黄颡鱼MHC class II基因全长的克隆及饲料维生素D3对其组织表达的影响

为进一步丰富鱼类MHC class II基因的研究, 同时也为进一步探讨低磷饲料中添加维生素D₃对鱼类免疫功能可能的影响, 实验利用RACE (Rapid-amplification of cDNA ends) 即cDNA末端快速扩增技术, 成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex, MHC) class II基因, 全长1074 bp, 其中ORF (Open reading frame)708 bp, 编码236个氨基酸, 5′UTR (5′端非翻译区)78 bp, 3′UTR (3′端非翻译区)259 bp。进行氨基酸序列比对分析得到: 黄颡鱼MHC class II基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%, 与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用qPCR对黄颡鱼MHC class II基因进行组织表达分析, 结果表明MHC class II在小肠、肝脏、鳃中表达较高; 在肌肉、鳍条中表达较低; 而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D₃显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class II基因的分子结构、组织表达以及维生素D₃的作用, 在降低磷排放的同时, 为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。     

中国美利奴绵羊MHC222G18BAC克隆的基因筛选与结构分析

绵羊主要组织相容性复合体(MHC)是与控制绵羊抗病性和易感性紧密连锁的基因簇.为了深入了解该类基因的组成与结构,利用中国美利奴绵羊细菌人工染色体( BAC)文库MHC区段克隆222G 18,经BsaJ Ⅰ酶切后制备α-32p放射性探针,通过噬菌斑原住杂交技术筛选中国美利奴绵羊cDNA文库,经过两轮杂交筛选,获得12个cDNA阳性克隆,经测序、比对等生物信息学分析确定获得7条与免疫相关的序列,其中3条具有完整的编码序列.利用SIM4软件将7条序列定位到BAC克隆上,结果显示绵羊MHC区段的表达序列多为断裂基因且跨度很大,可能是形成其基因多样性的重要原因之一.     

光棘球海胆(Mesocentrotus nudus)TGF-β基因克隆及其对海水酸化的响应

旨在明确光棘球海胆(Mesocentrotus nudus)转化生长因子-β(transforming growth factorβ,TGF-β)基因的序列及结构信息,探明该基因在海水酸化胁迫下的表达模式。利用同源克隆和cDNA末端快速扩增(RACE)技术首次获得光棘球海胆TGF-β基因(命名为MnTGF-β)的全长cDNA序列。结果显示:(1)光棘球海胆TGF-β基因的cDNA全长为2 299 bp,其中5'非编码区长度为745 bp,3'非编码区长度为261 bp;开放阅读框(ORF)长度为1 290 bp,编码430个氨基酸,相对分子量为48.31 kD,理论等电点为5.34。(2)同源性及系统进化分析显示,光棘球海胆MnTGF-β蛋白序列与紫球海胆(Strongylocentrotus purpuratus)SpTGF-β2蛋白序列高度保守(同源性97.69%)。(3)实时荧光定量PCR(qRT-PCR)检测发现,MnTGF-β基因在光棘球海胆性腺、体腔液、肠、围口膜、管足5种组织中均有表达,其相对表达量从高到低依次为:管足围口膜肠体腔液性腺。(4)与自然海水组(pH 8.06±0.01)相比,当海水△pH为-0.3时,随酸化时间延长,MnTGF-β基因在光棘球海胆性腺中的相对表达量呈先升高而后极显著降低(P0.01)再极显著升高(P0.01)趋势,在管足中的相对表达量呈现极显著上调趋势(P0.01),而在肠组织中的相对表达量则先极显著降低(P0.01)而后显著升高(P0.05);当海水△pH为-0.4和-0.5时,随酸化时间的延长,MnTGF-β基因在光棘球海胆性腺和管足中的相对表达量呈现先降低后升高的趋势,而在肠组织中的相对表达量则呈现极显著降低趋势(P0.01)。     

蒙古绵羊 tnp1基因cDNA全编码区的克隆及序列分析

过渡蛋白1基因(tnp1)是圆形精子细胞特异表达的基因.绵羊tnp1基因的DNA序列至今尚未报道.为了开展绵羊圆形精子细胞标记基因的研究,根据其他物种tnp1基因cDNA的保守序列设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp1基因cDNA全编码区.该基因cDNA 长246 bp,包含一个168 bp的ORF,编码含有54个氨基酸的多肽链.DNA序列测定结果与牛的核苷酸序列比对,同源性为94.0%.绵羊tnp1基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础.     

绵羊BTG1基因的半定量RT-PCR及生物信息学分析

B细胞易位基因1(BTG1基因)是BTG/TOB基因家族的成员之一,在动物细胞的增殖和分化中起重要的作用．利用牛BTG1基因的mRNA序列与绵羊的EST数据库进行Blast检索,并通过序列拼接和逆转录RT-PCR方法首次获得绵羊BTG1基因的部分cDNA序列（GenBank登录号FJ444829）,其片段长度为1 358 bp,包括完整的开放阅读框516 bp,编码171个氨基酸．半定量PCR研究结果表明：BTG1基因在小尾寒羊和陶赛特羊的10种组织中均表达,并具有一致的表达趋势．同源分析结果表明,绵羊BTG1蛋白的氨基酸序列中存在BTG/TOB的保守结构域,并且该蛋白在不同物种间具有很高的保守性．通过生物信息学预测BTG1蛋白功能,发现绵羊BTG1蛋白存在1个跨膜结构域、8个磷酸化位点和1个特异性蛋白激酶磷酸化位点．蛋白质结构同源建模分析表明,绵羊BTG1蛋白具有BTG/TOB蛋白家族的典型空间结构．     

藏羚羊PGC-1α基因编码区的克隆与分析

以藏羚羊(Pantholops hodgsonii)及同海拔分布的藏系绵羊(Tibetan Sheep)的心肌组织为材料,提取总RNA,利用逆转录聚合酶链反应(RT-PCR)技术扩增出过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)的基因编码区cDNA片段,与载体连接构建重组质粒,经转化、扩增培养、鉴定后测序。利用生物信息学方法分析显示,藏羚羊和藏系绵羊的PGC-1α基因编码区长度均为2 349 bp,编码797个氨基酸(GenBank登录号分别为:JF449959和JF449960);与其他脊椎动物PGC-1α基因的核苷酸及氨基酸序列相似性达到90%以上;其包含RNA/DNA结合位点、RNA识别基序(RRM)、与核呼吸因子1(NRF-1)及肌细胞增强因子2C(MEF2C)相互作用的区域、富含丝氨酸/精氨酸的结构域、负调节功能结构域、LXXLL模体以及TPPTTPP和DHDYCQ两个保守序列,14个氨基酸差异性位点位于以上部分功能结构域中;此外,磷酸化位点的预测提示藏羚羊可能存在一个潜在的蛋白激酶G的磷酸化位点(第329位的苏氨酸)。本研究成功克隆出了藏羚羊PGC-1α基因的编码区序列,为从能量代谢角度深入探讨藏羚羊适应高原的分子生物学机制提供了新的思路。     

Molecular Cloning and Sequence Analysis of PGC-1α cDNA in Tibetan Antelope

以藏羚羊(Pantholops hodgsonii)及同海拔分布的藏系绵羊(Tibetan Sheep)的心肌组织为材料,提取总RNA,利用逆转录聚合酶链反应(RT-PCR)技术扩增出过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)的基因编码区cDNA片段,与载体连接构建重组质粒,经转化、扩增培养、鉴定后测序.利用生物信息学方法分析显示,藏羚羊和藏系绵羊的PGC-1α基因编码区长度均为2 349 bp,编码797个氨基酸(GenBank登录号分别为:JF449959和JF449960);与其他脊椎动物PGC-1α基因的核苷酸及氨基酸序列相似性达到90％以上;其包含RNA/DNA结合位点、RNA识别基序(RRM)、与核呼吸因子1( NRF-1)及肌细胞增强因子2C(MEF2C)相互作用的区域、富含丝氨酸/精氨酸的结构域、负调节功能结构域、LXXLL模体以及TPPTTPP和DHDYCQ两个保守序列,14个氨基酸差异性位点位于以上部分功能结构域中;此外,磷酸化位点的预测提示藏羚羊可能存在一个潜在的蛋白激酶G的磷酸化位点(第329位的苏氨酸).本研究成功克隆出了藏羚羊PGC-1α基因的编码区序列,为从能量代谢角度深入探讨藏羚羊适应高原的分子生物学机制提供了新的思路.     

一个麻疯树RING型锌指蛋白基因JcRFP1的克隆及表达

首次从麻疯树胚乳cDNA丈库中克隆得到一个RJNG型锌指蛋白基（GenBank登录号为JF920726）,命名为JcRFP1。该cDNA长度为728bp,包含编码JcRFP1蛋白的完整开放阅读框（516bp）。脚,基因在麻疯树各器官中均检测到表达且表达量依次为：叶〉茎〉花〉果实〉种子〉根。将克隆到的JcRFP1基因的cDNA序列连接到表达载体pET32a（＋）上,导入BL21（DE3）pLysS菌株,成功诱导表达相对分子质量为33．2kDa的可溶性融合蛋白。该融合蛋白免疫新西兰大白兔,得到效价为1：6500的抗血清。研究表明,JcRFP1蛋白具有体外泛素连接酶E3活性,在麻疯树体内可能参与油菜素甾醇信号转导途径。     

藏系绵羊BMP2基因克隆及组织表达分析

本研究旨在克隆藏系绵羊BMP2基因序列,并获得其生物学特征,同时阐明其组织表达规律。提取藏系绵羊皮肤组织的总RNA,利用RT-PCR技术克隆BMP2基因序列,同时利用荧光定量PCR技术检测该基因在不同组织中的表达情况。结果表明,获得藏系绵羊BMP2基因序列1 217 bp,其中CDS区长度为1 188 bp,编码395个氨基酸。BMP2蛋白有36个磷酸化位、6个糖基化位点和1个跨膜结构,属于不稳定不溶性碱性蛋白,主要在细胞核和线粒体中发挥生物学作用。藏系绵羊BMP2氨基酸序列与山羊、绵羊和牛等的氨基酸同源性很高。BMP2基因在藏系绵羊肺脏组织中表达水平最高,极显著高于其他组织(p0.01)。本研究结果将为进一步研究藏系绵羊BMP2基因的结构和功能提供参考资料。     

草地藏系绵羊IGF-Ⅰ基因的克隆及序列分析

采用Trizol法从草地藏系绵羊肝脏中提取总RNA,用反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增并克隆测序,获得长度为589 bp的胰岛素样生长因子(IGF-Ⅰ)cDNA序列,共编码70个氨基酸的成熟肽.分析显示,草地藏系绵羊与其他绵羊IGF-Ⅰ的cDNA编码区序列存在1个碱基差异,同源性达99.78%.     

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。      

Analysis of sheep T-cell receptor β-chain heterogeneity

 We analyzed nucleotide and deduced amino acid sequence heterogeneity of sheep T-cell receptor β-chain cDNAs isolated from an anchored-polymerase chain reaction library. Evaluation of 34 individual rearrangements has defined 18 new β-chain variable region sequences which have been clustered into 13 families. Presumptive allelic polymorphisms of four of these variable regions have been defined, as well as ten distinct β-chain joining region sequences. The present analysis indicates that sheep T-cell receptor β-chains are composed of characteristic leader, variable, joining, and constant region sequences, and that imprecise joining and N-region addition contribute significantly to diversity in the third hypervariable region. Thus, it appears that sheep, like all other mammals studied to date, employ somatic rearrangement of multiple germline genes to create β-chain heterogeneity. These findings have allowed us to estimate the diversity of the sheep T-cell receptor β-chain variable region repertoire, and they provide information that will permit the evaluation of the role that specific T-cell populations play in naturally occurring and experimental diseases of sheep. Received: 20 October 1997 / Revised: 20 April 1998     

Effects of the sheep-chewing louse (Damalinia ovis) on the epidermis of the Australian merino

Frozen longitudinal skin sections taken from the dorsal thoraco-lumbar region of adult Merino sheep that were infested with the sheep-chewing louse were examined by light microscopy. The epidermis of infested sheep exhibited acanthosis due to hyperplasia of the stratum spinosum, and orthokeratosis. The thickness of the uncornified epidermis, the stratum corneum, and the sudanophilic region were significantly greater (P less than 0.005) than equivalent regions in louse-free Merinos and the results suggest that a positive correlation exists between the thickness of each region and the level of louse infestation. The results indicate that the variance in region thickness was greater in lousy than in louse-free sheep (P less than 0.005).     

Assignment of the growth hormone gene locus to 19q26-qter in cattle and to 11q25-qter in sheep by in situ hybridization

The growth hormone gene locus (GH) of cattle and sheep was mapped to a chromosomal region in each species by using in situ hybridization. The probe employed was an 830-bp cDNA sequence from the ovine growth hormone gene. Based on QFQ chromosome preparations, our results show that the GH locus is on cattle chromosome 19 in the region of bands q26-qter and in sheep on chromosome region 11q25-qter. The GH assignments together with previous localizations of type I cytokeratin genes (KRTA) and one homeobox (HOX2) gene in cattle and one type I cytokeratin gene (KRTA) in sheep identify a strongly conserved chromosomal segment on human chromosome 17, bovine chromosome 19, and sheep chromosome 11.     

Influence of hydrothermal factors on wool development of Tan sheep in China

Hydrothermal factors in Ningxia, an arid and semi-arid area in Northwestern China, were examined to determine their influence on the geographical differentiation of the wool quality of Tan sheep lambskin. Prospective areas for extension of the Tan sheep industry were also investigated. Two sheep groups were sampled; one consisted of artificial insemination flocks (1241 lambs sampled from 25 flocks) and the other of natural mating flocks (1009 lambs from 38 flocks). Six phenotypical traits were measured for each lamb and six hydrothermal factors were collected from the meteorological stations located in the sampling areas. A significant correlation was found between wool characteristics of the lambskin and hydrothermal conditions. Data further indicated that among all the hydrothermal factors measured, those causing the geographical differentiation of soil and vegetation were the major factors responsible for the corresponding differentiation of the lambskin and wool quality of Tan sheep. Thermal factors were mostly positively correlated with wool quality, while the influence of moisture was negative. Three eco-geographical regions were defined based on a combination of the hydrothermal conditions and corresponding wool characters: (i) typical region or super-suitable region, (ii) sub-typical region or suitable region and (iii) transitional region. The delimitation could be used as a basis for the extension of the Tan sheep industry. It is also suggested that the wool quality of lambskin of Tan sheep in these three regions could be improved by means of controlled breeding and selection.     

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

11个绵羊品种MSTN基因非翻译区的变异

利用PCR-RFLP技术对特克塞尔羊、夏洛莱羊、小尾寒羊、蒙古羊、乌珠穆沁羊、阿勒泰羊、呼伦贝尔羊、塔什库尔干羊、多浪羊、湖羊和岗巴羊11个品种的345个个体的肌肉生长抑制素(Myostatin, MSTN)基因非翻译区(UTR)的变异进行了多态性分析。结果表明大小为271 bp和1 003 bp的扩增片段经限制性内切酶MboⅡ和BsaⅠ酶切后表现多态, 经卡方检验所有品种在该基因座位均处于平衡状态(P>0.05), 3种基因型在11个绵羊品种中的分布差异极显著(P<0.01)。通过限制性内切酶HpyCH4Ⅳ 酶切实验, 证明我国9个地方绵羊品种不存在特克塞尔绵羊中发现的导致肌肉发达的SNP位点, 并在3′UTR区发现了个别碱基突变位点能够形成miRNA作用的靶基序, 测序表明3′UTR区的突变频率较高。     

Polyceraty (multi‐horns) in Damara sheep maps to ovine chromosome 2

Polyceraty (presence of multiple horns) is rare in modern day ungulates. Although not found in wild sheep, polyceraty does occur in a small number of domestic sheep breeds covering a wide geographical region. Damara are fat‐tailed hair sheep, from the south‐western region of Africa, which display polyceraty, with horn number ranging from zero to four. We conducted a genome‐wide association study for horn number with 43 Damara genotyped with 606 006 SNP markers. The analysis revealed a region with multiple significant SNPs on ovine chromosome 2, in a location different from the mutation for polled in sheep on chromosome 10. The causal mutation for polyceraty was not identified; however, the region associated with polyceraty spans nine HOXD genes, which are critical in embryonic development of appendages. Mutations in HOXD genes are implicated in polydactly phenotypes in mice and humans. There was no evidence for epistatic interactions contributing to polyceraty. This is the first report on the genetic mechanisms underlying polyceraty in the under‐studied Damara.     

威宁绵羊GHR基因克隆及组织表达

本试验以6月龄、1周岁和2周岁的威宁绵羊公母羊为研究对象,利用分子手段对威宁绵羊GHR基因编码区序列进行克隆,并进行生物信息学分析,此外,本试验同时采用实时荧光定量PCR的手段对GHR基因在威宁绵羊不同性别及不同生长阶段心脏、肝脏、脾脏、肺脏、肾脏及背最长肌6个组织中mRNA水平上的表达规律进行探究,结果显示:威宁绵羊GHR基因CDS区序列长度为1 905 bp,发现2个SNP位点。GHR基因在威宁绵羊各组织均有不同程度的表达,在不同性别、不同生长阶段相同组织中的表达具有一定的显著性差异。     

A single nucleotide polymorphism on chromosome 10 is highly predictive for the polled phenotype in Australian Merino sheep

The aim of this study was to fine map the genomic location of the Horns locus in the Australian Merino sheep population and to identify markers that can be used to predict the horn phenotype. A linkage disequilibrium analysis of horn data from Australian Merino sheep mapped the Horns locus to a small region on chromosome 10. A single nucleotide polymorphism in the region was found to be highly predictive for the polled phenotype in an experimental population of Merino sheep. This was owing to a dominance effect of one of the alleles when inherited maternally. It was suggested that a genetic test would provide a good predictor of the polled phenotype. Finally, an evaluation of industry data showed that the SNP is at very different frequencies in Poll Merino sheep that have been bred for polledness (based on phenotype alone) compared with the Merino sheep breed.