Application of miR-3032-z in preparation of product for inhibiting expression of CYP15A1 gene of brown planthopper

Abstract

The application belongs to the technical field of application of miR-3032-z in preparation of products for inhibiting expression of CYP15A1 gene of brown planthopper, and particularly relates to the technical field of pest control. The application provides application of miR-3032-z or a reagent for overexpressing miR-3032-z in preparation of products for inhibiting expression of CYP15A1 gene of brown planthopper; the products include drugs or kits. miR-3032-z regulates expression of a target gene CYP15A13'UTR region through targeted combination, overexpression of miR-3032-z can inhibit expression of cytochrome P450 family gene CYP15A1, and can significantly reduce honeydew secretion and weight gain of biotype Y brown planthopper on insect-resistant rice YHY15, and obviously inhibit feeding behavior, so that the harmful type brown planthopper restores sensitivity to resistant rice YHY15.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pest control, and particularly relates to application of miR-3032-z in preparation of products for inhibiting gene expression of Nilaparvata lugens. CYP15A1 BACKGROUND

[0002] Nilaparvata lugens is a kind of monophagous pest which feeds on rice. It can cause serious economic losses by sucking a large amount of phloem sap of rice, leading to loss of nutrients of rice plants, yellowing and wilting of rice leaves, and yield reduction. Planting of rice varieties containing resistance genes against Nilaparvata lugens is the most economical and environmentally friendly measure for Nilaparvata lugens control. The interaction between phytophagous insects and host plants is dynamic. For a long time, rice and Nilaparvata lugens have co-evolved, and rice has evolved a complex defense system to resist the invasion of Nilaparvata lugens. In recent years, more and more rice genes against Nilaparvata lugens have been discovered (Cheng X, Wu Y, Guo J, et al. 2013a. A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination. The Plant Journal, 76: 687-698). However, Nilaparvata lugens has strong adaptability. With the extensive planting of resistant rice varieties, Nilaparvata lugens gradually overcomes and adapts to the defense mechanism of resistant rice varieties under the pressure of natural selection, establishes new host relationship, evolves new biotype, and forms new population (Kobayashi T, Yamamoto K, Suetsugu Y, et al. 2014. Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens. Proceedings-Royal Society. Biological Sciences, 281: 20140726). With the emergence of new biotype population, the resistance of original pest-resistant rice varieties is overcome, and the damage of Nilaparvata lugens to resistant rice varieties gradually increases, which brings severe challenges to the breeding of resistant rice varieties.

[0003] ​The enhancement of the damage of resistant rice by the brown planthopper is a heritable change. The genetic variation mechanism of this evolution is mainly divided into two categories: one is gene mutation, and the other is the change of gene expression level. Gene mutation will cause the binding ability of the brown planthopper to the defense-related metabolic substances produced by the brown planthopper to feed on resistant rice to change, thereby causing the brown planthopper to be insensitive to resistant rice (Simon JC, D'Alencon E, Guy E, et al. 2015. Genomics of adaptation to host–plants in herbivorous insects. Briefings in Functional Genomics&Proteomics, 14: 413–423). The change of gene expression level refers to the content of gene products with specific functions in the organism will change over time, living environment, physiological behavior, etc., and this change is mostly related to metabolic resistance-related genes (Wang R, Zhu Y, Deng L, et al. 2017. Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2. Insect Biochemistry and Molecular Biology, 26: 543–551. Wu YQ, Xu HF, Pan YO, et al. 2018. Expression profile changes of cytochrome P450 genes between thiamethoxam susceptible and resistant strains of Aphis gossypii Glover. Pesticide Biochemistry and Physiology, 149: 1–7).There are researchers who use high-throughput sequencing technology to conduct transcriptome sequencing analysis on the salivary glands and fat bodies of two different populations of brown planthoppers that can cause damage to TN1 and Mudgo rice, respectively. The results show that the differentially expressed genes between the two populations are mainly involved in metabolic, digestive, secretory protein and immune expression pathways, and it is speculated that these genes may be closely related to the evolution of the virulence of brown planthoppers (Ji R, Yu H, Fu Q, et al. 2013. Comparative transcriptome analysis of salivary glands of two populations of rice brown planthopper, Nilaparvata lugens, that differ in virulence. PLoS One, 8: e79612. Yu H, Ji R, Ye W, et al. 2014. Transcriptome analysis of fat bodies from two brown planthopper (Nilaparvata lugens) populations with different virulence levels in rice. PLoS One, 9: e88528). Later, another group of researchers analyzed the salivary glands of two groups of brown planthoppers that fed on resistant rice B5 and susceptible rice TN1, and found that the population of brown planthoppers that fed on resistant rice B5 had up-regulated expression of genes related to sugar metabolism (Wang X, Zhang M, Feng F, et al. 2015. Differentially regulated genes in the salivary glands of brown planthopper after feeding in resistant versus susceptible rice varieties. Archives of Insect Biochemistry and Physiology, 89: 69-86). Similarly, the fat bodies of two groups of brown planthoppers that fed on resistant rice B5 and susceptible rice TN1 were analyzed, and it was found that the population of brown planthoppers that fed on resistant rice B5 had up-regulated expression of genes related to the immune system (Wang X, Zhang M, Feng F, et al. 2015. Differentially regulated genes in the salivary glands of brown planthopper after feeding in resistant versus susceptible rice varieties. Archives of Insect Biochemistry and Physiology, 89: 69-86). Bph6Transcriptome sequencing of BPH feeding on transgenic resistant rice lines and comparing with the transcriptome data of BPH feeding on wild type Nipponbare, it was found that the expression of genes involved in metabolism, detoxification, autophagy and other processes in BPH feeding on resistant rice were up-regulated (Zhang J, Guan W, Huang C, et al. 2019. Combining next-generation sequencing and single-molecule sequencing to explore brown plant hopper responses to contrasting genotypes of japonica rice. BMC Genomics, 20: 682).

[0004] Cytochrome P450 family genes involved in regulating BPH metabolic resistance are commonly involved in regulating detoxification metabolism. It can participate in the metabolism of endogenous and exogenous compounds in insects, including ingested plant secondary metabolites and exogenous compounds such as pesticides, with a very wide range of action, and is one of the most important detoxification systems in insects. It plays a very important role in the process of insect and plant co-evolution (Wang R, Zhu Y, Deng L, et al. 2017. Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2. Insect Biochemistry and Molecular Biology, 26: 543-551). Studies have found that overexpression of P450 family genes mediates metabolic resistance of many pests including BPH to insect growth regulators, carbamates, organophosphates and other substances.

[0005] MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded RNAs approximately 22 nt in length. They are small molecule regulators that participate extensively in the regulation of various life processes, including embryonic development, cell growth, differentiation, proliferation, apoptosis, and metabolism. They play an important role in post-transcriptional gene regulation (Bushati N, Cohen SM. 2007. MicroRNA functions. Annual Review of Cell and Developmental Biology, 23: 175–205). miRNAs are involved in various physiological processes in insects, including phenotypic differentiation, genetic development, immune defense, and stress resistance. Studies have found that miRNAs participate in insect pesticide resistance by regulating the expression of genes related to resistance and detoxification functions, including the cytochrome P450 family, GST, Bt toxin, esterases, superoxide dismutase, and ABC transporters (Zhu B, Li XX, Liu Y, et al. 2017. Global identification of microRNAs associated with chlorantraniliprole resistance in diamondback moth Plutella xylostella (L.). Scientific Reports, 7: 4071). In summary, miRNAs and resistance-related genes play a crucial role in the evolution of insect resistance and can serve as important targets for pest control. However, effective methods for controlling the harmful brown planthopper are currently lacking. Summary of the Invention

[0006] The purpose of this invention is to provide miR-3032-z in the preparation of products that inhibit brown planthoppers. CYP15A1 Applications of gene expression in products. miR-3032-z and its corresponding target genes. CYP15A1 It participates in detoxification and metabolic pathways in brown planthoppers and plays an important role in the evolution of the harmfulness of brown planthoppers and the formation of new biotypes.

[0007] This invention provides miR-3032-z or reagents that overexpress miR-3032-z in the preparation of drugs that inhibit brown planthoppers. CYP15A1 Application in products that express genes; said products include pharmaceuticals or kits.

[0008] This invention also provides a method for inhibiting brown planthoppers. CYP15A1 The application of gene expression reagents in the preparation of drugs to restore the sensitivity of brown planthoppers to insect-resistant rice; the inhibition of brown planthoppers CYP15A1The reagent for gene expression includes a reagent for overexpression of miR-3032-z.

[0009] The present application also provides a method for inhibiting the feeding behavior of Nilaparvata lugens. CYP15A1 The present application also provides a use of a reagent for gene expression in the preparation of a medicine for breaking the detoxification metabolism ability of Nilaparvata lugens against insect-resistant rice. CYP15A1 The reagent for gene expression includes a reagent for overexpression of miR-3032-z.

[0010] Preferably, the insect-resistant rice includes insect-resistant rice YHY15.

[0011] The present application also provides a method for inhibiting the feeding behavior of Nilaparvata lugens. CYP15A1 The present application also provides a use of a reagent for gene expression in the preparation of a medicine for reducing the honeydew secretion and / or the weight gain of Nilaparvata lugens. CYP15A1 The reagent for gene expression includes a reagent for overexpression of miR-3032-z.

[0012] The present application also provides a method for inhibiting the feeding behavior of Nilaparvata lugens. CYP15A1 The present application also provides a use of a reagent for gene expression in the preparation of a medicine for inhibiting the feeding behavior of Nilaparvata lugens. CYP15A1 The reagent for gene expression includes a reagent for overexpression of miR-3032-z.

[0013] Preferably, the Nilaparvata lugens includes a virulent type of Nilaparvata lugens.

[0014] Preferably, the virulent type of Nilaparvata lugens includes a biological Y type of Nilaparvata lugens.

[0015] Preferably, the reagent for overexpression of miR-3032-z includes a miR-3032-z agonist.

[0016] Preferably, the nucleotide sequence of the sense strand of the agomiR-3032 is preferably as shown in SEQ ID NO. 2, and the nucleotide sequence of the antisense strand of the agomiR-3032 is preferably as shown in SEQ ID NO. 3.

[0017] The present application provides a use of miR-3032-z or a reagent for overexpression of miR-3032-z in the preparation of a product for inhibiting the detoxification metabolism of Nilaparvata lugens. CYP15A1 The product includes a medicine or a kit. The present application finds that miR-3032-z can inversely regulate CYP15A1 this detoxification metabolism gene. CYP15A1 The gene is a cytochrome P450 family gene, and cytochrome P450 can participate in the metabolism of endogenous and exogenous compounds in insects. The present application first finds that miR-3032-z and its corresponding target gene CYP15A1The miRNA (miR-3032-z) capable of regulating detoxification metabolism of the brown planthopper is identified based on the miRNA as a breakthrough point and the expression difference of the miRNA in the two different biotypes of the brown planthopper (biotype 1 and biotype Y), which reveals the important role of the miRNA in the evolution of the brown planthopper and the formation of a new biotype, and provides a new direction for in-depth study of the gene expression regulation network of the brown planthopper responding to and adapting to the resistant rice. Figure 1 The test results show that the miR-3032-z regulates the expression of the gene by targeting the 3'UTR region of the target gene, the expression of the cytochrome P450 family gene is inhibited by injecting the agomir-3032, and after the agomir-3032 is injected, the honeydew secretion amount and the weight gain of the biotype Y brown planthopper on the insect-resistant rice YHY15 are significantly reduced, the feeding behavior is obviously inhibited, and the harmful type brown planthopper restores the sensitivity to the resistant rice YHY15. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0019] Figure 2 The pmirGLO plasmid map and enzyme digestion site schematic diagram provided by the present application;

[0020] CYP15A1 The qRT-PCR verification of the expression results of the miRNA and the target gene provided by the present application; wherein, A: the expression amount of miR-3032-z of the two biotypes of the brown planthopper; B: the expression amount of the target gene of the two biotypes of the brown planthopper; data represent mean (three biological replicates) ± standard error, statistical analysis is performed by T test: Figure 3 <0.01, *P <0.001; *P <0.001;

[0021] Figure 4The results of verifying miRNA binding to target genes using the dual-luciferase reporter system provided in this invention are shown in the figure; where the data represent the mean (three biological replicates) ± standard error, and a one-way ANOVA Tukey test was used. Different letters above the bars indicate significant differences. P <0.05);

[0022] CYP15A1 The miR-3032-z pair provided by this invention CYP15A1 The graph shows the regulatory results of expression; where A: expression of miR-3032-z after injection of agomir-3032; B: expression of miR-3032-z after injection of agomir-3032. CYP15A1 A: Expression of miR-3032-z after antagomir-3032 injection; C: Expression of miR-3032-z after antagomir-3032 injection; D: Expression of miR-3032-z after antagomir-3032 injection. Figure 5 The expression is shown in the figure; CK represents the blank control group without microinjection, and NC represents the negative control group; data are expressed as mean (three biological replicates) ± standard error, and statistical analysis was performed using a t-test: * indicates a significant difference between the experimental group and the negative control group, ** *P <0.001;

[0023] CYP15A1 The following figures illustrate the effects of miR-3032-z on the feeding behavior of brown planthoppers, as provided by this invention. A: Honeydew secretion by female adult brown planthoppers injected with agomir-3032 on YHY15 rice plants after 48 hours of feeding; B: Body weight change of female adult brown planthoppers injected with agomir-3032 on YHY15 rice plants after 48 hours of feeding. Data represent the mean (honeydew secretion from 30 brown planthoppers) ± standard error, and statistical analysis was performed using a t-test. Detailed Implementation

[0024] This invention provides miR-3032-z or reagents that overexpress miR-3032-z in the preparation of drugs that inhibit brown planthoppers. CYP15A1 Application in gene expression products; said products include pharmaceuticals or kits. In this invention, the nucleotide sequence of miR-3032-z is as shown in SEQ ID NO.1: TGTTAGTATAACTCTTAGTAACA. In this invention, the... CYP15A1The gene number is XM_039430744.1. miRNAs, as important post-transcriptional regulatory factors in insects, form miRNA-target gene regulatory networks that directly or indirectly participate in almost all biological processes, including embryonic development, digestion and metabolism, apoptosis, and maintaining homeostasis. One of the main strategies for herbivorous insects to respond to plant defenses is through the evolution of sophisticated metabolic and digestive systems, which detoxify ingested plant secondary metabolites through oxidation, hydrolysis, and molecular cross-linking. Cytochrome P450 participates in the metabolism of endogenous and exogenous compounds in insects, including ingested plant secondary metabolites and exogenous compounds such as pesticides. Its scope of action is very broad, making it one of the most important detoxification systems in insects and playing a crucial role in the co-evolution of insects and plants. This invention discovers that miR-3032-z is related to the cytochrome P450 family of genes. CYP15A1 Related. Cytochrome P450 family genes CYP15A1 This is a target gene of miR-3032-z. This invention reveals that miR-3032-z can reversely regulate... CYP15A1 The expression of this detoxification metabolic gene mediates the adaptation of the brown planthopper to insect-resistant rice. In this embodiment of the invention, a dual-luciferase assay confirmed in vitro that miR-3032-z could significantly inhibit... CYP15A1 The expression of miR-3032-z is described. In this invention, the product preferably includes a product that enhances the sensitivity of different biotypes of brown planthopper to different insect-resistant rice varieties; more preferably, it includes a product that enhances the sensitivity of biotype Y brown planthopper to insect-resistant rice YHY15. In this invention, the reagent for overexpressing miR-3032-z preferably includes a miR-3032-z agonist. In this invention, the miR-3032-z agonist preferably includes agomiR-3032. In this invention, the nucleotide sequence of the sense strand of agomiR-3032 is preferably as shown in SEQ ID NO. 2, and the nucleotide sequence of the antisense strand of agomiR-3032 is preferably as shown in SEQ ID NO. 3.

[0025] This invention also provides a method for inhibiting brown planthoppers. CYP15A1 The application of gene expression reagents in the preparation of drugs to restore the sensitivity of brown planthoppers to insect-resistant rice; the inhibition of brown planthoppers Bph15 The gene expression reagent includes a reagent for overexpressing miR-3032-z. In this invention, the insect-resistant rice preferably includes insect-resistant rice YHY15. In this invention, the brown planthopper preferably includes a pathogenic brown planthopper. In this invention, the pathogenic brown planthopper preferably includes the biological Y-type brown planthopper. The biological Y-type brown planthopper is forcibly reared in an environment containing... CYP15A1 Laboratory biotypes gradually formed on YHY15 insect-resistant rice containing the brown planthopper resistance gene were able to infest YHY15 rice. A reagent overexpressing miR-3032-z inhibited brown planthopper growth.CYP15A1 gene expression can restore the sensitivity of biotype Y of Nilaparvata lugens to resistant rice YHY15. The present application uses the in vitro synthesized miR-3032-z agonist agomir-3032 to inject the brown planthopper, and finds that the agomir-3032 can inhibit the expression of CYP15A1 gene expression can restore the sensitivity of biotype Y of Nilaparvata lugens to resistant rice YHY15. The present application uses the in vitro synthesized miR-3032-z agonist agomir-3032 to inject the brown planthopper, and finds that the agomir-3032 can inhibit the expression of CYP15A1 , which proves that miR-3032-z can regulate the detoxification metabolism of the brown planthopper and affect the adaptation process of the brown planthopper to resistant rice.

[0026] The present application also provides a reagent for inhibiting the expression of CYP15A1 gene expression in the preparation of a drug for disrupting the detoxification metabolism of the brown planthopper to resistant rice; the reagent for inhibiting the expression of CYP15A1 gene expression includes a reagent for overexpressing miR-3032-z.

[0027] The present application also provides a reagent for inhibiting the expression of CYP15A1 gene expression in the preparation of a drug for reducing the honeydew secretion and / or weight gain of the brown planthopper; the reagent for inhibiting the expression of CYP15A1 gene expression includes a reagent for overexpressing miR-3032-z. Overexpression of miR-3032-z can reduce the honeydew secretion and / or weight gain of biotype Y of the brown planthopper on resistant rice YHY15.

[0028] The present application also provides a reagent for inhibiting the expression of CYP15A1 gene expression in the preparation of a drug for inhibiting the feeding behavior of the brown planthopper; the reagent for inhibiting the expression of CYP15A1 gene expression includes a reagent for overexpressing miR-3032-z. Overexpression of miR-3032-z can significantly inhibit the feeding behavior of biotype Y of the brown planthopper on resistant rice YHY15.

[0029] In specific embodiments of the present application, the in vitro synthesized miR-3032-z agonist agomir-3032 is injected into the brown planthopper, which inhibits the expression of CYP15A1 gene in the brown planthopper, disrupts the detoxification metabolism of the brown planthopper to resistant rice, and further inhibits the feeding behavior of the brown planthopper, reduces the honeydew secretion and weight gain of the brown planthopper, and restores the sensitivity of biotype Y of the brown planthopper to resistant rice YHY15.

[0030] In order to further illustrate the present application, the application of miR-3032-z provided by the present application in the preparation of a product for inhibiting the expression of Bph15 gene in the brown planthopper will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0031] The application relies on the national key research and development plan, and the project information is as follows:

[0032] Project type: national key research and development plan.

[0033] Project number: 2021YFD1401100.

[0034] Project name: Mechanism of rice planthopper catastrophe and sustainable prevention and control technology.

[0035] Example 1

[0036] Materials and methods

[0037] 1. Brown planthopper and rice materials

[0038] 1.1 Test insects and feeding

[0039] The source of biotype 1 brown planthopper and biotype Y brown planthopper is a conventional source. Biotype 1 brown planthopper is collected from natural populations in the field in Wuhan and has been bred on a non-pest-resistant rice variety TN1 for a long time. Biotype Y brown planthopper is derived from biotype 1 brown planthopper after forced feeding on YHY15 rice material containing anti-brown planthopper gene for 33 generations. Bph15

[0040] The test brown planthoppers were bred in the greenhouse of Wuhan University, and the breeding temperature was controlled at 27±1°C, the relative humidity was between 70~80%, and the light cycle was 16 h light / 8 h dark per day. Brown planthoppers were bred on rice seedlings in the vegetative growth stage and in good condition, and the rice seedlings were replaced in time according to the density of brown planthoppers and the growth state of the fed rice.

[0041] 1.2 Rice materials

[0042] The pest-susceptible rice material used in the present application is TN1 rice variety without any anti-brown planthopper gene; the pest-resistant rice material is YHY15 rice strain containing anti-brown planthopper gene Figure 1

[0043] The test rice materials were planted in the Institute of Genetics, Wuhan University, and cultivated under natural conditions from May to October, and cultivated in the plant greenhouse for the rest of the month, with the cultivation temperature controlled at 28°C, the relative humidity between 70~80%, and the light cycle of 16 h light / 8 h dark per day.

[0044] 2. qRT-PCR verification of miRNA expression

[0045] 2.1 Extraction of total RNA from samples

[0046] ​​RNA was extracted using RNAiso Plus (TaKaRa #9109) reagent, and the specific operation was as follows:

[0047] (1) The single head of the brown planthopper sample was placed in a 1.5 mL RNase-free centrifuge tube, 500 μL of RNAiso Plus reagent was added, and the brown planthopper body was ground thoroughly using a tissue grinder soaked with chloroform. Then, 500 μL of RNAiso Plus reagent was added to rinse the tissue grinder, and it was mixed thoroughly by shaking. It was placed in a-80℃ refrigerator and was kept at low temperature for more than 30 min. The whole body of the brown planthopper sample obtained was washed clean in RNase-free PBS buffer, and was dissected under a stereomicroscope. The tissue sample obtained was transferred to a 1.5 mL RNase-free centrifuge tube containing 500 μL of RNAiso Plus reagent, and the subsequent operation was the same as that of the single head of the brown planthopper sample;

[0048] (2) 15000 rpm, 4℃ centrifugation for 15 min, 800 μL of supernatant was transferred to a new 1.5 mL RNase-free centrifuge tube;

[0049] (3) 200 μL of chloroform was added, and it was shaken vigorously for 1 min to form a milky liquid. It was kept at room temperature for 15 min to separate the layers, and was centrifuged at 15000 rpm and 4℃ for 15 min. 400-500 μL of supernatant was taken to a new 1.5 mL RNase-free centrifuge tube;

[0050] (5) An equal volume of isopropanol pre-cooled at-20℃ was added to the remaining supernatant, and it was mixed by inverting. It was kept at low temperature at-20℃ for more than 10 min, and was centrifuged at 15000 rpm and 4℃ for 15 min. The supernatant was discarded;

[0051] (6) 1 mL of 75% ethanol prepared using RNase-free H2O was added to wash the precipitate, and it was centrifuged at 15000 rpm and 4℃ for 15 min. The supernatant was discarded;

[0052] (7) 1 mL of 75% ethanol prepared using RNase-free H2O was added again to wash the precipitate, and it was centrifuged at 15000 rpm and 4℃ for 15 min. The supernatant was discarded;

[0053] (8) The precipitate was placed in a clean bench and was air-dried for 5 min;

[0054] (9) 50 μL of RNase-free H2O was added to dissolve the precipitate. A small amount was used for agarose electrophoresis and Nanodrop2000 ultraviolet spectrophotometer to detect the integrity and concentration of RNA. The rest was stored in a-80℃ refrigerator.

[0055] 2.2 Synthesis of miRNA first strand cDNA

[0056] The synthesis of miRNA first strand cDNA was performed using miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme #MR101) stem-loop kit, and the subsequent operation was performed according to the instructions:

[0057] (1) Removal of genomic DNA: Prepare the reaction system on ice, and the main components are shown in Table 1.

[0058] Table 1 Reaction system

[0059]

[0060] Gently mix the prepared mixture with a pipette, centrifuge it instantly, and put it into a PCR instrument, incubate at 42°C for 2 min, and store at 4°C.

[0061] (2) Synthesis of first strand cDNA: Design and synthesize miRNA reverse transcription special stem-loop structure primer according to Table 2.

[0062] Table 2 miRNA reverse transcription primer sequence

[0063]

[0064] Prepare the reaction mixture on ice according to the system shown in Table 3, gently mix it with a pipette, and to ensure the accuracy of the content of each component in the reaction system, first prepare a premix according to the reaction number, then dispense 10 μL into the reaction tube of the previous step to reduce error;

[0065] Table 3 Reaction system

[0066]

[0067] After centrifuging the prepared reaction system, immediately put it into a PCR instrument for reaction, and the reaction conditions are: 25°C, 5 min; 50°C, 15 min; 85°C, 5 min; 4°C storage.

[0068] 2.3 qRT-PCR

[0069] qRT-PCR experiments were performed using miRNA Universal SYBR qPCR Master Mix (Vazyme #MQ101) kit. First, take out 5 μL of cDNA obtained in the previous step, dilute it 10 times with RNase Free H2O, and store the remaining cDNA at -20°C for future use. Design and synthesize miRNA quantitative primers according to Table 4.

[0070] Table 4 miRNA quantitative primer sequence

[0071]

[0072] Prepare the reaction solution on ice according to the system described in Table 5. To ensure the accuracy of the content of each component in the reaction system, first prepare the premix solution according to the number of reactions, mix thoroughly, and then dispense into each reaction well to reduce errors.

[0073] Table 5 Reaction system

[0074]

[0075] Centrifuge the prepared reaction system, and place it in a CFX96 Real-Time System (Bio-Rad) instrument for detection. The reaction conditions are shown in Table 6:

[0076] Table 6 Reaction conditions

[0077]

[0078] After the reaction, use Bio-Rad CFX Manager software for data analysis.

[0079] 3 qRT-PCR verification of gene expression

[0080] 3.1 Synthesis of cDNA

[0081] Use the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa #RR047A) kit to perform reverse transcription reaction on the extracted total RNA to synthesize cDNA. Follow the instructions to perform the following operations:

[0082] (1) Remove genomic DNA reaction: Prepare the reaction system on ice. To ensure the accuracy of the content of each component in the reaction system, first prepare the premix solution according to the number of reactions, mix the prepared premix solution thoroughly, and then dispense into 200 μL RNase-free PCR tubes to reduce errors. Finally, add the RNA sample. The main components of the reaction solution are shown in Table 7:

[0083] Table 7 Main components of the reaction solution

[0084]

[0085] Gently mix the prepared mixture with a pipette, centrifuge it, and place it in a PCR instrument. Incubate at 42°C for 2 min, and store at 4°C.

[0086] (2) Reverse transcription reaction: the reaction mixture was prepared on ice according to the system as shown in Table 8. In order to ensure the accuracy of the content of each component in the reaction system, a premix solution was first prepared according to the reaction number, and 10 μL of the premix solution was dispensed into the reaction tube of the previous step after being fully mixed to reduce errors.

[0087] Table 8 Reaction system

[0088]

[0089] The prepared mixture was gently blown and mixed with a pipette, and then centrifuged instantly. Then, the mixture was immediately placed in a PCR instrument, and the reaction conditions were 37℃ for 15 min, 85℃ for 5 s, and 4℃ for storage.

[0090] 3.2 qRT-PCR

[0091] qRT-PCR experiments were performed using a TB Green Premix Ex TaqTM II (Tli RNaseH Plus) (TaKaRa #RR820A) kit. First, 5 μL of cDNA obtained by reverse transcription was taken out and diluted 10 times with RNase Free H2O. The remaining cDNA was stored at -20℃ for standby use. qRT-PCR primers were designed and synthesized according to Table 9.

[0092] Table 9 Quantitative PCR primers

[0093]

[0094] The reaction solution was prepared on ice according to the system as shown in Table 10. In order to ensure the accuracy of the content of each component in the reaction system, a premix solution was first prepared according to the reaction number, and then dispensed into each reaction well after being fully mixed to reduce errors.

[0095] Table 10 Reaction system

[0096]

[0097] The prepared reaction system was centrifuged instantly and placed in a CFX96 Real-Time System (Bio-Rad) instrument for detection. The reaction conditions are shown in Table 11.

[0098] Table 11 Reaction conditions

[0099]

[0100] After the reaction, Bio-Rad CFX Manager software was used for data analysis.

[0101] 4 Verification of the binding of miRNA and target genes by dual luciferase method

[0102] 4.1 pmirGLO vector construction

[0103] 4.1.1 PCR amplification

[0104] Dual-luciferase reporter gene verification was performed on the predicted target genes of miRNA. Referring to the sequence of the target gene, enzyme cleavage site recognition sequences and protective bases were added to the 3' UTR target fragment containing the miRNA binding site, and the primers were designed and synthesized using SnapGene software according to the primer design principle (see Table 12 for details).

[0105] Table 12 Primers required for dual-luciferase reporter gene detection

[0106]

[0107] Note: The underlined part indicates the position of the restriction enzyme cleavage site.

[0108] The brown planthopper cDNA was used as a template for PCR amplification, and a 50 μL reaction system was prepared as shown in Table 13:

[0109] Table 13 Reaction system

[0110]

[0111] After mixing, centrifuge for a moment and place in a PCR instrument, and react according to the conditions shown in Table 14:

[0112] Table 14 Reaction conditions

[0113]

[0114] 4.1.2 PCR product gel recovery

[0115] 0.7 g of agarose was weighed, 70 mL of 0.5×TBE buffer was added, and after mixing, the agarose was heated to completely dissolve, cooled to about 50℃, 7 μL of nucleic acid dye Gelred was added, and gently shaken. Note that bubbles are avoided during shaking. Pour the gel liquid into a mold of appropriate size, make it evenly cover the entire gel plate, insert a sample comb of appropriate size and number of holes, and stand for 15-20 min. After the agarose gel is completely solidified, remove the comb. Add an appropriate amount of 6×LoadingBuffer to the PCR reaction well, mix well, and set the voltage of the electrophoresis instrument to 180 V, and the electrophoresis time to 15 min. After electrophoresis, quickly cut out the gel containing the target band under ultraviolet light, weigh it, and place it in a 2 mL centrifuge tube.

[0116] The PCR product was recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme #DC301) kit, and the following operations were performed according to the instructions:

[0117] (1) Add 100 μL Buffer GDP to every 100 mg agarose gel, 50~55℃ water bath for 7~10 min, during which time invert and mix 2 times until the gel is completely dissolved;

[0118] (2) Place the FastPure DNA Mini Columns-G adsorption column in the Colledtion Tubes 2 mL collection tube, and transfer the gel liquid to the adsorption column, centrifuge at 12000 rpm for 30~60 s;

[0119] (3) Discard the filtrate, and place the adsorption column back into the collection tube, add 300 μL Buffer GDP, and centrifuge at 12000 rpm for 30~60 s;

[0120] (4) Discard the filtrate, and place the adsorption column back into the collection tube, add 700 μL Buffer GW to which anhydrous ethanol has been added in advance along the wall of the adsorption column, close the tube cap, invert gently 2~3 times, and centrifuge at 12000 rpm for 30~60 s;

[0121] (5) Repeat step (4);

[0122] (6) Discard the filtrate, and place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min;

[0123] (7) Place the adsorption column in a new 1.5 mL centrifuge tube, add 30 μL 55℃ preheated ddH2O to the center of the adsorption column, stand for 2 min, centrifuge at 12000 rpm for 1 min, discard the adsorption column, and obtain the purified PCR product;

[0124] (8) Measure the concentration and purity of the recovered product using a Nanodrop 2000 ultraviolet spectrophotometer.

[0125] 4.1.3 Double enzyme digestion reaction of target fragment and vector

[0126] The vector pmirGLO (Promega #E1330) was selected for the dual luciferase test, and the target fragment and pmirGLO empty vector (Promega #E1330) were subjected to double enzyme digestion reaction according to the system described in Table 15, to obtain the target fragment with sticky ends and the linearized vector large fragment; CYP15A1

[0127] Table 15 Reaction system ​

[0128]

[0129] Reaction condition: 37°C, 3 h, after reaction, add appropriate amount of 10x Loading Buffer to terminate reaction, then perform agarose gel electrophoresis to purify and recover the enzyme digestion product.

[0130] 4.1.4 Ligation of target fragment and vector

[0131] Ligate pmirGLO vector and enzyme digestion product of target fragment according to the system as shown in Table 16:

[0132] Table 16 Reaction system

[0133]

[0134] Reaction condition: 16°C overnight, 4°C storage.

[0135] 4.1.5 Transformation and bacterial liquid PCR

[0136] Take out DH5α E. coli competent cells from -80°C refrigerator, completely dissolve on ice, and transfer to clean bench. Add 10 μL ligation product to 100 μL competent cells, shake the tube wall to mix thoroughly, and stand on ice for 30 min. Heat shock at 42°C for 90 s, and after heat shock, quickly place the centrifuge tube on ice for 2 min. Then add 300 μL LB liquid medium without antibiotic to the tube, and shake culture at 37°C, 220 r / min for 1 h. Take 100 μL culture liquid and evenly spread on LB solid culture dish containing ampicillin, and shake culture at 37°C overnight.

[0137] Pick 12 single colonies per dish, and inoculate in 300 μL LB liquid medium containing ampicillin, and shake culture at 37°C, 220 r / min for more than 3 h. Take 1 μL bacterial liquid to perform bacterial liquid PCR reaction as shown in Table 17:

[0138] Table 17 Reaction system

[0139]

[0140] After mixing, centrifuge instantaneously, and place in PCR instrument to perform reaction according to the conditions as shown in Table 18:

[0141] Table 18 Reaction condition

[0142]

[0143] Detect by agarose gel electrophoresis, and take 200 μL bacterial liquid of positive clone with correct band for testing.

[0144] 4.1.6 Plasmid extraction

[0145] 10 μL of the bacterial liquid of the positive clone with correct sequencing results was inoculated in 30 mL of LB liquid medium containing ampicillin, and cultured at 37°C, 220 r / min for 12-16 h.

[0146] The plasmid was extracted using FastPure Plasmid Mini Kit (Vazyme #DC201) kit, and the following operations were performed according to the instructions:

[0147] (1) The bacterial liquid of the overnight culture was divided into centrifuge tubes, each tube not more than 5 mL, 10000 rpm centrifugation for 1 min, and the supernatant was discarded;

[0148] (2) 250 μL of Buffer P1 pre-added with RNase A was added to the centrifuge tube, and shaken to mix;

[0149] (3) 250 μL of Buffer P2 was added, and gently inverted to mix for 8-10 times to obtain a viscous clear mixture;

[0150] (4) 350 μL of Buffer P3 was added, and immediately gently inverted to mix for 8-10 times, and a white flocculent precipitate was produced, and centrifuged at 12000 rpm for 10 min;

[0151] (5) The FastPure DNA Mini Columns adsorption column was placed in the Colledtion Tube 2 mL collection tube, and the supernatant was transferred to the adsorption column, and centrifuged at 12000 rpm for 30-60 s;

[0152] (6) Discard the waste liquid, add 500 μL of Buffer PW1 to the adsorption column, and centrifuge at 12000 rpm for 30-60 s;

[0153] (7) Discard the waste liquid, add 600 μL of Buffer PW2 pre-diluted with anhydrous ethanol to the adsorption column, and centrifuge at 12000 rpm for 30-60 s;

[0154] (8) Repeat step (7);

[0155] (9) Discard the waste liquid, put the adsorption column back into the collection tube, and centrifuge at 12000 rpm for 1 min to completely dry the adsorption column;

[0156] (10) Put the adsorption column into a new 1.5 mL centrifuge tube, add 50 μL preheated ddH2O to the center of the adsorption column, stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the adsorption column, and obtain the recombinant plasmid;

[0157] (11) Measure the concentration and purity of the recombinant plasmid using a Nanodrop 2000 ultraviolet spectrophotometer.

[0158] 4.2 Cell culture

[0159] 4.2.1 Preparation of S2 cell complete culture medium

[0160] FBS was thawed in advance at 4℃ overnight, and 5 mL FBS was transferred into 45 mL Schneide's Drosophila culture medium in a clean bench, and 500 μL 10000 U / mL penicillin-streptomycin was added, and mixed gently. The prepared complete culture medium was stored at 4℃ in the dark, and was used within a week. It needs to be incubated in advance before use.

[0161] 4.2.2 S2 cell recovery

[0162] The S2 cell cryopreservation tube was quickly taken out of the liquid nitrogen tank and immediately placed in a clean 30℃ water bath for 1 min for rapid thawing. The cryopreservation tube was gently shaken during thawing to accelerate thawing, and then transferred to the clean bench after disinfecting the outer wall of the cryopreservation tube with 75% alcohol. The cell liquid in the cryopreservation tube was completely aspirated and transferred to a centrifuge tube containing 5 mL preheated Schneide's Drosophila culture medium, and centrifuged at 200 rpm for 5 min. The supernatant was discarded. An appropriate amount of preheated complete culture medium was added, the cell suspension was gently blown with a pipette, and then transferred to a cell culture bottle. The cell culture bottle was placed in a cell incubator and cultured at 28℃.

[0163] 4.2.3 S2 cell passage

[0164] S2 cells are semi-suspended cells. Use a pipette to gently blow and make the cells completely suspended. Count the cells using a hemocytometer. When the cell density in the culture bottle is about 2×10 7 absorb 4 mL of cell suspension and transfer it to 20 mL of preheated complete culture medium. Gently blow the cell suspension with a pipette, transfer it to a cell culture bottle, and gently shake the bottle to evenly distribute the cells on the bottom of the bottle. Place the cell culture bottle in a cell incubator and culture at 28℃.

[0165] 4.3 Cell transfection

[0166] 4.3.1 Cell Seeding

[0167] Approximately 24 hours before transfection, cells were passaged and seeded in 24-well cell culture plates at a density of 0.3–1 × 10⁶ cells / well. 5 Cells were cultured at 28°C for approximately 24 hours using one cell per well. Transfection was performed using the ExFect Transfection Reagent (Vazyme#T101) kit, and subsequent procedures were followed according to the manufacturer's instructions.

[0168] 4.3.2 Formation of the EcFect / DNA complex

[0169] Add 50 μL of serum-free cell culture medium to a 1.5 mL sterile centrifuge tube, then add 1 μL of ExFect, and gently pipette to mix.

[0170] Take another 1.5 mL sterile centrifuge tube, add 50 μL serum-free cell culture medium, then add 0.5 μg pmirGLO recombinant plasmid and 1 μL 100 nM miRNA agomir / antagomir, and gently pipette to mix.

[0171] Add the mixed ExFect to the DNA mixture, gently pipette to mix, and let stand at room temperature for 15-20 minutes before use. The resulting ExFect / DNA complex solution should be used within 30 minutes.

[0172] 4.3.3 Transient transfection of cells

[0173] Transfect cells when they reach 70%–80% confluence. Add the ExFect / DNA complex solution dropwise to the culture medium, gently agitating the dish to ensure even dispersion. Incubate at 28°C for 24–48 hours, then harvest cells for subsequent experiments.

[0174] 4.4 Dual-luciferase assay

[0175] The Dual Luciferase Reporter Assay Kit (Vazyme#DL101) was used to detect the fluorescence intensity of Luciferin substrate after cell transfection, determine the expression level of Luciferase, and then infer the regulatory role of the gene. The procedure was performed according to the instructions as follows:

[0176] 4.4.1 Pretreatment of test reagents

[0177] Transfer all the components of Reaction Buffer II (Luciferase) to the Luciferase Substrate component and mix well. Aliquot as required and store at -80°C in the dark.

[0178] Mix 5x Cell Lysis Buffer with ddH2O at a ratio of 1:4 before each experiment, and store on ice for later use.

[0179] After centrifugation of the Renilla Substrate component, mix with Stop & Reaction Buffer at a ratio of 1:50 as required, and use at room temperature in the dark.

[0180] Before adding the sample for detection, the reagent should be equilibrated to room temperature to prevent changes in the enzymatic reaction caused by temperature changes, which can cause errors in the measurement results.

[0181] 4.4.2 Cell lysis

[0182] Collect the cell culture solution in the 24-well plate into a 1.5 mL centrifuge tube, centrifuge at 11200 rpm for 2 min, discard the supernatant; wash with PBS twice, centrifuge at 11200 rpm for 2 min, discard the supernatant; add 100 μL of diluted 1x Cell Lysis Buffer to the tube, mix gently, incubate at room temperature for 5 min, centrifuge at 11200 rpm for 2 min, and take the supernatant for subsequent detection.

[0183] 4.4.3 Firefly luciferase reaction detection

[0184] Take 20 μL of cell lysis supernatant into a black enzyme plate, add 100 μL of Luciferase Substrate equilibrated to room temperature to each well, mix quickly, and immediately detect the Firefly luciferase reporter gene activity on a full-wavelength enzyme marker.

[0185] 4.4.4 Renilla luciferase reaction detection

[0186] Add 100 μL of Renilla Substrate equilibrated to room temperature to the reaction solution from the previous step, mix quickly, and immediately detect the Renilla luciferase reporter gene activity on a full-wavelength enzyme marker.

[0187] Calculate the ratio of Firefly luciferase fluorescence expression and Renilla luciferase fluorescence expression to determine the dual luciferase activity.

[0188] 5 Microinjection of miRNA agomir and antagomir

[0189] The miRNA agomir and antagomir were synthesized by Wuhan Tianyi Huayu Gene Technology Co., Ltd. according to Table 19.

[0190] Table 19 miRNA agomir and antagomir sequences

[0191]

[0192] The agomir is a modified double-stranded RNA molecule that can mimic the functional mode of the miRNA complex in the body, and up-regulate the expression of miRNA by transfecting cells or administering in the animal body. Generally, one strand of the agomir is almost completely identical to the sequence of the miRNA molecule, and the 3' end of the antisense strand is modified with high affinity cholesterol and 4 thio backbones, and the 5' end is modified with 2 thio backbones, and the whole chain is methylated. The antagomir can interfere with the expression of endogenous miRNA, and is one of the important tools for studying the loss of function of miRNA. It is a modified single-stranded RNA molecule, and its sequence is almost completely complementary to the miRNA reverse complementary sequence, and the modification method is exactly the same as that of the modified chain of the agomir.

[0193] 5.1 Microinjection of agomir and antagomir

[0194] 5.1.1 Preparation before experiment

[0195] Weigh 1.5 g of agar powder, add 100 mL of ddH2O, and heat in a microwave oven until the agar powder is completely dissolved. Transfer the dissolved agar gel solution to a clean glass dish, and let it stand at room temperature until it is completely solidified.

[0196] Place the capillary glass needle used for microinjection on a glass slide and observe under a stereomicroscope. Use a clean scalpel to gently cut the needle tip to make the needle tip pass through and form a suitable size port and an angle suitable section. Use a syringe to inject liquid paraffin from the tail end of the capillary glass needle until liquid droplets can be seen overflowing from the needle tip. Fix the capillary glass needle on the microinjection instrument to ensure that the sealing plug is tightly plugged and no air enters to ensure air tightness.

[0197] 5.1.2 Microinjection

[0198] According to the experimental requirements, select the appropriate size of the brown planthopper, and put the brown planthopper into a clean glass test tube, and pass in 15 s CO2, or stand on ice for 30 s, so that the brown planthopper temporarily loses the ability to move. The brown planthopper is gently transferred to the agar gel platform with the abdomen upwards, and the angle of the micro-injection instrument is adjusted, so that the needle tip gently pierces the intersegmental membrane between the prothorax and the mesothorax of the brown planthopper. Each test insect is injected with 50 nL of 10 μM miRNA agonist or antagonist. After injection, the brown planthopper is transferred to a clean dish containing clean filter paper and fresh rice plants for recovery for 24 h, and then subsequent experiments are performed.

[0199] 5.3 Detection of brown planthopper weight gain and honeydew secretion after microinjection

[0200] Select several 5th instar biotype Y brown planthopper nymphs to inject miRNA agonists, and after recovery, transfer them to clean rice plants for rearing until they emerge. Before the experiment, the weight of the just emerged brown planthoppers is measured using an electronic balance and recorded, and then the weighed brown planthoppers are placed in pre-prepared wax bags, and the wax bags are fixed to the stems of YHY15 rice plants at the base. After the brown planthoppers have fed on the rice material for 48 h, the wax bags are removed from the stems, and the brown planthoppers are removed from the wax bags, which contain the honeydew secreted during feeding. Careful operation is required during the experiment to avoid scratching. The weights of the brown planthoppers and the wax bags after feeding are measured and recorded. The change in the weight of the brown planthoppers and the wax bags before and after feeding is the weight gain of the brown planthoppers and the amount of honeydew secretion.

[0201] 5.4 Detection of changes in target gene and miRNA expression levels after injection by fluorescence quantitative PCR

[0202] Select several just emerged biotype Y brown planthopper short-winged female adults to inject miRNA agonists and antagonists, and after recovery, sample, freeze in liquid nitrogen, and store in a -80°C refrigerator. RNAiso Plus (TaKaRa #9109) reagent is used to extract RNA from each sample. Fluorescence quantitative PCR is used to detect changes in miRNA and target gene expression levels after injection of miRNA agonists and antagonists.

[0203] Results analysis

[0204] 1. Screening of differentially expressed miRNAs and target genes and qPCR verification

[0205] The regulation of various physiological and biochemical processes in insects is involved in miRNA, and the expression of miRNA in two different biotypes of brown planthopper is sequenced by high-throughput sequencing, the differentially expressed miRNA is identified, and the function of the sequenced gene is classified by bioinformatics analysis means; and the miRNA and the target gene are matched one by one, according to the research on the molecular mechanism of brown planthopper adapting to insect-resistant rice, the miR-3032-z related to detoxification metabolic pathway is selected for research. MiRNA mainly exercises biological function by targeting and regulating the expression of target genes, so qRT-PCR technology is used to detect the expression of miR-3032-z and the corresponding target gene cytochrome P450 family gene Figure 2 In two biotypes of brown planthopper, the expression is detected, and the results show that, compared with biotype 1, the expression of miR-3032-z in biotype Y brown planthopper with higher virulence is significantly decreased (A in CYP15A1 ), and the expression of the corresponding target gene Figure 2 is significantly increased (B in CYP15A1 ), miR-3032-z is negatively correlated with CYP15A1 . The qRT-PCR verification result is basically the same as the sequencing result, so it is speculated that miR-3032-z and the corresponding target gene Figure 3 may be involved in the detoxification metabolic pathway of brown planthopper, and play an important role in the process of brown planthopper virulence evolution and formation of new biotype.

[0206] 2. Dual luciferase verification of miRNA and target gene binding

[0207] In order to verify the binding of miRNA and target gene, the dual luciferase reporter gene detection system is used for analysis. The 3' UTR region containing the target gene and the miRNA binding site is inserted into the pmirGLO vector to construct the recombinant plasmid, and co-transfected with miRNA agonist agomir into S2 Drosophila embryonic cells as experimental group, the relative fluorescence value of which is significantly lower than that of the blank control group transfected with empty plasmid pmirGLO and the negative control group transfected with agomir-NC (A in Figure 3 ), CYP15A1 The results show that miR-3032-z and CYP15A1 gene target binding, specifically, miRNA regulates the expression of gene by targeting and binding to the 3' UTR region of target gene.

[0208] 3. miR-3032-z regulates the expression of CYP15A1

[0209] In order to further reveal whether the change of the expression amount of miR-3032-z in brown planthopper affects the expression of CYP15A1 ​expression of miR-3032-z, the expression levels of miR-3032-z and target genes were detected CYP15A1 The results showed that, compared with the NC control, the expression level of miR-3032-z in the brown planthopper injected with agomir-3032 increased by 3.462 times at 24 h, while the expression level of the target gene Figure 4 significantly decreased by 0.239 times (A in Figure 4 and B in CYP15A1 ); the expression level of miR-3032-z in the brown planthopper injected with antagomir-3032 decreased significantly, which was 0.672 times of the NC control, while the expression level of the target gene Figure 4 increased by 1.48 times (C in Figure 4 and D in Figure 5 ).

[0210] 4. Effect of miR-3032-z on the feeding behavior of the brown planthopper

[0211] To further clarify whether the increase in the expression level of miR-3032-z can affect the feeding behavior of the brown planthopper, the expression level of miR-3032-z in the brown planthopper was elevated by injecting the artificially synthesized miRNA agonist into the newly emerged female adult brown planthoppers, and then the amount of honeydew excreted and the weight gain of the brown planthoppers after feeding on YHY15 rice materials for 48 h were detected. As shown in A in Figure 5 and B in Figure 5 , after microinjection of agomir-NC, the weight gain of the brown planthoppers and the amount of honeydew excreted after feeding on rice plants for 48 h were 0.3263 mg and 12.074 mg, respectively. After injection of agomir-3032, the weight gain of the brown planthoppers and the amount of honeydew excreted after feeding on rice plants for 48 h were 0.1497 mg and 9.2733 mg, respectively CYP15A1 .

[0212] Compared with injection of agomir-NC, the weight gain and honeydew secretion of the planthoppers injected with agomir-3032 were reduced, indicating that the increase of the expression of miR-3032-z would affect the feeding behavior of the planthoppers. The measurement of the amount of honeydew secretion and the change of the weight of the planthoppers can reflect the amount of the feeding of the planthoppers, and then reflect the resistance of the rice varieties, which is an important index for the phenotype identification of the planthoppers and the rice varieties. The less the honeydew secretion of the planthoppers, the lower the weight gain, and even the negative weight gain, indicating that the resistance of the rice material is stronger, and the feeding behavior of the planthoppers is inhibited. The reduction of the honeydew secretion indicates that the digestion and metabolism of the planthoppers are affected, and the reduction of the weight gain indicates that the growth and development process of the planthoppers is inhibited. The results show that, by inhibiting the expression of the cytochrome P450 family gene through injection of agomir-3032, the honeydew secretion and the weight gain of the biotype Y planthoppers on the insect-resistant rice YHY15 are reduced, and the feeding behavior is obviously inhibited, so that the virulent planthoppers restore the sensitivity to the resistant rice YHY15. CYP15A1

[0213] Conclusion: Functional miRNAs can target and bind to the 3'UTR region of target genes to inhibit or degrade mRNA, and then regulate the expression of target genes (Asgari S. 2013. MicroRNA functions in insects. Insect Biochemistry and Molecular Biology, 43: 388-397. Bartel DP. 2009. MicroRNAs: target recognition and regulatory functions. Cell, 136: 215-233. Fabian MR, Sonenberg N, Filipowicz W. 2010. Regulation of mRNA translation and stability by microRNAs. Annual Review of Biochemistry, 79: 351-379.). In order to explore the post-transcriptional regulation mechanism of the functional genes involved in the digestion and metabolism and detoxification of the planthoppers, the present application uses the experimental methods of fluorescence quantification and dual luciferase reporter gene detection, and confirms that miR-3032-z targets CYP15A1 ​The 3' UTR region of a gene is thus regulated to regulate the expression of the gene. In order to further reveal the action relationship between the miRNA and the target gene, the expression amount of the miRNA in the brown planthopper is regulated by artificially synthesizing the agonist (agomir) and the antagonist (antagomir) of the miRNA to inject the miRNA, and the results show that after the agonist is injected, the expression amount of the miRNA in the brown planthopper is obviously increased, and the expression amount of the corresponding target gene is obviously decreased. On the contrary, after the miRNA antagonist is injected, the expression amount of the corresponding target gene is obviously increased. At the same time, it is found through further phenotype identification experiments of the brown planthopper that the honeydew secretion amount and the weight gain of the brown planthopper after the injection of the agonist are obviously decreased. The above results show that the miR-3032-z inversely regulates the expression of the target gene and further mediates the adaptation process of the brown planthopper to the resistant rice. ​

[0214] The present application provides new insights into the molecular genetic mechanism of the participation of miRNA in the damage caused by the brown planthopper, deepens the understanding of the digestion and metabolism and detoxification process of the brown planthopper from the perspective of miRNA, provides a reference for the research on the detoxification and metabolism of the brown planthopper, provides an important theoretical basis for revealing the adaptation of the brown planthopper to the resistant rice, deepens the understanding of the molecular mechanism of the plant-insect interaction, and provides a reference for formulating the prevention and control strategy for the brown planthopper in the future.

[0215] Although the above embodiment makes a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained by people according to the present embodiment without creativity, and these embodiments all belong to the protection scope of the present application.​

Claims

1. miR-3032-z or reagents overexpressing miR-3032-z were used in the preparation of drugs that inhibit brown planthoppers. CYP15A1 Application in gene expression products; the product is a kit; the nucleotide sequence of miR-3032-z is shown in SEQ ID NO.1; CYP15A1 The gene number is XM_039430744.

1.

2. Inhibiting Nilaparvata lugens CYP15A1 The use of a reagent for gene expression in the preparation of a product for restoring the sensitivity of Nilaparvata lugens to insect-resistant rice; the inhibition of Nilaparvata lugens CYP15A1 The reagent for gene expression includes a reagent for overexpressing miR-3032-z; the nucleotide sequence of the miR-3032-z is shown as SEQ ID NO. 1; the CYP15A1 The gene number is XM_039430744.

1.

3. Inhibiting Nilaparvata lugens CYP15A1 The use of a reagent for gene expression in the preparation of a product for disrupting the detoxification metabolic capacity of Nilaparvata lugens against insect-resistant rice; the inhibition of Nilaparvata lugens CYP15A1 The reagent for gene expression includes a reagent for overexpressing miR-3032-z; the nucleotide sequence of the miR-3032-z is shown as SEQ ID NO. 1; the CYP15A1 The gene number is XM_039430744.

1.

4. Use according to claim 2 or 3, characterized in that, The insect-resistant rice includes insect-resistant rice YHY15.

5. Inhibiting Nilaparvata lugens CYP15A1 The use of a reagent for gene expression in the preparation of a product for reducing the amount of honeydew secretion and / or the weight gain of Nilaparvata lugens; the inhibition of Nilaparvata lugens CYP15A1 The reagent for gene expression includes a reagent for overexpressing miR-3032-z; the nucleotide sequence of the miR-3032-z is shown as SEQ ID NO. 1; the CYP15A1 The gene number is XM_039430744.

1.

6. Inhibiting Nilaparvata lugens CYP15A1 The use of a reagent for gene expression in the preparation of a product for inhibiting the feeding behavior of Nilaparvata lugens; the reagent for inhibiting the feeding behavior of Nilaparvata lugens CYP15A1 The reagent for gene expression includes a reagent for overexpressing miR-3032-z; the nucleotide sequence of the miR-3032-z is shown as SEQ ID NO. 1; the reagent for overexpressing the miR-3032-z includes a reagent for overexpressing the miR-3032-z in the body of Nilaparvata lugens. CYP15A1 The gene number is XM_039430744.

1.

7. The use according to any one of claims 1 to 3, 5 or 6, wherein the compound is of formula (I) ###0001### (I) or a pharmaceutically acceptable salt thereof. The brown planthopper includes a virulent brown planthopper.

8. Use according to claim 7, characterized in that, The virulent brown planthopper includes a biological Y-type brown planthopper.

9. The use according to any one of claims 1 to 3, 5 or 6, wherein, The reagent for overexpressing miR-3032-z includes a miR-3032-z agonist.

10. Use according to claim 9, characterized in that, The miR-3032-z agonist includes agomiR-3032; a nucleotide sequence of a sense strand of the agomiR-3032 is shown in SEQ ID NO. 2, and a nucleotide sequence of an antisense strand of the agomiR-3032 is shown in SEQ ID NO. 3.

Images