猪肉嘌呤含量表型变异及其遗传基础研究
发布时间：2018-09-05 07:55
【摘要】：过去几十年,随着饮食结构的改变和人均嘌呤摄入量不断提升,中国高尿酸血症和痛风的患病率大约升高了十倍,因此食品中的嘌呤含量受到消费者和食品研究者的重视。肉品的嘌呤含量较高,且猪肉占中国总肉品消费的65%左右,但目前国内外有关猪肉嘌呤含量的表型变异程度和遗传基础研究尚为空白。本文选取杜洛克(6头)、杜长大(18头)、巴马香(18头)、二花脸(18头)、莱芜(18头)和嵌合家系(377头)六个不同种群共455头猪的背最长肌样品,测定了其4种嘌呤碱基含量(包括鸟嘌呤、腺嘌呤、次黄嘌呤和黄嘌呤)和多种肉质性状;并对其中377头嵌合家系F6代个体进行基因组全序列测序(7×深度)和40头极端表型个体的肌肉组织转录组测序;另外,随机选取嵌合家系三头阉公猪,测定其12种组织嘌呤含量。进而系统地分析了猪肉嘌呤含量在品种间及品种内、组织间和性别间的差异,并开展了基于全基因组重测序的关联研究(GWAS),定位了各嘌呤和总嘌呤含量的数量性状位点(QTL),得到以下主要结果:(1)宰后猪肉嘌呤含量的动态变化:宰后猪肉中腺嘌呤不断向次黄嘌呤转化,至24h趋于稳定;但总嘌呤和鸟嘌呤含量基本维持恒定。(2)品种间差异显著:除杜洛克外,莱芜猪平均总嘌呤含量(114.15 mg/100g)极显著低于其他四个品种(P0.01),而二花脸猪显著低于巴马香(P0.05)。(3)性别间差异显著:嵌合家系中阉公猪的肌肉平均嘌呤含量(137.71 mg/100g)显著低于母猪(139.90 mg/100g)。(4)组织间差异显著:肺、肝和肾脏的嘌呤含量超过200 mg/100g,为高嘌呤组织;肌肉(包括心肌、骨骼肌、平滑肌)嘌呤含量大约为130 mg/100g,为中嘌呤组织;背膘、皮肤和蹄部结缔组织嘌呤含量低于30 mg/100g,为低嘌呤组织。(5)总嘌呤含量与pH、肉色、肌内脂肪含量(IMF)等多种肉质性状显著的相关(P0.05),相关系数达0.2-0.4之间。相关性结果表明低嘌呤含量的猪肉具有较高的终pH、IMF和系水力,且肉色趋向鲜红、嫩度和口感较好。因此培育低嘌呤优质肉种猪,不仅不会与传统关注的重要肉质性状的选育方向发生冲突,甚至可能在一定程度上有助于改善这些肉质性状。(6)GWAS分析鉴别到40个与总嘌呤含量显著关联的SNP位点(P5.00×10-8),它们可能代表了11个QTLs,其中最显著关联的SNP rs32337327(P=2.75×10-9)位于猪8号染色体(SSC8)的LDB2和QDPR基因之间,而SSC3、SSC11和SSC14上的最强关联SNPs则分别落在TGFBRAP1、MPHOSPH8和C12orf43的内含子区域内。同时还检出263个与鸟嘌呤含量显著关联SNPs,它们分布于26个QTLs区域,其中最显著关联SNP rs343436977(P=9.72×10-13)位于ABCC4基因的内含子上,另有9个QTLs区间内的最显著关联SNPs也位于不同基因的内含子上。本研究未检测到与腺嘌呤和次黄嘌呤含量显著关联信号,但发现SSC7和SSC8上可能存在影响它们的潜在QTLs(P5.00×10-6),其最强关联SNPs分别位于PAQR8和SLC10A7基因内。腺嘌呤与次黄嘌呤之和的GWAS结果和总嘌呤GWAS结果相似。(7)通过分析肌肉嘌呤含量极端个体的转录组数据,共鉴别到25个差异表达基因或者序列标签,其中包含8个已注释基因的转录本,分别为RYK、ORAOV1、MTERF1、MOSPD1、SLCO1A2、B3GNTL1、ZDHHC7和FAM92A。未发现GWAS信号落在差异表达基因区域内,提示猪肉嘌呤含量可能受复杂遗传因子调控。(8)通过对GWAS最显著SNP位点附近已注释的70个候选基因和8个表达差异基因进行GO分析,发现了11个功能富集条目,其中钙离子结合富集条目包含8个相关基因。KEGG分析发现ABCC4、ADCY2和GRIA1等基因参与cAMP信号通路。此外,这78个基因编码的蛋白中,有10个具有ATP结合位点,有41个属于膜蛋白。该结果提示钙离子结合蛋白和cAMP信号通路很可能参与调控肌细胞中嘌呤物质代谢与转运,从而影响猪肌肉嘌呤含量。本研究首次定位了影响猪肉嘌呤含量的QTL位点,鉴别了多个相关候选基因,并揭示钙离子和cAMP信号通路对肌肉嘌呤含量有重要影响。这些结果为深入揭示肌肉嘌呤含量的遗传机制奠定了坚实基础,也为今后培育低嘌呤优质猪种提供了理论与技术支持。
[Abstract]:Over the past few decades, the prevalence of hyperuricemia and gout in China has risen tenfold as dietary changes and per capita intake of purine have increased. As a result, the purine content in foods has attracted the attention of consumers and food researchers. The study on the phenotypic variation and genetic basis of pork purine content at home and abroad is still blank. In this paper, the samples of longissimus dorsi from 6 Duo (18 head), Bama (18), Bama (18), two face (18), Laiwu (18) and inlay families (377 *) six pigs were collected. Guanine, adenine, hypoxanthine and xanthine, and a variety of meat traits; 377 chimeric F6 generations were sequenced (7 x depth) and 40 extreme phenotypic individuals were sequenced in muscle tissue transcriptomes; in addition, three castrated boars from chimeric families were randomly selected to determine the purine content of 12 tissues. Quantitative trait loci (QTLs) of purines and total purines in pork were identified based on genome-wide sequencing (GWAS). The main results were as follows: (1) Dynamic changes of purine content in postmortem pork: Adenine content in postmortem pork However, the contents of total purine and guanine were basically constant. (2) there was a significant difference among varieties. Except for Duroc, the average total purine content (* 114.15 mg/100g) of Laiwu pigs was significantly lower than that of the other four varieties (P0.01), while two * Hualian pigs were significantly lower than those of Bama (P0.05). (3) the difference between the sexes was significant: 24h. The average purine content (137.71 mg/100g) in the muscle of castrated boars was significantly lower than that of sows (139.90 mg/100g). (4) There was significant difference between tissues: the purine content in lung, liver and kidney exceeded 200 mg/100g, which was high purine tissue; the purine content in muscle (including myocardium, skeletal muscle, smooth muscle) was about 130 mg/100g, which was middle purine tissue; the purine content in backfat, skin and kidney was about 130 mg/100g. (5) Total purine content was significantly correlated with pH, meat color, intramuscular fat content (IMF) and other meat quality traits (P 0.05), the correlation coefficient was between 0.2 and 0.4. The correlation results showed that pork with low purine content had higher final pH, IMF and waterpower, and the meat color tended to bright red. Therefore, the breeding of low purine quality broiler pigs will not conflict with the breeding direction of important meat quality traits of traditional concern, and may even help to improve these meat quality traits to a certain extent. (6) GWAS analysis identified 40 SNPs (P5.00 *10-8) which were significantly associated with total purine content, and they may be progenies. 11 QTLs were listed, among which the most significant SNP rs32337327 (P=2.75 * 10-9) was located between LDB2 and QDPR genes of porcine chromosome 8 (SSC8), while the strongest correlation between SSC3, SSC11 and SSC14 was located in the intron region of the SSC3, SSC11 and SSC14 respectively, and 263 of them were significantly associated with guanine content. Among 26 QTLs, the most significant association SNP rs343436977 (P = 9.72 *10-13) was located on the intron of ABCC4 gene, and the most significant association SNPs in nine QTLs were also located on the intron of different genes. Their potential QTLs (P5.00 *10-6), the strongest associated SNPs were located in PAQR8 and SLC10A7, respectively. The GWAS results of the sum of adenine and hypoxanthine were similar to those of the total purine GWAS. (7) Twenty-five differentially expressed genes or sequence tags were identified by analyzing the transcriptome data of individuals with extreme muscle purine content. The transcripts of the annotated genes were RYK, ORAOV1, MTERF1, MOSPD1, SLCO1A2, B3GNTL1, ZDHHC7 and FAM92A. No GWAS signals were found in the differentially expressed gene regions, suggesting that pork purine content might be regulated by complex genetic factors. (8) 70 candidate genes and 8 differentially expressed genes were annotated near the most significant SNP site of GWAS. By GO analysis, 11 functional enrichment entries were identified, of which 8 related genes were found. KEGG analysis revealed that ABCC4, ADCY2 and GRIA1 were involved in the cAMP signaling pathway. In addition, 10 of the 78 genes encoded proteins had ATP binding sites and 41 belonged to membrane proteins. The cAMP signaling pathway is likely to participate in regulating the metabolism and transport of purine * in muscle cells, thereby affecting the purine content of porcine muscles. For the first time, this study locates the QTL locus affecting pork purine content, identifies multiple candidate genes, and reveals that calcium and cAMP signaling pathways have important effects on muscle purine content. It lays a solid foundation for revealing the genetic mechanism of purine content in muscle and provides theoretical and technical support for breeding low * purine quality pig breeds in the future.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
本文编号：2223660
[Abstract]:Over the past few decades, the prevalence of hyperuricemia and gout in China has risen tenfold as dietary changes and per capita intake of purine have increased. As a result, the purine content in foods has attracted the attention of consumers and food researchers. The study on the phenotypic variation and genetic basis of pork purine content at home and abroad is still blank. In this paper, the samples of longissimus dorsi from 6 Duo (18 head), Bama (18), Bama (18), two face (18), Laiwu (18) and inlay families (377 *) six pigs were collected. Guanine, adenine, hypoxanthine and xanthine, and a variety of meat traits; 377 chimeric F6 generations were sequenced (7 x depth) and 40 extreme phenotypic individuals were sequenced in muscle tissue transcriptomes; in addition, three castrated boars from chimeric families were randomly selected to determine the purine content of 12 tissues. Quantitative trait loci (QTLs) of purines and total purines in pork were identified based on genome-wide sequencing (GWAS). The main results were as follows: (1) Dynamic changes of purine content in postmortem pork: Adenine content in postmortem pork However, the contents of total purine and guanine were basically constant. (2) there was a significant difference among varieties. Except for Duroc, the average total purine content (* 114.15 mg/100g) of Laiwu pigs was significantly lower than that of the other four varieties (P0.01), while two * Hualian pigs were significantly lower than those of Bama (P0.05). (3) the difference between the sexes was significant: 24h. The average purine content (137.71 mg/100g) in the muscle of castrated boars was significantly lower than that of sows (139.90 mg/100g). (4) There was significant difference between tissues: the purine content in lung, liver and kidney exceeded 200 mg/100g, which was high purine tissue; the purine content in muscle (including myocardium, skeletal muscle, smooth muscle) was about 130 mg/100g, which was middle purine tissue; the purine content in backfat, skin and kidney was about 130 mg/100g. (5) Total purine content was significantly correlated with pH, meat color, intramuscular fat content (IMF) and other meat quality traits (P 0.05), the correlation coefficient was between 0.2 and 0.4. The correlation results showed that pork with low purine content had higher final pH, IMF and waterpower, and the meat color tended to bright red. Therefore, the breeding of low purine quality broiler pigs will not conflict with the breeding direction of important meat quality traits of traditional concern, and may even help to improve these meat quality traits to a certain extent. (6) GWAS analysis identified 40 SNPs (P5.00 *10-8) which were significantly associated with total purine content, and they may be progenies. 11 QTLs were listed, among which the most significant SNP rs32337327 (P=2.75 * 10-9) was located between LDB2 and QDPR genes of porcine chromosome 8 (SSC8), while the strongest correlation between SSC3, SSC11 and SSC14 was located in the intron region of the SSC3, SSC11 and SSC14 respectively, and 263 of them were significantly associated with guanine content. Among 26 QTLs, the most significant association SNP rs343436977 (P = 9.72 *10-13) was located on the intron of ABCC4 gene, and the most significant association SNPs in nine QTLs were also located on the intron of different genes. Their potential QTLs (P5.00 *10-6), the strongest associated SNPs were located in PAQR8 and SLC10A7, respectively. The GWAS results of the sum of adenine and hypoxanthine were similar to those of the total purine GWAS. (7) Twenty-five differentially expressed genes or sequence tags were identified by analyzing the transcriptome data of individuals with extreme muscle purine content. The transcripts of the annotated genes were RYK, ORAOV1, MTERF1, MOSPD1, SLCO1A2, B3GNTL1, ZDHHC7 and FAM92A. No GWAS signals were found in the differentially expressed gene regions, suggesting that pork purine content might be regulated by complex genetic factors. (8) 70 candidate genes and 8 differentially expressed genes were annotated near the most significant SNP site of GWAS. By GO analysis, 11 functional enrichment entries were identified, of which 8 related genes were found. KEGG analysis revealed that ABCC4, ADCY2 and GRIA1 were involved in the cAMP signaling pathway. In addition, 10 of the 78 genes encoded proteins had ATP binding sites and 41 belonged to membrane proteins. The cAMP signaling pathway is likely to participate in regulating the metabolism and transport of purine * in muscle cells, thereby affecting the purine content of porcine muscles. For the first time, this study locates the QTL locus affecting pork purine content, identifies multiple candidate genes, and reveals that calcium and cAMP signaling pathways have important effects on muscle purine content. It lays a solid foundation for revealing the genetic mechanism of purine content in muscle and provides theoretical and technical support for breeding low * purine quality pig breeds in the future.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828
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