Internal reference gene of Cephalopoda spp. and its detection primers and applications

Abstract

The present invention discloses internal reference genes for the grain borer, as well as detection primers and applications thereof. Using the grain borer as the experimental material, the present invention utilizes RT-qPCR technology combined with ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder internal reference gene stability analysis software to screen for internal reference genes that are stably expressed in the grain borer under different growth and development stages, temperatures, starvation treatments, and sexes, as well as under all of the aforementioned experimental conditions. This provides a reference for further research on key functional genes in the grain borer and an effective calibration tool for studies of gene differential expression.

Description

Technical Field

[0001] The present invention relates to the field of insect molecular biology, and more specifically to an internal reference gene of a grain borer, a detection primer thereof, and an application thereof. Background Art

[0002] The grain borer, Rhyzopertha dominica (Fabricius), belongs to the family Bostrichidae in the order Coleoptera. It is a significant stored-grain borer. Both larvae and adults can infest grains, posing a threat to my country's grain storage security. It is distributed worldwide, with the most severe outbreaks occurring in tropical and subtropical regions, primarily in Australia, Oceania, India, the Arabian Peninsula, the Yangtze River Basin, and the humid and hot regions of southern my country. Its diet is complex, primarily consisting of rice, wheat, corn, sorghum, beans, medicinal herbs, dried fruits, books, furs, and leather products. It can also bore into wood, where it prefers to hide, metamorphose, and overwinter, severely damaging the wooden structure of warehouses. It is one of the world's most important stored-grain pests.

[0003] With the advancement of molecular biology techniques, research on the grain borer has expanded beyond morphological characteristics, control measures, and traditional biological studies to include molecular biology studies of its essence. Real-time quantitative polymerase chain reaction (RT-qPCR) is a highly efficient method for measuring gene expression and is currently widely used in disease diagnosis, gene function identification, and temporal and spatial gene expression profiling. This method offers advantages such as low cost, ease of use, accurate quantification, reproducibility, and high sensitivity. The use of RT-qPCR requires the availability of stably expressed reference genes for calibration and normalization to improve quantitative accuracy. Commonly used reference genes are genes encoding proteins essential for maintaining basic cellular activities. Ideally, reference genes should be relatively consistently expressed across insect developmental stages, treatments, and sexes. However, studies have shown that in real-world experiments, no reference genes consistently express under all conditions. Direct use of unselected reference genes can lead to biased experimental data and compromise the reliability of target gene expression results. Therefore, it is important to select reference genes with relatively stable expression based on specific experimental conditions. So far, there has been no report on stable internal reference genes corresponding to the grain borer under different experimental conditions, nor has there been any work on functional gene verification of the grain borer-related genes. Summary of the Invention

[0004] In response to the above-mentioned problems existing in the prior art, one object of the present invention is to screen out internal reference genes of the grain borer that can be expressed relatively stably, and provide the application of the above-mentioned internal reference genes in fluorescence quantitative analysis under conditions such as different developmental stages, different treatments and / or different sexes of the grain borer.

[0005] Another object of the present invention is to provide detection primers for the above-mentioned internal reference gene.

[0006] Another object of the present invention is to provide a fluorescence quantitative kit for the grain borer using the above-mentioned internal reference gene and detection primers.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an internal reference gene for P. glutinosus, wherein the internal reference gene for P. glutinosus comprises at least one of the following (1)-(6):

[0009] (1) Rps3 gene of Ceropegia glutinosus, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0010] (2) Rps6 gene of Ceropegia glutinosus, the nucleotide sequence of which is shown in SEQ ID NO. 2;

[0011] (3) Rps13 gene of Ceroplastes glutinis, the nucleotide sequence of which is shown in SEQ ID NO. 3;

[0012] (4) Agromyces actin gene, the nucleotide sequence of which is shown in SEQ ID NO. 4;

[0013] (5) the gadph gene of the grain borer, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0014] (6) Tubulin gene of Ceropegia glutinis, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0015] In a second aspect, the present invention provides an application of the above-mentioned internal reference gene of the grain borer for fluorescence quantitative analysis under conditions such as different developmental stages, different treatments and / or different sexes of the grain borer, specifically:

[0016] Application of at least one of the rice borer rps3 gene, rice borer rps6 gene and rice borer rps13 gene as an internal reference gene in fluorescence quantitative analysis of rice borer at different growth and development stages;

[0017] Preferably, the rice borer rps3 gene and the rice borer rps6 gene are used together as internal reference genes in the fluorescence quantitative analysis of rice borer at different growth and development stages.

[0018] Application of at least one of the rice borer rps3 gene, rice borer rps6 gene, rice borer rps13 gene, rice borer actin gene, rice borer gadph gene, rice borer tubulin gene and rice borer 18S rRNA gene as an internal reference gene in fluorescence quantitative analysis of rice borer under different temperature conditions;

[0019] Preferably, the rice borer rps13 gene and the rice borer 18S rRNA gene are used together as internal reference genes in the fluorescence quantitative analysis of rice borer under different temperature conditions.

[0020] Use of at least one of the rice borer rps3 gene, rice borer rps6 gene, rice borer rps13 gene, rice borer actin gene, rice borer gadph gene, rice borer tubulin gene and rice borer 18S rRNA gene as an internal reference gene in fluorescence quantitative analysis of rice borer under starvation conditions;

[0021] Preferably, the rice borer rps6 gene and the rice borer gadph gene are used together as internal reference genes in the fluorescence quantitative analysis of the rice borer under starvation conditions.

[0022] Application of at least one of the following genes: rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA gene of the grain borer as an internal reference gene in fluorescence quantitative analysis of different sex conditions of the grain borer;

[0023] Preferably, the rice borer rps6 gene and the rice borer actin gene are used together as internal reference genes in the fluorescence quantitative analysis of different sex conditions of rice borers.

[0024] The application of the rice borer rps3 gene and / or the rice borer rps13 gene as internal reference genes in the fluorescence quantitative analysis of the rice borer in different growth and development stages, different temperature conditions, starvation conditions and different sex conditions, that is, the rice borer rps6 gene and / or the rice borer rps13 gene can be used alone or together as internal reference genes for all the above experimental conditions (different growth and development stages, different temperature conditions, starvation conditions and different sex conditions).

[0025] In a third aspect, the present invention also provides detection primers for the internal reference genes of the grain borer. The detection primers are based on the sequences of the above-mentioned internal reference genes and are designed according to the principles of fluorescent quantitative PCR primer design to obtain detection primers for each internal reference gene.

[0026] According to a specific embodiment of the present invention, the nucleotide sequences of the detection primers for the rps3 gene of the grain borer are shown as SEQ ID NO.7 and SEQ ID NO.8; the nucleotide sequences of the detection primers for the rps6 gene of the grain borer are shown as SEQ ID NO.9 and SEQ ID NO.10; the nucleotide sequences of the detection primers for the rps13 gene of the grain borer are shown as SEQ ID NO.11 and SEQ ID NO.12; the nucleotide sequences of the detection primers for the actin gene of the grain borer are shown as SEQ ID NO.13 and SEQ ID NO.14; the nucleotide sequences of the detection primers for the gadph gene of the grain borer are shown as SEQ ID NO.15 and SEQ ID NO.16; the nucleotide sequences of the detection primers for the tubulin gene of the grain borer are shown as SEQ ID NO.17 and SEQ ID NO.18.

[0027] In a fourth aspect, the present invention provides the use of the above-mentioned rice borer internal reference gene or the above-mentioned detection primer in the preparation of a rice borer fluorescence quantitative kit, and provides a rice borer fluorescence quantitative kit, the kit comprising at least one of the above-mentioned detection primers;

[0028] Furthermore, the kit may also include detection primers for the 18S rRNA gene of the grain borer, the nucleotide sequences of which are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0029] Furthermore, the kit also includes reagents required for fluorescent quantitative RT-qPCR reaction.

[0030] The beneficial effects of the present invention are as follows:

[0031] The lack of effective, stably expressed reference genes in the prior art has greatly limited functional research on the major stored-food pest, the grain borer. To identify reference genes that are stably expressed under all experimental conditions, including different growth and development stages, temperatures, starvation treatments, and sex, the present invention analyzed the expression stability of seven candidate reference genes for the grain borer (rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA) using RT-qPCR combined with five reference gene stability analysis software (ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder). Using geNorm software, we determined that two reference genes—the rps3 and rps13 genes—were suitable for all five experimental conditions. Based on the analysis results of the five softwares, the present invention screened out reference genes with relatively stable expression at different growth and development stages, relatively stable expression at different temperature conditions, relatively stable expression at starvation conditions, relatively stable expression at different sex conditions, and internal reference genes with stable expression across all experimental conditions (different growth and development stages, different temperature conditions, starvation conditions, and different sex conditions). The present invention screened out relatively stable expression reference genes, which can provide a reference for further research on key functional genes of the grain borer and an effective calibration tool for gene differential expression research. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0033] Figure 1 The dissolution curves of candidate internal reference genes are shown.

[0034] Figure 2 The graph shows the cycle threshold (Ct) value analysis of 7 candidate reference genes in all grain borer samples.

[0035] Figure 3 The stability ranking diagram of seven candidate internal reference genes analyzed by RefFinder is shown.

[0036] Figure 4 The figure shows the geNorm analysis of the V values ​​of the seven candidate internal reference genes. From left to right, each cluster of histograms is: V2 / V3, V3 / V4, V4 / V5, V5 / V6, and V6 / V7. DETAILED DESCRIPTION

[0037] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and drawings. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0038] Example 1 Screening of internal reference genes and primer design

[0039] 1. Sources

[0040] Grain borers were collected from Guangdong Province in 2016 and reared in a laboratory-grown wheat-based incubator (Memmert, Germany) at a constant temperature and humidity of 27°C and 65% relative humidity. Day-old eggs of the grain borer were collected, observed under a microscope, and impurities removed with a brush to obtain pure eggs. A small amount of flour was added to the collected eggs and placed in an incubator for approximately 10 days to obtain first-instar larvae.

[0041] The samples at different growth and development stages included about 300 grain borer eggs, 200 first-instar larvae within 3 days of hatching, 20 fourth-instar larvae, 5 pupae and 5 adults;

[0042] For samples at different temperatures, adults of the grain borer were placed at 5, 15, 27, 35, and 40°C for 48 hours and then taken out;

[0043] For samples of different sexes, the male and female grain borer pupae were collected and then separated into male and female. After the pupae emerged, 5 female and 5 male grain borers of the same age were collected.

[0044] For starvation stress treatment samples, adults of the same age were placed in glass bottles without any food, and 5 adults were taken out as a sample at 24h, 72h and 120h.

[0045] The samples of D. gracilis were quickly frozen in liquid nitrogen and then stored in an 80°C ultra-low temperature freezer until RNA extraction. Three biological replicates were performed for each sample.

[0046] 2. Internal reference gene screening and primer design

[0047] Based on the transcriptome data of the grain borer, alignment was performed based on databases such as NT, NR, and Swissprot to obtain annotation information of the full-length transcripts. The high-quality full-length transcripts after de-redundancy were predicted using TransDecoder software, and six genes were selected as candidate internal reference genes, namely ribosomal protein (rps3, nucleotide sequence shown in SEQ ID NO.1), ribosomal protein (rps6, nucleotide sequence shown in SEQ ID NO.2), ribosomal protein (rps13, nucleotide sequence shown in SEQ ID NO.3), actin (actin, nucleotide sequence shown in SEQ ID NO.4), glyceraldehyde-3-phosphate dehydrogenase (gadph, nucleotide sequence shown in SEQ ID NO.5), and tubulin (tubulin, nucleotide sequence shown in SEQ ID NO.6). The 18S ribosomal RNA (18S rRNA, GenBank accession number KP419272.1) reported as an internal reference gene for the grain borer was combined with the 18S rRNA. Primer Premier 5 software was used to design RT-qPCR primers for candidate internal reference genes (Table 1). To investigate the amplification efficiency of the designed primers for the candidate genes, a standard curve for each gene was calculated by serial dilution of the samples.

[0048] Table 1 Primer pair sequences of internal reference genes of Ceropegia glutinosus

[0049]

[0050] 3. RNA Extraction and cDNA Reconversion

[0051] According to the kit instructions, use Total RNA from all samples was extracted using the Qiagen Plus Mini Kit (Qiagen, Germany). The purity and concentration of all RNA samples were determined spectrophotometrically. First-strand cDNA was then synthesized using the HiScript III RTSuperMix for qPCR (with gDNA wiper) reverse transcription kit (Novozymes, China) according to the manufacturer's instructions. Prior to use in RT-qPCR reactions, cDNA samples were diluted 100-fold with sterile water and stored at 20°C.

[0052] 4. RT-qPCR Reaction Procedure

[0053] RT-qPCR reactions were prepared using ChamQ Universal SYBR qPCR Master Mix (Novozymes, China). A 20 μL reaction system consisted of 10 μL 2× ChamQ Universal SYBR qPCR Master Mix, 5 μL 1:100 diluted cDNA, 0.4 μL forward and reverse primers (10 μM), and 4.2 μL RNase-free ddH2O. RT-qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The initial denaturation step was 95°C for 30 s. The cycling program consisted of 40 cycles of 95°C for 10 s and 60°C for 30 s. After the amplification reaction, product specificity was assessed using a melting curve. After cycling, a melting curve was generated at 55°C and 95°C, with fluorescence signals collected at 0.5°C increments for 5 s. After the completion of the quantitative PCR, Ct values ​​for the seven candidate internal reference genes were obtained.

[0054] 5. Data Processing and Analysis

[0055] The expression levels of all candidate internal reference genes were assessed using gene cycle threshold (Ct) values ​​generated by Bio-Rad CFX Manager 3.1 software (Bio-Rad, USA). Ct values ​​for each gene in different samples were obtained by RT-qPCR, and expression stability was assessed using five evaluation methods: ΔCt, geNorm, BestKeeper, NormFinder, and RefFinder. The ΔCt method estimates the relative expression of gene pairs in each sample, and reference genes with lower standard deviation (SD) values ​​are more stable. geNorm (v3.0) calculates a stability value (M) using the mean squared deviation of each gene relative to all other genes. Genes with lower M values ​​are more stable, while genes with M values ​​>1.5 are not suitable as stable internal reference genes. BestKeeper uses Ct values ​​directly for calculations and primarily assesses the stability of candidate internal reference genes through the standard deviation (SD) coefficient of variation. Gene expression is more stable when SD < 1 and the SD value is the lowest. Normfinder evaluates the stability value (SV) of candidate reference gene expression by calculating the changes within and between different sample groups. RefFinder (https: / / www.heartcure.com.au / reffinder) integrates ΔCt, BestKeeper, Normfinder, and geNorm4 algorithms to evaluate the stability of reference genes. The gene with the lowest weighted geometric mean is the most stable. RefFinder is used to sort the results of the above four analysis methods. n / V (n+1) It can be used to determine the optimal number of internal reference genes. n / V (n+1) When ρ<0.15, the optimal number of internal reference genes is n.

[0056] 6. Results Analysis

[0057] 6.1 Evaluation of primer specificity and qRT-PCR amplification efficiency

[0058] In this application, seven genes (rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA) were selected as candidate internal reference genes, and the length of the amplified products was 100-200 bp. Samples of grain borers at different developmental stages (eggs, 1st instar larvae, 4th instar larvae, pupae, and adults), grain borers treated at different temperatures (5°C, 15°C, 27°C, 35°C, and 40°C), grain borers of different sexes (female and male), and grain borers starved (non-starved and starved) were used as templates to amplify the candidate internal reference genes. The specificity of the primers was evaluated by RT qPCR melting curves. The results showed that all target internal reference genes had specific single peaks, that is, all primers had good specificity and could be used for RT qPCR gene expression level analysis ( Figure 1 According to the results of the standard curve, the gene with the lowest amplification efficiency was 18S rRNA (96.66%), and the highest was tubulin (103.36%). The regression coefficients (R 2 ) ranged from 0.9887 to 0.9997 (p<0.05). The results showed that the seven candidate primer pairs were stable and specific (Table 1).

[0059] 6.2 Analysis of cycle threshold (Ct) values ​​of candidate reference genes in all samples

[0060] The expression levels of seven candidate reference genes in different insect samples were evaluated by comparing Ct values. The analysis results showed that the Ct values ​​in all samples (294) ranged from 9.23 to 33.40 ( Figure 2 Tubulin had an average Ct value of 27.53 ± 2.00, showing low expression across all experimental samples, while 18S rRNA had an average Ct value of 11.61 ± 2.07, showing the highest expression among all candidate reference genes. The expression levels of each gene under different conditions indicated that these genes generally remained stable under these conditions.

[0061] 6.3 Stability assessment of the internal reference gene of P. gracilis under different conditions

[0062] 6.3.1 Stability Assessment of Reference Genes in Different Growth and Development Stages of P. glutinosus

[0063] For the five growth and development stages of grain borers, namely eggs, 1st instar larvae, 4th instar larvae, pupae and adults, the Ct values ​​were further analyzed using five softwares, namely ΔCt, geNorm, BestKeeper, NormFinder and RefFinder. Among them, the ranking results of ΔCt and geNorm were similar. According to geNorm analysis, the M values ​​of the seven genes were all less than 1.5, which showed good stability. The BestKeeper analysis showed that the rps3, rps13 and rps6 genes were stably expressed, but the stability values ​​of actin, 18SrRNA, gadph and tubulin were greater than 1, which were not suitable as internal reference genes. The rps3 and rps6 genes were also highly stable in the NormFinder analysis (Table 2). Finally, RefFinder was used to calculate the analysis results of the above four software for comprehensive ranking: rps3>rps6>rps13>gadph>actin>18S rRNA>tubulin( Figure 3 Therefore, the rps3, rps6, and rps13 genes can be used as reference genes for the stable expression of P. glutinosa at different growth and development stages based on the results of the five algorithms, and the order is rps3>rps6>rps13.

[0064] Table 2 Stability of internal reference genes of P. glutinosus under different conditions

[0065]

[0066]

[0067] 6.3.2 Stability Assessment of Reference Genes in P. gracilis Treated at Different Temperatures

[0068] The effect of temperature on the stability of the internal reference genes of the grain borer was studied by treating them at 5℃, 15℃, 27℃, 35℃ and 40℃. According to the analysis results (Table 2), the ΔCt and NormFinder ranking results were similar, and the top three internal reference genes were rps13, actin and 18S rRNA. According to the geNorm analysis, the M values ​​of the 7 genes were all less than 1.5, and the stability values ​​of the BestKeeper analysis were all less than 1, indicating that the 7 genes had good stability under different temperature conditions. The analysis results of the above four software were calculated using RefFinder for comprehensive ranking: 18SrRNA>rps13>rps3>actin>rps6>tubulin>gadph( Figure 3Therefore, the comprehensive analysis of the five algorithms showed that rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA could be used as reference genes for the stable expression of P. glutinosa under different temperature treatments, and the order was 18S rRNA>rps13>rps3>actin>rps6>tubulin>gadph.

[0069] 6.3.3 Stability Assessment of Reference Genes in Different Sexes of P. gracilis

[0070] To investigate the effect of sex on the stability of the internal reference genes of the grain borer, female and male adults of the same age were selected for analysis (Table 2). The M values ​​of the seven candidate genes in the geNorm calculation results were all lower than 1.5, and the stability values ​​of the BestKeeper analysis were all less than 1, indicating that the seven genes had good stability under different sex conditions. The analysis results of the above four software were comprehensively ranked according to RefFinder: rps6>actin>rps13>tubulin>rps3>gadph>18S rRNA ( Figure 3 C in the figure). Therefore, based on the comprehensive analysis of the five algorithms, rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA can all be used as stable reference genes in different experiments on the sex of grain borer, and the order is rps6>actin>rps13>tubulin>rps3>gadph>18S rRNA.

[0071] 6.3.4 Stability Assessment of Reference Genes in P. glutinosus Under Starvation Conditions

[0072] The effect of starvation treatment on the stability of the internal reference genes of the grain borer was analyzed by non-starvation and starvation treatment. According to the analysis results (Table 2), there were certain differences in the ΔCt, geNorm, BestKeeper and NormFinder rankings, but the M values ​​of the 7 genes according to geNorm analysis were all less than 1.5, and the stability values ​​of BestKeeper analysis were all less than 1, indicating that the 7 genes had good stability under different temperature conditions. RefFinder was used to calculate the comprehensive ranking of the analysis results of the above four software: rps6>gadph>actin>rps3>rps13>tubulin>18S rRNA ( Figure 3 D in the figure). Therefore, the seven genes from the five algorithms can be used as reference genes for the stable expression of P. glutinosus under different temperature treatments, and the order is rps6>gadph>actin>rps3>rps13>tubulin>18S rRNA.

[0073] 6.3.5 Stability Assessment of Reference Genes under All Experimental Conditions

[0074] For all samples under the above four conditions, rps6, rps3, and rps13 are the top three genes in terms of stability in the ΔCt, GeNorm, and BestKeeper analysis results. The geNorm results show that the M values ​​of the seven genes are all less than 1.5, but in the BestKeeper calculation results, only rps3 and rps13 have stability values ​​less than 1, making them suitable as internal reference genes. The stability order of RefFinder is: rps6>rps3>rps13>actin>gadph>18S rRNA>tubulin ( Figure 3 Therefore, based on the five algorithms, rps3 and rps13 can be used as internal reference genes for all experimental conditions, and rps3>rps13.

[0075] 6.4 Analysis of the number of most suitable internal reference genes

[0076] The geNorm software was used to analyze the number of the most suitable reference genes under different experimental conditions for the seven reference genes, and the pairwise variation V value of the normalization factor after the introduction of a new gene was calculated. n / V n+1 The value of V is used to determine the optimal number of internal references required. 2 / 3 The ratio is less than the recommended value of 0.15 ( Figure 4 ), indicating that the optimal number of internal reference genes is always 2. Therefore, in experiments on different growth and development stages of P. granarium, the rps3 and rps6 genes can be used as internal reference genes; in experiments on different temperature treatments, the 18S rRNA and rps13 genes can be used as internal reference genes; in experiments on different sexes, rps6 and actin can be used as internal reference genes; in starvation experiments, rps6 and gadph can be used as internal reference genes; and in all experimental conditions, rps3 and rps13 can be used as internal reference genes.

[0077] In summary, in order to standardize RTqPCR gene expression data, the present invention attempts for the first time to screen a group of candidate internal reference genes with high stability in different developmental stages, different temperature conditions, different sexes, starvation conditions and all experimental conditions of the grain borer. Based on the comprehensive analysis of ΔCt, geNorm, BestKeeper, NormFinder and RefFinder, at least one of the rps3, rps6 and rps13 genes can be selected in experiments on different growth and development stages of the grain borer, and rps3 and rps6 genes are recommended. In experiments on different temperature treatments, at least one of the rps3, rps6, rps13, actin, gadph, tubulin and 18S rRNA genes can be selected as internal reference genes for the grain borer, and 18S rRNA and rps13 genes are recommended. In experiments on different sexes, at least one of the rps3, rps6, rps13, actin, gadph, tubulin and 18S rRNA genes can be selected as internal reference genes for the grain borer, and rps6 and actin are recommended. In experiments on starvation conditions, rps3, rps6, rps13, actin, gadph, tubulin and 18S rRNA genes can be selected. Any two rRNA genes are used as internal reference genes for the grain borer, with rps6 and gadph being recommended. Under all experimental conditions, rps3 and rps13 are used in combination as internal reference genes. This invention lays the foundation for accurate gene expression analysis of the grain borer in the future.

[0078] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Grain borer rps3 Gene, Cephalopod rps6 Genes and Cereal Borers rps13 The use of at least one of the genes as an internal reference gene in fluorescence quantitative analysis of different growth and development stages of grain borer is characterized in that: The grain borer rps3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The grain borer rps6 The nucleotide sequence of the gene is shown in SEQ ID NO. 2; The grain borer rps13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The grain borer rps3 Genes and Cereal Borers rps6 The application of these genes as internal reference genes in fluorescence quantitative analysis of grain borer at different growth and development stages.

3. Grain borer rps3 Gene, Cephalopod rps6 Genes and Cereal Borers rps13 The use of at least one of the genes as an internal reference gene in fluorescence quantitative analysis of rice borer under different temperature conditions is characterized in that: The grain borer rps3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The grain borer rps6 The nucleotide sequence of the gene is shown in SEQ ID NO. 2; The grain borer rps13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that The grain borer rps13 Genes and Cereal Borers 18S rRNA The application of these genes as internal reference genes in fluorescence quantitative analysis of grain borer under different temperature conditions.

5. Grain borer rps3 Gene, Cephalopod rps6 Genes and Cereal Borers rps13 The use of at least one of the genes as an internal reference gene in fluorescence quantitative analysis of starvation conditions of grain borer is characterized in that: The grain borer rps3 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; The grain borer rps6 The nucleotide sequence of the gene is shown in SEQ ID NO. 2; The grain borer rps13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that The grain borer rps6 Genes and Cereal Borers gadph The application of the genes as internal reference genes in the quantitative fluorescence analysis of the starvation condition of the grain borer, the grain borer gadph The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

7. Grain borer rps3 Gene, Cephalopod rps6 Gene, Cephalopod rps13 The use of at least one of the genes as an internal reference gene in fluorescence quantitative analysis of different sex conditions of grain borer is characterized in that: The grain borer rps3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The grain borer rps6 The nucleotide sequence of the gene is shown in SEQ ID NO. 2; The grain borer rps13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

8. The use according to claim 7, characterized in that The grain borer rps6 Genes and Cereal Borers actin The application of the genes as internal reference genes in the fluorescence quantitative analysis of different sex conditions of the grain borer, the grain borer actin The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

9. Grain borer rps3 Genes and / or grain borers rps13 The application of the gene as an internal reference gene in fluorescence quantitative analysis of grain borer at different growth and development stages, different temperature conditions, starvation conditions and different sex conditions is characterized in that: The grain borer rps3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The grain borer rps13 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

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