Reference gene combination stably expressed in Hyphantria cunea (Drury) under different conditions and its application

Abstract

The application discloses a combination of reference genes of Dendrolimus punctatus Walker stably expressed under different conditions and application thereof. The application screens and obtains reference genes of Dendrolimus punctatus Walker stably expressed under different development stages, different host feeding conditions, different temperature conditions, starvation conditions, different mating states and different tissues, which are suitable for correction of RT-qPCR detection results of Dendrolimus punctatus Walker under different experimental conditions. Meanwhile, the application also provides amplification primers for detecting the reference genes of Dendrolimus punctatus Walker, which are high in specificity and amplification efficiency. The application lays a preliminary foundation for quantification of related functional genes of Dendrolimus punctatus Walker under different experimental conditions and related molecular biology research.

Description

Technical Field

[0001] This invention relates to the technical field of insect molecular biology, specifically to the combination of internal reference genes stably expressed under different conditions by the fig clearwing moth and its application. Background Technology

[0002] Real-time quantitative PCR (RT-qPCR) is a highly efficient molecular method for analyzing target gene expression profiles. Due to its high sensitivity, reliability, and specificity, RT-qPCR is widely used in research on disease diagnosis, gene function identification, and spatiotemporal gene expression characteristics. Before using RT-qPCR, it is necessary to obtain stably expressed internal reference genes for calibration and homogenization to improve the accuracy of PCR quantification results, including sample quantity, RNA quality and quantity, and PCR efficiency.

[0003] Some internal reference genes, including 18S ribosomal RNA (18S rRNA), actin, arginine kinase (AK), ribosomal proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin (TUB), and elongation factor (EEF-1α), are often classified as housekeeping genes (HKGs). However, studies have shown that there is currently no universally applicable internal reference gene that can be expressed relatively consistently under different experimental conditions, such as developmental stages, different treatment stages, and sexual stages. In non-model organisms, due to limited genetic and genomic information, the identification of internal reference genes is usually based on the homologous sequences of widely recognized housekeeping genes in model insect species. However, misusing commonly used housekeeping genes as internal reference genes in non-model insects can also lead to errors or mistakes. Therefore, the screening of internal reference genes in a specific species must be conducted under specific experimental conditions.

[0004] The fig clearwing moth, belonging to the order Lepidoptera and family Lymantriidae, is a significant deciduous pest in Southeast Asia and southern East Asia, primarily damaging banyan trees (Ficus spp.). Research on the fig clearwing moth mainly focuses on pest surveys, prediction of potential distribution areas, host selection, integrated pest management, and the study of chemical communication substances. With the development of molecular biology techniques, research on the fig clearwing moth will gradually expand into the field of molecular biology, aiming to reveal the nature of its life cycle. Current reports do not include the evaluation of the internal reference genes of the fig clearwing moth, which limits research on its gene expression. Summary of the Invention

[0005] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a combination of internal reference genes stably expressed by the fig clearwing moth under different conditions and their applications.

[0006] The first objective of this invention is to provide a combination of internal reference genes.

[0007] A second objective of this invention is to provide a composition of amplification primers for internal reference genes RPS3, TBP, and 18S rRNA.

[0008] A third objective of this invention is to provide a composition of amplification primers for the internal reference genes RPL28 and RPS3.

[0009] A fourth objective of this invention is to provide a composition of amplification primers for the internal reference genes RPL13 and BACT.

[0010] A fifth objective of this invention is to provide a composition of amplification primers for the internal reference gene 25S rRNA and BACT.

[0011] A sixth objective of this invention is to provide a composition of amplification primers for the internal reference genes RPL28, RPS3, and TBP.

[0012] A seventh objective of this invention is to provide a composition of amplification primers for the internal reference genes TUB, 18S rRNA, and GAPDH.

[0013] An eighth object of the present invention is to provide any of the above-described compositions for use in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing moth or in the fluorescent PCR quantitative detection of the fig clearwing moth.

[0014] The ninth objective of this invention is to provide a fluorescent PCR quantitative detection kit.

[0015] The tenth objective of this invention is to provide a method for quantitative detection of the fig clearwing moth using fluorescent PCR.

[0016] Therefore, the present invention claims protection for the following:

[0017] An internal reference gene combination, wherein the internal reference gene combination is any one of the following combinations:

[0018] RPS3, TBP, and 18S rRNA;

[0019] RPL28 and RPS3;

[0020] RPL13 and BACT;

[0021] 25S rRNA and BACT;

[0022] RPL28, RPS3, and TBP;

[0023] TUB, 18S rRNA, and GAPDH.

[0024] A composition of amplification primers for internal reference genes RPS3, TBP and 18S rRNA, wherein the nucleotide sequence of the amplification primer for RPS3 is shown in SEQ ID NO: 15-16;

[0025] The nucleotide sequences of the TBP amplification primers are shown in SEQ ID NO: 23-24;

[0026] The nucleotide sequences of the amplification primers for the 18S rRNA are shown in SEQ ID NO: 17-18.

[0027] A composition of amplification primers for internal reference genes RPL28 and RPS3, wherein the nucleotide sequence of the amplification primer for RPL28 is shown in SEQ ID NO: 13-14;

[0028] The nucleotide sequences of the amplification primers for RPS3 are shown in SEQ ID NO: 15-16.

[0029] A composition of amplification primers for internal reference genes RPL13 and BACT, wherein the nucleotide sequence of the amplification primers for RPL13 is shown in SEQ ID NO: 11-12;

[0030] The nucleotide sequences of the amplification primers for BACT are shown in SEQ ID NO: 3-4.

[0031] A composition of amplification primers for the internal reference gene 25S rRNA and BACT, wherein the nucleotide sequence of the amplification primers for the 25S rRNA is shown in SEQ ID NO: 19-20;

[0032] The nucleotide sequences of the amplification primers for BACT are shown in SEQ ID NO: 3-4.

[0033] A composition of amplification primers for internal reference genes RPL28, RPS3 and TBP, wherein the nucleotide sequence of the amplification primer for RPL28 is shown in SEQ ID NO: 13-14;

[0034] The nucleotide sequences of the amplification primers for RPS3 are shown in SEQ ID NO: 15-16;

[0035] The nucleotide sequences of the amplification primers for the TBP are shown in SEQ ID NO: 23-24.

[0036] A composition of amplification primers for the internal reference gene TUB, 18S rRNA and GAPDH, wherein the nucleotide sequence of the amplification primer for TUB is shown in SEQ ID NO: 21-22;

[0037] The nucleotide sequences of the amplification primers for the 18S rRNA are shown in SEQ ID NO: 17-18;

[0038] The nucleotide sequences of the GAPDH amplification primers are shown in SEQ ID NO: 9-10.

[0039] Application of any of the above compositions in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing tussock moth or in the quantitative detection of the fig clearwing tussock moth using fluorescent PCR:

[0040] The application of the combination of amplification primers for internal reference genes RPS3, TBP and 18S rRNA in the preparation of a fluorescent PCR quantitative detection kit for Ficus clearwing moth at different developmental stages, or in the fluorescent PCR quantitative detection of Ficus clearwing moth at different developmental stages.

[0041] The application of the combination of amplification primers for internal reference genes RPL28 and RPS3 in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing moth under different host rearing conditions, or in the fluorescent PCR quantitative detection of the fig clearwing moth under different host rearing conditions.

[0042] The application of the combination of amplification primers for internal reference genes RPL13 and BACT in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing moth under different temperature conditions, or in the fluorescent PCR quantitative detection of the fig clearwing moth under different temperature conditions.

[0043] The application of the combination of amplification primers for the internal reference gene 25S rRNA and BACT in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing moth under starvation conditions, or in the fluorescent PCR quantitative detection of the fig clearwing moth under starvation conditions.

[0044] The application of the combination of amplification primers for internal reference genes RPL28, RPS3 and TBP in the preparation of a fluorescent PCR quantitative detection kit for the fig clearwing moth under different mating conditions, or in the fluorescent PCR quantitative detection of the fig clearwing moth under different mating conditions.

[0045] The application of a combination of amplification primers for internal reference genes TUB, 18S rRNA and GAPDH in the preparation of a quantitative fluorescent PCR detection kit for different tissues of the fig clearwing moth, or in the quantitative fluorescent PCR detection of different tissues of the fig clearwing moth.

[0046] A fluorescent PCR quantitative detection kit, the kit containing amplification primers for each internal reference gene in the above-mentioned internal reference gene combination.

[0047] Preferably, the kit contains any of the above-described compositions.

[0048] More preferably, the fluorescent PCR quantitative detection kit is a kit for detecting the fig clearwing moth at different developmental stages, using a combination of amplification primers for internal reference genes RPS3, TBP and 18S rRNA.

[0049] The fluorescent PCR quantitative detection kit is a kit for detecting the fig clearwing moth under different host rearing conditions, using a combination of amplification primers for the internal reference genes RPL28 and RPS3.

[0050] The fluorescent PCR quantitative detection kit is a kit for detecting the fig clearwing moth under different temperature conditions, using a combination of amplification primers for the internal reference genes RPL13 and BACT.

[0051] The fluorescent PCR quantitative detection kit is a kit for detecting the fig clearwing moth under starvation conditions, using a combination of amplification primers for the internal reference gene 25S rRNA and BACT.

[0052] The fluorescent PCR quantitative detection kit is a kit for detecting the fig clearwing moth under different mating conditions, using a combination of amplification primers for internal reference genes RPL28, RPS3 and TBP.

[0053] The fluorescent PCR quantitative detection kit is a kit for detecting different tissues of the fig clearwing moth, using a combination of amplification primers for the internal reference genes TUB, 18S rRNA, and GAPDH.

[0054] A method for quantitative detection of the fig clearwing moth using fluorescent PCR, using any of the above-described compositions.

[0055] Preferably, the method is a method for detecting the fig clearwing moth at different developmental stages, using a combination of amplification primers for internal reference genes RPS3, TBP, and 18S rRNA;

[0056] The method described is for detecting the fig clearwing moth under different host rearing conditions, using a combination of amplification primers for the internal reference genes RPL28 and RPS3;

[0057] The method described is for detecting the fig clearwing moth under different temperature conditions, using a combination of amplification primers for the internal reference gene RPL13 and BACT.

[0058] The method described is for detecting the fig clearwing moth under starvation conditions, using a combination of amplification primers for the internal reference gene 25S rRNA and BACT.

[0059] The method described is for detecting the fig clearwing moth under different mating conditions, using a combination of amplification primers for the internal reference genes RPL28, RPS3, and TBP.

[0060] The method described is for detecting different tissues of the fig clearwing moth, using a combination of amplification primers for the internal reference genes TUB, 18S rRNA, and GAPDH.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] This invention discloses a combination of internal reference genes stably expressed by the fig clearwing tussock moth under different conditions and their applications. This invention screened and obtained stable internal reference genes of the fig clearwing tussock moth expressed in different developmental stages, under different host rearing conditions, under different temperature conditions, under starvation conditions, under different mating states, and in different tissues. These genes are suitable for correcting RT-qPCR detection results of the fig clearwing tussock moth under different experimental conditions. Simultaneously, this invention also provides amplification primers for detecting these fig clearwing tussock moth internal reference genes. These primers have high specificity and high amplification efficiency. This invention lays a preliminary foundation for the quantification of relevant functional genes of the fig clearwing tussock moth under different experimental conditions and related molecular biological research. Attached Figure Description

[0063] Figure 1 The Ct values ​​of the candidate internal reference gene under different treatment conditions are: A: different developmental stages, B: different host feeding conditions, C: different temperature conditions, D: starvation conditions, E: different mating states, F: different tissues, G: all samples.

[0064] Figure 2 The overall ranking of the expression stability of internal reference genes under different treatment conditions is as follows: A: different developmental stages, B: different host feeding conditions, C: different temperature conditions, D: starvation conditions, E: different mating states, F: different tissues, G: all samples.

[0065] Figure 3 These are paired variation values. Detailed Implementation

[0066] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0067] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0068] Example 1: Design and Detection of Candidate Internal Reference Gene Primers

[0069] I. Experimental Methods

[0070] 1. Extraction of RNA from the fig clearwing moth and preparation of cDNA template

[0071] use Total RNA was extracted from the fig clearwing moth using the Total RNA Extraction Kit (brand: Seven, catalog number: SM132). The purity and concentration of all RNA samples were then determined using Nanodrop. Following the manufacturer's instructions, 1 μg of RNA was transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+gDNAwiper) (brand: Vazyme, catalog number: R323-01). The synthesized cDNA was diluted 100-fold with RNase-free ddH2O and stored at -20°C for subsequent RT-qPCR experiments.

[0072] 2. Design of primers for internal reference genes

[0073] Actin (ACT), β-actin (BACT), arginine kinase (AK), elongation factor-1α (EEF1-α), glyceraldehyde-3-phosphate (GAPDH), 18S ribosomal RNA (18S rRNA), 25S ribosomal RNA (25S rRNA), tubulin (TUB), ribosomal protein L13 (RPL13), ribosomal protein L28 (RPL28), ribosomal protein S3 (RPS3), and TATA-binding protein (TBP) were selected as candidate internal control genes, and primers were designed for RT-qPCR. The primer sequences for the candidate internal control genes are shown in Table 1.

[0074] Table 1. Primer sequence information for candidate internal reference genes RT-qPCR

[0075]

[0076]

[0077] 3. RT-qPCR amplification

[0078] Using cDNA synthesized from the fig clearwing moth via reverse transcription as a template, RT-qPCR reactions were performed sequentially using the candidate internal reference gene primers listed in Table 1. Standard curves for each gene were plotted using Bio-Rad CFX Manager 2.1 software to obtain the primer slope (k), PCR amplification efficiency, and correlation coefficient (R). 2 ).

[0079] The reaction system (20 μL) consisted of 1 μL of cDNA template, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, and 8.2 μL of RNase-free water.

[0080] Reaction program: 95℃ for 2 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles; 95℃ for 5 s, 60℃ for 5 s, 95℃ for 0.5 s.

[0081] II. Experimental Results

[0082] The results are shown in Table 2, with correlation coefficients (R) 2 The PCR amplification efficiencies were 0.9655–0.9998 and 95.07%–116.84%, respectively. The results indicate that the primers for all 12 candidate internal reference genes possess stability and specificity.

[0083] Table 2. PCR amplification efficiency, correlation coefficient, and slope of candidate internal reference genes.

[0084]

[0085]

[0086] Example 2: Screening of the most stable internal reference gene of the fig clearwing moth under different conditions

[0087] I. Experimental Methods

[0088] 1. Collection of samples of the fig clearwing moth under different treatment conditions

[0089] (1) Sample collection at different developmental stages

[0090] Samples of the fig clearwing moth at different developmental stages were collected, including the egg stage (200 eggs), larval stage (including 100 first instar larvae, 50 second instar larvae, 20 third instar larvae, 10 fourth instar larvae, and 10 fifth instar larvae, on the second day after molting), pupal stage (10 male pupae and 10 female pupae, on the second day after pupation), and adult stage (10 male moths and 10 female moths, on the second day after emergence). The fig clearwing moth samples were quickly frozen in liquid nitrogen and stored at -80°C.

[0091] (2) Sample collection under different host rearing conditions

[0092] Healthy fifth-instar Ficus clearwing moth larvae were starved for 24 hours and then fed with four different host plants—Ficus goldenii, Ficus altissima, Ficus microcarpa, and Ficus weepingii—for two days. The moths were then rapidly frozen in liquid nitrogen and stored at -80°C. Ten replicates were performed for each host plant.

[0093] (3) Sample collection under different temperature conditions

[0094] Healthy fifth-instar larvae of the fig clearwing moth were reared for 24 hours in artificial incubators at 10℃ (low temperature), 24℃ (optimal temperature), and 40℃ (high temperature), and then the fig clearwing moths were rapidly frozen in liquid nitrogen and stored at -80℃. Each temperature condition was repeated 10 times.

[0095] (4) Sample collection under starvation conditions

[0096] Healthy fifth-instar larvae of the fig clearwing moth, randomly selected from those of similar size, were starved for 24 hours, then rapidly frozen in liquid nitrogen and stored at -80°C. This treatment was repeated 10 times.

[0097] (5) Sample collection under different mating conditions

[0098] Adult moths were selected one day after mating (mating group) and adults that had not mated one day after emergence (non-mating group). Ten pairs of male and female moths were selected from each group and quickly frozen in liquid nitrogen and stored at -80°C.

[0099] (6) Sample collection from different tissues

[0100] Healthy fifth-instar larvae of the fig clearwing moth were starved for 24 hours before being dissected. The larvae were washed 1-2 times with distilled water, then disinfected with 75% alcohol (v / v), and any residual alcohol was removed with distilled water. After anesthetizing the larvae on ice for 5-30 minutes, the larval tissues, including the head, fat body, intestines, Malpighian tubules, and hemolymph, were separated using dissecting scissors. The base of the larval legs was cut open to allow the hemolymph to drain, and the hemolymph was immediately placed in centrifuge tubes containing citrate and physiological saline. Tissues of the adults (such as antennae, head, thorax, abdomen, legs, and wings) were dissected from 10 male moths and 10 female moths (on the second day after emergence). Each tissue sample was washed three times with PBS solution (140 mM NaCl, 2.70 mM KCl, 10 mM Na2HPO4, 1.80 mM KH2PO4, pH 7.40) after dissection, and the tissue samples were rapidly frozen in liquid nitrogen and stored at -80°C. Each tissue was subjected to 10 replicates.

[0101] 2. RT-qPCR amplification

[0102] Following the method in Example 1, RNA was extracted, cDNA templates were prepared, and RT-qPCR amplification was performed on samples collected under different treatment conditions. After the reaction, the cycle threshold (Ct value) of each candidate internal reference gene in each treated sample was derived. The stability of the candidate internal reference genes under different treatment conditions was evaluated using the ΔCt method, GeNorm, BestKeeper, and NormFinder. The overall expression stability was ranked using RefFinder.

[0103] II. Experimental Results

[0104] 1. Expression profiles of candidate internal reference genes

[0105] The expression of 12 internal reference genes was evaluated under seven different treatment conditions using Ct values. The Ct values ​​of these internal reference genes ranged from 15.02 to 32.39. AK showed the lowest expression level across all samples, with a mean Ct value of 27.39 ± 2.01, while BACT showed the highest expression level, with a mean Ct value of 18.66 ± 2.02. Figure 1 ).

[0106] 2. Different developmental stages

[0107] For different developmental stages (Table 3), RPS3 expression was the most stable and EEF1A expression was the least stable in the ΔCt method and the BestKeeper method. In the BestKeeper analysis, the SD values ​​of ACT, AK, 25S rRNA, EEF1A, GAPDH and TUB were greater than 1, indicating that these six genes are not suitable as internal reference genes.

[0108] In the GeNorm method, RPS3 and 18S rRNA expression were the most stable; AK expression was the least stable and its M value was greater than 1.5, indicating that AK is not suitable as an internal reference gene.

[0109] In the NormFinder method, TBP expression is the most stable, while AK expression is the least stable.

[0110] The stability ranking generated by RefFinder is as follows: RPS3 > TBP > 18S rRNA > TUB > RPL28 > RPL13 > BACT > 25S rRNA > AK > GAPDH > ACT > EEF1A Figure 2 (A) in the middle.

[0111] Table 3 Expression stability of candidate internal reference genes at different developmental stages

[0112]

[0113] 3. Raising different hosts

[0114] Under different host conditions (Table 4), ACT expression was the least stable among the four methods. In contrast, 18S rRNA expression was the most stable in the ΔCt and BestKeeper methods.

[0115] In the GeNorm method, RPL28 and AK showed the most stable expression with M values ​​less than 1.5, indicating that all candidate internal reference genes had high stability.

[0116] In the NormFinder method, RPL28 expression is the most stable;

[0117] In the BestKeeper analysis, the SD values ​​of ACT and EEF1A were greater than 1, indicating that they are not suitable as internal reference genes.

[0118] The stability ranking generated by RefFinder is as follows: RPL28 > RPS3 > 18S rRNA > AK > BACT > RPL13 > TUB > 25S rRNA > GAPDH > TBP > EEF1A > ACT. Figure 2 (B in the middle).

[0119] Table 4. Expression stability of candidate internal reference genes under different host feeding conditions.

[0120]

[0121]

[0122] 4. Different temperatures

[0123] After treatment at different temperatures (Table 5), 25S rRNA expression was the most unstable among the four methods. In the ΔCt and BestKeeper methods, TBP expression was the most stable. Furthermore, in the BestKeeper analysis, the SD values ​​of AK and 25S rRNA were greater than 1, indicating that they are not suitable as internal reference genes.

[0124] In the GeNorm method, BACT and RPL13 were the most stable in expression, and their M values ​​were both less than 1.5, indicating that all candidate internal reference genes had high stability.

[0125] In the NormFinder method, RPL13 expression is the most stable.

[0126] The stability ranking generated by RefFinder is as follows: RPL13 > BACT > TBP > ACT > EEF1A > RPS3 > RPL28 > TUB > 18S rRNA > GAPDH > AK > 25S rRNA Figure 2 (C in the middle).

[0127] Table 5. Expression stability of candidate internal reference genes under different temperature treatment conditions.

[0128]

[0129]

[0130] 5. Starvation conditions

[0131] After starvation treatment (Table 6), AK expression was the most stable and RPS3 expression was the least stable in the ΔCt and BestKeeper methods. In the BestKeeper analysis, the SD values ​​of ACT, RPS3 and RPL13 were greater than 1, indicating that they are not suitable as internal reference genes.

[0132] In the GeNorm method, BACT and 18S rRNA expression were the most stable, while EEF1A expression was the least stable, and all M values ​​were less than 1.5, indicating that they are all suitable as internal reference genes.

[0133] In NormFinder, 25S rRNA expression was the most stable, while EEF1A expression was the least stable.

[0134] The stability ranking generated by RefFinder is as follows: 25S rRNA > BACT > 18S rRNA > TUB > RPL28 > AK > TBP > GAPDH > ACT > RPS3 > RPL13 > EEF1A Figure 2 (D in the middle).

[0135] Table 6 Expression stability of candidate internal reference genes under starvation conditions

[0136]

[0137]

[0138] 6. Different mating states

[0139] Under different mating states (Table 7), TUB expression was the most stable in both the ΔCt method and the BestKeeper method. In the BestKeeper method, only EEF1A and TUB had SD values ​​less than 1, indicating that only these two genes are suitable as internal reference genes under this algorithm.

[0140] In the GeNorm method, the M values ​​of all 12 candidate internal reference genes were less than 1.5, indicating that they were all suitable as internal reference genes. Among them, RPS3 and 18S rRNA expression was the most stable.

[0141] In NormFinder, RPL28 expression was the most stable, while EEF1A expression was the least stable.

[0142] The stability ranking generated by RefFinder is as follows: RPL28 > RPS3 > TBP > AK > 18S rRNA > 25S rRNA > TUB > EEF1A > RPL13 > BACT > GAPDH > ACT Figure 2 (E in the text)

[0143] Table 7 Expression stability of candidate internal reference genes under different mating states

[0144]

[0145]

[0146] 7. Different organizations

[0147] In different tissues (Table 8), ACT expression was the most unstable under all four methods. TUB expression was the most stable under both the ΔCt and BestKeeper methods. Furthermore, in the BestKeeper method, the SD values ​​of BACT, ACT, AK, RPL28, and EEF1A were all greater than 1, indicating that they are not suitable as internal reference genes.

[0148] In the GeNorm method, TUB and TBP showed the highest expression stability, and only ACT, RPL28, and EEF1A had M values ​​greater than 1.5, indicating that the remaining 9 genes were suitable as internal reference genes.

[0149] In NormFinder, GAPDH expression is the most stable.

[0150] The stability ranking generated by RefFinder is as follows: TUB > 18S rRNA > GAPDH > TBP > 25S rRNA > RPL13 > RPS3 > BACT > AK > RPL28 > EEF1A > ACT Figure 2 (F in the middle).

[0151] Table 8 Expression stability of candidate internal reference genes in different tissues

[0152]

[0153] 8. All samples

[0154] The cycle thresholds (Ct values) of each candidate internal reference gene were summed under different developmental stages, different host feeding conditions, different temperature conditions, starvation conditions, different mating states, and different tissue samples. Then, the stability of the candidate internal reference genes was evaluated using the ΔCt method, GeNorm, BestKeeper, and NormFinder. The overall expression stability was ranked using RefFinder.

[0155] In all samples (Table 9), AK expression was the most unstable in the ΔCt, GeNorm, and NormFinder methods, while ACT expression was the most unstable in the BestKeeper method. TUB expression was the most stable in the ΔCt and BestKeeper methods, and in the BestKeeper method, only TUB and TBP had SD values ​​less than 1, making them suitable as internal reference genes. In the GeNorm method, RPS3 and RPL13 expression was the most stable.

[0156] The stability ranking generated by RefFinder is as follows: 18S rRNA > RPL13 > RPL28 > BACT > RPS3 > GAPDH > 25S rRNA > AK > TUB > EEF1A > ACT > TBP Figure 2 (G in the middle).

[0157] Table 9. Expression stability of candidate internal reference genes in all samples.

[0158]

[0159] 9. Determining the optimal number of internal reference genes

[0160] The optimal number of internal reference genes was determined by calculating the paired variance value using GeNorm and based on whether it was below 0.15. Figure 3 It can be seen that under different host conditions, different temperatures, and starvation conditions, the V2 / 3 value is lower than 0.15, indicating that the optimal number of internal reference genes under these three conditions is 2; while under different developmental stages, different mating states, different tissues, and all sample conditions, the V2 / 3 value is higher than 0.15, indicating that the optimal number of internal reference genes is 3.

[0161] Therefore, the combination of internal reference genes used under different developmental stages is RPS3, TBP and 18S rRNA;

[0162] The internal reference gene combination used under different host rearing conditions is RPL28 and RPS3;

[0163] The internal reference gene combination used under different temperature conditions was RPL13 and BACT;

[0164] The internal reference gene combination used under starvation conditions was 25S rRNA and BACT;

[0165] The internal reference gene combination used under different mating conditions was RPL28, RPS3, and TBP;

[0166] The combination of internal reference genes used in different tissues is TUB, 18S rRNA, and GAPDH.

[0167] Example 3: An RT-qPCR kit for detecting the fig clearwing moth

[0168] 1. Composition

[0169] Includes amplification primers, RT-qPCR reaction reagents, and ddH2O for the internal reference genes RPS3, TBP, 18S rRNA, RPL28, RPL13, BACT, 25S rRNA, TUB, and GAPDH;

[0170] The nucleotide sequences of the amplification primers for the internal reference gene RPS3 are shown in SEQ ID NO: 15-16.

[0171] The nucleotide sequences of the primers for amplifying the internal reference gene TBP are shown in SEQ ID NO: 23-24;

[0172] The nucleotide sequences of the primers for amplifying the internal reference gene 18S rRNA are shown in SEQ ID NO: 17-18;

[0173] The nucleotide sequences of the amplification primers for the internal reference gene RPL28 are shown in SEQ ID NO: 13-14;

[0174] The nucleotide sequences of the primers for amplifying the internal reference gene RPL13 are shown in SEQ ID NO: 11-12;

[0175] The nucleotide sequences of the amplification primers for the internal reference gene BACT are shown in SEQ ID NO: 3-4;

[0176] The nucleotide sequences of the primers for amplifying the 25S rRNA of the internal reference gene are shown in SEQ ID NO: 19-20;

[0177] The nucleotide sequences of the primers for amplifying the internal reference gene TUB are shown in SEQ ID NO: 21-22;

[0178] The nucleotide sequences of the primers for amplifying the internal reference gene GAPDH are shown in SEQ ID NO: 9-10.

[0179] 2. How to use

[0180] (1) Extract total RNA from the sample to be tested and reverse transcribe it to synthesize cDNA template;

[0181] (2) RT-qPCR was performed using the synthesized cDNA as a template, and the combination of internal reference genes was selected according to different treatment conditions:

[0182] The combination of internal reference genes used under different developmental stages was RPS3, TBP and 18S rRNA;

[0183] The internal reference gene combination used under different host rearing conditions is RPL28 and RPS3;

[0184] The internal reference gene combination used under different temperature conditions was RPL13 and BACT;

[0185] The internal reference gene combination used under starvation conditions was 25S rRNA and BACT;

[0186] The internal reference gene combination used under different mating conditions was RPL28, RPS3, and TBP;

[0187] The combination of internal reference genes used in different tissues is TUB, 18S rRNA, and GAPDH.

[0188] The reaction system (20 μL) consisted of 1 μL of cDNA template, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, and 8.2 μL of RNase-free water.

[0189] Reaction program: 95℃ for 2 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles; 95℃ for 5 s, 60℃ for 5 s, 95℃ for 0.5 s.

[0190] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Internal reference gene RPS3 , TBP and 18S rRNA The application of the amplification primer composition in the preparation of a fluorescent PCR quantitative detection kit for *Ficus clearwing tussock moth* at different developmental stages, or in the fluorescent PCR quantitative detection of *Ficus clearwing tussock moth* at different developmental stages, is characterized by, The internal reference gene RPS3 The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 15-16; The internal reference gene TBP The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 23-24; The internal reference gene 18S rRNA The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 17-18.

2. A method for quantitative detection of the fig clearwing moth at different developmental stages using fluorescent PCR, characterized in that, Using internal reference genes RPS3 , TBP and 18S rRNA The amplification primer composition was used for detection; The internal reference gene RPS3 The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 15-16; The internal reference gene TBP The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 23-24; The internal reference gene 18S rRNA The nucleotide sequences of the amplification primers are shown in SEQ ID NO: 17-18.

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