Study on Complete Mitochondrial Genome of Oula Sheep (Ovis aries)

Mitochondrial genome has been widely used in species identification and gene conservation. In the present study, the complete mitochondrial genome of Oula sheep (Ovis aries ) was determined using next-generation sequencing. This genome was 16618 bp (NCBI accession number: KU575248) and contained 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a typical control region. The overall nucleotide composition was 33.7％ A, 27.4％ T, 25.8％ C, and 13.1％ G, with a total A+T content of 61.1％. The phylogenetic analysis of selected sheep breeds showed that Oula sheep were clustered within branch A and originated from approximately 6 ka. This mitochondrial genome will provide valuable information for molecular genetic research of Oula sheep.     

Microbial Community in the Forestomachs of Alpacas (Lama pacos) and Sheep (Ovis aries)

Four 2-yr old alpacas ((48±2.3) kg) and four 2-yr old sheep ((50±1.7) kg) were used to study the pH and microbial community of forestomach from alpacas (Lama pacos) and sheep (Ovis aries) fed fresh alfalfa as the sole forage at low altitude (793 m). The forestomach fluid was taken anaerobically via the esophagus. The electric pH meter and quantitative polymerase chain reaction systems were used to study the the pH and microbial community of forestomach. The results showed that the mean pH of forestomach fluid from alpacas was higher than that from sheep (P<0.01). The percentages of methanogens and Ruminococcus flavefaciens to total bacterial were lower in the forestomach of alpacas than that in the rumen of sheep, while the percentage of fungi and Fibrobacter succinogenes were higher. The percentage of protozoa was similar in the forestomach of alpacas and sheep. These differences can partly explain the reason that alpacas were lower methane production than sheep.     

Study on the Complete Mitochondrial Genome of Qaidam Cattle(Bos taurus)

Qaidam cattle(Bos taurus) is an important endemic breed in Northwest China, which is mainly distributed in the northwest of Qinghai-Tibet Plateau. It has strong adaptive ability to plateau and swamp conditions, such as cold tolerance and insect resistance. In this study, the first complete mitochondrial genome of Qaidam cattle was reported. The circular double-stranded genome is 16 340 bp in size, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a D-loop region. The overall nucleotide composition is 33.4% A, 27.2% T, 26.0% C and 13.4% G, with a total A +T content of60.6%. The gene order and composition are similar to those of other B. taurus breeds. Molecular phylogenetic analysis indicated that Qaidam cattle was split as an independent clade and nested within Asian cattle breeds.     

玻璃化冻存对羊软骨细胞的影响

对玻璃化冻存后羊Ovis aries软骨细胞的存活率和琥珀酸脱氢酶（SDH）活性进行了初步研究。结果表明：细胞存活率随着冻存时间的延长而逐渐下降,但下降速度不明显;SDH活性在玻璃化冻存1 ~ 20 d变化幅度较大,20 ~ 30 d下降趋势减弱,保持相对稳定。从各项指标来看,认为玻璃化溶液VSb组的冻存效果最好,在经过5,10, 15,20和30 d的冻存后,其存活率分别为85.47% ± 1.78%,80.73% ± 1.81%,78.62% ± 2.06%, 76.35% ± 2.58%和73.83% ± 1.49%,细胞SDH活性吸光度值分别为0.49 ± 0.064,0.444 ± 0.073,0.394 ± 0.039,0.354 ± 0.082和0.339 ± 0.053。该玻璃化冻存方法对长期冻存羊软骨细胞具有一定的实际应用意义。图4表2参17     

建昌鸭线粒体基因组全序列分析

参照近源物种线粒体基因组序列设计引物15对,用PCR产物直接测序法测得建昌鸭(Anas platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示:建昌鸭线粒体基因组全长16 606 bp,碱基A、T、G、C的含量分别为29.21%、22.19%、15.77%、32.83%,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码调控区(D-loop),基因组结构和已知雁形目鸟类的完全相同.基于线粒体13个蛋白质编码基因序列,采用邻接法构建雁形目8个物种的系统进化树,所得结果与传统的系统分类基本一致.     

建昌鸭线粒体基因组全序列分析

参照近源物种线粒体基因组序列设计引物15对,用PCR产物直接测序法测得建昌鸭(Arms platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示:建昌鸭线粒体基因组全长16 606 bp,碱基A、T、G、C的含量分别为29.21%、22.19%、15.77%、32.83%,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码调控区(D-loop),基因组结构和已知雁形目鸟类的完全相同.基于线粒体13个蛋白质编码基因序列,采用邻接法构建雁形目8个物种的系统进化树,所得结果与传统的系统分类基本一致.

Abstract：

The complete mitochondrial genome of Jianchang duck(Anas platyrhychos)was determined by direct DNA sequencing based on the PCR products amplified with 15 pairs of primers designed on related species.The entire genome was found to be 16 606 bp in length and contain 37 genes(13 protein coding genes,2 rRNA and 22 tRNA)and a non-coding control region(D-loop).The content of the nucleotides A,T,G and C was 29.19%,22.20%,15.80%and 32.81%,respectively.The characteristics of the mtDNA of Jianchang duck appeared to be almost identical to those of the other birds of the Anseriformes known.A phylogenetie tree of 8 bird species based on the 13 protein coding genes was then constructed by neighbor-joining analysis.The results were largely consistent with the conclusion in traditional morphological and taxonomic studies.     

五指山猪线粒体基因组全序列测定与分析

采用PCR及TA克隆测序技术,获得五指山猪线粒体基因组全序列(KJ909516),并对其特征进行具体分析。结果表明,五指山猪线粒体基因组序列全长16 689bp,碱基组成为A(34.7%)、C(26.2%)、G(13.3%)、T(25.8%),包含13个蛋白编码区,2个rRNA,22个tRNA和1个非编码控制区(D-Loop区)。13个蛋白质编码基因中,ND2、ND3和ND5起始密码子为ATA,ND4L起始密码子为GTG,其余均为ATG。蛋白编码基因ND1和ND2终止密码子为TAG,Cyt b终止密码为AGA,COXⅡ、COXⅢ、ND3和ND4的终止信号为不完全密码子T,其余均为TAA。22个tRNA中除tRNASer(AGY)缺少DHU臂外,其余tRNA均可形成典型三叶草结构。线粒体控制区全长1 254bp,含有22个长度为10bp的串联重复序列ACGTGCGTAC。5种中国小型猪蛋白质编码基因的比对结果说明,五指山猪在ND2和ND5基因上与其他4个猪种存在明显差异,可为能量代谢差异研究提供分子学依据。     

福建黄兔线粒体基因组全序列测定与分析

福建黄兔是中国优良的小型兼用品种，从线粒体水平分析福建黄兔在兔形目动物中的系统发育地位，对探究其起源、进化和种群分类具有重要意义。利用PCR高保真扩增技术和生物信息学软件对福建黄兔线粒体基因组进行获取、拼接与注释。结果发现福建黄兔线粒体基因组全长17 000 bp(Genbank No.MN518689)，主要包括tRNA基因（22个）、rRNA基因（2个）、蛋白编码基因（13个）和控制区（1个）4 部分，基因排列紧密。除控制区，共存在37个编码基因，有9个基因由轻链（L链）编码，其余28个编码基因均由重链（H链）编码。22个tRNA基因中，除 tRNA-Ser(AGY)基因由于缺失DHU臂不能形成三叶草结构外，其余21种tRNA基因均可折叠形成经典的三叶草结构。控制区含有3种结构域：延长终止序列区（ETAS1和ETAS2）、中央保守区、保守序列区（CSB1、CSB2和CSB3），其中，CSB1和CSB2间存在200 bp的短重复序列，CSB3和tRNA-Phe间存在306 bp的长重复序列。基于线粒体基因组控制区序列构建10 种兔形目动物的系统进化树，结果表明福建黄兔起源于穴兔，支持将福建黄兔划归为穴兔属。     

iTRAQ-based quantitative proteomic analysis reveals key pathways responsible for scurs in sheep (Ovis aries)

Scurs is a horn phenotype that exhibits as small corneous structures on the skull due to the deformed development of horn tissues. Previous genome-wide association analysis of scurs in Soay sheep showed a significant association to the polled locus, relaxin-like receptor 2(RXFP2). However, the molecular mechanism underlying the development of scurs remains largely unknown. In the present study, we performed an i TRAQ-based quantitative proteomic analysis of horn tissues from both scurs and normal two-horned and four-horned individuals among Altay sheep to identify the differentially expressed proteins(DEPs) responsible for the scurs phenotype. In total, 232 proteins showed significant differential expression, and the most significant Gene ontology categories were the adhesion processes(biological adhesion(P=4.07×10–17) and cell adhesion(P=3.7×10–16)), multicellular organismal process(single-multicellular organism process(P=2.06×10–11) and multicellular organismal process(P=2.29×10–11)) and extracellular processes(extracellular matrix organization(P=4.77×10–16) and extracellular structure organization(P=4.93×10–16)). Kyoto encyclopedia of genes and genomes(KEGG) analysis showed that extracellular matrix(ECM)-receptor interactions and focal adhesion pathways were the most significant pathways. This finding is consistent with the reduced formation of extracellular matrix in scurs and the development of deformed horn tissues. Our study helps to elucidate the inheritance pattern of sheep horn traits from the perspectives of downstream expressed proteins.     

凭祥睑虎线粒体基因组序列测定与分析

采用PCR扩增方法测定了凭祥睑虎(Goniurosaurus luii)线粒体基因组全序列。经测序线粒体基因组全长16 519 bp,含有13个蛋白质编码基因、2个r RNA基因、22个t RNA基因和1个控制区。凭祥睑虎线粒体基因组碱基组成分别为A(34.11%)、T(27.43%)、C(26.01%)、G(12.45%)。除t RNA-Ser(AGY)外,其余21个t RNA基因的二级结构均为典型的三叶草结构。13个蛋白质编码基因中,7个基因(ND1、ND4、ND5、COⅡ、COⅢ、ATP8、ATP6)的起始密码子为ATG,2个基因(ND2、ND3)为ATA,2个基因(ND4L、Cytb)为ACA,剩余2个分别为ATC(COI)和GTG(ND6)。终止密码子一般都是TAA、AGA或者TAA,ND2、ND3、ND4、ND6、ATP6、COⅢ6个基因由不完全终止密码子终止(TA或T)。控制区全长1 147 bp,含有7个串联重复序列、6个终止相关序列TAS和2个保守序列(CBS-2,CBS-3)。     

High gene flows promote close genetic relationship among finewool sheep populations(Ovis aries) in China

The aim of our present study was to construct genetic structure and relationships among Chinese fine-wool sheep breeds.46 individuals from 25 breeds or strains were genotyped based on the Illumina Ovine 50K SNP array.Meanwhile,genetic variations among 482 individuals from 9 populations were genotyped with 10 microsatellites.In this study,we found high genetic polymorphisms for the microsatellites,while 7 loci in the Chinese superfine Merino strain（Xinjiang types）（CMS）and 5loci in Gansu alpine superfine-wool sheep strain（GSS）groups were found deviated from Hardy-Weinberg equilibrium（HWE）.Genetic drift F_ST=0.019（P<0.001）and high gene flows were detected in all the 7 fine-wool sheep populations.Phylogenetic analysis showed fine-wool sheep populations were clustered in a group independent from the Chinese indigenous breeds such that the 7 fine-wool sheep clustered distinct from Liangshan semifine-wool sheep（LS）and Hu sheep（HY）reflected by different population differentiation analyses.Overall,our findings suggested that all fine-wool sheep populations have close genetic relationship,which is consistent with their breeding progress.These populations,therefore,can be regarded as open-breeding populations with high levels of gene flows.Furthermore,the two superfine-wool strains,viz.,CMS and GSS,might be formed by strong artificial selection and with frequent introduction of Australian Merino.Our results can assist in breeding of superfine-wool sheep and provide guidance for the cultivation of new fine-wool sheep breeds with different breeding objectives.     

赤琥珀螺线粒体全基因组测定与系统发育关系的研究

旨在通过高通量测序技术获得赤琥珀螺（Succinea erythrophana）线粒体基因组全序列,对其结构及组成特征进行分析,并结合NCBI数据库已公布的琥珀螺科及柄眼目物种序列,以最大似然法和贝叶斯法分别进行系统发育分析。结果显示：①赤琥珀螺线粒体基因组全长 14 023 bp（GenBank No.ON533899）,由37个基因和一段富含AT的非编码控制区组成,有20处基因间隔,13处基因重叠。A＋T平均含量为77.3%,表现出明显的AT偏向性。基因的排列顺序、结构与组成、密码子使用情况与琥珀螺科已报道种类相似。 ②除tRNA-Phe、tRNA-His、tRNA-Ser1、tRNA-Ser2外,其余 tRNAs呈典型三叶草结构。③Ka/Ks选择压力分析显示其受到纯化选择作用。④系统发育研究揭示赤琥珀螺与同属的腐败琥珀螺（Succinea putris）亲缘关系最近,然后与同科其他物种形成姐妹群关系。     

和田羊线粒体DNA D-loop区序列研究

[目的]利用线粒体DNA D-loop区序列分析和田羊遗传多样性.[方法]采用测序法获得和田羊线粒体DNA D-loop区核苷酸序列,比对分析和田羊与Genbank中收录的藏绵羊、蒙古羊、哈萨克羊、巴什拜羊的绵羊线粒体DNA D-loop区序列.[结果]和田羊mtDNA D-loop区序列为1 184 bp,均含有4个75 bp重复序列,A+T含量62.50;,G+C含量37.50;.和田羊与藏绵羊遗传距离最近为0.001,与哈萨克羊遗传距离最远为0.004,同源性分析结果与遗传距离分析结果相同.[结论]和田羊与藏绵羊遗传距离近、同源性高的结果与两个品种在地理分布上可能存在一定关系.     

夏季放牧补饲对欧拉型藏羊羔羊育肥效果的研究

[目的]为藏羊羔羊夏季的育肥提供参考。[方法]选用6月龄欧拉型藏羊羔羊90只分为放牧组(对照组)和放牧补饲组(试验组),在夏季牧草旺盛期进行补饲育肥,研究青藏高原夏季不同育肥方式对藏羊羔羊育肥性能和肉品质的影响。[结果]试验组羔羊的净增重和平均日增重显著高于对照组(P0.05)。试验组羔羊的宰前活重、胴体重、屠宰率、胴体净肉重和胴体净肉率均极显著高于对照组(P0.01),而试验组羔羊的骨重、肉骨比、花油重和板油重均显著高于对照组(P0.05)。试验组羔羊的肌肉内脂肪含量为3.43%,显著高于对照组(P0.05)。试验组欧拉型藏羊羔羊的肉色评分、大理石和熟肉率均显著高于对照组(P0.05)。试验组和对照组羔羊肉中异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸酪氨酸的含量均高于FAO/WHO的标准模式。[结论]通过夏季补饲可进一步提高欧拉型藏羊羔羊的育肥效果。     

绵羊BMPR-IB基因密码子使用偏好和适应性进化分析

探讨BMPR-IB编码特点可为提高外源基因在受体物种中稳定、高效表达提供重要依据。本研究运用CHIPS、CUPS和CodonW分析绵羊BMPR-IB基因序列,与不同物种BMPR-IB、模式生物基因组及其他多胎主效基因比较,构建进化树后,利用PAML分析BMPR-IB对环境的适应性。结果表明:绵羊BMPR-IB基因与多胎性状相关的主效基因及除人类、倭黑猩猩和家犬外的不同物种CDS序列中大部分偏好使用G/C结尾密码子,对17种密码子具有偏好性,偏好性较强的密码子为GTG(2.02);绵羊多胎性状相关的BMP-15、GDF-9主效基因,对27种密码子具有偏好性,使用频率最高的为TGT(2.79)。密码子偏好性的聚类结果类似于进化结果,且不同物种的BMPR-IB基因中性选择的作用比例较高;绵羊与3种模式生物基因组密码子使用频率比较发现,小鼠基因组优于酵母和大肠杆菌。本研究结果为今后进一步开展绵羊新基因的发现、功能基因表达调控研究等提供了理论基础。     

欧拉羊胎儿皮肤细胞的培养、保存与鉴定

用胰蛋白酶对欧拉羊胎儿皮肤组织进行热消化分离并培养原代细胞，通过差速消化和差速贴壁法纯化成纤维细胞，扩大培养至第3代时用液氮保存，复苏后进行活力、形态、生长曲线、微生物污染检测和连续传代培养试验。结果显示，欧拉羊胎儿皮肤细胞呈成纤维型，复苏活力93.5%以上，生长良好，细胞生长曲线呈“S”型，最大增殖量为3.31×10⁵ mL^-1，倍增时间为26.2 h；细菌、真菌、病毒、支原体检测呈阴性，连续传代培养在10代内生长正常。表明欧拉羊胎儿皮肤细胞分离培养成功，使这一重要种质资源在细胞水平上得以保存。     

野生盘羊与巴什拜羊第二代杂种羔羊生长发育规律的研究

对野生盘羊与巴什拜羊第二代杂种羔羊生长发育进行测定,通过对第二代杂种羔羊初生前的生长发育、哺乳期的生长发育测定,并且绘制了累积生长、绝对生长和相对生长曲线图.计算分析生长发育的速度、强度等指标,并与纯巴什拜羔羊比较.测定结果表明:第二代杂种羔羊在胚胎期四肢生长强度比躯体强,这一点继承了盘羊的遗传特性;从初生到断奶期间躯体的生长速度和强度较好,这方面继承了巴什拜羊早期生长发育快的遗传特征,但在体尺指标和外部特征上野生基因占优势,为今后巴什拜羊与野生盘羊杂交改良工作提供科学依据.     

绵羊BMPR1A基因组织表达及其多态性与产羔数的关联分析

为探索BMPR1A基因在不同生理时期(卵泡期和黄体期)和不同繁殖力(单羔组和多羔组)小尾寒羊组织表达特征及其多样性与小尾寒羊产羔数的关系,采用qPCR技术检测BMPR1A基因在小尾寒羊14种组织中的表达特征。同时,采用Sequenom MassARRAY~?SNP技术对BMPR1A基因3个SNPs位点在大群中的多态性进行检测,并与小尾寒羊产羔数进行关联。qPCR结果显示,BMPR1A在14种组织中均有表达,其中在大脑、下丘脑和甲状腺组织高表达;BMPR1A在卵泡期和黄体期多羔组下丘脑和卵巢组织表达量均高于单羔组,但未达到显著水平。分型发现BMPR1A基因g.41128335AT和g.41127600CT位点在单、多羔绵羊品种间的基因频率和基因型频率达到显著水平。卡方适合性检验结果显示,3个SNPs在大多数绵羊品种中均处于Hardy-Weinberg平衡状态。关联分析结果发现,3个SNPs多态性与小尾寒羊各胎产羔数均无显著相关,但g.41128335AT和g.41127598AG位点突变型产羔数基本上均高于野生型。以上结果初步表明,BMPR1A基因表达与小尾寒羊产羔数增加存在一定程度的正相关,但3个SNPs与小尾寒羊各胎产羔数均无显著关联,说明它们可能均不是影响BMPR1A基因功能的关键位点。     

欧拉羊胚肾细胞的分离培养和鉴定

[目的]分离培养并鉴定欧拉羊胚肾细胞,为该品种基因组文库构建和遗传多样性研究提供生物学材料。[方法]用胰蛋白酶热消化法分离并培养欧拉羊胚肾原代细胞,通过差速消化和差速贴壁法纯化成纤维细胞,扩大培养至第3代用液氮保存,细胞复苏后进行活力、形态、生长曲线、微生物污染检测和连续传代培养试验。[结果]欧拉羊胚肾细胞呈成纤维型,复苏活力90%以上,生长良好,生长曲线呈"S",最大增殖浓度为4.61×105/ml,倍增时间26 h;细菌、真菌、病毒、支原体检测呈阴性,连续传代培养在10代内生长正常。[结论]欧拉羊胚肾细胞分离培养成功,使这一重要种质资源在细胞水平上得以保存。     

细胞色素b基因标记绵羊种内亲缘关系的初步研究

[目的]从群体的角度,初步探讨细胞色素b（Cytb）基因标记绵羊种内亲缘关系。[方法]用PCR方法扩增出滩羊和洼地绵羊2个地方绵羊品种共112个个体的线粒体DNA（m tDNA）Cytb基因,用限制性内切酶EcoRΙ对其进行限制性长度多态性（RFLP）分析。[结果]在56个滩羊样品中,有51个个体检测到1个酶切位点,5个个体没有检测到切点,呈现出2种限制性态型。在56个洼地绵羊样品中均能检测到酶切位点,表现为1种限制性态型。[结论]受试绵羊品种线粒体DNA多态性较贫乏,且m tDNA Cytb基因在绵羊品种内、品种间都有很强的保守性。因此,Cytb基因标记绵羊种内亲缘关系有一定的局限性。