东北肉羊LPL基因多态性与肉质性状的关联分析
发布时间：2019-02-08 18:15
【摘要】：本试验以脂蛋白脂肪酶(Lipoprotein Lipase,LPL)作为东北肉羊肉质性状的候选基因,研究其外显子上的单核甘酸多态性(single nucleotide polymorphisms,SNPs)位点及其与肉质性状的关联,为改善肉羊肉质及其遗传育种提供分子生物学基础。本试验以48头成年健康的东北肉羊肌肉组织样本为材料,提取总RNA与基因组DNA,采用RT-PCR和TA克隆方法成功克隆了东北肉羊的LPL基因c DNA序列并对其进行生物信息学分析;将LPL基因作为肉质性状的候选基因,应用直接测序和PCR-RFLP相结合的方法对LPL基因部分外显子进行SNPs筛选,并通过一般线性模型分析研究了LPL基因多态性与肉质性状的关联。主要试验结果如下:1.提取了总RNA,通过RT-PCR的方法,克隆了东北肉羊LPL基因的完整编码序列,并对其进行了序列测定。对东北肉羊LPL基因完整编码序列进行生物信息学分析,结果表明,东北肉羊LPL基因ORF(开放阅读框)长1 437bp,共编码了478个氨基酸。2.通过直接测序及PCR-RFLP技术,对东北肉羊LPL基因第3、4、5和6外显子进行序列测定与多态性分析,发现LPL基因第3和4外显子上存在SNPs位点,而第5和6外显子则未发现SNPs位点。这些SNPs位点分别是:第3外显子46 bp处T/C(T304C),第3外显子126 bp处A/T(A384T),第4外显子24bp处T/C(T462C)。这3个SNP位点都没有引起氨基酸变异,属于同义突变。3.对东北肉羊第3外显子A384T突变位点进行酶切与基因分型,并统计基因频率、基因型频率、χ2-Hardy-Weinberg平衡状态、基因纯合度(Ho)、基因杂合度(He)、有效等位基因数(Ne)、多态信息含量(PIC)等数据。结果表明,东北肉羊群体在该位点的遗传变异为低度多态(PIC0.25),并处于Hardy-Weinberg平衡状态(P0.05)。4.对第3外显子A384T突变位点各基因型与肉质性状进行关联性分析,结果表明该位点TT型滴水损失极显著高于TA型和AA型(P0.01);TT型p H1值显著高于TA型(P0.05),与AA型则差异不显著(P0.05);压榨损失、熟肉率和剪切力性状在各基因型间差异不显著(P0.05)。TT型肉豆蔻酸含量显著低于TA型和AA型(P0.05);TT型棕榈酸含量显著低于TA型(P0.05),与AA型则差异不显著(P0.05);其余脂肪酸含量在各基因型间差异不显著(P0.05)。TT型甘氨酸含量显著高于TA型(P0.05),与AA型差异不显著(P0.05);TT型精氨酸含量显著高于TA型和AA型(P0.05);其余氨基酸含量在各基因型间差异不显著(P0.05)。
[Abstract]:In this study, lipoprotein lipase (Lipoprotein Lipase,LPL) was used as a candidate gene for meat quality traits in Northeast Sheep. The polymorphism of mononuclear glycolylate (single nucleotide polymorphisms,SNPs) locus in the exon and its association with meat quality traits were studied. To provide molecular biological basis for improving meat quality and genetic breeding of meat sheep. The total RNA and genomic DNA, were extracted from the muscle tissue samples of 48 adult healthy Northeast meat sheep. The c DNA sequence of LPL gene was cloned successfully by RT-PCR and TA cloning method and bioinformatics analysis was carried out on the LPL gene of Northeast Meat Sheep. LPL gene was used as candidate gene for fleshy traits. SNPs was used to screen some exons of LPL gene by direct sequencing and PCR-RFLP. The association between LPL gene polymorphism and fleshy traits was studied by general linear model analysis. The main results are as follows: 1. The complete encoding sequence of LPL gene of Northeast Meat Sheep was cloned and sequenced by the method of total RNA, extracted by RT-PCR. The complete coding sequence of LPL gene of Northeast Meat Sheep was analyzed by bioinformatics. The results showed that the LPL gene ORF (Open Reading frame) of Northeast Meat Sheep was 1 437 BP long and encoded 478 amino acids. By direct sequencing and PCR-RFLP analysis, the exons 3, 4 and 6 of LPL gene were sequenced and polymorphic analysis. It was found that there were SNPs loci in exons 3 and 4 of LPL gene. No SNPs loci were found in exons 5 and 6. These SNPs loci were T / C (T304C) at exon 3 46 bp, A / T (A384T) at exon 126 bp and T / C (T462C) at 24bp at exon 4. None of the three SNP loci caused amino acid mutation, which belonged to synonymous mutation. The mutation site A384T in exon 3 of Northeast Meat Sheep was digested and genotyped. The gene frequency, genotype frequency, 蠂 2-Hardy-Weinberg equilibrium state, homozygosity (Ho), gene heterozygosity (He), were analyzed. Effective allele number (Ne), polymorphism information content (PIC) et al. The results showed that the genetic variation at this locus was low polymorphic (PIC0.25) and Hardy-Weinberg equilibrium (P0.05). The relationships between the genotypes of A384T mutation site and meat quality traits were analyzed. The results showed that the drip loss of TT type was significantly higher than that of TA type and AA type (P0.01). The value of pH1 in TT type was significantly higher than that in TA type (P0.05), but there was no significant difference between AA type and TT type (P0.05). Squeezing loss, cooked meat rate and shear stress were not significantly different among genotypes (P0.05) the content of myristic acid in). TT type was significantly lower than that in TA type and AA type (P0.05). The content of palmitic acid in TT type was significantly lower than that in TA type (P0.05), but there was no significant difference between AA type and TT type (P0.05). There was no significant difference in the content of other fatty acids among genotypes (P0.05). The content of glycine in). TT type was significantly higher than that in TA type (P0.05), but there was no significant difference with AA type (P0.05). The content of arginine in TT type was significantly higher than that in TA type and AA type (P0.05), while the other amino acid contents had no significant difference among genotypes (P0.05).
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
本文编号：2418661
[Abstract]:In this study, lipoprotein lipase (Lipoprotein Lipase,LPL) was used as a candidate gene for meat quality traits in Northeast Sheep. The polymorphism of mononuclear glycolylate (single nucleotide polymorphisms,SNPs) locus in the exon and its association with meat quality traits were studied. To provide molecular biological basis for improving meat quality and genetic breeding of meat sheep. The total RNA and genomic DNA, were extracted from the muscle tissue samples of 48 adult healthy Northeast meat sheep. The c DNA sequence of LPL gene was cloned successfully by RT-PCR and TA cloning method and bioinformatics analysis was carried out on the LPL gene of Northeast Meat Sheep. LPL gene was used as candidate gene for fleshy traits. SNPs was used to screen some exons of LPL gene by direct sequencing and PCR-RFLP. The association between LPL gene polymorphism and fleshy traits was studied by general linear model analysis. The main results are as follows: 1. The complete encoding sequence of LPL gene of Northeast Meat Sheep was cloned and sequenced by the method of total RNA, extracted by RT-PCR. The complete coding sequence of LPL gene of Northeast Meat Sheep was analyzed by bioinformatics. The results showed that the LPL gene ORF (Open Reading frame) of Northeast Meat Sheep was 1 437 BP long and encoded 478 amino acids. By direct sequencing and PCR-RFLP analysis, the exons 3, 4 and 6 of LPL gene were sequenced and polymorphic analysis. It was found that there were SNPs loci in exons 3 and 4 of LPL gene. No SNPs loci were found in exons 5 and 6. These SNPs loci were T / C (T304C) at exon 3 46 bp, A / T (A384T) at exon 126 bp and T / C (T462C) at 24bp at exon 4. None of the three SNP loci caused amino acid mutation, which belonged to synonymous mutation. The mutation site A384T in exon 3 of Northeast Meat Sheep was digested and genotyped. The gene frequency, genotype frequency, 蠂 2-Hardy-Weinberg equilibrium state, homozygosity (Ho), gene heterozygosity (He), were analyzed. Effective allele number (Ne), polymorphism information content (PIC) et al. The results showed that the genetic variation at this locus was low polymorphic (PIC0.25) and Hardy-Weinberg equilibrium (P0.05). The relationships between the genotypes of A384T mutation site and meat quality traits were analyzed. The results showed that the drip loss of TT type was significantly higher than that of TA type and AA type (P0.01). The value of pH1 in TT type was significantly higher than that in TA type (P0.05), but there was no significant difference between AA type and TT type (P0.05). Squeezing loss, cooked meat rate and shear stress were not significantly different among genotypes (P0.05) the content of myristic acid in). TT type was significantly lower than that in TA type and AA type (P0.05). The content of palmitic acid in TT type was significantly lower than that in TA type (P0.05), but there was no significant difference between AA type and TT type (P0.05). There was no significant difference in the content of other fatty acids among genotypes (P0.05). The content of glycine in). TT type was significantly higher than that in TA type (P0.05), but there was no significant difference with AA type (P0.05). The content of arginine in TT type was significantly higher than that in TA type and AA type (P0.05), while the other amino acid contents had no significant difference among genotypes (P0.05).
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826
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