Dsrna of brown planthopper gene nlugt7 and its application and method for preventing and treating brown planthopper

Abstract

The application belongs to the field of agricultural biotechnology, and particularly relates to a dsRNA of a brown planthopper gene and application of the dsRNA in preventing and treating the brown planthopper and related methods. NlUGT7 The dsRNA of the gene is prepared through in vitro transcription, and RNA interference treatment is performed on the brown planthopper through microinjection. NlUGT7 The results show that after the dsRNA is injected, the expression amount of the gene in the brown planthopper of biotype Y and biotype 1 is significantly reduced. NlUGT7 After the gene is silenced, the survival rate of the brown planthopper of biotype Y feeding on YHY15 rice containing a Bph15 resistance gene or the brown planthopper of biotype 1 feeding on TN1 rice not containing the resistance gene is significantly reduced. NlUGT7 The gene plays an important role in the survival process of the brown planthopper, and can be used as an effective target for preventing and treating the brown planthopper. NlUGT7 The gene and the dsRNA thereof provided by the application can be applied to research and development of biological pesticides for the brown planthopper and green prevention and control, and have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and specifically relates to a dsRNA and its application in the control of brown planthoppers. Background Technology

[0002] Brown planthoppers are a major pest of rice, characterized by short generation cycles, high reproductive capacity, and strong adaptability. Currently, control of brown planthoppers still relies primarily on chemical insecticides. However, the long-term and extensive use of insecticides has led to a series of problems, such as increased pesticide resistance, increased pesticide residues, and the "3R" problem (rats, insects, and their resurgence). Therefore, actively developing and applying biological control technologies for brown planthoppers is of great significance for the scientific and effective control of brown planthopper damage and for ensuring food security and ecological environmental safety.

[0003] Currently, research on the biological control of brown planthoppers has encompassed various aspects, including the protection and utilization of natural enemies, the breeding of insect-resistant varieties, the development of biological pesticides, and ecological regulation. Biological control technologies, due to their environmental friendliness, high sustainability, and high safety to non-target organisms, have become an important approach to replace chemical control and achieve the scientific control of brown planthoppers. Among these, molecular biology techniques, with their efficient and specific targeted control methods, show promising application prospects.

[0004] RNA interference (RNAi) is a highly efficient and specific degradation phenomenon of homologous mRNA induced by double-stranded RNA (dsRNA). Due to its high efficiency and specificity, RNAi technology has been widely used in insect gene function research and pest control. Microinjection is one of the commonly used methods of RNAi. Compared with other delivery methods, this method is simple to operate, has stable effects, can precisely control the dosage of dsRNA introduced into the insect, and only requires a small amount of dsRNA to induce a significant gene silencing effect, while producing no pesticide residues, meeting the development needs of green pest control. For example, patent CN202510948972.1 discloses the use of RNAi technology to suppress brown planthoppers. DGAT1 Technical solutions to significantly reduce the survival rate of brown planthoppers and effectively control their population size by adjusting gene expression levels.

[0005] RNAi technology provides an efficient means for studying the gene function of brown planthoppers. By screening key genes closely related to the survival, development, and reproduction of brown planthoppers, important targets can be provided for developing novel RNAi-mediated brown planthopper control technologies. This helps to promote the development of brown planthopper biological control technology from traditional methods to molecularly targeted control, and has important theoretical significance and application value for the scientific and effective control of brown planthoppers, ensuring safe rice production and ecological environment safety. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a brown planthopper gene. NlUGT7 The dsRNA of brown planthoppers and its application and methods in the control of brown planthoppers.

[0007] The technical solution of this invention is implemented as follows:

[0008] On one hand, the present invention provides a dsRNA that targets and silences brown planthoppers. NlUGT7 Gene, NlUGT7 The gene sequence is shown in SEQ ID No. 1. NlUGT7 Encoding 521 amino acids, the amino acid sequence is shown in SEQ ID No. 2; the dsRNA includes the nucleotide sequence shown in SEQ ID No. 3 and its reverse complementary nucleotide sequence.

[0009] Secondly, the present invention also provides a method for synthesizing the above-mentioned dsRNA, comprising the following steps:

[0010] (1) Using brown planthopper cDNA as a template, primers ds with a T7 promoter were used. NlUGT7 -F and primer ds NlUGT7 -R is used for PCR amplification to obtain an amplification product containing the T7 promoter sequence;

[0011] (2) The amplified product was purified and used as a transcription template for in vitro transcription to synthesize dsRNA.

[0012] Preferably, the above primer ds NlUGT7 The -F sequence is shown in SEQ ID No. 4, primer ds NlUGT7 The sequence of -R is shown in SEQ ID No. 5.

[0013] Thirdly, this invention applies to protect a biological material, wherein the biological material is recombinant DNA containing the above-mentioned dsRNA, an expression cassette, an expression vector, or an engineered bacterium.

[0014] Fourthly, this invention also seeks to protect a reagent for controlling brown planthoppers, comprising the aforementioned dsRNA or the aforementioned biological material.

[0015] Preferably, the dosage of dsRNA in the above reagents is 40-230 ng / head.

[0016] Fifthly, this invention also seeks to protect the use of the above-mentioned dsRNA, the above-mentioned biological material, or the above-mentioned reagent in the control of brown planthoppers.

[0017] Sixthly, this invention claims a method for controlling brown planthoppers, in which the above-mentioned reagent is injected into the body of brown planthoppers to control them.

[0018] Preferably, the dosage of dsRNA in the above-mentioned injection reagent is 40-230 ng / head. The injection site is the ventral part connecting the prothorax and mesothorax of the fourth instar nymph of the brown planthopper, wherein the brown planthopper is biotype Y brown planthopper or biotype I brown planthopper.

[0019] The present invention has the following beneficial effects:

[0020] This invention provides a brown planthopper NlUGT7 The application of dsRNA of the gene in the control of brown planthoppers was obtained through in vitro transcription. NlUGT7 The double-stranded RNA (dsRNA) of the gene was injected via microinjection into either brown planthopper biotype Y (which can infect the YHY15 rice variety containing the Bph15 resistance gene) or susceptible brown planthopper biotype 1 (which can only infect the TN1 rice variety) that had already undergone pathogenic mutation. The results showed that the injection... NlUGT7 The expression levels of target genes in biotype Y or biotype 1 brown planthoppers were significantly decreased; silencing NlUGT7 After gene injection, ds NlUGT7 The mortality rate of YHY15 rice was significantly increased when biotype Y brown planthopper fed on YHY15 rice, or when TN1 brown planthopper fed on TN1 rice, thereby reducing the direct impact of brown planthopper feeding on rice and achieving the goal of controlling brown planthoppers. This invention provides... NlUGT7 The gene, its encoded protein, and dsRNA can be used as targets for the research and development of biopesticides and the biological control of brown planthoppers, showing promising application prospects and playing an important role in the green control of brown planthoppers. Attached Figure Description

[0021] 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.

[0022] Figure 1 Brown planthopper NlUGT7 Spatiotemporal expression patterns of genes; where Figure A represents different developmental stages and Figure B represents different tissue sites.

[0023] Figure 2 Injecting ds into biotype Y brown planthopper EGFP and ds NlUGT7 back NlUGT7 The results of gene expression levels and their survival rate on YHY15 rice are shown in Figure A. NlUGT7Relative gene expression level, B represents the survival rate of brown planthopper on YHY15 rice; *** indicates P <0.001, ** indicates P <0.01, * indicates P <0.05.

[0024] Figure 3 Inject ds into biotype 1 brown planthopper EGFP and ds NlUGT7 back NlUGT7 The results of gene expression levels and their survival rate on TN1 rice are shown in Figure A. NlUGT7 Relative gene expression levels, where B represents the survival rate of brown planthoppers on TN1 rice; *** indicates P <0.001, ** indicates P <0.01, * indicates P <0.05.

[0025] Figure 4 Inject ds into biotype 1 brown planthopper EGFP and ds NlUGT7 Phenotypic diagram of mortality on TN1 rice. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] Brown planthoppers tested: The tested brown planthopper population consisted of biotype 1 brown planthoppers reared on susceptible rice TN1 and those containing [unclear text - likely a specific species or species]. Bph15 Biotype Y brown planthopper reared on YHY15 rice with resistance gene.

[0029] The rearing temperature was 28℃, the humidity was 75%, and the photoperiod was 16L:8D.

[0030] The main reagents of this invention are shown in Table 1:

[0031] Table 1 Main Reagents

[0032]

[0033] Table 2 Primers used

[0034]

[0035] The primers used in this invention are shown in Table 2:

[0036] Example 1: NlUGT7 dsRNA of genes

[0037] 1. Brown planthopper NlUGT7 Gene cloning

[0038] Fourth-instar nymphs of the brown planthopper were placed into 1.5 mL centrifuge tubes using a homemade trematode tube, and total RNA was extracted using the TRIzol method. Using 1 μg of total RNA as a template, cDNA was synthesized according to the instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa) kit. The cDNA was obtained from the NCBI database. NlUGT7 The nucleotide sequence of the gene, with the sequence number LOC111058748, was used to design PCR amplification primers using Primer Premier 5.0 (Table 2).

[0039] PCR amplification was performed using the above-mentioned cDNA and primers. The PCR system consisted of 10×Buffer 5 μL, dNTP 4 μL, primers 1 μL each, cDNA 1 μL, Ex Taq 0.25 μL, and H2O 37.75 μL. The PCR program was set as follows: 95℃ for 5 min; 95℃ for 30 s, 50.8℃ for 30 s, 72℃ for 1 min 47 s, 35 cycles; 72℃ for 10 min. After electrophoresis of the PCR products using 1% agarose gel, they were ligated into the pMD-18T vector (TaKaRa) and transformed into E. coli DH5α competent cells. The cells were plated on ampicillin (Amp) resistant plates and cultured overnight. Single colonies were picked for PCR verification. Correct colonies were sent to the company for sequencing. Sequencing results were compared and analyzed with sequences in the NCBI database using Bioedit software. Figure 1 ).

[0040] Obtained from the NCBI database NlUGT7 Gene information was used to design primers for gene cloning to obtain the CDS (sequence as shown in SEQ ID No. 1) and its encoded amino acid (sequence as shown in SEQ ID No. 2).

[0041] Example 2: NlUGT7 spatiotemporal expression

[0042] Biotype 1 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 rapidly frozen in liquid nitrogen. Simultaneously, samples were taken from brown planthoppers (nymphs of different instars and males / females) using a homemade trematode, and rapidly frozen in liquid nitrogen, then stored in an ultra-low temperature freezer. Detection was performed using quantitative real-time PCR. NlUGT7 The expression of the gene at different developmental stages and in different tissues was investigated using quantitative primers designed with Primer Premier 5.0 (Table 2). Following the instructions for 2×M5 HiPer SYBR Premix Es Taq, 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 1 min; 95℃ for 10 s, 60℃ for 1 min, for 45 cycles. The 18S ribosomal RNA gene was used as an internal control gene, and the relative expression level of the target gene was determined using a 2:1 ratio. -△△Ct The method is used for calculation.

[0043] Table 3. NlUGT7 Expression levels at different developmental stages of biotype 1 brown planthopper

[0044]

[0045] Table 4. NlUGT7 Expression levels in different tissues

[0046]

[0047] Detection of brown planthoppers using real-time PCR NlUGT7 The expression patterns of genes in different developmental stages and tissues of the brown planthopper were studied. The results showed that... NlUGT7 The gene was expressed more highly in males and 5th instar larvae. Figure 1 (A, Table 3) The highest expression was found in the fat body, followed by the salivary glands, and expression was also found in other tissues. Figure 1 B, Table 4).

[0048] Example 3: Synthesis and purification of dsRNA

[0049] Plasmids were extracted from bacterial cultures with correct sequences using the M5 plasmid miniprep plus kit (Polymer Biotech). dsRNA primers were designed using Primer Premier 5.0, with the T7 promoter sequence added to the front end of the primers (Table 2). The plasmids were amplified by PCR using the dsRNA primers, and the amplified products were purified using the MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (TaKaRa). The purified DNA template, once its concentration and quality were deemed acceptable, became the template for dsRNA synthesis (SEQ ID No. 3).

[0050] The synthesis of dsRNA was performed according to the instructions of the MEGAscript™ T7 Transcription Kit (Thermo Scientific), as follows: Add the reagents to a 200 μL centrifuge tube in the following proportions: 2 μL ATP, 2 μL CTP, 2 μL GTP, 2 μL UTP, 2 μL 10× Reaction buffer, 2 μL Enzyme mix, 200 ng dsRNA template, and bring the volume to 20 μL with Nuclease-free Water. Gently mix, centrifuge briefly, and incubate at 37°C for 16 h. After the reaction, add 1 μL TVRBO DNase and place in a PCR instrument. The reaction conditions are: 37°C for 15 min; 65°C for 10 min, then terminate the reaction. Dilute the product to the desired concentration.

[0051] Example 4: NlUGT7 dsRNA of the gene was introduced into the brown planthopper

[0052] Using microinjection technology NlUGT7 The dsRNA was introduced into the brown planthopper as follows:

[0053] Instrument setup: Use a disposable syringe to introduce mineral oil into the glass capillary. Install the mineral oil-filled glass capillary into the microinjector. Click MANUAL to drain the mineral oil to 50%, then click MANUAL to aspirate dsRNA to 100%. Injection parameters are 46 nL, 23 nL / s.

[0054] Injecting dsRNA: Using a homemade trematode tube, collect fourth-instar nymphs of the brown planthopper, anesthetize them with carbon dioxide for 3-5 seconds, place them on a 1% agarose gel, and then inject dsRNA into the ventral region connecting the prothorax and mesothorax of the fourth-instar nymphs. After the brown planthoppers awaken, transfer them to fresh rice paddies.

[0055] Example of implementation effect 1: NlUGT7 Gene silencing effect detection

[0056] Samples were taken at 24, 48, and 72 h after injection of brown planthoppers. Total RNA was extracted from the brown planthoppers using the TRIzol method, and cDNA was reverse-engineered. qRT-PCR was used to detect whether the expression of target genes was inhibited. The brown planthoppers designed using the methods described in Table 2 above... NlUGT7 Quantitative primers for genes.

[0057] In the biotype Y brown planthopper, the expression levels of the target gene decreased by 88.57%, 75.49%, and 81.19% at 24 h, 48 h, and 72 h after treatment, respectively. Figure 2 A); In biotype 1 brown planthopper, the expression levels of the target gene decreased by 81.37%, 91.35%, and 82.35% at 24 h, 48 h, and 72 h after treatment, respectively. Figure 3 A). The above results indicate that NlUGT7 dsRNA has a significant gene silencing effect on both the harmful variant Y brown planthopper and the common biotype 1 brown planthopper, and can effectively inhibit the expression of target genes in their bodies.

[0058] Example of implementation effect 2: NlUGT7 Survival rate determination of brown planthoppers on rice after gene silencing

[0059] To clarify NlUGT7 The function of genes in the brown planthopper-rice interaction process was investigated by silencing genes in brown planthoppers using RNA interference (RNAi) technology. NlUGT7 Gene expression was studied, and its survival rate on different rice materials was determined.

[0060] The specific method is as follows: In vitro synthesized... NlUGT7 dsRNA was injected into brown planthoppers to inject dsRNA. EGFP Brown planthoppers were used as a control. After treatment, biotype 1 brown planthoppers were inoculated onto the susceptible rice variety TN1, and biotype Y brown planthoppers were inoculated onto rice varieties containing resistance genes. Bph15 The experiment was conducted on rice variety YHY15 for rearing and observation. From the date of inoculation, the number of surviving brown planthoppers of different biotypes on different rice varieties was investigated and recorded daily until the end of the experiment.

[0061] The rearing conditions for brown planthoppers are: temperature 28℃, relative humidity 75%, and photoperiod of 16 hours of light / 8 hours of darkness.

[0062] Table 5. Injection of biotype Y brown planthopper nymphs with ds NlUGT7 and ds EGFP Survival rate of YHY15 rice

[0063]

[0064] Experimental results show that, compared with ds EGFP Compared with the control group, at each observation time point, the injection of ds NlUGT7 The survival rate of the post-biotype Y brown planthopper on YHY15 rice was low, and the differences between groups gradually increased with the extension of treatment time. Figure 2 B, Table 5). Specifically, days 1-10 ds EGFP The survival rates of the groups were 98.04%, 94.78%, 92.19%, 89.11%, 86.70%, 82.78%, 80.89%, 78.89%, 78.15%, and 78.15%, respectively; while the ds NlUGT7 The percentages for the groups were 93.83%, 90.19%, 83.91%, 68.25%, 49.59%, 27.43%, 13.61%, 6.95%, 4.02%, and 2.05%, respectively. (Compared to ds) EGFP Compared to the group, ds NlUGT7 The survival rates of the groups on days 1-10 were 4.21, 4.59, 8.28, 20.86, 37.11, 55.35, 67.28, 71.94, 74.13, and 76.10 percentage points lower, respectively. These results indicate that ds NlUGT7 It can significantly reduce the survival rate of biotype Y brown planthopper on YHY15 rice, and the inhibitory effect increases with time.

[0065] Table 6. Injection of ds into biotype 1 brown planthopper nymphs NlUGT7 and ds EGFP Survival rate of TN1 rice

[0066]

[0067] Experimental results show that, compared with ds EGFP Compared with the control group, injection of ds NlUGT7 The survival rate of brown planthoppers at each observation time point generally showed a decreasing trend, and the differences between groups gradually increased with the extension of treatment time. Figure 3 B, Table 6). Specifically, days 1-10 ds EGFP The survival rates of the groups were 96.60%, 91.24%, 84.81%, 80.03%, 77.49%, 73.68%, 70.82%, 70.82%, 70.82%, and 70.82%, respectively; ds NlUGT7 The percentages for the groups were 95.62%, 89.52%, 84.54%, 80.28%, 66.83%, 50.90%, 30.10%, 13.40%, 5.88%, and 4.07%, respectively. (Compared to ds) EGFP Compared to the group, ds NlUGT7The survival rates of the group on days 1, 2, 3, 5, 6, 7, 8, 9, and 10 were 0.98, 1.72, 0.27, 10.66, 22.78, 40.72, 57.42, 64.94, and 66.75 percentage points lower, respectively. NlUGT7 The mortality phenotypes in the treatment group mainly included abdominal shrinkage, inability to molt normally, or death during the molting process. Figure 4 The above results indicate that ds NlUGT7 Treatment effectively reduced the survival rate of biotype 1 brown planthopper on TN1 rice, and the inhibitory effect was more pronounced in the later stages of treatment.

[0068] The above results indicate that NlUGT7 The brown planthopper plays an important role in the adaptation of different biotypes of brown planthopper to rice hosts, and its genes and encoded products have potential application value in the control of brown planthopper.

[0069] 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. A dsRNA, characterized in that: The dsRNA targets and silences brown planthoppers NlUGT7 Gene, NlUGT7 The gene sequence is shown in SEQ ID No. 1, and the dsRNA includes the nucleotide sequence shown in SEQ ID No. 3 and its reverse complementary nucleotide sequence.

2. The method for synthesizing dsRNA according to claim 1, characterized in that: The steps are as follows: (1) Using brown planthopper cDNA as a template, primers ds with a T7 promoter were used. NlUGT7 -F and primer ds NlUGT7 -R is used for PCR amplification to obtain an amplification product containing the T7 promoter sequence; (2) The amplified product was purified and used as a transcription template for in vitro transcription to synthesize dsRNA.

3. The synthesis method according to claim 2, characterized in that: The primer ds NlUGT7 The -F sequence is shown in SEQ ID No. 4, primer ds NlUGT7 The sequence of -R is shown in SEQ ID No.

5.

4. A biomaterial, characterized in that: The biological material is selected from expression cassettes, expression vectors, or engineered bacteria containing the dsRNA of claim 1.

5. A reagent for controlling brown planthoppers, characterized in that: It comprises the dsRNA of claim 1 or the biological material of claim 4.

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

7. The use of the dsRNA of claim 1, the biological material of claim 4, or the reagent of claim 6 in the control of brown planthoppers.

8. A method for controlling brown planthoppers, characterized in that: The reagent described in claim 6 is injected into the brown planthopper to control it.

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

10. The method according to claim 9, characterized in that: The brown planthoppers mentioned are either biotype Y brown planthoppers or biotype 1 brown planthoppers.

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