SNP (Single Nucleotide Polymorphism) related to key rate-limiting enzyme-cytochrome CYP4A21 for synthesizing hyocholic acid of adult domestic pig, detection method and application of SNP

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) related to hyocholic acid synthesis key rate-limiting enzyme-cytochrome CYP4A21 of adult domestic pigs, a detection method and application thereof, and relates to the field of animal breeding. Gene markers related to hyocholic acid synthesis key rate-limiting enzyme of Min pigs and large white pigs are screened out, CYP4A21 is selected as a candidate gene for high-meat-quality breeding of adult pigs, and the CYP4A21-cytochrome CYP4A21-cytochrome CYP4A21-cytochrome CYP4A21-cytochrome By searching genetic variation of a pig CYP4A21 gene promoter region, a molecular marker and a detection method thereof are provided and applied to pig breeding, a pig molecular marker assisted breeding method is established, and a gene marker and a technical method are provided for breeding of a new high-meat-quality pig strain. The SNP locus is located in rs99141509 of a promoter region of the CYP4A21 gene of the pig, and the polymorphism of the SNP locus is T / C. The SNP locus can be used for detecting the CYP4A21 gene of the pig. The method is applied to identifying the quality of the adult pork. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1. The method is applied to the breeding field.

Description

Technical Field

[0001] The present invention relates to the field of animal breeding, and in particular to a SNP for selecting the bile acid synthesis ability of adult domestic pigs, a detection method and an application thereof. Background Art

[0002] Min pigs are the only local pig breed in Northeast my country, and a high intramuscular fat content is considered an important factor in the excellent quality of its meat products. However, due to the influence of factors such as operator experience and pig development level, the accuracy of intramuscular fat estimation of Min pigs by detection technologies such as live B-ultrasound in actual production is much lower than that of post-slaughter measurement. However, for local breeds such as Min pigs with extremely small population sizes, live seed retention should be prioritized over slaughter sampling. Therefore, the development of minimally invasive (or non-invasive) meat quality phenotype measurement technology and multi-dimensional genetic markers is extremely important for discovering Min pig meat traits, breeding and improving new varieties, and improving breeding accuracy and efficiency.

[0003] Hyocholic acid (3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholanic acid, HCA) is the only trihydroxycholic acid in adult domestic pigs. Before the 1960s, HCA was considered a trihydroxybile acid unique to domestic pigs and did not receive widespread attention in clinical practice and application. Although there were reports as early as 1964 that adding HCA to the diet improved cholesterol metabolism disorders in mice, there has been a lack of attention to the relationship between HCA and glycolipid homeostasis in the past half century, and related reports have rarely been seen. Until the past decade, with the rapid development of multi-omics sequencing and joint analysis, the mechanism of HCA as a key signaling molecule involved in regulating glycolipid metabolic homeostasis in mammals has gradually been discovered and paid attention to. Targeted metabolome sequencing of 20 pigs showed that the IMF content of the longissimus dorsi muscle was positively correlated with the serum HCA level. Adding HCA to livestock feed may help regulate the fat metabolism of livestock, thereby controlling the level of IMF, improving meat quality and maintaining the health of livestock. In the breeding process, pig bile acid can be used as a good candidate marker molecule to select those breeds that can naturally produce higher HCA levels, which can improve the fat metabolism ability of future generations of livestock to a certain extent. HCA is a primary bile acid produced by limiting C6α-hydroxylation by CYP4A21 in the liver of domestic pigs. CYP4A21 can directly participate in the synthesis of HCA and is a key enzyme in the HCA production pathway. CYP4A21 may also indirectly affect the production of HCA by affecting other related metabolic pathways.

[0004] In 2021, two groundbreaking studies by Professor Jia Wei's team at Shanghai Jiao Tong University provided a basis for the negative correlation between HCA and metabolic syndrome derived from glucose and lipid disorders in clinical practice. The study analyzed the molecular mechanism by which HCA increases the secretion of glucagon-like peptide-1 by activating intestinal epithelial cell TGR5 and inhibiting farnesoid X receptor signaling, thereby accelerating blood sugar clearance and maintaining blood sugar homeostasis under a high-sugar diet. It clarified the physiological basis for HCA to produce strong resistance to diet-induced diabetes in domestic pigs during the fattening stage. In the past two years, in the livestock industry, Zhang B's team has reported across species (Tan sheep) that HCA regulates fat distribution and improves lipid metabolism disorders. Mitsuhiro Watanabe, Velazquez-Villegas LA and Broeders EP et al. found in clinical and mouse models that the content of HCA bile acids in the circulatory system is significantly negatively correlated with the occurrence of individual obesity and metabolic syndrome. In-depth studies have confirmed that after TGR5 activation, it can promote aerobic respiration and energy consumption of brown fat cells by upregulating the expression of iodothyronine deiodinase II, and can also increase mitochondrial fission and β-oxidation by activating the downstream ERK / DRP1 pathway, increasing energy consumption and transdifferentiation of white fat cells. Yuan Zhang et al. found that after LXR activation, it can activate downstream SREBP-1c (Sterol regulatory elementbinding protein 1c, SREBP-1c), increase cholesterol and fatty acid synthesis in hepatocytes, and promote fat deposition. Kajimura S, Chen A and Marcinkiewicz A et al. showed that FTO can modify the maturation of precursor mRNA of key proteins / enzymes for lipid synthesis and deposition through m6A demethylation, thereby promoting the differentiation of precursor adipocytes into mature white adipocytes. Professor Wang Yizhen's team conducted experiments on primary porcine adipocytes and 3T3-L1 cells and found that after specific knockout or targeted interference of FTO, the methylation level of autophagy-related proteins 5 and 7 mRNA was increased, and the level of recognition and binding by methyladenosine RNA-binding protein 2 was increased, thereby causing mRNA degradation and translation inhibition, reducing cellular autophagy and increasing fat deposition. Summary of the invention

[0005] The present invention provides a SNP related to cytochrome CYP4A21, a key rate-limiting enzyme in bile acid synthesis of adult domestic pigs, a detection method and an application thereof. The present invention selects CYP4A21 as a candidate gene for breeding high meat quality in adult pigs by screening out gene markers related to the key rate-limiting enzyme in bile acid synthesis of Min pigs and Large White pigs, and provides molecular markers and a detection method thereof by searching for genetic variations in the promoter region of the pig CYP4A21 gene. The molecular markers and a detection method thereof are applied to pig breeding, and a pig molecular marker-assisted breeding method is established, thereby providing gene markers and technical methods for the selection and breeding of new strains of high meat quality pigs (high bile acid).

[0006] The present invention discloses an application of a SNP related to cytochrome CYP4A21, a key rate-limiting enzyme in bile acid synthesis in adult domestic pigs, in assisted breeding. The SNP is applied in assisted breeding to improve pork quality; the SNP is a polymorphic site NC_010448.4:rs99141509 in the promoter region of the CYP4A21 gene, and the nucleotide sequence of the SNP is shown in SEQ ID NO:1.

[0007] Furthermore, the polymorphism is T / C.

[0008] Furthermore, the assisted breeding for improving pork quality is achieved by selecting individual piglets with relatively high bile acid synthesis ability from the pig population to be bred.

[0009] Furthermore, the assisted breeding for improving pork quality is achieved by selecting piglets with relatively high bile acid synthesis ability from the pig population to be bred, and the selection of piglets with relatively high bile acid synthesis ability is to eliminate CC genotype individuals and retain TT genotype individuals as piglets with relatively high bile acid synthesis ability; wherein, CC genotype individuals are individuals whose bile acid synthesis amount is lower than that of TT genotype individuals.

[0010] Furthermore, the domestic pig is a Min pig or a Large White pig.

[0011] Furthermore, the assisted breeding method is as follows:

[0012] (1) Using primers CYP4A21F and CYP4A21R, the PCR product of the gene fragment of the promoter region of the CYP4A21 gene of the pig population to be bred is obtained by the PCR method. After successful sequencing verification, the obtained CYP4A21 gene promoter region gene has a C allele at the -1234th position, which is recorded as a CC genotype individual; the obtained CYP4A21 gene promoter region gene has a T allele at the -1234th position, which is recorded as a TT genotype individual;

[0013] (2) removing individuals with CC genotype from the pig population to be selected and retaining individuals with TT genotype, thereby completing the assisted breeding;

[0014] Among them, the nucleotide sequences of CYP4A21F and CYP4A21R primers are as follows:

[0015] CYP4A21F: ATTATACCCTTGGGTCGGTGCACT;

[0016] CYP4A21R: TCAGTGGACTTGTAGGGCGA.

[0017] The primers for detecting SNPs related to cytochrome CYP4A21, a key rate-limiting enzyme in bile acid synthesis in adult domestic pigs, are CYP4A21F and CYP4A21R, and the nucleotide sequences are as follows:

[0018] CYP4A21F: ATTATACCCTTGGGTCGGTGCACT;

[0019] CYP4A21R: TCAGTGGACTTGTAGGGCGA.

[0020] A kit containing the primers of the present invention.

[0021] The molecular marker developed based on the SNP site of the present invention is achieved by detecting the genotype of the SNP (NC_010448.4:rs99141509) site in the promoter region of the CYP4A21 gene in the pig genome, that is, in the process of individual genetic assessment, individuals with the SNP (NC_010448.4:rs99141509) site as TT are screened out, thereby achieving molecular marker-assisted selection for improving the key rate-limiting enzyme CYP4A21 in bile acid synthesis of adult domestic pigs.

[0022] The present invention carried out population genetic parameter analysis on the polymorphism of SNP (NC_010448.4:rs99141509) site, and found that there were obvious differences in the genotype frequency and allele frequency of this site between Min pigs and Large White pigs: in the adult domestic pig group with strong swine bile acid synthesis ability, the frequency of C allele at position -1234 was higher than that of T allele (0.530vs 0.469), indicating that this site is related to the swine bile acid synthesis ability of domestic pigs.

[0023] The study of this invention shows that the frequency of T allele in the Large White pig population is higher than that of C allele (0.5125 vs 0.4875); while in the Min pig population, the frequency of C gene is higher than that of T allele (0.6 vs 0.4), suggesting that this site is related to the ability of Large White pigs and Min pigs to synthesize bile acid.

[0024] The present invention conducts trait association analysis on the polymorphism of SNP (NC_010448.4:rs99141509) locus in Min pig and Large White pig populations, and explores the correlation between molecular markers and the bile acid synthesis index of adult pigs. Statistical analysis shows that SNPrs99141509 is significantly correlated with the bile acid synthesis score of Min pig and Large White pig (P<0.05), among which the bile acid synthesis amount of TT genotype individuals is significantly higher than that of CC genotype individuals (P<0.01); therefore, in actual breeding work, eliminating CC type individuals can improve the bile acid synthesis ability of Min pig and Large White pig.

[0025] The present invention found that the T to C mutation at position -1234 of the SNP (NC_010448.4:rs99141509) in the promoter region of the porcine CYP4A21 gene (the CC mutation is shown in the -1234th base in the sequence table SEQ ID NO: 1) changed the activity of the CYP4A21 promoter in an in vitro test, and the difference between the two was significant. It can be seen that the detection of molecular markers associated with mutations is not only simple and fast, but also not affected by the environment, and can achieve early seed selection. The present invention selects seeds by judging the polymorphism of the mutation site in the promoter region of the porcine CYP4A21 gene, and uses the mutation site to select piglets with higher porcine bile acid synthesis ability, thereby accelerating the breeding process of the new strain of high meat quality of the Min pig. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis diagram of PCR amplification of porcine CYP4A21 gene promoter; in the figure, lane M is DL2000 marker, and lanes 1 and 2 are target bands;

[0027] Figure 2 The sequencing results of the SNP (NC_010448.4:rs99141509) site in the promoter region of the porcine CYP4A21 gene; Figure A is a TT type individual; Figure B is a CT type individual; Figure C is a CC type individual;

[0028] Figure 3 Schematic diagram of HCA targeted metabolomics sequencing results for individuals with CC genotype and TT genotype;

[0029] Figure 4 Schematic diagram of the luciferase reporter system used to detect changes in vector expression after the mutation of the SNP (NC_010448.4:rs99141509) site. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0031] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, but are not intended to limit the present invention.

[0032] The beneficial effects of the present invention are verified by the following examples:

[0033] Example 1

[0034] Analysis of the Promoter Region of the Porcine CYP4A21 Gene by PCR

[0035] 1. Genome extraction: Take pig ear tissue and extract genomic DNA using the conventional phenol-chloroform method.

[0036] 2. Primer design: The pig CYP4A21 gene information (transcript ID: NM_214425.1) was queried through the Ensemble database of NCBI, and the 2600bp nucleotide sequence upstream of the first exon was downloaded for primer design. The primers are CYP4A21F and CYP4A21R. The primer sequences are as follows:

[0037]

[0038]

[0039] 3. PCR amplification: This example uses conventional PCR to amplify the -2564 to -894 fragment of the promoter region of the Min pig CYP4A21 gene. The amplification system is: 1 μL of genomic DNA, 5 μL of 2×Papid Taq Mister Mix, 0.4 μL of each upstream and downstream primer, and ddH2O to make up to 10 μL. The amplification program is: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58.3℃ annealing for 15 s, 72℃ extension for 41 s, 31 cycles; 72℃ final extension for 5 min; 4℃ storage.

[0040] 4. PCR product detection and gel recovery and purification of target fragments:

[0041] After the PCR reaction is completed, a 1.0% agarose gel is prepared to detect the PCR amplification product. The setting conditions are: 110V, electrophoresis for 30 minutes. After the electrophoresis is completed, the PCR amplification results are observed on the C150 gel imaging system. If a single, bright and correct length target fragment is observed, the target fragment area is gel cut. The gel recovery test is performed according to the instructions of the gel recovery kit of BioTeke. After the gel recovery product is detected by agarose gel electrophoresis, it is uniformly stored in a -20℃ refrigerator.

[0042] 5. Sequencing of PCR amplification products: The gel-recovered products were tested by 1.0% agarose gel electrophoresis, and the single band solution with the correct length was selected and sent to Liuhe BGI (Beijing) Gene Technology Co., Ltd. for sequencing. The sequencing results are shown in Seq ID No: 1, and the Seq ID No: 1 is the SNP of the present invention, and the specific nucleotide sequence is as follows:

[0043] ATTATACCCTTGGGTCGGTGCACTGCTTGCAAAGATTTTCTCCCATTCTGTGGGT

[0044] TGTCTTTGCATTCTTTGAAGGGTTCCCATTGCTGTGCAAAAGCTTTCGAATTTAGTTG

[0045] GGTCCCATTTGTTTATTTTGTTCTTATTGTCATTATTCTAGGAGGTGGATCAAATAAGA

[0046] TGTTGCTGAGATTTATGTCAGAGAGCATTCTGCCTGTGTTTTCCTCTAAGAGTTCTAG

[0047] AGTATCTCACCTTACATTTATGCCTTTAATCCATTTTGAGATTATTTTGTGTACGATGTT

[0048] AGAGATTGTTCTAACTTCATTCTTTTGCTTGTAGCTGTCCCGTTTTTGCCAGAACCACT

[0049] TGGTTTTGTTGTTGTTGCTGTTGTTGTTGTTGTTGTTGTCGTTGTTTGTCTTTTTGCCA

[0050] TTTCCATGGGCTGCTCCTGCGGCATATGGAGGTTCCGAGGCTAGGGGGTCGAATTGGA

[0051] GCTGTGGCTGCCGGCCTACGCCAGAGCCACAGCAACGCGGGATCTGAGCTACATCT

[0052] GCCACCTACACCACAGCTCACGGCAACGCTGGATCCTCAACCCACTGAGCAAGGCC

[0053] AGGGATCAAACCCGCAACCTCATGGTTCCTAGTCGGATTCGTTACCCACTGAGCCAC

[0054] AACGGGACCTCCAGAACCACTTGTTGAAGAGAGTCTCTTTCCCTCACTGTGTGTTCT

[0055] TGGATACTTTGCTTAGGTTACTCTTAACCATAGGTGCATCGGTGTATTTCTAGGCTTTC

[0056] CATCTTGTTCCATTGATCTCTATTTCTGCTTTTGTTATGTGAACTACATGGGTGTGATA

[0057] ACTGTATTTGTGTATTATAGTCTGAAGTCAGAGAGACTGATTCCTGTTGTTCTTTTTTT

[0058] TTTCTCCCAACATTTCTTTGGCTATTTGGGGTCTTTGAATTCCATAAAAAAAATTTAA

[0059] AGATTTTGTTATAGTTCAGTGAAGAGTGTCATTGGTCCTTTGATAAGGATGGCATTGA

[0060] ACCTGAAGATAACCTTGGGTAGGATATTCATTTTGACAATATTGATTCTTGCAAACTT

[0061] TCCATCTGTTTGTATCATCTTTGCTTTCTTTCATCAGTGTCTTATAGTTTTCAGTGCAG

[0062] AGGTCTTTTGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTAAT

[0063] TATTTATTTATTTATTTATCTATTTGGTCTTTTTGGGGCCAAACCCACAGCATATGGAAG

[0064] TTCCCAGGCTAGTGGTTGAGTCAGAGCTGTAGCCCCTGGCCTACACCACAGCCACA

[0065] GCAACGCCAGATCCAAGTAGACTCTGTGAAGTACACCACAGCTCATGGCAATGCTA

[0066] GATCATTAACCCACTGACTGAGGCCAGGGATAGAACCTGCGCCCTCACGGAAGCTA

[0067] GACAGAATCATTTCCACTGAGCCATGGTGGGAAATCCTAGGTCTTTTGCCTGTTTAG

[0068] GTAAGTTTATTCTTAAGTGTTTTAATCTTTTCGATGTGATGCTAAATGGGATGGTTCCC

[0069] CTAAATTCTCAATCTCATCTCCCCATGTAATTACATAGAAATGCCTTTGACTTCTGAGT

[0070] TTTAATTTGGTATCTGGCAACTTACTGATGCCCATGACTTTTTAGGCCCCATCCCTCTA

[0071] GAAGCACTCTCTGCCTGGTGAGTTCCTATTCGCCCTACAAGTCCACTGAATTTTTTTA

[0072] CTTTTCCCCTTCCCTGGTTGTCCCAGTCACTCTTCATTCTTCCAGGACAACCTTTGTC

[0073] ATTTTTATTGTACTAATACCATTTTCGGCCTTTACCTGTAGAGCCAGAGAAATATTTGC

[0074] TGAGTGAATGACTGAATGAACGAATGAGTAGTCACAGGAGGTACACAGGAAATGTA

[0075] CTAACATGCCAACTTTCAAAGGAGCAACGGAGGCTCAGAGAGTACATGTTGTTAGC

[0076] TGCATGTGTGGTGAGGCTGCCCTGGGGCTGGGGCTCCTGATTCCAAGCCCAGAGCT

[0077] CCGTCCACTGGCACAGTCTCCCAGGACACAACCACACAGTGGACTGGGACAAGCC

[0078] GTGGGGGGACCTGGCAGAGGCAGCTGAGGCTGGCAGTCTGAGGTCCAACAGAGAA

[0079] GGGCTGCGAGCTCAAGGATTTCAGCTGGGACTCTCAAAGGCCGTGTTCCTCTGGCT

[0080] ACATCACCTCTCCTCTCTGAAATGGGTTTTGACCTCTGAAAGGGGGATAACGATAGA

[0081] GGTAACCGCACCTACTGCACGTAGGGCAAATGAGCCAGTGAATGTGTGGGAGTCAA

[0082] GCAAAGTTCATGTCTGCTTTGCTCGCAGCTGTGTCCCAGCACCTAGCCGACTGCCTG

[0083] GCAGGTTGCAGATGCTCATGAACTCTTTGGCGAGTGATGTGGAGAAGGAAAAGTGG

[0084] GTTTTGCACGGCCTTTGTCCCTCTCGCGGACAAAGCTTTGCGCACATATTTTCTCCAT

[0085] AGTTAGCATATCTGGAGGCCCAGCTCTGACCTCTACCCTCCTCACCACCCTCCCCCA

[0086] GGTGGGCGGGGCCATCCTCCCTGACTAGGCTGGATGCTCAGGGGAGGTCAGCCCCA

[0087] GTCAGGGACAGTCAGCCATCCAGAAGCCCGTGCACCATGACGGTCCCTGCCCTGGC

[0088] CAGTGTTCTCTGGGCTCCTGCAGGTGGCCTCCCTGCTCGGGCTGCTGCTACTTCTCCT

[0089] CAAGGCAGCACAGCTCTACCTGCGCAGACAGTGGCTGCTCAAAGCCCTCCAGCAGT

[0090] TCCCGTCCCCTCCTTCCCACTGGCTCTATGGACACAGCAGGGAG.

[0091] 6. Identification of polymorphic sites: 50 individuals were randomly selected from the Minzhu adult pig population and 80 individuals were randomly selected from the Large White adult pig population. Their genomic DNA was used as a template and PCR amplification was performed using primers CYP4A21F / R. The PCR amplification products were gel-recovered, purified, and sequenced as described above. SnapGene software was used to perform multiple sequence alignment analysis on 130 sequencing results to find polymorphic sites. A total of 2 SNPs were detected in this region, including the SNP (NC_010448.4:rs99141509) site.

[0092] 7. Genotype and allele frequency analysis

[0093] The genotype and allele frequency analysis was performed on the sequencing results of 130 individuals randomly selected from the Min pig adult pig population and the Large White pig adult pig population. As shown in Table 1, there are 9 individuals with TT genotype in the Min pig population, with a genotype frequency of 0.07, 17 individuals with CC genotype, with a genotype frequency of 0.13, and 104 individuals with CT genotype, with a genotype frequency of 0.80. The C allele frequency of the -1234 site is 0.530, and the T allele frequency is 0.469.

[0094] Table 1 Genotype frequency and allele frequency of SNP rs99141509 of porcine CYP4A21 gene in Min pig population and Large White pig population

[0095]

[0096] Example 3

[0097] Site-directed mutagenesis of SNP (NC_010448.4:rs99141509) in the promoter region of porcine CYP4A21 gene and detection of luciferase activity

[0098] According to the experimental results of Example 2, the promoter region of the CYP4A21 gene -2564 to -894 segment was analyzed using JASPAR prediction software, and combined with polymorphism analysis, it was found that after the SNP (NC_010448.4:rs99141509) site mutated from T to C, a SREBP transcription factor binding site was generated. The overlap extension PCR method was used with the CYP4A21-pGL3-CC vector as a template, and the -1234 site was subjected to site-directed mutagenesis to construct a mutant vector CYP4A21-pGL3-TT in which the gene at the site was TT.

[0099] 1. Enzyme digestion of target fragment and pGL3-basic vector

[0100] The target fragment obtained in Example 2 and the desired plasmid empty vector were digested with enzymes. The target fragment and PGL3Basic were digested with Sma I. The reaction system was as shown in the figure below. The reaction conditions were: incubation in a 37°C water bath for 60 min, and gel recovery and purification were performed after agarose gel electrophoresis.

[0101]

[0102] 2. Ligation of target fragment and pGL3-basic vector:

[0103] The vector was connected according to the instructions of TaKaRa's pMD18-T Vector Cloning Kit. The total connection system for 10 μL was: 5 μL of Solution I, 4 μL of the target fragment gel recovery product, and 1 μL of pMD18-T vector. The reaction solution was mixed evenly in a 0.2 ml EP tube and connected in a 16°C metal bath for 6 hours.

[0104] 3. Transformation of recombinant plasmid:

[0105] Transformation was performed according to the instructions of TreliefTM 5α Chemically Competent Cell from Qingke Biotechnology Co., Ltd. The specific steps are as follows:

[0106] (1) Take out the competent cells from the -80°C refrigerator and thaw them in an ice bath for 5 min.

[0107] (2) Use a pipette to add 50 μL of competent cells to 10 μL of ligation product, mix gently, and let stand on ice for 30 min.

[0108] (3) Heat shock at 42°C in a water bath for 45 seconds, quickly transfer to an ice bath, and let stand for 2 minutes.

[0109] (4) Add 200 μL of resistance-free LB culture medium to the centrifuge tube and resuscitate at 37°C / 200 rpm for 30 min.

[0110] (5) Take the resuscitation solution and spread it evenly on the culture medium containing the corresponding antibiotics, and culture it upside down in a 37°C incubator overnight.

[0111] 4. Screening and identification of positive clones:

[0112] (1) In a clean bench, use a sterilized pipette tip to pick up monoclonal colonies with smooth and round edges and of moderate size from a glass plate. Place the pipette tip into a 1.5 ml EP tube (1 ml of LB liquid culture medium supplemented with ampicillin is added to the tube). Pick 10-15 colonies from each plate.

[0113] (2) Place the EP tube in a 37°C constant temperature air bath shaker and incubate at 180 rpm for 2 h.

[0114] (3) PCR amplification was performed using bacterial solution as template. The amplification system was: 5 μL of 2×Taq Master Mix (Dye Plus), 0.4 μL of upstream and downstream primers, 1 μL of bacterial solution DNA template, and dd H2O was added to 10 μL. The PCR amplification product was detected by 1.0% agarose gel electrophoresis, and the bacterial solution with a single band of the correct length was selected and sent to Heilongjiang Jiansu Gene Technology Co., Ltd. for sequencing. The sequencing results are shown in Seq ID No: 1.

[0115] 5. High-purity, endotoxin-free plasmid extraction:

[0116] The bacterial solution with correct sequencing and containing the luciferase reporter gene was inoculated into LB liquid medium containing 50 μg / mL ampicillin at a ratio of 1:500, and shaken vigorously at 200 rpm for 16 hours. The endotoxin-free plasmid was extracted using the TIANpure MidiPlasmid Kit from Tiangen Company. The specific operation was carried out strictly according to the instructions, and the plasmid concentration was measured using a UV spectrophotometer.

[0117] 6. Using the overlap extension PCR method and the CYP4A21-pGL3-CC vector obtained in the above step as a template, site-directed mutagenesis was performed on the -1234 site to construct a mutant vector CYP4A21-pGL3-TT in which the gene at this site is TT. The primer sequences are:

[0118]

[0119] Note: The horizontal line indicates the mutated base.

[0120] The site-directed mutagenesis method was overlap extension PCR, which included two rounds of reactions. In the first round, the primer pairs CYP4A21F / CYP4A21MR and CYP4A21MF / CYP4A21R were used as templates for CYP4A21-pGL3-CC vectors to amplify the fragments with point mutations at both ends and overlapped with each other. In the second round, the appropriate dilutions of the PCR products of the above two reactions were used as templates, and CYP4A21F / CYP4A21R was used as primers for PCR amplification to introduce the point mutation into the interior of the PCR product. The first round of PCR amplification system was: 1 μL of CYP4A21-pGL3-CC vector DNA, 5 μL of 2× PrimeSTAR Max Premix, 0.3 μL of each upstream and downstream primer, and ddH2O was added to 10 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 98°C denaturation for 10 s, 58.3°C annealing for 15 s, 72°C extension for 1 min 8 s, and 31 cycles. The second round of PCR amplification system was: 1 μL of the two PCR product dilutions from the first round, 5 μL of 2× PrimeSTAR Max Premix, 0.3 μL of upstream and downstream primers, and ddH2O to 10 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min; 98°C denaturation for 10 s, 58.3°C annealing for 15 s, 72°C extension for 1 min 38 s, 31 cycles; 72°C final extension for 5 min; 4°C storage.

[0121] 7. After the second round of PCR products were recovered and purified by agarose gel electrophoresis, a single band with the correct length was selected and sent to Heilongjiang Jiansu Gene Technology Co., Ltd. for sequencing. After confirming that the results were correct, the mutant target fragment obtained was digested with the pGL3-basic vector according to the above steps and then connected to the pGL3-basic vector. After transformation, screening and identification of positive clone colonies, a single band with the correct length was selected and sent to Heilongjiang Jiansu Gene Technology Co., Ltd. for sequencing. The CC mutation of the CYP4A21 gene SNP (NC_010454.4:rs99141509) site was identified as TT, and the CYP4A21-pGL3-TT mutant vector plasmid was constructed.

[0122] 8. Cell Culture and Transfection

[0123] 293T cells were cultured in complete medium containing 90% DME / F12, 10% fetal bovine serum and 1% double antibody (penicillin + streptomycin mixed solution) and grown in a 5% CO2 incubator at 37°C to 80-90% confluence. 293T cells were seeded on a 24-well cell culture plate and transfected with the reporter gene vector when the monolayer cell confluence reached 30-50% under a microscope. Cells were harvested 24 hours later. Transfection was performed according to the instructions for PEI transfection reagent from Polysciences:

[0124] (1) Add 0.5 μg of recombinant plasmids CYP4A21-pGL3-TT, CYP4A21-pGL3-CC and empty plasmid pGL3-basic into 50 μL Opti-MEM medium, and set up a blank control. Set up 3 replicates for each group. Note that the RL-TK internal reference plasmid must be added.

[0125] (2) Add 0.5 μL of liposomes to 50 μL of Opti-MEM medium, gently mix the transfection reagent / DNA complex, and let it stand at room temperature for 15 min.

[0126] (3) Take the cell culture dish out of the 37°C CO2 incubator and add 50 μL of the premixed transfection reagent / DNA complex to each well.

[0127] (4) The cell culture dish was placed in a 5% 37°C carbon dioxide incubator and taken out after 10 hours to observe the cell status. The cells were collected 24 hours after transfection to detect the dual luciferase activity.

[0128] 9. Dual luciferase activity assay

[0129] Use Yisheng's Dual Luciferase Reporter Assay Kit to detect dual luciferase activity. The steps are as follows:

[0130] (1) Cell lysis: Remove the cell culture plate, discard the culture medium, wash twice with cold PBS, add 100 μL 1× Cell Lysis Buffer to each well of the 24-well plate, lyse at room temperature for 15 min, pipette the cell lysate and transfer it to a 1.5 ml centrifuge tube, centrifuge at 12,000 rpm for 5 min at room temperature, and use the supernatant for subsequent experiments.

[0131] (2) Prepare firefly luciferase reaction working solution and Renilla luciferase reaction solution. Dilute firefly luciferase substrate (50×) and Renilla luciferase substrate (50×) to 1× working solution with corresponding buffers, and incubate at room temperature.

[0132] (3) Firefly luciferase reaction test: Pipette 10ul of cell lysis supernatant into a 1.5ml EP tube, then carefully add 15ul of firefly luciferase reaction solution that has been equilibrated to room temperature. Mix quickly and evenly, and immediately detect the firefly luciferase reporter gene activity in a luminescence detector.

[0133] (4) Renilla luciferase reaction test: Add 15ul of Renilla luciferase reaction solution to the mixture in the previous step, mix quickly and evenly, and immediately detect the activity of the Renilla luciferase reporter gene.

[0134] The results of dual luciferase assay were as follows Figure 4 As shown in the figure, the activity of TT type promoter was significantly higher than that of CC type promoter (P<0.01).

Claims

1. An application of a SNP associated with cytochrome CYP4A21, a key rate-limiting enzyme in bile acid synthesis in adult domestic pigs, in assisted breeding, characterized in that The SNP is used in auxiliary breeding to improve pork quality; the SNP is a polymorphic site NC_010448.4:rs99141509 in the promoter region of the CYP4A21 gene, and the nucleotide sequence of the SNP is shown in SEQID NO:

1.

2. The use according to claim 1, characterized in that The polymorphism is T / C.

3. The use according to claim 1, characterized in that The auxiliary breeding for improving pork quality is achieved by selecting individual piglets with relatively high bile acid synthesis ability from the pig population to be bred.

4. The use according to claim 1 or 3, characterized in that The assisted breeding for improving pork quality is achieved by selecting piglets with relatively high bile acid synthesis ability from the pig population to be bred. The selection of piglets with relatively high bile acid synthesis ability is to eliminate CC genotype individuals and retain TT genotype individuals as piglets with relatively high bile acid synthesis ability; wherein, CC genotype individuals are individuals whose bile acid synthesis amount is lower than that of TT genotype individuals.

5. The use according to claim 1, 2 or 3, characterized in that The domestic pig is a Min pig or a Large White pig.

6. The use according to claim 1, 2 or 3, characterized in that The method of assisted breeding is as follows: (1) Using primers CYP4A21F and CYP4A21R, the PCR product of the gene fragment of the promoter region of the CYP4A21 gene of the pig population to be bred is obtained by the PCR method. After successful sequencing verification, the obtained CYP4A21 gene promoter region gene has a C allele at the -1234th position, which is recorded as a CC genotype individual; the obtained CYP4A21 gene promoter region gene has a T allele at the -1234th position, which is recorded as a TT genotype individual; (2) removing individuals with CC genotype from the pig population to be selected and retaining individuals with TT genotype, thereby completing the assisted breeding; Among them, the nucleotide sequences of CYP4A21F and CYP4A21R primers are as follows: CYP4A21F: ATTATACCCTTGGGTCGGTGCACT; CYP4A21R: TCAGTGGACTTGTAGGGCGA.

7. A primer for detecting SNPs associated with cytochrome CYP4A21, a key rate-limiting enzyme in bile acid synthesis in adult domestic pigs, characterized in that The primers are CYP4A21F and CYP4A21R, and the nucleotide sequences are as follows: CYP4A21F: ATTATACCCTTGGGTCGGTGCACT; CYP4A21R: TCAGTGGACTTGTAGGGCGA.

8. A kit comprising the primer according to claim 7.

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