绵羊GDF9基因PCR-SSCP分析

生长分化因子9（GDF9）是由卵母细胞分泌的一种生长因子,它对早期卵泡的生长和分化起重要的调节作用。采用PCR－SSCP技术分析了GDF9基因在小尾寒羊、湖羊、多赛特羊和萨福克羊4个绵羊品种的多态性。结果表明：GDF9基因在两对引物扩增片段中均存在PCR－SSCP多态性。对于引物1扩增片段,4个绵羊品种均检测到AA基因型,AB基因型只出现在湖羊、多赛特羊和萨福克羊中,仅在萨福克羊中检测到BB基因型;在4个绵羊品种中,A等位基因频率明显高于B等位基因频率。对于引物2扩增片段,4个绵羊品种均检测到AA基因型,AB基因型只出现在湖羊、多赛特羊和萨福克羊中,4个绵羊品种均没有检测到BB基因型;在4个绵羊品种中,AA基因型频率最高,A等位基因频率明显高于B等位基因频率。引物1的多态性片段测序分析表明：位于GDF9基因cDNA第152处发生了单碱基的改变（A→G）,并导致了氨基酸的改变（天冬酰胺→天冬氨酸）。     

乐至黑山羊PRLR基因外显子10多态性与产羔数的关系研究

设计2对特异性引物对乐至黑山羊PRLR基因第10外显子进行了PCR-SSCP检测,并研究该基因与产子性能的相关性。结果表明,P1引物扩增片段不存在多态性;P2引物扩增片段存在多态性,表现为AA,AB,AD和CD 4种基因型,测序结果表明,4种基因型都在该片段第89、94、146和157位存在C→T、A→C、C→G、G→C的突变;此外AA型还在61位发生C→T的突变;AD型还在175位发生A→G的突变;CD型还在24位发生T→C的突变,96位发生C→T的突变,通过统计分析发现AD型平均产羔数优于其他3种基因型,并且与AB型差异达到显著水平(P<0.05)。因此认为PRLR基因对于乐至黑山羊产子性能有一定的影响。     

FSHβ 基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究

采用PCR-SSCP技术检测促卵泡素b亚基(follicle-stimulating hormone β, FSHβ)基因5′调控区、外显子1和外显子2在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊、安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 山羊与绵羊的FSHβ 基因该段核苷酸序列同源性为98%。9对引物中, 只有P9的扩增片段存在多态性。P9的扩增片段在济宁青山羊和辽宁绒山羊中检测到AA、AB和AC 3种基因型; 在波尔山羊中检测到AA、CC和AC 3种基因型; 在安哥拉山羊中检测到AA、BB、CC、AB、AC和BC共6种基因型。测序分析发现BB型与AA型相比在外显子2的第94 bp处有G→A突变, 并引起氨基酸改变(丙氨酸→苏氨酸); CC型与AA型相比在外显子2的第174 bp有一处C→T沉默突变。济宁青山羊AA、AB和AC基因型频率分别为0.686、0.137和0.177。AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.78只(P<0.05), 比AC基因型的多0.64只(P<0.05)。     

山羊生长激素基因5调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5^'区的26－239bp以及225－429bp片段,扩增产物经SSCP分析发现均存在多态性。在26－239bp片段上,波尔山羊及杂交后代以 AA型个体占多数,而鲁北白山羊则BB型个体较多;在225－429bp片段上,所有种群均以 CC型个体较多。对两个片段的纯合型（AA,BB;CC,DD）分别克隆测序发现：（1）26－239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,（2）225－429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T。上述结果为首次实验证实山羊生长激素5^'调控区存在序列多态性。     

催乳素受体基因外显子10多态性及其与济宁青山羊高繁殖力关系的研究

设计5对引物, 采用PCR-SSCP技术检测催乳素受体(prolactin receptor, PRLR)基因外显子10及部分3′非翻译区在高繁殖力山羊品种(济宁青山羊)和低繁殖力山羊品种(辽宁绒山羊、波尔山羊和安哥拉山羊)中的单核苷酸多态性, 同时研究该基因对济宁青山羊高繁殖力的影响。结果表明: 首次拼接出的山羊PRLR基因外显子10及部分3′非翻译区的核苷酸序列长度为1,118 bp, 与已公布的绵羊、牛、人PRLR基因mRNA相应序列的同源性分别为98.33%、93.92%、74.52%, 与已公布的羊驼PRLR基因部分序列的同源性为78.29%。引物P1、P2与P4扩增片段具有多态性, 其余2对引物的扩增片段不存在多态性。对于P1扩增片段, 在济宁青山羊和辽宁绒山羊中检测到AA型和AB型, 在安哥拉山羊中检测到AA型和AC型, 在波尔山羊中只检测到AA型; 克隆测序表明AB型与AA型相比有两处突变(186G→A和220T→C), 分别导致氨基酸由天冬氨酸变为天冬酰胺、亮氨酸变为脯氨酸; AC型与AA型相比有1处突变(140A→G), 该突变没有导致氨基酸变化; 济宁青山羊AA和AB基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P2扩增片段, 在济宁青山羊、辽宁绒山羊和波尔山羊中都检测到DD型和DE型, 而在安哥拉山羊中只检测到DD型; 克隆测序表明DE型和DD型相比有两处突变(52G→A和122G→A), 其中122 bp处的突变导致氨基酸由精氨酸变为甘氨酸; 济宁青山羊DD和DE基因型之间产羔数的最小二乘均值差异不显著(P>0.05)。对于P4扩增片段, 在济宁青山羊中检测到FF型和FG型, 在辽宁绒山羊中检测到FF型和GG型, 在波尔山羊中只检测到FF型, 在安哥拉山羊中检测到FF型、FG型和GG型; 克隆测序表明GG型和FF型相比在扩增片段的143 bp处发生1处碱基突变(A→G), 并导致氨基酸由蛋氨酸变为缬氨酸; FG基因型济宁青山羊产羔数最小二乘均值比FF基因型的多0.76只(P<0.05)。研究结果初步表明: PRLR基因可能是控制济宁青山羊多胎性能的一个主效基因或是与之存在紧密遗传连锁的一个标记。     

甘肃肉羊新品种选育群GDF9基因外显子2多态性及其与产羔性状关联分析

根据GenBank发布的绵羊GDF9基因外显子2的序列设计4对引物,采用PCR-SSCP技术分析GDF9基因外显子2在甘肃内羊新品种选育群羊中的单核苷酸多态性,并与产羔性状进行关联分析.结果表明,GDF9基因的扩增片段在所检测的新品种群羊中存在PCR-SSCP多态性,检测到3种基因型(AA、AB和BB),而在32只无角陶赛特母羊群中只检测到AA和AB基因型.测序结果显示,GDF9基因编码区第978位碱基发生A→G突变,但没有导致氨基酸的改变;第994位碱基发生G→A突变,导致Ⅴ变成Ⅰ(缬氨酸→异亮氨酸).新品种选育群羊产羔数的最小二乘均值关系为AB＞ AA＞ BB,统计分析结果初步表明3种基因型之间差异不显著(P＞0.05).故该区域可能不是影响新品种群羊繁殖力的功能结构区城.     

山羊生长激素基因5′调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5′区的26～239bp以及225～429bp片段,扩增产物经SSCP分析发现均存在多态性.在26～239bp片段上,波尔山羊及杂交后代以AA型个体占多数,而鲁北白山羊则BB型个体较多;在225～429bp片段上,所有种群均以CC型个体较多.对两个片段的纯合型(AA,BB;CC,DD)分别克隆测序发现:(1)26～239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,(2)225～429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T.上述结果为首次实验证实山羊生长激素5′调控区存在序列多态性.     

猪H-FABP基因第三内含子的遗传变异

以马身猪、大白猪、长白猪和杜洛克猪四个品种共190头猪为实验动物,成功扩增出猪H-FABP基因intron 3的全序列,全长1 350 bp,已向GenBank提交,检索号为DQ 002993。根据序列测定结果设计引物P2,扩增其中255bp的片段,应用PCR-SSCP分析发现P2-SSCP多态位点,该位点上具有两个等位基因A和B。在4个猪种中,AA基因型频率最高,A等位基因频率明显高于B等位基因频率。序列测定的结果表明,P2-SSCP的变异是由碱基A→G的替换造成的。     

猪PRLR基因PCR—SSCP多态性与产仔性能的关联分析

采用PCR-SSCP技术分析了杜洛克猪、长白猪、大白猪、马身猪、山西黑猪和山西白猪等6个品种472个个体催乳素受体基因(PRLR)的多态性,检测到A、B、C 3个等位基因和AA、AB、AC、BB、CC 5种基因型.在马身猪中, B等位基因为优势基因,频率为0.55;其他品种中,优势基因为A等位基因,频率分布在0.79～0.89之间;C等位基因除在大白猪频率略高外(0.20),在其他品种中频率都很低,在0～0.09之间.对AA、BB、CC三种纯合子进行克隆测序和同源序列比较,发现在扩增片段内有6处SNP,都发生在PRLR基因的第8内含子,分别是内含子8第26位、54位和99位的C→T突变,47位和68位的A→G突变,63位的G→A突变.利用最小二乘分析研究了PRLR基因型对母猪头胎总产仔数和产活仔数的影响,结果表明PRLR基因不同基因型母猪的头胎总产仔数和产活仔数差异均不显著.     

MC1R是控制鸡黑色素形成的候选主效基因

黑素皮质素受体1 (melanocortin 1-receptor, MC1R)基因是控制动物黑色素合成的重要基因.采用多聚酶链反应-单链构象多态性分析(PCR-SSCP)以及DNA测序的方法,在由丝羽乌骨鸡与明星肉鸡为亲本建立的中国农业大学资源家系群体鸡MC1R基因的编码区检测到3个单核苷酸多态位点,并对该单核苷酸多态性进行了分析.结果显示,鸡MC1R基因编码区引物3扩增片段多态性是由G→A（867位）点突变引起的,引物5扩增片段的多态性是由C→T（1 292位）与C→G（1 377位）两个点突变引起的,最后对单核苷酸多态性与肤色、肉色、胫色与内脏膜色等黑色素性状进行了卡方独立性分析,结果显示,MC1R基因编码区867处突变与鸡的肤色性状显著相关（P＜0.05）,1 292处突变与鸡的活体胫色性状显著相关（P＜0.05）,1 377处突变与鸡的肉色性状显著相关（P＜0.05）.研究表明,MC1R基因可能是鸡黑色素性状的主效基因或者与鸡控制黑色素性状的主效基因连锁.     

Chicken pars distalis development

Summary Anlagen of the pars distalis (Rathke's pouch), adjacent ectoderm, endoderm, infundibulum and mesenchyme left in situ were put in 3% glutaraldehyde between 1–3 pm and fixed overnight. Epon sections of this material from six White Leghorn, Gallus gallus, embryos fixed at each stage, and of two control partes distales from laying hens of the same flock were examined.At stage 17 (12 hours after formation) Rathke's pouch cells were stratified, uninnervated, non-vascularized and stellate, with high nucleo-cytoplasmic ratios and few organelles. Except for lipid inclusions, pouch cells did not appear appreciably different by stage 27, either regionally within the pouch wall or from the adjacent epithelioid cells.Apparent major changes indicative of cytoplasmic maturation by stage 27 included: reduction in number of polysomes; appearance of single-membraned, dumb-bell shaped to rounded, dense granula which were usually in basal position but also in areas of the Golgi apparatus; greater prominence of rough endoplasmic reticulum with cisternae containing dense material, and of the Golgi apparatus, notably with variously dense and/or coated vesicles and material; reduction in lipid inclusions by stage 24. Non-terminating axons in the infundibulum were first seen at stage 27.We found no convincing evidence for any possible morphogenetic or other relationship between the pars distalis and surrounding tissues. Melanophore-stimulating hormone-activity, reported to appear on day 5 of incubation, stored thyroid-stimulating hormone or pars distalis hormone granula or activities could not be identified. Rathke's pouch and other epithelioid structures may have been secreting, taking up nutrients and/or differentiating, activities which could be important for yolk sac development, nutrition and/or cytodifferentiation, respectively.Some results were reported at the Seventh Conference of European Comparative Endocrinologists in Budapest Hungary, Aug., 1973 (Betz and Jarskär, 1974).Part of work done on T. W. Betz's sabbatical leave awarded by Department of Biology, Carleton University, Ottawa, Canada, K 1 S 5 B 6 while additionally sustained by a North Atlantic Treaty Organization (NATO) Post-doctoral Fellowship in Science administered by National Science Foundation, Washington D.C., USA, 20550. My referees, Professors D. S. Farner, A.V. Nalbandov, R. L. Watterson and K. G. Wingstrand deserve special thanks. Professor Anders Enemar was a gracious host in so many important ways making my stay enormously worthwhile. Additionally supported by Swedish Natural Science Research Council (grant B 2180-22) and National Research Council of Canada (grants A 2822, T 541). We are grateful for the competent help of Ulla Wennerberg, Professor Björn Afzelius of Wenner-Grens Institut, Stockholm provided expert encouragement by endorsing our techniques.     

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50–70%.     

逆转录病毒与转基因鸡

摘要：随着生物制药业的迅速发展,转基因鸡输卵管生物反应器正逐渐成为人们关注的焦点,转基因鸡以其卓越的优势必将成为科学研究的热点和生物制药领域的新兴产业之一。目前,转基因鸡最成功的制备方法即逆转录病毒介导法,并已成功制备出表达标记基因的转基因鸡。本文主要综述了逆转录病毒载体制备转基因鸡的优势,逆转录病毒制备转基因鸡的方法和转基因鸡的研究进展,同时也讨论了转基因鸡的意义和存在问题。