猪TLR4基因2个突变位点的PCR-SSCP分析

为了研究Toll样受体4(TLR4)的多态性与动物对相关病原的免疫力的相关性,试验选择北京黑猪、长白猪、野猪、杜洛克猪、大白猪、民猪6个中外猪种,利用PCR-SSCP和克隆测序方法对TLR4基因第3外显子1 824位点的G/A和3’UTR 208位点的T/C多态进行群体遗传学分析。结果表明:1 824位点北京黑猪与野猪、民猪、杜洛克猪、长白猪和大白猪的基因型分布存在显著差异(P<0.05);3’UTR 208位点基因型分布在猪种间存在极显著差异(P<0.01)。     

猪TLR4基因SNP位点筛选及生物信息学分析

通过构建基因组DNA混合池对猪TLR4基因进行扩增和测序,并对其编码区的SNP位点进行筛选,运用生物信息学分析软件对猪TLR4基因的理化特性和编码蛋白质的二、三级结构进行预测。结果发现,在扩增的TLR4基因上没有发现SNP位点,猪TLR4基因片段一共编码118个氨基酸,其中缬氨酸(Val)最多,甲硫氨酸(Met)最少,编码产物不稳定指数大于40,说明编码产物具有不稳定性。编码蛋白质片段的亲水性平均值为0.385,说明该基因编码的蛋白质呈现疏水性,猪TLR4基因编码的蛋白质是一种不溶性蛋白质。研究结果为猪TLR4基因深入研究提供理论基础。     

猪TLR4基因的变异位点分析

Toll样受体4(TLR4)主要识别革兰氏阴性菌表面的脂多糖,其基因变异可能改变宿主对脂多糖(LPS)的免疫反应.研究利用克隆、测序的方法在TLR4基因的外显子3寻找变异位点,并利用PCR-SSCP方法对寻找到的部分错义突变进行群体遗传学分析,确定不同基因型在猪种间的分布规律.结果表明:共寻找到11个点突变,其中8个是...     

不同鸡种TLR1和TLR2基因SNP检测分析

本研究利用DNA直接测序技术对9个地方鸡种(北京油鸡、白耳鸡、溧阳鸡、河南斗鸡、泰和乌骨鸡、大骨鸡、狼山鸡、仙居鸡、茶花鸡)和1个引进品种(白来航蛋鸡)鸡Toll样受体1(chTLR1-1、chTLR1-2)、Toll样受体2(chTLR2-1、chTLR2-2)基因的全部外显子、部分内含子及5′调控区的序列进行单核苷酸多态性(SNPs)检测。结果共发现了27个SNP位点,96%的SNPs位于编码区,其中33%的SNPs为错义突变。chTLR2-1基因在所有品种中均未发现SNP位点,chTLR1-1、chTLR1-2和chTLR2-2基因在9个地方品种中突变位点较为丰富,且不同品种间SNP数量差别较大,但在白来航蛋鸡中均未发现SNPs位点。本试验为进一步开展地方鸡种chTLR1和chTLR2基因多态性与抗病研究提供了理论依据。     

猪TLR3基因变异位点分析

为了研究TLR3基因突变对介导机体天然免疫和获得性免疫能力的影响及各等位基因在猪群体间的分布规律,试验采用PCR产物克隆测序的方法在TLR3基因外显子3上寻找多态位点,并利用PCR-SSCP和PCR-RFLP方法对选定的突变位点进行群体遗传学分析.结果表明:在所检测出的8个SNPs中,有A1116T和C1647T 2个...     

部分小型猪与大型猪IGF-1基因的SNP分析

利用PCR-SSCP技术对版纳微型猪、西藏小型猪、军牧猪和大白猪4个品种的IGF-1基因进行了多态性分析.结果表明,在外显子3上存在1个SNP(G201A),在外显子4上存在2个SNPs(A440G和T455C).各SNP位点基因及基因型频率统计结果显示,在G201A位点,A为大型猪的优势等位基因;在A440G和T455C位点,AT为大型猪的优势等位基因,小型猪的上述2个位点均没有共同的总体特征;2个等位基因型分布差异极为显著(P＜0.01),西藏小型猪的多态信息含量最低,而军牧猪的杂合程度最高.     

高黎贡山猪DECR基因外显子1的克隆及SNP检测

根据已报道猪线粒体2,4-双烯脂酰辅酶A还原酶(mitochondrial 2,4-dienoyl-CoA reductase,DECR)基因全序列(GenBank登录号:NC_010446),用Oligo软件设计扩增DECR外显子1引物,通过对高黎贡山猪、明光小耳猪、大河猪、滇南小耳猪、迪庆藏猪、长白猪、大约克夏和杜洛克猪的外显子1片段的克隆,经EcoRⅠ和HindⅢ双酶切鉴定,符合理论值后测序。序列分析结果显示,在外显子1筛选到一个SNP(C237T),高黎贡山猪为T,其他猪为C。通过271头猪检测,该突变基因频率为79%,其他群体尚未检测到,可能为高黎贡山猪高含量的多不饱和脂肪酸的分子标记。该试验结果为验证高黎贡山猪含较高高多不饱和脂肪酸由遗传因素决定奠定了基础。     

猪TLR4基因可变剪接体的鉴定

为合理、有效地利用民猪资源进行抗病育种,本研究利用重叠延伸RT-PCR结合巢式PCR法扩增民猪TLR4基因,寻找可变剪接体;利用竞争性RT-PCR方法研究各可变剪接体的组织表达情况。获得了猪TLR4基因3个可变剪接体(其中2个是未见报道的),并且鉴定出一类特殊的剪接模式——外显子内部的部分片段被单独剪除,该类型在动物中尚属首次发现;组织表达分析表明3个可变剪接体普遍表达。TLR4基因存在着复杂的剪接机制,也暗示着其功能的复杂性。     

大白猪TLR4基因外显子1遗传变异及其与细胞因子水平的关系

本实验运用PCR-SSCP技术对大白猪TLR4基因外显子1遗传变异进行分析,同时利用ELISA方法对外周血重要细胞因子(IL-2、IL-4、IL-6、IFN-Beta、IL-10、IL-12)的水平进行测定,旨在分析大白猪TLR4基因外显子1多态性及其与重要细胞因子水平间的关系,探讨将其作为抗F18大肠杆菌病遗传标记的可行性。结果表明:大白猪TLR4基因外显子1存在G93C同义突变位点,共检测到3种基因型,分别定义为BB、BC和CC;关联分析结果表明,大白猪TLR4外显子1变异位点对机体重要细胞因子水平没有显著影响(P>0.05),表明基于该位点的分子选育不会对机体免疫应答和一般抗病力产生不利影响。结果提示,有必要将TLR4基因外显子1的变异位点作为抗大肠杆菌病的重要遗传标记进行深入研究。     

4个品种鸡Toll样受体4基因第2内含子的多态性分析

本研究采用PCR-SSCP和DNA测序的方法检测了4个品种鸡(仙居鸡、白耳鸡、清远麻鸡和安卡鸡)Toll样受体4(Toll-like receptor 4,TLR4)基因第2内含子的单核苷酸多态性。结果表明,4个品种鸡TLR4基因的第2内含子上均发现了G1894C的突变,共产生了AA、AB和BB 3种基因型,4个品种鸡该位点的多态信息含量(PIC)分别为0.570、0.597、0.583和0.588,均属于高度多态性,且处于Hardy-Weinberg平衡状态(P>0.05);4个品种鸡G1894C多态位点的基因型在感染鸡白痢沙门氏菌阳性组和阴性组间的分布差异均不显著(P>0.05);G1894C多态位点在仙居鸡与白耳鸡2个品种间基因型分布差异极显著(P<0.01)。     

扬州鹅PRL基因5'调控区SNP检测及其与产蛋性状的相关分析

催乳素(Prolactin,PRL)是影响禽类就巢性和产蛋性能的重要候选基因,本试验以扬州鹅为研究素材,采用PCR-SSCP方法检测催乳素基因5′调控区序列的SNP位点并分析其与扬州鹅产蛋性状的相关性,以期为扬州鹅产蛋性状标记的辅助选择提供一定的理论依据。结果表明,在试验群体中检测到CC、DD两种基因型,未检测到CD型;对不同基因型个体测序分析发现,在外显子1上游﹢11处发现了一个G→T突变;等位基因C和D的频率分别为0.7692和0.2308。供试群体在5′调控区检测位点上处于Hardy-Weinberg不平衡状态。PRL基因5′调控区的碱基突变对扬州鹅的产蛋量和开产日龄有极显著的影响,CC型群体在一个产蛋周期内的平均产蛋量极显著高于DD型(P0.01);开产日龄也是CC型极显著早于DD型(P0.01)。     

Single strand conformation polymorphism detection in alpha-lactalbumin gene of Indian Jakhrana milk goats

Abstract

Alpha-lactalbumin (α-LA) (ALA), one of the two major whey proteins, is strongly correlated with the nutritional value and the functional properties of whey and whey products. The genetic variations of ALA gene in Jakhrana Goat breed (Capra hircus) were investigated using an optimized non-radioactive polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) analysis of four amplified fragments covering all four exons of the gene. A total of eight SSCPs patterns were detected in three of these exons in a sample of 50 Jakhrana goats, indicating that this breed has high genetic variability in the ALA gene. This result opens interesting prospects for future breeding programs and conservation strategies. These ALA gene variants can be sequenced and screened in the entire population to develop single nucleotide polymorphisms for association studies with different productive and reproductive performances and marker assisted selection. In addition, our data show that PCR–SSCP is an appropriate tool for evaluating genetic variability.     

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea‐specific primers, Ar1000F and Ar1500R, or methanogen‐specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α‐cyclodextrin‐horseradish oil complex (CD‐HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea‐specific and methanogen‐specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD‐HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR‐SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD‐HR to the rumen.     

猪TLR2基因Bi-PASA遗传标记的建立及多态性研究

根据人和小鼠TLR2基因序列设计了特异性PCR引物,优化PCR条件后扩增到中国梅山猪、欧洲约克夏猪、PIC-2系及PIC-3系商品猪的TLR2基因690 bp的基因片段,并对其进行序列分析。序列分析结果显示,猪TLR2基因多态性程度低,发现在猪TLR2基因编码区第1 255位点上存在1个单碱基突变位点。利用双向特定等位基因PCR扩增法(B i-PASA)建立了检测猪TLR2基因变异的遗传标记。利用猪TLR2基因的B i-PASA标记,分析了TLR2基因在大河乌猪、撒坝猪和黔邵花猪中的基因频率和多态性。研究建立的B i-PASA遗传标记和基因变异信息,将为进一步分析猪TLR2基因变异与疾病抵抗力及经济性状的相关分析提供基础资料。     

四川白鹅催乳素基因Exon5克隆及SNP分析

以四川白鹅为材料、运用POE—SSCP技术检测催乳素（PRL）基因外显子5上可能存在的SNP位点。结果表明,四川白鹤PEL基因外显子5存在1个SNP（TI55C）位点。等位基因A频率为0．8585,B频率为0．1615。x2分析表明,两种基因型在群体内分布差异不显著（P〉0．05）。     

中国荷斯坦牛Toll样受体4基因的遗传多态性与体细胞评分的关联分析

试验旨在研究中国荷斯坦牛Toll样受体4(toll-like receptor 4,TLR4)基因的遗传多态性及其与体细胞评分(somatic cell score,SCS)的关联性,寻找与乳房炎相关的分子标记,加快中国荷斯坦牛的抗病育种。利用PCR-RFLP和PCR-SSCP技术对30个公牛家系的610头中国荷斯坦牛TLR4基因进行多态性检测,用最小二乘均数法对TLR4基因的多态位点与SCS进行相关分析。结果发现,试验共检测到2个单核苷酸多态(single nucleotide polymorphisms,SNPs),TLR4基因5'侧翼区存在SNP -226 G>C突变,经PCR-RFLP检测发现3种基因型:GG、GC和CC,基因型频率分别为0.208、0.482和0.310;外显子3存在SNP 1760 C>T突变,经PCR-SSCP检测发现3种基因型:CC、TC和TT,基因型频率分别为0.738、0.225和0.007,以上2位点均偏离Hardy-Weinberg 平衡。对于SNP -226 G>C位点,基因型个体的SCS差异不显著;对于SNP 1760 C>T位点,CC基因型个体的SCS最小二乘均值极显著低于TT和TC基因型个体(P<0.01)。SNP 1760 C>T的CC基因型对于中国荷斯坦牛的SCS有较大的遗传效应,可作为分子标记应用于奶牛乳房炎抗性筛选。     

Identification and functional analysis of a novel single nucleotide polymorphism (SNP) in the porcine Toll like receptor (TLR) 5 gene

Nine suckling calves were allowed to afterstimulate their dams at three levels, ad libitum , for 3 min or none, at each suckling meal twice daily in a repeated 3 2 3 Latin square experimental design. Each experimental unit consisted of one cow-calf pair for 6 days. Milk yield was recorded and samples were taken at the morning meal on day 7 by hand-milking one quarter while the calf suckled the other three. Samples were analysed for fat content and 19 fatty acids (FA), summarized in five groups: locally synthesized FA, FA deriving mainly from blood serum (BSFA), saturated FA (SFA), unsaturated FA (USFA) and essential FA (ESFA). Variables were expressed as g g -1 fat, kJ g -1 fat, g kg -1 milk delivered at the recorded suckling meal and kJ kg -1 delivered milk. The only factors affected were milk yield, and BSFA, SFA, USFA and ESFA when related to the delivered milk. The relationship between levels of AS and milk yield was a second-degree polynomial.     

石歧杂鸡催乳素基因外显子4变异与产蛋就巢性状的关联分析

利用PCR-SSCP技术和测序技术对催乳素（prolactin,PRL）基因外显子4进行SNP检测,并分析了该基因与石歧杂鸡产蛋、就巢性状的相关性。结果表明,PRL基因外显子4存在1个多态性位点,该位点在6017位点发生G→A的碱基突变,产生了AA、AB和BB 3种基因型,AA基因型为优势基因型,等位基因A为优势等位基因,该突变未导致氨基酸的变异;该位点的变异与开产日龄、首次就巢日龄呈现一定的显著差异趋势,BB基因型开产日龄、首次就巢日龄都晚于AA和AB基因型。     

子宫内膜中TLR4的定位及pcDNA3.1-TLR4载体的构建

试验旨在研究TLR4(Toll like receptor4)蛋白在子宫内膜组织中具体的分布与表达,从而构建TLR4真核表达载体,为TLR4在细胞水平上的功能研究提供一种技术手段.选择早期妊娠绵羊(9d、13d、17d、21d、25d)子宫组织,采用免疫组织化学和细胞免疫学抗体着色法检测各阶段子宫组织中TLR4蛋白的分布与表达,确定受体蛋白的表达部位.同时将构建的pcDNA3.1-TLR4质粒转染到确定的受体细胞,检测TLR4蛋白的表达水平.结果表明:TLR4蛋白在子宫内膜各部位均有表达,呈一定的动态时空模式,且第17d胚胎附植后主要表达于内膜基质细胞;重组质粒转染到基质细胞后,TLR4蛋白的表达水平明显增强.此研究成功构建pcDNA3.1-TLR4表达载体,为TLR4在生殖细胞免疫学功能的研究奠定了一定的理论基础.     

Analysis of a polymorphism in the DGAT1 gene in 14 cattle breeds through PCR-SSCP methods

The diacylglycerol O-acyltransferase (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. Recent work have evidenced a significant association between lysine at amino acid position 232 with elevated milk fat content, while an alanine at this position is associated with lowered milk fat content. The aim of the present work was to develop a simple and inexpensive PCR-SSCP assay in order to discriminate the CG/AA alleles in exon 8 of the DGAT1 gene. In addition, this method was used to analyze the polymorphism of the DGAT1 through PCR-SSCP methods in 14 populations of cattle from Argentine, Bolivia and Uruguay. The PCR primers were designed from GenBank reported sequences. In this study, we found three PCR-SSCP variants, which were denominated from "A" to "C". However, DNA sequencing analysis showed that "A" variant corresponded with the A allele, while both "B" and "C" observed pattern have the motif AA at positions 10,433-10,434 (K allele), being two alternative conformations of the same DNA sequence. Both variants were detected within each breed with the exception of Hereford, and the heterozygosity varied between 0.000 and 0.524. The gene frequency analysis evidenced significant differences among the studied breeds (F(ST) = 0.325, p = 0.000). European Bos taurus breeds, with the exception of Jersey breed, showed the lowest frequency of the K allele, while highest K allele frequencies were harboured by Bos indicus type cattle. In addition, unselected South American Creole cattle breeds and the synthetic Brangus breed had intermediate allele frequencies.