合作猪SLA-DQA基因第4外显子多态性分析

合作猪的MHC-DQA基因的适应性变异,其抗原识别区域(即外显子4)通过PCR扩增和随后的单链构象多态性(SSCP)和序列分析,结果显示在439个合作猪个体,SLA-DQA第4外显子检出4个等位基因和6个基因型(AA、BB、DD、AB、AC和AD),其中A等位基因和AA基因型的频率最高,为优势基因和优势基因型。对不同型的PCR-SSCP条带测序分析,发现7个突变位点(5 068 bp T→C,5 109 bp和5 149 bp处缺失C,5 131 bp A→G导致丝氨酸变为甘氨酸,5 135 bp C→T,5 234 bp G→A,5 136 bp处插入A)。遗传学分析发现,合作猪多态信息含量(PIC)为0.240 1,属于低度多态,各种基因型的分布不显著。研究结果证实,合作猪SLA—DQA基因第4外显子为低度多态。     

猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制

目的:研究猪Mx1和牛Mx1蛋白在PK-15细胞中的表达并检测其是否对伪狂犬病病毒(PRV)具有抑制作用。方法:从IBRS-2细胞和MDBK细胞中分别调取猪Mx1和牛Mx1基因,并克隆到pc DNA3.1/myc-His(-)B,构建得到真核重组表达质粒,以脂质体转染的方法将其分别导入到PK-15细胞,从mRNA水平和蛋白质水平鉴定重组质粒在细胞内的表达情况,然后用细胞毒性试剂盒检测这两种蛋白是否对PK-15细胞具有毒性。之后,通过荧光定量PCR检测猪Mx1和牛Mx1在攻毒后不同时间、不同攻毒剂量的条件下对PRV的抑制情况,并观察100TCID50病毒攻击细胞72h后的病变程度。结果:成功克隆了猪Mx1和牛Mx1基因,经mRNA水平和蛋白质水平证实,两种重组质粒在PK-15细胞内能够正常表达。从荧光定量PCR和细胞病变的角度来看,细胞内表达的Mx1蛋白对PRV具有显著性的抑制(P0.001)。结论:猪Mx1和牛Mx1基因在PK-15细胞中表达的Mx1蛋白能够抑制PRV在胞内的复制。     

猪GH基因部分突变位点对生产性能的影响

本试验利用PCR－RFLPs技术,用MspⅠ,ApaⅠ两种限制酶,检测了猪生长激素基因＋206－＋711位共506bp片段中[ 295- 300, 563- 566]2处突变位点,分析各多态位点在姜曲海猪中的分布及其与生产性能的关系。发现：姜曲海猪中,MspⅠ酶切突变位点中的G1G2基因型和G2G2基因基因型个体的生产性能没有差异,不同日龄体重在两组间变化没有规律。ApaⅠ酶切突变位点G4G4基因型个体的20个日龄体重、45日龄体重高于G3G3／G3G4基因型个体,但差异不显。G4G4基因型个体的70日龄体重极显高于G3G3／G3G4基因型个体（P＜0．01）。G4G4基因型个体的0－70日龄日增重也极显高于G3G3／G3G4基因型个体（P＜0．01）。这些结果显示GH基因有可能作为数量性状基因座的侯选基因。     

猪激素敏感脂肪酶基因外显子1的遗传多态性分析

采用PCR-SSCP方法检测猪激素敏感脂肪酶（hormone sensitive lipase,HSL）基因外显子1的多态性,并分析其与初生重、断奶重、6月龄重和背膘厚的关联性。根据猪HSL基因的DNA序列（AJ000483）设计5对引物,结果在P5引物对扩增的片段上发现了多态性,并对纯合子进行测序,发现外显子1的874bp处存在G-A转换,且存在3种基因型（AA、AB、BB）。统计结果表明,3种基因型在各品种中的分布不一致,长白猪和大白猪与莱芜猪和沂蒙黑猪比较差异极显著（P〈0．01）,长白猪与大白猪比较,莱芜猪与沂蒙黑猪比较差异均不显著（P〉0．05）。固定效应模型分析结果表明,背膘厚基因型间差异显著（P〈0．05）,而初生重、断奶重和6月龄重基因型间差异均不显著（P〉0．05）。最小二乘分析结果表明,BB基因型个体同AB和AA基因型个体比较背膘厚的差异显著（P〈0．05）,3种基因型在背膘厚的大小排列顺序为BB〈AB〈AA。因此,推测HSL基因对个体胴体瘦肉率存在一定的影响将HSL某因应用于猪育种过程中的标记辅助选择可以加快猪的育种进程。     

猪TLR4基因外显子1新等位基因的分离及遗传变异分析

文章采用PCR-SSCP方法对亚洲野猪、3个引进的商业化品种和10个中国地方猪品种共893个个体TLR4基因外显子1的遗传变异进行了检测,旨在系统分析国内外猪种TLR4基因的多态性,为探讨该基因在免疫和防御系统中发挥的作用提供依据。结果,在猪TLR4基因外显子1中分离到新的等位基因,共检测到3个等位基因,6种基因型。其中杜洛克检测到AA、BB、CC、AB、AC、BC基因型,有杜洛克血统的苏太猪中检测到BB、CC、BC基因型,长白猪、约克夏中检测到CC、BC基因型,野猪及所有10个中国地方猪品种TLR4基因外显子1高度保守,只检测到CC基因型,中国地方猪品种和引进品种TLR4基因外显子1多态性存在极显著的差异。3种基因型中CC型与GenBank中的序列一致,BB和AA基因型分别存在G93C同义突变位点和G194A无义突变位点,这2个变异位点与抗逆性和一般抗病力的关系值得进一步深入研究。     

猪AMPD1基因的克隆与变异位点分析

腺苷一磷酸脱氨酶基因(AMPD1)在嘌呤代谢过程中起着重要的作用。AMPD1基因主要在骨骼肌中高水平表达, 与肌肉中肌苷酸代谢有关。还发现AMPD1与猪的胴体性状有关。对AMPD1基因CDS进行了克隆、测序, 获得2 195 bp的CDS序列, 并利用PCR-SSCP方法对其进行分子扫描, 寻找多态位点, 分析不同基因型在不同猪种间的分布规律。χ2独立性检验表明, 民猪、大白猪、杜洛克间不同基因型的分布存在着极显著的差异(P<0.01)。     

猪CAPN1基因部分外显子及3'UTR区的SNPs检测

以野猪.民猪和大白猪为研究对象,根据网上公布的序列设计了7对引物,采用测序,PCR-SSCP和PCR-RFLP方法对CAPN1基因的部分外显子和3'UTR区进行了单核苷酸多态性检测和基因型分析,探讨CAPN1基因多态性与瘦肉率和嫩度的关系.研究发现11个SNPs,其中5个位于外显子,4个位于内含子,2个位于3'UTR区,外显子中的突变有一处是错义突变,导致了蛋白质多肽链第260位氨基酸发生了M/V的替代.群体遗传学分析表明,在所检测的各多态位点上,野猪、民猪、大白猪3个品种间不同基因型的分布都存在着极显著的差异(P<0.01),而野猪和民猪之间各基因型的分布差异不显著(P>0.05),民猪和大白猪之间各基因型的分布存在着极显著的差异(P<0.01).结合品种特性分析表明,P4、P6引物和3'UTR区Hinf1位点所检测的不同基因型和瘦肉率具有一定的相关性.     

西藏小型猪SLA-DQA基因外显子2的PCR-RFLP多态性分析

目的分析西藏小型猪SLA-DQA基因第2外显子不同酶切位点的基因型多态性以及等位基因的多态性,检验这些酶切位点上的基因频率是否达到Hardy-Weiberg平衡态。方法采用EcoRⅠ和AluⅠ两种限制性内切酶对西藏小型猪SLA-DQA目的基因的第1和第2内含子部分序列以及完整的外显子2进行PCR-RFLP分析。结果经EcoRⅠ酶切后,以纯合子BB基因型居多,BB、AB、AA基因型频率分布为45.000%、31.667%和23.333%,其中B为优势等位基因（60.833%）;经AluⅠ酶切后,MN基因型频率（50.000%）分别高于MM型（30.000%）和NN型（20.000%）,     

SIM1基因第8外显子的SNP对猪背膘厚的影响

采用PCR-SSCP方法检测了约克夏、杜洛克、皮特兰、长白猪、嘉兴黑猪和金华猪6个品种共169头猪的SIM1基因外显子8的SNP及其基因型频率。结果共发现CC、CT、TT 3种基因型, 其基因型频率在国内外猪品种之间具有较大差异。其中, 国内猪种嘉兴黑猪和金华猪只存在TT基因型, 而国外猪种约克夏、杜洛克、皮特兰、长白猪则都存在3种基因型。用最小二乘法分析SNP对长白猪、约克夏猪和杜洛克猪的背膘厚的效应的结果表明, 纯合基因型个体的背膘厚大于杂合基因型个体。SIM1基因型对国外猪种背膘厚有显著效应(P< 0.05), 并且不同部位效应不同。     

Three types of polymorphisms in exon 14 in porcine Mx1 gene

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistibility to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)–restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1 ^c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1 ^b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1 ^a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1 ^c deletion is associated with variation in resistance to the myxovirus family in the pig.     

Molecular Characterization of Mx1 Gene in Native Indian Breeds of Chicken

The genetic polymorphism of Mx1 gene was explored in Indian chicken breeds. PCR-RFLP analysis in 102?bp fragment of partial intron 13 and partial exon 14 of Mx1 gene revealed two genotypes viz. RS and SS with two alleles viz. R and S both in Naked Neck and Tellicherry breeds of chicken. The homozygous genotype RR was not identified. When deduced amino acid sequences were compared, the asparagine amino acid was found to be substituted in “R” allele for serine in “S” allele. PCR-SSCP analysis of 284?bp fragment in 5′-UTR and partial promoter region revealed three genotypes viz. CC, CG, and CH with three different alleles viz. C, G, and H in Naked Neck breed of chicken and five genotypes viz. DI, JK, KK, KL, and KM with six different alleles viz. D, I, J, K, L, and M in Tellicherry breed of chicken. The homozygous genotypes viz. GG and HH in Naked Neck and DD, II, JJ, LL, and MM in Tellicherry chicken was not identified. The nucleotide substitution rate estimated to be in the range of 0.004–0.011. The identified genetic variation can be helpful for better insight to disease resistance property of the Mx1 gene.     

A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility to Influenza Virus In Vitro

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10–100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.     

表达猪流行性腹泻病毒S1基因植物乳酸杆菌工程菌的免疫原性分析

为探究表达猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S1基因的植物乳杆菌工程菌是否对动物产生保护力,利用构建的含重组质粒pVE5523-S1乳杆菌口服免疫豚鼠3次,每次间隔14d,每次2×108 CFU/只.并以植物乳杆菌及含表达质粒pVE5523植物乳杆菌为阴性对照,...     

眼皮肤白化病患者酪氨酸酶基因突变的研究

目的：对临床诊断为眼皮肤白化病（OCA）患者的酪氨酸酶（TYR）基因进行突变筛查,了解我国大陆OCA患者TYR基因突变类型,探讨基因突变对人TYR蛋白结构和功能的影响。方法：应用PCR技术,扩增患者及其父母的TYR基因外显子、外显子-内含子交界区及启动子区;以DNA序列测定技术,进行突变筛查与鉴定;利用生物信息学方法,对突变引起蛋白结构和功能的改变进行预测与分析。结果：在15名患者的30个TYR等位基因内,查明11种突变;其中错义突变5种（W400L、R299H、E294K、R77Q和K142M）,无义突变3种（R116X、R278X和G295X）,插入突变2种（929insC和232insGGG）,剪切位点突变1种（IVS1-3 C〉G）;对4个突变W400L、R299H、929insC、232insGGG的生物信息学分析显示,突变的致病性与蛋白结构和功能的改变相关。结论：W400L占本研究所检出全部OCA1突变等位基因的30．0％（9／30）,可能为中国大陆人群中较常见的TYR基因突变类型;应用生物信息学分析方法对TYR基因突变的致病性做出一些合理可能的解释是可行的。     

MX1 exon 13 polymorphisms in broiler breeder chickens and associations with commercial traits

The Mx1 gene in mice is induced by type I interferons and is the major determinant of resistance to influenza virus and related orthomyxoviruses. It has been previously shown that a SNP in exon 13 of the chicken MX1 gene determines differential antiviral activity of the protein. We evaluated this SNP and two additional SNPs in elite broiler lines by PCR amplification and sequence analysis. Associations between MX1 exon 13 SNPs and several traits of economic interest were evaluated. Significant associations were found between the SNP determining antiviral activity and mortality in one line and leg defects in another line.     

Holt-Oram syndrome: a new mutation in the TBX5 gene in two unrelated families

Holt-Oram syndrome (HOS) is a specific developmental defect involving upper limb malformations and cardiac defects. Mutations in the TBX5 gene, located on chromosome 12q24.1, were demonstrated as the underlying molecular defect in several families with this disorder. We report on two unrelated families with HOS. Affected members of both families have the same truncation mutation in exon 5 of the TBX5 gene (Y136X). This mutation has not been reported before in HOS. The spectrum of defects is similar in both families, displaying an ASD, hypoplastic deltoid muscles and hypoplastic or absent thumbs extending to radial defects in one case. So far, only a single genotype-phenotype analysis in HOS has been done which is not sufficient to explain the high inter- and intrafamilial variability of expression. Our observation further supports that the position of the mutation in the TBX5 gene is related to the phenotype expression of HOS.