation time points, and the differences between groups gradually increased with the extension of treatment time. Specifically, the survival rate of ds from day 1 to day 9 was... ​ The survival rates of the groups were 93.33%, 85.83%, 83.00%, 77.17%, 68.67%, 68.67%, 68.00%, 67.17%, and 66.33%, respectively; while the ds ​ The percentages for the groups were 90.21%, 84.11%, 75.56%, 62.63%, 38.23%, 29.30%, 27.58%, 26.41%, and 25.09%, respectively. (Compared to ds) ​ Compared to the group, ds ​ The survival rates of the groups on days 1-9 were 3.12, 1.72, 7.44, 14.54, 30.44, 39.37, 40.42, 40.76, and 41.24 percentage points lower, respectively. These results indicate that ds ​ Treatment significantly reduced the survival rate of brown planthoppers, and the inhibitory effect gradually increased with prolonged treatment time. ​ B, Table 6).

[0087] Table 6. Injection of ds into 3rd instar nymphs of the brown planthopper ​ and ds ​ Survival rate on rice

[0088]

[0089] Further measurements were taken of honeydew secretion and body weight gain in emerging female brown planthoppers. The results showed that, compared with ds ​ Compared with the control group, silence ​ After gene administration, the weight gain of brown planthoppers on rice decreased significantly, by 42.22%, which was highly significant (P=0.00005). ​ C). Meanwhile, with ds ​ Compared with the control group, silence ​ Following gene modification, the honeydew secretion of brown planthoppers on rice also decreased significantly, by 65.41% (P=0.026). ​ D).

[0090] Example 4: Brown planthopper L4440- ​ -dsRNA expression and its RNAi effect

[0091] 1. Brown planthopper L4440- ​ Construction of prokaryotic expression vectors

[0092] (1) Fragment amplification

[0093] Primers were designed using Primer primier5, with the upstream primer cL- ​ -F adds the SacⅠ site, downstream primer cL- ​ -R adds a KpnⅠ site. The target gene was amplified using brown planthopper cDNA as a template. The reaction system was as follows: Phanta Flash Master Mix 25 μL, PCR primers 1 μL each, cDNA 1 μL, and double-distilled water to 50 μL. The PCR reaction steps were: 98℃ pre-denaturation for 30 sec; 98℃ denaturation for 10 s; 60℃ annealing for 5 s, 72℃ extension for 2 s, 35 cycles, and a final extension at 72℃ for 1 min. The amplified product was purified using the FastPure Gel DNA Extraction Mini kit. The purified gene fragment was digested with SacⅠ and KpnⅠ, and then purified by gel extraction. The above primer cL- ​ The -F sequence is shown in SEQ ID No. 5, and the primer cL- ​ The sequence of -R is shown in SEQ ID No. 6.

[0094] (2) Expression vector L4440 double enzyme digestion

[0095] After transforming the L4440 plasmid into Escherichia coli i HT115, the culture was streaked overnight (LB solid medium containing ampicillin and tetracycline). Single colonies were picked and cultured overnight at 37°C with shaking at 220 rpm on LB liquid medium containing ampicillin and tetracycline. The L4440 plasmid was extracted, digested with SacⅠ and KpnⅠ, and purified by gel extraction.

[0096] Brown planthopper ​ The ligation reaction system of the fragment and expression vector L4440: double digestion of linear L4440 plasmid; double digestion ​ Fragment; 10× ligation buffer; T4 DNA ligase.

[0097] The product is converted to ​ HT115 (DE3), L4440- ​ plasmid maps as follows ​ As shown.

[0098] 2. Prokaryotic expression of brown planthopper L4440- ​ -dsRNA and dsRNA extraction

[0099] Induction of dsRNA:

[0100] Take the identification as ​ Strains of the HT115(DE3) positive clone were streaked overnight on LB solid medium containing ampicillin and tetracycline. Single colonies were picked and cultured in 5 mL LB liquid medium containing ampicillin and tetracycline at 37°C with shaking at 220 rpm until the OD value reached 0.5. After induction with IPTG (final concentration 1 mmol / L) for 4 h, dsRNA was extracted using the phenol-chloroform method.

[0101] (1) Collection of bacteria. Collect 5 mL of the induced bacterial culture by centrifugation at 4℃ and 10,000 rpm for 10 min;

[0102] (2) Lysis. After centrifugation, add 100 μL of PBS buffer to the precipitate to resuspend the E. coli in the PBS buffer, then add 1 mL of RNA extraction buffer (Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1), and vortex the test tube for 3 min to fully lyse the E. coli cells and release dsRNA;

[0103] (3) Extracting aqueous dsRNA. Centrifuge the above mixture at 4℃ and 12000rpm for 15 min, then transfer the upper aqueous phase to a new RNase-free centrifuge tube, add an equal volume of isopropanol, vortex thoroughly, let stand for 1 h, and centrifuge at 4℃ and 10000rpm for 10 min;

[0104] (4) Washing. Discard the supernatant, add 500 μL of 75% anhydrous ethanol (prepared fresh for use), gently invert, and wash the precipitate. Centrifuge at 7500 rpm for 5 min at 4℃;

[0105] (5) Repeat step (4);

[0106] (6) Drying;

[0107] (7) Add 10 μL of RNase-free H2O to dissolve the RNA.

[0108] RNA quality was assessed using agarose gel electrophoresis, and 1 μL of RNA was used for purity and concentration analysis using a micro-spectrophotometer.

[0109] L4440- ​ -dsRNA electrophoresis diagram as shown below ​ As shown, by ​ It can be seen that IPTG successfully induced E. coli HT115 to produce dsRNA.

[0110] 3. Artificial feeding of brown planthoppers L4440- ​ RNAi effect after -dsRNA

[0111] L4440- after induction as described above ​ E. coli with -dsRNA were enriched at room temperature. RNase-free H2O was added at a ratio of 200:1 to dissolve the enriched cells, and the mixture was thoroughly mixed.

[0112] Thirty uniformly sized third-instar brown planthopper nymphs were selected and placed in a small plastic container with a diameter of 2.5 cm and a height of 7 cm. The artificial feed was wrapped in double-layered sealing film, and the plastic container was covered with a black cloth. Due to phototaxis, the brown planthoppers approached the rim of the container to feed on the artificial feed. 20 μL of bacterial solution was added to every 100 μL of artificial feed. The artificial feed was changed regularly each day, and the feeding was continued. Total RNA was extracted from the brown planthopper nymphs after the first three days of feeding, and the expression level of NlVHA-c was quantitatively analyzed. Survival rates were also recorded.

[0113] The results showed that feeding brown planthoppers ​ After dsRNA administration, samples were taken at 24 h, 48 h, and 72 h for detection. ​ Gene expression levels. Results showed that, compared to ds ​ Compared with the control group, at 48 h and 72 h after treatment ​ Gene expression levels were significantly downregulated, decreasing by 57.96% and 73.60%, respectively, indicating that the dsRNA had a good silencing effect on the target genes. ​ A).

[0114] Reduce brown planthoppers by feeding dsRNA ​ Gene expression was observed, and the organism was placed on rice plants to monitor growth and development. The results were as follows: ​ As shown, starting from day 8, ds ​ The survival rate of the control group was significantly lower than that of the control group, with a decrease of 50.35% on day 8 and a decrease of 48.98% on day 9. ​ B).

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Brown planthopper NlVHA-c The dsRNA of a gene is characterized by: The dsRNA includes a nucleotide sequence as shown in SEQ ID No. 3 and a nucleotide sequence that is inversely complementary to it.

2. An engineered strain expressing the dsRNA of claim 1 in prokaryotes, characterized in that, The method for constructing the engineered strain is as follows: (1) Using brown planthopper cDNA as a template, primers containing the SacⅠ site cL- NlVHA-c -F and primers containing the KpnⅠ site cL- NlVHA-c -R was used for PCR amplification, and the amplification product was purified and recovered after double enzyme digestion to obtain the target fragment; (2) After double enzyme digestion, the prokaryotic expression vector is ligated with the target fragment in step (1), and the ligation product is transformed into RNase III-deficient Escherichia coli. The positive clone strains obtained by screening are the engineered strains that express dsRNA in prokaryotes.

3. The engineered strain according to claim 2, characterized in that: The primer cL- NlVHA-c The -F sequence is shown in SEQ ID No. 5, and the primer cL- NlVHA-c The sequence of -R is shown in SEQ ID No. 6; the prokaryotic expression vector is vector L4440.

4. A reagent for controlling brown planthoppers, characterized in that: Includes the brown planthopper as described in claim 1 NlVHA-c The dsRNA of the gene or the engineered strain as described in claim 3.

5. The reagent according to claim 4, characterized in that: The dosage of dsRNA used in the reagent is 40-230 ng / head.

6. The use of the dsRNA of claim 1 or the reagent of claim 5 in the control of brown planthoppers.

7. A method for controlling brown planthoppers, characterized in that: The reagent described in claim 5 is injected into the brown planthopper or artificially fed to the brown planthopper for control.

8. The method according to claim 7, characterized in that: The dosage of dsRNA in the reagent used during injection is 40-230 ng / head.

9. The method according to claim 7, characterized in that: The artificial feeding method involves adding the bacterial solution of the engineered strain in the reagent to artificial feed and feeding it to brown planthoppers.

10. The method according to claim 9, characterized in that: When artificially feeding, add 20 μL of engineered bacterial solution to every 100 μL of brown planthopper artificial feed.

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