Gene for regulating migration and reproduction behaviors of cnaphalocrocis medinalis and application of gene

Abstract

The invention discloses a gene for regulating migration and reproduction behaviors of cnaphalocrocis medinalis and application of the gene, and belongs to the technical field of agricultural biology. On one hand, the invention provides a gene for regulating migration and reproduction behaviors of cnaphalocrocis medinalis, and on the other hand, the invention provides application of the gene. The gene for regulating and controlling migration and reproduction behaviors of the rice leaf rollers is used for designing double-stranded RNA to inject the rice leaf rollers, so that the migration and takeoff proportion of the rice leaf rollers is remarkably reduced, and ovary development is promoted. The expression of the Period gene in the migratory individual is obviously higher than that of the resident individual. By monitoring the expression quantity of the Period gene, the population characteristics of the cnaphalocrocis medinalis can be judged. The invention discloses the application of the Period gene in rice leaf roller damage monitoring for the first time, and the generation characteristics of the local rice leaf roller population can be defined, so that the prediction and the prevention and control of the rice leaf rollers can be better guided.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a gene for regulating the migration and reproductive behavior of rice leaf folder and an application thereof. Background Art

[0002] The rice leaf folder, Cnaphalocrocis medinalis, is one of the most destructive migratory pests on rice. It has a strong migratory ability and is significantly affected by changes in the photoperiod. It has multiple migratory damages in my country every year, and in severe cases, it can cause grain yield losses of up to 63% to 80%. Since the outbreak of the rice leaf folder depends largely on the migrating population, and the occurrence is explosive and sudden, it makes its monitoring and prevention more difficult. At present, there is an urgent need to explore effective rice leaf folder population monitoring methods to distinguish between migratory and resident types, and then clarify the occurrence characteristics of local rice leaf folder populations, so as to provide guidance for the prediction and prevention of rice leaf folders.

[0003] Previous studies have found that CRY1 is involved in the regulation of the migration and take-off behavior of the rice leaf folder. The Period (PER) gene is a circadian clock gene downstream of CRY1. The accumulation, translocation, and degradation of PER in the cytoplasm and nucleus are also regulated by CRY1. It has been found in many insects that the expression of the per gene is involved in regulating physiological behaviors such as emergence, mating, reproduction, and migration. The expression of the per gene may be related to migration behavior. Silencing or knocking out the per gene in the organism can significantly reduce the night flight activity of the oriental armyworm and delay its egg-laying period, and can also interrupt the egg-laying rhythm of the silkworm and lose diurnal movement activity. This shows that per is involved in a variety of physiological processes such as flight and reproduction of Lepidoptera insects. However, it is not yet clear whether per is involved in regulating the migration and reproductive behavior of the rice leaf folder. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to design and provide a gene for regulating the migration and reproductive behavior of rice leaf folder and a technical solution for its application.

[0005] The technical solution specifically adopted in the present invention is:

[0006] In one aspect, the present invention provides a gene for regulating the migration and reproductive behavior of rice leaf folder, the nucleotide sequence of the gene is:

[0007] 1) the nucleotide sequence shown in SEQ ID No. 1; or

[0008] 2) The nucleotide sequence shown in SEQ ID No. 1 is replaced by one or several nucleotides to obtain a nucleotide sequence encoding Period.

[0009] The second aspect of the present invention provides the use of the above gene in regulating the migration and reproductive behavior of rice leaf folder.

[0010] The third aspect of the present invention provides the use of the above gene in monitoring damage caused by rice leaf folder.

[0011] In a fourth aspect, the present invention provides a use of a Period gene or its encoded protein as a target for preparing a preparation for regulating the migration and reproductive behavior of rice leaf folder, wherein the nucleotide sequence of the Period gene is:

[0012] 1) the nucleotide sequence shown in SEQ ID No. 1; or

[0013] 2) The nucleotide sequence shown in SEQ ID No. 1 is substituted with one or more nucleotides to obtain a nucleotide sequence encoding Period;

[0014] The amino acid sequence of the encoded protein is:

[0015] 1) the amino acid sequence shown in SEQ ID No. 2; or

[0016] 2) An amino acid sequence with equivalent function formed by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.

[0017] The fifth aspect of the present invention provides dsRNA obtained by transcribing the above gene template.

[0018] A sixth aspect of the present invention provides the use of the above dsRNA in regulating the migration and reproductive behavior of rice leaf folder.

[0019] A seventh aspect of the present invention provides the use of the above dsRNA in monitoring damage caused by rice leaf folder.

[0020] An eighth aspect of the present invention provides a kit for regulating the migration and reproductive behavior of rice leaf folder, the kit comprising the dsRNA prepared as above.

[0021] A ninth aspect of the present invention provides a method for regulating the migration and reproductive behavior of rice leaf folder, wherein the method uses the above-mentioned dsRNA to treat the rice leaf folder.

[0022] The present invention discovered a key gene Period that can regulate the migration and reproductive behavior of rice leaf folder. The present invention uses the Period gene to design double-stranded RNA for injection into rice leaf folder, which significantly reduces the migration and take-off ratio of rice leaf folder and promotes ovarian development. The expression of the Period gene in migratory individuals is significantly higher than that in resident individuals. By monitoring the expression level of the period gene, the population characteristics of the rice leaf folder can be determined. The present invention discloses for the first time the application of the Period gene in monitoring the damage of rice leaf folder, which can clarify the occurrence characteristics of the local rice leaf folder population, thereby better guiding the prediction and prevention of the rice leaf folder. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Cmedper gene sequence and protein sequence;

[0024] Figure 2 Cmedper protein structure;

[0025] Figure 3 Cmedper protein sequence alignment;

[0026] Figure 4 Cmedper phylogenetic tree;

[0027] Figure 5 Expression of Cmedper in different parts of adult worms;

[0028] Figure 6 24 h expression profile of Cmedper under different nutrient conditions and temperatures;

[0029] Figure 7 The take-off ratios of migratory and resident rice leaf folders and the relative expression of Cmedper at different times and in different migration types;

[0030] Figure 8 Silencing efficiency after interference with Cmedper at 2 and 3 days of age;

[0031] Fig. 9 Interference with Cmedper resulted in differences in take-off ratio, ovarian development and expression of related genes. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0033] Example 1

[0034] 1 Materials and methods

[0035] 1.1 Insect farming

[0036] The source of rice leaf roller was collected from the rice experimental field of Baima Base of Nanjing Agricultural University and raised in the laboratory for ten generations to form a laboratory population. The method of indoor rearing of rice leaf roller refers to Guo et al. (2019). Rice leaf roller larvae were raised with wheat seedlings 10-15 cm high until they pupated. The pupae were transferred to a plastic box with moistened cotton wool on the bottom for rearing, and the adults were collected after they emerged. Healthy female adults that emerged within 2 hours of the peak emergence period (0:00-2:00 every day) were selected for the experiment. All test insects were placed in an incubator with a constant temperature of 27°C, RH80-90%, and a photoperiod of 14L:10D (photoperiod of 5:00-19:00, BJT, the same below) and entered the photoperiod at ZT0 (zeitgebertime).

[0037] 1.2 Total RNA extraction and cDNA acquisition

[0038] The wings and legs of 2-day-old female adults were cut off and the hair was scraped off, then quickly frozen in liquid nitrogen and stored at -80°C. The total RNA of the insects was extracted using the TRIzol method. The specific method is as follows:

[0039] 1) Transfer the rice leaf folder to a centrifuge tube (1.5 mL imported RNase-free centrifuge tube), add 1000 mL of RNA extraction buffer (TRIzol reagent: 100 mM Tris pH 7.5, 100 mM LiCl, 20 mM DTT, 10% sodium dodecyl sulfate (SDS)), then add grinding beads, and grind with a grinder for 2 minutes;

[0040] 2) The centrifuge tube was left to stand at room temperature for 5 minutes; then centrifuged at 12000 rpm at 4°C for 5 minutes;

[0041] 3) Add 200 μL of chloroform, shake vigorously, and place on ice for 10 min; centrifuge at 12,000 rpm at 4°C for 15 min;

[0042] 4) Carefully pipette 400 μL of supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of isopropanol solution, mix by inverting, and let stand at room temperature for 10 min; centrifuge at 12,000 rpm for 10 min at 4°C;

[0043] 5) Pour off the supernatant solution, add 1 mL of 75% ethanol solution to the precipitate, and centrifuge at 12,000 rpm for 5 min at 4°C; repeat 2-3 times;

[0044] 6) Pour off the supernatant, open the centrifuge tube to dry the residual ethanol solution; add 20 μL-60 μL DEPC water to dissolve RNA; 1% agarose gel electrophoresis to detect the integrity of the obtained RNA, and spectrally quantify the RNA sample at OD260nm on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Then store in a -80°C refrigerator for future use.

[0045] The total RNA solution was obtained using Polestar 1 st cDNA was obtained by reverse transcription using cDNA Synthesis Kit (gDNA removal) (Beijing Baoying Tonghui Biotechnology Co., Ltd.). The reaction system was: Total RNA: 2 μg; 5× Polestar RT Master Mix (with dsDNase): 4 μL; 20× OligodT (25) &RandomPrimer:1μL; RNase Free H 2 O: Up to 20μL.

[0046] The reaction procedure is:

[0047] 25°C 10 min (primer pairing);

[0048] 55°C for 30-60 min (cDNA synthesis);

[0049] 85℃5min (inactivate MLV).

[0050] The obtained cDNA was stored in a -20°C refrigerator for cloning.

[0051] 1.3 Cmedper gene cloning

[0052] Based on the conserved amino acid sequence between homologous genes of Cmedper (GeneID: Cmed076210.1) obtained from the reference genome of rice leaf folder (http: / / v2.insect-genome.com / Organism / 192), primers targeting the conserved region of the gene were designed (see Table 1). TM DNA Polymerase high-fidelity enzyme (Bao Ri Yi Biotechnology Co., Ltd.) was used for polymerase chain reaction. The high-fidelity enzyme reaction system was:

[0053]

[0054] The polymerase chain reaction conditions were: 94°C for 3 min, then 94°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 30 s, for 30 cycles. The PCR product was electrophoresed in a 1% TAE Buffer agarose gel (Gel Red added), and the agarose region with bright bands was cut out and purified using the Omega EZNA Gel Extraction Kit (Omega Bio-Tek). The specific steps were as follows:

[0055] 1) Place the PCR product analyzed by agarose gel / ethidium bromide electrophoresis into a clean 1.5 mL centrifuge tube and weigh it to determine the volume of the gel slice.

[0056] 2) Add an equal volume of XP 2Binding Buffer. Incubate at 60°C for 7 min or until the gel is completely melted. Vortex or shake the centrifuge tube every 2-3 min.

[0057] 3) Insert the DNA Mini column into a 2 mL collection tube. Add no more than 700 μL of the DNA / agarose solution from step 2 to the DNA Mini column. Centrifuge at 10,000 × g for 1 min at room temperature. Discard the filtrate and reuse the collection tube. Repeat 2-3 times until all the sample has been transferred to the column.

[0058] 4) Add 300 μL XP2 Binding Buffer. Centrifuge at maximum speed (≥13000×g) for 1 min at room temperature. Discard the filter and reuse the collection tube.

[0059] 5) Add 700 μL of SPW buffer diluted with 100% ethanol. Centrifuge at maximum speed for 1 min. Discard the filtrate and reuse the collection tube. Repeat step 8 for a second SPW Buffer wash step.

[0060] 6) Centrifuge the empty column at maximum speed for 2 minutes to dry the column. Transfer the DNA Mini column to a clean 1.5 mL centrifuge tube.

[0061] 7) Add 30-50 μL Elution Buffer directly to the center of the column matrix. Incubate at room temperature for 2 minutes. Centrifuge at maximum speed for 1 minute. Store DNA at -20°C.

[0062] The purified products were sequenced (Qingke Biotechnology Co., Ltd.) and then compared in DNA MAN.

[0063] Table 1 Primer sequences of genes

[0064]

[0065] 1.4 Bioinformatics analysis

[0066] The nucleic acid sequence was translated using Expasy (https: / / web.expasy.org / translate / ) to obtain the open reading frame (ORF). Post-translational modification sites were predicted using ProSite Scan (https: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_proscan.html). The secondary structure was determined using SOPMA (https: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) (Geourjon and Deléage, 1995). Phyre2 (http: / / www.sbg.bio.ic.ac.uk / phyre2 / phyre2_output / fd82f25425609a0c / summary.html#domain_analysis) was used to construct protein 3D models, and InterPro (http: / / www.ebi.ac.uk / interpro / result / InterProScan / iprscan5-R20230608-041709-0815-8539751-p1m / ) was used for domain analysis. Amino acid sequences of known insects were obtained from NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and amino acid sequence alignment and phylogenetic tree construction were performed using MEGA11.

[0067] 1.5 Expression of circadian clock genes in different parts of female moths

[0068] Antennae (20 pairs / group), head (5 heads / group), thorax (5 / group), and abdomen (5 / group) of 2-day-old rice leaf folders were collected, and 3 biological replicates were collected for each part.

[0069] 1.6 Effects of adult nutrition and temperature on the expression pattern of Cmedper

[0070] Take newly emerged female adults and subject them to two treatments with different adult nutrition and temperature:

[0071] (1) Temperature treatment: newly emerged female adults were placed in an environment of 16°C, 26°C, and 36°C and fed with clean water;

[0072] (2) Nutritional treatment: The starvation treatment used cotton balls soaked in water at the bottom of a plastic cup, while the nutritional supplementation treatment used 5% honey water. Female adults were collected at 0:00, 4:00, 8:00, 12:00, 16:00, and 20:00 on the 2nd day of the peak takeoff age (Yang et al., 2015), with 3 adults per sample and 3 biological replicates at each time.

[0073] 1.7 Analysis of Cmedper expression between migratory and resident individuals

[0074] Female adults of rice leaf rollers at 2 days old after eclosion were collected for observation of take-off behavior. The light intensity change treatment simulated the light intensity change at dusk at sunset in the field, referring to the method of Yang Fan et al. (2013). The light source consisted of 12 rows of monochromatic non-flickering LCD lamps (35V / W) (Ningbo Jiangnan Instrument Factory, Ningbo, China), with a total brightness of 1000lx. During the experiment, the light intensity in the climate chamber was gradually reduced from 1000lx to less than 0.1lx within 30min by gradually weakening the light intensity (100lx / 3min), so as to simulate the light intensity change at dusk. The light change treatment started at 19:00 (the same below) and ended 30min after entering darkness (20:00). The insects were placed in a take-off cage made of transparent acrylic with a height of 1.2m and a diameter of 1m. The top was covered with a gauze mesh for ventilation, and the bottom was a white acrylic base. During the dark period, moths that spiral upward and reach the top of the take-off cage or the cage wall more than 1m in one go are regarded as migratory individuals with the intention to migrate, while moths that do not have any flying behavior during the entire experiment are regarded as resident individuals and do not have the intention to migrate. At the same time, moths that fly irregularly at the bottom of the take-off cage or lie on the side wall of the flight cage for a long time after a short flight during the experiment are regarded as diffusing individuals and do not have the intention to migrate. The entire test environment is kept quiet to avoid human interference. All adults to be tested are transferred to the climate room 2h before the start of the observation experiment to adapt to the surrounding environment. During this period, the light intensity is kept constant at 1000lx.

[0075] After the take-off experiment, the migratory individuals and resident individuals at 2 days old were obtained. After the experiment, the migratory individuals and resident individuals were raised to 3 days old and the take-off experiment was conducted again to record the take-off ratio. Samples were collected every 15 minutes until 20:00 during the light reduction and dark phases. All samples were set up with 3 replicates, and each replicate had 3 adults.

[0076] 1.8 Real Time-qPCR

[0077] All the above samples were immediately frozen in liquid nitrogen after collection and stored in a -80°C refrigerator. cDNA was obtained by referring to the method in 1.2, and the relative expression level of Cmedper was detected by fluorescence quantitative PCR. iTaq TM Universal Green Supermix kit (Bio-Rad Biomedical Products Co., Ltd.) was used to configure the reaction system, specifically 10uLiTaq TM Universal Green Supermix (2×); 1uL upstream and downstream primers (primer sequences see Table 1); 7uL RNase free H 2 O and 1 μL cDNA. The reaction system is:

[0078]

[0079]

[0080] Quantification of gene expression was performed on a real-time polymerase chain reaction system (Bio-Rad Biomedical Products Co., Ltd.). β-actin and RPS3 were used as reference genes in the same run for each sample (see Table 1). Separate reactions were used to quantify each RNA level in a given cDNA sample, and the average Ct of three replicate reactions in the same run was used for quantification. Relative gene expression was calculated using 2 －ΔΔCt The method was followed (Livak et al., 2001).

[0081] 1.9 Preparation of DsRNA

[0082] According to the sequence of Cmedper, dsRNA primers were designed using NCBI primer-BLAST, and T7 promoter sequences were added to the 3' end of the primers (primer sequences are shown in Table 1). DNA fragments containing T7 promoters at the ends were obtained by PCR. The PCR reaction system and procedures were all as in 1.3. The presence of a single bright band was verified by agarose gel electrophoresis, and purified DNA was obtained by gel recovery. The purification steps are shown in 1.3. The purified DNA obtained was sent to Qingke (Qingke Biotechnology Co., Ltd.) for sequencing to confirm that the primers were not off-target.

[0083] The DsGFP primer sequence was referenced to (Li et al., 2022), and DNA was extracted from Escherichia coli transcribed with GFP plasmid and PCR was performed in the same way as above.

[0084] dsRNA was prepared using T7 RNAi Transcription Kit (Nanjing Novogene Biotech Co., Ltd., Nanjing, China). The preparation method is as follows:

[0085] 1) Configure the template reaction system:

[0086]

[0087]

[0088] 2) Incubate in PCR machine at 37°C for 2 h;

[0089] 3) Dilute 100U / μL RNaseTi to 10U / μL using RNaseT1 Dilution Buffer;

[0090] 4) Configure the double enzyme digestion system:

[0091]

[0092] After mixing, gently pipette to mix, and briefly centrifuge the reagent at the bottom of the tube;

[0093] 5) Incubate at 37°C for 30 min;

[0094] 6) Detection of transcription products by electrophoresis;

[0095] 7) Add 200 μL of chloroform to the purified product, shake it vigorously, and place it on ice for 10 min; centrifuge it at 12,000 rpm at 4°C for 15 min;

[0096] 8) Carefully pipette 400 μL of supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of isopropanol solution, mix by inverting, and let stand at room temperature for 10 min; centrifuge at 12,000 rpm for 10 min at 4°C;

[0097] 9) Pour off the supernatant solution, add 1 mL of 75% ethanol solution to the precipitate, and centrifuge at 12,000 rpm for 5 min at 4°C; repeat 2-3 times;

[0098] 10) Pour off the supernatant and leave the centrifuge tube open to dry the remaining ethanol solution; add 20 μL-60 μL RNase-free H 2 O to dissolve dsRNA, measure the concentration of dsRNA sample on NanoDrop 2000 spectrophotometer (instrument brand), add RNase-free H 2 O until the concentration is adjusted to 1000 ng / μL. Then store in a -80℃ refrigerator for later use.

[0099] The nucleotide sequence of the effective interference sequence of the Cmedper gene, that is, the sequence of dsRNA is shown in SEQ ID NO.3.

[0100] 1.10 RNA interference

[0101] RNA interference was performed on the newly emerged female adults of rice leaf roller. Before the experiment, dsRNA was taken out of the refrigerator and thawed on ice. After the solution melted, 1 μL of liposome RFect was added. PM Primary cell plasmid DNA transfection reagent (Bio-Bio Co., Ltd., Changzhou, China) was used to protect dsRNA from degradation by nucleases in insects. After mixing, the mixture was incubated on ice for 30 min before use.

[0102] Select newly emerged female adults within 2 hours after emergence. After anesthesia on ice, use Nanojection for microinjection. Inject 1μL dsRNA into each test insect. Inject the same amount of dsGFP as the control group. Do not damage the fragile parts of the test insects such as wings, legs, and antennae during injection. After the injection, transfer the test insects to a 500mL plastic cup, place a soaked cotton ball at the bottom of the cup, cover the top of the cup with plastic wrap, and poke holes for ventilation. Then, transfer to a climate chamber for breeding, and replace the cotton balls every day. Detect the interference efficiency at 2 or 3 days of age.

[0103] 1.11 Effects of interfering with Cmedper on ovarian development

[0104] After interfering with Cmedper, ovarian dissection was performed at 17:00 on the 3rd day of age. After the test insects were anesthetized on ice, the wings and legs were removed, and the villi on the dorsal plate and abdomen were scraped off with a brush. The test insects were then placed on a dissecting wax box, and 10 mmol / L PBS buffer (pH = 7.2) was added to the wax box. Under a microscope, the test insects were fixed with the abdomen facing up with an insect pin. The tail of the female moth was fixed with tweezers in the left hand, and the right hand used an insect pin to cut the dorsal-ventral junction along the tail direction, tear open the epidermis, and separate the ovaries. Use an insect pin to remove the fat granules around the ovaries so that the ovarian tubules are clearly visible. The ovaries of the rice leaf folder were graded according to Guo et al. (2022). 15 test insects were dissected in the DsPER and dsGFP treatment groups, respectively.

[0105] 1.12 Effects of Cmedper disruption on the expression of circadian clock-related genes and juvenile hormone-related genes

[0106] After interfering with Cmedper, the relative expression levels of circadian clock-related genes Cmedcry1, Cmedtim and Cmedper and juvenile hormone-related genes Cmedjhamt and Cmedjhe were analyzed in 2- and 3-day-old adults. All samples were replicated three times, with three adults in each replicate. The specific methods of total RNA, reverse transcription to obtain cDNA and fluorescence quantitative PCR are shown in 1.3.

[0107] 1.13 Data Analysis

[0108] The statistical software SPSS16.0 was used to perform statistical analysis on the data obtained from each experimental step, and the mean value and standard error were obtained. The one-way general linear model (one-way ANOVA) was used to analyze the 24h expression pattern of Cmedper. The two-way general linear model (two-way ANOVA) was used to analyze the effect of Cmedper on the daily expression pattern under different temperatures and different nutritional conditions. Tukey's HSD method was used to compare the expression levels of the target genes in different tissues. All daily expression data were analyzed by cosine curve fitting to determine whether there was oscillation. The difference in the take-off ratio of dsPER and dsGFP was tested using Fisher's Exact Test. The interference efficiency and the relative expression differences of Cmedcry1, Cmedtim and Cmedper, as well as Cmedjhamt and Cmedjhe between the interference group and the control group were compared using t-test.

[0109] 2 Results

[0110] 2.1 Cloning and alignment of Cmedper gene

[0111] The Cmedper gene has an open reading frame of 3597 bases, encoding a polypeptide of 1199 amino acid residues ( Figure 1 The deduced relative molecular mass of CmedPER protein was 133.2 kDa, and the isoelectric point was 6.58. The post-translational modification sites inferred by PROSITESCAN showed that it had 6 N-glycosylation sites, 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 12 protein kinase C phosphorylation sites, 16 caseinkinase II phosphorylation sites, and 5 N-myristoylation sites. Figure 1 ). SOPMA was used to construct the secondary structure, and the results showed that alpha helix accounted for 20.60%, extended strand accounted for 11.18%, beta turn accounted for 3.17%, and random coil accounted for 65.05%. Phyre2 was used to construct the CmedPER3D model, and domain analysis was compared with Drosophila PER protein, with a confidence level of 100% ( Figure 2 ).

[0112] Using NCBI protein BLAST to compare the amino acid sequence of CmedPER, it was found that it had high homology with other insect PERs. The highest homology was with Ostrinia furnacalis (GenBank accession number: XP_028177217.1, 71.43% identity), Spodoptera frugiperda (GenBank accession number: XP_050560396.1, 71.66% identity), Spodoptera litura (GenBank accession number: AWK22769.1, 67.40% identity), Trichoplusia ni (GenBank accession number: XP_026727843.1, 70.18% identity), Mamestra brassicae (GenBank accession number: AAS49924.1, 66.46% identity), Chrysodeixis spp. Includens (GenBank accession number: CAH0578061.1, 67.80% identity) and Manduca sexta (GenBank accession number: XP_030039653.2, 66.87% identity) also have good homology. MEGA multiple sequence comparison results showed that PER has multiple highly conserved domains in Lepidoptera insects, and domain analysis was performed using IntroPro. Two PAS domains (PAS-A and PAS-B, PAS, Per-Arnt-SIM) domains and a C-terminal region (framed in red, Figure 3 ). The phylogenetic tree constructed by the neighbor-joining method based on the amino acid sequences of PER in 15 insects. The results showed that Lepidoptera insects clustered into a large branch, which was far away from the PER of other orders of insects such as fruit flies and aphids, which was consistent with the results of traditional taxonomy ( Figure 4 ).

[0113] The nucleotide sequence of the Cmedper gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2:

[0114] MYAVQYFAHQSEYKTVVTSPSPLHLCGKKSITPTCPDIEMDNLDDSENNAKISDSAYSNSCSNSQSRRSHSSKSTHSGSNSSGSSGYGGQPSTSGSSNQPPEKRIKEKEPKTKKQVQVDSAATDTETECRPPDPVPEDDPKEEECDVDQTPSLELAHSEDGPQSMDISSPETLGGKDDGCVVPCSQPLGNASVNFVTSRPGALVNCSDGFSCVISMHDGVVMYTTSSLTTTLGFPKDMWMGRSFIDFIHPRDRSTFASQITSGLAVPKIVNGTQEAVSNIRNLHYRLPVQIPSNTQLSTMVCRIRRYRGLTAGFGVKERVVTFMPFLLKLSFKNVNDERGESRTKWCSNPYPFVMRHAANGNLEYLDPESVPYLGYLPQDVIGEDALKFYHPDDLCYLRQVYETIVKEGGLTRSKSYRMMTQNGDYLRLETEWSSFINPWSRKLEFVIAKHRILEGPNNPDVFQTPEIKPKTKVKEEEENKQQSMRDSIIKILNEVLTKPAELAKQQMSKRCQDLASFMETLLEEQPKTDEELRLEIQDADHSYYERDSMLGGISPHHDFNDSKSSTETPLSYNQLNYNENLQRYFESHQPFSFEDHNTVTGENVLGLKDLKPISSNCLSPMAQQSGDSVDVNSSCESGLVVRDTSPVAPLGDYQPIRLTENLINKHNTEMEKELLRAHRETRSSSKGEREKTSNENRQKKKEHLARCNASFQPAAVVATSVEAPAEKKSHGVKRSKQLDSDPAHKHHCSSRQARRRHVT2SAAPAHPSATITTTVAATHWHPNPVNNMNTFILGVGMPQQMSIISPVMSQQVAHMSGMFPMYYVPSPAPVTSMPHNHNVPTSSTANMSQNDPSKNNPHTPQPCNSQFQAPAMQCMMYGQAVYGSPFMYS PIAPSPQFPTQQTMMAQTMQYTNSLYPLGLASSNYEEACKPSVPLRPTKSSGGWRDKKRGESQAASSKTDRGSGDSNSSSNALNRSTEAVPTSKASRDAMSKNK MRTSDETIDRTDGESSYSSFYSSFFKTDSGSAEDSGDLKKGRPKMRASTSEFWDKNTPDDQSSVEANSAYHEPKKHKKVTRRKMEPPWMEQVCVTSELIYKYQ ILTKNMEEVLTADKQKMSTFTQPSLVNEQLGQLYLDLQLEGVAARLTLEEGITSSSSSGEETPTVATKGSAKRKREYSKLVMIYEEDAPLPPPSTDDEASAMT*

[0115] 2.2 Expression characteristics of Cmedper in different parts

[0116] The expression levels of Cmedper in different parts of Cnaphalocrocis medinalis were significantly different (F 3,8 =19.41, P<0.001). Among them, the expression level of Cmedper was significantly higher in the head and significantly lower in the abdomen. The relative expression level of Cmedper in the head and antennae was significantly higher than that in the chest and abdomen ( Figure 5 ), which is consistent with the results that the biological clock operates in the head (Patke et al., 2020).

[0117] 2.3 Effects of adult nutrition and temperature on the periodic expression of circadian clock genes

[0118] Cmedper was oscillatory in C. medinalis regardless of hunger (hunger: F 5,17 =8.185, P = 0.001, R 2 =0.872, Nutritional supplement: F 5,17 =25.917, P<0.001, R 2 =0.999), while maintaining peak-to-valley synchronization ( Figure 6 A). However, starvation has a significant effect on the biological clock. At ZT8 and ZT12, the relative expression of Cmedper under nutritional supplementation was significantly upregulated (F 1,24 =19.71, P<0.001)( Figure 6A). The results showed that starvation of Cnaphalocrocis medinalis adults did not affect the periodicity and peak-trough timing of Cmedper expression, but its expression increased significantly during the transition from light to dark.

[0119] The expression of Cmedper at 16°C was lower than that of the control group (26°C) at all times, and the rhythmic oscillation disappeared. The peak expression was detected at ZT8, and the lowest point was at ZT0. There was no significant difference in the expression of Cmedper within 24 hours (F 5,17 =3.070, P = 0.52), indicating that Cmedper lost its rhythmic expression under low temperature ( Figure 6 B). At 36°C, the rhythmic expression of Cmedper still operates normally, and the expression pattern is similar to that at 26°C (F 5,17 =4.421, P=0.016, R 2 =0.0883)( Figure 6 B). The results showed that the expression of Cmedper in rice leaf folder lost its oscillation under low temperature and its expression was significantly reduced under low temperature.

[0120] The above results show that the periodic expression of Cmedper is affected by the nutrition and temperature of the adults, especially the changes at the time of day and night. Both the nutrition and temperature of the adults are environmental factors that affect the migration of the rice leaf folder, and the migration of the rice leaf folder occurs at sunset, which means that Cmedper may be related to the migration behavior of the rice leaf folder.

[0121] 2.4 Differences in takeoff ratio and Cmedper expression between migratory and resident morphs

[0122] At 3 days old, after the take-off period began, the expression of Cmedper in migratory individuals slowly increased, and by 20:00, it showed a significant difference from the resident type ( Figure 7 Whether or not the insect takes off has a significant effect on the expression of Cmedper, which indicates that PER may be involved in the formation of the migration intention of Cnaphalocrocis medinalis.

[0123] 2.5 Effects of interfering with Cmedper on take-off behavior and expression of clock-related genes in Cnaphalocrocis medinalis

[0124] After injection of dsPER, the expression of Cmedper in 2-day-old (t=9.733, df=4, P<0.001) and 3-day-old (t=9.630, df=4, P<0.001) rice leaf folders decreased significantly by 75% and 25% ( Figure 8 In the take-off experiment, after interfering with the expression of Cmedper, the take-off rate of the rice leaf folder decreased significantly ( Fig. 9A). After interfering with Cmedper, the expression of Cmedtim also decreased, significantly decreasing by 50% and 20% at 2 days and 3 days, respectively (2 days: t = 9.094, df = 4, P = 0.0008; 3 days: t = 6.657, df = 4, P = 0.0026) ( Fig. 9 B), indicating that the expression of Cmedper and Cmedtim in rice leaf folder is synchronous. However, no significant difference was found in the expression of cryptochrome ( Fig. 9 B), indicating that abnormal biological clock does not affect the expression of Cmedcry1 (2 days old: t = 0.05306, df = 4, P = 0.9602; 3 days old: t = 1.729, df = 4, P = 0.1588).

[0125] 2.6 Effects of Cmedper interference on ovarian development and juvenile hormone expression in Cnaphalocrocis medinalis

[0126] The results of ovarian dissection at 3 days old showed that the ovaries of Cnaphalocrocis medinalis treated with dsPER were mainly developed at level III (11 / 15), while the ovaries of the control group (dsGFP) were mostly at level II, with only a small number developing to level III (4 / 15). The results showed that there was a significant difference in the ovarian development level between the two groups (W=63.5, P=0.02399) ( Fig. 9 C). After interfering with Cmedper, it was found that the ovarian development of the rice leaf folder was significantly accelerated.

[0127] Considering that the ovarian development of rice leaf folder is related to juvenile hormone, the expression difference of genes related to juvenile hormone regulation after interfering with Cmedper was determined to verify the results of ovarian anatomy. After interfering with Cmedper, the relative expression of Cmedjhamt at 2 days old was significantly upregulated, and the expression returned to normal at 3 days old (2 days old: t = 6.318, df = 4, P = 0.0032; 3 days old: t = 0.7508, df = 4, P = 0.4945). On the contrary, the relative expression of Cmedjhe decreased significantly at 2 days old, and it also increased at 3 days old as the interference efficiency decreased ( Fig. 9 D) (2 days old: t = 8.139, df = 4, P = 0.0012; 3 days old: t = 1.495, df = 4, P = 0.2093). The expression of the Cmedjhe gene (juvenile hormone esterase), which is involved in inhibiting the secretion of juvenile hormone, was significantly downregulated. The expression of Cmedjhamt (juvenile hormone acid methyltransferase), a key regulatory enzyme involved in the last pathway of the juvenile hormone synthesis pathway, was significantly increased. It is speculated that interfering with the expression of Cmedper will promote the secretion of juvenile hormone to promote the development of the insect reproductive system. This shows that after disrupting the operation of the biological clock, the rice leaf folder tends to stay in the habitat, give up migration, and turn to the occurrence of reproductive behavior.

Claims

1. A gene that regulates the migration and reproductive behavior of rice leaf folder, characterized in that The nucleotide sequence of the gene is: 1) the nucleotide sequence shown in SEQ ID No. 1; or 2) The nucleotide sequence shown in SEQ ID No. 1 is replaced by one or several nucleotides to obtain a nucleotide sequence encoding Period.

2. Use of the gene as claimed in claim 1 in regulating the migration and reproductive behavior of rice leaf folder.

3. Use of the gene as claimed in claim 1 in monitoring damage caused by rice leaf folder.

4. A use of a Period gene or its encoded protein as a target in the preparation of a preparation for regulating the migration and reproductive behavior of rice leaf folder, characterized in that The nucleotide sequence of the Period gene is: 1) the nucleotide sequence shown in SEQ ID No. 1; or 2) The nucleotide sequence shown in SEQ ID No. 1 is substituted with one or more nucleotides to obtain a nucleotide sequence encoding Period; The amino acid sequence of the encoded protein is: 1) the amino acid sequence shown in SEQ ID No. 2; or 2) An amino acid sequence with equivalent function formed by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.

2.

5. A dsRNA obtained by transcribing the gene template according to claim 1.

6. Use of the dsRNA according to claim 5 in regulating the migration and reproductive behavior of rice leaf folder.

7. Use of the dsRNA according to claim 5 in monitoring damage caused by rice leaf folder.

8. A kit for regulating the migration and reproductive behavior of rice leaf folder, characterized in that Containing the dsRNA prepared according to claim 5.

9. A method for regulating the migration and reproductive behavior of rice leaf folder, characterized in that The dsRNA prepared according to claim 5 is used to treat rice leaf folder.

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