从江香猪DRD1基因多态及其生物信息学分析

为揭示猪DRD1基因多态性,本研究以从江香猪为研究对象,以外三元(杜×长×大)杂交猪及贵州宗地花猪为对照,采用DNA池和直接测序技术,筛查DRD1基因所有外显子区SNPs位点;利用生物信息学软件估算等位基因频率并预测SNPs位点对m RNA二级结构的影响。结果表明,在DRD1基因中共筛查到1个SNPs位点,位于5'UTR区为T-182G;从等位基因频率角度分析,从江香猪与贵州宗地花猪估算得到的等位基因频率较一致,与外三元(杜×长×大)杂交猪却存在较大差异;利用RNA secondary structure prediction软件预测,发现m RNA二级结构发生明显变化     

从江香猪MC4R基因多态性及生物信息学分析

[目的]旨在对3个群体猪MC4R基因进行SNPs筛选,为从江香猪选种选育提供一定的理论基础。[方法]以从江香猪作为研究对象,野猪×从江香猪二元杂交猪和杜×大×长外三元杂交猪作为对照,构建品种DNA池,PCR扩增MC4R基因外显子、内含子和3’非编码区序列,采用直接测序法对3个群体的MC4R基因进行单核苷酸多态性检测,利用生物信息学软件预测不同多态性位点对MC4R基因mRNA二级结构和蛋白质二级结构的影响。[结果]在3个群体中共检测出10个SNPs位点,其中,C860T、G1392A和G1577A位于第2外显子区;G1699A、T1702A、A1870C、G1875A、C2025T、G2073A和A2111T位于3’非编码区序列。C860T为同义突变,G1392A和G1577A为错义突变,分别导致了精氨酸变为组氨酸、天冬氨酸变为天冬酰胺。经分析软件预测,处于第二外显子中的3个SNPs位点对MC4R基因的mRNA二级结构、蛋白质二级结构有一定的影响。[结论]DNA池结合测序技术检测到MC4R基因外显子、内含子和3’非编码区序列共10个SNPs。     

贵州白山羊GOLA-DQA1基因多态性及生物信息学分析

目的:通过筛选贵州白山羊GOLA-DQA1基因SNPs位点,为进一步研究MHC基因多态性与山羊免疫性状的相关性提供依据。方法:选取同一生长环境中的贵州白山羊母羊157只构建品种DNA池并进行测序,结合SNPs位点测序峰高比值估算等位基因频率,利用软件对不同基因型的RNA和蛋白质二级结构进行预测。结果:在贵州白山羊DQA1基因发现6个SNPs位点G+2930A(同义突变)、T+2959C(Asn→Ser)、G+3061A(Pro→Leu)、A+3082G(Me→Thr)、C+3463A(Trp→Cys)、C+3525T(Arg→His)。生物信息学分析发现,C+3463A变异位点使RNA二级结构更加稳定;A+3082G变异导致自由能增加,稳定性降低。蛋白质结构预测发现除A+3082G基因型与原序列具有相同的二级结构外,其他基因型均引起蛋白质二级结构的改变。结论:贵州白山羊GOLA-DQA1基因具有丰富的多态性,但SNPs位点等位基因频率差异较大。     

不同牛种STAT5B基因启动子多态性分析

目的:试验旨在筛选STAT5B基因启动子区SNP及研究其对启动子功能元件的影响,为地方牛种选种选育提供一定理论依据.方法: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,直接测序筛选SNP位点.结果:STAT5B基因5'调控区及第1外显子存在3个SNPs位点,分别为:T-181C、C-31G、T+119C.生物信息学软件预测得到STAT5B基因核心启动子区和转录因子结合位点,SNP位点导致5个转录因子结合位点消失,而产生8个新的转录因子结合位点.软件未发现可能的CpG岛范围,但STAT5B基因RNA二级结构和最小自由能在突变后显著改变.结论:DNA池结合测序技术可快速筛选SNP位点,STAT5B启动子区存在功能性SNP位点.     

贵州山羊品种DQA1基因第3外显子遗传变异分析

目的:通过筛选3个贵州地方山羊种DQA1基因Exon3的SNPs位点并做相应的遗传变异分析,为今后山羊抗病育种及地方山羊品种的选育提供理论依据。方法:采用构建品种DNA池与直接测序的方法,对三个贵州地方山羊种群共514只个体的DQA1基因外显子3进行遗传变异分析。结果:在3个山羊品种中共筛选得到4个SNPs,G71A(同义突变)、T100C(Asn→Ser)、G202A(Pro→Leu)、A223G(Met→Thr)。生物信息学分析发现,G202A位点变异虽未引起mRNA二级结构的变化,但最小自由能降低,结构稳定性增强;DQA1基因Exon3的不同基因型的蛋白质二级结构中均不含有α螺旋。结论:三个贵州地方山羊种DQA1基因外显子3中含有较丰富的多态性,G202A位点变异在mRNA二级结构及蛋白质二级结构中与其他基因型具有较明显的差异。     

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。     

贵州黑山羊、贵州白山羊TFB1M基因第7外显子SNP研究

目的:研究TFB1M基因编码区多态性,筛选出对山羊肉质有显著影响的SNPs位点,以期为山羊品种选种选育提供科学依据。方法:采用DNA池结合PCR直接测序技术分析TFB1M基因在贵州本地山羊的多态性,估算等位基因频率,利用在线软件分析TFB1M基因RNA二级结构及其蛋白二级、三级结构。结果:在TFB1M基因中筛选到2个SNPs:A76G和T221G,其中A76G为同义突变;T221G错义突变,导致编码的半胱氨酸(Cys)变为甘氨酸(Gly)。RNA二级结构最小自由能改变,贵州黑山羊RNA二级结构稳定性增强。贵州黑山羊β折叠链增加1%,α螺旋增加4%,混乱度也上升1%。β转角减少4,α螺旋增加6,自由卷曲减少3,延伸链增加1。结论:SNPs位点对TFB1M基因RNA二级结构及其蛋白结构均有影响。     

癫痫相关基因SCN1A启动子区多态性位点的生物信息学分析

SCN1A是多种神经系统疾病的致病基因,该基因的精确表达对于维持神经系统正常功能非常重要.为了认识SCN1A启动子区多态性位点(single nucleotide polymorphisms,SNPs)的保守性及其功能意义,应用生物信息学方法分析了目前已发表位于SCN1A启动子区的11个SNPs,分别命名S1～S11.分析结果表明,S3、S5和S7等位基因频率没有人种差异性,其余SNP等位基因频率均有人种差异性;接近核心启动子的SNPs要比远离核心启动子SNPs的保守程度高,提示靠近核心启动子的SNPs影响SCN1A基因表达的概率可能越大;大部分SNPs祖传等位基因在哺乳动物中是保守的(S3除外),暗示这些SNPs新生等位基因有可能在人类进化过程起到一定的作用:启动子分析软件预测发现,含S2、S4、S8及S9等4个SNPs不同等位基因的同一序列分别存在不同转录因子结合元件,而含S1和S11不同等位基因的同一序列都只能预测到含其中一个等位基因的序列存在转录因子结合元件,这些差异可能是SNPs影响SCN1A表达的重要原因之一.这些分析将为进一步研究SCN1A启动子区SNPs与神经系统疾病的相互关系打下基础.     

贵州黑山羊和黔北麻羊LEPR基因第7、8、10和18外显子的多态性分析

为探究LEPR基因的遗传多态性,丰富山羊LEPR基因的研究,本研究以贵州黑山羊和黔北麻羊为试验材料,运用DNA池结合直接测序方法进行LEPR基因SNPs位点的筛选,从而对突变的SNPs位点进行RNA二级结构以及所编码蛋白质的二级结构和三级结构进行生物信息学分析。在试羊LEPR基因中共发现4个SNPs,分别为Exon7-T81C(同义突变)、Exon8-C39T(同义突变)、Exon10-A70G(Asn-Ser)和Exon18-C94T(Ser-Leu)。分析表明,4个位点突变前后的等位基因频率、mRNA二级结构的最小自由能、LEPR蛋白质二级结构和三级结构均有改变。结果表明,LEPR基因拥有较为丰富的遗传多态性。     

猪基质Gla蛋白基因序列多态及生物信息学分析

选择具有明显品种差异的3个猪品种(可乐猪、白香猪和大约克)构建品种DNA池,采用PCR产物直接测序法研究公猪MGP基因5’侧翼调控区、外显子1共1 112 bp范围内可能与精液品质相关的序列多态性,快速筛查到两个SNPs(C-849T、C-895T).进一步利用生物信息学分析MGP基因5’侧翼调控序列.预测出评分为95分以上的转录因子结合位点有31个,发现了CpG岛.C-849T和C-895T的变异则使白香猪比可乐猪和大约克多出了两个HSF可能的结合位点(85.9分).1个TATA可能的结合住点(91.4分),1个cdxA可能的结合位点(92.9分).研究结果为进一步研究MGP转录效率以及对猪精液品质的影响奠定基础.     

五指山猪IGF2基因5′调控区单核苷酸多态性分析

利用PCR产物直接测序法, 对五指山猪、滇南小耳猪、香猪、梅山猪和大白猪共60个样本的IGF2基因5＇调控区部分片段的单核苷酸多态性进行了研究。找到13个SNP, 分别是: C5872T、C5888T、A5976G、C6010T、T6029A、C6037T、C6043T、C6063T、C6112T、C6164T、G13520A、G13563A和G13669A。T6029A为T←→A碱基颠换, A5976G、G13520A、G13563A和G13669A为A←→G转换, 其他均为C←→T转换。针对13个SNP位点得到23种组合基因型。统计各位点等位基因和基因型以及各组合基因型在总群体与各品种内的分布频率, 发现3个小型猪在A5976G、C6164T和G13669A位点上的优势等位基因均分别为G、T和A, 而梅山猪和大白猪的优势等位基因均分别为A、C和G; H19型为3个小型猪的特征组合基因型, 而另两个猪品种为H15型。同时对123头五指山猪IGF2基因C5888T位点进行了PCR-RFLP分析, 研究表明该位点C为优势等位基因(0.8536), CC为优势基因型(0.7235)。卡方检验表明该位点处于Hardy-Weinberg平衡状态。这些结果可为五指山猪等小型猪的生长发育规律、矮小机制等方面的研究提供遗传学依据。     

Identification of Mx gene nucleotide dimorphism (G/A) as genetic marker for antiviral activity in Egyptian chickens

Egyptian chickens, representing 2 breeds and 7 strains, were genotyped using the PCR-RFLP and sequencing techniques for detection of a non-synonymous dimorphism (G/A) in exon 14 of chicken Myxovirus resistance (Mx) gene. This dimorphic position is responsible for altering Mx protein’s antiviral activity. Polymerase Chain reactions were performed using Egyptian chickens DNA and specific primer set to amplify Mx DNA fragments of 299 or 301?bp, containing the dimorphic position. Amplicons were cut with restriction enzyme Hpy81. Genotype and allele frequencies for the resistant allele A and sensitive allele G were calculated in all the tested chickens. Results of PCR-RFLP were confirmed by sequencing. The three genotypes AA, AG, GG at the target nucleotide position in Mx gene were represented in all the studied Egyptian chicken breeds and strains except Baladi strain which showed only one genotype AA. The average allele frequency of the resistant A allele in the tested birds (0.67) was higher than the sensitive G allele average frequency in the same birds (0.33). Appling PCR-RFLP technique in the breeding program can be used to select chickens carrying the A allele with high frequencies. This will help in improving poultry breeding in Egypt by producing infectious disease-resistant chickens.     

Polymorphisms in BMP-2 gene and their associations with growth traits in goats

BMPs plays an important role in skeletal development. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of caprine BMP-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of BMP-2 gene were investigated, only 3′ flanking region of BMP-2 gene showed polymorphism. The alignment between nucleotide sequences of AY714781.1 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (AY714781.1: g.A172G) in 3′ flanking region of BMP-2 gene, which constructed three genotypes (AA, AB, BB). The frequencies of allele A and genotype AA were dominant in all three breeds. Frequency of allele B in Haimen goat breed was significantly lower than that of the other two breeds. The genotype distributions were in good agreement with Hardy-Weinberg equilibrium (P>0.05) in each breed. The PIC (Polymorphism Information Content) values in three populations were 0.3261, 0.2558, 0.1663 for Boer goat, Xuhuai White goat and Haimen goat respectively. It was indicated that individuals of Boer goats with genotype BB were significantly higher than those of individuals with genotype AA in body trunk index (P<0.01). No polymorphism was detected in the exon3.     

Porcine skeletal muscle differentially expressed gene CMYA1: isolation, characterization, mapping, expression and association analysis with carcass traits

To investigate the differences in gene expression between some obese and lean pig breeds, differential display of mRNA was employed in our previous research. One differentially expressed EST ( BI596262 ) was further identified as the porcine cardiomyopathy associated 1 ( CMYA1 ) gene because of its homology to the human CMYA1 gene. The full-length DNA of the porcine CMYA1 gene encompasses 9379 bp, including a complete open reading frame encoding 1839 amino acid residues, a 158-bp 5'-untranslated region and a 630-bp 3'-untranslated region. The porcine CMYA1 gene was assigned to chromosome 13 by the radiation hybrid panel (IMpRH). The porcine CMYA1 gene was expressed only in the striated muscle. Single nucleotide polymorphism (SNP) scanning in the coding region identified one synonymous mutation (c.1053C>T) and three missense mutations, c.1394A>G (p.His465Arg), c.1751A>G (p.Asp582Gly) and c.3290C>A (p.Thr1097Asp). The allele frequencies were tested among about 200 unrelated pigs from several pig breeds. Linkage mapping was further conducted with the SNP c.1751A>G (p.Asp582Gly) in a Berkshire × Yorkshire resource family and this confirmed that porcine CMYA1 is closely linked with Sw344 (distance  =  2 cM, LOD score is 129.47), an interesting region harbouring a QTL for back fat thickness. Association analysis in our experimental pig population showed that different genotypes of CMYA1 gene were associated with different back fat thicknesses ( P  <   0.05). Our results suggest that the porcine CMYA1 gene has effects on porcine back fat deposition and further investigation will be necessary to illustrate the underlying mechanisms.     

Single nucleotide polymorphism analysis of exons 3 and 4 of IGF-1 gene in pigs

The IGF-1 gene has been implicated as a candidate gene for the regulation of pig growth traits. We analyzed exons 3 and 4 of IGF-1 gene polymorphisms of the Banna mini-pig (28), the Tibetan mini-pig (30), the Junmu pig (55), and L. Yorkshire species (50) using PCR-SSCP. Three genotypes in exon 3 and 6 genotypes in exon 4 were observed, among which, one single nucleotide polymorphism, G201A, on exon 3 and two single nucleotide polymorphisms, A440G and T455C, on exon 4 were found. Statistical analysis of genotype frequencies revealed that the A allele was dominant in the large pig at the G201A locus (PIC = 0.20-0.34), and the AT alleles were dominant in the large pig at the A440G and T455C loci (PIC = 0.30-0.60). The genotype distribution between the various groups was significantly different (P< 0.01), with the highest heterozygosity seen in Junmu pigs at 0.223 and the lowest seen in L. Yorkshire at 0.098. The genetic distance of the Junmu pig from the L. Yorkshire is the smallest, the distance from the Tibetan miniature pigs is larger, and the distance from the Banna mini-pig is the largest. The IGF-1 gene polymorphism and heterozygosity results from various pig breeds indicate that IGF-1 is substantially polymorphic with significant difference of the polymorphic distribution and expression levels among various pig breeds. This information provides a theoretical basis for the genetic background of miniature pigs but also provides means to breed improved pig varieties.     

猪CAST基因的单核苷酸多态性及其对肉质性状的效应

CAST基因作为肉质性状的主要候选基因.以80头外来猪和190头地方猪为材料,在CAST基因内含子24上检测到两个多态性位点(A916G 和C1633G ).在916位点上,长白猪和大白猪以A基因为优势基因,其频率分别为0.88和1.00;莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪以B基因为优势基因,其频率分别为0.93, 0.97, 0.78和0.68.在1633位点上,长白猪和大白猪以C基因为优势基因,其频率分别为0.82和0.79:莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪以D基因为优势基因,其频率分别为1.00, 1.00, 0.88, 0.78.在试验猪种中,共检测到6种单倍型(AACC,AACD,AADD,ABCC,BBCC,BBDD).单倍型分布的多重比较结果表明,外来猪种(长白猪和大白猪)与地方猪种(莱芜猪,大薄莲猪,沂蒙黑猪和里岔黑猪)比较差异极显著(P < 0.01).固定效应模型分析结果表明,嫩度,屠宰45 min后pH值和滴水损失单倍型间差异显著(P < 0.05).最小二乘分析结果表明,外来猪种与地方猪种在嫩度,屠宰45 min后pH值和滴水损失间差异显著(P < 0.05).BBDD单倍型个体与其它单倍型个体比较,嫩度及滴水损失差异显著(P < 0.05);AADD,BBCC,BBDD单倍型个体与其它单倍型个体比较,屠宰45 min后pH值差异显著(P < 0.05).因此,在育种过程中将CAST基因应用于标记辅助选择,将有利于改善猪肉品质,加快育种进程.     

猪前列腺素内过氧化物酶2(PTGS2)基因的遗传变异及其与繁殖性状相关性研究

前列腺素内过氧化物酶2是花生四烯酸合成前列腺素的限速酶,在包括排卵、受精、着床、分娩等一系列生殖过程中起着重要作用,因而编码该酶的基因是影响繁殖性状的重要候选基因。通过PCR—RFLP分析前列腺索内过氧化物酶2基因在15个中外不同繁殖性能猪种中的遗传变异,结果表明,不同类型中国地方猪种和外来商业猪种往此摹因位点上存在丰富的多态性,繁殖性能相埘较好的江海型、华北型和华中型猪种中A等位基因表现为优势等位基因,卡方检验显示其基因频率分布与西方商业猪种及繁殖性能较低的高原型藏猪和华南型猪种差异均极为显著（P〈0．001）。利用二花脸x杜洛克资源家系F2群体分析该基因与繁殖性状的相关性,在180头F2代母猪群体中,未能进一步证实该基凶位点对总产仔数、产活仔数和死胎数3个繁殖性状存在显著影响（P〉0．05）,但携带优势等位基因4的个体趋向于拥有较高的总产仔数、产活仔数和偏低的死胎数,鉴于该基因的重要作用,基于全基因序列的SNP扫描和大样本群体的相关性分析仍很有必要。     

猪Mx1基因第14外显子多态性分析及新突变位点的 发现

采用PCR-RFLP方法对国内外7个猪种Mx1基因第14外显子的多态性进行分析, 共检测到3个等位基因, 6种基因型。其中杜洛克中仅存在AA基因型, 苏太猪中存在全部基因型, 只有在梅山猪和具有梅山猪血统的苏太猪中出现基因型BB。所有猪种中, 只有在地方猪种和培育猪种中出现等位基因B, 所有猪种除松辽黑猪外均以A为优势等位基因。卡方检验结果表明, 不同猪种间基因型分布差异较大, 梅山猪和松辽黑猪与其他所有猪种的基因型频率差异极显著(P＜0.01) , 苏太猪与除皮特兰猪外的所有猪种的基因型频率差异也极显著(P＜0.01) , 淮猪与杜洛克和约克夏这两个国外猪种基因型频率差异不显著(P＞0.05), 而与皮特兰和其他地方猪种的基因型频率均存在极显著差异(P＜0.01) 。通过测序在扩增片段中新发现了3种类型的碱基突变, 前2个分别导致了Thr和Glu向Ala和Arg的替换, 最后一个突变不引起氨基酸的变化, 且后两个突变位点为BB基因型所特有。     

The porcine fat mass and obesity associated (FTO) gene is associated with fat deposition in Italian Duroc pigs

In humans, common variants in the fat mass and obesity associated ( FTO ) gene are associated with body mass index and obesity. Here we sequenced exon 4, parts of introns 3 and 4 and two portions of the 3'-untranslated region of the porcine FTO gene in a panel of nine pigs of different breeds and identified three SNPs. Allele frequencies of the g.276T>G ( AM931150 ) mutation were studied in seven pig breeds. This mutation was used to linkage-map FTO to SSC6. Association analyses between the g.276T>G polymorphism and several traits [pH of semimembranosus muscle and estimated breeding values (EBV) for average daily gain, back fat thickness, lean cuts, ham weight and feed:gain ratio] were carried out in 257 sib-tested Italian Large White pigs. Only feed:gain ratio showed P  <   0.05. A selective genotyping approach was applied, analysing two extreme and divergent groups of Italian Large White pigs selected on the basis of back fat thickness EBV (50 with most positive and 50 with most negative values). Fisher's exact test (two-tailed) was not significant when comparing the allele frequencies of these two groups. The same approach was used in the Italian Duroc breed for which two extreme and divergent groups of animals were selected according to visible intermuscular fat EBV. Differences of allele frequencies between these two groups were highly significant ( P  <   0.00001, P  <   0.001 and P  <   0.0001, considering all animals or only two- or three-generation unrelated animals respectively), indicating association between the analysed FTO marker and intermuscular fat deposition.