黑素皮质素受体4(MC4R)基因多态性与动物生长性能的相关性研究

黑素皮质素受体4(Melanocortin 4 receptor,MC4R)是G蛋白偶联受体(G Protein coupled receptors,GPCRs)超家族的一个成员,在动物的体重、能量稳态和采食量的调控中具有重要作用。综述了MC4R基因的结构与定位、生物学功能以及MC4R基因与动物生长性能的相关性研究进展。     

Rapid Detection of SNPs in Buffalo MC4R Gene by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The study used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for rapid determination of single nucleotide polymorphisms (SNPs) in buffalo MC4R gene,which provided a foundation for constructing the marker-assisted selection(MAS)strategy.The cDNA sequence of buffalo MC4R gene was screened rapidly based on PCR amplification and sequencing method,and in which 13 SNPs were found.To further identify the reliability of screening system,8 SNPs were genotyped in 380 buffaloes using the MALDI-TOF-MS technology.The results revealed that 8 SNPs had an average of 98.0% in call rate,and in which three genotypes could be accurately distinguished.The average minor allele frequency (MAF),average heterozygosity and polymorphism information content (PIC) value of the 8 markers was 0.24,0.28 and 0.23,respectively.Our findings indicated that the rapid determination method of MALDI-TOF-MS with PCR and sequencing with highly accurate properties was a useful tool for identifying the SNPs of buffalo MC4R gene that laid the foundations for studying SNP genotype in buffalo genes associated production traits.     

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。     

鹅黑素皮质素受体-4基因的克隆与序列分析

黑素皮质素受体-4（Melanocortin receptor-4,MC4R）是其黑素皮质素受体MCR（MC1-5R）家族成员之一,属G蛋白偶联受体。它可与瘦蛋白、神经肽、α-黑素细胞刺激素等一起调节动物体重和采食量。参考鸡MC4R基因序列设计引物,克隆并测序了鹅MC4R基因。结果表明,鹅MC4R基因编码区全长996 bp,其核苷酸序列与鸡的同源性为95.3%,与人、牛、猪等哺乳动物同源性在75%-79%;其氨基酸序列与鸡的同源性达到98.5%。构建哺乳类、鸟类和鱼类MC4R基因核苷酸进化树显示,鹅较早地与哺乳类动物分化开来。分析MC4R蛋白的氨基酸残基特性参数表明,MC4R的7次跨膜结构与MC4R的亲水性区域、电荷密度以及氨基酸残基位于表面概率的变化规律相一致。     

黑素皮质素受体4基因多态性及与肌内脂肪含量相关性研究

利用PCR-RFLP技术检测75头乌金猪和41头长白猪黑素皮质素受体4(MC4R)基因的第7跨膜结构功能域D298N主效位点的遗传变异,并用最小二乘法模型分析MC4R基因对猪肌内脂肪含量的遗传效应。结果:乌金猪和长白猪MC4R基因第7跨膜结构功能域TaqⅠ-RFLP位点均存在多态性,基因型为AA、Aa和aa。长白猪AA基因型肌内脂肪含量高于Aa和aa基因型,但差异不显著(P0.05);AA基因型对PCR-RFLP的最小二乘值最大,证明该基因在该位点对肌内脂肪(IMF)含量的效应值最大。乌金猪肌内脂肪含量aa基因型显著高于AA和Aa基因型(P0.05),aa基因型对PCR-RFLP的最小二乘值最大,证明该基因在该位点对IMF含量的效应值最大。提示,乌金猪和长白猪的MC4R基因对肌内脂肪的调控是由不同的基因型所控制的。乌金猪可通过提高MC4R基因aa基因型的频率来增加肌内脂肪含量,达到改善乌金猪猪肉品质的目的。     

野猪黑素皮质素受体3基因的克隆及变异初步研究

为了揭示黑素皮质素受体3群体遗传变异,寻找新的遗传标记,对野猪黑素皮质素受体3基因进行了克隆和序列分析,并对发现的突变位点进行PCR—SSCP多态性检测。序列分析结果表明：野猪与家猪相比存在5个SNPs,其中有3个是在家猪中没有发现的;利用PCR—SSCP方法在各多态位点均检测到3种基因型,且基因型分布符合Hardy-weinberg定律,说明野猪具有独特的遗传信息。     

黑素皮质素受体1（MC1R）基因的研究进展

黑素皮质素受体1(melanocortin 1 receptor,MC1R)基因,也称为促黑素细胞激素受体(MSHR)基因,是由扩散位点(extension locus,E) 编码的,是控制动物黑色素合成的重要基因。作者对MC1R基因结构功能、作用机理、基因的定位与多态性检测进行了综述。     

金华猪促黑激素皮质素受体4基因多态性分析

应用PCR—RFLP方法对金华猪Ⅰ系(154头)、金华猪Ⅱ系(41头)和金华猪Ⅲ系(53头)的MC4R基因的1269∽1494bp区段进行扩增，并用TaqⅠ酶进行酶切，比较了MC4R酶切后基因型频率分布情况。结果表明，AA基因型频率在金华猪上较高达到0．9194，在Ⅱ系中最高达到0．9512，Ⅲ系次之达到0．9434，Ⅰ系最低达到0．9026，而金华猪的AB基因型和BB基因型频率都较低分别为0．0565和0．0242。     

4个黄牛群体黑素皮质素受体1(MC1R)基因变异研究

为了评价E(extension)座位对牛毛色性状的影响,采用DNA序列分析和PCR-RFLP技术对云南地区4个黄牛群体(3个本地群体和1个引进品种)的黑素皮质素受体1(MC1R)基因的编码区进行了基因特征分析和群体变异检测。结果,在文山黄牛、昭通黄牛和短角牛中发现E+和e2种等位基因,检测到E+/E+、E+/e和e/e3种基因型。在文山黄牛中E+和e等位基因的频率分别为0.54和0.46,在昭通黄牛中分别为0.70和0.30;而在迪庆黄牛中共检测到E+、ED和e3种等位基因,它们的频率分别为0.76、0.14和0.10,该群体存在E+/E+、ED/ED、E+/ED、E+/e和ED/e5种基因型,未发现e/e型个体。与所研究个体的毛色性状相联系,发现E座位对牛的毛色有重要影响,E+和ED等位基因与黑色表型有关,而e等位基因与红色表型有关,但黄色、棕色和红色表型还与其它座位基因相关联。     

冷应激下民猪黑素皮质素受体基因的表达变化

黑素皮质素受体3（melanocortins 3 receptors,MC3R）是G-蛋白耦联受体超家族的成员之一。研究采用Real-time PCR的方法,对冷应激下民猪腿肌内MC3R基因的表达变化情况进行了研究。结果表明,冷应激下,MC3R基因的表达出现了显著地上升（P〈0.05）。据此推测,民猪很可能是通过上调MC3R基因的表达来增加能量消耗而抵御寒冷气候的。     

基于飞行时间质谱法的单核细胞增生李斯特氏菌定性质控样品研制

通过真空冷冻干燥技术成功制备出单核细胞增生李斯特氏菌定性质控样品,并系统分析质控样品的均匀性和稳定性。通过优化冻干基质条件,得到制备质控样品的最佳条件。通过培养计数和基质辅助激光解吸电离飞行时间质谱技术,验证定性质控样品的均匀性和稳定性。结果表明：制备的单增李斯特菌定性质控样品为白色、质地均匀小球,均匀性验证实验质控样品培养计数结果F=0.567,小于临界值,表明均匀性一致;运输稳定性实验验证了质控样品在37、25?℃环境下含量几乎没有下降,计数稳定;贮藏稳定性实验验证了质控样品在－20?℃贮藏28?d后的复苏率为101.5%,在4?℃条件下贮藏28?d后的复苏率为99.6%,说明该样品均匀性和稳定性良好,可以作为阳性质控样品用于单增李斯特菌检测和质量控制。     

基质辅助激光解析电离飞行时间质谱在动物病原菌检测中的应用

应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）技术,对厦门口岸进口的1000多份动物及其产品的样品进行致病菌的检测和鉴定,共检出20多种致病菌,并对这些菌株同时用传统生化鉴定方法进行确认。结果表明MALDI-TOF-MS对未知细菌进行鉴定,较传统方法更加快速、准确,而且可以进行高通量检测,可以广泛应用于口岸动物检疫以及微生物检验实验室的日常检验。此外,将国内分离鉴定的各种参考菌株建立的常见致病菌MALDI-TOF-MS数据库与布鲁克公司的MALDI Biotyper数据库进行病原菌鉴定的比较,结果表明,实验室自建的MALDI-TOF-MS数据库可以取得更加准确地结果。     

MC4R基因研究进展

黑素皮质素受体-4(melanocortin-4 receptor,MC4R)是下丘脑腹内侧核分泌的一类肽类物质,为黑素皮质素受体家族5个亚型(MC1-5R)之一。在哺乳动物中,MC4R具有介导瘦蛋白(leptin)的功能,是一个调节能量平衡与能量动态平衡的重要信号分子,可与其内源性配体黑素皮质素激素(melanocortin,MC)或刺鼠色蛋白(agouti protein,agouti蛋白)和agouti相关蛋白(agouti related protein,AGRP)相结合,从而在控制食欲和体重稳态中起关键作用。因此,MC4R在人类肥胖研究中作为重要的调节因子倍受关注。最近有研究表明:MC4R的1232位点的G→A的突变与绵羊背膘厚度间存在关联,AG和AA型较GG型具有较高的背膘厚度。     

水牛黑色素皮质素1受体基因克隆、生物信息学分析及表达模式研究

本试验旨在对水牛黑色素皮质素1受体(MC1R)基因进行克隆、生物信息学分析及表达模式研究。参考牛MC1R基因(GenBank登录号:JN123363.1)序列设计引物,以本地沼泽水牛、白沼泽水牛、摩拉水牛和黄牛基因组DNA为模板,应用PCR方法扩增克隆MC1R基因片段并进行测序分析。运用QRT-PCR方法检测摩拉水牛、沼泽水牛、白沼泽水牛和黄牛皮肤组织中MC1R基因的表达模式,并通过Western blotting方法检测沼泽水牛和白沼泽水牛MC1R基因的蛋白表达差异。结果表明,应用PCR方法成功克隆了水牛MC1R基因,其编码区全长954 bp,共编码317个氨基酸。测序分析后发现沼泽水牛、白沼泽水牛、摩拉水牛和黄牛MC1R基因的核苷酸序列和氨基酸序列相似性很高。沼泽水牛与白沼泽水牛在476、618、881、930和931 bp位点上分别发生T→C、G→C、G→A、G→A和A→G突变,导致了沼泽水牛和白沼泽水牛第159位氨基酸由丝氨酸变成苯丙氨酸,第310位氨基酸由谷氨酸变成丙氨酸,第294位氨基酸由天冬氨酸变成丙氨酸,发生了非同义突变。QRT-PCR结果发现,MC1R基因在摩拉水牛、沼泽水牛和黄牛皮肤组织中的相对表达量均显著高于白沼泽水牛(P<0.05);Western blotting分析结果显示,沼泽水牛皮肤组织中MC1R蛋白的表达量高于白沼泽水牛。综上所述,白沼泽水牛MC1R基因的编码区发生氨基酸位点突变,且相对表达量和蛋白表达量均低于沼泽水牛,推测此为白沼泽水牛体内合成的黑色素缺失而导致毛色白化的主因。     

Cloning,Bioinformatics and Expression Pattern Analysis of Buffalo MC1R Gene

The aim of this study was to clone and analyze the expression pattern of buffalo MC1R gene.A pair of specific primers was designed according bovine MC1R sequence (GenBank accession No.:JN123363.1),with genome DNA of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle as template,MC1R was amplified by PCR.Then relative expression level of MC1R gene of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle was analyzed using QRT-PCR,and protein expression was detected by Western blotting method.The results showed that the 954 bp coding region of buffalo MC1R gene was successfully cloned and sequenced,which code for 317 amino acids.The MC1R gene nucleotide sequences and amino acid sequences of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle were highly conserved.Five polymorphic sites were found between White swamp buffalo and swamp buffalo of MC1R gene,including 476 T→C,618 G→C,881 G→A,930 G→A and 931 A→G,which caused three nonsynonymous mutation sites of Phe159Ser,Glu310Ala and Asp294Ala.The QRT-PCR result showed that relative expressions of MC1R gene of Murrah buffalo,swamp buffalo and Yellow cattle were significant higher than that of White swamp buffalo (P<0.05).The Western blotting results revealed that MC1R protein expression level in swamp buffalo was higher than that of White swamp buffalo.In conclusion,there were amino acids mutation in White swamp buffalo MC1R gene,and MC1R gene relative expression of White swamp buffalo was lower than that of other buffalo,which was the main reason of lacking of melanin production in White swamp buffalo.     

黔北麻羊MC4R基因多态性及其与生长性状的关联分析

【目的】探究黔北麻羊黑素皮质素受体-4（melanocortin receptor-4,MC4R）基因多态性及其与生长性状的关联性,为黔北麻羊的选种选育提供理论依据。【方法】选取196只6月龄黔北麻羊,利用DNA混合池结合Sanger测序筛选MC4R基因单核苷酸多态性（SNP）位点,并采用SPSS 22.0软件中的一般线性模型（GLM）对MC4R基因SNP位点与黔北麻羊生长性状进行关联分析。【结果】在黔北麻羊MC4R基因中发现4个SNPs位点：g.59345469 C>A和g.59346773 T>C位点分别位于5'-和3'-端非编码区（UTR）;g.59345871 A>C位点突变导致第37位天冬氨酸（Asp）变为丙氨酸（Ala）,引起RNA二级结构及蛋白质二级结构、三级结构发生明显改变;g.59346263 A>G位点突变导致第168位异亮氨酸（Ile）变为缬氨酸（Val）,对RNA二级结构和蛋白质二级结构有明显影响。关联分析结果显示,g.59345469 C>A位点对黔北麻羊体重、胸围和管围均有显著影响（P<0.05）;g.59345871 A>C位点对黔北麻羊体重、体高、管围和胸围均有显著影响（P<0.05）;g.59346263 A>G位点对黔北麻羊体高、体重和胸围均有显著影响（P<0.05）;g.59346773 T>C位点对黔北麻羊体重和胸围均有显著影响（P<0.05）。4个SNPs位点联合共检测到9种单倍型和21种双倍型,其联合对黔北麻羊体重、体高、胸围和管围均产生显著遗传效应（P<0.05）,纯合双倍型H5H5（AAAAGGCC）个体的生长性状优于其他双倍型。【结论】黔北麻羊MC4R基因存在4个SNPs位点,与黔北麻羊体重和胸围均存在显著关联,可作为黔北麻羊体重和胸围的遗传标记用于分子育种。     

陆川猪黑皮质激素受体4基因克隆及序列分析

试验旨在对陆川猪黑皮质激素受体4（melanocortin-4 receptor,MC4R）基因进行克隆及相关信息学分析。通过提取陆川猪背最长肌总RNA,采用RT-PCR、克隆等方法获得含目的基因MC4R的质粒pMD18-T-MC4R,经菌落PCR和测序鉴定正确后,应用相关生物信息学软件对陆川猪MC4R基因的理化性质、蛋白质的结构、修饰结构和亚细胞定位等进行预测分析。结果表明,MC4R基因CDS区长999 bp,编码332个氨基酸,与NCBI上公布的野猪MC4R基因序列中的CDS区存在4个碱基差异,其中175和906 bp处为同义突变,110和278 bp处为错义突变,分别引起第37位谷氨酸变为甘氨酸和第93位缬氨酸变为丙氨酸。同源性比对结果发现,MC4R基因在不同物种及进化的过程中具有较高的保守性。陆川猪MC4R蛋白有明显的疏水区域,不存在信号肽,但有7个跨膜结构域,其编码蛋白的二级结构元件有α-螺旋、延伸链、β-转角和无规则卷曲。修饰结构预测表明,MC4R蛋白存在多处N糖基化位点,但无O糖基化位点,可能主要分布于内质网和囊泡。本研究成功克隆了陆川猪MC4R基因,为更好地开发利用地方品种陆川猪及其繁育奠定理论基础。     

Application of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Identification of Stallion Semen Bacterial Contamination

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the cutting-edge methods currently applied in medical bacteriologic diagnostics. The aim of the study was to prove the possibility of applying MALDI-TOF MS to identify bacterial contamination in the ejaculate of stud stallions, which may cause infections to reproductive organs of mares following artificial insemination with cooled semen. A partial aim was to determine changes in the total count of microorganisms in long-term storage of ejaculate after its treatment with gentamicin and also without antimicrobial medication. Aerobic cultivation on Columbia agar was used to examine 26 semen samples from 13 horses; 31 different species of bacteria were isolated, which were identified by MALDI-TOF MS. The most frequently detected species came from Aerococcaceae, Staphylococcaceae, and Micrococcaceae families. The results of our work confirm that MALDI-TOF MS is a quick alternative method for identifying bacterial species that may contaminate stallion semen.     

The Correlation Analysis Between the SNPs of MC4R Gene and Meat Quality Traits in Yanbian Yellow Cattle

In order to explore the effects of the key gene of regulating cattle fat deposition on meat quality traits, screening candidate gene which affect beef fattening traits. This study employed 157 cattles (24 months old) with similar weight as research object, analyzed the correlation between SNPs of MC4R gene and meat quality traits in Yanbian Yellow cattle by PCR direct sequencing method. The results indicated that the mutation site of C→G had been found in MC4R gene 1 069 bp, which resulted in L (leucine) translated into V (valine). Protein secondary structure lost α-spiral and β-fold at the 166, 243 and 280 amino acid position, which resulted in the changes of spatial position of β-turns in tertiary structure. This site showed different degrees correlation with backfat thickness, lrightness, yellowness of Yanbian Yellow cattle. The backfat thickness and yellowness (3 d) of GG genotype were significantly higher than CC and CG genotypes (P <0.05). The lrightness of CC and CG genotypes were significantly higher than CC genotype (P <0.05). These results declared that the site had remarkably correlatived with Yanbian Yellow cattle relevant economic traits, and the mutation site could be used as a potential molecular marker.