Method for judging mycoplasma gallisepticum resistance of chicken based on MMP7 gene

Abstract

The invention belongs to the technical field of genetic engineering of animal molecular breeding, and aims to disclose a method for judging the mycoplasma gallisepticum resistance of chicken based on an MMP7 gene. Through accurate screening and analysis of chicken embryo lung tissue sequencing data, the MMP7 gene is successfully identified as a typical immune gene related to body weight and mucosal thickness. The discovery provides important theoretical support and reference basis for the development of molecular auxiliary markers and biological disease-resistant breeding of mycoplasma gallisepticum.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering of animal molecular breeding, and in particular relates to a method for judging the strength of chicken's resistance to Mycoplasma gallisepticum based on MMP7 gene. Background Art

[0002] Disease resistance breeding is the use of genetic basis to cultivate poultry breeds with strong disease resistance. The genetic basis of disease resistance breeding refers to the genes or genomic regions that have disease resistance in poultry. These genes or genomic regions can affect the infection, transmission and reproduction of pathogens in poultry, thereby enhancing their disease resistance. Therefore, identifying the expression characteristics of relevant immune genes is the research basis of disease resistance breeding.

[0003] MMP7 is the smallest and simplest member of the MMPs family. Its inactive form, pro-MMP7, is only 28 kDa in size (19 kDa in active form). Except for the absence of the C-terminal hemoglobin-binding protein-like region, the basic structure and physicochemical properties of MMP7 are basically similar to those of other MMPs. MMP7 can not only degrade ECM components such as type IV collagen, laminin and elastin, but also has the ability to cleave Fas ligand, growth factors and cytokines. MMP7 is a major regulator of innate and acquired immunity, and it plays an important role in maintaining innate immunity in organs such as the lungs and intestines. A large number of literatures have confirmed that MMP7 is expressed in tumor tissues of every organ, especially in lung tumors. MMP7 has a high expression level in lung tissue and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and pulmonary nodules. . Summary of the invention

[0004] The invention provides a method for judging the strength of chicken's anti-Mycoplasma gallisepticum ability based on MMP7 gene. Molecular biological technology and bioinformatics analysis method are used to study the expression of MMP7 gene mRNA in chicken embryo lung tissue and lungs of five varieties of chickens. It is found that the MMP7 gene is related to the anti-Mycoplasma gallisepticum ability. The strength of chicken's immunity to Mycoplasma gallisepticum is judged through the correlation between gene expression and body weight and mucosal thickness. Chickens with high expression in lungs are easily infected by Mycoplasma gallisepticum and have weak immune function.

[0005] In order to achieve the above object, the present invention proposes the following technical solutions:

[0006] A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene comprises the following specific steps:

[0007] 1) RNA extraction

[0008] ① Take out the chicken embryo lung tissues of the MG infection group and the control group at -80℃, take 100 mg from them, add 1 mL of TRIzol, and homogenize them in a homogenizer 4 times, each time for 20 seconds to ensure that the tissues are completely broken. After the homogenization, place the samples at -80℃ for low-temperature lysis for more than 0.5 h;

[0009] ② Centrifuge at 4°C, 12,000 rpm for 10 min;

[0010] ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4℃ for 5 minutes, and centrifuge at 4℃ and 12,000 r / min for 15 minutes; ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20℃ for 15 minutes, centrifuge at 4℃ and 12,000 r / min for 15 minutes, and discard the supernatant;

[0011] ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice;

[0012] ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction.

[0013] ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio.

[0014] 2) PCR primer design

[0015] According to the relevant gene sequence of Gallus gallus in GenBanK, primers were designed and synthesized using primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis; the primer sequence is

[0016] F:5'-CCC AAGCTT ATGCAATACCTCCTACTTTGTGCT-3';

[0017] R:5'-AAATAT GCGGCCGC TCACGAGTTTGCTGAGTTTCT-3'

[0018] 3) Screening of the core regulatory candidate immune gene MMP7

[0019] In order to further screen the core regulatory immune genes of MG infection, we used Venn diagrams to comprehensively compare and analyze the candidate genes screened in this study with the Top30 significantly upregulated DEGs reported by previous researchers to obtain 11 candidate immune genes, and used STRING to analyze the interaction network between the proteins encoded by these 11 candidate genes.

[0020] 4) Detection of MMP7 gene expression

[0021] The expression of MMP7 was detected in AECII cells infected with different concentrations of MG;

[0022] The expression of MMP7 was further detected in the immune organs of chicken embryos;

[0023] Further detection of MMP7 expression in chicken immune organs

[0024] The reaction conditions of fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s, for 38 cycles.

[0025] The beneficial effects of the present invention are:

[0026] The present invention designs primers for the MMP7 gene of chicken, performs PCR amplification, detects the expression changes of the mRNA of the MMP7 gene in AECII cells infected with different MG concentrations, and the correlation between its expression and the body weight and tracheal mucosal thickness of MG infected with different breeds. The expression shows that chickens with high MMP7 expression are more susceptible to infection with Mycoplasma gallisepticum, which provides an immune theoretical basis for the formulation of MG infection prevention and control strategies and the use of molecular marker-assisted selection methods to cultivate disease-resistant breeding strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a comparative analysis diagram of the core immune candidate genes of the chicken embryo lung tissue infected with MG of the present invention.

[0028] Figure 2 It is the expression level of the MMP7 gene of the present invention in AECII cells infected with different doses of MG.

[0029] Figure 3 This is a comparative analysis diagram of the expression of the MMP7 gene of the present invention in chicken embryo immune organs.

[0030] Figure 4 This is a comparative analysis diagram of the expression of the MMP7 gene of the present invention in the immune organs of chicks.

[0031] Figure 5This is a comparative analysis diagram of the expression of the MMP7 gene of the present invention in different breeds of chickens.

[0032] Figure 6 This is an analysis diagram of the correlation between the MMP7 gene expression level in the lung tissue and the chicken body weight gain of the present invention.

[0033] Figure 7 This is an analysis diagram of the correlation between the MMP7 gene expression level in the lung tissue and the chicken tracheal mucosal thickness of the present invention. DETAILED DESCRIPTION

[0034] like Figure 1-4 As shown, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention.

[0035] Rather than all the embodiments, all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene comprises the following specific steps:

[0038] 1) RNA extraction

[0039] ① Select chicken embryo lung tissues from the MG infection group and the control group of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken, take 100 mg from them, add 1 mL of TRIzol, and homogenize in a homogenizer 4 times, each time for 20 seconds to ensure that the tissue is completely broken. After the homogenization, place the sample at -80℃ for low-temperature lysis for more than 0.5h;

[0040] ② Centrifuge at 4°C, 12,000 rpm for 10 min;

[0041] ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4℃ for 5 minutes, and centrifuge at 4℃ and 12,000 r / min for 15 minutes; ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20℃ for 15 minutes, centrifuge at 4℃ and 12,000 r / min for 15 minutes, and discard the supernatant;

[0042] ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice;

[0043] ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction.

[0044] ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio.

[0045] 2) PCR primer design

[0046] According to the relevant gene sequence of Gallus gallus in GenBanK, primers were designed and synthesized using primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis; the primer sequence is

[0047] F:5'-CCC AAGCTT ATGCAATACCTCCTACTTTGTGCT-3';

[0048] R:5'-AAATAT GCGGCCGC TCACGAGTTTGCTGAGTTTCT-3'

[0049] 3) Detection of MMP7 gene expression

[0050] The expression of MMP7 gene was detected in the lung tissues of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken. The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, and 38 cycles.

[0051] 4) Body weight measurement of MG infection group and negative group

[0052] The body weights of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken were measured during MG infection, and the negative ones were used as the control group.

[0053] 5) Determination of tracheal mucosal thickness in MG-infected chicks

[0054] The thickness of tracheal mucosa of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken after MG infection was measured.

[0055] like Figure 5-Figure 7 The expression level of MMP7 gene was detected in the lung tissues of Jingfen No. 6, Hailanbai, Qingjiao Ma chicken, Tianlu Ma chicken and Guangxi native chicken, and its correlation with body weight and mucosal thickness.

[0056] The present invention designs primers for the MMP7 gene of chicken, performs PCR amplification, detects the expression changes of the mRNA of the MMP7 gene in AECII cells infected with different MG concentrations, and the correlation between its expression and the body weight and tracheal mucosal thickness of MG infected with different breeds. The expression shows that chickens with high MMP7 expression are more susceptible to infection with Mycoplasma gallisepticum, which provides an immune theoretical basis for the formulation of MG infection prevention and control strategies and the use of molecular marker-assisted selection methods to cultivate disease-resistant breeding strains.

[0057] The above embodiments of the present invention provide a detailed introduction to the method for different breeds of chickens with MMP7 genes to resist Mycoplasma gallisepticum. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, but are not limited to the above five chicken breeds. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene, characterized in that: The specific steps include: 1) RNA extraction ① Take out the chicken immune organs bursa of Fabricius, thymus and spleen from the MG infection group and the control group at -80℃, take 100 mg from them, add 1 mL of TRIzol, and homogenize in a homogenizer 4 times, 20 seconds each time, to ensure that the tissue is completely broken. After the homogenization, place the sample at -80℃ for low-temperature lysis for more than 0.5h; ② Centrifuge at 4°C, 12,000 rpm for 10 min; ③ Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at 4°C for 5 minutes, and centrifuge at 4°C and 12,000 r / min for 15 minutes; ④ Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix by inversion several times, let stand at -20°C for 15 min, centrifuge at 4°C and 12,000 r / min for 15 min, and discard the supernatant; ⑤ Add 1 mL of 75% anhydrous ethanol, gently flick the precipitate to rinse the precipitate, centrifuge at 4°C and 12,000 r / min for 5 min, and discard the supernatant; repeat this step twice; ⑥ According to the amount of precipitation, add an appropriate amount of DEPC water to dissolve the RNA precipitation. After gently mixing and dissolving, NanoDrop-2000 was used to determine the concentration and purity of the extracted total RNA. Take 2 μL of total RNA and perform electrophoresis on 1.2% agarose gel. The remaining RNA was stored in a -80℃ refrigerator for later use. The three bands of 28s rRNA, 18s rRNA, and 5.8s rRNA were observed to determine the quality of RNA extraction. ⑦ Determination of RNA purity and concentration: RNA has the largest absorption peak at 230nm wavelength. The concentration of total RNA is measured by 230nm wavelength spectrophotometry. The OD260 / OD280 ratio of pure RNA samples is about 2.0, so the purity of RNA can be estimated based on the OD260 / OD280 ratio. 2) PCR primer design According to the relevant gene sequence of Gallus gallus in GenBanK, primers were designed and synthesized using primer design software primer5.0, and then fluorescent quantitative PCR was performed after synthesis; The primer sequence is F: 5'-CCC AAGCTT ATGCAATACCTCCTACTTTGTGCT-3'; R: 5'-AAATAT GCGGCCGC TCACGAGTITGCTGAGTTTCT-3' 3) Detect the expression of MMP7 gene in cells The expression of MMP7 gene was detected in AECII cells infected with different concentrations of MG.The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles. 4) Detection of the expression of MMP7 gene in chicken embryos The expression of MMP7 gene was detected in the thymus and bursa of chicken embryo immune organs.The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles. 5) Detection of the expression of MMP7 gene in chicks The expression of MMP7 gene was detected in the immune organs spleen, thymus and bursa of Fabricius in chicks. The reaction conditions of fluorescence quantitative PCR were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 38 cycles.

2. A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene, characterized in that: Step 2) The primer sequence of the MMP7 gene used for fluorescence quantification in PCR primer design is: F: 5'-CCC AAGCTT ATGCAATACCTCCTACTITGTGCT-3'; R: 5'-AAATAT GCGGCCGCTCACGAGTTTGCTGAGTTTCT-3' 3. A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene, characterized in that: The cells are AECII cells, and the dosages of MG are 50ul, 100ul, 200ul, 300ul and 500ul / ml respectively.

4. A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene, characterized in that: The chicken embryo immune organs are the thymus and the bursa of Fabricius.

5. A method for judging the ability of chickens to resist Mycoplasma gallisepticum based on the MMP7 gene, characterized in that: The chicken immune organs are spleen, thymus and bursa of Fabricius.

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