東北肉羊LPL基因多態(tài)性與肉質(zhì)性狀的關(guān)聯(lián)分析
發(fā)布時(shí)間：2019-02-08 18:15
【摘要】：本試驗(yàn)以脂蛋白脂肪酶(Lipoprotein Lipase,LPL)作為東北肉羊肉質(zhì)性狀的候選基因,研究其外顯子上的單核甘酸多態(tài)性(single nucleotide polymorphisms,SNPs)位點(diǎn)及其與肉質(zhì)性狀的關(guān)聯(lián),為改善肉羊肉質(zhì)及其遺傳育種提供分子生物學(xué)基礎(chǔ)。本試驗(yàn)以48頭成年健康的東北肉羊肌肉組織樣本為材料,提取總RNA與基因組DNA,采用RT-PCR和TA克隆方法成功克隆了東北肉羊的LPL基因c DNA序列并對(duì)其進(jìn)行生物信息學(xué)分析;將LPL基因作為肉質(zhì)性狀的候選基因,應(yīng)用直接測(cè)序和PCR-RFLP相結(jié)合的方法對(duì)LPL基因部分外顯子進(jìn)行SNPs篩選,并通過(guò)一般線性模型分析研究了LPL基因多態(tài)性與肉質(zhì)性狀的關(guān)聯(lián)。主要試驗(yàn)結(jié)果如下:1.提取了總RNA,通過(guò)RT-PCR的方法,克隆了東北肉羊LPL基因的完整編碼序列,并對(duì)其進(jìn)行了序列測(cè)定。對(duì)東北肉羊LPL基因完整編碼序列進(jìn)行生物信息學(xué)分析,結(jié)果表明,東北肉羊LPL基因ORF(開(kāi)放閱讀框)長(zhǎng)1 437bp,共編碼了478個(gè)氨基酸。2.通過(guò)直接測(cè)序及PCR-RFLP技術(shù),對(duì)東北肉羊LPL基因第3、4、5和6外顯子進(jìn)行序列測(cè)定與多態(tài)性分析,發(fā)現(xiàn)LPL基因第3和4外顯子上存在SNPs位點(diǎn),而第5和6外顯子則未發(fā)現(xiàn)SNPs位點(diǎn)。這些SNPs位點(diǎn)分別是:第3外顯子46 bp處T/C(T304C),第3外顯子126 bp處A/T(A384T),第4外顯子24bp處T/C(T462C)。這3個(gè)SNP位點(diǎn)都沒(méi)有引起氨基酸變異,屬于同義突變。3.對(duì)東北肉羊第3外顯子A384T突變位點(diǎn)進(jìn)行酶切與基因分型,并統(tǒng)計(jì)基因頻率、基因型頻率、χ2-Hardy-Weinberg平衡狀態(tài)、基因純合度(Ho)、基因雜合度(He)、有效等位基因數(shù)(Ne)、多態(tài)信息含量(PIC)等數(shù)據(jù)。結(jié)果表明,東北肉羊群體在該位點(diǎn)的遺傳變異為低度多態(tài)(PIC0.25),并處于Hardy-Weinberg平衡狀態(tài)(P0.05)。4.對(duì)第3外顯子A384T突變位點(diǎn)各基因型與肉質(zhì)性狀進(jìn)行關(guān)聯(lián)性分析,結(jié)果表明該位點(diǎn)TT型滴水損失極顯著高于TA型和AA型(P0.01);TT型p H1值顯著高于TA型(P0.05),與AA型則差異不顯著(P0.05);壓榨損失、熟肉率和剪切力性狀在各基因型間差異不顯著(P0.05)。TT型肉豆蔻酸含量顯著低于TA型和AA型(P0.05);TT型棕櫚酸含量顯著低于TA型(P0.05),與AA型則差異不顯著(P0.05);其余脂肪酸含量在各基因型間差異不顯著(P0.05)。TT型甘氨酸含量顯著高于TA型(P0.05),與AA型差異不顯著(P0.05);TT型精氨酸含量顯著高于TA型和AA型(P0.05);其余氨基酸含量在各基因型間差異不顯著(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).
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S826
本文編號(hào)：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).
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S826
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