Correlation of the A-FABP Gene Polymorphism and mRNA Expression with Intramuscular Fat Content in Three-Yellow Chicken and Hetian-Black Chicken

The adipocyte-type fatty acid-binding protein (A-FABP) is considered a candidate gene for fat metabolism; thus, it affects fat deposition in chickens. The present study was designed to examine the polymorphism and mRNA abundance of the A-FABP gene with intramuscular fat (IMF) in the pectoralis muscles (PM) and leg muscles (LM) of Three-yellow Chicken (TYC) and Hetian-black Chicken (HTBC). In total, 60 TYCs and 60 HTBCs were sacrificed using exsanguination at market age. The IMF contents of the PM and LM in the HTBC were significantly higher than those in the TYC. Three genotypes of the A-FABP gene first exon, AA, AB, and BB, were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and a C51 T mutational site, which is a silent substitution mutation, was revealed. The IMF contents of the AA genotype in the PM of the HTBC were significantly higher than those in the AB genotype; thus, the C51 T mutable site is a gene marker for selecting a higher IMF content in the PM of the HTBC. The relative expression of the A-FABP mRNA in the LM of the HTBC, which was measured by quantitative real-time PCR, was significantly higher than in the TYC. A significantly positive association was detected between A-FABP expression with the IMF contents of the PM and LM of both the TYC and the HTBC. These results provide basic data that might be helpful to further research the role of the A-FABP gene in fat deposition and fatty acid metabolism in chickens.     

西部地区主要猪种H-FABP基因多态性,IMF含量及不同基因型脂肪细胞脂滴量的关系

H-FABP是FABP家族成员之一,在长链脂肪酸的吸收和代谢平衡中发挥关键作用,但它对猪IMF含量的影响还知之甚少,对中国西部地区的猪种更是如此.文章利用PCR-RFLP（HinfⅠ、HaeⅢ和Msp Ⅰ 3种限制性内切酶）分子标记技术,检测了杜洛克猪、长白猪、大白猪、内江猪、荣昌猪、汉江黑猪、汉中白猪、八眉猪和野猪共计265头猪H-FABP基因5＇-上游区和第二内含子区的遗传变异,利用最小二乘模型分析了H-FABP基因对猪肌内脂肪含量的遗传效应,并运用猪脂肪细胞培养,油红O染色和TG测定等技术检测了H-FABP基因不同基因型脂肪细胞内脂滴的形态和沉积的量.结果表明：（1）在HinfⅠ-RFLP位点上,上述品种和野猪均存在多态性,其中大白猪、八眉猪、汉江黑猪、汉中白猪和野猪表现为低度多态,杜洛克、长白猪、内江猪和荣昌猪为中度多态;除汉江黑猪（P＜0.05）和野猪（P＜0.01）外,其他猪种基因频率和基因型频率都处于Hardy-Weinderg平衡状态（P＞0.05）;而在HaeⅢ-RFLP和Msp Ⅰ-RFLP位点上,仅内江猪、荣昌猪、汉江黑猪和八眉猪为单态;（2）9种基因型对肌内脂肪（IMF）含量的影响,HH＞Hh＞hh,DD＜Dd＜dd,AA＜Aa＜aa,遗传效应值分别为：3.89,3.42,3.17,2.27,2.49,2.91,2.28,2.70,2.95,H-FABP基因可显著地提高IMF含量（P＜0.05）;（3）aaddHH型的脂肪细胞脂滴的形态,密度和含量与其他基因型细胞差异显著（P＜0.05）.结果提示：可通过提高＂aaddHH＂基因型的频率来增加IMF含量,达到改善猪肉质的目的.     

猪H-FABP基因多态性与肌内脂肪含量的关联分析

以山西白猪为实验材料,采用索氏脂肪抽提法对其背最长肌的肌内脂肪含量进行了测定。根据心脏脂肪酸结合蛋白(heart fatty acid-binding protein,H-FABP)基因的PCR-RFLP多态性,应用SPSS软件,用最小二乘法拟合一般线性模型(GLM)分析了H-FABP基因的不同位点的基因型对肌内脂肪含量的影响。结果表明,基因型hh、dd、BB对肌内脂肪含量影响的效应值最大。证明H-FABP基因可以作为肌内脂肪含量的侯选基因。     

A single nucleotide polymorphism in the porcine cathepsin K (<Emphasis Type="Italic">CTSK</Emphasis>) gene is associated with back fat thickness and production traits in Italian Duroc pigs

Cathepsin K (CTSK) was selected as a candidate gene for fat deposition in pigs because recently, in human and mouse, it was shown that this lysosomal proteinase is an obesity marker. A single nucleotide polymorphism (SNP) was identified in intron 4 of the porcine CTSK gene (g.15G>A; FM209043). Allele frequencies of this polymorphism were analysed in seven pig breeds. Radiation hybrid mapping confirmed the localization of CTSK to porcine chromosome 4, close to the FAT1 QTL region. Three populations of pigs (one Italian Large White and two Italian Duroc groups of pigs) were selected for association analysis. In the Italian Large White breed the g.15G>A SNP was not informative. Association analysis including all Italian Duroc pigs showed that the CTSK marker was associated with back fat thickness and lean cuts (P < 0.01), and average daily gain and feed:gain ratio (P < 0.05) estimated breeding values.     

猪L-FABP基因的克隆、表达特征及遗传多态性研究

FABPs属于脂结合蛋白超家族成员,是一类分子量较小而对脂肪酸有高亲和力的蛋白质,广泛存在于脊椎动物和非脊椎动物的细胞质中.FABPs担当细胞内脂肪酸的运输任务,它们与脂肪酸结合将其运输到脂肪酸氧化的位置、脂肪酸脂化成甘油三醋或磷脂的位置,或者进入细胞核内发挥其可能的调控功能.因此FABPs对脂类代谢具有重要的调控作用.本研究把L-FABP基因作为影响猪肌内脂肪含量的候选基因.为此,利用cDNA末端快速扩增(RACE)和PCR技术,克隆到猪肝脏型脂肪酸结合蛋白基因(L-FABP)的全长cDNA序列(GenBank登录号AY960623)和部分基因组序列(GenBank登录号DQ182323).猪L-FABP基因的cDNA序列全长518 bp,该序列包括起始密码子TGA和38 bp的5'末端非编码区(5'URT),终止密码子TAG和99 bp的3'末端非编码区(3'URT),在3'URT结构区域中包含polyA加尾信号序列AATAAA.猪L-FABP基因与其他FABPs基因一样,也由4个外显子(67 bp、173 bp、93 bp和51 bp)和3个内含子组成,内含子1和3的大小是1 679bp和565 bp,没有获得内含子2的序列,外显子和内含子剪接处符合GT/AG规律.应用Clustal W/X程序对猪L-FABP与其他物种的L-FABP进行多重序列比对,发现猪L-FABP与人、大鼠、鸡的L-FABP的相似性分别为89.8%、81.9%和72.4%.亲水性分析表明,猪L-FABP也是一个潜在的跨膜蛋白,在氨基酸残基57-65之间有一个明显的跨膜α螺旋.应用半定量RT-PCR分析发现,猪L-FABP在猪体组织中广泛存在,但在肝脏和小肠组织中表达量最为丰富.分析还发现,所克隆得到的编码区核苷酸序列与已知猪L-FABP基因的编码区核苷酸序列存在一定的变异,分别是外显子2中T→C(116位)、C→T(231位)、C→A(236位)和A→C(258位),演绎成氨基酸在Leu74Met存在差异.为进一步证实这些突变位点在猪群中真实存在,利用PCR-SSCP检测方法对4个猪种(藏猪、大河猪、雅南猪和约克夏)的157头个体的外显子2全序列进行SNP位点多态性片段的基因型分型,结果发现一个C→T的单核苷酸多态,等位基因频率在中国地方猪种(藏猪、大河猪、雅南猪)与国外约克夏猪种间存在极显著的差异(P＜0.01).连锁分析发现,基因型CC的肌内脂肪含量(4.86±0.22%)显著的高于基因型CT(4.16±0.23%)和TT(4.05±0.27%)的肌内脂肪含量(P＜0.05).因此,推测L-FABP基因可能是影响猪肌内脂肪含量的主效基因或与主效基因紧密连锁的标记基因,并且能够在分子标记辅助选择中用于对猪肌内脂肪含量的遗传改良.     

Functional and association studies on the pig HMGCR gene, a cholesterol-synthesis limiting enzyme

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme in the biosynthesis of cholesterol. We have studied the role of the HMGCR gene in pig lipid metabolism by means of expression and structural analysis. We describe here the complete coding region of this gene in pigs and report two synonymous single nucleotide polymorphisms in the coding region. We have, additionally, studied the association of one of these polymorphisms (HMGCR:c.807A>C) with several lipid deposition- and cholesterol-related traits in a half-sib population generated from a commercial Duroc line, showing in some families a positive relationship of HMGCR:c.807A allele with serum low-density lipoprotein (LDL)-bound cholesterol and triglyceride levels, and also with intramuscular fat (IMF) content of gluteus medius muscle. We have also assessed the expression levels in muscle and in liver from 68 Duroc individuals corresponding to the most extreme animals for the analysed traits. Liver HMGCR expression correlated negatively with the serum high-density lipoprotein (HDL) levels, carcass lean percentage and stearic acid content, while muscle expression correlated also negatively with the carcass lean percentage, stearic and linoleic acids content, but showed a positive correlation with the serum lipid cholesterol (HDL, LDL and total cholesterol), IMF and muscle oleic and palmitic fatty acid content. With this information, we have performed an association analysis of expression data with lipid metabolism phenotypic levels and the HMGCR genotype. The results indicate that HMGCR expression levels in muscle are different in the two groups of pigs with extreme values for fat deposition and total cholesterol levels, and also between animals with the different HMGCR genotypes.     

西部地区主要猪种和野猪H-FABP基因分子标记

利用PCR-RFLP(Hinf I、Hae Ⅲ和Msp I 3种限制性内切酶)分子标记技术,检测了杜洛克猪、长白猪、大白猪、内江猪、荣昌猪、汉江黑猪、汉中白猪、八眉猪和野猪共计265头猪H-FABP基因5′-上游区和第二内含子区的遗传变异,并利用最小二乘模型分析了H-FABP基因对猪肌内脂肪含量的遗传效应。结果表明：（1）在Hinf I-RFLP位点上,上述品种和野猪均存在多态性,其中大白猪、八眉猪、汉江黑猪、汉中白猪和野猪表现为低度多态,杜洛克、长白猪、内江猪和荣昌猪为中度多态;除汉江黑猪（P<0.05）和野猪（P<0.01）外,其他猪种基因频率和基因型频率都处于Hardy-Weinderg平衡状态（P>0.05）;而在Hae Ⅲ-RFLP和Msp I-RFLP位点上,仅内江猪、荣昌猪、汉江黑猪和八眉猪为单态;（2）9种基因型对肌内脂肪（IMF）含量的影响,HH>Hh>hh,DD相似文献   

H-FABP、MC4R、ADD1基因多态性在3个猪群中分布及其与肌内脂肪和背膘的相关研究

具有不同遗传特性的猪种具有不同的肉质性状。尤其是地方品种和引进品种间在肉质性状存在极大的差异。在已有的研究中H-FABP,MC4R,ADD1基因同肌内脂肪或背膘相关。利用梅山猪、苏太猪和杜×长×大猪为试验动物,研究上述3个基因的多态性分布和多态性同肌内脂肪和背膘的相关性。结果表明：3个基因的多态性分布在不同猪种间存在极显著的差异,这种差异可能是肌内脂肪（IMF）或背膘（BF）不同的主要原因之一。连锁分析表明：H-FABP和ADD1基因多态性同IMF有显著的相关,但是同BF没有显著的相关;MC4R基因的多态性同IMF和BF都有显著相关性。说明：H-FABP和ADD1基因多态性有可能应用到提高IMF,同时不影响BF的育种实践中。      

Evaluation of quantitative trait loci affecting intramuscular fat and reproductive traits in pigs using marker‐assisted introgression

We investigated the effects of previously identified quantitative trait loci (QTL) in an experimental backcross (BC) between Chinese Meishan pigs and commercial Duroc pigs. We performed marker‐assisted introgression of two QTL for intramuscular fat (IMF) content (IMF population) and three QTL for reproductive traits (reproduction population) from a donor Meishan pig into a recipient Duroc pig. At the fourth BC generation of the IMF population and third BC generation of the reproduction population, carrier animals were selected for the production of animals homozygous for the QTL. Our previous studies have shown that the presence of a Meishan allele on the IMF QTL is associated with low IMF values, and the Meishan allele on the reproductive QTL is associated with large litters. In this study, the presence of a Duroc allele at the IMF QTL on SSC9 resulted in a 0.27% increase in IMF (additive effect = 0.27 ± 0.08), whereas the presence of a Meishan allele at the IMF QTL on SSC7 resulted in a 0.34% increase in IMF (additive effect = ?0.34 ± 0.09). The presence of the Meishan allele at the IMF QTL on SSC7 thus had the opposite effect to our previous studies, that is, increased IMF. In the reproduction population, we observed no differences between the genotypes of the three QTL in regard to number of corpora lutea or litter size. Marker‐assisted introgression at these QTL is thus unlikely to result in an associated increase in litter size. These results show that it is possible to introgress alleles from other breeds into a selection population using molecular markers; any unexpected results might be associated with the genetic background.     

Relationship Among H-FABP Gene Polymorphism, Intramuscular Fat Content, and Adipocyte Lipid Droplet Content in Main Pig Breeds with Different Genotypes in Western China

H-FABP (Heart fatty acid-binding protein), a member of FABP family, plays an essential role in long-chain fatty acid uptake and metabolic homeostasis. Its role in pig intramuscular fat content remains poorly understood, especially in local pig breeds in western China. In this study, the genetic variations of 5′-upstream region and the second intron in porcine H-FABP gene were investigated by PCR-RFLP in 256 pigs including Duroc, Large White, Landrace, Neijiang, Rongchang, Bamei pig, Hanjiang Black, Hanzhong White, and the wild ones. The effect of H-FABP gene on the IMF content was analyzed by the least square method. Lipid droplet morphology and content in adipocytes cultured from pigs with different H-FABP genotypes, were studied by oil red O staining and a triglyceride assay kit. Results showed a Hinf I -RFLP in these eight pig breeds and wild pigs, among which Large white, Bamei pig, Hanjiang Black, Hanzhong White, and wild pigs presented with low polymorphism while the other breeds had intermediate polymorphism. There was no Hae III or Msp I -RFLPs in the four Chinese local pig breeds tested, but Duroc, Landrace, Large White, Hanzhong White and wild pig had polymorphism. Landrace, Large White and wild pigs had low levels of Hae III-and Msp I -RFLP, whereas others had intermediate polymorphism. H-FABP genotypes significantly affected the IMF content (P<0.05). The IMF content ordered by H-FABP genotypes were HH>Hh>hh, DD<Dd<dd, and AA<Aa<aa. The genetic effect values were 3.89, 3.42, 3.17, 2.27, 2.49, 2.91, 2.28, 2.70, and 2.95, respectively. Fat deposition in adipocytes was stronger in the HH, dd and aa genotypes than in others. The results suggest that porcine meat quality may be improved by increasing the frequency of genotype aa-dd-HH in pig breeds.     

Cloning and characterization of chicken adipocyte fatty acid binding protein gene

Fatty acid binding proteins (FABPs) are members of a superfamily of lipid-binding proteins and occur intracellularly in vertebrates and invertebrates. This study was designed to clone and characterize the adipocyte fatty acid binding protein (A-FABP) gene in the chicken. PCR primers were designed according to mammalian A-FABP gene sequence to amplify partial cDNA of A-FABP gene from chicken adipose tissues, and the full length of the gene was cloned by 5'RACE and 3'RACE. Analysis of sequence showed that the cDNA of the chicken A-FABP gene was 74 and 73% homologous with porcine and human A-FABP gene, respectively. The similarity was 77, 28, and 23% at the predicted amino acid level with human A-FABP, human L-FABP, and human I-FABP, respectively. RT-PCR and Northern blot analysis indicated that the chicken A-FABP gene, similar to that of the mammal, is only expressed in fat tissues. This is the first report to identify and characterize A-FABP gene in the chicken.     

Genome-wide identification of quantitative trait loci for carcass composition and meat quality in a large-scale White Duroc × Chinese Erhualian resource population

Carcass and meat quality traits are economically important in pigs. In this study, 17 carcass composition traits and 23 meat quality traits were recorded in 1028 F₂ animals from a White Duroc × Erhualian resource population. All pigs in this experimental population were genotyped for 194 informative markers covering the entire porcine genome. Seventy-seven genome-wide significant quantitative trait loci (QTL) for carcass traits and 68 for meat quality were mapped to 34 genomic regions. These results not only confirmed many previously reported QTL but also revealed novel regions associated with the measured traits. For carcass traits, the most prominent QTL was identified for carcass length and head weight at 57 cM on SSC7, which explained up to 50% of the phenotypic variance and had a 95% confidence interval of only 3 cM. Moreover, QTL for kidney and spleen weight and lengths of cervical vertebrae were reported for the first time in pigs. For meat quality traits, two significant QTL on SSC5 and X were identified for both intramuscular fat content and marbling score in the longissimus muscle, while three significant QTL on SSC1 and SSC9 were found exclusively for IMF. Both LM and the semimembranous muscle showed common QTL for colour score on SSC4, 5, 7, 8, 13 and X and discordant QTL on other chromosomes. White Duroc alleles at a majority of QTL detected were favourable for carcass composition, while favourable QTL alleles for meat quality originated from both White Duroc and Erhualian.