Internal reference genes, primers, and applications of citrus psyllids

Abstract

This invention discloses the internal reference gene of the citrus psyllid, its primers, and applications, belonging to the field of genetic engineering technology. Through screening, this invention identifies Actin, the most stable and superior internal reference gene for the citrus psyllid. This invention finds that Actin exhibits the lowest expression stability in different sexes, different tissues and organs, different developmental stages, and different disease states of the citrus psyllid, making it the most stable internal reference gene for related gene expression studies in the citrus psyllid. This lays the foundation for research on the biological characteristics and disease transmission mechanisms of the citrus psyllid and also provides a theoretical reference for the study of internal reference genes in other homoptera insects.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the internal reference gene of citrus psyllid, its primers, and applications. Background Technology

[0002] Citrus Huanglongbing (HLB), also known as citrus green fruit disease, can occur year-round and is a devastating disease threatening citrus production worldwide, causing serious economic losses to the citrus industry.

[0003] The citrus psyllid (Diaphorina citri Kuwayama), belonging to the family Psyllididae in the suborder Hemiptera, is a bisexual oviparous insect, laying eggs in clusters. The eggs are pointed at the tip and rounded at the end, resembling a mango, and are mostly laid on the tender shoots of the host plant. It is the vector insect for citrus Huanglongbing (HLB). Although a range of control methods exist for citrus psyllids, including chemical control (broad-spectrum insecticides such as neonicotinoids and pyrethroids), physical control (yellow sticky traps), and biological control (predatory enemies such as fungi, parasitic wasps, and ladybugs), an effective fungicide that can completely cure HLB has not yet been developed. Current technology has revealed that citrus psyllids can transfer the HLB pathogen, Candidatus Liberibacter asiaticus (CLas), into the citrus plant by feeding on citrus branches and leaves. CLas can continuously proliferate and metastasize within the citrus psyllid, and electron microscopy reveals a wide distribution of the pathogen, found in the salivary glands, digestive tract filter chambers, fat bodies surrounding the salivary glands, and cells of the midgut and hindgut. Furthermore, the bacterial titer increases with each instar of the citrus psyllid, indicating that pathogen replication accompanies the growth and development of the vector insect. Therefore, research on the citrus psyllid is a crucial component of citrus Huanglongbing (HLB) control research. While the physiological habits of the citrus psyllid are relatively well understood, the molecular biology of the citrus psyllid is still in its early stages. Therefore, it is necessary to establish a set of molecular biological research methods specifically for the citrus psyllid.

[0004] Internal reference genes are minimally affected by intrinsic factors and external environmental factors, and are genes that can be relatively stably expressed in any organ, tissue, or cell of the same organism. Therefore, when detecting changes in gene expression levels, internal reference genes can be used as a reference for correction. The relative expression level is obtained by converting the Ct value of the target gene and the Ct value of the internal reference gene, and experimental data is homogenized. This effectively reduces and corrects experimental errors between samples, ensuring accurate and reliable experimental results. Recent findings indicate that the selection of internal reference genes is not unique; their expression levels vary under different sexes, developmental stages, tissues and cells, and treatment conditions in insects. Therefore, to improve the accuracy of real-time quantitative PCR (qPCR) detection and ensure the reliability of gene expression analysis, it is best to selectively introduce one or more internal reference genes with high copy numbers and stable expression to correct the expression level of the target gene.

[0005] To date, there have been no reports of research related to the internal reference genes of citrus psyllids. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide specific primers for detecting the expression of the internal reference gene Actin in citrus psyllids of different sexes, different tissues and organs, different developmental stages, and different disease states.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides specific primers for detecting the expression of a reference gene in citrus psyllids, wherein the reference gene is Actin, and the forward and reverse primers for Actin are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] Preferably, the nucleotide sequence of the internal reference gene Actin is shown in SEQ ID NO.3.

[0010] Preferably, the citrus psyllid includes both adult citrus psyllids carrying the pathogen and healthy adults.

[0011] Preferably, the citrus psyllid includes both female and male citrus psyllids.

[0012] Preferably, the citrus psyllid includes adult citrus psyllids and nymphs.

[0013] This invention also provides the application of the above-mentioned specific primers in the preparation of reagents for detecting the internal reference gene of citrus psyllid.

[0014] The present invention also provides a kit for detecting the internal reference gene of citrus psyllid, the kit comprising the above-mentioned specific primers.

[0015] This invention also provides the application of the above-mentioned specific primers in the study of gene expression and function of citrus psyllids using quantitative real-time PCR technology.

[0016] This invention also provides the application of the above-mentioned specific primers in studying the infection of citrus psyllids by Huanglongbing fungus or the effects of Huanglongbing fungus on different developmental stages of citrus psyllids.

[0017] Preferably, the internal reference gene Actin is used as a correction gene.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention screened and obtained the Actin internal reference gene for the citrus psyllid. The Actin gene screened in this invention has a relatively suitable expression abundance, with a CT value of around 20. Furthermore, the Actin gene can be stably expressed in different sexes of adult citrus psyllids, different tissues and organs, different developmental stages, and different disease states. It is suitable for related gene expression studies of the citrus psyllid, laying the foundation for studying the biological characteristics and disease transmission mechanisms of the citrus psyllid, and also providing a theoretical reference for the study of internal reference genes in other homoptera insects.

[0020] This invention provides specific primers for detecting the expression of the internal reference gene Actin in citrus psyllids. These primers have significant specificity and can establish a real-time fluorescence quantitative PCR detection method for citrus psyllids under different conditions. This greatly improves detection efficiency, shortens detection time, enhances the reliability of detection results, and improves the stability, reliability, and reproducibility of gene expression analysis studies in citrus psyllids. Attached Figure Description

[0021] Figure 1 : Electrophoresis image of RNA from a citrus psyllid sample;

[0022] Figure 2 Gene expression abundance of 7 internal reference genes in different citrus psyllid experimental groups;

[0023] Figure 3 Expression stability values ​​(M) of various internal reference genes in different organs and tissues of female citrus psyllids obtained by GeNorm software analysis;

[0024] Figure 4 Expression stability values ​​(M) of each internal reference gene obtained by GeNorm software analysis in healthy and infected female citrus psyllids;

[0025] Figure 5Expression stability values ​​(M) of each internal reference gene in healthy male and female citrus psyllids obtained by GeNorm software analysis;

[0026] Figure 6 Expression stability values ​​(M) of each internal reference gene in healthy citrus psyllid nymphs and adults obtained by GeNorm software analysis. Detailed Implementation

[0027] This invention provides specific primers for detecting the expression of a reference gene in citrus psyllids, wherein the reference gene is Actin, and the forward and reverse primers for Actin are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0028] The nucleotide sequence of the internal reference gene Actin described in this invention is shown in SEQ ID NO.3.

[0029] This invention is based on molecular biology experiments on the citrus psyllid and uses real-time quantitative PCR to screen for stably expressed genes in the citrus psyllid. Seven housekeeping genes from the citrus psyllid reference gene sequence (GCA_000475195.1) were selected: actin, wingless protein (Wg), α-tubulin (α-Tub), 18S ribosomal protein (RPS18), transcription elongation factor (EF1a), heat shock protein (HSP), and mitochondrial protein (Cox1). Four different experimental groups were set up, representing different organs, sexes, ages, and bacterial carrier states. cDNA of the citrus psyllid was amplified using real-time quantitative PCR, and the expression stability value (M value) of the internal reference gene was calculated. This allowed for the screening of relatively stable internal reference genes in each experimental group, with Actin, which was stably expressed in each group, being identified as the internal reference gene.

[0030] The specific primers for the Actin internal reference gene of the citrus psyllid described in this invention can be used to detect gene expression in both bacteria-carrying and healthy citrus psyllid adults.

[0031] The specific primers for the Actin internal reference gene of the citrus psyllid described in this invention can be used to detect gene expression in female and male citrus psyllids.

[0032] The specific primers for the Actin internal reference gene of the citrus psyllid described in this invention can be used to detect gene expression in adult and nymphal citrus psyllids.

[0033] This invention allows for the selection of different organs of the citrus psyllid for gene expression level detection, including the digestive tract, hemolymph, and salivary glands. Actin can be used as an internal reference gene when different organs of the citrus psyllid are used as samples.

[0034] The citrus psyllid described in this invention is preferably the Asian citrus psyllid.

[0035] The present invention also provides the application of the above-mentioned Actin-specific primers for the citrus psyllid internal reference gene in the preparation of reagents for detecting the citrus psyllid internal reference gene, which can accurately detect the expression level of the internal reference gene Actin.

[0036] This invention also provides a kit for detecting the citrus psyllid reference gene Actin, the kit comprising specific primers for the aforementioned citrus psyllid reference gene Actin. This invention does not limit the selection of other reagents in the kit.

[0037] This invention also provides the application of the above-mentioned Actin-specific primers for the citrus psyllid internal reference gene in the study of gene expression and function of the citrus psyllid using real-time PCR technology. The Actin internal reference gene described in this invention is used as a correction gene.

[0038] This invention also provides the application of the above-mentioned Actin-specific primers for the citrus psyllid internal reference gene in the study of the infection status of citrus Huanglongbing fungus on citrus psyllids or the effects of citrus Huanglongbing fungus on different developmental stages of citrus psyllids. The Actin internal reference gene described in this invention is used as a correction gene.

[0039] To detect the relative expression levels of genes at the transcriptional level and eliminate potential differences in RNA yield, quality, and reverse transcription efficiency among different samples to obtain the true differences in the specific expression of the target gene, it is usually necessary to select a suitable internal reference gene for correction. The Actin primer sequence selected in this invention exhibits high specific amplification efficiency and good linear correlation, and can be used to detect changes in gene expression levels in different individuals of the citrus psyllid, correcting for the expression levels of the target gene, in order to study the biological characteristics and disease transmission mechanisms of the citrus psyllid.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] The reagents and instruments used in the specific embodiments of this invention are as follows:

[0042] (1) Artificial climate chamber RXZ-380A, manufactured by Ningbo Jiangnan Instrument Factory;

[0043] (2) 1.5mL centrifuge tubes;

[0044] (3) Test tube: 12cm, used for collecting adult insects;

[0045] (4) Anhydrous ethanol: purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0046] (5) DEPC: an enzyme that degrades RNA;

[0047] (6) Chloroform / isoamyl alcohol (24:1): Purchased from Guangzhou brand chemical reagents;

[0048] (7) Nucleic acid concentration measuring instrument: Qubit 2.0 Fluorometer (Invitrogen by lifetechnologies);

[0049] (8) PCR instrument: GeneAmp PCR System 9700, manufactured by PE Company;

[0050] (9) Real-time quantitative PCR: Bio-RAD CFX Connect™ Real-time System;

[0051] (10) DNA concentration analyzer: Qubit 2.0 Fluorometer;

[0052] (11) High-speed refrigerated centrifuge: Sigma Laboratory Centrifuges 3K18, manufactured by Sigma Corporation;

[0053] (12) Sterilizer: High-pressure moist heat sterilizer, model HIRAYMA;

[0054] (13) Palm-sized centrifuge: Palm-sized centrifuge, model Lx-100, manufactured by Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd.;

[0055] (14) Constant temperature water bath: DK-8D type electric heating constant temperature water bath, produced by Shanghai Medical Constant Temperature Equipment Factory.

[0056] Unless otherwise specified, the following embodiments are all conventional methods.

[0057] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0058] Example 1

[0059] 1. Test plant materials

[0060] The tangerine (Citrus reticulata cv. Shatangju) is a 3-year-old, 50cm tall seedling infected with Huanglongbing (HLB). The diseased buds were obtained from the Citrus Research Institute of Boluo County, Huizhou City, Guangdong Province.

[0061] Murraya exotica is a healthy seedling that is 2 years old.

[0062] The disease status of all the plant materials was determined by real-time quantitative PCR and then placed in a net room.

[0063] 2. Test insect source

[0064] Adults and nymphs of the Asian citrus psyllid (C. truncatula) were selected from F2 generation seedlings infected with Huanglongbing (HLB) in Satsuma mandarin oranges and healthy Murraya paniculata plants. Four experimental treatments were set up:

[0065] (1) Dissecting the organs of 5 female citrus psyllids: digestive tract, hemolymph, salivary glands;

[0066] (2) Female citrus psyllids carrying the pathogen and healthy female citrus psyllids;

[0067] (3) Male and female citrus psyllids;

[0068] (4) Citrus psyllid nymphs and adults.

[0069] In the experimental group (2), some of the citrus psyllids were infected with the fungus. Each experimental treatment was set up with 10 replicates.

[0070] 3. Experimental Methods

[0071] 3.1 Insect Dissection

[0072] In experimental group (2), the citrus psyllids were starved for 3 hours at room temperature to remove their digestive tract contents, and only surviving citrus psyllids were dissected. The citrus psyllids were placed on a glass slide containing 200 μL of 0.01 mol / L PBS buffer for microscopic dissection. The dissection method was as described in (Ammar et al., 2011a; 2011b). The digestive tract of the citrus psyllid is located roughly between the thoracic cavity and the abdomen. The salivary glands are located in the anterior part of the thoracic cavity, adjacent to the head. Hemolymph was collected using a 10 μL pipette with a 0.1–2.5 μL tip.

[0073] 3.2 Insect RNA Extraction and cDNA Synthesis

[0074] To prevent RNase contamination, wear disposable gloves and activated carbon masks during experiments. Turn on the centrifuge in advance, set the temperature for pre-cooling, disinfect the work surface and experimental equipment with alcohol, and wipe the work surface and experimental equipment with DEPC water.

[0075] (1) Pre-cooling Trizol; After dissecting the psyllid, the tissues (digestive tract, salivary glands, hemolymph) were quickly placed in 1.5mL centrifuge tubes, and 400μL of Trizol was added to each tube (80μL was added first, the tissue was ground and broken with a grinding rod, and then 320μL was added). The tubes were placed on liquid nitrogen and ground thoroughly with a grinding rod. The tubes were left at room temperature for 5 minutes to allow the tissues to fully lyse.

[0076] (2) Add 150 μL of chloroform to each tube (operate in a fume hood), invert vigorously to mix for 10 seconds, let stand at room temperature for 3 minutes, and repeat the inversion 3 times.

[0077] (3) Centrifuge at 12000xg for 10 min at 4℃, take the supernatant, transfer it to a new 1.5mL tube, and discard the precipitate;

[0078] (4) Add 0.6 times the volume of pre-cooled isopropanol to the tube (operate in a fume hood), gently aspirate and mix, and let stand at room temperature for 10 minutes.

[0079] (5) Centrifuge at 4℃ for 12000xg for 10min, discard the supernatant, then centrifuge briefly, and use a small pipette tip to remove the supernatant (be careful not to aspirate the precipitate at the bottom).

[0080] (6) Add 1 mL of 75% ethanol to the tube, gently invert (do not use too much force) to suspend the precipitate (if the precipitate is small, suspension is not necessary), and let stand at room temperature for 3 min.

[0081] (7) Centrifuge at 12000xg for 5 min at 4℃, discard the supernatant, perform a simple instantaneous centrifugation, and place in a clean bench to air dry at room temperature;

[0082] (8) Add 25 μL of RNase-free water to the tube and incubate briefly;

[0083] (9) RNA purification was performed according to the instructions of the Recombinant DNase I (RNase-free) kit. After RNA purification, the concentration of extracted RNA was determined by electrophoresis and analyzed by a Nanodrop 2000 micro UV spectrophotometer (Thermo), and the RNA concentration was standardized.

[0084] Total RNA was extracted from four experimental treatments of citrus psyllids using the Trizol method. After DNase digestion, the RNA was detected by 1.2% agarose gel electrophoresis. The results are shown in the figure below. Figure 1 .Depend on Figure 1 It can be seen that the electrophoretic bands are clear and bright, without diffusion, and the 28S rRNA band is clearer and brighter than the 18S rRNA band, indicating that the RNA is intact and has not been degraded.

[0085] The concentration and purity of RNA were detected using a Nanodrop 2000 micro-ultraviolet spectrophotometer. The results showed that A260 / 230 and A260 / 280 were both between 2.0 and 2.1, indicating that the RNA purity was high and it could be used as a suitable sample for amplification reactions, meeting the requirements of subsequent experiments. It could be used as a template for real-time quantitative PCR analysis.

[0086] (10) The cDNA strand was synthesized according to the instructions of the Takara PrimeScript II 1st Strand cDNA Synthesis Kit. After the cDNA was synthesized, the cDNA concentration was detected using a Qubit real-time fluorescence instrument (Invitrogen by Thermo Fisher Scientific), and the cDNA concentration was standardized to 200 ng / μL.

[0087] 3.3 Selection of internal reference genes and primer design

[0088] The seven internal reference genes are the encoding genes for actin, wingless protein (Wg), α-tubule, 18S ribosomal protein (RPS18), transcription elongation factor (EF1a), heat shock protein (HSP), and mitochondrial protein (Cox1). The sequences of the internal reference genes were downloaded from the GenBank database. The nucleotide sequences of actin, wingless protein, α-tubule, 18S ribosomal protein, transcription elongation factor, heat shock protein, and mitochondrial protein are shown in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively. Seven pairs of specific primers and probes for the internal reference gene were designed using Primer 3web (4.0.0) (see Table 1). The parameters were set as follows: amplification length of 80–200 base pairs, primer sequence length of 17–25 base pairs, GC content of 45%–55%, and the TM values ​​of the forward and reverse primers should not differ too much.

[0089] 3.4 Real-time quantitative PCR analysis of internal reference genes

[0090] Seven pairs of internal reference genes were quantitatively analyzed using the TransStart Top Green qPCR SuperMix real-time quantitative PCR kit from Beijing TransGen Biotech Co., Ltd. Specific reagent dosages were as per the manufacturer's instructions, and each sample was repeated three times. The amplification reaction program was: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing for 15 seconds, and 72℃ extension for 10 seconds, for a total of 40 cycles. After amplification, the specificity of the amplified products was determined by analyzing the melting curves. The temperature was increased from 65℃ to 95℃ every 10 seconds, and the fluorescence intensity of the samples was continuously measured to obtain the melting curves. The experimental data were analyzed using relative quantification. Equal amounts of cDNA template from nine experimental samples were taken, mixed, and then serially diluted six times, each time by a 5-fold dilution (1, 1 / 5, 1 / 25, 1 / 625, 1 / 3125, and 1 / 15625). Each reaction was performed in triplicate. The melting temperature and amplification efficiency E were calculated using the CFX Manager™ Software built into the PCR instrument (see Table 1).

[0091] Table 1 Primer sequences of 7 internal reference genes for the Asian citrus psyllid.

[0092]

[0093]

[0094] In Table 1, the forward and reverse primers for the internal reference gene Actin are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the forward and reverse primers for the internal reference gene Wg are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively; the forward and reverse primers for the internal reference gene α-Tub are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively; the forward and reverse primers for the internal reference gene RPS18 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; the forward and reverse primers for the internal reference gene EF1a are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively; the forward and reverse primers for the internal reference gene HSP are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively; and the forward and reverse primers for the internal reference gene Cox1 are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively.

[0095] Table 1 shows the analysis results of PCR amplification efficiency and standard curve slope for the seven candidate internal reference genes. The amplification efficiency of the seven internal reference genes was good, ranging from 90% to 110%, indicating good quantification results and that the selected primers could specifically amplify the DNA sequences. The slopes of the standard curves were all greater than 0.98, close to 1, indicating that the dilution results were accurate and reliable, showing a good linear relationship.

[0096] 3.5 Data Processing and Analysis

[0097] Stability analysis of candidate genes was performed using the ΔCt value method and the GeNorm internal reference gene screening software. The ΔCt value method sequentially selected the candidate internal reference genes with the lowest Ct value for each gene's signal expression data. Then, the minimum Ct value was subtracted from the other Ct values ​​of that candidate internal reference gene to obtain the ΔCt value. The obtained ΔCt value was then input into the software to calculate the average expression stability value (M) of each internal reference gene. Genes were then sorted according to the size of their M values; the smaller the M value, the higher the stability.

[0098] Ct values ​​are inversely proportional to expression abundance; higher Ct values ​​indicate lower expression abundance, and lower Ct values ​​indicate higher expression abundance. The expression levels of the seven internal reference genes varied across different sexes, tissues, organs, developmental stages, and disease states in citrus psyllids. The results are shown in [Figure 1]. Figure 2 The expression abundance of the seven candidate reference genes in nymphs, females, and males, from highest to lowest, was Cox1 > Actin > HSP > RPS18 > EF1a > α-Tub > Wg; in the digestive tract, the expression abundance of the seven candidate reference genes, from highest to lowest, was Cox1 > Actin > HSP > RPS18 > EF1a > Wg > α-Tub; in hemolymph and salivary glands, the expression abundance of the seven candidate reference genes, from highest to lowest, was Cox1 > Actin > EF1a > RPS18 > HSP > Wg = α-Tub; and in diseased females, the expression abundance of the seven candidate reference genes, from highest to lowest, was Cox1 > HSP > Actin > RPS18 > EF1a > α-Tub > Wg. It can be seen that the expression abundance of the internal reference genes Cox1, HSP and Actin is moderate, with Ct values ​​around 20 (a Ct value between 15 and 25 indicates moderate expression abundance and can be used as an expression for internal reference gene stability); the expression abundance of the internal reference genes α-Tub and Wg is relatively low, with Ct values ​​around 30.

[0099] The expression stability (M) of the reference genes in different sexes, tissues, organs, and experimental treatments of the citrus psyllid was analyzed using GeNorm software. The results showed that the M values ​​of all seven reference genes were less than 1.5, indicating that the expression of all seven genes was relatively stable. In different tissues and organs of female citrus psyllids, the expression stability of the seven reference genes, from highest to lowest, was as follows: Actin (0.014) = α-Tub (0.014) > RPS18 (0.015) = Cox1 (0.015) > HSP (0.018) > Wg (0.023) > EF1a (0.025) (see results below). Figure 3 In healthy and infected female citrus psyllids, the stability of expression of the seven internal reference genes, from highest to lowest, was: Actin (0.023) > RPS18 (0.025) = Cox1 (0.025) > HSP (0.029) > EF1a (0.032) > α-Tub (0.033) > Wg (0.035) (see results). Figure 4 In healthy male and female citrus psyllids, the stability of expression of the seven internal reference genes, from high to low, was Actin (0.028) = RPS18 (0.028) > Cox1 (0.031) > α-Tub (0.033) > HSP (0.037) > EF1a (0.04) > Wg (0.05) (see results). Figure 5 In citrus psyllid nymphs and adults, the stability of expression of the seven internal reference genes, from high to low, was Actin (0.022) = HSP (0.022) > Cox1 (0.023) > α-Tub (0.024) > RPS18 (0.033) > EF1a (0.034) > Wg (0.045) (see results). Figure 6 ).

[0100] The experimental results show that: (1) The expression stability of the internal reference gene EF1a was slightly poor in different tissues and organs of female citrus psyllids, while the expression stability of the internal reference gene Wg was slightly poor in the other experimental groups; (2) The M values ​​of the seven internal reference genes were moderate in different sexes, different tissues and organs, different developmental stages, and different disease states of citrus psyllids, indicating that the gene expression was stable. However, due to the low expression abundance of Wg and α-Tub (the quantitative results were more accurate when the Ct value was between 10 and 30), it was impossible to obtain more accurate and reliable quantitative results; (3) The expression stability of the internal reference gene Actin was better in different health conditions; the expression stability of the internal reference genes Actin and α-Tub was better in different organs and tissues; the expression stability of the internal reference genes Actin and RPS18 was better in different sexes of citrus psyllids; and the expression stability of Actin and HSP was the best in different developmental stages; (4) In all experimental groups, the internal reference gene Actin was the most stable and excellent internal reference gene. Therefore, Actin can be used as the most stable internal reference gene and is suitable for related expression studies of citrus psyllids.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of specific primers for detecting the expression of reference genes in citrus psyllids using quantitative real-time PCR technology to detect gene expression and function in citrus psyllids under different health conditions, in different organs and tissues, in different sexes, and / or at different developmental stages, characterized in that... The internal reference gene is Actin, which is used as a correction gene. The forward and reverse primers of the internal reference gene Actin are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. The application of specific primers for detecting the expression of the internal reference gene in citrus psyllids in detecting the infection status of citrus Huanglongbing fungus on citrus psyllids or the effects of citrus Huanglongbing fungus on different developmental stages of citrus psyllids, characterized in that, The internal reference gene is Actin, which is used as a correction gene. The forward and reverse primers of the internal reference gene Actin are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

3. The application according to claim 1 or 2, characterized in that, The nucleotide sequence of the internal reference gene Actin is shown in SEQ ID NO.

3.

4. The application according to claim 1 or 2, characterized in that, The citrus psyllids include both adult citrus psyllids carrying the pathogen and healthy adults.

5. The application according to claim 1 or 2, characterized in that, The citrus psyllids include both female and male citrus psyllids.

6. The application according to claim 1 or 2, characterized in that, The citrus psyllids include adult citrus psyllids and nymphs.

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