绵羊雌激素受体基因外显子4多态性分析

根据GenBank发表的人、鸡、大鼠雌激素受体(estrogen receptor,ESR)基因外显子4的序列设计1对引物,采用PCR SSCP技术分析ESR基因外显子4在高繁殖力绵羊品种（小尾寒羊和湖羊）和低繁殖力绵羊品种（特克塞尔、中国美利奴、考力代和杜泊）中的单核苷酸多态性,同时研究该基因对小尾寒羊高繁殖力的影响。结果表明,ESR基因的此对引物扩增片段在所检测的6个绵羊品种中均不存在PCR SSCP多态性,说明所检测的ESR基因外显子4序列比较保守,该区域可能不是影响绵羊高繁殖力的功能结构域。     

天祝白牦牛Leptin和SCD1基因单核苷酸多态性与肌肉脂肪酸含量的关联性分析

试验旨在研究Leptin和SCD1基因多态性对天祝白牦牛肌肉脂肪酸含量的影响。通过对PCR产物直接测序,在Leptin基因外显子2和3共发现7个SNPs位点(其中4个是错义突变);在SCD1基因第5外显子区发现1个SNP位点(错义突变),对所有SNPs等位基因替代效应对25种单一脂肪酸、单一不饱和度指数、多不饱和度指数和去饱和效应指数的影响进行了检测。结果发现,SCD1基因SNP和Leptin基因3个SNPs位点在不同程度上影响到由饱和脂肪酸FA到单一不饱和脂肪酸MUFA的去饱和作用。说明除了SCD1基因对FA去饱和作用的影响外,Leptin基因错义突变也会对肌肉脂肪中FA的组成产生影响。     

小尾寒羊雌激素受体基因外显子4的克隆与序列分析

根据GenBank发表的人、鸡、大鼠雌激素受体(estrogen receptor,ESR)基因外显子4的序列设计1对引物,采用PCR 技术扩增出小尾寒羊ESR基因外显子4的DNA片段,将该片段克隆到pGEM-T Easy 质粒中,重组质粒用PCR 扩增进行阳性克隆鉴定,然后测定其核苷酸序列并推导氨基酸序列,同时将测定的小尾寒羊ESR基因外显子4序列与人、牛、猪、大鼠、鸡的外显子4序列进行比较。结果表明：克隆测序所得的核苷酸和翻译后的氨基酸序列与人、牛、猪、大鼠、鸡相比,同源性分别为77.68%～97.28%和71.82%～98.18%,显示了很强的保守性;小尾寒羊的核苷酸序列与人、牛、猪、大鼠、鸡相比存在1处特有变异,氨基酸序列23～36存在高变异区。     

Expression and characterisation of equine interleukin 2 and interleukin 4

In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycosylation modifications. The biological activities of both cytokines were tested by proliferation assays using leukoagglutinin (LAG) prestimulated equine PBMC. Both, equine IL2 and IL4 induced dose-dependent lymphocyte proliferation. In contrast to IL4, IL2 supported the proliferation of B cells.     

水牛FASN基因外显子37多态性及其生物信息学分析

[目的]脂肪酸合成酶(Fatty acid synthase,FASN)是影响哺乳动物脂肪酸合成的关键酶,但有关水牛FASN基因的群体遗传组成特征还不清楚。[方法]本研究采用PCR产物直接测序法和PCR-SSCP方法对88头河流型和122头沼泽型水牛、54头牦牛和40头大额牛FASN基因外显子37进行群体变异检测,并结合已发表的牛科物种序列进行生物信息学分析。[结果]结果表明,仅在水牛中发现2个SNP位点,为c.6363CT和c.6372CT,均为同义替换。河流型和沼泽型水牛共享这2个SNP位点,但它们在两类水牛中的群体遗传组成不同。确定水牛特有的核苷酸位点3个,即c.6183A,c.6255T和c.6394A,其中c.6394A导致水牛FASN蛋白第2132位氨基酸与其它牛科物种不同,在水牛中为M而其它牛科物种中为V;山羊特有的位点2个,为c.6189A和c.6267T。普通牛、山羊和绵羊中具有异义替换SNP位点,分别为1个、3个和7个。普通牛的异义替换SNP位点c.6365GA对蛋白质功能影响不显著(subPSEC-3);山羊的这些异义替换SNP位点c.6296TC、c.6301CT和c.6341TC对其蛋白质功能影响均有显著影响(subPSEC-3);绵羊7个异义替换SNP位点中的2个,即c.6286AC和c.6406AG对FASN功能有显著影响(subPSEC-3)。[结论]这些核苷酸差异引起的氨基酸差异可能引起不同牛科物种间FASN功能的差异。     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.     

白羽番鸭脂联素基因SNP与肌内脂肪和血清总胆固醇的关联分析

采用PCR-SSCP法对白羽番鸭脂联素基因全编码区和内含子进行多态性检测,并分析其多态对肌内脂肪(IMF)和血清总胆固醇(TC)的遗传效应,为番鸭分子标记辅助选择提供参考.结果表明:3个SNP分别在外显予1和外显子2的编码区发生A167G和G711A突变,为沉默突变,在内含子发生C290T突变;3个SNP位点均处于Ha...     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

绵羊DECR1基因exon 5部分序列多态性分

试验根据GenBank登录的牛2,4-双烯-CoA还原酶1（2,4-dienoyl-CoA reductase1,DECR1）基因序列（NP_001068891）设计引物,应用PCR技术对德国美利奴绵羊、杜泊绵羊、特克塞尔绵羊DECR1基因的exon 5部分序列进行克隆测序;利用PCR-SSCP技术检测了3个绵羊群体exon 5单链构象多态性（SNP）,所获序列与GenBank中其他物种exon 5序列进行了同源性比较,并构建了亲缘关系聚类分析图。结果表明,绵羊DECR1基因exon 5长为137 bp,3个绵羊群体中exon 5均不存在SNPs,各种动物之间DECR1基因exon 5的同源性较高,在81.02%（鸡）～97.08%（牛）之间,其中绵羊和牛同源性最高,达到97.08%;与鸡的同源性最低,为81.02%;聚类分析结果显示,绵羊首先与牛聚为一类,再分别与兔子、狗聚为一类,最后分别与人、猪聚为一类,绵羊与牛的亲缘关系最近,与鸡的亲缘关系最远,与传统分类相一致,说明DECR1基因exon 5在动物进化过程中高度保守。     

陕西地区荷斯坦牛Mx1基因外显子多态性分析

牛Mx1基因位于1号染色体上,含有14个外显子,编码648个氨基酸。研究以346头陕西地区荷斯坦牛为研究对象,根据GenBank所提供的牛Mx1基因序列,设计15对引物,采用混合DNA池扩增测序和PCR-RFLP的方法对牛Mx1基因的14个外显子进行多态性分析。结果表明,4个SNPs,分别位于外显子3（g.843330T〉C,g.843411A〉G）、外显子4（g.844048C〉T）和外显子7（g.851325T〉C）。其中,外显子4（g.844048C〉T）为同义突变;外显子3（g.843330T〉C,g.843411A〉G）两个位点的突变分别导致了异亮氨酸（Ile）向苏氨酸（Thr）、谷氨酸（Glu）向精氨酸（Arg）的突变;外显子7（g.851325T〉C）位点的突变导致亮氨酸（Leu）变为脯氨酸（Pro）。4个SNPs位点都存在三种基因型,但纯合子的突变频率较低。4个SNPs位点共构建了11种单倍型,其中以H6为最主要的单倍型。结果显示,陕西地区荷斯坦牛Mx1基因存在较丰富的多态性,这将为Mx1基因结构和功能的研究以及将来在育种实践中的应用提供基础资料。     

中国美利奴羊内皮型一氧化氮合酶基因3个外显子多态性的PCR-SSCP鉴定

本试验旨在研究中国美利奴羊内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS）基因第7、13、14外显子上的单核苷酸多态性（SNPs）和单氨基酸多态性（SAPs）。利用生物信息学方法对人、小鼠和中国美利奴羊eNOS基因上的第7、13、14外显子进行了相似性比较,并对GenBank SNP数据库上已公布的人和小鼠eNOS基因外显子多态性进行了统计分析,然后采用PCR-SSCP方法对120只中国美利奴羊eNOS基因的这3个外显子的扩增片段进行分析,通过分析发现在120只中国美利奴羊这3个外显子上并没有SNPs位点,说明中国美利奴羊eNOS基因上的这3个外显子区域相对保守。     

Identification of genetic variation in 11 candidate genes of canine mammary tumour

The incidence of canine mammary tumours (CMTs) differs significantly between breeds, strongly supporting an influence of genetic risk factors. We aimed at identifying germline genetic variations in mammary tumour-associated genes in dogs and survey whether these might alter the encoded proteins. We sequenced 11 genes (BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EGFR, ESR1, HER2, PTEN, STK11 and TP53) and screened for genetic variations. Sixty-four single nucleotide polymorphisms (SNPs) were identified. Nine of the coding SNPs were non-synonymous, of which four were located in gene regions conserved across four species. Three of the non-synonymous SNPs might be damaging according to PolyPhen predictions. One of the indels identified has previously been associated with CMTs. Because of the founder effects, genetic drift and inbreeding in many dog breeds the allele frequencies of the genes studied are likely to vary significantly between breeds and contribute to the considerable difference in genetic risk associated with cancer.     

Polymorphism of LAMB1 Gene and Its Association Analysis with the Fiber Diameter in Fine-wool Sheep

This study aimed to research the single nucleotide polymorphism (SNPs) of LAMB1 gene exon and its correlation with the fiber diameter in Fine-wool sheep.Based on DNA pools with the Re-sequencing technology to gain SNPs data,totally screened 20 SNPs of LAMB1 gene exon regions,at the same time,combining with direct sequencing method,PCR-SSCP and bioinformatics software to verify the accuracy of 10 missense mutations.The genetic effects of LAMB1 on fiber diameter at Xinjiang Kunes farm were analyzed by the GLM of SAS,totally 300 sheep.The results showed that 20 SNPs of LAMB1 gene were screened,which included 10 synonymous mutation SNPs and 10 non-synonymous mutation SNPs,after the validation,there were 7 missense mutation SNPs which led to the nature of protein change,3 SNPs were not mutated.LAMB1 protein became increasingly hydrophobic after mutating,predicting that was insoluble protein.TT genotype in the fiber diameter of the individual value was significantly higher than TC and CC genotypes on SNP5(P<0.05),GG and TT genotypes in the fiber diameter of the individual value was significantly higher than GT genotype on SNP9 (P<0.05).Although,all of the SNPs were completely detected by DNA pool with the Re-sequencing technology and minimized the false positives,it still need to be validated by direct sequencing.The results revealed that LAMB1 gene existed highly genetic diversity,mutated SNPs were unevenly distributed in exons,SNP5(rs159769941) and SNP9(rs159769901) could be considered as an effective genetic markers of Fine-wool sheep on fiber diameter.     

细毛羊LAMB1基因外显子多态性及其与羊毛纤维直径的关联性分析

试验旨在研究LAMB1基因外显子在细毛羊中的多态性及其与羊毛纤维直径的关联性。基于DNA池重测序技术获得的SNPs数据,共筛选LAMB1基因外显子区域20个SNPs,利用直接测序法、PCR-SSCP及生物信息学软件对10个错义突变SNPs的准确性进行验证,利用SAS 8.1的GLM程序分析其对新疆巩乃斯种羊场育种核心群300只细毛羊的遗传效应及与被毛纤维直径的关联性。结果表明,20个SNPs中10个是同义突变SNPs;10个错义突变SNPs验证后7个发生错义突变,导致蛋白性质改变,3个呈假阳性。突变后LAMB1蛋白疏水性更强,预测是不可溶性蛋白。SNP5中TT基因型个体纤维直径显著高于TC和CC基因型个体（P<0.05）,SNP9中GG和TT基因型个体纤维直径显著高于GT基因型个体（P<0.05）。虽然DNA混池全基因组重测序技术完整地检测到基因的SNPs,并将假阳性最小化,但还需要对结果进行验证。研究揭示LAMB1基因外显子多态性丰富,突变SNPs在外显子中分布不平衡,LAMB1基因SNP5（rs159769941）和SNP9（rs159769901）可以考虑作为影响细毛羊被毛纤维直径的有效遗传标记。     

5个家兔群体白细胞介素10基因外显子的遗传多样性分析

本研究旨在分析家兔IL-10基因外显子的多态性,以期为进一步研究家兔IL-10与疾病抗性的相关性提供理论依据。根据GenBank上收录的IL-10基因序列设计5对特异性引物,采用PCR-SSCP方法对海狸色獭兔、白色獭兔、皖系长毛兔、闽西南黑兔、九疑山兔这5个家兔群体IL-10基因的5个外显子序列进行多态性分析。结果表明:在IL-10基因外显子3上检测到4种等位基因,10种基因型,存在3个SNPs位点;在外显子4上检测到2种等位基因,3种基因型,存在1个SNP位点。而外显子1、2、5对于实验群体未发现有遗传多态性。在外显子3中,D等位基因只在闽西南黑兔和九疑山兔中检测到,除海狸色獭兔和闽西南黑兔外其余群体均处于哈代-温伯格平衡,各群体不同基因型分布存在极显著差异(P<0.01)。在外显子4中,A1B1基因型在獭兔群体中没有检测到;除海狸色獭兔外其余各群体均处于哈代-温伯格平衡,海狸色獭兔跟白兔獭兔不同基因型分布差异不显著(P>0.05),而其余群体彼此不同基因型分布存在显著性差异(P<0.05或P<0.01)。结果提示:5个家兔群体在IL-10基因外显子3和4中存在遗传多态性,不同家兔群体在遗传基础上存在着一定的差异。     

Molecular cloning and sequencing of equine interleukin 4

We have cloned equine interleukin 4 (IL-4) cDNA using the polymerase chain reaction (PCR) and primers based on the human IL-4 sequence. The cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology with IL-4 sequences from other species.     

4个绵羊品种Leptin基因部分片段的SNPs分析

利用PCR-SSCP技术对萨福克、道塞特、特克塞尔及滩羊4个绵羊品种358个个体Leptin基因2、3外显子进行多态性分析,共检测到7个SNPs,其中新发现5个SNPs。测序结果表明,在外显子2上无突变。内含子2上存在99位碱基由A突变为G;115位碱基由G突变为A;150位碱基由C突变为T;171位碱基由C突变为T。外显子3上,存在271位碱基由G突变为A,使编码的Arg转变为Gln;316位碱基由C突变为A,使编码的Pro转变为Gln;387位碱基由G突变为T,使编码的Val转变为Leu。     

鸽黑素皮质素受体4基因多态性与生长性状及体组成的相关研究

通过比对GenBank中鸡、鹅黑素皮质素受体4（MC4R,melanocortin receptor）基因DNA序列,找出保守序列设计引物,克隆了鸽的MC4R基因编码区985bp序列,与鹅、鸡、短尾猊、北极狐和犬类的同源性分别为94%、94%、91%、84%和84%。利用PCR-SSCP技术和DNA测序方法检测两群体鸽MC4R基因编码区序列的单核苷酸多态性,共检测出两个多态位点,最小二乘分析表明,T933C基因型对肉鸽群体活重、屠体重、全净膛有显著影响（P＜0.05）,对野生鸽群体生长性状无影响（P＞0.2）,多重比较显示,该位点AA基因型个体的活重、屠体重、全净膛显著高于BB基因型个体（P＜0.05）。MC4R基因可能是影响鸽生长性状和体组成的主效基因或与主效基因相连锁。     

4个绵羊品种Leptin基因部分片段的SNPs多态性与生长发育性状的相关性分析

利用PCR-SSCP技术对萨福克、陶赛特、得克塞尔及滩羊4个绵羊品种358个个体Leptin基因等2、3外显子进行多态性分析,共检测到7个SNPs,其中新发现5个SNPs。测序结果表明,在外显子2上无突变。内含子2上存在A99G、G115A、C150T、C171T位点。外显子3上,存在G271A;C316A;G387T位点。外显子3上的SNPs使编码的氨基酸发生变化。统计分析表明A99G、C150T和A99G＋C150T位点与生长发育性状存在相关性。在A99G位点,Aa基因型初生质量、日增质量、体高、胸围和尻宽指标上均高于AA基因型,初生质量、日增质量和体高指标差异显著（P〈0．05）,胸围和尻宽指标差异极显著（P〈0．01）。C150T和A99G＋C150T位点结果一致,突变基因型日增重、体高、体长、胸围和尻宽指标均高于野生基因型,差异显著（P〈0．05）。