CN113136439B - Method for detecting sheep LIPE gene single nucleotide polymorphism and application thereof - Google Patents
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Abstract
The invention discloses a method for detecting sheep LIPE gene single nucleotide polymorphism and application thereof, comprising the following steps: extracting DNA samples of Hu sheep tissues and typing single nucleotide polymorphism of LIPE gene mutation sites g.4819A > G; through the correlation analysis of the single nucleotide polymorphism of the Hu sheep LIPE gene and the intramuscular fat content, the typing detection of the single nucleotide polymorphism sites of the Hu sheep LIPE gene is shown to obtain the molecular marker for improving the intramuscular fat content of the Hu sheep, and the molecular marker can be used for breeding sheep varieties with good meat quality.
Description
Technical Field
The invention belongs to the field of molecular genetics detection, and relates to a method for detecting single nucleotide polymorphism of a sheep LIPE gene by using a DNA mixed pool sequencing combined with an imLDR technology and application thereof.
Background
Single Nucleotide Polymorphism (SNP) refers to a variation at the genomic level due to a transition, transversion, insertion, deletion of a Single base. SNP is widely existed in genome, and has wide application in aspects of genetic map construction, gene accurate positioning, genetic breeding and the like. SNPs are classified into coding SNPs (cnps) of coding regions of genes and non-coding SNPs according to the positions of mutations, and cnps are classified into synonymous cnps and non-synonymous cnps. Synonymous cSNP means that no change in the amino acid sequence of the protein is caused after a base mutation. Non-synonymous cSNP refers to a change in the amino acid sequence of a protein caused by a base mutation, for example, a SNP in an intron region does not normally alter the amino acid sequence of a protein encoded by a gene, but these SNPs may change the cleavage site to produce a different transcript, thereby affecting the phenotype. The fluorescent multiple enzyme ligation reaction (imLDR) technology is an improved detection and typing technology based on the principle of LDR technology, has the advantages of high accuracy, high detection speed and the like, can improve the flux of SNP typing, and reduces the typing cost.
Intramuscular fat (IMF) is the main source of volatile compounds, a species-specific flavour precursor. The presence of IMF on muscle fibers, which can separate muscle fiber bundles and thus improve meat tenderness, is one of the most important factors affecting meat quality. Too low an IMF content makes meat dry and hard. The IMF content is influenced by factors such as nutrition, heredity, sex, age of day, feeding method, etc. Research shows that the mutton quality with the IMF content of 4-5% is the best, so that the proper IMF content is one of the important indexes for promoting the meat quality improvement.
Liu et al showed that down-regulation of lipolytic genes in the PPAR signaling pathway increased IMF levels in chicken pectoralis major muscle tissue. Schenkel et al performed correlation analysis on genotyping of 5 SNPs of Leptin gene and dorsolongisimus fat content. Jing et al performed correlation analysis on 2 SNPs in the Leptin gene coding region of Simmental hybrid cattle and meat quality traits (backfat thickness, IMF content and polyunsaturated fatty acid content). And (3) performing correlation analysis on SNP (single nucleotide polymorphism) of a second exon of the duck L-FABP gene and IMF (internal reference frame) content by using He Jun. Therefore, meat quality (flavor, juiciness and tenderness) can be improved by genetic selection (e.g., marker-assisted selection) based on candidate genes affecting the content of IMF, thereby satisfying human consumption needs.
The hormone-sensitive lipase (HSL) gene, also known as LIPE gene, is located on sheep chromosome 14, has a total length of 12799bp, and HSL is a rate-limiting enzyme involved in fat catabolism in animals, and has the function of hydrolyzing triglyceride into glycerol and fatty acid in the fat catabolism process to provide energy for the metabolism of the animal body. Researches show that the expression levels of LIPE genes in the biceps femoris muscle, the longissimus dorsi muscle and the psoas major muscle of the Hu sheep have positive correlation with the IMF content, the mRNA expression level of the LIPE genes of the Kazakh sheep and the IMF content have negative correlation, and the mRNA expression level of the LIPE genes of the Xinjiang fine hair sheep and the IMF content have no obvious correlation, and the researches show that the expression levels of the LIPE genes in different sheep varieties have certain difference. Therefore, reports of LIPE gene molecular marker sites which are obviously related to the content of IMF in sheep are not seen at present, and meanwhile, the early screening of sheep individuals with meat quality trait advantages by using molecular markers still belongs to the technical problem.
Disclosure of Invention
The invention aims to provide a method for detecting single nucleotide polymorphism of a sheep LIPE gene and application thereof, which can accelerate breeding of sheep varieties with good meat quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting sheep LIPE gene single nucleotide polymorphism comprises the following steps:
extracting sheep individual genome DNA as a nucleic acid sample, and analyzing the extracted nucleic acid sample to determine the genotype of individual LIPE gene single nucleotide polymorphism sites, wherein the single nucleotide polymorphism sites are LIPE gene mutation sites g.4819A > G (the reference sequence is NC-040265.1).
Preferably, the analysis of the nucleic acid sample employs DNA sequencing, or the analysis of the nucleic acid sample employs imLDR techniques.
Preferably, the LIPE gene mutation site is determined by mixing DNAs extracted from testis tissues of individuals of different sheep (e.g., Hu sheep), and amplifying and sequencing a target fragment from a pool of the mixed DNAs.
Preferably, the extraction method of the testis tissue DNA is a high-salt method, the operation of extracting the testis tissue DNA by the high-salt method is simple and short in time consumption, the integrity of the extracted DNA (genome DNA) is good, the OD (260/280) is 1.6-1.8, and the imLDR technical typing requirement can be met.
Preferably, the primer used for amplifying the target fragment is a primer pair P1 (the amplification product contains mutation sites g.4819A > G, i.e. the single nucleotide polymorphism site of the amplified LIPE gene), and the primer pair P1 specifically comprises:
an upstream primer F: 5'-TCACCGAGATCCAGGTGCTA-3'
A downstream primer R: 5'-TCACTCCTCCGAGAGACGG-3' are provided.
The method for detecting the single nucleotide polymorphism of the sheep LIPE gene is applied to sheep marker-assisted selection.
The application of sheep LIPE gene mutation site g.4819A > G (reference sequence NC-040265.1) in sheep marker-assisted selection.
Preferably, individuals with GG genotype at the LIPE gene mutation site g.4819A > G (reference sequence NC-040265.1) in the sheep (e.g., Hu sheep) population are superior in meat quality traits.
Preferably, the meat quality trait is selected from the group consisting of intramuscular fat (IMF) content.
A kit for detecting single nucleotide polymorphism of LIPE gene of sheep, which comprises necessary reagents for completing analysis of the nucleic acid sample.
Preferably, the necessary reagent specifically includes the primer pair P1 described above.
The invention has the beneficial effects that:
according to the invention, through carrying out typing detection on newly found SNP sites (specifically g.4819A > G) of the LIPE gene, a candidate molecular marker is provided for the early screening of the sheep excellent meat quality traits, so that a theoretical research and practical basis is provided for the breeding of sheep varieties with excellent meat quality by marker-assisted selection.
Furthermore, in the invention, the SNP site of the LIPE gene (specifically g.4819A > G) can be effectively genotyped by adopting the imLDR technology, and the sheep population with excellent meat quality character can be quickly established, thereby accelerating the breeding process of the fine sheep.
Drawings
FIG. 1 is an electrophoresis diagram of the PCR amplification product (483bp fragment) of the target fragment of Hu sheep LIPE gene (containing the fifth exon, part of the fourth intron, and part of the fifth intron).
FIG. 2 is a diagram of sequencing peaks of DNA pool amplification products of different individuals of Hu sheep; the arrow in the figure indicates the 4819 th mutation site of LIPE gene.
Detailed Description
The invention is described in further detail below with reference to the figures and examples. The examples are only used to explain the technical solution of the present invention, and do not limit the protection scope of the present invention.
Screening new Hu sheep LIPE gene single nucleotide mutation sites by DNA pool sequencing
And S1 sample selection:
in 2018, 8-2020, 1, 921 (four batches) of testis tissues and longisimus dorsi tissues of 6-month-old healthy Hu sheep from the same breeding condition of the Ministry of Denfu agriculture technology Limited company are collected, and the intramuscular fat content of the longisimus dorsi of the individual is measured.
Construction of S2 DNA pool:
a) 921 Hu sheep individual testis tissue DNA (genome DNA) is extracted by using a high-salt method, and the method comprises the following specific steps:
i) grinding a tissue sample under liquid nitrogen, and taking 0.1g of tissue powder into a centrifugal tube;
II) adding 20% SDS (100 mu L) and DNA extraction solution (400 mu L) as lysis solution, standing for 5min, whirling to completely dissolve precipitate (tissue powder) in the lysis solution, adding 20 mu L proteinase K, and reversing and mixing;
III) placing the uniformly mixed sample in a 56 ℃ water bath kettle, and digesting overnight;
IV) adding saturated saline solution (300 mu L) into the digested sample, reversing for 3min, and standing at 4 ℃ for 10 min;
v) centrifuging at 12000r for 10min, and transferring the supernatant into a new centrifuge tube;
VI) adding precooled absolute ethyl alcohol (1mL), reversing and uniformly mixing for 1min, centrifuging at 12000r for 2min, removing supernatant, and keeping DNA precipitate;
VII) adding 75% ethanol (500 mu L) into the centrifuge tube, shaking gently for mixing uniformly, centrifuging at 12000r for 1min, discarding the supernatant, retaining the DNA precipitate, adding 75% ethanol (500 mu L) into the centrifuge tube again, shaking gently for mixing uniformly, centrifuging at 12000r for 1min, discarding the supernatant, retaining the DNA precipitate, uncovering the cover and standing for 5h to completely volatilize the residual ethanol;
VIII) Add 56 ℃ TE buffer (200. mu.L) to the centrifuge tubes, uncap for 10min at 56 ℃ (dissolve the ethanol-evaporated precipitate), and dissolve DNA overnight at 4 ℃.
b) The integrity of the extracted DNA sample (DNA dissolved in TE buffer) is detected by agarose gel electrophoresis, 260/280nmOD value and DNA concentration are measured by a nucleic acid protein concentration determinator Nanodrop 2000, and the DNA sample with the concentration of more than 50 ng/. mu.L and the OD 260/280 of 1.6-1.8 is judged as a qualified sample.
c) After the DNA samples of 921 Hu sheep are qualified through quality detection, 100 DNA samples are randomly selected and uniformly diluted to 50 ng/mu L, 1 DNA mixed pool (namely a DNA pool consisting of 10 individual genome DNAs) is constructed for every 10 diluted DNA samples, and 10 DNA mixed pools are constructed in total.
S3 PCR amplification:
using the DNA sequence of the ovine LIPE gene published by NCBI (NC-040265.1) as a reference, a Primer pair P1 for amplifying the LIPE gene fragment (the fifth exon and a part of the fourth intron and a part of the fifth intron) was designed by using Primer-Blast software:
an upstream primer F: 5'-TCACCGAGATCCAGGTGCTA-3'
A downstream primer R: 5'-TCACTCCTCCGAGAGACGG-3'
Carrying out PCR amplification by taking the DNA mixed pool as a template;
the reaction system is 25 μ L: 12.5. mu.L Mix, 10.5. mu.L sterile distilled water, 0.5. mu.L forward primer (10 pmol/. mu.L), 0.5. mu.L reverse primer (10 pmol/. mu.L), and 1. mu.L DNA template (50 ng/. mu.L).
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62.7 ℃ for 30s, and extension at 72 ℃ for 40s for 34 cycles; further extension was carried out at 72 ℃ for 5 min.
Referring to FIG. 1, 483bp of the target fragment was obtained by agarose gel electrophoresis of the product amplified using primer pair P1, which showed that primer pair P1 was specific and could be used to conveniently obtain a sufficient number of the target fragment by PCR amplification.
Sequencing of S4 amplification products:
the PCR product amplified by the DNA mixed pool in the step S3 is subjected to bidirectional sequencing. Referring to FIG. 2, the amplified target fragment was sequenced, and the 4819 th single nucleotide site of the Hu sheep LIPE gene was found to be a mutation site (g.4819A > G).
(II) typing candidate SNP sites by a fluorescent multiplex enzyme ligation reaction (imLDR) technology
Uniformly diluting 921 Hu sheep DNA samples qualified in quality detection to 50 ng/. mu.L, subpackaging the diluted Hu sheep DNA samples into 1mL centrifuge tubes, taking the mutation sites as LIPE gene candidate single nucleotide polymorphism sites, taking 15bp DNA sequence information of the upstream and downstream of the mutation sites to provide to Shenzhen Tianhao science and technology Limited company, and typing the candidate nucleotide polymorphism sites of the diluted 921 DNA samples by utilizing imLDR technology.
(III) Hu sheep LIPE genotyping and intramuscular fat content data analysis
3.1 Hu sheep LIPE Gene mutation site frequency statistics
Gene frequency calculation formula:
PB＝(2NBB+NBb1+NBb2+NBb3+NBb4+……+NBbn)/2N
in the formula, PBIndicates allele B frequency, NBBRepresenting the number of individuals in the population with the BB genotype, NBbiIndicates having B in the populationbiThe genotype individual number, B1-bn is the N different multiple alleles of allele B, and N is the total individual number of the detection population.
Genotype frequency calculation formula:
PBB＝NBB/N
in the formula, PBBRepresenting the BB genotype frequency, N, of a certain siteBBRepresenting the number of individuals having a BB genotype in the population, N being the total number of individuals in the test population;
after the analysis of the alleles and genotypes of different Hu sheep individuals is completed, the frequencies of the alleles and genotypes of the Hu sheep population LIPE gene mutation sites are calculated according to the formula, and the results are shown in Table 1:
TABLE 1 Hu sheep LIPE Gene mutation site g.4819A > G allele frequency and genotype frequency
According to the results in Table 1, it can be determined that Hu sheep LIPE gene mutation site g.4819A > G (A is dominant allele) belongs to SNP site.
3.2 Hu sheep LIPE gene Single Nucleotide Polymorphism (SNP) locus and intramuscular fat (IMF) content correlation analysis data to be analyzed: the typing results of the imLDR technique; intramuscular fat content phenotype data
The data are analyzed by adopting SPSS 25 software, and the correlation analysis of the SNP locus typing result and the intramuscular fat content phenotype data specifically adopts the following analysis model:
Yijk＝μ+Gj+Eijk
in the formula, YijkIs a phenotype of the individual; μ is the population mean; gjThe genotype effect for each site; eijkIs a random error. The analytical results are shown in table 2:
TABLE 2 Association analysis of Hu sheep LIPE gene single nucleotide polymorphism and intramuscular fat content
Note: different shoulder-marked letters show significant difference (P <0.01), and the same shoulder-marked letters show insignificant difference (P > 0.05); the IMF content is the data after using R3.6.0 to remove the batch effect, and the negative sign is caused by the removal of the batch effect.
The correlation analysis result shows (Table 2), the SNP of g.4819A > G site of Hu sheep LIPE gene can affect the individual IMF content phenotype, and the difference between the IMF content of the individual with the G.4819A > G site and the IMF content of the individual with GG genotype is very obvious (P < 0.01). According to the analysis result, the g.4819A > G site can be used as a potential genetic marker for improving the IMF content, and by selecting the 4819 mononucleotide polymorphism site of the LIPE gene as an individual of GG genotype, a Hu sheep population with the advantage of meat quality traits can be quickly established, so that the breeding process of the excellent sheep variety is accelerated.
The single nucleotide polymorphism site of the LIPE gene detected by the invention is a mutation site g.4819A > G located in the fourth intron of the sheep LIPE gene, and the mutation of the g.4819A > G site does not cause the change of codons and amino acids. The research shows that SNP of the intron region can change the cutting site to generate different transcripts, influence the translation efficiency, thereby influencing the LIPE gene expression amount of sheep (such as Hu sheep), the protein space structure of the hormone sensitive lipase and the like, and further influencing the intramuscular fat content of individuals.
In a word, the IMF content is one of important reference indexes for promoting the mutton quality improvement, molecular markers (SNP markers) for improving the intramuscular fat content of sheep (such as Hu sheep) can be obtained through typing detection of single nucleotide polymorphism sites of the LIPE gene, early screening of sheep individuals is realized, the sheep individuals can be used for quickly establishing sheep populations with excellent meat quality characters, and therefore breeding of sheep varieties with good meat quality is accelerated, and the method has important significance for improving the mutton quality in the aspect of molecular breeding.
<110> Lanzhou university
<120> method for detecting sheep LIPE gene single nucleotide polymorphism and application thereof
<160> 2
<210> 1
<211> 20
<212> DNA
<213> F
<400> 1
tcaccgagat ccaggtgcta 20
<210> 2
<211> 20
<212> DNA
<213> R
<400> 2
tcactcctcc gagagacgg 19
Claims (6)
1. Detection sheepLIPEThe application of the gene single nucleotide polymorphism method in sheep marker-assisted selection is characterized in that:
detecting sheepLIPEA method for gene single nucleotide polymorphism comprising the steps of:
extracting sheep genome DNA for analysis and determinationLIPEThe genotype of a single nucleotide polymorphic site of a gene, said single nucleotide polymorphic site comprisingLIPEGene mutation site NC-040265.1 g.54064878A>G；
LIPEGene mutation site NC-040265.1 g.54064878A>The sheep individual with the genotype of G being GG is better in meat quality character;
the meat quality character is better, namely the intramuscular fat content is high;
the sheep is selected from Hu sheep.
2. Use according to claim 1, characterized in that: the above-mentionedLIPEThe genotype of the gene single nucleotide polymorphism site is analyzed and determined by adopting a DNA sequencing method or an imLDR technology.
3. Use according to claim 1, characterized in that: the above-mentionedLIPEThe gene mutation sites are determined by mixing DNA extracted from different sheep individuals, and amplifying and sequencing target fragments of the mixed DNA pools.
4. Use according to claim 3, characterized in that: the extraction method of the DNA is a high-salt method.
5. Use according to claim 3, characterized in that: the primer adopted for target fragment amplification is a primer pair P1, and the primer pair P1 is:
an upstream primer F: 5'-TCACCGAGATCCAGGTGCTA-3'
A downstream primer R: 5'-TCACTCCTCCGAGAGACGG-3' are provided.
6. SheepLIPEGene mutation site NC-040265.1 g.54064878A>The application of G in sheep marker-assisted selection is characterized in that:LIPEgene mutation site NC-040265.1 g.54064878A>The sheep individual with the genotype of G being GG is better in meat quality character;
the meat quality character is better, namely the intramuscular fat content is high;
the sheep is selected from Hu sheep.
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| "湖羊肌内脂肪含量特征及相关分子标记筛选";刘星;《中国优秀硕士学位论文全文数据库农业科技辑》;20210915(第09期);D050-115 * |
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