Application of EEF1A1 gene and / or FTH1 gene as chicken reference gene

Abstract

The invention relates to the technical field of molecular biology. The invention provides a chicken reference gene EEF1A1 gene and / or FTH1 gene, and also provides an application of the EEF1A1 gene and / or FTH1 gene as a reference gene in chicken gene qualitative detection, and an application of the EEF1A1 gene and / or FTH1 gene as a reference gene in fluorescent quantitative detection of chicken in different development stages or different tissues. The new chicken reference gene disclosed by the invention has the characteristics of stability, extensiveness and sensitivity, can be effectively used for identifying and proving the functions of the gene and other regulatory molecules in biological pathways, and is beneficial to deeply understanding the functions of poultry genomes; biological characteristics and genetic mechanisms of poultry can be deeply understood; the accuracy of disease diagnosis is improved, and errors of a traditional method are reduced; in agricultural breeding, the method is helpful for identifying genes related to various production traits and screening and optimizing chicken varieties. The invention provides an important tool for biological research, agricultural breeding, transgenic animal research and the like.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to the application of EEF1A1 gene and / or FTH1 gene as chicken internal reference genes. Background Art

[0002] With the development of poultry genetic breeding technology, the research on chicken genome has been continuously deepened. As an important component of the genome, housekeeping genes are used as molecular markers for accurate species identification and classification due to their sequence conservation; key genes that affect growth and meat quality in genetic breeding are identified; the genetic basis of chicken adaptability to the environment is revealed; chicken genome resources are improved to support genetic breeding and functional gene research; immune adaptability is demonstrated in disease defense, which is crucial for poultry disease research; expression pattern research is used to enhance the understanding of chicken biology and genetics; and genomic mechanisms for improving nutritional efficiency are explored in nutritional genomics. These applications reflect the core position of housekeeping genes in poultry scientific research.

[0003] However, in the field of poultry research, especially chicken research, the existing reference gene system still has significant limitations, mainly including: (1) Variation in expression stability: Although housekeeping genes are generally considered to be stably expressed, their expression levels may vary in different tissues, developmental stages or environmental conditions. This variation may affect the accuracy and reliability of gene expression analysis. (2) Limitations in gene selection: In poultry, ideal housekeeping genes may not always be easy to identify. Some housekeeping genes may have low expression levels in specific tissues or developmental stages, limiting their applicability as reference genes. (3) Challenges in detection technology: In the diagnosis of poultry diseases, the detection of reference genes may be inhibited by various substances in the sample, resulting in false negative results. Therefore, the reaction system containing the internal reference target can not only detect the presence of inhibitors in the template DNA, but also evaluate the quality of sample collection to a certain extent, thereby improving the credibility of the test results. (4) Complexity of gene expression: The expression of housekeeping genes may be affected by many factors, including genetic background, environmental factors and physiological state, which increases the complexity of using housekeeping genes as internal references.

[0004] Based on the above problems, developing a stable, extensive and sensitive chicken internal reference gene system has important theoretical significance and application value. Summary of the invention

[0005] The purpose of the present invention is to provide the use of EEF1A1 gene and / or FTH1 gene as chicken internal reference genes. The chicken internal reference genes of the present invention are stable, extensive and sensitive, and provide important tools for biological research, agricultural breeding, disease diagnosis and transgenic animal research.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a chicken internal reference gene, comprising an EEF1A1 gene or an FTH1 gene, wherein the nucleotide sequence of the EEF1A1 gene is shown as SEQ ID NO:1, and the nucleotide sequence of the FTH1 gene is shown as SEQ ID NO:2.

[0008] The invention provides the application of the chicken EEF1A1 gene as an internal reference gene in the qualitative detection of chicken genes.

[0009] The invention provides the application of the chicken FTH1 gene as an internal reference gene in the qualitative detection of chicken genes.

[0010] The present invention provides the application of the chicken EEF1A1 gene and FTH1 gene as internal reference genes in chicken gene qualitative detection.

[0011] The present invention also provides the use of the chicken EEF1A1 gene as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0012] Preferably, the primer set used in the fluorescent quantitative detection process includes an EEF1A1 upstream primer and an EEF1A1 downstream primer, the nucleotide sequence of the EEF1A1 upstream primer is shown in SEQ ID NO:3, and the nucleotide sequence of the EEF1A1 downstream primer is shown in SEQ ID NO:4.

[0013] The present invention also provides the application of the chicken FTH1 gene as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0014] Preferably, the primer set used in the fluorescent quantitative detection process includes an FTH1 upstream primer and an FTH1 downstream primer, the nucleotide sequence of the FTH1 upstream primer is shown in SEQ ID NO:5, and the nucleotide sequence of the FTH1 downstream primer is shown in SEQ ID NO:6.

[0015] The present invention also provides the use of the chicken EEF1A1 gene and the FTH1 gene as internal reference genes in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects:

[0017] The EEF1A1 gene and FTH1 gene screened in the technical solution of the present invention are stable, extensive and sensitive as chicken internal reference genes, which provide important tools for biological research, agricultural breeding, disease diagnosis and transgenic animal research.

[0018] The chicken internal reference gene of the present invention can be used to develop a new poultry gene kit to improve the selectivity of the internal reference gene. It can be used to identify and prove the functional role of genes and other regulatory molecules in biological pathways, which is helpful for in-depth understanding of the function of the poultry genome; it can also deeply understand the biological characteristics and genetic mechanisms of poultry; improve the accuracy of disease diagnosis and reduce the error of traditional methods.

[0019] In agricultural breeding, the internal reference gene of the present invention helps to identify genes related to various production traits and screen and optimize chicken varieties.

[0020] In disease prevention and control, the internal reference gene of the present invention can be used for rapid diagnosis of diseases and development of vaccines, which is of great significance for reducing the occurrence of diseases and controlling the spread of epidemic diseases, and helps to improve the disease management efficiency of the aquaculture industry. In addition to developing molecular diagnostic kits, it can also improve the detection capabilities of food safety and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis diagram of the PCR amplification product of 3-month-old female Lueyang black-bone chicken;

[0022] Figure 2 This is the Ct value result diagram of the candidate internal reference gene in 9 tissues of 3-month-old female Lueyang black-bone chicken;

[0023] Figure 3 The Ct value results of the candidate internal reference gene in nine tissues of 7-month-old female Lueyang black-bone chicken and the electrophoresis of the PCR amplification products;

[0024] Figure 4 The Ct value results of the candidate internal reference gene in 9 tissues of 7-month-old male Lueyang black-bone chicken and the electrophoresis of PCR amplification products;

[0025] Figure 5 The Ct value results of the candidate internal reference gene in nine tissues of one-month-old female Liangfenghua broiler chickens and the electrophoresis of the PCR amplification products;

[0026] Figure 6 The Ct value results of the candidate internal reference gene in nine tissues of one-month-old male Liangfenghua broiler chickens and the electrophoresis of the PCR amplification products;

[0027] Figure 7 The Ct value results of the candidate internal reference gene in nine tissues of 2-month-old female Liangfenghua broiler chickens and the electrophoresis of the PCR amplification products;

[0028] Figure 8 The Ct value results of the candidate internal reference gene in 9 tissues of 2-month-old male Liangfenghua broiler chickens and the electrophoresis of PCR amplification products;

[0029] Fig. 9 The Ct value results of the candidate internal reference gene in nine tissues of 4-month-old female Liangfenghua broiler chickens and the electrophoresis of the PCR amplification products;

[0030] Fig.10 The Ct value results of the candidate internal reference gene in 9 tissues of 4-month-old male Liangfenghua broiler chickens and the electrophoresis of PCR amplification products;

[0031] Fig.11 The Ct value results of the candidate internal reference gene in nine tissues of 2-month-old female silky chicken and the electrophoresis of PCR amplification products;

[0032] Fig.12 The Ct value results of the candidate internal reference gene in 9 tissues of 2-month-old male Silky Chicken and the electrophoresis of PCR amplification products;

[0033] Fig.13 The coefficient of variation statistics of EEF1A1, RPS24 and FTH1 genes in 11 chickens. DETAILED DESCRIPTION

[0034]

[0035]

[0036] The invention provides the application of the chicken EEF1A1 gene as an internal reference gene in the qualitative detection of chicken genes.

[0037] The invention provides the application of the chicken FTH1 gene as an internal reference gene in the qualitative detection of chicken genes.

[0038] The present invention provides the application of the chicken EEF1A1 gene and FTH1 gene as internal reference genes in chicken gene qualitative detection.

[0039] The present invention also provides the use of the chicken EEF1A1 gene as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0040] In the present invention, the primer set used in the process of the fluorescent quantitative detection includes an EEF1A1 upstream primer and an EEF1A1 downstream primer. The nucleotide sequence of the EEF1A1 upstream primer is shown in SEQ ID NO:3, and the specific sequence is 5'-GGACAACATGCTG GAGCCTA-3'; the nucleotide sequence of the EEF1A1 downstream primer is shown in SEQ ID NO:4, and the specific sequence is 5'-TCTTGA AGAGGCAGACGCAG-3'.

[0041] The present invention also provides the application of the chicken FTH1 gene as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0042] In the present invention, the primer set used in the process of fluorescent quantitative detection includes an FTH1 upstream primer and an FTH1 downstream primer. The nucleotide sequence of the FTH1 upstream primer is shown in SEQ ID NO:5, and the specific sequence is 5'-TACGCCTCCTACGTGTAC CT-3'; the nucleotide sequence of the FTH1 downstream primer is shown in SEQ ID NO:6, and the specific sequence is 5'-TCTCAGCATGTTC ACGCTCC-3'.

[0043] The present invention also provides the use of the chicken EEF1A1 gene and the FTH1 gene as internal reference genes in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] All Lueyang black-bone chickens involved in the present invention are from Heihe Renjia Agricultural Development Co., Ltd., Lueyang County, Hanzhong City, Shaanxi Province, and other varieties of chickens are purchased from Hanzhong Market, Hanzhong City, Shaanxi Province.

[0046] Example 1 (one)

[0048] Three normal and healthy female Lueyang black-bone chickens aged 3 months were selected, and the corresponding 9 tissues and organs including bursa of Fabricius, pectoral muscle, heart, kidney, liver, lung, ovary, skin, and spleen were taken for transcriptome sequencing. HKGs (genes constitutively expressed in all tissues and cell types) were screened based on the transcriptome sequencing data, and the highly expressed HKGs were selected as candidate housekeeping genes. The screening criteria were: (1) TPM value > 1000 in specific tissues; (2) CV of candidate genes < 0.15.

[0049] Table 1 Expression levels of candidate housekeeping genes in nine tissues of 3-month-old female Lueyang black-bone chickens

[0050]

[0051] Note: All data in this table are calculated accurately and retained to two decimal places.

[0052] The gene ID of the EEF1A1 gene (eukaryotic translation elongation factor 1alpha 1) is ENSGALG00000015917, the gene ID of the FTH1 gene (ferritin heavy chain 1) is ENSGALG00000007220, the gene ID of the RPS24 gene (ribosomal protein S24) is ENSGALG00000004871, the gene ID of the ACTB gene (actin β) is ENSGALG00000009621, and the gene ID of the GAPDH gene (glyceraldehyde-3-phosphate dehydrogenase) is ENSGALG00000014442.

[0053] Based on the above operations, EEF1A1, RPS24, and FTH1 genes were preliminarily screened as candidate housekeeping genes. (two)

[0055] The RNA from the above nine tissues was extracted by Trizol method and reverse transcribed into cDNA.

[0056] Take 50 mg of each of the nine tissues, including the bursa of Fabricius, pectoral muscle, heart, kidney, liver, lung, ovary, skin, and spleen, of the above-mentioned fresh 3-month-old female Lueyang black-bone chicken, quickly transfer them to a mortar pre-cooled with liquid nitrogen, and grind the tissues with a pestle while continuously adding liquid nitrogen until they are ground into powder.

[0057] Place the fully ground tissue in a sterile, enzyme-free centrifuge tube, add 500μL Trizol to each tube, and let it stand on ice for 5 minutes. Centrifuge at 12000rpm, 4℃ for 10 minutes. Aspirate the supernatant and transfer it to a new centrifuge tube. Add 100μL chloroform, cover the tube tightly, shake vigorously for 15s, and let it stand on ice for 5 minutes. Centrifuge at 12000rpm, 4℃ for 15 minutes. Aspirate the supernatant and transfer it to a new centrifuge tube. Add an equal volume of isopropanol to the supernatant, turn it upside down, mix it thoroughly, and let it stand on ice for 10 minutes. Centrifuge at 12000rpm, 4℃ for 10 minutes. A white precipitate appears at the bottom, which is RNA. Discard the supernatant and keep the precipitate. Slowly add 1mL of 75% ethanol along the bottom of the tube, slightly turn it upside down, and wash the precipitate. Centrifuge at 12000rpm, 4℃ for 10 minutes. Discard the supernatant and keep the precipitate, put it in a fume hood, and air-dry it for 5 minutes. Add 20 μL of RNase-free water to resuspend the precipitate. If necessary, gently pipette and stir until it is completely dissolved. Measure the concentration and set up the subsequent reverse transcription system based on the measured concentration.

[0058] Then, the total RNA extracted above was reverse transcribed into cDNA using a reverse transcription kit (purchased from Quanshijin Biotechnology Co., Ltd., catalog number AE311-02). The reverse transcription reaction system is shown in Table 2.

[0059] Table 2 Reverse transcription reaction system

[0060]

[0061] After adding the above system, mix gently, then incubate at 42°C for 30 min, and heat at 85°C for 5 s to inactivate, to obtain cDNA. (three)

[0063] According to the CDs region sequence, gene-specific primers were designed. The primer information of three candidate housekeeping genes and two internal reference genes is shown in Table 3.

[0064] Table 3 Primer information of candidate housekeeping genes and internal reference genes

[0065]

[0066] The gene sequences were amplified by PCR using the above primers, and the amplification system of each gene is shown in Table 4. The PCR amplification program was: ① pre-denaturation at 95°C for 5 min; ② (denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 30 s) for 34 cycles; ③ extension at 72°C for 5 min.

[0067] Table 4 PCR amplification system

[0068]

[0069] (Four)

[0071] After amplification, the product was placed on a 2% agarose gel and electrophoresed for 40 minutes at 100 V to analyze the PCR amplification product and observe whether the target band exists. DNAMarker is a 2000 bp DNALadder molecular weight standard.

[0072] The results are as follows Figure 1 As shown, the positions of the electrophoresis bands are consistent, indicating that the amplified products are consistent. (five)

[0074] RT-qPCR validation was performed using cDNA from 3-month-old female Lueyang black-bone chickens, with 3 technical replicates for each sample and ACTB as an endogenous control. RT-qPCR reaction conditions: ① pre-denaturation at 95℃ for 30s; ② (denaturation at 95℃ for 5s; annealing at 60℃ for 30s) for 40 cycles; ③ extension at 50℃ for 1min.

[0075] Table 5 RT-qPCR reaction system

[0076]

[0077] The Ct value (Cycle Threshold) is an important parameter in real-time quantitative PCR (qPCR) experiments. The Ct value is inversely proportional to the initial amount of template DNA, that is, the more the initial template amount, the fewer cycles required to reach the threshold, and the lower the Ct value. Therefore, the Ct value can be used to compare the relative expression of genes in different samples.

[0078] The results are as follows Figure 2 As shown, it shows that EEF1A1 and FTH1 genes are highly expressed in various tissues, and the expression differences are small.

[0079] Example 2 (one)

[0081] After the verification of 3-month-old female Lueyang black-bone chickens was completed, PCR and RT-qPCR verification were performed on chickens of different breeds and at different times to ensure the stability and universality of the housekeeping gene. The PCR and RT-qPCR verification process, reaction system and reaction procedure were the same as above.

[0082] The test subjects selected were: 10 chickens, one male and one female, 7-month-old Lueyang black-bone chicken, 1-month-old Liangfenghua broiler chicken, 2-month-old Liangfenghua broiler chicken, 4-month-old Liangfenghua broiler chicken and 2-month-old Silky black-bone chicken.

[0083] The Ct value results of candidate reference genes in 9 tissues of 7-month-old Lueyang black-bone female and male chickens and the electrophoresis results of PCR amplification products are shown in Figure 3-4As shown in the figure, the Ct value results of candidate reference genes in 9 tissues of 1-month-old female and male chickens and the electrophoresis results of PCR amplification products are as follows Figure 5-6 As shown in the figure, the Ct value results of candidate reference genes in 9 tissues of 2-month-old female and male chickens and the electrophoresis results of PCR amplification products are as follows Figure 7-8 As shown in the figure, the Ct value results of candidate reference genes in 9 tissues of 4-month-old female and male chickens and the electrophoresis results of PCR amplification products are shown in Figure 9-10 As shown in the figure, the Ct value results of candidate reference genes in 9 tissues of 2-month-old silky black roosters and roosters and the electrophoresis results of PCR amplification products are shown in the figure. Figure 11-12 shown. (two)

[0085] The coefficient of variation (CV) of genes in different tissues was calculated based on the Ct value, and this was used to evaluate the stability of the internal reference gene. Fig.13 shown.

[0086] The calculation formula is CV = σ / μ, where σ represents the standard deviation and μ represents the mean value.

[0087] From the analysis of the above experimental results, it can be seen that a large number of experiments have verified that the EEF1A1 and FTH1 genes are highly and stably expressed in chicken heart, liver, spleen, lung, kidney, skin, ovary, testis, pectoral muscle and bursa of Fabricius tissue, which is in line with the expected results and meets the requirements of stability and universality as internal references. However, the coefficient of variation of the RPS24 gene is large, more discrete, and less stable, which does not meet the requirements of being an internal reference.

[0088] In summary, it can be seen that the EEF1A1 gene and FTH1 gene screened in the technical solution of the present invention are stable, extensive and sensitive as chicken internal reference genes, which also provides important tools for biological research, agricultural breeding, disease diagnosis and transgenic animal research.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Chicken internal reference gene, characterized in that, It comprises EEF1A1 gene or FTH1 gene, wherein the nucleotide sequence of the EEF1A1 gene is shown as SEQ ID NO:1, and the nucleotide sequence of the FTH1 gene is shown as SEQ ID NO:

2.

2. Use of the chicken EEF1A1 gene according to claim 1 as an internal reference gene in qualitative detection of chicken genes.

3. Use of the chicken FTH1 gene according to claim 1 as an internal reference gene in qualitative detection of chicken genes.

4. Use of the chicken EEF1A1 gene and FTH1 gene as claimed in claim 1 as internal reference genes in qualitative detection of chicken genes.

5. Use of the chicken EEF1A1 gene according to claim 1 as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

6. The use according to claim 5, characterized in that: The primer set used in the fluorescent quantitative detection process includes an EEF1A1 upstream primer and an EEF1A1 downstream primer. The nucleotide sequence of the EEF1A1 upstream primer is shown in SEQ ID NO:3, and the nucleotide sequence of the EEF1A1 downstream primer is shown in SEQ ID NO:

4.

7. Use of the chicken FTH1 gene according to claim 1 as an internal reference gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

8. The use according to claim 7, characterized in that: The primer set used in the fluorescent quantitative detection process includes an FTH1 upstream primer and an FTH1 downstream primer. The nucleotide sequence of the FTH1 upstream primer is shown in SEQ ID NO:5, and the nucleotide sequence of the FTH1 downstream primer is shown in SEQ ID NO:

6.

9. Use of the chicken EEF1A1 gene and FTH1 gene as claimed in claim 1 as internal reference genes in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

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