Application of ARF1 as an internal reference gene in real-time quantitative PCR detection in Western honeybees

Abstract

This invention relates to the field of molecular biology technology, and specifically discloses... ARF1 Application of a reference gene in real-time quantitative PCR detection in Western honeybees. This invention has found that... ARF1 It can be effectively used as an internal reference gene in real-time quantitative PCR detection of adult worker bees in two specific subspecies of Honeybee in different tissues and developmental stages, improving the reliability of detection results and the stability, reliability and reproducibility of gene expression analysis studies. Its wide application lays an important foundation for the study of differential gene expression in Honeybee.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to... ARF1 Application of the gene as an internal reference in real-time quantitative PCR detection in Western honeybees. Background Technology

[0002] Western honeybee ( Apis mellifera Bees are important pollinators and typical social insects, with a mature social division of labor established within the colony. Adult worker bees exhibit age-dependent division of labor, with younger workers primarily engaged in tasks such as cleaning the cells and feeding larvae, while older workers mainly handle fieldwork such as pollination and nectar collection. The transition from nursery to foraging behavior in worker bees is malleable and can be flexibly accelerated, delayed, or reversed according to the needs of the colony. The structure, function, and gene expression of related tissues and organs are adjusted accordingly, such as the development and secretion activity of the hypopharyngeal glands and the sensitivity of antennae to larval pheromones. Furthermore, from the Italian honeybee (Apis spp.) A. m. ligustica The high-yielding royal jelly production line (royal jelly bee) bred in this group exhibits a significantly higher royal jelly production capacity than unselected Italian honeybees and other subspecies of Western honeybees. Changes in gene expression patterns, adapted to the high-yielding royal jelly phenotype, were observed in the hypopharyngeal glands, antennae, and brains of its newly emerged cells, nurse bees, and foraging bees. Scientific questions concerning honeybee gland development, division of labor, and the mechanisms of high royal jelly production have long been research hotspots in insect biology. Accurate quantification of gene expression levels is crucial for identifying relevant functional genes and regulatory networks.

[0003] Real-time quantitative PCR (RT-qPCR) is a crucial technique for quantifying gene expression levels. Accurate quantification relies on the normalization of target gene expression data using stably expressed internal reference genes. Different internal reference genes may exhibit varying expression levels under different treatment conditions, necessitating the screening and validation of internal reference genes stably expressed under specific conditions. While functional gene studies in different subspecies of adult worker bees at various developmental stages and tissues have garnered significant attention, the use of internal reference genes under these conditions still faces challenges requiring improvement. Firstly, previous studies frequently used… Actin and GAPDHDifferent reference genes can affect the comparability of research results, and these reference genes exhibit poor expression stability under certain conditions, such as in honeybees treated with pesticides (Kim S, Cho S, Lee SH. Selection of stable reference genes for real-time quantitative PCR in honeybee pesticide toxicity studies. Journal of Apicultural Research, 2022, 61(1): 26-36). Secondly, existing comparative studies on the expression stability of reference genes in honeybees mainly use the whole bee or simply divide it into three parts: head, thorax, and abdomen, which cannot reflect the true level of reference gene expression in specific tissues. Finally, current screening of reference genes in honeybees is mostly concentrated under single conditions, lacking a comprehensive analysis of the expression stability of reference genes under various conditions such as different subspecies, different developmental stages, and different tissues of adult worker honeybees. Therefore, it is urgent to develop a reference gene that is applicable to all these conditions to provide scientific support for in-depth research on scientific issues such as differences in bee production performance, gland development, and behavioral plasticity. Summary of the Invention

[0004] One of the objectives of this invention is to provide an internal reference gene for genetic research on adult worker bees of the Western honeybee, which can improve the reliability of detection results, and its application.

[0005] This invention provides ARF1 As an application of internal reference genes in real-time quantitative PCR detection of different tissues in adult worker bees of the Western honeybee, the Western honeybee is the Italian honeybee and / or the Carniolan honeybee, and the different tissues include the pharyngeal glands, antennae and / or brain.

[0006] The present invention also provides ARF1 As an internal reference gene, it is used to analyze the different gene expression in different tissues of adult worker bees of the Western honeybee, wherein the Western honeybee is the Italian honeybee and / or the Carniolan honeybee, and the different tissues include the pharyngeal gland, antennae and / or brain.

[0007] This invention proposes for the first time to... ARF1 The gene was used as an internal reference gene for RT-qPCR in different tissues of adult worker bees of a specific subspecies of Honeybee to normalize the expression level of the target gene, which solved the problem of the general use of internal reference genes in Honeybee research under different conditions. At the same time, it laid an important foundation for the study of gene expression in different developmental stages and tissues of adult worker bees of different subspecies of Honeybee.

[0008] This invention also provides amplification ARF1The application of primer sets for real-time quantitative PCR detection of different tissues in adult worker honeybees of the Western honeybee, wherein the Western honeybee is the Italian honeybee and / or the Carniolan honeybee, and the different tissues include the pharyngeal glands, antennae and / or brain.

[0009] This invention also provides amplification ARF1 Application of primer sets in screening or identifying differential genes at different developmental stages of adult worker honeybees in the Western honeybee, wherein the Western honeybee is the Italian honeybee and / or the Carniolan honeybee.

[0010] This invention also provides amplification ARF1 The application of primer sets for the preparation of products for screening or identifying differential genes at different developmental stages of adult worker honeybees, or for the preparation of real-time fluorescence quantitative PCR detection reagents or kits for different tissues of adult worker honeybees, wherein the honeybees are Italian honeybees and / or Carniolan honeybees.

[0011] In the application of this invention, the screening or identification of differential genes at different developmental stages of adult worker bees of the Western honeybee is achieved by detecting the pharyngeal glands, antennae, and / or brain of adult worker bees of the Western honeybee at different developmental stages.

[0012] In the application of this invention, adult worker bees of the Western honeybee at different developmental stages refer to newly emerged bees (emerging from the cell in less than 12 hours), nurse bees (whose heads are inserted into the larval cells for more than 10 seconds), and / or foraging bees (honeybees returning to their nests with pollen in their hind legs).

[0013] In the application of the present invention, ARF1 The nucleotide sequence is shown in SEQ ID NO.21.

[0014] In the application of this invention, the primer set includes: forward primer: 5'-CTGTATGGGACGTAGGTGG-3' and reverse primer: 5'-CATTCATTGCATTTGGGAG-3'.

[0015] The beneficial effects of this invention are at least as follows:

[0016] This invention provides ARF1 The application of genes as internal reference genes in RT-qPCR detection of gene transcriptional expression levels in specific subspecies of Honeybee can be used to study the expression of key differentially expressed genes in different developmental stages and tissues of adult worker bees from different subspecies of Honeybee. This improves the reliability of the detection results and the stability, reliability, and reproducibility of gene expression analysis studies. Its wide application lays an important foundation for the study of differentially expressed genes in Honeybee. Attached Figure Description

[0017] Figure 1The image shows the agarose gel electrophoresis results of the routine PCR products of the candidate internal reference gene.

[0018] Figure 2 For 10 candidate internal reference genes C T Box plot.

[0019] Figure 3 The expression stability ranking of candidate reference genes was calculated using five software programs, where (A) is the result of BestKeeper analysis, (B) is the result of geNorm analysis, (C) is the result of NormFinder analysis, (D) is the result of ΔCt value analysis, and (E) is the result of RefFinder analysis.

[0020] Figure 4 For the target gene Mblk1 and MRJP2 The validation results for the stability of the screening internal reference gene, where (A) is... MRJP2 The relative expression levels, (B) are... Mblk1 Relative expression levels. Detailed Implementation

[0021] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0023] Example 1

[0024] This embodiment provides a method for screening reference genes in Western honeybees, including the following steps:

[0025] 1. Experimental materials

[0026] All bee materials were obtained from the experimental apiary of the Institute of Apiculture, Chinese Academy of Agricultural Sciences, using dwarf bees. A. m. ligustica and Carniola bees A. m. carnicaNewly emerged bees, nurse bees, and foraging bees were collected from three bee colonies with roughly the same population strength. Adult worker bees whose heads were inserted into the larval cells for more than 10 seconds were collected as nurse bees, and foraging bees with pollen baskets in their hind legs were collected at the hive entrance as foraging bees. Capped combs about to emerge from the colonies were transferred to a constant temperature incubator (34℃ ± 1℃, 50% relative humidity) to collect newly emerged adult worker bees (less than 12 hours old). The collected adult worker bee samples were immediately frozen in liquid nitrogen and stored at -80℃ for RNA extraction.

[0027] 2. Total RNA extraction and cDNA synthesis

[0028] Frozen worker bee samples were placed on ice, and brain, pharyngeal gland, and antennae samples were dissected. Five biological replicates were formed by mixing brains from 10 bees, pharyngeal glands from 5 bees, and antennae from 18 bees, with five replicates for each tissue. RNA was extracted using the TRIzol method, and RNA concentration and purity were determined using a NanoDrop 2000 ultraviolet spectrophotometer. One μg of RNA was used to synthesize the first strand of cDNA using the PrimeScript™ RT reagent Kit (Perfect Real Time). The synthesized product was stored at -20 °C for later use.

[0029] 3. Selection of internal reference genes and synthesis and validation of primers

[0030] Ten candidate internal reference genes were selected from those commonly used in Western honeybees and other insects. Actin , EF1 , RPS18 , GAPDH , RPS5 , α-TUB , Rab1 , ARF1 , RP49 , RPL32 Nucleotide sequence information for 10 internal reference genes was obtained from the NCBI database, and primers for these genes were precisely designed using Primer Premier 5. TaKaRa Taq was then used. TMThe amplification specificity of 10 candidate internal control genes was verified by PCR, using cDNA as the PCR template. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 15 s, for 35 cycles. The reaction mixture consisted of: TB Green Premix Ex Tag 11 (Tli RNaseH Plus), 10 μl; forward primer (10 μM), 1 μl; reverse primer (10 μM), 1 μl; DNA template, 2 μl; and enzyme-free sterile water, 6 μl. After obtaining the amplification products, 1% agarose gel electrophoresis was performed to determine the amplification specificity of the primers. The agarose gel containing the target band was purified using the AxyPrep DNA Gel Extraction Kit, and the purified product was subcloned into the T vector using the pMD™ 19-T vector cloning kit. The plasmid was then transformed into Trans1-T1 chemicompetent cells. Plasmid DNA was isolated using the AxyPrep™ Plasmid Miniprep kit to calculate amplification efficiency as a template for RT-qPCR. A standard curve was constructed containing five concentration gradients at 10-fold dilutions to calculate the amplification efficiency. After obtaining the standard curve, the amplification efficiency value for each gene was calculated based on the slope of the standard curve using the formula: E = 10^2 / 20^2. −1 / 斜率 The agarose gel electrophoresis results of the candidate internal reference gene's routine PCR products are shown in the figure. Figure 1 The primer sequences (SEQ ID No. 1-20) and amplification efficiencies of the 10 candidate internal control genes are shown in Table 1. All PCR products showed single bands on 1% agarose gels, with bands of the expected size and no primer dimers. Furthermore, the amplified bands were verified as correct by Sanger sequencing. In summary, the primers for all candidate internal control genes exhibited high specificity, meeting the conditions for RT-qPCR experiments. In the primer amplification efficiency calculation, the linear regression coefficients (R²) of the 10 candidate internal control genes were used. 2 All values ​​were >0.990, and the amplification efficiency ranged from 87.2% to 114.5%.

[0031] Table 1

[0032]

[0033] 4. RT-qPCR

[0034] All RT-qPCR reactions were quantitatively analyzed using a BIOER FQD 96A Linegene 9600 Plus in a three-step fluorescence method for the 10 candidate internal reference genes in all cDNA templates obtained in step 2. TB Green was used. ®RT-qPCR analysis was performed using Premix ExTaq™ II (Tli RNaseH Plus) under the following conditions: pre-denaturation (95℃ for 30 s), PCR (95℃ for 5 s, 55℃ for 30 s, 72℃ for 30 s, 40 cycles), and melting (95℃ for 5 s, 60℃ for 1 min). The specificity of each primer was verified by melt curve analysis. The cycle threshold (C) was determined at the same fluorescence threshold line. T The C value for each gene is obtained by calculating the arithmetic mean of repeated iterations using computational techniques. T Value. 10 candidate internal reference genes C T See the box plot. Figure 2 .

[0035] This invention uses a cyclic threshold (C) T This reflects the expression levels of 10 candidate reference genes at different developmental stages and in different tissues of two honeybee subspecies. The analysis results show that the C of most reference genes... T The values ​​ranged from 16 to 24, with variations in expression abundance among different internal reference genes. The gene with the highest expression level was... Actin Its average C T The value was 17.01, while the lowest expression level was the average C. T The value is 23.79 α-TUB Among the 10 candidate internal reference genes, α-TUB , Actin and GAPDH C T The values ​​varied considerably, with ranges of 9.62, 9.45, and 9.12 respectively; while ARF1 C T The range of the values ​​is the smallest, at 5.4. Therefore, α-TUB , Actin and GAPDH The expression stability is the worst under the current experimental conditions, while ARF1 The expression is more stable.

[0036] RT-qPCR was used to identify the C values ​​of 10 candidate genes at different developmental stages and in different tissues. T Values, organize the original C T The values ​​were determined using gene stability evaluation software (geNorm, NormFinder, BestKeeper) and ΔC. T The stability of 10 candidate reference genes was comprehensively evaluated using the online analysis tool RefFinder, and the optimal reference genes for two subspecies of Honeybee in different developmental stages and tissues were selected. The ranking of expression stability of candidate reference genes calculated by five software programs is shown below. Figure 3 The analysis results of the five algorithms are shown in Table 2. The specific analysis methods are as follows:

[0037] (1) BestKeeper analysis

[0038] The algorithm considers C T The smaller the standard deviation (SD) of the value, the better the stability of the internal reference gene, and vice versa. BestKeeper identification. EF1 The gene is the most stable gene, with an SD value of 0.99. α-TUB The gene with the lowest stability has an SD value of 1.84. The average stability of the genes is ranked as follows: EF1 > ARF1 > RPS5 > RP49 > RPL32 > RPS18 > Rab1 > Actin > GAPDH > α-TUB See details Figure 3 (A) in the middle.

[0039] (2) geNorm analysis

[0040] The geNorm program screens internal reference genes for those with better stability by calculating the M value of each gene's stability. Using M=1.5 as the threshold, the smaller the M value, the better the stability of the internal reference gene, and vice versa. The geNorm results show that the stability ranking of the internal reference genes is as follows: RPL32 / RP49 > RPS5 > EF1 > ARF1 > RPS18 > Rab1 > Actin > GAPDH > α-TUB See details Figure 3 (B) in the analysis shows that... RPL32 and RP49 These are the two most stable internal reference genes, with an M value of 0.603. α-TUB The gene has the lowest stability, with an M value of 1.128.

[0041] (3) NormFinder analysis

[0042] NormFinder calculates stability values ​​based on the expression levels of candidate genes to determine the most stable internal reference gene. Using the NormFinder algorithm, the gene with the lowest stability value is identified as the most stable gene. NormFinder analysis results show that... ARF1 The gene exhibited the strongest stability, with a stability value of 0.317, followed by... RPL32 (Stability value 0.385)Rab1 (Stability value 0.421) RP49 (Stability value 0.458). The remaining genes are as follows: RPS18 , RPS5 , EF1 , Actin , GAPDH , α-TUB See details Figure 3 (C) in the middle.

[0043] (4) ΔCt value analysis

[0044] ΔC T The algorithm evaluates the stability of the internal reference gene using the mean standard deviation (mSD). The magnitude of mSD indicates the stability of the internal reference gene; a smaller value indicates a more stable gene, and vice versa. In this invention, ΔC T Value analysis results indicate that the most stable gene is ARF1 The mSD is 0.948. α-TUB The gene exhibited the worst stability, with an mSD of 1.564. (See details...) Figure 3 (D) in the middle.

[0045] (5) RefFinder analysis

[0046] To avoid errors caused by evaluating a single internal reference gene, RefFinder integrates the four algorithms mentioned above into a network-based tool for assessing the stability and reliability of internal reference genes. Based on the gene stability ranking obtained from the four algorithms, RefFinder assigns appropriate weights to each gene and calculates the geometric mean of these weights to arrive at the final ranking. According to the analysis results, RefFinder indicates... ARF1 Genes are the most stable genes, with a geometric mean of 1.778. The average stability of each gene is ranked as follows: ARF1 > RPL32 > RP49 > EF1 > RPS5 > Rab1 > RPS18 > Actin > GAPDH > α-TUB See details Figure 3 (E) in the middle.

[0047] Table 2

[0048]

[0049] After evaluation of five algorithms, including RefFinder, ΔCt value analysis, and NormFinder results, it was found that... ARF1It ranked first among 10 candidate internal reference genes. Meanwhile, BestKeeper showed... ARF1 It is the second most stable gene. Therefore, it is believed... ARF1 The gene is the optimal internal reference gene in different developmental stages and tissues, and α-TUB , GAPDH , Actin It ranked last in all five algorithm analyses, therefore it is not suitable as an internal reference gene for RT-qPCR.

[0050] 5. Verification of the stability of the internal reference gene

[0051] To verify the stability of the screened internal reference genes, this invention uses RT-qPCR to detect differentially expressed genes in the hypopharyngeal glands at different developmental stages. Mblk1 (NM_001011629) and MRJP2 The expression patterns of (NM_001011580) (studies have shown that both are highly expressed in the hypopharyngeal glands of foraging bees and nurse bees, respectively) were investigated. Using cDNA from the hypopharyngeal glands of different developmental stages (newly emerged bees, foraging bees, and nurse bees) as templates, RT-qPCR was used to select the two candidate internal control genes with the best stability (NM_001011580). ARF1 , RPL32 ) and two unstable internal reference genes ( GAPDH , α-TUB To provide a control, the target genes were normalized separately, and 2... -ΔΔCT Legal analysis Mblk1 and MRJP2 The relative expression levels of the gene in the hypopharyngeal glands at different developmental stages were analyzed to verify the stability of the internal control gene. The reaction system and procedure are as described above. Target gene Mblk1 and MRJP2 The validation results for the stability of the screening internal reference gene are shown in [link to relevant documentation]. Figure 4 .

[0052] Validation results of the selected internal reference genes showed that different internal reference genes could affect the expression analysis of target genes. Using genes with better stability... ARF1 or RPL32 When used as an internal reference gene MRJP2 The expression pattern is consistent with existing literature ([1] Feng M, Fang Y, Li J. Proteomic analysis of honeybee worker ( Apis mellifera)hypopharyngeal gland development. BMC Genomics, 2009, 10: 1-12. [2]Ueno T, Nakaoka T, Takeuchi H, et al. Differential gene expression in thehypopharyngeal glands of worker honeybees ( Apis mellifera The results reported in *L.* (associated with an age-dependent role change. Zoological Science, 2009, 26(8): 557-563) are consistent with those of the study; however, the results using less stable methods are inconsistent. GAPDH or α-TUB hour, MRJP2 The relatively high dispersion of expression levels confirms the poor stability of the two internal reference genes. (See details...) Figure 4 (A) in the middle. For Mblk1 For details, see Figure 4 In (B), when using GAPDH or α-TUB When used as an internal reference gene, the expression level of the target gene is relatively high. ARF1 or RPL32 When used as an internal reference gene, significant differences emerge, deviating from the conclusions of the aforementioned studies. Therefore, unstable internal reference genes can lead to deviations in the expression levels of the target gene, affecting experimental accuracy, further demonstrating… ARF1 The reliability of the internal reference gene.

[0053] In summary, based on RT-qPCR results, this invention utilizes NormFinder, geNorm, BestKeeper, ΔCt, and RefFinder to reveal the expression stability of 10 candidate internal reference genes in different developmental stages and tissues of adult worker bees from two subspecies. Experimental results show that... ARF1 The gene exhibits the most stable expression across all samples and can serve as the optimal internal reference gene for RT-qPCR. Applying the normalization strategy proposed in this invention, accurate quantitative gene expression results can be obtained, thereby facilitating research on gene function in different developmental stages and tissues of adult worker bees in Western honeybees.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. ARF1 As an application of internal reference genes in real-time quantitative PCR detection of different tissues of adult worker honeybees in Western honeybees, the Western honeybee is Carniolan honeybee, and the different tissues are pharyngeal glands, antennae and / or brain; ARF1 The nucleotide sequence is shown in SEQ ID NO.

21.

2. ARF1 As an internal reference gene, it is used to analyze the different gene expression in different tissues of adult worker bees of the Western honeybee, wherein the Western honeybee is Carniolan bee, and the different tissues are the pharyngeal gland, antennae and / or brain; ARF1 The nucleotide sequence is shown in SEQ ID NO. 21.

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