Marker gene of four tissues of chicken breast muscle, heart, liver and lung and application of marker gene

Abstract

The invention relates to the technical field of molecular biology. The invention provides marker genes of four tissues of pectoral muscle, heart, liver and lung of a chicken. The marker genes comprise a PGM1 gene, a TNNT2 gene, a PIT54 gene and an SFTPC gene. The invention also provides an application of the marker gene as a screening gene in chicken gene qualitative detection and an application of the marker gene as a screening gene in fluorescent quantitative detection of chicken in different development stages or different tissues. The marker gene screened by the invention provides an important tool for biological research, agricultural breeding, disease diagnosis and transgenic animal research; and screening and optimization of chicken varieties are facilitated, the specific tissue of the transgenic chicken can be marked, and the research reliability is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to marker genes of four tissues of chicken breast muscle, heart, liver and lung, and applications thereof. Background Art

[0002] Marker genes are important molecular tools in modern biological research. As molecular markers for tracking, locating and identifying specific cells or organisms, they play a key role in basic and applied research. Such genes can be endogenous or exogenous, and usually carry easily detectable features such as fluorescent proteins, specific enzyme activity or drug resistance, enabling researchers to conduct precise molecular-level observation and analysis. With its advantages of convenient detection, versatility, high efficiency and accuracy, marker gene technology has been widely used in many fields such as cell tracing, gene expression regulation, genomic analysis, immunohistochemistry and drug screening.

[0003] However, in the field of poultry research, especially chicken research, the existing tissue-specific marker gene system still has significant limitations, which are mainly reflected in the following aspects: (1) Insufficient tissue specificity. Most known marker genes have overlapping expression between different tissues, which reduces their application value as tissue-specific markers. For example, some skeletal muscle and cardiac muscle tissues share similar gene expression profiles, which affects the accurate identification of tissue types. The expression pattern of marker genes often changes dynamically with the developmental stage and pathological state, resulting in a significant reduction in their tissue specificity, which limits their application in developmental biology and pathological research; (2) Limited detection sensitivity. The basal expression level of some marker genes is low, which makes it difficult to achieve stable detection in trace tissue samples or rare cell populations, affecting the reliability of experimental results. External environmental factors (such as nutritional status, stress response, disease, etc.) can significantly affect gene expression levels, resulting in fluctuations in test results, reducing the practicality of the marker gene system; (3) Significant individual differences. There are obvious gene expression differences between chickens of different strains, ages and physiological states, which affects the universal application of marker genes. Gene polymorphism further exacerbates the expression differences between individuals and reduces the repeatability and comparability of experimental results.

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

[0005] The purpose of the present invention is to provide marker genes for four tissues of chicken breast muscle, heart, liver and lung and their applications. The marker genes screened by the present invention are stable, specific and sensitive, and can be used to accurately identify chicken breast muscle, heart, liver and lung tissues. It also provides an important tool 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 invention provides marker genes for four tissues of chicken breast muscle, heart, liver and lung, including PGM1 gene, TNNT2 gene, PIT54 gene and SFTPC gene, wherein the gene ID of the PGM1 gene is ENSGALG00000011016, the gene ID of the TNNT2 gene is ENSG ALG00000000302, the gene ID of the PIT54 gene is ENSGALG00000046217, and the gene ID of the SFTPC gene is ENSGALG00000053247.

[0008] The invention provides the application of the marker gene as a screening gene in the qualitative detection of chicken genes.

[0009] The invention provides the application of the marker gene as a screening gene in fluorescence quantitative detection in different development stages or different tissues of chickens.

[0010] Preferably, the primer set used in the fluorescence quantitative detection process includes a PGM1 primer set, a TNNT2 primer set, a PIT54 primer set and a SFTPC primer set.

[0011] Preferably, the PGM1 primer set comprises a PGM1 upstream primer and a PGM1 downstream primer, the nucleotide sequence of the PGM1 upstream primer is shown in SEQ ID NO:1, and the nucleotide sequence of the PGM1 downstream primer is shown in SEQ ID NO:2.

[0012] Preferably, the TNNT2 primer set comprises a TNNT2 upstream primer and a TNNT2 downstream primer, the nucleotide sequence of the TNNT2 upstream primer is shown in SEQ ID NO:3, and the nucleotide sequence of the TNNT2 downstream primer is shown in SEQ ID NO:4.

[0013] Preferably, the PIT54 primer set includes a PIT54 upstream primer and a PIT54 downstream primer, the nucleotide sequence of the PIT54 upstream primer is shown in SEQ ID NO:5, and the nucleotide sequence of the PIT54 downstream primer is shown in SEQ ID NO:6.

[0014] Preferably, the SFTPC primer set comprises a SFTPC upstream primer and a SFTPC downstream primer, the nucleotide sequence of the SFTPC upstream primer is shown in SEQ ID NO:7, and the nucleotide sequence of the SFTPC downstream primer is shown in SEQ ID NO:8.

[0015] The present invention also provides the application of the PGM1 gene as a screening gene in the fluorescence quantitative detection of chicken breast muscle tissue.

[0016] The present invention also provides the use of the TNNT2 gene as a screening gene in fluorescence quantitative detection in chicken heart tissue.

[0017] The present invention also provides the application of the PIT54 gene as a screening gene in fluorescence quantitative detection in chicken liver tissue.

[0018] The present invention also provides the application of the SFTPC gene as a screening gene in fluorescence quantitative detection in chicken lung tissue.

[0019] By adopting the above technical solution, the present invention has the following beneficial effects:

[0020] The marker gene screened by the technical solution of the present invention is stable, specific and sensitive, and can be used to accurately identify chicken breast muscle, heart, liver and lung tissues. The chicken tissue marker gene of the present invention provides an important tool for biological research, agricultural breeding, disease diagnosis and transgenic animal research. The marker gene can accurately distinguish different tissues of chickens, improve the accuracy of disease diagnosis, and reduce the error of traditional methods. In agricultural breeding, it is helpful to screen and optimize chicken varieties. At the same time, it can also mark specific tissues of transgenic chickens to ensure the reliability of research. In addition, the system can significantly improve the early identification and prevention capabilities of chicken diseases and improve the disease management efficiency of the breeding industry. Based on this technology, molecular diagnostic kits can also be developed to improve the detection capabilities of food safety and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Agarose gel electrophoresis analysis of PCR amplification products from nine tissues of 3-month-old female Lueyang black-bone chicken ( Figure 1 A represents PGM1 gene, B represents TNNT2 gene, C represents PIT54 gene, and D represents SFTPC gene);

[0022] Figure 2 The expression levels of PGM1, TNNT2, PIT54 and SFTPC genes in nine tissues of 3-month-old female Lueyang black-bone chickens ( Figure 2 A represents PGM1 gene, B represents TNNT2 gene, C represents PIT54 gene, and D represents SFTPC gene);

[0023] Figure 3 The expression of PGM1 gene in nine tissues of 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 ( Figure 3A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0024] Figure 4 The expression of PGM1 gene in 9 tissues of male 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 ( Figure 4 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0025] Figure 5 The expression of TNNT2 gene in nine tissues of female 7-month-old Lueyang Silky Chicken, 1-month-old Liangfenghua Broiler Chicken, 2-month-old Liangfenghua Broiler Chicken, 4-month-old Liangfenghua Broiler Chicken and 2-month-old Silky Chicken ( Figure 5 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0026] Figure 6 The expression of TNNT2 gene in 9 tissues of male 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 ( Figure 6 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0027] Figure 7 The expression of PIT54 gene in 9 tissues of 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 ( Figure 7 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0028] Figure 8 The expression of PIT54 gene in 9 tissues of male 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 ( Figure 8 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0029] Fig. 9 The expression of SFTPC gene in 9 tissues of female 7-month-old Lueyang Silky Chicken, 1-month-old Liangfenghua Broiler Chicken, 2-month-old Liangfenghua Broiler Chicken, 4-month-old Liangfenghua Broiler Chicken and 2-month-old Silky Chicken ( Fig. 9 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken);

[0030] Fig.10 The expression of SFTPC gene in 9 tissues of male 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 ( Fig.10 A represents 7-month-old Lueyang black-bone chicken, B represents 1-month-old Liangfenghua broiler chicken, C represents 2-month-old Liangfenghua broiler chicken, D represents 4-month-old Liangfenghua broiler chicken and E represents 2-month-old Silky black-bone chicken). DETAILED DESCRIPTION

[0031] The invention provides marker genes for four tissues of chicken breast muscle, heart, liver and lung, including PGM1 gene, TNNT2 gene, PIT54 gene and SFTPC gene.

[0032] In the present invention, the marker gene of the chicken pectoral muscle tissue is preferably the PGM1 gene, the marker gene of the chicken heart tissue is preferably the TNNT2 gene, the marker gene of the chicken liver tissue is preferably the PIT54 gene, and the marker gene of the chicken lung tissue is preferably the SFTPC gene.

[0033] In the present invention, the gene ID of the PGM1 gene is ENSGALG00000011016, the gene ID of the TNNT2 gene is ENSGALG00000000302, the gene ID of the PIT54 gene is ENSGALG00000046217, and the gene ID of the SFTPC gene is ENSGALG 00000053247.

[0034] The invention provides the application of the marker gene as a screening gene in the qualitative detection of chicken genes.

[0035] The invention provides the application of the marker gene as a screening gene in fluorescence quantitative detection in different development stages or different tissues of chickens.

[0036] In the present invention, the primer sets used in the process of the fluorescence quantitative detection include a PGM1 primer set, a TNNT2 primer set, a PIT54 primer set and a SFTPC primer set.

[0037] In the present invention, the PGM1 primer set includes a PGM1 upstream primer and a PGM1 downstream primer, the nucleotide sequence of the PGM1 upstream primer is shown in SEQ ID NO: 1, and the specific sequence is 5'-ACACGGTTGAGAAAGCCGAT-3'; the nucleotide sequence of the PGM1 downstream primer is shown in SEQ ID NO: 2, and the specific sequence is 5'-GGAAGATGATGCGAGAGCCA-3'.

[0038] In the present invention, the TNNT2 primer set includes a TNNT2 upstream primer and a TNNT2 downstream primer, the nucleotide sequence of the TNNT2 upstream primer is shown in SEQ ID NO:3, and the specific sequence is 5'-TGAACATCGACCACCTCAGC-3'; the nucleotide sequence of the TNNT2 downstream primer is shown in SEQ ID NO:4, and the specific sequence is 5'-TGATCTCGTACTTCTGCCGC-3'.

[0039] In the present invention, the PIT54 primer set includes a PIT54 upstream primer and a PIT54 downstream primer, the nucleotide sequence of the PIT54 upstream primer is shown in SEQ ID NO:5, and the specific sequence is 5'-GTCCTGCTGGCAGAAGATGTA-3'; the nucleotide sequence of the PIT54 downstream primer is shown in SEQ ID NO:6, and the specific sequence is 5'-CCCGTAAATCCCAACCTTCA-3'.

[0040] In the present invention, the SFTPC primer set includes a SFTPC upstream primer and a SFTPC downstream primer, the nucleotide sequence of the SFTPC upstream primer is shown in SEQ ID NO:7, and the specific sequence is 5'-CGGTGGTGGTGTTCGACTA-3'; the nucleotide sequence of the SFTPC downstream primer is shown in SEQ ID NO:8, and the specific sequence is 5'-CAAAATGGAGCGGTCAGCG-3'.

[0041] The present invention also provides the application of the PGM1 gene as a screening gene in the fluorescence quantitative detection of chicken breast muscle tissue.

[0042] The present invention also provides the use of the TNNT2 gene as a screening gene in fluorescence quantitative detection in chicken heart tissue.

[0043] The present invention also provides the application of the PIT54 gene as a screening gene in fluorescence quantitative detection in chicken liver tissue.

[0044] The present invention also provides the application of the SFTPC gene as a screening gene in fluorescence quantitative detection in chicken lung tissue.

[0045] The technical solution provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] 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 the market in Hanzhong City, Shaanxi Province.

[0047] Example 1 (one)

[0049] Three normal and healthy female Lueyang black-bone chickens aged three months were selected, and the corresponding 9 tissues including bursa of Fabricius, pectoral muscle, heart, kidney, liver, lung, ovary, skin and spleen were taken for transcriptome sequencing. TSGs (genes that are highly expressed in a specific tissue type and not expressed or expressed at a very low level in other tissues) were screened based on the transcriptome sequencing data. Tissue-specific expression of genes means that they have tissue-related functions and can be used as marker genes for specific tissues. Therefore, TSGs highly expressed in each tissue were used as marker genes for each tissue. The screening criteria are: (1) TPM value in a specific tissue is >1000 and is more than 3 times the expression in other tissues; (2) TPM value of the target tissue of the candidate marker gene is greater than 50% of the average expression level of all other tissues; (3) The expression level of the candidate marker gene ranks in the top 25% of all genes in each tissue.

[0050] Table 1 Expression levels of candidate marker genes in nine tissues of 3-month-old female Lueyang black-bone chickens

[0051]

[0052]

[0053] Note: The values ​​in this table are calculated accurately and retained to two decimal places.

[0054] The reference genomes used were all ensembl_release_106, https: / / ftp.ensembl.org / pub / release-106 / gtf / gallus_gallus / . The gene ID of the PGM1 gene (phosphoglycerate mutase 1) is ENSGALG00000011016, the gene ID of the TNNT2 gene (troponin T2) is ENSGALG00000000302, the gene ID of the PIT54 gene (haptoglobin) is ENS GALG00000046217, and the gene ID of the SFTPC gene (surfactant protein C) is ENSG ALG00000053247.

[0055] Based on the above operations, PGM1 gene, TNNT2 gene, PIT54 gene and SFTPC gene were preliminarily screened out as candidate marker genes for pectoral muscle, heart, liver and lung. (two)

[0057] The total RNA from the above nine tissues was extracted by Trizol method and reverse transcribed into cDNA.

[0058] Take 50 mg each of the 9 tissues including 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.

[0059] 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, 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.

[0060] 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.

[0061] Table 2 Reverse transcription reaction system

[0062]

[0063] 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)

[0065] Gene-specific primers were designed according to the CDs region, and the primer information of four candidate marker genes and one internal reference gene is shown in Table 3.

[0066] Table 3 Primer information of candidate marker genes and internal reference genes

[0067]

[0068] The above primers were used to amplify the gene sequences by PCR, and the amplification system of each gene is shown in Table 4. The PCR amplification program of PGM1 and TNNT2 genes was as follows: 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.

[0069] The PCR amplification procedure of PIT54 gene was as follows: ① pre-denaturation at 95℃ for 5 min; ② (denaturation at 95℃ for 30 s; annealing at 53℃ for 30 s; extension at 72℃ for 30 s) for 34 cycles; ③ extension at 72℃ for 5 min.

[0070] The PCR amplification procedure of SFTPC gene was as follows: ① pre-denaturation at 95℃ for 5 min; ② (denaturation at 95℃ for 30 s; annealing at 54℃ for 30 s; extension at 72℃ for 30 s) for 34 cycles; ③ extension at 72℃ for 5 min.

[0071] Table 4 PCR amplification system

[0072] (Four)

[0074] After amplification, the product was placed on a 2% agarose gel and electrophoresed at 100 V for 40 min to analyze the PCR amplification product and observe whether the target band exists. DNA Marker is a 2000 bp DNA Ladder molecular weight standard; ACTB is used as a control.

[0075] The results are as follows Figure 1As shown, A is the electrophoresis of PCR amplification products of PGM1 gene in 9 kinds of tissues, and the results show that there is a clear and bright band with the same size as the target fragment in the pectoral muscle tissue; B is the electrophoresis of PCR amplification products of TNNT2 gene in 9 kinds of tissues, and the results show that there is a clear and bright band with the same size as the target fragment in the heart tissue; C is the electrophoresis of PCR amplification products of PIT54 gene in 9 kinds of tissues, and the results show that there is a clear and bright band with the same size as the target fragment in the liver tissue; D is the electrophoresis of PCR amplification products of SFTPC gene in 9 kinds of tissues, and the results show that there is a clear and bright band with the same size as the target fragment in the lung tissue. (five)

[0077] 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.

[0078] Table 5 RT-qPCR reaction system

[0079]

[0080] The quantitative analysis results of RT-qPCR were analyzed by 2 -ΔΔCt Methods The Ct value was transformed to calculate the expression fold difference of the target gene between the experimental group and the control group. ANOVA was used to determine whether there were significant differences in the means between different groups. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0081] ΔCt value: ΔCt = Ct (target gene) - Ct (reference gene)

[0082] ΔΔCt value: ΔΔCt = ΔCt (experimental group) - ΔCt (control group)

[0083] The expression levels of four candidate marker genes, PGM1, TNNT2, PIT54 and SFTPC, in nine tissues of 3-month-old female Lueyang black-bone chickens Figure 2 shown.

[0084] Example 2 (one)

[0086] 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 different periods to ensure the stability and universality of the candidate marker genes. The PCR and RT-qPCR verification process, reaction system and reaction procedure were the same as above.

[0087] 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.

[0088] The results showed that the four candidate marker genes PGM1, TNNT2, PIT54 and SFTPC were specifically expressed in chicken breast muscle, heart, liver and lung, which was in line with the expected results and could be used as tissue marker genes. The expression of the PGM1 gene in nine tissues of female 7-month-old Lueyang Silky Chicken, 1-month-old Liangfenghua Broiler Chicken, 2-month-old Liangfenghua Broiler Chicken, 4-month-old Liangfenghua Broiler Chicken and 2-month-old Silky Chicken was as follows: Figure 3 As shown in Figure 2, the expression of PGM1 gene in 9 tissues of each male test subject is as follows Figure 4 The expression of the above TNNT2 gene in 9 tissues of each female test subject is shown in Figure 5 As shown in Figure 2, the expression of TNNT2 gene in 9 tissues of each male test subject is as follows Figure 6 The expression of the above PIT54 gene in 9 tissues of each female test subject is shown in Figure 7 As shown in Figure 2, the expression of PIT54 gene in 9 tissues of each male test subject is as follows Figure 8 The expression of the above SFTPC gene in 9 tissues of each female test subject is shown in Fig. 9 As shown in Figure 2, the expression of SFTPC gene in 9 tissues of each male test subject is as follows Fig.10 shown.

[0089] In summary, it can be seen that the PGM1 gene, TNNT2 gene, PIT54 gene, and SFTPC gene screened by the technical scheme of the present invention are stable, extensive, and sensitive marker genes for four tissues of chicken breast muscle, heart, liver, and lung, which also provides an important tool for biological research, agricultural breeding, disease diagnosis, and transgenic animal research.

[0090] 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. A marker gene for four tissues of chicken breast muscle, heart, liver and lung, characterized in that: It includes PGM1 gene, TNNT2 gene, PIT54 gene and SFTPC gene, the gene ID of the PGM1 gene is ENSGALG00000011016, the gene ID of the TNNT2 gene is ENSGALG00000000302, the gene ID of the PIT54 gene is ENSGALG00000046217, and the gene ID of the SFTPC gene is ENSGALG00000053247.

2. Use of the marker gene according to claim 1 as a screening gene in qualitative detection of chicken genes.

3. Use of the marker gene according to claim 1 as a screening gene in fluorescence quantitative detection in different developmental stages or different tissues of chickens.

4. The use according to claim 1, characterized in that: The primer sets used in the fluorescent quantitative detection process include a PGM1 primer set, a TNNT2 primer set, a PIT54 primer set, and a SFTPC primer set; The PGM1 primer set includes a PGM1 upstream primer and a PGM1 downstream primer, the nucleotide sequence of the PGM1 upstream primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the PGM1 downstream primer is shown in SEQ ID NO: 2; The TNNT2 primer set includes a TNNT2 upstream primer and a TNNT2 downstream primer, the nucleotide sequence of the TNNT2 upstream primer is shown in SEQ ID NO:3, and the nucleotide sequence of the TNNT2 downstream primer is shown in SEQ ID NO:4; The PIT54 primer set includes a PIT54 upstream primer and a PIT54 downstream primer, the nucleotide sequence of the PIT54 upstream primer is shown in SEQ ID NO:5, and the nucleotide sequence of the PIT54 downstream primer is shown in SEQ ID NO:6; The SFTPC primer set includes a SFTPC upstream primer and a SFTPC downstream primer, the nucleotide sequence of the SFTPC upstream primer is shown in SEQ ID NO:7, and the nucleotide sequence of the SFTPC downstream primer is shown in SEQ ID NO:

8.

5. Use of the PGM1 gene according to claim 1 as a screening gene in fluorescence quantitative detection of chicken breast muscle tissue.

6. Use of the TNNT2 gene according to claim 1 as a screening gene in fluorescence quantitative detection in chicken heart tissue.

7. Use of the PIT54 gene according to claim 1 as a screening gene in fluorescence quantitative detection in chicken liver tissue.

8. Use of the SFTPC gene according to claim 1 as a screening gene in fluorescence quantitative detection in chicken lung tissue.

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