基于测序技术的海南地方猪TLR4基因多态性分析

为了了解海南地方猪TLR4基因的多态性,试验采用PCR技术对五指山猪、临高猪和屯昌猪的TLR4基因组序列进行了克隆、测序,应用DNAStar和BioEdit软件分析其序列,SMART、SOPMA、SWISS-MODEL在线软件分析TLR4蛋白的结构,并预测二级结构和三级结构。结果表明:获得的三个品种海南地方猪TLR4基因DNA序列全长均为10 435 bp,其中编码区长2 526 bp,编码841个氨基酸。三个品种猪TLR4基因序列种内比对时,五指山猪在种内有1个核苷酸位点存在多态性;屯昌猪在种内有6个核苷酸位点存在多态性,2处位于编码区;临高猪在种内有3个核苷酸位点存在多态性,1处位于编码区。三个品种猪TLR4基因序列种间比对时,有27个核苷酸位点存在多态性,其中2个为错义突变,导致氨基酸改变。三个品种猪的TLR4基因序列的同源性在99.8%～99.9%之间,高度保守。海南猪TLR4基因在7 209位点的G→T突变和7 781位点的G→A位点突变引起了TLR4蛋白二级结构和三级结构发生改变。说明海南地方猪TLR4基因存在多态性,并且其多态性引起的结构改变可能使TLR4基因功能发生变化。     

西藏小型猪线粒体DNA控制区的分子遗传学研究

通过扩增102头西藏小型猪以及16头巴马小型猪、17头贵州香猪的线粒体DNA控制区,测序并与国内其他猪种进行比较,研究西藏小型猪的遗传标记以及与其他国内地方猪种的亲缘关系.结果显示,两藏小型猪线粒体DNAD-loop区分3个区域.串联重复序列区处于中间位置,包含有15～29个10 bp的重复片段,分为A、B 2种类型.3'端340 bp,与国内其他猪种的序列相同,比较保守;D-loop 5'端704 bp,共有22个变异位点.由22个变异位点中归纳出25个单倍型,其中有2种主要的单倍型,分别占34.4%和36.6%.根据3个转换位点:305、500、691,将西藏小型猪分成了2组,几乎与串联重复序列所分的A、B 2组类型相对应.与西藏小型猪相比,巴马小型猪和贵州香猪D-loop 5'端变异位点较少,分别只有4种和2种单倍型,串联重复区也只有1个类型.说明西藏小型猪可能有2个母系祖先,并且与我国西南地区的猪种有较近的亲缘关系;不同的串联重复片段类型和5'端的变异位点可以联合组建西藏小型猪的遗传标记.     

蕨麻猪生长激素基因的同源性和多态性分析

为了探索蕨麻猪的遗传多样性,对蕨麻猪生长激素基因序列的多态性进行研究,并选择GenBank中14条序列作为比较研究对象;首次成功克隆了7条蕨麻猪生长激素基因,测出5条蕨麻猪生长激素基因DNA序列,其多态性是内含子1有1个转换和1个颠换;内含子2有2个转换。说明蕨麻猪之间的差异可能与内含子的控制有关。蕨麻猪与其他14种猪之间在编码区和非编码区共检测到核苷酸的变异为155个,内含子有119个,外显子有28个,5′端有8个。其中,颠换104个,转换14个,缺失23个,插入14个。出现碱基取代的位点中,大多数是颠换型突变,颠换与转换之比为7.43∶1,存在颠换偏倚现象。对蕨麻猪生长激素基因同源性百分数和趋异数分析,结果发现,蕨麻猪与小型猪相比同源性较近,蕨麻猪与大型猪同源性较远。蕨麻猪等15 种猪生长激素基因序列树状图分析,结果表明,蕨麻猪与Vize猪有较远的亲缘关系;蕨麻猪与贵州榕江香猪亲缘关系最近。     

中国地方猪种线粒体基因组遗传多样性及其系统发育分析

为从分子水平上探究中国地方猪种遗传多样性和分类地位,本试验采用生物信息学方法比较了6个类型共22个中国地方猪种的线粒体基因组全序列,分析了其多态性,并构建了6个类型猪种线粒体D-loop区单倍型的网络中介图以及基于线粒体D-loop序列、Cytb基因、完整编码区序列的系统进化树。结果表明,6个类型22个猪种中共检测到了144个多态位点,22种单倍型,说明地方猪种具有丰富的遗传多样性;地方猪线粒体基因组序列中核苷酸变异以转换为主,且Ti/Tv大于转换/颠换比临界值（2.0）,变异位点均符合中性突变。6个类型猪种间遗传距离均较小,且有共享单倍型。系统进化树结果表明,6种类型地方猪种主要聚为两个支系。表明线粒体D-loop序列及Cytb基因均可作为研究种内系统发育、起源进化的分子标记。     

中国6个地方猪种与3个外种猪氟烷基因PCR产物序列比较研究

本文应用PCR方法 ,体外扩增了中国地方猪和外种猪共 16个个体的氟烷基因 1814 9～ 1876 0位之间 6 12bp的片段 (包含完整的外显子 17和cDNAC1843 →T1843 突变位点 ) ,并进行了序列测定。结合网上下载的 5个中外猪种相同区段的序列进行了比较研究。结果表明 ,本文所测序列比网上公布的序列少 2bp ,在 1815 3,1815 4处有两胞嘧啶(C)缺失 ;皮特兰猪在cDNA的C1843 位点存在C1843 →T1843 突变 ;6 12bp的片段中共有 14个变异位点 ,皆为转换型突变 ,内含子 16中有 3个位点存在转换型杂合子 ;所有突变中 ,有 4处发生在外显子 17内 ,除香猪和皮特兰猪外均为同义突变 ;这些变异位点共形成 12种单倍型 ,其中扩增片段 5 14位 (全序列中为 186 6 2位 )的C→T突变为内江猪独有 ,构成了内江猪独特的单倍型 ,C18662 →T18662 突变具有品种特异性     

基于线粒体细胞色素b基因(mtDNA Cytb)序列分析吉林山鸡的遗传多样性

为了解吉林山鸡种群的遗传多样性,采用PCR技术及测序技术对20只吉林山鸡的mtDNA Cytb基因序列进行了分析。结果表明,吉林山鸡mtDNA Cytb基因序列中,共检测到核苷酸多态位点11个,20个个体具有3种单倍型;吉林山鸡与美国山鸡亲缘关系较近,与北京山鸡和甘肃山鸡亲缘关系较远。     

11个猪种SLC6A14基因3个SNPs的群体遗传变异研究

本研究旨在了解猪种的遗传变异、种群间的亲缘关系和遗传分化。以白色杜洛克×二花脸资源家系F0代的17头二花脸母猪和2头白色杜洛克公猪的DNA池为模板,通过直接测序在猪SLC6A14基因内识别4个SNPs,以3个突变位点(g.7944AT、c.1438GA、g.21063GT)为基础,通过PCR-RFLP技术,对11个中外猪种进行多态性检测。结果表明:在SLC6A14g.7944AT和g.21063GT2个突变位点上,所有检测猪种都出现变异,其中槐猪、二花脸猪在g.7944AT位点上变异显著(0.01P0.05);而里岔黑猪、槐猪、玉山黑猪、合作藏猪、八眉猪在g.21063GT内变异极显著(P0.01),以GT基因型为主,且二花脸猪中出现TT基因型;在SLC6A14c.1438GA突变位点上,3个欧洲猪种(长白、大白、杜洛克)没有出现变异,均为GG纯合型,而其他猪种在此位点上均存在变异;再根据11个猪种3个位点的多态性信息计算Nei氏遗传距离,由UPGMA法构建聚类图,说明中国地方猪种与外引猪种存在明显的遗传分化;由此得出中国地方猪种比外引猪种有高的遗传多样性和变异,且存在明显的遗传分化,品种间多数猪种符合品种地域分布和品种特性。     

三个国外猪种线粒体COXⅠ基因SNPs筛选及序列分析

本研究对三个国外猪种216个样本(大白猪、长白猪和杜洛克)mt DNA COXⅠ基因构建混合池,利用直接测序技术获得3个猪种COXⅠ基因的序列,进行SNPs筛选及序列分析,并采用MEGA 6.0分析软件基于Kimurk双参数模型应用邻接法构建系统发生树。结果表明,3个猪种mt DNA COXⅠ基因序列共存在16个突变位点,且有3个突变位点为3个猪种共有。长白猪与杜洛克猪的多态性较丰富,含有13个相同位点的突变,核苷酸的转换数(si)和颠换数(sv)的比值(R)分别为12和15,序列替换远未达到饱和。系统进化树和遗传距离分析显示长白猪及杜洛克亲缘关系较近,先聚为一支,再与大白猪、及野猪聚为一支。大白猪与中国东北地区野猪聚为一支,这可能与大白猪在培育过程中亲本遗传背景较复杂有关。本研究在一定程度上为了解大白猪的育成史及合理利用国外猪种提供了理论依据。     

吉林省圈养梅花鹿种群遗传资源多样性分析

为了探讨吉林省圈养梅花鹿的遗传分化情况,利用DNA直接测序技术测定其线粒体细胞色素mt DNA Cytb基因全序列,结合Gen Bank收录的其他梅花鹿亚种同源序列,利用生物学软件进行分析。结果表明,15头样本mt DNA Cytb基因序列中,共检测到核苷酸多态位点38个,吉林省圈养梅花鹿种群的遗传多样性水平较低,与俄罗斯、韩国、朝鲜亚种亲缘关系较近,与四川亚种亲缘关系较远。     

安徽省五华鸡J亚群禽白血病病毒gp85基因分子序列特征分析

为进一步了解J亚群禽白血病病毒(avian leukosis virus subgroup J,ALV-J)gp85基因的结构、功能及其遗传变异趋势,采用PCR方法克隆了安徽省地方品种五华鸡ALV-J gp85基因序列ALV-J-WH,并将该序列与GenBank数据库中登录的15个gp85基因序列进行了比对分析。结果显示,ALV-J-WH与所比对的氨基酸序列同源性介于85.9%~96.2%之间,与其同源性最高的是来自于国内ALV-J毒株的JN378888基因序列,进化树也证实两者亲缘关系较近;此外,相对其他ALV-J gp85氨基酸序列,ALV-J-WH在223位氨基酸后有10个氨基酸的缺失;对所有全长序列分析结果表明,共有71个可变氨基酸位点,10个高度变异位点,尤其是hr1和hr2区域分布较多。结果表明,ALV-J-WH与国内部分ALV-J毒株分离gp85基因来自共同的原始病毒,但其具有独特的序列特征;ALV-J gp85基因高度易变,部分高频率突变的氨基酸位点可能是ALV-J在抗体免疫选择压下的作用位点。     

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).     

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.     

靶向大肠杆菌(E.coli)黏肽合成酶PBP1b的抗菌先导化合物的虚拟筛选

大肠杆菌(E.coli)是一种常见的致病菌,大肠杆菌的黏肽合成酶PBP1B对于它的细胞壁合成和存活具有至关重要的作用,是β-内酰胺类抗生素的作用靶点。但是,由于抗生素滥用等原因,目前已造成了大肠杆菌对β-内酰胺类抗生素较强的耐药性。本研究针对大肠杆菌的黏肽合成酶PBP1B,通过分子对接软件DOCK6.5进行虚拟筛选,以期得到具有全新结构的高亲和力的先导化合物,用作新型抗菌药物。我们对含104万个化合物的ZINC数据库进行了虚拟筛选,以grid score进行了第1轮打分,得到分值在-30kcal/mol以下的化合物约6万个,从中挑选出打分高的600个化合物进入第2轮筛选,采用amber score进行第2轮打分,筛出分值在-20kcal/mol以下的化合物约200个。对这些化合物进行分析,最终挑选2种打分高并且结构新颖的先导化合物,并进行了有机合成。采用MH肉汤稀释法检验了先导化合物的抗菌活性,发现先导化合物对常见革兰阳性菌和革兰阴性菌均有抗菌活性,并且抗菌活性比具有相似结构的磺胺嘧啶更强。因此,这2种先导化合物有望进一步开发为新型抗菌药物。     

Xanthophylls increased HDLC level and nuclear factor PPARγ, RXRγ and RARα expression in hens and chicks

This study was designed to investigate effects of xanthophylls on serum lipid profile (triglyceride, TG; cholesterol, CHO; high‐density lipoprotein cholesterol, HDLC; and low‐density lipoprotein cholesterol, LDLC) and nuclear factor (peroxisome proliferator‐activated receptor gamma, PPARγ; PPAR gamma coactivator 1 alpha, PGC1α; retinoid X receptor gamma, RXRγ; and retinoic acid receptor alpha, RARα) gene expression of breeding hens and chicks. In experiment 1, 432 hens were divided into three groups and fed diets supplemented with 0 (as control group), 20 or 40 mg/kg xanthophylls. Blood was sampled at 7, 14, 21, 28 and 35 days of trial. Liver, duodenum, jejunum and ileum were sampled at 35 days of trial. Results showed that serum HDLC level of hens was increased after dietary 40 mg/kg xanthophyll addition for 21, 28 and 35 days, while serum TG, CHO and LDLC were not affected. Xanthophyll addition also increased PPARγ expression in jejunum, RXRγ expression in duodenum and jejunum, and RARα expression in liver and duodenum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg/kg xanthophyll diet of hens were fed diet containing either 0 or 40 mg/kg xanthophylls. Liver, duodenum, jejunum and ileum were sampled at 0, 7, 14 and 21 days after hatching. Blood samples were also collected at 21 days. Results showed that in ovo xanthophylls elevated PPARγ in duodenum and jejunum, and RXRγ and RARα in liver of chicks mainly within 1 week after hatching, while dietary xanthophylls increased serum HDLC level and PPARγ and RXRγ in liver from 2 weeks onwards. In conclusion, our research suggested xanthophylls can regulate serum lipid profile and nuclear factor expression in hens and chicks.     

bta-miR-34b/c和bta-miR-449a/b/c靶基因预测及生物信息学分析

【目的】试验旨在对bta-miR-34b/c和bta-miR-449a/b/c的序列保守性、转录因子和靶基因进行生物信息学预测和分析,探究其影响精子发生的作用机制,为研究其生物学功能及作用机制提供指导和思路。【方法】利用miRBase数据库获取不同物种的bta-miR-34b/c和bta-miR-449a/b/c序列并进行相似性分析;通过TargetScan、miRWalk、miRDB等方法预测bta-miR-34b/c和bta-miR-449a/b/c的靶基因,对获得的靶基因集合分别进行GO功能和KEGG通路富集分析;通过AnimalTFDB获取共同调控bta-miR-34b/c和bta-miR-449a/b/c的转录因子,并构建TF-miRNA-mRNA作用网络。【结果】bta-miR-34b/c和bta-miR-449a/b/c在不同物种间高度保守。预测得到2个转录因子和49个候选靶基因,这些靶基因主要富集在精子发生、钙离子依赖性胞吐的调节、胰岛素分泌的调节等GO条目,以及HIF-1信号通路、甲状腺激素信号通路、Wnt信号通路等KEGG通路。bta-miR-34b/c、bta-m...     

α- and β-adrenoceptors in the sheep urinary bladder

A study was undertaken to determine the presence and distribution of alpha- and beta-adrenoceptors in the sheep bladder body and base. In the bladder body, noradrenaline and isoproterenol induced relaxation which was significantly inhibited by propranolol, pafenolol and butoxamine. In the presence of propranolol (10(-5) M), noradrenaline induced a small contraction, as well as phenylephrine, but B-HT 920 failed to cause any effect on the bladder body. In the bladder base, noradrenaline caused a contraction that was significantly inhibited by prazosin but not by yohimbine. Phenylephrine also induced a contractile response in this structure which was inhibited by prazosin. Isoproterenol caused a relaxation that was significantly inhibited by propranolol and pafenolol but not by butoxamine. Relaxation was mediated by both beta 1 and beta 2-adrenoceptors in the detrusor muscle and by beta 1-adrenoceptors in the bladder base. Alpha 1-adrenoceptors contributed to maintain the detrusor tone and contract the bladder base.     

家鹅细胞色素b基因系统发育研究

本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(1143 bp),与Genbank库中白额雁细胞色素b基因序列合并进行比对分析,结果表明各序列同义替换速率(dS)和非同义替换速率(dN)分别为0.0787和0.0011,dS与dN值间的差异极显著(Z=4.713,P＜0.01),dS＜0.5,可使用dS值来构建分子系统发育关系树,构建的最大简约树与邻接树拓扑结构一致,支持中国家鹅的双起源学说,即除伊犁鹅外的其它中国鹅品种起源于鸿雁,伊犁鹅和欧洲的郎德鹅、莱茵鹅起源于灰雁.