藏绵羊生长激素释放激素基因内含子Ⅱ部分序列的分析

对藏绵羊生长激素释放激素(GHRH)基因内含子Ⅱ部分序列进行了PCR扩增和测序,用Genbank中的Blastn方法与普通牛相应基因序列进行了比对分析。结果表明,藏绵羊与普通牛GHRH基因内含子Ⅱ部分序列间同源性为92%;2个物种该基因内含子Ⅱ部分序列间的碱基变异类型主要表现为碱基转换和颠换,亦存在碱基插入和缺失。     

6个品种马DRD4基因克隆与序列比较分析

从6个品种马的血液中提取基因组DNA,根据GenBank中已发表的人、鼠、雪貂的DRD4 cDNA基因保守区序列,设计3对特异性引物,另1对由文献所得,对6个品种马的DRD4基因分4段进行PCR扩增,经电泳检测,分别呈4条特异条带,将其分别克隆入pMD19-T载体中,进行序列测定并拼接序列,得到包括所有内含子的DRD4基因序列。分析结果表明,马DRD4基因含有4个外显子和3个内含子,所得序列与人、鼠和狗同源区比对,发现其同源性都达到了75%以上。对6个品种马DRD4基因序列比较发现,不同品种马之间,第1内含子和第3外显子存在一定的变异,包括转换、颠换、插入/缺失和VNTR。     

野猪生长激素基因的克隆与序列分析

参考家猪GH基因序列[M17704.1],设计1对特异引物,用PCR方法从野猪基因组中扩增出1条长约1.8kb的DNA片段。PCR产物经PMD18-TVector载体转化感受态DH5α株大肠杆菌,获得重组克隆子。结果表明:野猪生长激素基因DNA序列长1792bp,含完整的5个外显子和4个内含子,与家猪、牛、山羊、河马、狗、小鼠和恒河猴的GH基因cDNA序列同源性分别为99.19%、89.6%、89.7%、94.6%、92.9%、86.9%、77.3%。其cDNA所编码氨基酸与牛、山羊、河马、狗、小鼠和恒河猴同源性分别为98.74%、83.9%、83.9%、92.5%、90.7%、81.6%、65.5%。所测野猪生长激素基因序列与13个家猪品种的GH基因序列比较,检出54个核苷酸变异位点(外显子12个,内含子36个,侧翼区6个),其中24个为各家猪品种内所不具有的新变异位点,即野猪种群特有变异位点。在外显子的12个变异位点中,有8个可导致氨基酸残基的改变。这些结果,表明了野猪GH基因在进化过程中的保守性和多态性。     

藏猪生长激素基因核苷酸多态性分析

本文应用PC R方法,体外扩增了藏猪5个个体的生长激素(GH)基因64～2022位之间1959 bp的片段(包含完整的5个外显子和4个内含子),并进行了序列测定。结合网上下载的猪GH基因同源区段序列进行比较分析,结果表明:藏猪GH基因有较丰富的核苷酸多态性;检出的24个核苷酸突变位点中有6个发生在外显子区,外显子2内有4个突变位点,导致2个氨基酸变异,外显子3和外显子4内各有一个突变位点,特别是外显子4内这个突变位点引起了1个氨基酸变异;单核苷酸转换(21个位点)大于颠换(5个位点)。     

羊驼生长激素基因的克隆与序列分析

应用RT-PCR和PCR技术克隆得到了羊驼生长激素(Growth hormone,GH)基因,包括完整的编码区812 bp 的cDNA序列和1 800 bp 的DNA序列.两者比对后证明羊驼GH基因DNA序列由5个外显子和4个内含子组成,编码216个氨基酸的GH前体蛋白,其中包括26个氨基酸的信号肽和190个氨基酸的成熟肽.序列比较结果表明,羊驼GH基因序列与骆驼、猪、马、犬和猫等哺乳动物类似,但羊驼GH基因的启动子不是哺乳动物的典型TATA盒,而同骆驼一样为CATA盒.在DNA和cDNA水平以及推导的氨基酸水平,均与骆驼的同源性最高.通过GH氨基酸的功能位点分析推测,羊驼生长激素与人、猪等大多数动物的生长激素无明显功能上的差异.     

猪附红细胞体MSG1基因的克隆及序列分析

为研究猪附红细胞体MSG1基因的遗传变异特点,从上海地区3个屠宰场采集自然感染附红细胞体的猪血液,提取基因组DNA,根据GenBank已经发表的猪附红细胞体MSG1基因序列(登录号AM407404.1),设计引物进行PCR扩增,将扩增产物连接到pMD18-T载体上,进行序列测定和分析.结果共获得了82条猪附红细胞体MSG1基因序列,序列长度为1 011 bp,共编码336个氨基酸.同源性分析显示与GenBank中已经发表的猪附红细胞体MSG1基因核苷酸序列相似性为96.5％～98.3％,氨基酸的相似性为97.9％～99.1％0.MSG1基因核苷酸序列的系统进化分析表明,上海地区猪附红细胞体分离株与GenBank登录的德国54/96分离株序列的亲缘关系较远.     

羊MyoG基因内含子的扩增

以羊MyoG基因的内含子为研究对象,参考羊和猪MyoG基因的mRNA序列信息,选取保守基因序列设计上下游引物,采用PCR方法扩增羊MyoG基因内含子1和内含子2,克隆后进行序列测定.序列分析比较发现内含子1的片段与猪的同源性为71.9%,与人的同源性为65.7%;内含子2的片段与猪的同源性为77.9%,与人的同源性为62.7%.说明该基因的内含子在不同物种间具有较高的同源性.     

绵羊肺炎支原体溶血素TlyC基因的克隆及分子特征分析

根据GenBank报道的绵羊肺炎支原体(MO)基因序列,设计特异性引物,对MO新疆分离株(MO XJ)溶血素TlyC基因进行克隆及测序;并对该基因及其编码蛋白进行分子特征分析,并与其他支原体相应序列进行同源性分析,构建该基因系统进化树。结果表明,TlyC基因全长为1 239bp,编码426个氨基酸,其中第1~23位氨基酸残基为信号肽,编码区含有跨膜结构域和CBS结构域,二级结构以α-螺旋为主。系统发生分析表明,MO与猪肺炎支原体232株和絮状支原体的相应序列亲缘关系较近,而与其他种支原体的亲缘关系较远。研究首次克隆了MOTlyC基因,为了解MOTlyC生物学功能及其致病中的作用奠定了基础。     

含第1内含子的猪生长激素cDNA基因的构建

以 PCR方法扩增了猪生长激素 ( p GH)基因的第 1内含子 ,经测序表明 ,扩增的 p GH基因第 1内含子的序列与 Vize发表的序列存在 6个碱基的差异 ,同源性达 97.5%,说明 p GH基因第 1内含子具有多态性。此外 ,还发现此内含子存在有转录因子 ATF的结合位点。利用第 2外显子的 Hinf 位点 ,采用将 3个 DNA片段一步连接的方法 ,将扩增的第 1内含子插入到 p GHc DNA的正确位置上 ,从而构建出含第 1内含子的p GHc DNA。     

基于测序技术的海南地方猪TLR2基因多态性分析

为了解海南地方猪TLR2基因的多态性,本研究采用PCR法从五指山猪、临高猪和屯昌猪3种海南地方猪的血液中克隆Toll样受体2(TLR2)基因,并进行测序及序列分析。结果显示,3种海南地方猪TLR2基因的扩增长度均为2649bp,编码区长2358bp。3种猪TLR2基因序列的种内比对结果显示,五指山猪种内有7个核苷酸位点存在多态性,2处位于编码区;屯昌猪种内有5个核苷酸位点存在多态性,均在编码区外;临高猪种内有4个核苷酸位点存在多态性,1处位于编码区。种间比对结果显示3种猪有12个核苷酸位点存在多态性,其中3处为错义突变,导致氨基酸改变。同源性分析结果显示,3种猪的TLR2基因序列高度保守,同源性在99.6%~99.9%之间。蛋白质结构预测分析显示,海南猪TLR2基因在876、1454位点呈现的AG突变均引起了其蛋白质二级结构和三级结构的改变,提示可能存在TLR2功能的变化。本研究填补了海南地方猪TLR2基因多态性空白,为深入研究TLR2基因多态性与猪疫病易感性的关系提供基础研究资料。     

Adipocyte fatty-acid binding protein is closely associated to the porcine FAT1 locus on chromosome 4

We identified 22 polymorphisms in the adipocyte fatty-acid binding protein (FABP4) gene, a strong positional candidate gene for the FAT1 locus in porcine chromosome 4. The most informative polymorphism, an insertion/deletion in intron 1, together with a single nucleotide polymorphism in intron 3, was genotyped in a cross between Iberian and Landrace pigs. After performing QTL, single marker, and haplotype analyses, we showed that there were at least 2 quantitative trait genes in the FAT1 region and that the FABP4 polymorphism was tightly associated to fatness. A comparison of allelic frequencies in a panel of pig breeds suggested that the Del2634C polymorphism was under indirect selection. We also showed that FABP4 is tightly associated to fatness but not growth. Furthermore, a haplotype analysis suggests that there is genetic heterogeneity at the FAT1 locus within the Landrace breed.     

Polymorphism of IGFBP2 Gene and Its Association with Meat Quality Traits in Pigs

This study was aimed to analyze the genetic variation of insulin-like growth factor binding protein 2 (IGFBP2) gene effect on meat quality traits in pigs. A pair of primer was designed,which based on DNA sequence of IGFBP2 gene (GenBank accession No.:BV727778). The genetic polymorphism of IGFBP2 gene was detected by PCR-RFLP in Sujiang pig, Jiangquhai pig and Landrace pig, and the association between IGFBP2 gene polymorphism and meat quality traits in three pig breeds was analyzed. The results showed that three genotypes (AA, AB and BB) were detected. The Chi-square test results showed that the genotype distribution of three pig breeds was in Hardy-Weinberg equilibrium (P > 0.05). The polymorphism information content (PIC) were moderately polymorphic in three pig breeds. According to the association analysis of different genotypes, significant difference was found in color value, intramuscular fat content and marbling, the color value (except for Jianquhai pig) and intramuscular fat content of AA genotype were significantly higher than AB and BB genotypes (P < 0.05), the marbling of AA and AB genotypes was significantly higher than BB genotype (P < 0.05). In conclusion,the polymorphism of IGFBP2 gene intron 3 had significant effects on meat quality traits, and was candidate gene for the potential impact on meat quality traits of pigs.     

猪IGFBP2基因遗传多态性及其与肉质性状的关联分析

试验旨在探究胰岛素样生长因子结合蛋白2（insulin-like growth factor binding protein 2,IGFBP2）基因遗传变异对猪肉质性状的影响,为猪品种选育提供遗传学依据。参考GenBank中猪IGFBP2基因序列（登录号：BV727778）设计1对引物,采用PCR-RFLP技术检测IGFBP2基因在苏姜猪、姜曲海猪、长白猪中的遗传多态性,并分析基因座多态性与猪肉质性状的相关性。结果发现,3个猪种IGFBP2基因内含子3均具有Msp Ⅰ酶切多态性,分别检测到3种基因型：AA、AB和BB;χ²适合性检验显示,3个猪种IGFBP2基因基因型分布均处于Hardy-Weinberg平衡状态（P > 0.05）;多态信息含量（PIC）分析显示,3个猪种均为中度多态。IGFBP2基因不同基因型与肉质性状关联分析显示,3个猪种不同基因型肉色、肌内脂肪含量、大理石纹差异显著,AA基因型肉色（姜曲海猪除外）、肌内脂肪含量均显著高于AB、BB基因型（P < 0.05）;AA、AB基因型大理石纹显著高于BB基因型（P < 0.05）,其余指标间差异不显著（P > 0.05）。综上,苏姜猪、姜曲海猪、长白猪IGFBP2基因内含子3多态性对肉质性状影响显著,可作为潜在影响猪肉质性状的候选基因。     

松辽黑猪H-FABP基因位点多态性研究

为检测松辽黑猪H-FABP基因位点多态性，应用PCR-RFLP技术(HinfI、HaeIII和MspI 3种限制性内切酶)检测了62头松辽黑猪5’-上游区和第二内含子的遗传变异情况。结果发现松辽黑猪在MspI多态位点上表现为单一的AA型；而在HinfI多态位点上表现为多态性，等位基因H的基因频率为0.7177，在HaeIII位点上也表现出多态性，等位基因d的基因频率为0.6210。研究结果表明松辽黑猪H-FABP基因5’-上游区和第二内含子存在多态性。     

4猪种Nramp1基因第6内含子多态性研究

天然抗性巨噬蛋白(Nramp)基因是与人、鼠的一些病原微生物的易感性和抗性有关的重要候选基因。为了研究猪Nramp1基因的多态性,利用PCR-RFLP技术检测了杜洛克、大白猪、长白猪和合作猪共270头个体Nramp1基因第6内含子NdeⅠ酶切位点多态性。结果表明,4个猪种群共检测到3种基因型(AA、AB和BB),其中AB基因型为杜洛克、大白猪和长白猪的优势基因型;AA基因型为合作猪的优势基因型。经卡方适合性检测,杜洛克、合作猪和长白猪处于Hardy-Weinberg平衡状态(P>0.05),大白猪处于Hardy-Weinberg不平衡状态。多态信息含量分析显示,Nramp1基因的第6内含子NdeⅠ酶切位点在各猪种表现出中度多态性。     

Polymorphism of SNP Site rs80894395 and its Association with the Body Size of Xiang Pig

To explore the functional genes affecting the growth and development of Xiang pig, a single nucleotide polymorphism (SNP) site rs80894395, located at intron 10 of polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2) gene was screened out by Illumina Porcine SNP60K chip, which showed a weak association with the abdomen circumference of pig.Further, the genotypes of site rs80894395 in 520 female Xiang pigs were detected using allele-specific PCR(AS-PCR) method, taking 43 Yorkshire pigs and 36 Kele pigs as control groups.All of CC, CT and TT genotypes were detected from the three pig breeds.The dominant genotype of Xiang pig was CT genotype and it was positively weak-associated with its body measurement indexes including body weight, body length, and abdomen circumference.The CC genotype was dominant genotype in Yorkshire pig, and the frequency of T allele was extremely significant decreased compared with that of Xiang pig (P<0.01).There was no difference in the frequencies of CC and CT genotypes in Kele pig, and its allele frequencies was not significantly different compared with that of Xiang or Yorkshire pig breeds (P>0.05).A splice-site nearby site rs80894395 was found out using bioinformatics prediction software, which might affect the splicing process of GALNT2 gene.It suggested that site rs80894395 might have a connection with the growth and development of Xiang pig.     

香猪SNP位点rs80894395的多态性及其与体尺指标之间的关联研究

为了探究影响香猪生长发育的功能基因,本试验利用Illumina Porcine SNP60K芯片筛选出N-乙酰氨基半乳糖转移酶2(polypeptide N-acetylgalactosaminyltransferase 2,GALNT2)基因内含子10中一个与腹围呈弱相关的单核苷酸多态性(single nucleotide polymorphism,SNP)位点rs80894395。在此基础上应用等位基因特异性PCR(allele-specific PCR,AS-PCR)技术检测520头雌性香猪群体中该位点的基因型,并与43头大白猪、36头柯乐猪相比较。结果表明,从3个猪群体中检测到rs80894395位点的CC、CT和TT3种基因型;其中香猪群体以CT为优势基因型,且基因型与香猪的体重、体长和腹围呈弱的正相关;大白猪群体以CC基因型为主,T等位基因频率较香猪极显著减少(P<0.01);柯乐猪群体中CC与CT基因型频率相等,与香猪和大白猪的基因频率间的差异不显著(P>0.05)。生物信息学分析表明,rs80894395位点可能影响GALNT2基因的剪接过程。rs80894395与香猪的生长发育可能有一定的联系。     

猪水通道蛋白7基因第4内含子多态性分析

本研究旨在探讨国内外猪种水通道蛋白7(aquaporin 7,AQP7)基因多态性,以期为进一步研究猪AQP7基因与脂肪沉积性状的相关性提供理论依据。采用PCR-RFLP方法检测了猪AQP7基因第4内含子G/A在6个猪群中的多态性分布。检测结果表明,经PstⅠ酶切后发现了GG、AG和AA 3种基因型。在莱芜猪、里岔猪、鲁莱猪、杜洛克猪和大约克猪5个猪种中G均为优势等位基因,而在沂蒙猪中A为优势等位基因。χ²检验结果发现,基因型分布在莱芜猪、鲁莱猪和沂蒙猪群体达到了哈代－温伯格平衡(P>0.05),而里岔猪、杜洛克猪和大约克猪群体中均未达到平衡状态(P<0.05);不同基因型在群体间的分布差异极显著(P<0.01)。群体遗传特性分析表明,有效等位基因数在0.3084~0.4953之间,多态信息含量在0.2609~0.3726之间,属于中度多态。本研究结果提示,地方猪种、引进猪种和培育猪种AQP7基因内含子4的基因型分布存在明显差异,A等位基因作为脂肪沉积的有利等位基因尚需进一步验证。     

合作猪最佳饲养方式探讨

高原地方优良小型合作猪又叫蕨麻猪,在改变了传统的放牧到圈养的过程中肉品质更得到了关注,本文对不同饲养方式下合作猪的生长发育特性、屠宰性能和肉品质变化进行对比分析。结果表明,1-6月龄舍饲型合作猪生长发育较快,与放牧型合作猪比较,体重、体高、体长、胸围差异极显著（P〈0．01）;两种合作猪肉的肉色、大理石纹、PHlh、PH24h、失水率、系水力和风味物质含量二者无显著差异,舍饲合作猪肉的干物质、蛋白质含量显著高于放牧组（P〈0．05）;表明合作猪舍饲圈养方式对肉品质无显著影响,对合作猪进行舍饲圈养是可行的。     

Polymorphism and Tissue Expression Patterns of CKMT2 Gene in Tibetan and Yorkshire Pigs

In order to explore the polymorphism site and difference of expression level of creatine kinase,mitochondrial 2 (CKMT2) gene in muscle segments of Tibetan and Yorkshire pigs,in this experiment,Sanger sequencing method was used to screen and genotype the single nucleotide polymorphism (SNPs) in the 1 000 bp region upstream of the initiation codon of CKMT2 gene in 50 Tibetan and 60 Yorkshire pigs.The expression of CKMT2 gene was detected by Real-time quantitative PCR in heart,back fat and longissimus dorsi muscle of 180-day-old of 10 Tibetan and 10 Yorkshire pigs.The results showed that 5 SNPs (G-357A,T-469C,G-649T,A-784G and A-953G) were found in the upstream 1 000 bp region of the initiation codon of CKMT2 gene,the allele frequency of Tibetan pigs at all sites were significantly different from those of Yorkshire pigs.These SNPs were related to skeletal muscle growth and development,muscle energy metabolism and anti-hypoxia.The mRNA expression of CKMT2 gene in longissimus dorsi muscle and heart of Tibetan pigs were extremely significantly higher than Yorkshire pigs (P<0.01),and the mRNA expression in back fat of Tibetan pigs was extremely significantly lower than Yorkshire pig (P<0.01),which indicated that CKMT2 gene could positively regulate anti-hypoxia and muscle tissue production,and negatively regulate fat production,the analysis results were consistent with the breed characteristics of Tibetan and Yorkshire pigs.This experiment results provided some new ideas for further exploration of pig growth and development,fat deposition and hypoxia adaptability.