CN111763746B - Gene variation site for regulating and controlling Guizhou Xiang pig carcass index and application thereof - Google Patents
Gene variation site for regulating and controlling Guizhou Xiang pig carcass index and application thereof Download PDFInfo
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Abstract
The invention discloses a gene mutation site for regulating and controlling carcass indexes of Guizhou Xiang pigs and application thereof, belongs to the field of molecular biology, provides 10 SNPs (single nucleotide polymorphism) sites, provides application of the SNPs sites in regulation and control of animal carcass performance and regulation and control of pig fat deposition, and makes relevance analysis on the detected 10 SNPs sites and meat quality traits of 5 Guizhou Xiang Jiang pigs respectively to determine relevance between the 10 SNPs sites and the traits of a test population; the invention can provide molecular basis for the breeding of the carcass traits of pigs in the future, and also lays the foundation for the genetic improvement of the fat traits of the pigs by using the gene.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a gene variation site for regulating and controlling Guizhou Xiang pig carcass indexes and application thereof.
Background
In recent years, due to the excessive pursuit of high lean meat percentage in the breeding work of pigs, the quality of pork is seriously reduced. With the continuous improvement of living standard, people have higher and higher requirements on the meat quality characteristics of pork, such as color, tenderness, flavor and the like. In the past decade, pig breeding has been dominated by the need for pork production, such as increased fat deposition and carcass lean, but has often resulted in reduced fat storage and deposition. This mode of selection also results in a gradual reduction of the total lipid content in the muscle, especially in some species where one is inclined to select for lean mass deposition. For example, the breeding of big white pigs significantly reduces the marbling content from 2-4% in the past 1960 to 1% today. At present, with the continuous improvement of the pork quality requirements of consumers, the safe and high-quality pork can directly influence the purchasing power of the consumers and the like. Therefore, how to improve the pork quality has become a research hotspot of animal husbandry genetic breeding nowadays.
Intramuscular fat (IMF) is one of the important factors affecting meat quality, and is now increasingly recognized and valued. The deposition of intramuscular fat is a complex physiological and biochemical process, mainly dependent on the proliferation, differentiation and polyester maturation of intramuscular adipocytes. During differentiation and proliferation of adipocytes, the content of triglycerides is determined by the processes of synthesis, decomposition, transport, etc. of intracellular fat. Intracellular triglycerides are mainly present in lipid droplets, which are important organelles for maintaining cellular energy homeostasis. The core of the lipid drop is a lipid core formed by triglyceride and cholesterol ester, and the surface of the lipid drop is coated with a single layer of phospholipid membrane and is coated with various proteins. The process by which intracellular fat droplets store fat is the most fundamental biological process, but how intracellular fat is "packed" into lipid droplets, i.e., how cells regulate the phospholipoprotein layer to package fat into lipid droplets, has not been satisfactorily explained.
The Guizhou Yangxiang pig is famous for short body trunk, slow growth and development, tender meat, early sexual maturity, strong disease resistance, low genetic diversity and high gene purity, belongs to a special miniature local pig breed in China, is listed as a national secondary protection animal breed in 1993 by the Ministry of agriculture, and is mainly produced in villages and towns such as the Guizhou Yangxiang county and the like.
Sirtuin5 (SIRT 5) is a member of the silent signaling regulon family (Sirtuins) and is also one of the three mitochondrial proteins in the NAD family, and is a protein-dependent deacylase enzyme that controls a variety of metabolic pathways. Rardin et al indicate that SIRT5 regulates mitochondrial and peroxisomal fatty acid oxidation equilibrium in mouse proximal tubular epithelial cells. The Lijingya group firstly discovers an important role of SIRT5 in regulating brown fat differentiation, and confirms that SIRT5 is necessary for brown fat cell differentiation. The research finds that SIRT5 influences the concentration of a metabolite alpha-KG in cells, further leads to the change of methylation modification of PPAR gamma and PRDM16 gene promoter region H3K9 which are key differentiation transcription factors, and thus plays a regulating role.
At present, the influence and regulation mechanism of the SIRT5 gene on intramuscular fat deposition and lipid metabolism of the miniature Guizhou Xiang pig are not reported at home and abroad.
Disclosure of Invention
The invention aims to provide a gene variation site for regulating and controlling Guizhou fragrant pig carcass indexes and application thereof, which aim to solve the problems in the prior art and aims to disclose the influence of SIRT5 gene regulation and control on Guizhou fragrant pig carcass performance in the later period by taking Guizhou fragrant pigs as research animals and analyzing the genetic effect and relevance of the SIRT5 gene variation site.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a gene variation site for regulating and controlling Guizhou Xiang pig carcass indexes, which uses a pig SIRT5 gene complete sequence and uses a first transcription initiation site +1, and the gene variation sites are respectively as follows:
g.8306 A>G、g.14135 A>C、g.14247 C>A、g.14305 C>T、g.14335 C>T、g.16603 T>C、g.16613 T>C、g.16800 G>A、g.16812C>G、g.16916 A>G。
further, the positions of the gene variation sites on the complete sequence of the pig SIRT5 gene are respectively as follows:
further carrying out correlation analysis with Guizhou Xiang pig traits, and taking a pig SIRT5 gene complete sequence and a first transcription initiation site +1, the gene variation sites capable of adjusting the Guizhou Xiang pig carcass traits are respectively as follows:
g.16812C>G、g.16916 A>G。
the invention also provides application of the gene mutation site in regulation and control of animal carcass performance.
Further, the animal is Guizhou fragrant pig.
The invention also provides application of the gene variation locus in regulation of pig fat deposition or identification of pig fat deposition capability.
The invention discloses the following technical effects:
the invention provides 10 SNPs loci, provides application of the SNPs loci in regulation and control of animal carcass performance and regulation and control of pig fat deposition or identification of pig fat deposition capacity, and respectively performs relevance analysis on the detected 10 SNPs loci and 5 carcass meat quality traits of Xiang pigs from Jiang, guizhou to discover that the genotype of the g.14305C > T locus is obviously related to the skin thickness index of a test population, and CC is a dominant genotype; g, the genotype of 16812C > G locus is obviously related to the skin thickness and the eye muscle area of a test population, and CC is a dominant genotype; g.16916 The genotype of the A site and the G site is obviously associated with the intramuscular fat content of the test population, and AG is the dominant genotype.
In addition, the g.16812C > G and g.16916A > G sites have high heterozygosity in the Guizhou Xiang pig group and rich polymorphic information content, which indicates that the 2 SNPs sites have uniform allele distribution and large variation degree in the Guizhou Xiang pig group and can provide rich genetic information. Therefore, in later production and breeding practice, the selection strength of favorable genotypes of g.16812C > G and g.169916A > G sites should be increased, and the proportion of favorable genotypes in the population is continuously increased, so that the breeding improvement of the carcass performance of Guizhou Xiang pigs is accelerated.
The invention can provide molecular basis for the breeding of the pig fat content character in future. Meanwhile, a foundation is laid for genetic improvement of pig fat traits by using the gene.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram showing the electrophoresis detection of PCR amplification products of the exon regions of the SIRT5 gene of the Xiang pig, guizhou.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the reagents used in the examples of the present invention are commercially available, and the experimental methods used are conventional methods unless otherwise specified, and the databases used are public online databases. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
Example 1
1 materials and methods
1.1 test animals and sample Collection
107 blood samples of fattening pigs of Guizhou Yijiang Xiang pigs are collected by using a heparin sodium anticoagulant tube and are used for SIRT5 gene variation site genetic effect analysis, and experimental samples are collected from ecological welfare farms of green-source Xiang pigs in Guizhou Guiyang city.
1.2 carcass meat quality index determination
Weighing the weight of 107 test pigs before slaughtering, measuring the indexes of the thickness of the pigskin, the thickness of the back fat between the ribs and the area of the eye muscles after slaughtering, collecting 300g of the longissimus dorsi, wrapping the longissimus dorsi by using tin foil paper, and numbering the longissimus dorsi for measuring the indexes of the moisture content of the muscles and the content of the fat in the muscles. All indexes are executed according to the technical specification for measuring the muscle quality of pigs (NY/T821-2004) and the technical specification for measuring the carcass traits of lean pigs (NY/T825-2004) of the agricultural industry standard of the people's republic of China.
1.3 DNA extraction
The pig blood sample is extracted by Ezup column type genome DNA extraction kit (blood) produced by Shanghai biological engineering (Shanghai) corporation, and is stored at-20 ℃ for later use.
1.4 primer design Synthesis
With reference to the pig SIRT5 gene complete sequence (accession number: NC-010449.5), SIRT5 gene exon region amplification primers were designed using Primer premier5.0, as shown in Table 1, and synthesized by Biotechnology engineering (Shanghai) GmbH.
TABLE 1 primer information
1.5 PCR amplification reaction
The PCR amplification adopts a 25. Mu.L reaction system, and the reaction system is as follows: 2 × Es Taq MasterMix 12.5 μ L, ddH 2 O8.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers, and 2. Mu.L of the template DNA. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 72 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles as shown in Table 1; final extension at 72 deg.C for 2min, storing PCR amplification product at 4 deg.C, and inspecting by 1.2% agarose gel electrophoresisAnd (3) detecting, and selecting the PCR product with good specificity to be sent to the company Limited of the biological engineering (Shanghai) for sequencing.
1.6 data analysis
The SIRT5 gene polymorphism data of the Xiang pigs from Guizhou province, which are obtained by a direct sequencing method, utilize a General Linear Model (GLM) in SPSS 18.0 software to analyze the relevance between different genotypes and carcass and meat quality traits, and the mathematical model is as follows: yijk = μ + si + nj + eijk. In the formula, YIjk: a trait measure; μ: group mean value; si: a sex effect; nj: a genotype effect; eijk: random residual effects. The results are expressed as "mean ± standard deviation".
2 results and analysis
2.1 Guizhou fragrant pig SIRT5 gene SNPs locus and correlation analysis of the locus and characters
2.1.1 PCR amplification of SIRT5 Gene from Xiangjiang Guizhou pig and excavation of mutation site
1.2% agarose gel electrophoresis was used to detect the PCR amplification products of exons 1-8 of SIRT5 gene, and the results are shown in FIG. 1. As can be seen from the figure, the PCR amplification product has the same size with the target fragment, and the specificity is good, thereby achieving the sequencing standard.
Sequencing the PCR amplification product, comparing the sequencing sequence by using a Seqman program in DNAStar software, co-excavating an exon region and a partial intron region of the Guizhou Xiang SIRT5 gene to obtain 10 SNPs sites, referring to the complete sequence of the pig SIRT5 gene, and respectively naming 10 variant sites as a first transcription initiation site + 1: g.8306 A > G (intron 3), g.14135A > C (intron 6), g.14247C > a (intron 6), g.14305C > T (exon 7), g.14335C > T (exon 7), g.16603T > C (intron 7), g.16613T > C (intron 7), g.16800G > a (intron 7), g.16812c > G (intron 7), g.16a > G (exon 8).
2.1.2 population genetic Structure analysis
As can be seen from Table 2, the effective allele factors of the g.16603T > C, g.16613T > C and g.169916A > G loci are close to 2, which indicates that the 3 variant loci alleles are distributed more uniformly in Jiangxiang pigs in Guizhou, and the content of polymorphic information is higher than 0.35, indicating that the genetic marker loci can provide abundant genetic information. And the effective allelic factors and the heterozygosity value of the g.14135A > C, g.14247C > A and g.14305C > T sites are low, which indicates that the effective allelic factors and the heterozygosity value of the sites are low in distribution concentration in Guizhou Xiang pig groups, the variation degree is low, the polymorphic information content of the sites is 0.1023, 0.1023 and 0.0614 respectively, and the polymorphic information content of the sites belongs to low-degree polymorphisms (PIC < 0.25), which indicates that the genetic marker sites provide little genetic information. And g.16603T > C, g.16613T > C, g.14135A > C and g.14247C > A2 have consistency on the results of the genetic structure parameters of the variant loci.
TABLE 2 genetic parameters of SNPs loci of the SiRT5 gene of Guizhou Xiang pig
2.1.3 correlation analysis of variant sites and traits
As shown in the table 3, the index of skin thickness of the Guizhou Zhongjiang Xiang pig SIRT5 gene g.14305C > T site CC type individual is obviously higher than that of the TT type 14.04% (P < 0.05), the index of skin thickness and the index of eye muscle area of the g.16812C > G site CG type individual are obviously higher than that of the GG type 28.12% and 17.99% (P < 0.05), but the difference level of the AA type individual is not significant (P > 0.05), and the index of the G.169916A > G site AA type individual is obviously higher than that of the GG type individual (P < 0.05).
TABLE 3 correlation analysis of SNPs sites of SiRT5 gene of Guizhou Xiang pig and meat quality traits of carcass
3 conclusion
According to the invention, through carrying out correlation analysis on the detected 10 SNPs and the meat quality traits of 5 carcasses of Xiang pigs from Jiang in Guizhou, the genotype of the g.14305C > T site is obviously correlated with the skin thickness index of a test population, and CC is a dominant genotype; g, the gene type of 16812C > G locus is obviously related to the skin thickness and the eye muscle area of a test population, and CC is a dominant gene type; g.16916 The genotype of the site A and the site G is obviously associated with the intramuscular fat content of the test population, and AG is the dominant genotype. Although the g.16812C > G site is positioned on an intron, the g.16812C > G site cannot participate in the coding of the protein. However, the present invention demonstrates that mutations in non-coding regions have some effect on the stabilization, localization, and translation of mRNA.
In addition, the g.16812C > G and g.16916A > G sites have high heterozygosity in the Guizhou Xiang pig group and rich polymorphic information content, which indicates that the 2 SNPs sites have uniform allele distribution and large variation degree in the Guizhou Xiang pig group and can provide rich genetic information. Therefore, in later-stage production breeding practice, the selection strength of favorable genotypes of g.16812C > G and g.16916A > G sites should be increased, and the proportion of favorable genotypes in the group is continuously increased, so that the breeding improvement of the meat performance of the Guizhou Xiang pig carcass is accelerated.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (3)
1. The application of a gene variation site in regulation and control of meat quality performance of pig carcass is characterized in that a pig SIRT5 gene complete sequence is used, a first transcription initiation site +1 is used, the gene variation site is g.1682C > G, and the position of the gene variation site on the pig SIRT5 gene complete sequence is intron 7.
2. The use of claim 1, wherein the pig is a Guizhou fragrant pig.
3. Use of the genetic variation site of claim 1 for regulating pig fat deposition or identifying pig fat deposition ability.
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| CN104087595A (en) * | 2014-07-18 | 2014-10-08 | 湖北省农业科学院畜牧兽医研究所 | Pork carcass and meat-quality trait SNP genetic marker and applications |
| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
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| CN104087595A (en) * | 2014-07-18 | 2014-10-08 | 湖北省农业科学院畜牧兽医研究所 | Pork carcass and meat-quality trait SNP genetic marker and applications |
| CN105755131A (en) * | 2016-04-09 | 2016-07-13 | 华中农业大学 | Genetic marker associated with pig meat quality characters and carcass characters |
Non-Patent Citations (2)
| Title |
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| rs3475653930;佚名;《Ensembl genome browser 108 Sus_scrofa》;20191127;第1-2页 * |
| 猪SIRT5基因外显子区多态性研究;张雄等;《畜牧与兽医》;20200609(第06期);第10-14页 * |
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