家鹅细胞色素b基因序列进化研究

测定了中国家鹅15个品种、欧洲鹅2个品种共44个个体的细胞色素b基因全序列（1143bp），与Gen—Bank中白额雁细胞色素b基因序列合并在一起比对分析，结果表明：全序列中没有缺失、插入或移码突变，转换数（Ts）明显高于颠换数（Tv），密码子第3位点呈现了相当强烈的转换偏倚性；密码子第1位点的替换速率变异最大；同义密码子表现出一定程度的使用偏倚性；dS与dN值间的差异极显著（Z=4．713，P〈0．01），而ω（dS／dN）=0．0284，明显小于0．3，表明雁属鹅细胞色素b基因序列经历了中度净化选择作用；单步非同义替换分布模式与Grantharm’距离之间的关系说明密码子的3个位点所受的净化选择强度不同，第2位点的净化选择作用强度最大（Granthem距离为204）。     

家鹅线粒体细胞色素b跨膜螺旋结构分析

采用PCR和DNA测序技术测定了6个中国家鹅品种和2个欧洲鹅品种25个个体线粒体DNA（mitochondrial DNA，mtDNA）的部分序列，经测序分析表明该序列（1401bp）包括了细胞色素b（cytochrome b，Cytb）基因的完整序列（1143bp）。通过对家鹅线粒体基因组的研究，首次报道了家鹅线粒体Cytb的跨膜螺旋结构，并对家鹅Cytb跨膜螺旋结构及各区域序列的变异特征进行了分析。结果表明：鸿雁、灰雁和白额雁的Cytb具有完整而且完全相同的跨膜螺旋结构；家鹅Cytb基因的核苷酸序列在膜内、膜间和膜外的变异位点数分别为12、9和10，膜内、膜间的变异均属同义替换，而膜外有2个位点发生了非同义替换，表明跨膜螺旋的膜外区域变异程度高于膜间和膜内。研究的结果将为进一步探讨家鹅细胞色索b结构与功能的关系奠定基础。所测定的基因序列已登录国际GenBank数据库，序列号为AY427800-AY427805和AY427812-AY427814。     

中国家鹅起源和进化的分子研究进展

品种的起源和进化是研究生物遗传多样性的一个重要领域,对物种的分类鉴定、生物多样性保护和遗传育种都有重要的意义。中国家鹅的起源和进化一直倍受人们的关注。本文通过比较鹅核DNA和线粒体DNA及其所编码氨基酸序列与其它物种的同源性和分子标记(RFLP,RAPD和SSR)在鹅遗传多样性方面的研究阐述了中国家鹅的起源和进化,显示了中国的地方鹅种除伊犁鹅外都起源于鸿雁(Anser cygnoides),而伊犁鹅和欧洲鹅种起源于灰雁(Anser anser)。此外,还给出了一些物种起源和进化研究方面的建议。     

马头山羊线粒体细胞色素b基因遗传多样性研究

测定了马头山羊品种16个个体的细胞色素b基因全序列(1 140 bp),比较分析了群体中细胞色素b基因的碱基组成和序列间碱基的变异情况.结果显示:在该品种(群体)中细胞色素b基因序列中8个变异位点上观察到23次T-C间碱基转换,有11次T-G间碱基颠换发生在密码子第2位点,为非同义突变;观察到6种单倍型,单倍型多样度为0.808,核苷酸多样度为0.002 43.以绵羊为外群构建系统发生树(NJ tree).结果显示:马头山羊有两个母系起源,其中支系A占75%(12/16),支系B占25%(4/16).     

运用两类分子标记分析我国5个家鹅品种的遗传多样性和系统进化

首次运用33对微卫星标记(核内标记)和mtDNA D-环 521 bp 标记(核外标记),综合分析研究了我国丰城灰鹅、广丰白翎鹅、莲花白鹅、兴国灰鹅和伊犁鹅5个家鹅地方品种的遗传多样性和系统进化.结果表明:5个家鹅品种的杂合度,多态信息含量,单倍型多样度,核苷酸多样度分别为0.4 277～0.4 932,0.3 542～0.4 186,0～0.25 和 0～0.00 048,遗传多样度不丰富,两种标记构建的系统发生树均表明丰城灰鹅、广丰白翎鹅、莲花白鹅和兴国灰鹅等4个家鹅起源于鸿雁;伊犁鹅起源于灰雁.     

江苏地方山羊品种细胞色素b基因全序列的比较研究

对江苏2个地方山羊品种细胞色素b基因全序列进行测定,并引用国外10个山羊品种Cyt b基因序列构建山羊分子系统树。结果表明,长江三角洲白山羊和黄淮山羊Cyt b基因全序列均为1140 bp,而且碱基组成也相似,序列间的差异百分数较小,仅为0.09%;构建的分子系统树提示2个品种的亲缘关系较近,可能在较早的世代具有共同的祖先。     

8个山羊品种mtDNA细胞色素b基因序列的变异

本研究对8个山羊品种mtDNA细胞色素b(Cytb)基因克隆,并对序列的变异进行分析。结果表明:山羊的细胞色素b基因序列全长为1 140 bp,编码379个氨基酸和一个终止密码子。所有测定的序列中,共产生22个变异位点,其中包括19个单一多态位点和3个简约信息位点,这些变异定义了8个单倍型,单倍型的多样性为1.0%。根据Cyt b基因序列构建了系统发育树,8个山羊品种聚为两大类,每一类品种间存在生产性能上的联系。     

中国斗鸡mtDNA遗传多样性及起源研究

本研究旨在探究中国斗鸡的遗传多样性和起源.运用DNA测序的方法测定了河南斗鸡、鲁西斗鸡、吐鲁番斗鸡、西双版纳斗鸡和漳州斗鸡5个斗鸡品种40个个体的mtDNA细胞色素b(Cytb)的部分碱基序列(535 bp).结果表明,这5个斗鸡品种共有6种单倍型;单倍型多样度为0.428 57～0.785 71;核苷酸多样度为0.000 80～0.006 54;漳州斗鸡与其它斗鸡的遗传距离均较远;基于单倍型构建的系统发生树分析表明中国斗鸡母系起源于多个红色原鸡亚种.结论,漳州斗鸡的遗传多样性最丰富,而西双版纳斗鸡和吐鲁番斗鸡最贫乏;中国斗鸡母系起源于多个红色原鸡亚种.     

5个绵羊品种Cytb基因序列多态性及系统进化研究

利用克隆测序方法对杜泊羊、藏羊、小尾寒羊、哈萨克羊和阿勒泰羊DNA细胞色素b基因全序列进行克隆和测序,并对基因特点和系统发育进行分析。结果表明:5个绵羊品种线粒体细胞色素b基因全长1 140 bp,基因特点显示A+T含量(58.6%)比G+C(41.4%)含量高,表现出一定的碱基偏好,转换多于颠换,表现较高的转换偏向;基于线粒体细胞色素b基因构建了5个绵羊品种的NJ树,表明阿勒泰羊与哈萨克羊亲缘关系最近,杜泊羊与阿勒泰羊亲缘关系最远。     

我国十个鹅品种的遗传结构及遗传分化研究

利用多态性丰富的15个微卫星标记对列入国家级畜禽保护名录的10个鹅品种群体遗传结构、遗传分化及基因流进行研究。结果表明:10个鹅群体总近交系数(Fit)为-0.044,群体内近交系数(Fis)为-0.408,群体间基因分化系数(Fst)为0.257,3个固定指数只有Fst达到极显著水平(P<0.01)。鹅群体内近交系数(Fis)均为负值。各个鹅群体间,豁眼鹅与伊犁鹅的基因流值最小(Nm=0.416),四川白鹅与伊犁鹅间基因流值最大(Nm=1.430)。结合基因流和STRUCTURE分析结果,10个地方鹅品种可划分为四类:兴国灰鹅、豁眼鹅和太湖鹅为一类,四川白鹅、狮头鹅和乌鬃鹅为一类,酃县白鹅和皖西白鹅为一类,雁鹅和伊犁鹅为一类,这种聚类结果与各个品种的选育历史和地理分布等一致。     

大雁卵中磷脂含量的测定

对大雁卵 (鸿雁、灰雁 )卵黄和鹅蛋蛋黄中磷脂含量进行检测分析 ,试验结果表明 ,大雁卵卵黄中磷脂含量明显低于家鹅卵 ,而鸿雁和灰雁卵黄中磷脂含量无显著差异。     

Comparative antibacterial activity of avian egg white protein extracts

1. Egg white proteins from the eggs of domestic chicken (Gallus gallus), turkey (Meleagris gallopavo), duck (Anas platyrhynchos) and goose (Anser anser) were analysed in order to compare the antimicrobial activity of these products. 2. Albumen from each species was sampled and analysed by SDS-PAGE and Western blotting. Antimicrobial activity and lysozyme activity were measured. 3. Ovotransferrin and ovalbumin were identified in all species while c-type lysozyme was present in chicken, turkey and duck egg white samples, but not in goose. 4. Galliformes appear to possess albumens with greater antimicrobial activity than those of the Anseriformes. This can be attributed to higher concentrations of ovotransferrin and the broad acting c-type lysozyme.     

Functional analysis and induction of four novel goose (Anser cygnoides) avian β-defensins in response to salmonella enteritidis infection

In the current study, four novel avian β-defensins (AvBDs) (AvBD2, 5, 9, and 10) were identified in tissues from the Chinese goose (Anser cygnoides). The antibacterial activity of the AvBDs showed that all of these AvBDs exhibited antibacterial activity against most of the bacteria investigated (P<0.01). In addition, antibacterial activity of all of the AvBDs against Staphylococcus aureus and Pasteurella multocida decreased significantly or was completely abolished at 150mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. AvBD2 and AvBD10 were expressed widely, whereas AvBD5 and AvBD9 mRNAs were expressed in a limited number of geese tissues. AvBD9 was significantly induced in some immune tissues from geese after Salmonella enteritidis infection. The others were significantly upregulated in small intestine and some immune tissues of the geese (P<0.01). The present results suggest that the AvBDs are part of the host defense mechanism of the goose.     

鹅基因组微卫星富集文库的构建与分析

为分离鹅(Anser cygnoides)微卫星序列片段,提取鹅基因组DNA,用Hae III和Rsa I内切酶消化并连接接头,再用接头特异引物进行PCR扩增。扩增产物与生物素标记的(AC)12探针杂交,杂交复合物用链霉亲和素包裹磁珠进行结合,得到单链DNA目标片段。再经PCR扩增,连接pMD19-T载体,转化入感受态大肠杆菌,得到微卫星富集小插入片段DNA文库。用Colony-PCR法筛选获得318个阳性克隆,并进行测序分析。结果表明,所测的318个序列有242个含微卫星序列,197个为有效微卫星序列,其中完全型(perfect)占60.9%,非完全型(imperfect)20.8%,混合型(compound)18.2%。(CA)n重复最为常见。文章为鹅种资源遗传多样性、分子进化、遗传图谱的构建及重要经济性状基因座定位等研究奠定了基础。     

Antimicrobial activity of cuticle and outer eggshell protein extracts from three species of domestic birds

1. The eggshell cuticle is the proteinaceous outermost layer of the eggshell which regulates water exchange and protects against entry of micro-organisms. In this study, we investigated the hypothesis that the cuticle may also reduce microbial contamination by providing a chemical defence. 2. Outer eggshell and cuticle protein was extracted from domestic chicken (Gallus gallus), duck (Anas platyrhynchos) and goose (Anser anser) eggs by HCl and urea treatment, respectively. Antimicrobial activity of the extracts against Gram-positive and Gram-negative bacteria was evaluated. 3. C-type lysozyme, ovotransferrin and ovocalyxin-32 were identified in all extracts by Western blotting. All extracts from all species demonstrated lysozyme enzymatic activity. Immobilised c-type lysozyme retained some enzymatic activity. Protein extracts demonstrated activity against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis suggesting the action of antimicrobial proteins in addition to lysozyme. 4. The results suggest that the antimicrobial outer eggshell and cuticle proteins present in a number of avian species may be a mechanism which enhances avian reproductive success.     

Technical note: detection of chicken, turkey, duck, and goose tissues in feedstuffs using species-specific polymerase chain reaction

PCR method was applied for the qualitative identification of chicken (Gallus gallus), turkey (Meleagris gallipavo), duck (Anas platyrhynchos x Cairina muschata), and goose (Anser anser) tissues in feed-stuffs, on an individual basis. The assay uses oligonucleotide primers that are specific for each avian species, targeting the 12S rRNA mitochondrial gene. The primers designed generated amplicons of 95, 122, 64, and 98 bp length for chicken, turkey, duck, and goose, respectively. The specificity of the primers was tested against 29 animal species including mammals, birds, and fish, as well as 8 plant species. Analysis of experimental feedstuffs demonstrated the detection of each target species in the range of 0.1 to 100%. The performance of this method was not affected by prolonged heat-treatment (up to 133 degrees C for 20 min at 300 kPa), and consequently, it could be very useful for the accurate identification of tissues from these 4 avian species in products submitted to denaturing technologies, for which other methods cannot be applied.     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

利用微卫星标记分析伊犁鹅的保种效果

为分析某保种场伊犁鹅的保种效果,本研究利用9个微卫星标记对新疆伊犁鹅3个世代（0世代、1世代、2世代）的遗传多样性进行检测。试验以3个世代伊犁鹅血液为样本,使用苯酚-氯仿抽提法提取DNA,随后进行12%聚丙烯酰胺凝胶电泳检测、银染法显色及判型。结果表明,9个微卫星位点在伊犁鹅的3个世代中共检测到78个等位基因,0世代、1世代和2世代均检测到26个等位基因;3个世代的平均期望杂合度（He）分别为0.4322、0.4232和0.4516;平均多态信息含量（PIC）分别为0.3751、0.3707和0.3936,表明伊犁鹅的3个世代均处于中度多态,遗传变异处于中等水平,且3个世代间平均期望杂合度和平均多态信息含量均差异不显著（P>0.05）;3个世代的遗传距离分别为0.0097、0.0184和0.0074。综合来看,伊犁鹅具有较丰富的遗传多样性,且该保种场目前所采用的保种方法较好地保存了伊犁鹅原有的遗传多样性。     

伊犁鹅产蛋前后各期卵巢转录谱的构建及卵泡发育相关基因的分析

【目的】构建产蛋前后各期伊犁鹅卵巢转录组文库,结合生物信息学分析揭示不同产蛋期伊犁鹅卵巢组织差异表达基因,并鉴定出影响鹅卵巢发育的关键基因,为伊犁鹅的繁殖调控提供理论参考。【方法】选择处于开产期（KL组）、产蛋期（CL组）及休产期（XL组）的伊犁鹅各4只,屠宰后取其卵巢组织,用于构建卵巢转录组文库。通过新基因挖掘、基因差异表达、基因注释以及蛋白互作网络分析筛选出与鹅卵泡发育相关的候选基因;随机挑选8个差异表达基因,应用实时荧光定量PCR验证其表达情况。【结果】伊犁鹅卵巢组织学结果表明,在开产期时,伊犁鹅卵巢表面有大量初级卵泡,而产蛋期卵巢则显示出卵泡的层级性,在休产期卵巢中可观察到卵泡出现向内凹陷、闭锁的现象。通过转录组测序（RNA-Seq）,从构建的12个伊犁鹅卵巢cDNA文库中获得有效读数57 811 186~85 328 377条,Q30值均>93.38%,每个样品所产的测序读数于鹅参考基因组上的比对率在82.79%~89.24%。在卵巢中注释的新基因共有1 112个,单核苷酸多态性（SNP）位点总数为1 642 273~2 425 069个;SNP和插入缺失（InDel）均主要注释于内含子区域;各时期的可变剪切类型主要集中为最后1个外显子可变剪切（TSS）及第1个外显子可变剪切（TTS）。在KL vs CL、XL vs CL及XL vs KL组中分别有337、1 136和525个差异表达基因,共有差异表达基因为α2A肾上腺素能受体（ADRA2A）、表皮蛋白（CP）、非转移性黑色素瘤糖蛋白B （GPNMB）及α-1-抗胰蛋白酶样（LOC106033756）。GO功能富集分析发现,差异表达基因主要富集在肽基酪氨酸磷酸化的正调控、细胞黏附及质膜外侧等过程。KEGG通路富集分析发现,差异表达基因主要显著富集于神经活性配体-受体相互作用、ECM-受体相互作用及类固醇生物合成等。结合蛋白互作网络分析,筛选到与鹅卵巢发育相关的潜在调控因子Bruton’s酪氨酸激酶（BTK）、血小板源性生长因子受体α（PDGFRA）、整合素β3（ITGB3）等。实时荧光定量PCR结果显示,RNA-Seq结果准确可靠。【结论】本研究揭示了产蛋前后各期伊犁鹅卵巢组织中的基因表达差异,筛选到影响伊犁鹅卵巢发育的神经活性配体-受体相互作用、ECM-受体相互作用、类固醇生物合成等重要通路与BTK、PDGFRA和ITGB3等关键候选基因,为了解鹅卵巢组织调控产蛋性能的分子机制提供了理论支撑。     

Characterization of ovarian morphology and reproductive hormones in Zhedong white geese (Anser cygnoides domesticus) during the reproductive cycle

Zhedong white goose (Anser cygnoides domesticus) is a native Chinese breed with strong broodiness and low egg production, which is related to the physiology of reproduction. However, thus far, the physiology of goose reproduction has not been well elucidated. In the present study, the ovarian morphology and reproductive hormones of Zhedong white geese were investigated during the reproductive cycle (the laying and brooding periods). The results showed that the surface of the ovary was atrophied and follicular atresia appeared to some extent in the brooding period compared with the laying period. The concentrations of follicle-stimulating hormone, progesterone and luteinizing hormone were significantly higher than those in the brooding period (p < 0.05). In contrast, the concentrations of prolactin (PRL) and anti-Müllerian hormone (AMH) in the laying period were significantly lower than those in the brooding period (p < 0.05). In addition, the mRNA expression levels of PRL, AMH, dopamine-β-hydroxylase (DβH) and cytochrome P450 side-chain cleavage enzyme (P450scc) were detected in the hypothalamus, pituitary and ovaries by using real-time polymerase chain reaction. The results showed that AMH mRNA was expressed specifically in ovary tissue. The expression levels of DβH and PRL in the brooding period was significantly higher than those in the laying period in the three tissues, especially in the early and middle stages of the brooding period. Moreover, AMH mRNA expression in the ovaries presented the same trend. In addition, P450scc mRNA was highly expressed in both the ovary and pituitary in the laying period. These results revealed the remarkable features of ovarian morphology and characterized the hormonal pattern and expression profile during the reproductive cycle, all of which contribute to understanding the differences in reproductive physiology between the laying and brooding periods in Zhedong white geese.