滩羊裘皮毛色调控基因的研究进展

滩羊是中国裘皮来源的珍稀地方绵羊品种,以所产二毛裘皮而著称,二毛裘皮在国内外毛皮市场上均享有较高的声誉。毛色是宁夏滩羊重要的经济性状,滩羊多为体躯白色,头部有黑褐斑,个别个体黑头或体躯黑杂色,少数纯白。毛色与体内黑色素的数量、种类和分布等情况有关。掌握调控滩羊毛色基因的作用机制,可以有效控制其毛色性状。本文从滩羊的裘皮特性、毛色形成机理、毛色相关基因(MC1R基因、Agouti基因、TYR基因、MITF基因和KIT基因)的功能及选择信号检测等方面进行了综述。     

Leptin基因多态性与绵羊季节性发情的相关分析

根据leptin基因在GenBank中的已知序列设计两对引物,采用PCR-SSCP技术在常年发情的湖羊和季节性发情的阿勒泰羊群体中进行单核苷酸多态性(SNPs)检测,对筛查到的SNP位点进行基因型与绵羊季节性发情的关联分析。结果表明,与湖羊相比,阿勒泰羊leptin基因第1内含子上有3个连续碱基TTG的插入和C/T碱基突变;第3外显子3上发生G/T碱基突变,编码氨基酸由缬氨酸变成亮氨酸。Leptin外显子2扩增片段上检测到AA、AB、BB三种基因型,BB基因型在阿勒泰羊群体中属于优势基因型;对两个群体进行基因型频率独立性χ2检验,差异极显著(P0.001),说明BB基因型是影响季节性发情的有利基因型。研究结果提示,绵羊品种中Leptin基因序列的差异性可能是造成绵羊季节性发情的原因之一,可作为常年发情绵羊品种选育的辅助标记。     

MC1R是控制鸡黑色素形成的候选主效基因

黑素皮质素受体1 (melanocortin 1-receptor, MC1R)基因是控制动物黑色素合成的重要基因.采用多聚酶链反应-单链构象多态性分析(PCR-SSCP)以及DNA测序的方法,在由丝羽乌骨鸡与明星肉鸡为亲本建立的中国农业大学资源家系群体鸡MC1R基因的编码区检测到3个单核苷酸多态位点,并对该单核苷酸多态性进行了分析.结果显示,鸡MC1R基因编码区引物3扩增片段多态性是由G→A（867位）点突变引起的,引物5扩增片段的多态性是由C→T（1 292位）与C→G（1 377位）两个点突变引起的,最后对单核苷酸多态性与肤色、肉色、胫色与内脏膜色等黑色素性状进行了卡方独立性分析,结果显示,MC1R基因编码区867处突变与鸡的肤色性状显著相关（P＜0.05）,1 292处突变与鸡的活体胫色性状显著相关（P＜0.05）,1 377处突变与鸡的肉色性状显著相关（P＜0.05）.研究表明,MC1R基因可能是鸡黑色素性状的主效基因或者与鸡控制黑色素性状的主效基因连锁.     

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。     

湖羊、东湖杂种羊POMC基因外显子3单核苷酸多态性及其与生长性状的关联分析

阿黑皮素原(Pro-opiomelanocortin, POMC)在动物采食和能量平衡调控中发挥重要作用, 文章对绵羊POMC基因外显子3进行扩增和测序, 筛选多态性位点, 并分析多态位点与湖羊和东弗里生×湖羊杂种羊生长性状的相关性。测序后发现湖羊POMC基因外显子3有2个单碱基突变(g.273 T/C和g.456 G/A), 根据273位点处发生的T/C突变, 建立PCR-RFLP分析方法, 并对162只湖羊和130只东湖杂种羊进行检测分析。结果发现, 在湖羊群体中检测到TT(0.469)、TC(0.438)和CC(0.093)3种基因型, 而在东湖杂种羊群体中仅检测到TT(0.754)和TC(0.246)两种基因型。POMC基因外显子3的273位点多态性与生长性状的相关性研究结果显示：湖羊群体中CC基因型个体的2月龄断奶重、4月龄尻高及TC基因型个体4月龄体长和管围均显著高于TT型个体(P<0.05); CC基因型个体的4月龄重、6月龄重极显著高于TT和TC基因型个体(P<0.01); CC基因型个体的4月龄体高和体长极显著高于TT型个体(P<0.01), 且显著高于TC基因型个体(P<0.05)。此外, CC型个体的管围极显著高于TT基因型个体(P<0.01)。东湖杂种羊群体中TC基因型个体的2月龄断奶重、4月龄重及4月龄体高、体长、胸深和管围都显著高于TT型个体(P<0.05), TC型个体的6月龄重极显著高于TT型个体(P<0.01)。研究结果表明, POMC基因外显子3与绵羊生长性状相关, C等位基因对体重及体尺性状的增加更有利。该结果为进一步探讨POMC基因作为绵羊生长性状的辅助选育标记奠定了基础。     

羊FSHR基因5′端转录启动调控区生物学特性

文章对小尾寒羊、滩羊和澳洲绵羊等繁殖性状不同的3种绵羊与排卵有关的FSHR基因5′端转录启动调控区进行了克隆和分析,通过对FSHR基因的15个转录调控元件序列进行比较,结果表明,羊不同品种FSHR基因的转录调控元件序列之间没有差异。这说明绵羊的品种与FSHR基因5′端转录启动调控区的相关性不强,排除了因转录调控元件突变而影响转录调节能力的可能性。      

三种绵羊BMP15基因第二外显子多态性及与产羔数的相关性分析

绵羊存在影响多胎性状的不同主效基因,选择影响Romney Hanna绵羊和Cambridge绵羊高繁殖力的骨形态发生蛋白15 (bone morphogenetic protein 15, BMP15)为候选基因,采用PCR-SSCP的方法检测BMP15基因外显子Ⅱ第747位点(T747→C)和755位点(T755→C)在蒙古羊、甘肃高山细毛羊、小尾寒羊三种绵羊母羊中的多态性,同时还研究了上述两处突变对三种绵羊产羔数的影响。表明:(1)一共检测到野生纯合型AA、突变杂合型AB (T747→C)、AC (T755→C)三种不同的基因型,AA为优势基因型,A为优势等位基因;(2)三种基因型在甘肃高山细毛羊中均被检测到,而蒙古羊和小尾寒羊中未检测出AB基因型;(3)突变杂合型蒙古羊(AC)比野生纯合型(AA)的平均产羔数多0.27只(p<0.05)。(4)AC的基因型频率,双羔母羊和多羔母羊均高于单羔母羊。根据以上实验推测,BMP15第755位点发生的T→C突变(AC型)对蒙古羊一胎产双羔影响十分显著,甘肃高山细毛羊中AC基因型的绵羊其产羔数有比AA基因型和AB基因型多的趋势,因此该位点可能是一个影响绵羊高繁殖力潜在的DNA标记。     

新疆4个绵羊品种内皮型一氧化氮合酶基因第8外显子的PCR-SSCP鉴定

目的:对新疆4个绵羊品种内皮型一氧化氮合酶(eNOS)基因第8外显子的多态性进行鉴定。方法:利用PCR-SSCP和测序的方法对76只中国美利奴羊、51只无角陶赛特羊、57只萨福克羊、37只哈萨克羊共4个绵羊品种进行单核苷酸多态性(SNP)检测,并用生物信息学方法对检测出的SNP进行统计分析。结果:在中国美利奴羊、无角陶赛特羊、萨福克羊、哈萨克羊中共检测到AA、AB、BB等3种基因型,AA基因型的频率分别为0.0526、0.0980、0.1754和0.2973,BB基因型的频率分别为0.6316、0.1961、0.5614和0.1892,AB基因型的频率分别为0.3158、0.7059、0.2632和0.5135。通过测序,在eNOS基因第8外显子上发现了一个新的SNP位点(ss974768653),位于绵羊eNOS基因第8外显子142 bp处(A142G)。结论:中国美利奴羊和哈萨克羊的多态性位点(P0.05)处于Hardy-Weinberg平衡状态,萨福克和无角陶赛特羊的多态性位点(P0.05)不处于Hardy-Weinberg平衡状态。     

贵州地方山羊CAST基因外显子6、7和8的多态性分析

为分析山羊CAST基因的多态性,筛选出对山羊肉质性状有显著影响的SNPs位点,本研究以黔北麻羊和贵州黑山羊为试验对象,构建DNA池,采用PCR产物直接测序法对2个品种山羊该基因的外显子6、7和8进行单核苷酸多态性检测,估算各SNPs等位基因频率,并利用在线软件预测突变前后的m RNA二级结构及理化特性。结果显示,所设计引物的扩增片段发现1个同义突变,位于外显子8中的T81C。利用生物信息学软件对外显子8中的T81C位点进行分析,结果表明这个SNPs位点导致编码的m RNA二级结构以及理化特性的改变。     

贵州地方山羊ADIPOQ基因外显子1和3多态性研究

为分析山羊ADIPOQ基因的多态性,筛选出对山羊繁殖性状有显著影响的SNPs位点,本研究以黔北麻羊和贵州黑山羊为试验对象,构建池DNA,采用PCR产物直接测序法对2个品种山羊该基因的外显子1和3进行单核苷酸多态性检测,估算各SNPs等位基因频率,并利用在线软件预测不同基因型的m RNA二级结构。结果显示,4对引物扩增片段均存在多态性,共发现5个单碱基突变,分别位于内含子1中的C109G,外显子3中的A730G、G1055A、A1691T和A2244G。利用生物信息学软件对外显子3中的A1691T、A2244G进行分析,结果表明2个SNPs位点均导致编码的m RNA二级结构发生改变。表明ADIPOQ基因在黔北麻羊和贵州黑山羊群体中存在较高的遗传多样性,ADIPOQ位点有望丰富两个山羊品种繁殖性状的研究内容。     

一例白变猕猴的酪氨酸酶基因及病因分析

目的 对一例不同寻常的白变猕猴进行病因研究,以探讨其色素病变的分子机理。方法 利用DH—PLC分析及测序技术对酪氨酸酶基因进行突变研究。并检测了毛发黑色素含量、微量元素含量等;结果 白猴血液指标无异常变化,酪氨酸酶基因发生多处杂合突变。白猴黑色素含量仅为正常猴的67％,同时Fe元素含量高于正常值。结论 白猴色素变化与酪氨酶基因的多态杂合突变有关,又与血液中Fe元素含量升高有关,但二者之间是否有联系,有何种联系却尚待进一步研究。     

Physiological and genetic characteristics of black-boned sheep (Ovis aries)

Here we report for the first time the discovery of sheep that have black bones and black muscles. The spectral pattern of pigment extracted from tissues of these black-boned sheep is similar to that of black-boned Chinese silky fowl. Additionally, black-boned sheep have significantly higher plasma colour, tyrosinase activity and kidney function than normal sheep. Synonymous nucleotide substitutions in the tyrosinase (TYR) and melanocortin 1 receptor (MC1R) genes were detected in black-boned sheep when compared with the corresponding sequences in normal sheep. In addition, a missense mutation (215T>C) in exon 2 of tyrosinase-related protein 1 (TYRP1) was detected in black-boned sheep, and this resulted in a putative valine-to-alanine substitution at codon 68 (Val68Ala).     

黑线仓鼠及其白化突变系TYR、TYRP1的基因表达水平比较分析

目的本研究旨在研究TYR、TYRP1基因在黑线仓鼠与白化突变系皮肤组织中的表达情况,探索其与白化毛色性状的产生是否具有相关性。方法以黑线仓鼠和白化突变系皮肤组织为研究对象,采用实时荧光定量PCR技术,分别得到黑线仓鼠与白化突变系TYR和TYRP1基因的相对表达量。结果黑线仓鼠皮肤中的TYR和TYRP1基因的mRNA相对表达量分别是白化突变系皮肤组织中的2.5倍和5.3倍。结论表明黑线仓鼠皮肤中TYR、TYRP1的基因表达水平存在表达差异,白化突变系TYR、TYRP1基因发生表达下调,揭示出了TYR、TYRP1基因的表达量与白化突变系白化性状的产生有关。     

Identification of a tyrosinase (TYR) exon 4 deletion in albino ferrets (Mustela putorius furo)

Albinism is due to a lack of pigmentation in hair, skin and eye, and has been shown to occur in several animal species. Mutations of the tyrosinase (TYR) gene account for albinism in domestic cats, rabbits, cattle, mice and rats. In this study, we demonstrate that a TYR mutation accounts for albinism in the ferret (Mustela putorius furo). The coding sequence of the five exons of TYR was determined in genomic DNA from wild-type pigmented 'sable' coloured and albino ferrets. It was not possible to amplify TYR exon 4 in albino ferrets originating from different breeds. The deletion of exon 4 in albino ferrets was confirmed by Southern blot hybridization of genomic DNA from albino and pigmented ferrets. This is the first report of a deletion of a TYR exon in a non-human mammal.     

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.     

Molecular variation in pigmentation genes contributing to coat colour in native Korean Hanwoo cattle

Pigmentation genes such as TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (previously TYRP2, or tyrosinase-related protein 2), ASIP (agouti) and MC1R (melanocortin receptor 1) play a major role in cattle coat colour. To understand the genotypic profile underlying coat colour in native Korean Hanwoo cattle and Angus black cattle, portions of the above-mentioned genes were amplified. Sequence analysis revealed variation in the TYRP1 (exon 5) and MC1R genes. Restriction enzyme analysis of these two genes could distinguish between different colours of Hanwoo cattle. Quantitative estimates of melanin and eumelanin in hair from three different-coloured Hanwoo phenotypes and Angus black showed significant differences at the breed and phenotypic levels. Finally, sequence variants in MC1R were associated with total melanin and eumelanin in breeds as well as in Hanwoo phenotypes.     

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.