DNA polymorphism of 5' flanking region of prolactin gene and its association with litter size in sheep

A single nucleotide polymorphism of 5' flanking region of the prolactin gene was investigated in both high prolificacy breeds (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset and Suffolk sheep) using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). The results indicated that two genotypes (AA and AB) were detected in Small Tail Han sheep (n   =   239), only one genotype (AA) was detected in Hu (n   =   40), Dorset (n   =   50) and Suffolk sheep (n   =   39). The mutant homozygous genotype (BB) was not detected in four sheep breeds. In Small Tail Han sheep (n   =   239), the frequency of genotypes AA and AB was 0.91 and 0.09, the frequency of the A and B alleles was 0.95 and 0.05, respectively. The fitness tests showed that the Small Tail Han sheep population was in Hardy–Weinberg equilibrium. Sequencing revealed a mutation (G→T) at the position 63 bp of the 5' flanking region of prolactin gene in AB genotype compared with AA genotype in Small Tail Han sheep. The Small Tail Han ewes with AB genotype had 0.83 (p < 0.05) lambs more than those with AA genotype. These results preliminarily showed that the prolactin locus is either a major gene that influences the high prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

Identification and characterization of polymorphisms within the 5' flanking region, first exon and part of first intron of bovine GH gene

The aim of the present study was to identify and characterize polymorphisms within the 5′ flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental × Nelore (n = 30), Canchim × Nelore (n = 30) and Angus × Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648 , AY662649 and AY662650 , respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837 ), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652 , respectively). The allele sequences were compared with GenBank sequences ( M57764 , AF007750 and AH009106 ) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.     

褪黑激素受体MTNR1B5′调控区多态性及其与鸡产蛋性状的关联分析

用直接测序法检测了434只寿光鸡MTNR1B 5'调控区的SNPs,并与寿光鸡的产蛋性状进行关联分析。结果发现,在5'调控区有13个SNPs位点,9个位点中度多态,4个位点低度多态,所有位点处于哈代温伯格平衡状态。-836位点(C→T),-778位点(G→A)和-629位点(G→A)完全连锁,群体中有CGA和TAG两种单倍型。CGA使产蛋数增加,在前期、中期、后期和总产蛋数上,加性效应值分别为1.0,2.5,1.4,4.9枚。在中期、后期和总产蛋数上2种单倍型存在正向互作效应,显性效应分别达到了2.7,2.3,5.6枚。构建的包含TAG、CGA和TGG 3种单倍型启动活性试验表明,-778位点突变是单倍型效应差异的关键。综上所述,MTNR1B的5'调控区有丰富的SNPs位点,-836、-778和-629位点SNPs及单倍型对寿光鸡的产蛋量有显著影响,可作为寿光鸡产蛋量辅助选择的具有潜在应用价值的分子遗传标记。     

Novel SNP in 5' flanking region of EDG1 associated with marbling in Japanese Black beef cattle

Marbling, defined by the amount and distribution of intramuscular fat, is an economically important trait of beef cattle in Japan. The endothelial differentiation , sphingolipid G-protein-coupled receptor , 1 ( EDG1 ) gene has been considered as a positional functional candidate for the gene responsible for marbling. We have recently reported that 2 single nucleotide polymorphisms (SNPs), c.-312A > G in the 5' untranslated region (UTR) and c.*446G > A in the 3' UTR in EDG1 were associated with marbling in Japanese Black beef cattle, but this was not functional and a causal mutation for marbling. In the present study, we detected 2 novel SNPs, referred to as g.1475435G > A and g.1471620G > T , in the 5' flanking region of the EDG1 between low-marbled and high-marbled steer groups, which were previously shown to have EDG1 expression differences in musculus longissimus muscle. The g.1475435G > A SNP seemed not to segregate in Japanese Black beef cattle. The g.1471620G > T SNP was associated with the predicted breeding value for beef marbling standard number by the analyses using Japanese Black beef cattle population. Based on these findings, we hypothesized that the g.1471620G > T SNP might have an impact on EDG1 expression and also marbling.     

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)。     

水牛OCT4基因5'调控序列的克隆与功能分析

为探讨水牛OCT4基因的表达调控模式,本研究扩增克隆了水牛OCT4基因5'调控序列片段,并设计了-2 571 bp、-2 389 bp、-2 338 bp和-2 191 bp 4个不同长度的调控序列片段,分别构建了EGFP表达报告载体.通过生产转基因猪早期胚胎和转染水牛胎儿成纤维细胞分析了不同长度调控序列片段的转录活性.结果发现,在猪4.5d胚胎中各调控序列均能成功启动下游EGFP表达,但转染水牛胎儿成纤维细胞48 h后均表观察到荧光.QRT-PCR分析显示,-2 571 bp片段在4.5d猪胚胎细胞中的转录活性极显著高于其他调控序列(P＜0.01),-2 389 hp与-2 338 bp之间差异不显著(P＞0.05),二者均极显著高于-2 191 bp(P＜0.01).上述结果表明水牛源OCT4基因5 '调控序列在猪早期胚胎中具有转录活性,且翻译起始位点上游2 191 bp片段足以介导OCT4在多能性细胞中的特异性表达;-2 389～-2 338 bp片段上游的CR4亦参与了猪4.5d胚胎中OCT4基因的转录调控.     

牛α-乳清蛋白基因5'调控区多态性研究

试验选择品种差异较大的贵州荷斯坦奶牛和务川黑牛构建不同DNA池,设计1对引物分别扩增2个牛种α-乳清蛋白(alpha-lactalbumin, LALBA)基因5'调控区及第1外显子部分序列总长1126 bp。结果表明,LALBA基因5'调控区存在5个SNPs位点:T-114C、C-166T、A-225C、C-344T、T-778C,T-114C仅在务川黑牛表现多态性。生物信息学软件预测LALBA基因核心启动子区及转录因子结合位点,SNP位点导致11个转录因子结合位点消失,其中1个位于核心启动子区,产生5个新的转录因子结合位点。突变前后RNA二级结构发生明显改变,目标序列未发现CpG岛。     

鸡Hsc70基因5′侧翼区SNP与耐热性状的相关分析

为研究Hsc70基因作为鸡抗病育种中一种分子标记的可能性,本试验利用克隆测序技术对清远麻鸡、广东温氏矮脚黄鸡、灵山鸡和隐性白羽洛克鸡4个品种共20个个体Hsc70基因的5′侧翼区进行多态性筛查,发现10处变异,包括8个SNPs和2个插入/缺失突变。通过分析,选取C.-521A〉C位点,利用PCR-RFLP方法分析了此位点在灵山鸡和隐性白羽洛克鸡群体中的基因型分布,所得分型结果与测定的耐热性状（T3、皮质酮、CD3＋、CD4＋和CD8＋）进行关联分析,结果表明：常温状态下（15℃）,在隐性白羽洛克鸡群体中,位点C.-521A〉C与CD3＋、CD4＋和CD8＋显著相关（P〈0.05）,其中AA基因型个体CD3＋值显著低于AC基因型个体,而CD4＋与CD8＋值显著高于AC基因型个体;热应激状态下（35℃）,在灵山鸡群体中,位点C.-521A〉C与CD8＋极显著相关（P〈0.01）,CC基因型个体CD8＋值极显著高于AA和AC基因型。     

猪繁殖与呼吸综合征病毒5'非编码区前导转录调控序列及其侧翼序列的结构与功能解析

为解析猪繁殖与呼吸综合征病毒(PRRSV)5'非翻译区(5'UTR)的高级结构与功能,以北美株PRRSV感染性克隆为平台,通过定点突变PCR技术将其5'UTR中一特殊高级调控元件的一级序列进行突变,以改变其二级茎环结构,从而解析该结构的基因调控水平。将突变DNA克隆转染入BHK-21细胞后利用免疫荧光及RT-PCR试验来研究拯救后突变病毒的转录、翻译特性,及通过空斑形态学及病毒生长曲线分析突变病毒的生长特性。结果表明,PRRSV 5'UTR中该高级调控元件的顶端环结构为病毒复制所必需,其只可耐受2个核苷酸的突变;同时发现该调控元件中一保守的茎结构为病毒复制非必需。由此证实了PRRSV 5'UTR中调控病毒复制过程的必需高级结构,为进一步解析PRRSV复制调控元件奠定基础。     

褪黑激素受体MTNR1B5′调控区多态及其与鸡产蛋性状的关联分析

为研究褪黑激素受体基因MTNR1B对鸡产蛋性状的影响,本试验用直接测序法检测了434只寿光鸡MTNR1B5′调控区的SNPs,并与寿光鸡的产蛋性状进行关联分析。结果发现,在5′调控区有13个SNPs位点,9个位点中度多态,4个位点低度多态,所有位点处于哈代温伯格平衡状态。-836位点（C→T）,-778位点（G→A）和-629位点（G→A）完全连锁,群体中有CGA和TAG 2种单倍型。CGA使产蛋数增加,在前、中、后期和总产蛋数上,加性效应值分别为1.0、2.5、1.4和4.9枚。在中期、后期和总产蛋数上2种单倍型存在正向互作效应,显性效应分别达到了2.7、2.3和5.6枚。构建的包含TAG、CGA和TGG 3种单倍型启动活性试验表明,-778位点突变是单倍型效应差异的关键。综上所述,MTNR1B的5′调控区有丰富的SNPs位点,-836、-778和-629位点SNPs及单倍型对寿光鸡的产蛋量有显著影响,可作为寿光鸡产蛋量辅助选择的具有潜在应用价值的分子遗传标记。     

Polymorphisms of the PNPLA3 gene and their associations with chicken growth and carcass traits

1. An F₂ resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken PNPLA3 gene on growth and adipose accumulation.

2. Associations between three SNPs (g.40006G?>?T, g.42344T?>?C and g.42404A?>?T) and broiler traits were determined using linkage disequilibrium, haplotype construction and association analysis.

3. The g.40006G?>?T mutation was associated with body weights at 4, 6, 8, 10 and 12 weeks of age, carcass weight, evisceration weight and semi-evisceration weight (P? 4. Haplotypes of the g.42344T?>?C and g.42404A?>?T mutations were associated with body weight at 12 weeks, carcass weight, evisceration weight, and semi-evisceration weight (P? 5. The results suggest that the PNPLA3 gene may be in linkage with the causative mutation or a QTL controlling growth traits in chickens. In contrast to human studies, the polymorphisms were not associated with fat related traits.     

Three indel variants in chicken LPIN1 exon 6/flanking region are associated with performance and carcass traits

1. LPIN1 is a Mg²⁺-dependent phosphatidic acid phosphatase. Variation in chicken LPIN1 exon 6 and its flanking regions were identified and three indel variants in 6 breeds and their associations with performance traits were studied.

2. Seven variants were detected from 6 breeds, which contained a synonymous tri-allelic variant (c.924A/T/C) and three indels. The exon 6 variants detected from chicken breeds were conserved among bird species. The indel variation frequency presented clear differences among breeds.

3. Two coding indels (c.1014-1018del3 and c.1125-1138del12) were multiples of three nucleotides and maintained the open reading frames of LPIN1 proteins. However, they were predicted to result in the clear change of the RNA secondary structure of chicken LPIN1 exon 6 and LPIN1 protein conformation.

4. The association analysis showed that c.871–15-22del6 variation had a significant effect on body weight at hatch (BW0) and 2 weeks (BW2); c. 1014-1018del3 variation had a significant effect on BW4, BW6, caecum length and gizzard weight (GW) traits; c.1125-1138del12 variation had a significant effect on BW12, shank length at 4 weeks (SL4), carcass weight, lactate dehydrogenase traits (LDH), glucose (GLU) and albumin (ALB) traits. The genotype combination for c.1014-1018del3 and c.1125-1138del12 also presented significant effects on SL4, SL8, GW, leg muscle weight, ALB, GLU and LDH.

5. The study demonstrated that chicken LPIN1 has an important effect on body, carcass and organ weight, serum LDH, GLU and ALB level.

     

表达鸡TRAIL基因的重组5型腺病毒构建

本研究采用RT-PCR从鸡脾细胞扩增到TRAII,基因,测序验证后,将扩增的TRAIL基因开放阅读框克隆到pShuttle-CMV上,与pAdeasy-1载体在BJ 5183细菌内同源重组,获得含有TRAIL基因的5型腺病毒重组质粒,进而将Pac Ⅰ线性化重组质粒转染AD-293细胞,收获重组腺病毒.并对后者进行RT-PCR扩增,获得TRAIL基因片段,经western blot鉴定显示构建的重组腺病毒可表达TRAIL蛋白,表明TRAIL基因在构建的重组腺病毒rAd5-TRAIL中得到表达.本研究为下一步的动物试验和研究TRAIL基因的抑瘤作用奠定了基础.     

鹅GnRH基因5′端调控区单核苷酸多态性分析

运用PCR-SSCP方法对皖西白鹅GnRH基因5＇端调控区进行多态性检测。结果表明,GnRH基因5＇端调控区存在3个SNP位点,分别为GnRH基因5＇端调控区的-192位置存在G/C转换的多态性位点、-206位置存在A/T转换的多态位点、-417位置存在T/G转换的多态性位点。     

鹅GnRH基因5'端调控区单核苷酸多态性分析

运用PCR-SSCP方法对皖西白鹅GnRH基因5'端调控区进行多态性检测.结果表明,GnRH基因5'端调控区存在3个SNP位点.分别为GnRH基因5'端调控区的-192位置存在G/C转换的多态性位点、-206位置存在A/T转换的多态位点、-417位置存在T/G转换的多态性位点.     

Polymorphisms of the prolactin gene and their association with egg production traits in two Chinese domestic ducks

1. Prolactin (PRL) as a polypeptide hormone which plays a crucial role in egg production traits.

2. Polymorphisms of the PRL gene were analysed with DNA sequencing and polymerase chain reaction-single-strand conformation polymorphism methods in two Chinese domestic laying duck breeds (Jinding, n = 400, Youxian, n = 400, respectively).

3. The results showed that one polymorphism was detected (A-412G) in intron 1 of the PRL gene, with three genotypes: AA, AG and GG. Association analysis showed that the ducks with the GG genotype had significantly greater egg production and egg weight than those with AG and AA genotype (p < 0.05). Hence, the 412A > G polymorphism of the PRL gene in intron 1 is a potentially valuable genetic marker for laying duck breeding programmes.     

Polymorphisms in the promoter region of myostatin gene are associated with carcass traits in pigs

Higher average daily gain, more lean meat yield and less fat yield of porcine carcass increase selling profits for animal producers. Myostatin (MSTN), previously called GDF8, is a member of transforming growth factor‐β (TGF‐β) superfamily. It is a negative regulator for both embryonic development and adult homeostasis of skeletal muscle. In this study, the genotypes of the previously described SNPs MSTN g.435G>A and g.447A>G SNPs in 66 Duroc pigs, 33 Landrace pigs, 180 Duroc × Landrace (DL) pigs and 155 Duroc × Yorkshire × Landrace (DYL) pigs were determined by Taqman SNP Genotyping Assays. For Duroc and Landrace pigs, MSTN g.435GG/g.447AA individual had greater backfat thickness (p < 0.05) than g.435AA/g.447GG individual, whereas MSTN g.435AA/g.447GG had greater meat (p < 0.05) and meat percentage (p < 0.05) than g.435GA/g.447AG individual. For DL and DYL pigs, the MSTN g.435GG/g.447AA animals were greater in backfat at ultrasound 10th rib (p < 0.05) and carcass 10th rib (p < 0.01) than g.435AA/g.447GG individual. The MSTN g.435AA/g.447GG individual also had higher values than g.435GG/g.447AA for anterior‐end meat (p < 0.05), posterior‐end meat (p < 0.01), total meat weight (p < 0.01) and meat percentage (p < 0.01). This study confirmed evidence that MSTN g.435G>A and g.447A>G affected carcass traits in pigs. The effects of the mutated alleles were additive with the maximal effects resulting from two copies of the mutated allele. Selection for MSTN g.435A/g.447G allele is expected to increase muscle of limb and total meat production and decrease backfat thickness.