Housekeeping genes of pituitary, liver, ovary and liver fat storage cells of scatophagus argus and RT-qPCR (real-time quantitative polymerase chain reaction) amplification primer

Abstract

The invention discloses housekeeping genes of pituitary, liver, ovary and liver fat storage cells of scatophagus argus and RT-qPCR (real-time quantitative polymerase chain reaction) amplification primers, and belongs to the technical field of genes. 17 pituitary candidate genes, 4 ovary candidate genes, 4 liver tissue candidate genes and 4 liver fat storage cell candidate genes are amplified through an RT-qPCR method, stability analysis is carried out through four analysis methods of GeNorm, NormFinder, Ct and BestKeeper, finally, sequencing is carried out through RefFinder, and results show that gnb2l1 and rpl4 in pituitary, eef1a and hprt1 of ovary, actb and hprt1 of liver tissue, rpl1 and rpl2 in liver tissue, rpl3 and rpl4 in liver tissue, rpl3 and rpl4 in liver tissue, rpl3 and rpl4 in liver tissue, rpl3 and rpl4 in liver tissue, rpl4 and rpl4 in liver tissue b2m and actb of the liver fat storage cells can be used as housekeeping genes of corresponding tissues and cells of scatophagus argus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic technology, and particularly relates to housekeeping genes of pituitary, liver, ovary and hepatosteatosis cells of goldfish and RT-qPCR amplification primers. BACKGROUND

[0002] Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is one of the most commonly used techniques for detecting and evaluating gene expression, and the selection of appropriate housekeeping genes is the key to ensuring the accuracy and reliability of its analysis results. Generally, housekeeping genes are often referred to as home genes, such as 18S ribosomal RNA (18S), beta-actin (actb) and glyceraldehyde-3-phosphate dehydrogenase (gapdh). However, the expression of these genes may not be the same in different tissues, different experimental conditions or different developmental stages. For example, the expression of gapdh and actb in the gonads of female and male zebrafish (Danio rerio) is unstable, and there is a significant difference in expression. The results of studies in Monopterus albus show that the expression levels of gapdh, 18S and actb in the gonads are quite different, and are not suitable as housekeeping genes for the study of gonadal development. Therefore, in expression studies, especially under specific experimental treatment conditions, appropriate housekeeping genes are crucial.

[0003] So far, there has been no research report on the screening of housekeeping genes of goldfish. SUMMARY

[0004] One of the purposes of the present application is to provide the application of gene gnb2l1 or rpl4 as a pituitary housekeeping gene of goldfish.

[0005] The second purpose of the present application is to provide the application of gene actb or hprt1 as a liver housekeeping gene of goldfish.

[0006] The third purpose of the present application is to provide the application of genes eef1a and hprt1 as ovary housekeeping genes of goldfish.

[0007] The fourth purpose of the present application is to provide the application of genes b2m or actb as hepatosteatosis cell housekeeping genes of goldfish.

[0008] The fifth object of the present application is to provide the RT-qPCR amplification primer of the above-mentioned gene gnb2l1 or rpl4, wherein the RT-qPCR amplification primer sequence of the gene gnb2l1 is shown as SEQ ID NO. 11, 12; and the RT-qPCR amplification primer sequence of the gene rpl4 is shown as SEQ ID NO. 19, 20.

[0009] The sixth object of the present application is to provide the RT-qPCR amplification primer of the above-mentioned gene actb or hprt, wherein the RT-qPCR amplification primer sequence of the gene actb is shown as SEQ ID NO. 33, 34.

[0010] The seventh object of the present application is to provide the RT-qPCR amplification primer of the above-mentioned gene eef1a, wherein the RT-qPCR amplification primer sequence is shown as SEQ ID NO. 35, 36.

[0011] The eighth object of the present application is to provide the RT-qPCR amplification primer of the above-mentioned gene b2m, wherein the RT-qPCR amplification primer sequence is shown as SEQ ID NO. 3, 4.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] The present application identifies the suitable housekeeping genes suitable for the ovary, pituitary and liver of the different development periods (II, III and IV) of the golden fish, and the fat-storing cells (FSC, ITO) under different culture time conditions, which is of great significance for the next step of gene expression research. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the ovary tissue section diagram of the golden fish in Example 1.

[0015] Figure 2 It is the total RNA and cDNA quality detection result of the ovary in Example 1, wherein A: the total RNA quality detection result of the ovary; B: the cDNA quality detection result of the ovary.

[0016] Figure 3 It is the total RNA and cDNA quality detection result of the pituitary in Example 1, wherein A: the total RNA quality detection result of the pituitary; B: the cDNA quality detection result of the pituitary.

[0017] Figure 4 It is the total RNA and cDNA quality detection result of the liver in Example 1, wherein A: the total RNA quality detection result of the liver; B: the cDNA quality detection result of the liver.

[0018] Figure 5 Liver ITO cell total RNA quality test results for Example 1.

[0019] Figure 6 Ovary candidate gene melting curves for Example 1.

[0020] Figures 7-1 to 7-17 Pituitary candidate gene melting curves for Example 1.

[0021] Figure 8 Liver and ITO cell candidate gene melting curves for Example 1.

[0022] Figure 9 Specificity testing and expression levels of candidate housekeeping genes in ovary for Example 1.

[0023] Figure 10 Specificity testing and expression levels of candidate housekeeping genes in pituitary for Example 1.

[0024] Figure 11 Specificity testing and expression levels of candidate housekeeping genes in liver for Example 1.

[0025] Figure 12 Specificity testing and expression levels of candidate housekeeping genes in ITO cells for Example 1.

[0026] Figures 13-1 to 13-2 Stability of ovary candidate housekeeping genes across different ovary stages based on GeNorm (A-D), NormFinder (E-H), ACt (I-L), and BestKeeper (M-P) analysis for Example 1.

[0027] Figure 14 Ranking of ovary candidate housekeeping genes across different ovary stages using RefFinder for Example 1.

[0028] Figure 15 Stability of pituitary candidate housekeeping genes across different ovary stages based on GeNorm (A), NormFinder (B), ACt (C), and BestKeeper (D) analysis for Example 1.

[0029] Figure 16 Ranking of pituitary candidate housekeeping genes across different ovary stages using RefFinder for Example 1.

[0030] Figure 17Stability analysis of candidate housekeeping genes in liver tissue at different ovary development stages using GeNorm (A-D), NormFinder (E-H), ACt (I-L) and BestKeeper (M-P) programs in Example 1.

[0031] Figure 18 Ranking of candidate housekeeping genes in liver tissue at different ovary development stages for stability using RefFinder in Example 1.

[0032] Figure 19 Stability analysis of candidate housekeeping genes in liver adipocytes at different culture stages using GeNorm (A-D), NormFinder (E-H), ACt (I-L) and BestKeeper (M-P) programs in Example 1.

[0033] Figure 20 Stability analysis of candidate housekeeping genes in liver adipocytes at all culture stages using GeNorm (A), NormFinder (B), ACt (C) and BestKeeper (D) programs in Example 1.

[0034] Figure 21 Ranking of candidate housekeeping genes in liver adipocytes at different culture stages for stability using RefFinder in Example 1.

[0035] Figure 22 Best number of reference genes for normalization of gene expression in ovary (A) and pituitary (B) using GeNorm pairwise variation (V n / V n+1 , where "n" represents the number of reference genes).

[0036] Figure 23 Figure of best number of reference genes for liver and liver adipocytes in Example 1. DETAILED DESCRIPTION

[0037] Example 1

[0038] 1. Materials and methods

[0039] 1.1 Experimental materials

[0040] 1.1.1 Tissue sample collection

[0041] Twenty 2-year-old female G. atra (body weight: 242.83 ± 50.90 g; body length: 19.48 ± 1.13 cm) were selected from Donghai Island aquaculture base. The fish were fasted for 24 h before sampling and anesthetized with MS-222 (Sigma, St. Louis, MO, USA). The ovary, pituitary, and liver tissues were dissected and immediately transferred into liquid nitrogen and then stored at -80°C for subsequent RNA extraction. Meanwhile, a portion of the ovary was fixed in Bouin's solution overnight, and then paraffin sections were prepared from the fixed ovary samples and stained with hematoxylin-eosin. The stained sections were observed under a Nikon Eclipse Ti-E microscope (Tokyo, Japan) to determine the developmental stage of the ovary.

[0042] 1.1.2 Cell sample collection

[0043] The 3rd generation of primary cell line cells were removed from the culture fluid, washed with 1xPBS for 3 times; digested with 2 ml 0.25% trypsin for 10 min; the digestion solution was transferred to a 2 ml enzyme-free centrifuge tube and centrifuged at 3000 rpm / min for 10 min; the trypsin digestion solution was discarded, 1 ml PBS was added to resuspend the cells, which were centrifuged at 3000 rpm / min for 10 min, repeated 2 times, the liquid was discarded, and stored at -80°C for standby.

[0044] 1.2 Experimental methods

[0045] 1.2.1 Extraction of RNA and synthesis of cDNA

[0046] The total RNA of ovary, pituitary, and liver tissues and ITO cells was extracted using the Trizol kit (Invitrogen, CA, USA). The RNA integrity value (RIN) was evaluated using the Agilent 2100 Bioanalyzer (Agilent, CA, USA), and the RNA quality was detected by agarose gel electrophoresis. 1 μg of RNA was used for reverse transcription, and the cDNA first strand was synthesized using the Prime Script™ RT Reagent Kit with gDNA Eraser (RR047A; Takara Bio, Dalian, China) kit.

[0047] 1.2.2 Primer design

[0048] For pituitary, we selected 17 candidate housekeeping genes, including b2m, hprt1, gapdh, tubb4b, gusb, tbp, gnb2l1, which have been reported previously, and apoa1, cgba, rplp0, gnrhr, slc25a1, pla1a, ctfs, rpl4, rps2 and tfrc, which are not reported but highly expressed in the pituitary transcriptome of G. a. auratus. eef1a, b2m, gapdh and hprt1 are good housekeeping genes for ovary of goose, tilapia, mouse and sow, so we selected these 4 genes as candidate housekeeping genes for ovary for further analysis. For liver and ITO cells, we selected b2m, gapdh, hprt1 and actb which have been reported. We first obtained the mRNA sequences of the above candidate housekeeping genes based on the reference genome of G. a. auratus, and then designed primers using Oligo7 primer analysis software. Primers were synthesized by Shengong Biotechnology Co., Ltd. (Shanghai, China).

[0049] Table 1 Primer sequences of genes used in this study

[0050]

[0051] Note: “#” and “*” are the candidate genes for pituitary and ovary, respectively; “✭” is the candidate gene for liver tissue and ITO cells.

[0052] 1.2.3 Real-time fluorescent quantitative PCR (RT-qPCR)

[0053] SYBR Green PCR Mix b (TransGen Biotech, Beijing, China) was used for real-time fluorescent quantitative PCR. The RT-qPCR reaction system (total volume 20 mL) was composed of 10 ml of SYBR Green Mix, 1 ml of template, 0.5 ml of forward and reverse primers (100 mmol / mL), and 8 ml of water. The reaction conditions were denaturation at 95°C for 300 s, 40 cycles of 95°C for 30 s, annealing temperature for 20 s, and 72°C for 30 s. All reactions were analyzed by melting curve analysis to determine the specificity of PCR amplification products. The 1x, 10x, 100x and 1000x gradient dilution of cDNA template was used to draw standard curve, and the primer amplification efficiency (AE=(10[-1 / slope]-1) x 100) was determined. RT-qPCR was detected by LightCycler96 (Roche Diagnostics, Shanghai, China), with three replicates in each group.

[0054] 1.2.4 Stability analysis of candidate housekeeping genes

[0055] The stability of the candidate housekeeping genes was determined using the cDNA of the ovary, pituitary, liver and ITO cells of the zebrafish, respectively. To ensure data reliability, the RT-qPCR reactions were performed in triplicate. The stability of the candidate genes was analyzed using NormFinder, GeNorm, ACt and BestKeeper based on the mean Ct values of each sample. GeNorm calculated the M value for each gene, and the gene with the smallest M value was the most stable. NormFinder ranked the stability of the candidate genes based on the intergroup variation. The ACt method was used to determine the stability of each gene by calculating the standard deviation of the Cq difference within each sample and taking the average. BestKeeper selected the gene with the lowest variance and standard deviation (SD) as the most stable. Housekeeping genes with a standard deviation (SD) less than 1 were considered stable. Finally, RefFinder integrated the above four techniques and ranked the validated candidate housekeeping genes comprehensively. Pairwise variation (V n / n+1 ) was calculated using GeNorm, and the total number of housekeeping genes was selected for gene expression normalization. Generally, when (V n / n+1 ) was less than the threshold value of 0.15, n was the optimal number, indicating that n + 1 housekeeping genes were not needed for normalization.

[0056] 2. Results

[0057] 2.1 RNA extraction and quality detection

[0058] The ovary samples were staged using HE staining, and the results showed that a large number of primary growth stage oocytes (PG) occupied the II stage of ovarian development (n = 6), while previtellogenic oocytes (PV) and early vitellogenic oocytes (EV) appeared in the III stage of ovarian development (n = 3), and a large number of late vitellogenic oocytes (LV) were found in the IV stage of ovarian development (n = 11) (Fig. 1). Figure 1 .

[0059] The total RNA 28S: 18S ratio of all samples was greater than 1, and the OD260 / 280 ratio was 1.8-2.0. Except for one ovary sample in the IV stage, the RIN of the RNA of all other samples was > 7, indicating good RNA quality. The results of RT-PCR showed that the actb primers could amplify a single band of the cDNA of the ovary and pituitary at the same time, indicating that the cDNA could be used for further analysis (Fig. 2 and Fig. 3). Figure 2 and Figure 3 .

[0060] The 260 / 280 ratio of total RNA samples of liver was greater than 1.6, and only 2 samples had a 260 / 280 ratio less than 1.65. Four randomly selected samples of reverse-transcribed cDNA were used for cDNA quality testing using actb, and the results showed that the reverse-transcribed cDNA samples could be used for subsequent experiments. Figure 4 ).

[0061] The 260 / 280 ratio of total RNA samples of liver ITO cells was greater than 1.6, and only 1 sample had a 260 / 280 ratio less than 1.65. Figure 5 ).

[0062] 2.2 Specificity and efficiency of candidate primers

[0063] PCR amplification was used to identify the specificity of the primers, and agarose gel electrophoresis was used to detect the PCR products. The results showed that all primers could amplify a single band of ovary or pituitary. Melting curve analysis showed that all primers exhibited a single peak at the set primer annealing temperature, indicating good primer specificity. The AE values of all primer pairs were 92.71-110.27%, meeting the basic requirements of RT-qPCR. Figure 6 and Figures 7-1 to 7-17 ).

[0064] Melting curve analysis of liver tissue and ITO cells showed that all primers exhibited a single peak at the set annealing temperature, indicating good primer specificity. The AE values of all primer pairs were 92.71-110.27%, meeting the basic requirements of RT-qPCR. Figure 8 ).

[0065] 2.3 Expression profile of candidate housekeeping genes

[0066] RT-qPCR results showed that the average Ct values of ovary candidate housekeeping genes were 9.52-21.95 cycles, and the average Ct values of pituitary candidate housekeeping genes were 13.42-28.03 cycles. In ovary and pituitary tissues, the expression of candidate housekeeping genes changed with the development of ovary. Figure 9 and Figure 10 ).

[0067] RT-qPCR results showed that the average Ct values of liver tissue candidate housekeeping genes were 9.52-21.95 cycles, and the average Ct values of ITO cell candidate housekeeping genes were 16.42-32.15 cycles. In liver and ITO cells, the expression of candidate housekeeping genes changed with the development of ovary and the extension of ITO cell culture time. Figure 11 and Figure 12 ).

[0068] 2.4 Stability analysis of ovary candidate housekeeping genes

[0069] GeNorm analysis showed that the ranking of the stability of the housekeeping candidate genes was eef1a = hprt1 > actb > b2m in stage II, stage IV and all stages of ovarian development (Fig. 1A, C, D). While in stage III, the ranking of the stability was actb = hprt1 > eef1a > b2m (Fig. 1B). NormFinder analysis and △Ct analysis showed similar results: the ranking of the stability of the housekeeping candidate genes was eef1a > hprt1 > actb > b2m in stage II and stage IV (Fig. 1E, G, I, K), and actb > hprt1 > eef1a > b2m in stage III (Fig. 1F, J). In all stages of ovarian development, the ranking of the stability was hprt1 > eef1a > actb > b2m (Fig. 1H, L). BestKeeper analysis showed that the b2m gene in stage II and stage III, the actb gene in stage IV, and the eef1a, hprt1 and actb genes in all stages of ovarian development were unstable (SD > 1) (Fig. 1M-P). Therefore, according to BestKeeper analysis, the ranking of the stability of stage II was actb > eef1a > hprt1, stage III was hprt1 > eef1a > actb, and stage IV was b2m > hprt1 > eef1a > actb. Figure 13-1 Figure 13-1 Figure 13-1 Figure 13-2 Figure 13-1 Figure 13-2 Figure 13-1 Figure 13-2 Figure 13-2

[0070] Based on the results of GeNorm, NormFinder, BestKeeper and △Ct analysis, RefFinder was used to comprehensively rank the stability of the candidate genes in all stages. The results showed that the ranking of the stability of the candidate genes in stage II was eef1a > hprt1 > actb > b2m (Fig. 2A), in stage III was actb > hprt1 > eef1a > b2m (Fig. 2B), in stage IV was eef1a > hprt1 > b2m > actb (Fig. 2C), and finally, in all stages of ovarian development was hprt1 > eef1a > b2m > actb (Fig. 2D). Figure 14 Figure 14 Figure 14 Figure 14

[0071] 2.5 Stability analysis of pituitary candidate housekeeping genes​​​​​​​​​​​​​

[0072] NormFinder, ACt, BestKeeper and GeNorm analysis revealed that rpl4, gnb2l1, pla1a, tubb4b, gapdh, rps2, tbp and b2m were more stable than other candidate reference genes in pituitary, although their ranking order of stability was different in different developmental stages of ovary, but their comprehensive ranking of stability was always in the front ( Figure 15 A-D). GeNorm analysis showed that gapdh and tubb4b in phase II ovary, gnb2l1 and pla1a in phase III ovary, rpl4 and tubb4b in phase IV ovary and rpl4 and gnb2l1 in all stages were the most stable housekeeping genes in pituitary ( Figure 15 ). In BestKeeper analysis, the results showed that slc25a1, gnrhr, cgba in phase II, hprt1 and tfrc in phase III, hprt1, slc25a1 and gnrhr in phase IV and slc25a1, hprt1, gnrhr, cgba in all stages were unstable in pituitary (SD > 1) Figure 15 C).

[0073] According to RefFinder analysis, the comprehensive ranking of stability of pituitary candidate genes in phase II ovary was: gnb2l1> rpl4> pla1a> gapdh> tubb4b> tbp> ctfs> rps2> rplp0> b2m> apoa1> tfrc> gusb> hprt1> slc25a1> gnrhr> cgba Figure 16 A). The comprehensive ranking of stability of pituitary candidate genes in phase III ovary was: rpl4> tubb4b> gnb2l1> gapdh> pla1a> rps2> tbp> b2m> slc25a1> gusb> cgba> gnrhr> apoa1> rplp0> hprt1> tfrc Figure 16 B). The comprehensive ranking of stability of pituitary candidate genes in phase IV ovary was: rpl4> gnb2l1> rps2> tubb4b> pla1a> gapdh> tbp> b2m> rplp0> cgba> apoa1> ctfs> gusb> cgba> tfrc> hprt1> slc25a1> gnrhr Figure 16C). The overall ranking of the stability of the pituitary candidate genes in all ovarian stages was: rpl4>gnb2l1>rps2>pla1a>tubb4b>gapdh>b2m>tbp>ctfs>rplp0>apoa1>gusb>tfrc>hprt1>slc25a1>cgba>gnrhr Figure 16 D).

[0074] 2.6 Analysis of the stability of the liver tissue candidate housekeeping genes

[0075] GeNorm analysis showed that the ranking of the stability of the liver housekeeping candidate genes in stages III, IV and all stages of ovarian development was actb = hprt1 > b2m > gapdh Figure 17 B, C, D). While in stage II of ovarian development, the ranking of the stability of the liver housekeeping genes was b2m = hprt1 > actb > gapdh Figure 17 B). NormFinder analysis and the results of the ΔCt analysis were similar: the ranking of the stability of the liver housekeeping candidate genes in stage IV and all stages of ovarian development was b2m > actb > actb > b2m Figure 17 G, H, K, L), the ranking of the stability of the liver housekeeping in stage III of ovarian development was b2m > actb > hprt1 > gapdh Figure 17 F, J). In stage II of ovarian development, the ranking of the stability of the liver housekeeping genes was hprt1 > eef1a > actb > b2m Figure 17 E), the ranking of the liver in NormFinder in stage II of ovarian development was hprt1 > actb > b2m > gapdh Figure 17 E), the ranking in the ΔCt analysis was hprt1 > actb > b2m > gapdh Figure 17 I). The results of the BestKeeper analysis showed that the stable genes in stage IV of ovarian development and in all stages of ovarian development were the b2m gene, in stage II of ovarian development the hprt1 gene, in stage III the actb gene, and in stages III, IV and all stages the gapdh gene was unstable Figure 17 N-P). Thus, according to the BestKeeper analysis, the ranking of the stability of the liver housekeeping genes in stage II of ovarian development was hprt1 > actb > gapdh > b2m, in stage III of ovarian development was actb > b2m > hprt1 > gapdh, and in stage IV of ovarian development was b2m > hprt1 = eef1a > actb.

[0076] Based on the results of four analysis methods, GeNorm, NormFinder, BestKeeper, and △Ct, RefFinder was used to comprehensively rank the stability of candidate genes in all stages. The results showed that the stability ranking in liver tissue during ovarian development stage II was hprt1 > actb > b2m > gapdh ( Figure 18 A), the stability ranking in the liver during ovarian stage III is b2m > actb > hprt1 > gapdh ( Figure 18 B), the stability ranking in the liver of ovarian stage IV is actb>hprt1>b2m>gapdh ( Figure 18 C), finally, the stability of the liver during each ovarian developmental stage is ranked as actb>hprt1>b2m>gapdh ( Figure 18 D).

[0077] 2.7 Analysis of housekeeping gene stability in hepatic adipocytes (ITOs)

[0078] In hepatic adipocytes, GeNorm analysis showed that b2m and actb at the 12-h stage, and actb and hprt1 at the 24-h, 48-h, and 72-h stages were the most stable housekeeping genes in hepatic adipocytes. Figure 19 AD). In the NormFinder and △Ct analysis, the results showed that b2m was the most stable gene at different culture time stages, actb was the second most stable, and gapdh was the least stable ( Figure 19 EL). In the BestKeeper analysis, the results showed that the stability ranking of candidate genes at the 12 h stage of culture was actb>b2m>hprt1>gapdh ( Figure 19 M), the stability ranking of candidate genes at the 24 h stage was hprt1>actb>b2m>gapdh ( Figure 19 N), the stability ranking of candidate genes at 48 h and 72 h of culture was actb>hprt1>b2m>gapdh ( Figure 19 O, P).

[0079] Genorm analysis results showed that b2m and actb were the most stable, while gapdh was the most unstable in all culture stages of hepatic fat-storing cells. Figure 18 A). NormFinder and △Ct analysis showed that the gene stability ranking at all culture stages was b2m> actb> hprt1> gapdh ( Figure 18 B). BestKeeper analysis showed that actb gene had the highest stability ( Figure 20D). Four analysis software agreed that the stability of gapdh was the worst.

[0080] Based on the analysis results of the above four software, RefFinder was used to comprehensively rank the potential gene stability at each stage. The results showed that the gene stability ranking of liver lipid storage cells cultured for 12 h and all culture time stages was b2m> actb> hprt1> gapdh Figure 21 A, E), the gene stability ranking of culture for 24 h and 48 h was actb> b2m> hprt1> gapdh Figure 21 B, C), the gene stability ranking of culture for 72 h was hprt1> actb> b2m> gapdh Figure 21 D). The candidate gene stability ranking for all periods was b2m> actb> hprt1> gapdh.

[0081] 2.8 Determination of the number of optimal housekeeping genes

[0082] GeNorm analysis showed that the Vn / Vn+1of different ovarian development stages in ovary and pituitary tissues 2 / 3 were all lower than 0.15, indicating that the two housekeeping genes were reliable for accurate normalization of gene expression. Therefore, it was not necessary to introduce the next housekeeping gene for correction Figure 22 ). Combined with the housekeeping gene stability analysis, eef1a and hprt1, rpl4 and gnb2l1 could be used as the optimal housekeeping gene combination in stages II, IV and all stages in ovary and pituitary, respectively. In stage III, the optimal housekeeping gene combination of ovary and pituitary was actb and hprt1, rpl4 and tubb4b, respectively.

[0083] GeNorm analysis of Vn / Vn+1of liver and liver lipid storage cells showed that actb and hprt1 could be used as internal reference genes for liver in stage IV of ovarian development, and actb / hprt1 gene combination could be used as internal reference for data normalization in stages II, III and all stages of ovarian development. Actb and b2m could be used as stable internal reference genes for liver lipid storage cells cultured for 12 h, and actb / hprt1 gene combination was needed as internal reference for data normalization for culture for 24 h to all stages. See Figure 23 .

[0084] The above-described embodiments are only preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. Use of the gene gnb2l1 or rpl4 as a pituitary housekeeping gene of the goldfish.

2. Use of the gene actb or hprt1 as a liver housekeeping gene of the goldfish.

3. Use of the genes eef1a and hprt1 as ovary housekeeping genes of the goldfish.

4. Use of the gene b2m or actb as a liver lipid storage cell housekeeping gene of the goldfish.

5. A RT-qPCR amplification primer of the gene gnb2l 1 or rpl4 according to claim 1, characterized in that, The RT-qPCR amplification primer sequences of the gene gnb2l1 are shown as SEQ ID NO. 11, 12; the RT-qPCR amplification primer sequences of the gene rpl4 are shown as SEQ ID NO. 19, 20.

6. A RT-qPCR amplification primer of the gene actb or hprt according to claim 2, characterized in that, The RT-qPCR amplification primer sequences of the gene actb are shown as SEQ ID NO. 33, 34.

7. The RT-qPCR amplification primer of the gene eefla according to claim 3, characterized by, The RT-qPCR amplification primer sequences are shown as SEQ ID NO. 35, 36.

8. The RT-qPCR amplification primer of the gene b2m according to claim 4, characterized by, The RT-qPCR amplification primer sequences are shown as SEQ ID NO. 3, 4.

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