Real-time quantitative PCR reference genes in different tissues of mangrove goby, red-chinned goby (Odon valenciennei) and their screening method and application

Abstract

This invention discloses real-time quantitative PCR internal reference genes in different tissues of *Mulletodon zhuyi*, their screening methods, and applications. The invention involves collecting brain, gill, gonad, intestinal, and liver tissues from *Mulletodon zhuyi* under normal conditions and under conditions of exposure to bisphenol A, cadmium, and sulfamethoxazole, respectively. A systematic evaluation of 17 candidate internal reference genes was conducted. The results showed that the stable internal reference genes in brain, gill, intestinal, and liver tissues were, in descending order: rpl7 and rps4x , eif3h and stau1 , hsp90b and stau1 , rpl7 and mrpl3 The combination of these genes is recommended; four internal reference genes are recommended for use in gonads; and six internal reference genes are recommended for use in cross-tissue analysis. This invention lays an important methodological foundation for qRT-PCR research on *Mulletodon zhuyi* goby, and is applicable to research related to endocrine disruption, reproductive physiology, and marine ecotoxicology.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to real-time fluorescence quantitative PCR internal reference genes and their screening methods and applications in different tissues of the Chinese mullet goby. Background Technology

[0002] Zhu's mullet goby ( Mugilogobius chulae *Mulletus zhuyis* is a small, warm-water, benthic goby widely distributed in estuaries and brackish waters of Southeast Asia and the western Pacific. Due to its small size, short lifespan, ease of laboratory culture, and suitability for assessing pollutant toxicity, this species has gradually become a marine experimental fish of significant research value. With the establishment of artificial breeding techniques, closed laboratory populations, and supporting aquaculture systems, *Mulletus zhuyis* is increasingly used in environmental toxicology, physiology, and related biological process research, particularly in studying the toxic effects and mechanisms of environmental pollutants in estuarine ecosystems.

[0003] Real-time quantitative PCR (qRT-PCR) is an important research tool in environmental toxicology and physiology due to its high sensitivity, specificity, reproducibility, and ease of operation. It is one of the most widely used methods in gene expression analysis. Although the application of *Mulletodon spp.* in toxicological research is increasing, a systematic evaluation of suitable internal reference genes for this species under pollutant exposure conditions remains lacking. Accurate qRT-PCR analysis relies on the normalization of stable internal reference genes. Despite... β-actin , gapdh and 18S rRNA Traditional housekeeping genes are widely used for normalization, but numerous studies have shown that the expression stability of these genes can vary significantly across different species, tissues, developmental stages, sexes, and experimental treatments. For example, in the yellow-banded trevally... Pseudocaranx dentex Among the 10 types of adult fish tissues, the optimal internal reference gene is: rpl13 and ef-1α ,and β-actin and gapdh Its stability is relatively poor, and it adopts rpl13 and ef-1α When normalizing individually or jointly, myod1 Their expression patterns are relatively consistent, while β2m , β-actin and gapdh This would introduce significant bias. (Regarding Philippine clams) Ruditapes philippinarum The optimal combination of internal reference genes varies depending on the developmental stage and adult tissue. Adult tissue analysis recommends using two internal reference genes, while developmental stage analysis requires three. β-actin In both cases, it was identified as the most unstable gene. Similarly, under WSSV infection and matrine treatment conditions, *Procambarus clarkii*... P.clarkiiThe most stable internal reference gene also varies from tissue to tissue, among which eif It has the highest stability in the intestines, muscles, gills, and brain, while tbp The optimal selection was found in the hepatopancreas and gonads, with each tissue requiring only two internal reference genes. These results also indicate that internal reference genes cannot simply follow the common selection methods used in other species or other research systems, but should be empirically screened based on specific species and experimental contexts.

[0004] In *Mulletus zhurensis*, different types of pollutants may affect basal transcriptional homeostasis in different ways, and significant tissue heterogeneity may further affect the expression stability of candidate reference genes. Directly using reference genes commonly used in other fish species may not be suitable for accurate normalization analysis of *Mulletus zhurensis*. Therefore, screening and validating suitable reference genes is a necessary prerequisite for obtaining reliable transcriptional expression analysis results in different tissues of *Mulletus zhurensis* under different treatment conditions. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a real-time fluorescence quantitative PCR internal reference gene for different tissues of *Mulletus edulis*.

[0006] The second objective of this invention is to provide real-time quantitative PCR detection primers for internal reference genes of different tissues of the above-mentioned mullet goby.

[0007] A third objective of this invention is to provide a kit comprising the above-described real-time quantitative PCR detection primers.

[0008] The fourth objective of this invention is to provide the application of the above-mentioned real-time quantitative PCR internal reference gene of *Mulletus edulis* or the above-mentioned real-time quantitative PCR detection primers or the above-mentioned kit in the detection of gene expression levels in different tissues of *Mulletus edulis*.

[0009] The fifth objective of this invention is to provide a method for detecting gene expression levels in different tissues of the mullet goby.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention selected sexually mature female and male *Mulletodon zhubryanus* and collected brain, gill, gonad, intestinal, and liver tissues for analysis under normal conditions and under exposure to bisphenol A (BPA), cadmium (Cd), and sulfadiazine (SMX), respectively. Seventeen candidate internal reference genes were systematically evaluated using qRT-PCR to screen stable internal reference genes suitable for normalized analysis of all samples and individual tissues. Primer specificity and amplification performance were validated by agarose gel electrophoresis, melting curve analysis, sequencing, and standard curve analysis. The expression stability of candidate genes was evaluated using the comparative ΔCt method, NormFinder, geNorm, BestKeeper, and the comprehensive sequencing tool RefFinder. The results showed that the stability of the *Mulletodon zhubryanus* internal reference genes exhibited significant tissue specificity. In the pooled analysis of all samples… eif3h , rpl7 , rps4x , stau1 and ef1y It has the highest stability, while ube2 , hprt1l and aldob The stability was lowest. Further tissue-specific analysis showed that the optimal internal reference genes were not the same for the brain, gills, gonads, intestines, and liver. In brain tissue, the gene with higher stability was... actb2 , rps4x , eif3h and rpl7 In gill tissue, genes with higher stability are eif3h , stau1 , dera , b2m and mrpl3 In the gonads, ef1y , dera , eif3h , cyclophilin and stau1 It has high stability; in the intestine, mrpl3 , ef1y , stau 1. hsp90b and eif3h The stability is relatively high; in the liver, the gene with the highest stability is... rpl7 , ef1y , mrpl3 , eif3h and rps4x Using geNorm paired variant analysis, two internal reference genes are recommended for use in the brain, gills, intestine, and liver. The recommended combinations of internal reference genes are: brain tissue... rpl7 + rps4x gill tissue eif3h + stau1 Intestinal tissue hsp90b + stau1 ,liverrpl7 + mrpl3 Four internal reference genes are recommended for use in gonads. mrpl3 , ef1y , dera and staul In cross-organizational analyses, it is recommended to use 6 internal reference genes. rpl7, rps4x, eif3h, stau1, ef1y and rad18 The above results provide the first systematic basis for screening internal reference genes in *Mulletus zhurensis*, provide a basis for the accurate normalization of gene expression in *Mulletus zhurensis*, and lay an important methodological foundation for future qRT-PCR research on this species, especially applicable to endocrine disruption, reproductive physiology, and marine ecotoxicology research.

[0011] Therefore, this invention provides real-time quantitative PCR internal reference genes for different tissues of *Mulletodon zhuyi* goby, wherein the stable internal reference gene in the brain tissue is... rpl7 and rps4x The combination; the stable internal reference gene in gill tissue is eif3h and stau1 The combination; the stable internal reference gene in intestinal tissue is hsp90b and stau1 The combination; the stable internal reference gene in the liver is rpl7 and mrpl3 The combination; the stable internal reference gene in the gonads is mrpl3 , ef1y , dera and staul The combination; the stable internal reference gene in multi-tissue mixtures is rpl7, rps4x, eif3h, stau1, ef1y and rad18 The combination; the rps4x The nucleotide sequence is shown in SEQ ID No. 4; eif3h The nucleotide sequence is shown in SEQ ID No. 1; stau1 The nucleotide sequence is shown in SEQ ID No. 2; hsp90b The nucleotide sequence is shown in SEQ ID No. 7; rpl7 The nucleotide sequence is shown in SEQ ID No. 5; mrpl3 The nucleotide sequence is shown in SEQ ID No. 10; ef1y The nucleotide sequence is shown in SEQ ID No. 3; dera The nucleotide sequence is shown in SEQ ID No. 12; rad18 The nucleotide sequence is shown in SEQ ID No. 6.

[0012] Furthermore, the screening method for the internal reference gene includes the following steps: S1. Extract different tissue samples from the Chinese mullet goby, extract total RNA and reverse transcribe it to obtain cDNA; S2. Screen candidate internal reference genes and design qRCR primer pairs based on genome sequences; S3. Using the cDNA obtained in step S1 as a template, perform qRCR experiments with the qRCR primer pairs obtained in step S2, plot standard curves and melting curves, and determine the amplification efficiency and correlation coefficient of the qRCR primer pairs of the candidate internal reference gene. S4. Based on the expression CT values ​​of candidate internal reference genes in different tissue samples, perform expression stability analysis to determine the internal reference genes.

[0013] Furthermore, in step S2, the candidate reference genes are selected based on the functional gene annotation of the mullet goatfish genome and transcriptome data of the gonads, liver, brain, intestines and gills.

[0014] Furthermore, in step S3, the amplification efficiency of the qRCR primer pair is between 90% and 105%, and the correlation coefficient is between 0.99 and 1.

[0015] Furthermore, in step S4, the expression stability analysis is performed by using the ΔCt method, BestKeeper, NormFinder, geNorm, and RefFinder to conduct a comprehensive analysis of the candidate reference genes.

[0016] This invention also provides real-time quantitative PCR detection primers for the internal reference gene of different tissues of *Mulletodon spp.* and *Goblinus spp.*. rps4x The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 46-47; eif3h The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 40-41; stau1 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 50-51; hsp90b The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 44-45; rpl7 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 38-39; mrpl3 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 28-29; ef1y The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 42-43; dera The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 22-23; rad18The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 26-27.

[0017] The present invention also provides a kit comprising the above-mentioned real-time quantitative PCR detection primers.

[0018] The present invention also provides the application of the above-mentioned real-time quantitative PCR internal reference gene of *Mulletus edulis* or the above-mentioned real-time quantitative PCR detection primers or the above-mentioned kit in the real-time quantitative PCR for detecting gene expression levels in different tissues of *Mulletus edulis*.

[0019] Furthermore, the different tissues of *Mulletus zhurensis* are different tissues of *Mulletus zhurensis* under normal conditions or under stress conditions; the stress conditions are treatment with bisphenol A, cadmium, or sulfadiazine.

[0020] The present invention also provides a method for detecting gene expression levels in different tissues of *Mulletus zhurensis*, wherein the method employs real-time quantitative PCR and selects the aforementioned *Mulletus zhurensis* real-time quantitative PCR internal reference gene as the internal reference gene.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides real-time quantitative PCR reference genes for different tissues of *Mulletodon zhuyi*, along with their screening methods and applications. Sexually mature female and male *Mulletodon zhuyi* were selected, and brain, gill, gonad, intestinal, and liver tissues were collected for analysis under normal conditions and under conditions of exposure to bisphenol A, cadmium, and sulfadiazine, respectively. A systematic evaluation of 17 candidate reference genes was performed using qRT-PCR. The results showed that the stable reference gene in brain tissue was… rpl7 and rps4x The combination; the stable internal reference gene in gill tissue is eif3h and stau1 The combination; the stable internal reference gene in intestinal tissue is hsp90 b and stau1 The combination; the stable internal reference gene in the liver is rpl7 and mrpl3 The combination of internal reference genes is recommended; four internal reference genes are recommended for use in gonads; and six internal reference genes are recommended for use in cross-tissue analysis. This invention provides the first systematic basis for screening internal reference genes in *Mulletodon zhuyi*, provides a basis for the accurate normalization of gene expression in *Mulletodon zhuyi*, and lays an important methodological foundation for future qRT-PCR research on this species, especially applicable to endocrine disruption, reproductive physiology, and marine ecotoxicology research. Attached Figure Description

[0022] Figure 1 Agarose gel electrophoresis image of the PCR product of the candidate reference gene.

[0023] Figure 2 Melting curves for 17 candidate internal reference genes are shown. Note: Temperature is plotted against -ΔF / ΔT (fluorescence change / temperature change) to more clearly show the melting characteristics of each internal reference gene.

[0024] Figure 3 Standard curves for 17 candidate internal reference genes. Note: Amplification efficiency is determined by the slope of the log-linear portion of the calibration curve (Y function coefficient); the horizontal axis represents the initial template concentration (independent variable, i.e., the logarithmic value of 4- or 5-fold serial dilutions of mixed cDNA), and the vertical axis represents the corresponding Ct value (dependent variable).

[0025] Figure 4 The distribution of Ct values ​​for 17 candidate internal reference genes in 5 tissues under different treatment conditions is shown. Note: Each point represents the average Ct value of 3 replicates in a tissue under a certain treatment condition. The horizontal line represents the average Ct value of all samples under the same conditions, and the vertical line represents the standard deviation (SD).

[0026] Figure 5 The Ct values ​​of 17 candidate internal reference genes within the same tissue were compared.

[0027] Figure 6 This represents the optimal number of internal reference genes recommended by the geNorm software. Note: V2 / 3 indicates that the optimal number of internal reference genes is 2, V3 / 4 indicates that it is recommended to use 3 internal reference genes, and so on; a Vn / Vn+1 value below the 0.15 threshold (green dashed line) indicates that using n normalized internal reference genes is sufficient to meet the analysis requirements. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and 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.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1: Experimental Fish Farming and Pollutant Exposure Design Sexually mature mullet goby ( M.chulaeThe fish (18 months old) were provided by the Guangdong Institute of Biotechnology (Guangzhou, China). The average body length and weight of females were 3.44 cm and 0.97 g, respectively, while those of males were 3.97 cm and 1.46 g, respectively. The experimental fish were housed in a small-scale recirculating artificial seawater system under the following conditions: salinity 15 ppt, water temperature 26±1℃, and a photoperiod of 14 h light: 10 h darkness. After two weeks of acclimatization, healthy individuals were randomly divided into four groups: a solvent control group containing 0.1‰ DMSO, a BPA (bisphenol A) treatment group (200 μg / L), a Cd (cadmium) treatment group (100 μg / L), and an SMX (sulfamethoxazole) treatment group (1 mg / L). Each group was exposed for 7 days.

[0031] All animal experimental procedures followed the "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Animal Research and Ethics Committee of Guangdong Ocean University (Approval No.: 201903003).

[0032] Example 2: Selection of candidate internal reference genes and primer validation Following the exposure treatment in Example 1, four female and four male fish were randomly selected from each group for molecular analysis. Brain, gill, gonad, intestine, and liver tissues were dissected and immediately placed in RNA preservation solution for subsequent extraction of genomic DNA and total RNA. Gonad samples included testes and ovaries from sexually mature fish.

[0033] 1. Total RNA extraction and first-strand cDNA synthesis Following the kit instructions, genomic DNA and total RNA were simultaneously extracted from various tissue samples using an RNA / DNA co-extraction kit (Beyotime Biotechnology, Shanghai, China). RNA concentration and purity were detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was verified by 1.0% agarose gel electrophoresis. First-strand cDNA synthesis was performed using TransScript® All-in-One First-Strand cDNASynthesis SuperMix (TransGen Biotech, Beijing, China) as per the kit instructions. The resulting cDNA was diluted 10-fold with nuclease-free water and stored at -20°C for later use.

[0034] 2. Selection of candidate internal reference genes and primer validation Based on the genome annotation results of *Mulletodon zhuyi*, 17 candidate internal reference genes commonly used in fish and identifiable in this species were selected for stability evaluation. Gene-specific primers (Table 1) were designed using Primer Premier 5.0 software (Premier Biosoft International, USA) and synthesized by GENWIZ (Suzhou, China). Using the mixed cDNA of all experimental samples as a template, conventional PCR was first used to preliminarily detect primer specificity. Simultaneously, genomic DNA and ddH2O were used as templates to assess potential non-specific amplification and contamination. The total volume of the PCR reaction system was 20 μL, including: 10.0 μL 2×TransTaq®-T PCR SuperMix (with dye) (TransGen Biotech, Beijing, China), 7.0 μL ddH2O, 0.5 μL each of primers (10 μM), and 2.0 μL template. PCR amplification products were separated by 1.5% agarose gel electrophoresis to verify whether the amplified fragment size and specificity met expectations.

[0035] Table 1. Information on candidate genes and their primers

[0036] 3. Real-time quantitative PCR and amplification efficiency analysis A standard curve was established using serial dilutions of mixed cDNA (4-fold or 5-fold) to evaluate the amplification performance of each primer. The total volume of the qPCR reaction was 10 μL, including: 5.0 μL of 2×PerfectStart. TM Green SuperMix (TransGenBiotech, Beijing, China), 2.6 μL ddH2O, 0.2 μL primers (10 μM each), and 2.0 μL cDNA template were used. A template-free control was included in each reaction. qPCR was performed on a LightCycler 96 Real-Time PCR System (Roche, Switzerland) with the following amplification program: 94℃ pre-denaturation for 30 s; followed by 40 cycles: 94℃ for 5 s, 60℃ for 15 s, and 72℃ for 10 s. Melting curve analysis was performed after amplification using the following program: 95℃ for 10 s, 65℃ for 1 min, and 95℃ for 1 s, to verify the specificity of the amplified products. Each reaction was performed in triplicate. The slope of the standard curve was used, calculated using the formula E = (10^(-1 / slope)). 1) Calculate the amplification efficiency by multiplying by 100%, and simultaneously calculate the correlation coefficient (R²).2 Based on the standard curve results, all cDNA samples were diluted 20-fold and used for subsequent qPCR quantitative analysis of 17 candidate internal reference genes. All qPCR reactions were performed in triplicate.

[0037] II. Experimental Results 1. Identification of primer amplification efficiency Agarose gel electrophoresis results are as follows Figure 1 As shown, each primer pair amplified a single band of the expected size.

[0038] During qPCR quantitative analysis, the melting curve results are as follows: Figure 2 As shown, the melting curves all exhibited a single peak, further validating the specificity of the amplification. Sequencing and BLAST analysis results indicated that each amplified fragment corresponded to the expected target gene (Table 2). The amplified fragment lengths of the 17 candidate genes ranged from 91 to 194 bp, with amplification efficiencies of 90.2% to 104.3%, and correlation coefficients (R0 and R0) were [not specified in the original text]. 2 The values ​​are 0.9901-0.9998 (Table 1); Figure 3 This indicates that all primers can be used for subsequent expression stability analysis.

[0039] Table 2. Amplification fragment sequences of primers corresponding to 17 candidate genes.

[0040] 2. Expression characteristics of candidate internal reference genes under different pollutant treatment conditions and in different tissues The expression levels of 17 candidate internal control genes were expressed as cycle thresholds (Ct) obtained by qPCR. The expression levels of these 17 candidate internal control genes were detected in five tissue samples (brain, gills, gonads, intestine, and liver) from the control, BPA-treated, Cd-treated, and SMX-treated groups. Figure 4 As shown, the Ct values ​​of different candidate genes vary considerably, indicating significant differences in their transcript abundance. The average Ct value range for the 17 candidate genes is as follows: actb2 The lowest was 12.38; ube2 The highest was 33.93. Among the 17 genes, actb2 and b2m The relatively low Ct value indicates a high transcript abundance; while ube2 , mrpl3 and hprt1l The relatively high Ct value indicates a low expression level. The dispersion of Ct values ​​also varies significantly among different candidate genes. rpl7 and stau1 The Ct fluctuation was the smallest among all samples, while ube2 andaldob The largest fluctuations were observed, indicating significant differences in expression stability among different candidate genes. Furthermore, within the same tissue, the changes in Ct values ​​under different contaminant treatment conditions were generally smaller than the changes between different tissues. Figure 5 This indicates that tissue type is an important factor affecting the differences in the expression of internal reference genes.

[0041] Example 3: Stability analysis of candidate internal reference genes I. Experimental Methods Gene expression stability was evaluated on two levels: first, a pooled analysis of all samples (DMSO solvent control group, BPA treatment group, Cd treatment group, and SMX treatment group); and second, individual analysis of each tissue (different treatments included the same tissue from the control group). RefFinder (http: / / blooge.cn / RefFinder / ) was used for comprehensive stability analysis. This tool integrates four commonly used algorithms: the comparison ΔCt method, NormFinder, geNorm, and BestKeeper. RefFinder assigns a weight to each gene based on the ranking results of each algorithm and obtains the final comprehensive ranking by calculating the geometric mean.

[0042] Furthermore, geNorm (https: / / seqyuan.shinyapps.io / seqyuan_prosper / ) was used to evaluate the optimal number of internal parameters required for accurate normalization under different analytical scenarios. The paired variance value Vn / Vn+1 was calculated to determine whether adding an additional internal reference gene could significantly improve normalization accuracy, with 0.15 used as a commonly used threshold.

[0043] II. Experimental Results The expression stability of 17 candidate internal reference genes was evaluated under conditions of pooling all samples and analyzing each tissue separately. The results are shown in Tables 3-4. The specific analysis results are as follows: Table 3. Expression stability analysis of reference genes in *Mulletodon zhuyi* goby

[0044] Table 4. Stability analysis values ​​of 17 candidate internal reference genes calculated using four algorithms under different conditions.

[0045]

[0046] 1. ΔCt method analysis According to the comparison ΔCt method, a lower mSD value indicates higher expression stability. In the pooled analysis of all samples, eif3h The most stable gene was identified, followed by the following:stau1 , ef1y , rps4x and rpl7 Tissue-specific analysis showed that... eif3h It has the highest stability in the brain, gills, and liver, while ef1y It ranks first in the gonads and intestines. Conversely, aldob , hprt1l and ube2 Genes are usually classified as having poor stability.

[0047] 2. NormFinder Analysis NormFinder sorts candidate genes based on their stability value (SV), with lower SV values ​​indicating higher stability. In the analysis of all samples, eif3h , stau1 and rps4x These are among the top three most stable genes. In individual tissue analyses, the most stable gene in brain tissue is... actb2 , eif3h and rps4x ; gill tissue is eif3h , stau1 and dera ; in the gonads mrpl3 , dera and ef1y The intestines are mrpl3 , eif3h and ef1y ; in the liver ef1y , mrpl3 and rpl7 .

[0048] 3. geNorm Analysis geNorm ranked candidate genes based on their average expression stability value (M), with a lower M value indicating higher stability. In the pooled analysis of all samples, rpl7 and rps4x The most stable gene combination was identified, followed by eif3h and stau1 In individual tissue analyses, the most stable gene combination in brain tissue was: rpl7 and rps4x In the gill tissue eif3h and stau1 In the gonads mrpl3 and ef1y The intestines are hsp90b and stau1 The liver contains rpl7 and mrpl 3. In contrast, in the pooled analysis of all samples, aldob , hprt1l and ube2It consistently exhibits poor stability.

[0049] 4. BestKeeper Analysis BestKeeper primarily evaluates gene stability based on the standard deviation (SD) and coefficient of variation (CV) of the original Ct values. Compared to the other three algorithms, BestKeeper's ranking results show some differences. In the pooled analysis of all samples and brain tissue, cyclophilin It was identified as the most stable gene; while in the gills, gonads, intestines, and liver, the genes with the highest stability were respectively hsp90b , eif3h , actb2 and ef1y It is worth noting that in the pooled analysis of all samples, only cyclophilin The SD value was less than 1, indicating that the expression variation of the candidate internal reference gene was significantly increased when all tissues were analyzed together.

[0050] 5. RefFinder overall sorting To integrate the results of the four algorithms, RefFinder was used to perform a comprehensive ranking based on the geometric mean of the rankings from each algorithm. In the pooled analysis of all samples, the top 5 genes in terms of stability were: eif3h , rpl7 , rps4x , stau1 and ef1y ,and ube2 , hprt1l and aldo Gene b exhibits the lowest stability. The overall sequencing also shows significant tissue specificity; in brain tissue, genes with higher stability are... actb2 , rps4x , eif3h and rpl7 In gill tissue, the most prominent gene is... eif3h , stau1 , dera , b2m and mrpl3 In the gonads, mrpl3 , ef1y , dera , eif3h , cyclophilin and stau1 It has high stability; in the intestine, mrpl3 , ef1y , stau1 , hsp90b and eif3h The stability is relatively high; in the liver, the gene with the highest stability is... rpl7 , ef1y , mrpl3 , eif3h and rps4xThe above results indicate that there is no single optimal internal reference gene applicable to all tissues in *Mulletodon zhurensis*, and accurate normalization analysis requires targeted screening based on tissue type.

[0051] 6. The number of optimal intrinsic parameters required for normalization Paired variance analysis using geNorm was employed, with Vn / Vn+1=0.15 as the threshold, to determine the optimal amount of intrinsic parameters required for accurate normalization. Figure 6 In the brain, gills, intestine, and liver, the V2 / V3 values ​​were all below 0.15, indicating that reliable normalization can be achieved using two internal control genes in these tissues. Accordingly, the recommended dual internal control combinations are: brain tissue... rpl7 + rps4x gill tissue eif3h + stau1 Intestinal tissue hsp90b + stau1 ,liver rpl7 + mrpl3 However, in the gonads, both V2 / V3 and V3 / V4 were above 0.15, while V4 / V5 fell below the threshold for the first time, indicating that four internal reference genes were required for more rigorous normalization of this tissue. mrpl3 , ef1y , dera and staul (The need for more internal reference genes in gonads may be partly due to the strong heterogeneity of the gonad samples used in this invention, which include both testes from sexually mature males and ovaries from females. Since these two types of gonads differ significantly in cellular composition, developmental state, and transcriptional background, a more robust normalization strategy is needed to correct for their large biological variability.) For the dataset analyzed by pooling all tissues, the paired variance value only fell below 0.15 at V6 / V7, indicating that six internal reference genes are required when comparing gene expression across multiple tissues. rpl7、 rps4x, eif3h, stau1, ef1y and rad18 The above results indicate that, in *Mulletus zhurensis*, the number of intrinsic parameters required for normalization is significantly dependent on the specific analytical scenario, and cross-tissue analysis requires significantly higher robustness to normalization than single-tissue analysis.

[0052] In this invention, a certain degree of ranking difference exists between the ΔCt method, NormFinder, geNorm, and BestKeeper. This is to be expected, as these four algorithms are based on different statistical principles: the ΔCt method evaluates candidate genes by comparing pairwise differences in Ct values; NormFinder considers both intra- and inter-group variation; geNorm focuses on average paired expression stability; and BestKeeper primarily relies on the standard deviation (SD) and coefficient of variation (CV) of the original Ct values. Due to these different evaluation focuses, the ranking results obtained by these algorithms are often not entirely consistent. These comparisons demonstrate the practical significance of using comprehensive ranking tools like RefFinder, as they can reduce the bias introduced by a single algorithm, thus providing a more balanced basis for the final recommendation.

[0053] The significant tissue specificity shown in the results of this invention is biologically reasonable. The brain, gills, gonads, intestines, and liver exhibit significant differences in physiological function and respond differently to pollutant stress. The gills are crucial interfaces for pollutant uptake and ion regulation in aquatic bodies; the intestines and liver play a central role in substance absorption and xenobiotic metabolism; while the brain and gonads are closely related to neuroendocrine and reproductive regulation. Given this physiological context, it is expected that a gene may exhibit higher stability in one tissue and poorer performance in another. Similar tissue-dependent patterns have been reported in *Procambarus clarkii*, *Procambarus clarkii*, and *Mackerelia ensifolia*. These studies all indicate that accurate normalization should employ tissue-specific internal control screening strategies, rather than attempting to find a single internal control gene applicable to all tissues.

[0054] In summary, this invention systematically evaluated the expression stability of 17 candidate internal reference genes in the brain, gills, gonads, intestines, and liver of sexually mature mullet goby under control, BPA, Cd, and SMX exposure conditions. The results showed that the stability of the internal reference genes in mullet goby exhibited significant tissue specificity. Overall, eif3h , rpl7 , ef1y , rps4x , mrpl3 and stau1 It exhibits high stability, while aldob , hprt1l and ube2Generally unsuitable as internal reference genes. The optimal normalization strategy also varies across different analytical scenarios: two internal reference genes are sufficient for brain, gill, intestine, and liver, four are needed for gonads, and six are required for cross-tissue comparisons. This indicates that with increasing biological heterogeneity, the need for robust normalization strategies also increases significantly. In summary, this study provides the first systematic basis for screening internal reference genes in *Mulletodon zhuyi* and lays an important methodological foundation for future qRT-PCR research on this species, particularly applicable to endocrine disruption, reproductive physiology, and marine ecotoxicology studies.

Claims

1. Real-time fluorescence quantitative PCR internal reference genes for different tissues of *Mulletodon zhuyi* goby, characterized in that... The stable internal reference gene in the brain tissue is rpl7 and rps4x The combination; the stable internal reference gene in gill tissue is eif3h and stau1 The combination; the stable internal reference gene in intestinal tissue is hsp90b and stau1 The combination; the stable internal reference gene in the liver is rpl7 and mrpl3 The combination; the stable internal reference gene in the gonads is mrpl3 , ef1y , dera and staul The combination; the stable internal reference gene in multi-tissue mixtures is rpl7, rps4x, eif3h, stau1, ef1y and rad18 The combination; the rps4x The nucleotide sequence is shown in SEQ ID No. 4; eif3h The nucleotide sequence is shown in SEQ ID No. 1; stau1 The nucleotide sequence is shown in SEQ ID No. 2; HSP90B The nucleotide sequence is shown in SEQ ID No. 7; rpl7 The nucleotide sequence is shown in SEQ ID No. 5; mrpl3 The nucleotide sequence is shown in SEQ ID No. 10; ef1y The nucleotide sequence is shown in SEQ ID No. 3; dera The nucleotide sequence is shown in SEQ ID No. 12; rad18 The nucleotide sequence is shown in SEQ ID No.

6.

2. The real-time fluorescence quantitative PCR internal reference gene for different tissues of *Mulletodon spp.* according to claim 1, characterized in that, The screening method for the internal reference gene includes the following steps: S1. Extract different tissue samples from the Chinese mullet goby, extract total RNA and reverse transcribe it to obtain cDNA; S2. Screen candidate internal reference genes and design qRCR primer pairs based on genome sequences; S3. Using the cDNA obtained in step S1 as a template, perform qRCR experiments with the qRCR primer pairs obtained in step S2, plot standard curves and melting curves, and determine the amplification efficiency and correlation coefficient of the qRCR primer pairs of the candidate internal reference gene. S4. Based on the expression CT values ​​of candidate internal reference genes in different tissue samples, perform expression stability analysis to determine the internal reference genes.

3. The real-time fluorescence quantitative PCR internal reference gene for different tissues of *Mulletodon zhuyi* as described in claim 2, characterized in that, In step S2, the candidate reference genes were selected based on the functional gene annotation of the mullet goatfish genome and transcriptome data from the gonads, liver, brain, intestines, and gills.

4. The real-time fluorescence quantitative PCR internal reference gene for different tissues of *Mulletodon zhuyi* as described in claim 2, characterized in that, In step S3, the amplification efficiency of the qRCR primer pair is between 90% and 105%, and the correlation coefficient is between 0.99 and 1.

5. The real-time fluorescence quantitative PCR internal reference gene for different tissues of *Mulletodon spp.* according to claim 2, characterized in that, In step S4, the expression stability analysis is performed by using the ΔCt method, BestKeeper, NormFinder, geNorm, and RefFinder to conduct a comprehensive analysis of the candidate reference genes.

6. The real-time quantitative PCR detection primers for the internal reference gene of different tissues of *Mulletodon zhuyi* as described in claim 1, characterized in that... The rps4x The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 46-47; eif3h The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 40-41; stau1 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 50-51; HSP90B The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 44-45; rpl7 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 38-39; mrpl3 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 28-29; ef1y The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 42-43; dera The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 22-23; rad18 The primer sequences for real-time quantitative PCR detection are shown in SEQ ID No. 26-27.

7. A reagent kit, characterized in that, The kit includes the real-time quantitative PCR detection primers as described in claim 6.

8. The application of the real-time quantitative PCR internal reference gene of *Mulletus edulis* as described in claim 1, or the real-time quantitative PCR detection primers as described in claim 6, or the kit as described in claim 7, in the real-time quantitative PCR detection of gene expression levels in different tissues of *Mulletus edulis*.

9. The application according to claim 8, characterized in that, The different tissues of *Mulletus zhurensis* are different tissues of *Mulletus zhurensis* under normal conditions or under stress conditions; the stress conditions are treatment with bisphenol A, cadmium, or sulfadiazine.

10. A method for detecting gene expression levels in different tissues of *Mulletodon spp.*, characterized in that, The method employs real-time quantitative PCR, and selects the *Mulletus edulis* real-time quantitative PCR internal reference gene described in claim 1 as the internal reference gene.

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