The bovine fatty acid binding protein 4 gene is significantly associated with marbling and subcutaneous fat depth in Wagyu x Limousin F2 crosses

Fatty acid binding protein 4 (FABP4), which is expressed in adipose tissue, interacts with peroxisome proliferator-activated receptors and binds to hormone-sensitive lipase and therefore, plays an important role in lipid metabolism and homeostasis in adipocytes. The objective of this study was to investigate associations of the bovine FABP4 gene with fat deposition. Both cDNA and genomic DNA sequences of the bovine gene were retrieved from the public databases and aligned to determine its genomic organization. Primers targeting two regions of the FABP4 gene were designed: from nucleotides 5433-6106 and from nucleotides 7417-7868 (AAFC01136716). Direct sequencing of polymerase chain reaction (PCR) products on two DNA pools from high- and low-marbling animals revealed two single nucleotide polymorphisms (SNPs): AAFC01136716.1:g.7516G>C and g.7713G>C. The former SNP, detected by PCR-restriction fragment length polymorphism using restriction enzyme MspA1I, was genotyped on 246 F2 animals in a Waygu x Limousin F2 reference population. Statistical analysis showed that the FABP4 genotype significantly affected marbling score (P = 0.0398) and subcutaneous fat depth (P = 0.0246). The FABP4 gene falls into a suggestive/significant quantitative trait loci interval for beef marbling that was previously reported on bovine chromosome 14 in three other populations.     

Screening of indigenous goats for prolificacy associated DNA markers of sheep

The present study was undertaken to explore the genetic basis of caprine prolificacy and to screen indigenous goats for prolificacy associated markers of sheep in BMPR1B, GDF9 and BMP15 genes. To detect the associated mutations and identify novel allelic variants in the candidate genes, representative samples were collected from the breeding tract of indigenous goat breeds varying in prolificacy and geographic distribution. DNA was extracted and PCR amplification was done using primers designed or available in literature for the coding DNA sequence of candidate genes. Direct sequencing was done to identify the genetic variations. Mutations in the candidate genes associated with fecundity in sheep were not detected in Indian goats. Three non-synonymous SNPs (C818T, A959C and G1189A) were identified in exon 2 of GDF9 gene out of which mutation A959C has been associated with prolificacy in exotic goats. Two novel SNPs (G735A and C808G) were observed in exon 2 of BMP15 gene.     

Nucleotide variability and linkage disequilibrium patterns at the porcine FABP5 gene

Fatty acid binding protein 5 (FABP5) is a major positional and physiological candidate gene for the porcine FAT1 QTL on SSC4. Here we characterize the nucleotide polymorphism and haplotype variability of FABP5 and we compare it with that of FABP4, given their close physical location and similar metabolic roles. DNA resequencing of the FABP5 gene region in 29 pigs from 14 breeds and in European and Japanese wild boars revealed 36 polymorphisms in 5.2 kb, and a nucleotide diversity of 0.19%, comparable to values reported in other domestic species but sixfold lower than that previously found for FABP4. Remarkably, both the nucleotide variability and the haplotype structure of FABP5 and FABP4 were dramatically different, and the Hudson-Kreitman-Aguadé test was highly significant. Nevertheless, both genes also had similarities. The neighbour-joining trees of their haplotypes did not show a geographical arrangement for any of the genes. Besides, both genes presented a similar extent and pattern of linkage disequilibrium. Haplotype blocks did not extend for large stretches ( approximately 1 kb in both genes), and the number of tag SNPs required to capture all variability was higher than previously expected. Our findings indicate that FABP4 and FABP5 have undergone different selective or evolutive processes. The fact that haplotype blocks were so small may require us to increase the number of SNPs in prospective whole-genome association studies in the pig.     

Haplotyping using a combination of polymerase chain reaction–single-strand conformational polymorphism analysis and haplotype-specific PCR amplification

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR–SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.     

Association of the heart fatty acid-binding protein (FABP3) gene with milk traits in Manchega breed sheep

The ovine fatty acid-binding protein type 3 gene has been chosen as a functional candidate gene for milk traits. Two different single nucleotide polymorphisms (SNPs) of ovine FABP3 gene have been tested in a daughter design comprising 13 families. No association was found between estimated breeding values for milk yield, protein and fat contents (FC) and genotypes across families using anova and transmission disequilibrium test (TDT). In within-family analysis, one family showed a significant effect for FC. These results could indicate linkage disequilibrium between the FABP3 gene and a quantitative trait loci (QTL) for FC, with the heterozygous genotype associated with a positive effect in this trait.     

Polymorphisms of coding region of BMPR-IB gene and their relationship with litter size in sheep

The bone morphogenetic protein receptor IB (BMPR-IB) was studied as a candidate gene for the prolificacy of sheep. Nine pairs of primers (P1-P9) were designed to detect single nucleotide polymorphisms (SNPs) of exons 1-4 and 6-10 of the BMPR-IB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Texel and Chinese Merino sheep) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Only the products amplified by primers P2, P5, P6, P7, P8 and P9 displayed polymorphisms. The present study identified 22 SNPs in partial coding regions of ovine BMPR-IB, in which 20 SNPs were reported for the first time. In total of the 22 mutations, 18 DNA variations were originated from the Hu breed, three were found in the Small Tail Han breed (two of them were found in other sheep breeds), three in the Chinese Merino breed, and none in the Texel breed. These results preliminarily demonstrated that BMPR-IB is a major gene affecting the hyperprolificacy in Small Tail Han and Hu sheep, and could be used as a molecular genetic marker for early auxiliary selection for hyperprolificacy in sheep.     

Identification of genetic polymorphisms in FABP3 and FABP4 and putative association with back fat thickness in Korean native cattle

The aim of this study was to determine whether single nucleotide polymorphisms (SNP) in the beef cattle adipocyte fatty-acid binding protein 3 and 4 (FABP3 and FABP4) genes are associated with carcass weight (CW) and back fat thickness (BF) of beef cattle. By direct DNA sequencing in 24 unrelated Korean native cattle, we identified 20 SNPs in FABP3 and FABP4. Among them, 10 polymorphic sites were selected for genotyping in our beef cattle. We performed SNP, haplotype and linkage disequilibrium studies on 419 Korean native cattle with the 10 SNPs in the FABP genes. Statistical analysis revealed that 220AG (I74V) and 348+303TC polymorphisms in FABP4 showed putative associations with BF traits (P=0.02 and 0.01, respectively). Our findings suggest that the polymorphisms in FABP4 may play a role in determining one of the important genetic factors that influence BF in beef cattle.     

Silencing of FABP3 Inhibits Proliferation and Promotes Apoptosis in Embryonic Carcinoma Cells

Fatty acid–binding protein 3 (FABP3) facilitates the movement of fatty acids in cardiac muscle. Previously, we reported that FABP3 is highly upregulated in the myocardium of ventricular septal defect patients and overexpression of FABP3 inhibited proliferation and promoted apoptosis in embryonic carcinoma cells (P19 cells). In this study, we aimed to investigate the effect of FABP3 gene silencing on P19 cell differentiation, proliferation and apoptosis. We used RNA interference and a lentiviral-based vector system to create a stable FABP3-silenced P19 cell line; knockdown of FABP3 was confirmed by quantitative real-time PCR. Expression analysis of specific differentiation marker genes using quantitative real-time PCR and observation of morphological changes using an inverted microscope revealed that knockdown of FABP3 did not significantly affect the differentiation of P19 cells into cardiomyocytes. CCK-8 proliferation assays and cell cycle analysis demonstrated that FABP3 gene silencing significantly inhibited P19 cell proliferation. Furthermore, Annexin V-FITC/propidium iodide staining and the caspase-3 activity assay revealed that FABP3 gene silencing significantly promoted serum starvation–induced apoptosis in P19 cells. In agreement with our previous research, these results demonstrate that FABP3 may play an important role during embryonic heart development, and that either overexpression or silencing of FABP3 will lead to an imbalance between proliferation and apoptosis, which may result in embryonic cardiac malformations.     

FABP4基因在阿勒泰羊尾脂沉积与代谢模型中的表达变化规律

FABP4(Fatty acid binding protein 4)参与细胞内脂肪酸的转运和脂肪酸代谢,是当前研究动物脂肪沉积与代谢的热门候选基因。为了研究FABP4基因在绵羊尾脂沉积与代谢中的作用,文章采用生物信息学方法分析了FABP4氨基酸序列在各物种中的保守性;利用半定量RT-PCR方法检测了该基因在阿勒泰羊主要组织中的表达;采用饥饿法成功建立了模拟阿勒泰羊尾脂沉积与代谢的动物模型,利用qPCR和iTRAQ(isobaric tags for relative and absolute quantitation)技术同时验证FABP4基因mRNA和蛋白在阿勒泰羊非饥饿组与饥饿组尾脂中的表达变化。序列分析结果表明,绵羊FABP4氨基酸序列在物种间高度保守,提示FABP4基因可能由于其重要的生物功能而在进化中表现出其物种的保守性。组织表达谱结果显示,FABP4 mRNA在阿勒泰羊肠脂与尾脂中均高丰度表达,暗示FABP4基因可能在脂肪中行驶着重要的生理生化功能。qPCR与iTRAQ结果显示,FABP4 mRNA与蛋白在两种极端条件下尾脂中的表达差异均不显著(P>0.05),表明FABP4基因可能不是绵羊尾脂沉积与代谢两种极端差异表型的决定基因。以上研究结果为进一步研究FABP4基因在绵羊尾脂中的生物功能奠定了基础。     

Amelogenin cross-amplification in the family Bovidae and its application for sex determination

Sex-specific sequence variability of the amelogenin gene had been observed in a variety of mammalian species. In our study, the suitability of the amelogenin gene for sex determination in different species of the family Bovidae was examined. Based on a sequence insertion/deletion characteristic for X- and Y-specific amelogenin (AMELX and AMELY), PCR amplification on male and female genomic DNA from domestic and wild bovine species, sheep and goat, consistently displayed a sex-specific pattern. Thus, the amelogenin amplification by PCR proved to be a reliable method for sex determination not only in domestic and wild species of the tribe Bovini, but also in the related species sheep and goat. Sex determination using the amelogenin-based assay can be performed with at least 40 pg of genomic DNA. The assay enables the investigation of small amounts of DNA from meat, hair, bones, and embryo biopsies to identify species and sex for a number of applications in animal production, forensics, population research, and monitoring within the family Bovidae. Sequence comparison of the amplified amelogenin gene region specific for male and female animals from domestic and wild bovide species revealed further sequence variations within and between sexes as well as between species. Sequence variations in the AMELX gene can be applied to discriminate Bos and Bison individuals from other bovine species, and also from sheep and goat.     

Expression profiling of candidate genes for abdominal fat mass in domestic chicken Gallus gallus

The quantitative traits of mass and percentage of abdominal fat in chicken and various types of obesity in mammals are homologous and functionally similar. Therefore, the genes involved in obesity development in humans and laboratory rodents as well as those responsible for pig lard thickness could be involved in abdominal fat deposition in broilers. Expression of candidate genes FABP1, FABP2, FABP3, HMGA1, MC4R, PPARG, PPARGC1A, POMC and PTPN1 was studied in fat, liver, colon, muscle, hypophysis, and brain in chicken (broilers) using real-time PCR. Significant difference in the HMGA1 gene expression in the liver of broiler chicken with high (3.5 +/- 0.18%) and low (1.9 +/- 0.56%) abdominal fat concentration has been revealed. The expression of this gene was been shown to correlate with the amount (0.7, P < or = 0.01) and mass (0.7, P < or = 0.01) of abdominal fat. The PPARG gene expression in liver in the same chicken subsets was also significantly different. Correlation coefficients of the gene expression with the abdominal fat amount and mass were respectively 0.55 (P < or = 0.05) and 0.57 (P < or = 0.01). Based on these results, we suggest that the HMGA1 and PPARG genes are involved in abdominal fat deposition. The search for single nucleotide polymorphisms (SNPs) in the HMGA and PPARG regulatory regions could facilitate identifying genetic markers for broiler breeding according to the mass and percentage of abdominal fat.     

血管内皮生长因子基因3＇UTR不同基因型载体的构建与鉴定

目的：构建含SNP位点的血管内皮生长因子（VEGF）基因3＇UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3＇UTR的单核苷酸多态性（SNP）影响肺癌发病风险的分子机制奠定基础。方法：以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3＇UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果：单菌落质粒测序验证显示带有C/C单体型的VEGF基因3＇UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论：本研究成功构建了含有两个连锁SNP的VEGF基因3＇UTR的荧光素酶报告基因载体,为今后VEGF基因3＇UTR的功能研究奠定基础。     

血管内皮生长因子基因3'UTR 不同基因型载体的构建与鉴定

目的:构建含SNP位点的血管内皮生长因子(VEGF)基因3’UTR的荧光素酶报告基因载体,为进一步揭示VEGF基因3’UTR的单核苷酸多态性(SNP)影响肺癌发病风险的分子机制奠定基础。方法:以rs3025039和rs3025040两个位点均为C纯合子的非癌症病人血液DNA为模板,扩增出两位点为C/C单体型、长度为1448 bp的VEGF基因3’UTR目的片段,测序验证后将其克隆至pMIR-REPORT荧光素酶报告基因载体上,得到重组质粒pMIR-C/C。同时,我们以pMIR-C/C为模板定点突变两个SNP位点,得到具有T/T单体型的重组质粒pMIR-T/T。将各重组质粒转化大肠杆菌DH10B,筛选阳性克隆后提取质粒进行双酶切鉴定及DNA测序鉴定。结果:单菌落质粒测序验证显示带有C/C单体型的VEGF基因3’UTR重组质粒pMIR-C/C构建成功;经两次定点突变,成功将pMIR-C/C质粒转变为pMIR-T/T,经测序验证未引入任何其他突变。同时生物信息学预测还显示rs3025040位点位于miR-199a/b与VEGF基因mRNA的结合位置,其改变可以影响miRNA与mRNA的结合效率。结论:本研究成功构建了含有两个连锁SNP的VEGF基因3’UTR的荧光素酶报告基因载体,为今后VEGF基因3’UTR的功能研究奠定基础。     

SNP detection and haplotype analysis in partial sequence of MSTN gene in sheep

Polymerase chain reaction (PCR) products of the MSTN gene amplified from sixty sheep of nine Chinese indigenous sheep breeds and one imported sheep breed were sequenced to identify the single-nucleotide polymorphisms (SNPs) in a 378-bp fragment including intron 2 and exon 3 of the MSTN gene. A total of fifteen SNPs (A1937C, T1942G, C1956T, A1972C, A1990G, A2008C, A2011G, C2019T, A2025C, A2027C, T2085G, T2173C, C2198T, C2210T and C2213T) were detected among the sixty sequenced individuals and they were all located in intron 2. Twelve haplotypes were identified from these fifteen SNPs, of which haplotype I (CGTCGCGTCCGCTTT) and VIII (ATCAAAACAATTCCC) were the two major and basic ones with frequencies of 12.25% and 77.80%, respectively. Haplotype VIII was distributed in all sheep breeds and all individuals of the meat or meat-wool type sheep breeds were homozygous with respect to this haplotype. This suggests that haplotype VIII might be related to meat production traits in sheep. Haplotype I was only distributed in the fur, lambskin type and fur-meat type sheep breeds. This suggests that haplotype I may have some relationship with fur traits in sheep.     

Cloning and characterization of a cytolytic and mosquito larvicidal delta-endotoxin from Bacillus thuringiensis subsp. darmstadiensis

The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml).     

甘肃现代肉羊新品种群羊GH基因外显子1多态性与体重关联分析

根据GenBank发表的绵羊生长激素(GH)基因外显子1的序列设计一对引物,采用PCR-SSCP技术分析GH基因外显子1在甘肃现代肉羊新品种选育群羊中的单核苷酸多态性,并与3月龄前的体重进行关联分析。结果表明,GH基因外显子1在新品种群羊中存在多态性,检测到两种基因型(AA、AB),其301bp处有一个T/A突变和305bp处有一个G/A突变,初生重、1月、2月、3月龄体重AA、AB型都无显著性差异(P>0.05),但3月龄AB型个体的体重相对于AA型偏高,由此初步推断GH基因可能是影响绵羊体重性状的主基因或与主基因相连锁,可用以对绵羊体重性状进行标记辅助选择。     

Including endophenotypes as covariates in variance component heritability and linkage analysis

Background

Simple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous.

Results

A novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus ) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified.

Conclusion

PCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.     

甘肃肉羊新品种选育群GDF9基因外显子2多态性及其与产羔性状关联分析

根据GenBank发布的绵羊GDF9基因外显子2的序列设计4对引物,采用PCR-SSCP技术分析GDF9基因外显子2在甘肃内羊新品种选育群羊中的单核苷酸多态性,并与产羔性状进行关联分析.结果表明,GDF9基因的扩增片段在所检测的新品种群羊中存在PCR-SSCP多态性,检测到3种基因型(AA、AB和BB),而在32只无角陶赛特母羊群中只检测到AA和AB基因型.测序结果显示,GDF9基因编码区第978位碱基发生A→G突变,但没有导致氨基酸的改变;第994位碱基发生G→A突变,导致Ⅴ变成Ⅰ(缬氨酸→异亮氨酸).新品种选育群羊产羔数的最小二乘均值关系为AB＞ AA＞ BB,统计分析结果初步表明3种基因型之间差异不显著(P＞0.05).故该区域可能不是影响新品种群羊繁殖力的功能结构区城.