马传染性贫血病毒减毒疫苗诱导马外周血单个核细胞白细胞介素12的转录

目的 探讨马传染性贫血病毒(EIAV)驴白细胞减毒疫苗(DLV)免疫马后,外周血单个核细胞(PBMC)中白细胞介素12(IL-12)mRNA转录水平与免疫保护应答的关系,揭示DLV的免疫保护机制。方法 应用分子克隆及实时定量反转录-聚合酶链反应(RT-PCR)技术,建立马PBMC中IL-12 mRNA转录水平的定量检测方法,在不同时间点定期观察4组(疫苗免疫组、阴性对照组、强毒株阳性对照组和EIAV自然感染组)12匹马PBMC中IL-12 mRNA转录水平及分布特征,同时监测体温变化等指标。疫苗株免疫动物8个月后,用EIAV强毒株攻击,观察攻击前、后IL-12 mRNA转录水平的变化。结果 DLV免疫马在免疫期内,PBMC中IL-12 mRNA转录的量略高于阴性对照组及自然感染组,但免疫后8个月用EIAV强毒株攻击,IL-12 mRNA转录量显著升高,4匹免疫马获得完全保护;强毒株阳性对照组IL-12转录量随疾病进展波动,发热期下降。结论 本研究首次证明EIAV减毒疫苗可诱导马外周血PBMC中IL-12基因高效转录,其转录水平与DLV的免疫保护密切相关。此结果在分子水平为阐明DLV的免疫保护机制提供了新的实验依据。     

马

概述了马的起源,我国养马业的历史、地方马品种资源、繁育的主要优育马种和世界主要名贵马种,并分别介绍了它们的生物学特征及体质外貌的区别。     

马蛔虫受精卵有丝分裂的图像分析定量测定

用彩色图像分析仪,对马蛔虫受精卵有丝分裂各期进行形态学测量,每期测量50个细胞。从细胞水平测量计算了8项形态计量参数,其中5项参数的显著差异率在83％以上,反映了分裂各期的卵细胞的异型性。这些形态计量参数,为分析细胞有丝分裂形态变化的定量指标提供了参考。     

马链球菌马亚种IgG结合蛋白的原核表达和免疫原性

为研究马链球菌马亚种IgG结合蛋白(EAG)免疫原性和保护力,评价其作为马链球菌疫苗抗原的价值。采用PCR法扩增马链球菌马亚种EAG基因,将测序正确的EAG扩增产物与原核表达载体pET-28a(+)连接构建重组质粒,对转化后的大肠杆菌进行诱导表达,用诱导纯化后的重组蛋白作免疫原免疫小鼠,分析重组蛋白免疫小鼠后的抗体水平及对小鼠的免疫保护力。结果表明,诱导后可得到26 kDa的EAG重组蛋白,且该蛋白可与该菌阳性血清发生特异性反应。间接ELISA检测免疫小鼠的抗体效价可达1∶8 100,重组蛋白免疫后对小鼠保护力可达90%。该结果表明,表达的EAG蛋白具有良好的抗原性,可有效提高小鼠的体液免疫水平及免疫保护力。     

EIAV弱毒疫苗免疫马外周血单核细胞IL-2表达水平的研究

目的:通过定量监测马传染性贫血病毒(EIAV)弱毒疫苗免疫马外周血单核细胞(PBMC)IL-2表达水平的变化特征,探讨EIAV弱毒疫苗的免疫保护机制。方法:用实时定量RT-PCR技术建立了马外周血PBMCIL-2表达水平的定量检测方法。在不同的时间点定期对4组(疫苗免疫组、健康对照组、强毒攻毒组和EIAV自然感染组)12匹马的外周血PBMCIL-2表达水平进行了检测,同时观察了临床症状及体温变化等指标。疫苗株免疫动物8个月后用强毒攻毒,观察了攻毒前后IL-2表达水平的变化。结果:(1)疫苗免疫马外周血PBMCIL-2的表达量显著高于健康对照组及自然感染组(P<0.01),且免疫后攻毒IL2继续升高,4匹疫苗免疫马均获得完全保护;(2)强毒攻毒对照组IL2表达量随疾病进展波动,发热期明显下降。结论:首次证明EIAV弱毒疫苗可诱导马外周血PBMC表达高水平的IL-2,提示IL-2在疫苗的免疫保护应答中发挥了重要作用;IL-2表达水平还与EIAV感染后的疾病进展密切相关。     

基于基因组SNPs对东亚家马不同群体遗传多样性的评估

家马地方品种的遗传多样性是动态演变的, 并与其育种模式息息相关; 在过去约300年中, 育种者培育了具突出表型特征的标准化培育品种, 该类型品种对家马地方品种的繁育产生了巨大影响, 这是导致家马地方品种遗传多样性下降及遗传分化的主要因素之一。本研究采集了5个东亚家马地方品种(蒙古马、哈萨克马、河曲马、藏马和西南马)、2个西亚家马品种(阿拉伯马和阿哈尔捷金马)以及2个欧洲家马品种(设特兰矮马和克莱斯黛尔马)的70个样品, 并整合之前所发表的100匹内蒙古地区家马不同群体单核苷酸多态性(SNPs)数据集, 通过全基因组重测序和生物信息学方法分析了东亚家马不同群体的遗传多样性。研究发现东亚家马地方品种具有较丰富的遗传多样性, 相比欧洲和西亚品种产生了显著的遗传分化, 其中蒙古高原群体的遗传多样性最为丰富; 受杂交改良影响, 内蒙古不同群体间产生了一定程度的遗传分化; 河曲马和藏马的遗传背景最为单一, 受引种杂交繁育影响较小。本研究评估了东亚家马地方品种的遗传多样性分布格局和演化特征, 可为建立家马地方种质核心保护群体以及培育新品种提供遗传学理论支持。     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

云南文山黄牛和迪庆黄牛遗传多样性的蛋白电泳研究

采用水平淀粉胶蛋白电泳技术分析了云南文山黄牛和迪庆黄牛的３３种血液蛋白及同工酶,共计３７个遗传座位,其中６个座位检测到多态性。文山黄牛的多态座位百分比Ｐ＝０．１３８９,平均杂合度Ｈ＝０．０６１０,迪庆黄牛Ｐ＝０．１６６７,Ｈ＝０．０６９１。通过Ｎｅｉ氏遗传距离计算,运用ＰＨＹＬＩＰ３．５Ｃ软件包中的“ＵＰＧＭＡ”,“ＣＯＮＴＭＬ”和“ＮＥＩＧＨＢＯＲ”法,结合前人报道的数据对１０个黄牛品种进行聚类分析。结果得出：文山黄牛可能主要起源于瘤牛（Ｂｏｓｉｎｄｉｃｕｓ）,迪庆黄牛可能主要起源于普通黄牛（Ｂｏｓｔａｕｒｕｓ）。我们的结果提示,云南黄牛可能是由当地人驯化的野生牛群与外来的驯化牛群相结合并适应当地气候和环境逐渐形成的,因而在蛋白水平上具有较为丰富的多样性。     

马的起源驯化、种质资源与产业模式

介绍了马的起源及驯化历史,根据不同的分类方法对马品种进行分类,介绍了目前世界与中国的马种资源与登记管理状况,并对国内外马产业发展现状与趋势作了分析与展望。     

青海马

青海湖中有个梅心山,相传置牝马于其上,可得“龙种”。古吐谷浑曾将波斯草马放入其中,因得(马单)驹,能日行千里,被称做青海(马单)。惰炀帝为得“龙种”,曾置马在此放牧。这虽然是一些近乎神话的传道,但青海出名马,却是自古就闻名的了。 青海省有马三十五万多匹,按其体形和性能来说,约可分为河曲马、大通马、王树马和柴达木马四种,     

光叶珙桐的等位酶分析及其生物地理学意义

采用水平切片淀粉凝胶电泳实验方法,对光叶珙桐(Davidia involucrata var．vilmoriniana)4个居群、78个个体、11种酶系统,21个位点进行了等位酶分析。各居群的多态位点百分比P＝28.6％-47．6％,实际杂合度Ho＝0．177—0.308,平均每个位点的等位基因数A=1．3-1．6。在变种水平上,多态位点百分比P＝52．4％,平均每个位点的等位基因数A＝1．8,实际杂合度Ho＝0．272,期望杂合度Ho＝0．216。基因分化系数FST＝0．1928,说明光叶珙桐居群问分化小。各项指标表明光叶珙桐的遗传多样性水平较高,比原变种珙桐高,外部环境可能是影响珙桐分布格局的重要因素。昭通与宝兴的遗传多样性明显高于其他两个居群,遗传多样性保存较为完整,而处于分布区东、西两侧边缘(云南西北部和湖北五峰)遗传多样性则相对较低,推测四川盆地边缘山地是该种的遗传多样化中心,可能是在地质灾难中(如第四纪冰期),光叶珙桐真正的避难所。分布区东西两侧的居群可能是从四川盆地边缘山地扩散而来。     

Genetic variation of Polish endangered Biłgoraj horses and two common horse breeds in microsatellite loci

Genetic variation of endangered Bi?goraj horses and two common Polish horse breeds was compared with the use of 12 microsatellite loci (AHT4, AHT5, ASB2, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, VHL20). Lower allelic diversity was detected in all investigated populations in comparison to other studies. Large differences in the frequencies of microsatellite alleles between Bi?goraj horses and two other horse breeds were discovered. In all polymorphic loci all investigated breeds were in the Hardy-Weinberg equilibrium. Mean Fis values and the results of a test for the presence of a recent bottleneck were non-significant in all studied populations. Comparable values of observed and expected gene diversity indicate no substantial loss of genetic variation in the Bi?goraj population and two other breeds. The lowest variability observed in the investigated group of Thoroughbred horses was confirmed. About 10% of genetic variation are explained by differences between breeds. Values of pairwise Fst and two measures of genetic distance demonstrated that Bi?goraj horses are distantly related to both common horse breeds.     

拟环纹豹蛛不同地理种群遗传多样性的AFLP标记研究

为查明拟环纹豹蛛Pardosa pseudoannulata不同地理种群的遗传多样性机制,应用AFLP技术对6个拟环纹豹蛛地理种群的遗传多样性进行了研究分析。8对引物组合扩增出1 038个AFLP条带,其中多态性条带占86.622%,全部个体显示了各自独特的AFLP图谱。AFLP标记的遗传多样性分析结果表明： 拟环纹豹蛛无论在物种水平(P=86.62%,H=0.2622,I=0.3101),还是在种群水平（P=73.0%,H=0.2155,I=0.2554）都表现出较高的遗传多样性。其中湖南长沙雷锋镇种群内遗传变异最大,云南高黎贡山福贡种群内遗传变异最小,华南北部（湖南、湖北、江西）地区拟环纹豹蛛遗传多样性明显高于华南南部（云南、海南）种群。据种群变异来源分析,有35.77%的遗传变异来自种群间,64.23%的变异来源于种群内（N_m=0.898）,不同地理种群显示出一定的遗传分化。分析认为海拔是影响拟环纹豹蛛遗传分化的重要因素,这为进一步明确我国稻田狼蛛优势种群在农药胁迫下的遗传适应性机制提供了实验依据。     

竹叶眼子菜居群遗传多样性和克隆结构

采用ISSR技术对长江中游南岸豹澥湖和大冶湖不同生境中的竹叶眼子菜(Potamogeton malaianus)居群的遗传多样性及克隆结构进行了研究.结果表明,在两居群的106株个体中,利用6条ISSR引物共得到40条符合3/N标准并无连锁不平衡的清晰位点,竹叶眼子菜具有较高的遗传多样性,其多态位点百分率为75.0％,Shannon多样性指数为0.3736, 两居群的遗传分化很小.竹叶眼子菜的克隆多样性很高(D=0.9917),两居群间的克隆分化很大,不具有共有的基因型.竹叶眼子菜的基株分布为游击型构型,位于湖心的大冶居群克隆距离(3.0～31.5 m)明显地大于湖岸豹澥居群(2.4～6.7 m).     

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)

Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P(99)) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (H(exp)) of 0.294. At the population level, mean P(99) = 56%, mean A = 1.81, and mean H(exp) = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean F(IS) = 0.002). There was significant deviation from panmixia associated with population differentiation (mean F(ST) = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle F(ST) estimates, a pollen to seed flow ratio of 1.43?:?1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.     

RAPID GENETIC DIFFERENTIATION AND FOUNDER EFFECT IN COLONIZING POPULATIONS OF COMMON MYNAS (ACRIDOTHERES TRISTIS)

Populations of common mynas introduced to Australia, New Zealand, Fiji, Hawaii, and South Africa from India during the last century were compared genetically with the extant native population using isozyme electrophoresis of 39 presumptive loci. Average heterozygosity, mean number of alleles/locus, and the percentage of polymorphic loci are lower in the introduced populations, and the 18% loss of alleles involves only alleles that are rare in the native population. The native population is only weakly subdivided genetically (F_ST = 0.032) whereas the introduced populations are much more differentiated (F_ST = 0.123), and the mean genetic distance among them is significantly greater than among native samples. The reduction in mean number of alleles/locus and average heterozygosity is greatest in the South African population, consistent with a very small effective size in the founder population. In the introduced populations, random drift is implicated by the different subsets of polymorphic loci they possess, by their greater variance in allele frequencies, and by shifts either side of the native means. It is concluded that in the evolutionarily short period of 100–120 years, bottlenecks and random drift have promoted genetic shifts equal to those between different subspecies of birds.     

A microsatellite analysis of five Colonial Spanish horse populations of the southeastern United States

The domestic horse (Equus caballus) was re-introduced to the Americas by Spanish explorers. Although horses from other parts of Europe were subsequently introduced, some New World populations maintain characteristics ascribed to their Spanish heritage. The southeastern United States has a history of Spanish invasion and settlement, and this influence on local feral horse populations includes two feral-recaptured breeds: the Florida Cracker and the Marsh Tacky, both of which are classified as Colonial Spanish horses. The feral Banker horses found on islands off the coast of North Carolina, which include, among others, the Shackleford Banks, the Corolla and the Ocracoke, are also Colonial Spanish horses. Herein we analyse 15 microsatellite loci from 532 feral and 2583 domestic horses in order to compare the genetic variation of these five Colonial Spanish Horse populations to 40 modern horse breeds. We find that the Corolla horse has very low heterozygosity and that both the Corolla and Ocracoke populations have a low mean number of alleles. We also find that the Florida Cracker population has a heterozygosity deficit. In addition, we find evidence of similarity of the Shackleford Banks, Marsh Tacky and Florida Cracker populations to New World Iberian horse breeds, while the origins of the other two populations are less clear.     

Conservation genetics of an endemic and endangered epiphytic Laelia speciosa (Orchidaceae)

We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, P(p) = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity H(O) = 0.302, the average expected heterozygosity H(E) = 0.382). Moderate levels of inbreeding (?f = 0.216, 95% confidence interval = 0.029-0.381) were found. Low levels of genetic differentiation were observed among populations ((p) = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r(2) = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.     

华北2蝗区东亚飞蝗种群遗传结构的比较研究

利用水平淀粉凝胶电泳对采自天津北大港和河北黄骅两个相临蝗区的东亚飞蝗（Locusta migratoria manilensis)种群进行等位酶基因频率分析,比较了这两个种群的遗传结构,等位酶酶谱分析表明,19个基因座中4个基因座（Mdh-l,Pgm,Adk,G3pd)的等位基因频率变化很小,常见等位基因的频率均高于0．95,其他基因座有2－4个等位基因,但是两个种群的等位基因频率除两个基因座（Fbp,Got-2)外都很相似,多态位点的27个χ2检验表明,由于常见等位基因纯合子的高频率的和相应杂合子的缺乏,仅有北大港种群的2个基因座（Pgi,Got-1)符合Hardy-Weinberg平衡,在每个种群内的蝗虫存在明显的遗传变异,但在种群间遗传结构极为相似,多态位点的百分数P分别为73．7％和78．9％,每个基因座的平均等位基因数A为2．9和3．1,平均每个基因座的实际杂合度几乎相等（约为0．138）,F－统计量（FST＝0．053）也表明了两个种群间的遗传 一致性,遗传相似性系数（I）高达0．938,这些结果提示,这两个种群可能属于1 个大种群,在两个种群的一定位点上的遗传多态性和分化可能都与迁飞因素有关,因为东亚飞蝗的高度扩散能力有利于遗传结构的连续分布,高度的迁飞能力也导致个体暴露于各种不同的环境,而在种群水平上的遗传为异能增强种群在各种生态条件生存和繁殖能力,因此,迁飞有利于维持东亚飞蝗种群的遗传多态性的动态平衡。     

大王马先蒿云南五个居群遗传变异研究

采用随机扩增多态DNA(RAPD)分子标记方法,对大王马先蒿(PedicularisrexL.)分布在云南中甸、丽江、大理、武定和昆明的5个居群(其中中甸居群有3个亚居群)的遗传多样性和遗传结构进行了研究。结果表明,大王马先蒿居群具有较高的遗传多样性,多态位点百分率P、Shannon表型多样性指数I和Nei的基因多样性指数h分别为82.0%、0.361和0.240,遗传多样性水平与居群的地理分布范围相关;大王马先蒿居群间的遗传变异较高,遗传变异主要发生在居群之间,而不是居群内部;居群间遗传分化系数Gst为0.747,遗传分化明显,居群间基因交流较少。居群间的遗传距离与居群间的相对地理距离具有一定的相关性。对云南西北部中甸居群的3个亚居群的遗传多样性分析表明,居群的遗传多样性水平与海拔高度有一定的相关性。居群间遗传变异较高可能是由于大王马先蒿为短命多年生植物,繁育系统为混交型,且自交占较大比例等原因造成的。