Up-regulation of orphan nuclear estrogen-related receptor alpha expression during long-term caloric restriction in mice

The estrogen-related receptor alpha (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily that regulates a number of target genes encoding enzymes that participate in various metabolic pathways involved in maintaining energy balance in animals. In this study, whether long-term caloric restriction (alternate days of fasting for 3 months) in mice modulates the expression of ERRα in various tissues was investigated. Western blot analyses showed positive immunoreactive ERRα protein (53 kDa) band in various mice tissue extracts, though at varying levels. Heart, kidney, and skeletal muscles expressed significant levels of ERRα, with a comparatively lower level detected in the intestine, brain, and liver. Cardiac ERRα expression was the highest, with the least detected in the liver. Caloric restricted mice exhibited a significant increase in ERRα level in the heart (5.45-fold), kidney (3.70-fold), skeletal muscle (3.0-fold), small intestine (2.72-fold), and liver (2.44-fold) extracts as compared to ad libitum fed. However, caloric restriction could not evoke any detectable receptor level change in the brain. Notably, the highest ERRα up-regulation was detected in the heart. This up-regulation in ERRα level especially in highly oxidative tissues such as heart, kidney, small intestine, and skeletal muscle of caloric restricted mice may be helpful in modulating ERRα responsive genes that participates in maintaining energy balance. This may potentially strengthen the metabolic and biochemical adaptation in such tissues, which is necessary for animal survival under long-term caloric restriction.     

建鲤肠型脂肪酸结合蛋白基因的分离及其SNPs与增重的相关分析

文章采用PCR方法从建鲤 (Cyprinus carpio var. jian) 基因组分离到2个肠型脂肪酸结合蛋白基因 (Fatty acid binding protein 2, FABP2), ORF长度均为399 bp, 相似度为92.2%, 分别记为jlFABP2a、jlFABP2b, 和斑马鱼 (Danio rerio) FABP2 ORF的相似度分别为88.0%和90.5%。jlFABP2s基因结构与FABPs家族其他成员一致, 由4个外显子和3个内含子组成, 2a和2b间内含子序列和长度差异明显。系统树显示这2个基因对应斑马鱼的1个FABP2基因, 和鲤鱼染色体数是斑马鱼的2倍一致。实时荧光定量PCR结果显示, jlFABP2a、2b在建鲤肠中的表达量极显著高于脑、肝脏、肌肉、肾脏、心脏、性腺等其他组织 (P<0.01), 且2a表达量显著 (雄鱼, P<0.05) 或极显著 (雌鱼, P<0.01) 高于2b, 但在其他组织则2b表达量稍高, 暗示2a为肠特异性表达, 2b则为广谱表达。通过比对8尾建鲤的2a和2b基因序列, 在2a和2b上分别找到12个和4个SNP, 均位于内含子上。使用PCR-RFLP 法检测jlFABP2a 上4个SNP 位点I1-A15G、I1-A99G、I2-C487T和I3-A27T在建鲤选育群体中的基因型分布, 并进行了基因型与个体增重的关联分析, 结果表明, 4个位点与雌、雄成鱼阶段增重分别有极显著或显著相关。同时考虑4个位点的基因型与增重的关系, 结果基因型AGGGCCXX 和AGGGXXAT的个体平均增重比其他个体快15%, 这两种基因型个体在选育群中占了9%, 具有较大的选育空间, 可用于建鲤分子育种计划中。     

军曹鱼催乳素受体PRLR1基因的克隆及其在不同盐度条件下mRNA表达差异

催乳素受体通过结合催乳素,能调节鱼体渗透压。为研究催乳素受体1(PRLR1)在高盐水体和低盐水体中对军曹鱼(Rachycentron canadum)的渗透调节作用,利用cDNA末端快速扩增(RACE-PCR)技术,获得了军曹鱼PRLR1全长cDNA序列。该基因全长为2629 bp,包含1953 bp的开放阅读框ORF,可编码650个氨基酸。氨基酸序列包含了2个纤维连接蛋白3型结构域(FN3)、保守的WS区和box1。采用qRT-PCR技术,检测不同盐度(10‰、30‰和35‰)条件下鳃、肠、体肾中PRLR1基因mRNA表达情况。结果显示,PRLR1基因在军曹鱼的各个组织中均有表达,其中鳃表达量最高,其次是肌肉、体肾和肠,而在胃、脾、脑和心脏中则微量表达。低盐组、正常组和高盐组中,PRLR1基因的表达量均为鳃最高;肠次之;体肾最低。随着盐度提高,PRLR1基因的鳃、肠和体肾组织表达量变化规律均呈逐步下降趋势。以上结果反映了军曹鱼PRLR1在渗透压器官中的功能差异性,说明PRLR1在军曹鱼渗透压调节上具有重要作用。     

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。     

Rabbit oviductal glycoprotein 1 gene: genomic organization polymorphism analysis and mRNA expression

The OVGP1 is an oviductal glycoprotein that has positive effects on fertilization and early embryo development. We have amplified and sequenced the rabbit OVGP1 gene, which spans 13 kb and it is formed by 11 exons and 10 introns. To find polymorphisms, a region encompassing the promoter to intron 1 has been sequenced in 22 rabbits of the H, V, R, and A Spanish lines. We have identified five SNPs and one triallelic microsatellite in the promoter region, and three SNPs and one dinucleotide INDEL in intron 1. The 10 polymorphic sites cosegregate forming two haplotypes. The allelic frequencies of the microsatellite have been analyzed in 98 rabbits belonging to the four lines and the three alleles were found in all the lines. The relative quantification of the OVGP1 mRNA in liver, kidneys, lungs, skeletal muscle, ovary, uterus, and oviduct reveals that the OVGP1 expression in the oviduct is 5,500 higher than in the uterus or ovary, whereas a low level of basal expression is detected in non-reproductive tissues. We have also compared the mRNA expression between samples of oviducts taken from non-mated rabbit and samples of oviducts at different stages of the early pregnancy, but no significant differences were found.     

Expression of the bovine KLF6 gene polymorphisms and their association with carcass and body measures in Qinchuan cattle (Bos Taurus)

Kruppel-like factor 6 (KLF6) genes plays a significant role in the regulation of cell differentiation, proliferation and muscle development. The aim of this study is to investigate the genetic variation and the haplotype combination of the KLF6 gene in Qinchuan cattle and verify its contribution to bovine carcass traits and body measurements. The data were analyzed by real-time quantitative PCR (qPCR) to detect the expression profile of the KLF6 gene in the various tissues of Qinchuan cattle. PCR amplicons sequencing explored three novel SNPs at loci 3332C > G; 3413C > T and 3521G > A in the 2nd exon region of the KLF6 gene. The expression of KLF6 in the liver, kidney and lung was greater than that of other tissues. Allelic and genotypic frequencies of these SNPs were found to be in Hardy Weinberg equilibrium (P < 0.05). In SNP1, genotype CC, in SNP2, genotype CT and in SNP3 genotype GG were associated (P < 0.05) with larger body and carcass measurements. Association analysis results indicated that individuals with the Hap1/4 diplotype had a longer body and rump, were taller at the withers, and were wider at the hip than the other combinations. In terms of ultrasound carcass measures, Hap1/4 was associated with a larger muscle area and more intramuscular fat than other combinations. The bioinformatics study of the KLF6 protein showed a high degree of conservation in different mammalian species. The above results suggest that the KLF6 gene can used as potential candidate markers gene for the beef breed improvement through marker assisted selection of Qinchuan cattle.     

Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues

Two lactose-binding lectins from chicken tissues, chicken-lactose- lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL-11) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed not significant immunological cross-reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-1 contained only traces of CLL-11 whereas embryonic kidney, a very rich source of CLL-11 contained substantial CLL-1. In both muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-1 in liver and CLL-11 in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-1 and CLL-11 from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found.     

Cell type-specific expression of beta-carotene 9',10'-monooxygenase in human tissues.

The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.     

Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.     

京海黄鸡体重性状全基因组关联分析

体重性状是肉鸡重要的经济性状。为了寻找可用于京海黄鸡体重性状遗传改良的分子标记及候选基因,本文以400只京海黄鸡核心群母鸡为基础,测定了0~14周龄体重,利用简化基因组测序技术(Specific-locus amplified fragment sequencing, SLAF-seq)对京海黄鸡体重性状进行全基因组关联研究(Genome-wide association stndy, GWAS),筛选与京海黄鸡体重性状相关的SNPs位点。结果共检测到100个与京海黄鸡体重相关的SNPs位点,其中15个位点效应达到全基因组显著水平(P<1.87E-06),85个位点效应达到全基因组潜在显著水平(P<3.73E-05)。通过筛选每个显著SNP周围1 Mb区域内的基因,共找到9个可能的候选基因,其中FAM124A(Family with sequence similarity 124A)、QDPR(Quinoid dihydropteridine reductase)、WDR1(WD repeat domain 1)和SLC2A9(Solute carrier family 2 (facilitated glucose transporter), member 9) 4个基因可能是影响体重性状的重要候选基因。同时还发现,4号染色体75.6~80.7 Mb区域集中了大部分与京海黄鸡中后期体重性状显著相关的SNPs位点,该区域可能是影响京海黄鸡中后期生长体重的重要候选区域。     

SNP and INDEL detection in a QTL region on chicken chromosome 2 associated with muscle deposition

Genetic improvement is important for the poultry industry, contributing to increased efficiency of meat production and quality. Because breast muscle is the most valuable part of the chicken carcass, knowledge of polymorphisms influencing this trait can help breeding programs. Therefore, the complete genome of 18 chickens from two different experimental lines (broiler and layer) from EMBRAPA was sequenced, and SNPs and INDELs were detected in a QTL region for breast muscle deposition on chicken chromosome 2 between microsatellite markers MCW0185 and MCW0264 (105 849–112 649 kb). Initially, 94 674 unique SNPs and 10 448 unique INDELs were identified in the target region. After quality filtration, 77% of the SNPs (85 765) and 60% of the INDELs (7828) were retained. The studied region contains 66 genes, and functional annotation of the filtered variants identified 517 SNPs and three INDELs in exonic regions. Of these, 357 SNPs were classified as synonymous, 153 as non‐synonymous, three as stopgain, four INDELs as frameshift and three INDELs as non‐frameshift. These exonic mutations were identified in 37 of the 66 genes from the target region, three of which are related to muscle development (DTNA, RB1CC1 and MOS). Fifteen non‐tolerated SNPs were detected in several genes (MEP1B, PRKDC, NSMAF, TRAPPC8, SDR16C5, CHD7, ST18 and RB1CC1). These loss‐of‐function and exonic variants present in genes related to muscle development can be considered candidate variants for further studies in chickens. Further association studies should be performed with these candidate mutations as should validation in commercial populations to allow a better explanation of QTL effects.     

Genome-Wide Association Study Identifies Loci and Candidate Genes for Body Composition and Meat Quality Traits in Beijing-You Chickens

Body composition and meat quality traits are important economic traits of chickens. The development of high-throughput genotyping platforms and relevant statistical methods have enabled genome-wide association studies in chickens. In order to identify molecular markers and candidate genes associated with body composition and meat quality traits, genome-wide association studies were conducted using the Illumina 60 K SNP Beadchip to genotype 724 Beijing-You chickens. For each bird, a total of 16 traits were measured, including carcass weight (CW), eviscerated weight (EW), dressing percentage, breast muscle weight (BrW) and percentage (BrP), thigh muscle weight and percentage, abdominal fat weight and percentage, dry matter and intramuscular fat contents of breast and thigh muscle, ultimate pH, and shear force of the pectoralis major muscle at 100 d of age. The SNPs that were significantly associated with the phenotypic traits were identified using both simple (GLM) and compressed mixed linear (MLM) models. For nine of ten body composition traits studied, SNPs showing genome wide significance (P<2.59E−6) have been identified. A consistent region on chicken (Gallus gallus) chromosome 4 (GGA4), including seven significant SNPs and four candidate genes (LCORL, LAP3, LDB2, TAPT1), were found to be associated with CW and EW. Another 0.65 Mb region on GGA3 for BrW and BrP was identified. After measuring the mRNA content in beast muscle for five genes located in this region, the changes in GJA1 expression were found to be consistent with that of breast muscle weight across development. It is highly possible that GJA1 is a functional gene for breast muscle development in chickens. For meat quality traits, several SNPs reaching suggestive association were identified and possible candidate genes with their functions were discussed.