Molecular marker primer of FoxO1 gene related to tenderness of yak meat and application of molecular marker primer

Abstract

The invention discloses FoxO1 gene molecular marker primers related to tenderness of yak meat and application of the FoxO1 gene molecular marker primers, and relates to the technical field of biology, the number of the molecular marker primers is two pairs, and the nucleotide sequences of the molecular marker primers are shown as SEQ ID NO: 1-SEQ ID NO: 2 and SEQ ID NO: 3-SEQ ID NO: 4 respectively. The invention discloses the correlation between the yak FoxO1 gene mutation and the Gannan yak carcass and meat quality traits, and the FoxO1 gene mutation site can be used as a potential molecular genetic marker of the Gannan yak meat quality traits.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a FoxO1 gene molecular marker primer related to the tenderness of yak beef and an application thereof. Background Art

[0002] Yak ( Bosgrunniens ) is a rare breed of cattle in my country. It mainly lives in high-altitude areas such as Qinghai, Tibet, Gansu, and Sichuan. It has a living habit of cold resistance, roughage resistance, and hypoxia resistance. Yaks are an important source of income for local herders, providing living resources such as meat, milk, and wool. Yak meat is rich in minerals, trace elements, and amino acids, and has low fat and cholesterol content, and high nutritional value. Because yak beef has coarse muscle fibers, low intramuscular fat content, and poor tenderness, improving meat quality is a key task in yak breeding. Meat quality traits are affected by genetics, nutrition, fat, muscle fibers, and physiological structure, among which genetics is a key factor. The synthesis of muscle fibers and intramuscular fat is jointly regulated by genes and multiple regulatory factors, so screening candidate genes and transcription factors related to their synthesis is an important way to improve yak meat quality.

[0003] Transcription factors are a class of regulatory proteins with specific functions, including DNA binding domains and transcription activation domains. They regulate the development and metabolic processes of the body by regulating the expression of target genes. Since the forkhead box (Fox) transcription factor was first discovered in 1989, more than 100 family members have been identified in the Fox protein family, which is a large and widely used transcription factor family. There are four family members in the mammalian FoxO subfamily, namely FoxO1, FoxO3, FoxO4 and FoxO6. FoxO transcription factors have a wide range of functions and play an important role in the regulation of muscle, fat and liver tissues. In addition, FoxO transcription factors also play a key role in processes such as fat differentiation and muscle development.

[0004] Forkhead transcription factor 1 (FoxO1), also known as FKHR, belongs to the FoxO subfamily of the forkhead transcription factor family. It is mainly located in the nucleus and plays an important role in glucose transport and fat metabolism. It can promote adipocyte differentiation and inhibit skeletal muscle generation and the expression of type I myofiber genes. FoxO1 gene interacts with high mobility group protein A1 (HMGA1) to regulate the transcription level of genes closely related to insulin metabolism to maintain the balance of glucose metabolism in the liver. Zhang Hui et al. (Zhang Hui, Shi Xin'e, Yuan Yuan, et al. 2010. FoxO1 inhibits the expression of MyHCI in pig skeletal muscle. Chinese Journal of Biochemistry and Molecular Biology, 26(03), 283-289) used C2C12 myoblasts as a research model and found that overexpression of FoxO1 gene can induce the transformation of type I myofibers into type II myofibers and significantly reduce the activity of calcineurin. And the activity of FoxO1 gene is mainly affected by protein post-translational modification (phosphorylation / dephosphorylation, acetylation, ubiquitination). FoxO1 gene regulates fatty acid metabolism in skeletal muscle and participates in the regulation of skeletal muscle energy metabolism, which mainly relies on carbohydrates as energy sources to switch to fatty acid metabolism. FoxO1 gene is highly expressed in white adipose tissue and brown adipose tissue, and the expression level changes with fat intake. In monogastric animals and ruminants, FoxO1 gene can inhibit the proliferation and differentiation of adipocytes and muscle cells through dephosphorylation, thereby affecting meat quality. At present, there are few studies on FoxO1 gene and yak meat quality traits. This study takes yak FoxO1 gene as the research object to explore the effect of its gene mutation on meat quality traits, and provide a basis for improving and improving yak meat quality. Summary of the invention

[0005] In view of the above problems, the present invention provides a FoxO1 gene molecular marker primer related to the tenderness of yak beef and application thereof.

[0006] The invention provides FoxO1 gene molecular marker primers related to the tenderness of yak beef. The molecular marker primers include two pairs, and the nucleotide sequences thereof are shown in SEQ ID NO:1 to SEQ ID NO:2 and SEQ ID NO:3 to SEQ ID NO:4 respectively.

[0007] The present invention also provides an application of a FoxO1 gene molecular marker primer related to the tenderness of yak meat in yak meat quality trait screening and / or yak breeding.

[0008] Furthermore, the method for screening yak meat quality traits and / or breeding yaks comprises the following steps: S1: Extract genomic DNA from yak blood; S2: Using genomic DNA as a template, performing PCR amplification using the molecular marker primers shown in SEQ ID NO: 1 to SEQ ID NO: 2 or SEQ ID NO: 3 to SEQ ID NO: 4 in claim 1 to obtain an amplified product; S3: Perform mixed pool sequencing on the amplified products, detect the genotype at the 1122 bp or 1320 bp of the sequence, and retain the yak individuals with the CC genotype.

[0009] Furthermore, in step S2, the PCR reaction system is 20 μL, and the components in the system are: 0.8 μL DNA template, 0.8 μL upstream and downstream primers, 10 μL Taq DNA polymerase, and 7.6 μL ddH2O.

[0010] Furthermore, in step S2, the PCR amplification program is: 5 min 94°C pre-denaturation, 30 s 94°C denaturation, 30 s annealing, 30 s 72°C extension, a total of 35 cycles; 10 min 72°C extension, 4°C storage.

[0011] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, the mixed pool sequencing and competitive allele-specific PCR (KASP) technology are used to detect the yak FoxO1 Gene polymorphism was analyzed to determine the effect of mutation on the quality traits of Gannan yak meat. FoxO1 Two SNPs (Single Nucleotide Polymorphisms) were detected in the exon region of the gene, namely SNP1 (g.1122 T > C) and SNP2 (g.1320 C > T). Association analysis showed that SNP1 was significantly correlated with yak muscle shear force (P < 0.05). FoxO1 The two SNPs sites of the gene were used to construct three effective haplotypes and six haplotype combinations. The haplotype combination H2H3 had a significant negative effect on the shear force and water loss rate of yak meat, and individuals with this haplotype combination could be eliminated during the breeding process. FoxO1 Correlation between gene mutation and carcass and meat quality traits of Gannan yaks, FoxO1 The gene mutation sites can be used as potential molecular genetic markers for the meat quality traits of Gannan yaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The present invention FoxO1Gene PCR amplification results. Figure A and Figure B are agarose gel electrophoresis images of the PCR amplification products of primers P1 and P2, respectively. From left to right in the figure, each lane represents the amplification products of Maker and three samples; Figure 2 The yak of the present invention FoxO1 Gene SNPs sites; Figure 3 The Gannan yak of the present invention FoxO1 Figure 1. Genotyping results. DETAILED DESCRIPTION

[0013] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0014] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0015] Example 1 1 Materials and methods 1.1 Test samples Venous blood samples were collected from 545 Gannan yaks, and the carcass weight data were recorded after slaughter. A sample of the longest back muscle of each yak was collected to determine the meat quality traits. At the same time, information such as the ranch (household), gender, and age of each yak was recorded.

[0016] 1.2 Determination of beef quality traits of Gannan yak Meat quality traits were determined according to the method of Chen Jie (Chen Jie, 2016, Study on polymorphism of CAPN1 gene in yak and its association with carcass and meat quality traits. Master's degree thesis, Lanzhou: Gansu Agricultural University). Carcass weight refers to the hot carcass weight after slaughter. The cross section of the eye muscle between the 12th and 13th ribs of the carcass was traced with sulfuric acid paper to determine the eye muscle area. Muscle samples were taken from the 12th to 13th ribs of the yak carcass after slaughter. The muscle shear force, water loss rate and cooked meat rate were determined according to the "Objective Evaluation Method for Edible Quality of Meat" (NY / T 2793-2015). The shear force value was determined using a tenderizer (C-LM3), and the water loss rate (pressurized water loss rate) was determined using a steel ring expansion pressure meter.

[0017] 1.3 Amplification primers Reference Yak FoxO1The gene sequence was obtained from GeneBank (accession number: NW_005394190.1). Primers for exon 1 were designed using Primer Premier 5.0 software. The primer sequences are shown in Table 1.

[0018] Table 1 Primer sequence information

[0019] 1.4 Extraction of genomic DNA from blood of Gannan yaks After the yak blood samples were thawed, DNA was extracted according to the operating instructions of the animal blood genomic DNA extraction kit. The DNA integrity and concentration were measured by 1.5% agarose gel electrophoresis and ultra-micro spectrophotometer. Samples with complete and bright bands and meeting the requirements of concentration and purity were selected for gene mutation detection.

[0020] 1.5 Pooled sequencing, PCR amplification, and genotype detection Thirty unrelated samples were randomly selected from the genomic DNA of the Gannan yak population and amplified FoxO1 Gene exon region. PCR reaction system 20 μL: DNA template 0.8 μL, upstream and downstream primers 0.8 μL each, Taq DNA polymerase 10 μL, ddH2O 7.6 μL.

[0021] PCR amplification program: 5 min 94℃ pre-denaturation, 30 s 94℃ denaturation, 30 s annealing, 30 s 72℃ extension, 35 cycles; 10 min 72℃ extension, 4℃ storage. 2 μL of the amplified products of 30 samples were taken, and each pair of primers was amplified into a mixed pool, which was then sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequence determination. FoxO1 After the gene mutation site was identified, the competitive allele-specific PCR (KASP) technique was used to genotype the genomic DNA samples of the experimental Gannan yak population.

[0022] 1.6 Data Statistical Analysis Microsoft Excel 2016 software was used to analyze genotype frequency, allele frequency, gene homozygosity, gene heterozygosity, effective allele number and polymorphism information content. SHEsis online software (http: / / shesisplus.bio-x.cn / SHEsis.html) was used to analyze the linkage disequilibrium (LD) of gene mutation sites and construct haplotypes, and haplotypes with haplotype frequencies higher than 3% were retained. SPSS24.0 software was used for significance analysis, and P﹤0.05 was considered statistically significant.

[0023] SPSS26.0 software general linear model was used to analyze the correlation between genotype, haplotype and haplotype combination and carcass and meat quality traits. The model is as follows: y ijkl = μ + g i + a j + s k + f l + e ijkl ;y ijkl = μ + d i + a j + s k + F l + e ijkl ;y ijkl = μ + h i + a j + s k + f l + e ijkl , In the formula, y is the phenotypic value of the trait; μ is the overall mean; g i is the genotype effect; h i It is a haplotype effect; d i Haplotype combination effect; a j is the age effect; s k For gender effect; f l For field effect; e ijkl is a random error.

[0024] 2 Results 2.1 Agarose gel detection Yak FoxO1 The results of 1.5% agarose gel electrophoresis of the PCR amplification products were shown in Figure 1As shown, the length of the gene amplification product is consistent with the target fragment, and there is no non-specific band, so the next step of the experiment can be carried out.

[0025] 2.2 Yak FoxO1 Gene mutation site determination results Gannan Yak FoxO1 Gene exon sequencing revealed two SNPs, namely g.1122 T > C and g.1320 C > T. Figure 2 shown.

[0026] 2.3 Gannan Yak FoxO1 Genotyping test results Gannan Yak FoxO1 Genotyping results Figure 3 , FoxO1 The genotypes of the g.1122 T > C locus were TT, TC, and CC, and the genotypes of the g.1320 C > T locus were CC, CT, and TT.

[0027] 2.4 Gannan Yak FoxO1 Genetic polymorphism Gannan Yak FoxO1 The genetic polymorphism of the two SNPs loci of the gene is shown in Table 2. There are three genotypes at the g.1122 T > C locus: TT, TC and CC, with CC being the dominant genotype and C being the dominant allele; there are three genotypes at the g.1320 C > T locus: CC, TC and TT, with CC being the dominant genotype and C being the dominant allele. 2 The test showed that the g.1122 T > C and g.1320 C > T loci were both in Hardy Weinberg equilibrium in the Gannan yak population ( P > 0.05), and polymorphism information content 0.25 < PIC < 0.5 is considered moderately polymorphic.

[0028] Table 2 Gannan Yak FoxO1 Genetic polymorphism analysis

[0029] 2.5 Gannan Yak FoxO1 Association analysis between genotype and carcass and meat quality traits Gannan Yak FoxO1 The association analysis between genotype and carcass and meat quality traits is shown in Table 3. SNP1 locus has a significant effect on the muscle shear force of Gannan yaks, and the muscle shear force of TT genotype individuals is significantly higher than that of other genotype individuals ( P < 0.05), and there were no significant differences in other indicators ( P> 0.05). The three genotypes of SNP2 mutation site had no significant effect on the carcass weight and meat quality traits of Gannan yaks ( P > 0.05).

[0030] Table 3 Gannan Yak FoxO1 Association analysis between genotype and carcass and meat quality traits

[0031] Note: The values ​​in the table are "mean ± standard error"; different lowercase letters in the same column indicate significant differences ( P <0.05). 2.6 Gannan Yak FoxO1 Gene haplotype analysis Gannan Yak FoxO1 The two SNPs of the gene (D´= 0.99, r2 = 0.11) were in a weak linkage disequilibrium. The results of haplotype analysis are shown in Table 4. The two SNPs constructed a total of three haplotypes, with the H1 haplotype having the highest frequency of 48.9%, and the H2 and H3 haplotype frequencies being relatively low, at 23.1% and 29.6%, respectively. The three haplotypes formed six haplotype combinations (number of yak individuals), namely H1H1 (139), H1H3 (141), H2H1 (105), H2H2 (29), H2H3 (70), and H3H3 (61).

[0032] Table 4 Gannan Yak FoxO1 Haplotype analysis of gene mutation sites

[0033] 2.7 Gannan Yak FoxO1 Association analysis between gene haplotype combinations and meat quality traits Gannan Yak FoxO1 The association analysis between different haplotype combinations of genes (the number of individuals accounts for more than 2% of the sampling population) and carcass and meat quality traits is shown in Table 5. FoxO1 The muscle shear force of individuals with the H2H2 and H2H3 haplotype combinations was significantly higher than that of individuals with other haplotype combinations ( P < 0.05), the muscle water loss rate of individuals with the H2H3 haplotype combination was significantly higher than that of individuals with other haplotype combinations ( P <0.05, and the eye muscle area is lower than that of other haplotype combinations ( P > 0.05).

[0034] Table 5 Gannan Yak FoxO1 Effects of gene haplotype combinations on meat quality and carcass traits

[0035] 2.8 Gannan YakFoxO1 Association analysis between gene haplotypes and meat quality traits Gannan Yak FoxO1 The association analysis between the presence / absence of gene haplotypes and their carcass and meat quality traits is shown in Table 6. FoxO1 Gene haplotypes affect the muscle shear force and water loss rate of Gannan yaks. Among them, the muscle shear force of individuals carrying the H2 haplotype is significantly greater than that of individuals lacking H2 ( P < 0.05), the muscle water loss rate of individuals carrying the H1 haplotype was significantly lower than that of individuals not carrying the haplotype ( P < 0.05), the muscle water loss rate of individuals carrying the H3 haplotype was significantly higher than that of individuals not carrying the H3 genotype ( P < 0.05).

[0036] Table 6 Gannan Yak FoxO1 Correlation between the presence / absence of gene haplotypes and carcass and meat quality traits

[0037] 3 Discussion 3.1 Gannan Yak FoxO1 Genetic polymorphism is relatively rich at present, FoxO1 Gene polymorphism studies are mostly focused on humans and mice. FoxO1 There are relatively few studies on gene polymorphism. Wang Ling et al. (Wang Ling, Li Jian, Fu Chongyun, et al., 2010. cDNA cloning of the bovine FoxO1 gene and expression analysis in newborn calf tissues. Chinese Journal of Animal Husbandry, 46(13), 1-5.) used southern Sichuan mountain yellow cattle, Qin hybrid cattle, and Xi hybrid cattle as research objects and used Sanger sequencing to identify the genetic polymorphism of beef cattle populations. FoxO1 Eight mutation sites were detected in the gene, all located in the third exon region; Sun et al. (SUN Y, XUE J, GUO W, et al., 2013. Haplotypes of bovine FoxO1 genesequence variants and association with growth traits in Qinchuan cattle. J.Genet., 25;92(1): e8-14.) FoxO1 Five mutation sites were detected in the gene, of which four were located in the exon region and one in the 3' flanking region. Zhu Shubin et al. (Zhu Shubin, Zhou Chunbao, Han Dayong, et al., 2015. Genetic polymorphism of FoxO1 gene in Jiangquhai pig and its correlation with meat quality traits. Journal of Jiangsu Agricultural Sciences, 31(05), 1065-1069) analyzed 112 Jiangquhai pigs. FoxO1Genetic variation in exon 1 of the gene, polymorphism was found at the Bpil restriction site, and association analysis showed that it was significantly correlated with pig intramuscular fat content, marbling and shear force. The above research results show that FoxO1 The gene has rich genetic polymorphism in livestock. FoxO1 Two mutation sites were found in the exon region of the gene, and the results were similar to those of previous studies on ordinary cattle.

[0038] The higher the degree of genetic variation in a population, the higher the genetic richness of the population. The polymorphic information content (PIC) depends on the number and frequency distribution of the detected alleles. FoxO1 Two SNPs were detected in the exon region of the gene, both of which were moderately polymorphic, indicating that the genetic polymorphism was rich and had a good genetic variation basis. The Hardy-Weinberg equilibrium coefficient test results showed that g.1320 C > T was in a Hardy-Weinberg disequilibrium state in the Gannan yak population, and it was speculated that the Gannan yak population was greatly affected by natural selection and artificial selection at this site, which may also be related to the number of research subjects.

[0039] 3.2 FoxO1 Gene mutation affects carcass and meat quality traits of Gannan yaks Transcription factor FoxO1 is the earliest discovered member of the FoxO family and plays an important role in cell differentiation signaling pathways and transcriptional cascade reactions. Studies have shown that in livestock such as cattle, sheep, and pigs, FoxO1 The genes inhibit the proliferation and differentiation of adipocytes and muscle cells through dephosphorylation, thus further affecting meat quality. Zhu Shubin et al. (Zhu Shubin, Zhou Chunbao, Han Dayong, et al., 2015. Genetic polymorphism of FoxO1 gene in Jiangquhai pig and its correlation with meat quality traits. Journal of Jiangsu Agricultural Sciences, 31(05), 1065-1069.) et al. used PCR-RFLP molecular marker technology to study FoxO1 The polymorphism of the gene and its correlation with meat quality traits were found to significantly affect the intramuscular fat content, marbling and shear force of pigs; Ropka-Molik et al. (ROPKA-MOLIK K, BERETA A, ŻUKOWSKI K, et al., 2018. Screening for candidate genes related with histological microstructure, meat quality and carcass characteristic in pig based on RNA-seq data. Asian-Australas J Anim Sci., 31(10):1565-1574.) reported thatFoxO1 Gene polymorphism is significantly correlated with the diameter and lean meat percentage of muscle fiber types (IA and ⅡB) in Landrace, Large White, Pietrain and Duroc pigs; Wang Ling et al. (Wang Ling, Li Jian, Fu Chongyun, et al., 2010. cDNA cloning of bovine FoxO1 gene and expression analysis in newborn calf tissues. Chinese Journal of Animal Husbandry, 46(13), 1-5.) detected the FoxO1 gene in Qinchuan cattle FoxO1 The polymorphism of the gene was found to significantly affect the diameter and density of muscle fibers, shear force, and had a positive genetic effect on meat quality traits. FoxO1 The gene is a key candidate gene affecting the meat quality traits of livestock such as cattle and pigs. FoxO1 Two SNPs (g.1122 T > C, g.1320 C > T) were detected in the exon region of the gene, and the association analysis with the quality traits of Gannan yak meat was carried out from different perspectives of genotype, haplotype and haplotype combination. The genotype of SNP1 locus had a significant effect on muscle shear force and water loss rate; there were significant differences in muscle shear force and water loss rate between yaks with different haplotypes; the presence / absence of haplotype had a significant effect on muscle shear force and water loss rate. FoxO1 The gene mutation mainly affects the muscle shear force of Gannan yaks, and thus affects the meat quality traits of Gannan yaks.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A FoxO1 gene molecular marker primer related to the tenderness of yak beef, characterized by: There are two pairs of molecular marker primers, and their nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:2 and SEQ ID NO:3 to SEQ ID NO:4 respectively.

2. Use of the FoxO1 gene molecular marker primer associated with yak beef tenderness as claimed in claim 1 in yak beef quality trait screening and / or yak breeding.

3. The use according to claim 2, characterized in that: The method for screening yak meat quality traits and / or breeding yaks comprises the following steps: S1: Extract genomic DNA from yak blood; S2: Using genomic DNA as a template, performing PCR amplification using the molecular marker primers shown in SEQ ID NO: 1 to SEQ ID NO: 2 or SEQ ID NO: 3 to SEQ ID NO: 4 in claim 1 to obtain an amplified product; S3: Perform mixed pool sequencing on the amplified products, detect the genotype at the 1122 bp or 1320 bp of the sequence, and retain the yak individuals with the CC genotype.

4. The use according to claim 3, characterized in that: In step S2, the PCR reaction system is 20 μL, and the components in the system are: 0.8 μL DNA template, 0.8 μL upstream and downstream primers, 10 μL Taq DNA polymerase, and 7.6 μL ddH2O.

5. The use according to claim 3, characterized in that: In step S2, the PCR amplification program is: 5 min 94°C pre-denaturation, 30 s 94°C denaturation, 30 s annealing, 30 s 72°C extension, a total of 35 cycles; 10 min 72°C extension, 4°C storage.

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