新型鸭呼肠孤病毒NP03株S3基因序列分析

根据GenBank禽呼肠孤病毒(ARV)和番鸭呼肠孤病毒(MDRV)S3基因序列设计合成1对引物,特异性扩增新型鸭呼肠孤病毒NP03株的S3基因,并对其序列进行分析.结果克隆获得NP03株S3 cDNA包含完整的阅读框架,同源性分析NP03分离株S3基因核苷酸序列与ARV、火鸡呼肠孤病毒(TRV)和MDRV的同源性分别为60%～60.2%、61.9%和58.2%～62.7%,氨基酸的同源性分别为68.2%～69%、68.2%和63%～70.4%.表明NP03株的S3基因具有不同于ARV和MDRV的特征,分离毒NP03是一株新型鸭呼肠孤病毒.     

新型鸭呼肠孤病毒RT-PCR方法的建立与应用

研究建立检测新型鸭呼肠孤病毒(novel duck reovirus,NDRV)的RT-PCR方法,并运用建立的检测方法对分离毒与人工感染样品进行应用检测.根据NDRV-NP03株S3基因全序列(NDRV-NP03,GenBank登录号:GQ888710),设计合成了一对引物,以NDRV分离株为模板,建立了检测NDRV的RT-PCR方法.结果显示:该方法仅能从NDRV分离毒中扩增到与预期大小相符长度为586 bp的特异性目的片段,检测灵敏度达到2 pg病毒RNA,而其它病毒,番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)、禽呼肠孤病毒(Avian reovirus,ARV)、鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)、番鸭细小病毒(Muscovy duck parvovirus,MDPV)、鹅细小病毒(Goose parvovirus,GPV)、鸭副粘病毒(Duck paramyxovirus,DPMV)、鸭病毒性肝炎病毒(Duck hepatitis virus,DHV)等样品的扩增结果均为阴性.应用该方法对8株NDRV分离毒和3份人工感染鸭肝脾组织进行检测均为阳性.表明建立的RT-PCR方法特异性强、敏感度高,可用于NDRV的临床诊断和流行病学调查.     

稻褐飞虱呼肠孤病毒的核酸结合蛋白

采用Northwestern分子交杂方法，研究了稻褐飞虱呼肠孤病毒（NLRV）结构蛋白的功能，结果证明被推测为RNA聚合酶的P1蛋白、B突起的P2蛋白、NTP结合的P7蛋白和主要的病毒外层蛋白的P8蛋白是非特异性核酸结合蛋白，具有与单链或双链RNA和DNA4种核酸结构的能力，暗示了这4种蛋白在NLRV病毒核酸复制，病毒粒子包装中起作用。     

干扰水稻瘤矮病毒(RGDV)非结构蛋白(Pns12)的表达抑制病毒在介体昆虫培养细胞内的复制

水稻瘤矮病毒(Rice gall dwarf virus,RGDV)属呼肠孤病毒科(Reoviridae)植物呼肠孤病毒属(Phytoreovirus),由介体电光叶蝉(Recilia dorsalis)以持久增殖型方式传播,其基因组第12条片段编码的非结构蛋白(nonstructural protein,Pns12)是提供病毒复制和子代病毒粒体装配场所——病毒原质(viroplasm)的组分之一。为了明确Pns12在RGDV侵染介体电光叶蝉培养细胞中的功能,本研究通过原核表达的Pns12蛋白免疫注射兔(Oryctolagus cuniculus),制备Pns12抗体,并应用免疫荧光标记和RNA干扰(RNA interference,RNAi)技术研究Pns12在介体培养细胞内的定位和参与病毒原质形成的过程。共聚焦显微镜观察到,病毒侵染的细胞中,与荧光素交联的Pns12抗体特异地标记在病毒原质上。干扰Pns12蛋白表达后,可有效地阻碍病毒原质的形成、子代病毒粒体的组装和病毒非结构蛋白Pns12和外壳蛋白P8蛋白的表达,表明Pns12作为病毒原质的组分参与了RGDV在介体培养细胞内的复制。也表明,Pns12可作为理想的靶标用于阻断电光叶蝉携带和传播RGDV。     

利用RNAi技术研究水稻瘤矮病毒P8蛋白在病毒装配中的作用

水稻瘤矮病毒(Rice gall dwarf virus,RGDV)是呼肠孤病毒科(Reoviridae)植物呼肠孤病毒属(Phytoreovirus)的一个成员,由介体电光叶蝉(Recilia dorsalis)和黑尾叶蝉(Nephotettix cincticeps)以持久增殖型方式传播。其基因组编码6个结构蛋白和6个非结构蛋白。已知结构蛋白P8是病毒外层衣壳的主要成分,但其在病毒侵染介体细胞过程中的功能尚未弄清。本研究利用体外合成的ds RNA诱导的RNA干扰(RNA interference,RNAi)技术,研究RGDV P8蛋白在病毒侵染电光叶蝉培养细胞中的功能。通过免疫荧光标记技术对ds P8处理后的电光叶蝉培养细胞进行RGDV侵染观察,发现ds P8处理不仅抑制了P8在细胞内的正常表达,同时也抑制了病毒在细胞内的积累。随后通过SDS-PAGE凝胶电泳和Western blot分别检测病毒的ds RNA基因组和病毒蛋白水平的变化,发现ds P8处理显著降低了病毒双链RNA(double-stranded RNA,ds RNA)基因组的合成水平和病毒P8蛋白的表达量。由此推测RGDV P8蛋白参与了病毒粒体的组装过程和病毒在介体昆虫细胞内的有效增殖和侵染过程。本研究结果为利用RNAi技术控制水稻瘤矮病的传播提供了重要的理论依据。     

环介导等温扩增技术及其在水生动物病毒检测中的应用

水产养殖已成为国民经济重要组成部分,但经常受到水生动物病毒的困扰。相关病毒病原的检测已成为水生动物病原研究的热点之一。环介导等温扩增技术具有高特异性和灵敏度,并且扩增时间短,对仪器的要求较低,已广泛应用于水生动物病毒的检测中。本文从环介导等温扩增技术中引物的设计,扩增反应过程及反应产物的检测三个方面来阐述其基本原理,并综述了其在水生动物虹彩病毒、弹状病毒、呼肠孤病毒、疱疹病毒及对虾病毒检测中的应用,最后对其技术和应用上的发展前景进行了展望,以期为水生动物病毒检测相关研究提供参考。     

核酸探针检测禽呼肠孤病毒传播动态

用地高辛标记禽呼肠孤病毒(ARV)S1基因中编码σC蛋白的基因片段作为核酸探针,在斑点分子杂交中可检测到1.6pgARV的RNA。利用该核酸探针,通过检测鸡羽毛囊及体内病毒繁殖情况,比较研究了ARV感染后在鸡体内及鸡群中的传播动态。结果显示,研究建立的核酸探针检测方法灵敏度高、特异性强和操作简便,适于批量样品的检测。同时,用此方法检测发现,ARV感染24h后可侵染大部分器官,并且很快传播到同群未攻毒的鸡中,羽毛囊中的病毒检出率与鸡内脏器官中病毒的检出率一致。用核酸探针检测鸡羽毛囊中ARV的方法检测ARV的感染与流行情况,成本低,不影响鸡群生产。     

用核酸探针检测鸡脏器及羽毛囊中禽呼肠孤病毒

摘要：用地高辛标记禽呼肠孤病毒（ARV）S1基因中编码σ3蛋白的基因片段作为核酸探针，在斑点分子杂交中可检测到1.6pg的ARV RNA。利用该核酸探针，通过检测鸡羽毛囊及体内病毒繁殖情况，比较研究了 ARV感染后在鸡体内及鸡群中的传播动态。结果显示，ARV感染后在24小时时就可侵染大部分器官，并且很快传播到同群未攻毒的鸡中。羽毛囊中的病毒检出率与鸡内脏器官中病毒的检出率一致。该研究证明用核酸探针检测鸡羽毛囊中ARV的方法检测ARV的感染与流行情况，具有灵敏、特异性高，操作方便的特点，尤其适用于大量样品的同时检测。     

SRBSDV侵染的白背飞虱中肠酵母双杂交cDNA文库的构建和分析

南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)是呼肠孤病毒科(Reoviridae)斐济病毒属(Fijivirus)成员,主要由白背飞虱(Sogatella furcifera,WBPH)以持久增殖型方式进行传播.在病毒的侵染循回过程中,白背飞虱中肠上皮细胞是病毒的初侵染和主要增殖场所.而病毒的有效增殖是决定介体能否传毒的关键影响因素.为了更好地研究SRBSDV和介体白背飞虱的互作关系,尤其是了解白背飞虱中肠蛋白通过参与调控病毒的增殖过程,而使介体昆虫成功获毒并传毒,本研究以高带毒白背飞虱群体中肠组织为实验材料,构建了高带毒白背飞虱群体中肠的酵母双杂交cDNA文库.经过检测表明,构建的文库滴度为1.5×106 cfu/mL,平均插入片段主要分布在1.0～2.0 kb之间,文库质量较好,可用于研究SRBSDV编码蛋白和白背飞虱中肠蛋白的互作关系,并为开展SRBSDV和昆虫介体的互作研究奠定了基础.     

草鱼出血病及其疫苗研究进展

草鱼(Grass carp)是我国淡水养殖的四大家鱼之一,养殖范围广,经济价值高。但在草鱼养殖饲养过程中,易发生各种疾病,给草鱼养殖业带来巨大损失。其中,草鱼出血病是影响草鱼养殖的重要疾病之一,其病原为草鱼呼肠孤病毒。对于该病国内目前对其做了大量研究,化学药物难以达到治疗目的而免疫技术在该疾病的防治上取得了一定成就。基于此,首先总结了近年来关于草鱼出血病的研究,着重阐述草鱼出血病的病原生物学特征以及流行病学相关情况;而后就国内目前草鱼出血病疫苗研究情况做一介绍并提出该病防治的新展望。     

H5N1亚型高致病性禽流感病毒A/Duck/FuJian/01/02株感染性克隆构建

摘 要：1999-2002年本试验室从我国南方健康鸭体内分离到21株H5N1亚型禽流感病毒,对其进行系统地生物学特性和遗传演化分析发现,其中A/Duck/FuJian/01/02(简DKFJ)和A/Duck/Guangxi/53/02(简DKGX) 属于同一基因型, 对鸡都呈高致病性,但对哺乳动物模型Balb/c小鼠的致病性明显不同: DKGX对小鼠呈低致病性（MLD50>106.5）,DKGX株反向基因操作系统本试验室已经建立,而DKFJ对小鼠呈高致病性（MLD50<100.5）。为了研究这两株病毒对哺乳动物模型Balb/c小鼠致病性差异的分子机制,本研究构建了对DKFJ的8质粒反向基因操作系统,并通过细胞转染技术成功拯救了该病毒（R-DKFJ）。R-DKFJ在对哺乳动物模型Balb/c小鼠的致病性保持了与亲本野生毒（W-DKFJ, MLD50<100.5）一致的生物学特性,并且对小鼠呈全身感染,即106EID50鼻腔感染小鼠后第三天在脑、脾、肾、肺脏等脏器检测到病毒。DKFJ反向基因操作系统的成功建立为阐明H5N1亚型禽流感病毒对哺乳动物模型Balb/c小鼠的致病机制等方面奠定了基础。     

一株对小鼠具有高致病性的H5N1亚型禽流感病毒的拯救

禽流感病毒(Avianinfluenzavirus)正在逐渐获得突破种间屏障感染哺乳动物的能力,揭示其获得此种能力的分子机制已经成为禽流感病毒的研究热点。A/Chicken/Guangdong/04(H5N1)是一株高致病性禽流感病毒,同时对BALB/c小鼠也具有高致病力。实验通过反向遗传操作技术对该病毒进行拯救,获得了拯救毒R-A/Chicken/Guangdong/04(R-CG)。R-CG与其亲本毒A/Chicken/Guangdong/04(W-CG)在胚半数感染量(EID50)、细胞培养半数感染量(TCID50)、对SPF鸡和BALB/c小鼠致病力等生物学特性保持一致。即R-CG与W-CG对鸡都为高致病力,静脉接种指数分别为2.88和2.91;R-CG与W-CG一样,以106EID50鼻腔感染BALB/c小鼠后,均能引起小鼠死亡,在小鼠脑、肺、肾和脾脏中都能分离到病毒,说明拯救的病毒与亲本毒一样,都可在小鼠体内有效复制,对小鼠具有高致病性。本实验成功地拯救了A/Chicken/Guangdong/04,拯救病毒R-CG可作为背景毒株,为研究禽流感病毒突破种间屏障感染哺乳动物的分子机制奠定了实验基础。     

Effects of grazing practices and fossorial rodents on a winter avian community in Chihuahua, Mexico

Chihuahuan Desert grasslands are important wintering grounds for grassland and shrub-adapted birds. Many species belonging to these assemblages are currently exhibiting population declines. One area recognized for its importance to biological diversity, including grassland birds, is the Janos-Nuevo Casas Grandes black-tailed prairie dog (Cynomys ludovicianus) complex in northwestern Chihuahua, Mexico, an area containing 58 colonies with 30,000 ha of prairie dogs. This is one of the largest remaining prairie dog complexes and the only intact complex in the Chihuahuan Desert. In its current condition, a large percentage of this complex is of reduced value to wildlife. Overgrazing on communal (ejido) lands has resulted in areas being comprised of annual grasses and forbs. The density of active prairie dog burrows and banner-tailed kangaroo rat (Dipodomys spectabilis) mounds as well as avian diversity and abundance were lower on ejido lands than an adjacent private ranchland with and without prairie dogs. Few avian species used overgrazed portions of the prairie dog colony. Community similarity among plot types was low due to different management practices and differences on and off colony. To retain, and in many instances restore the biological diversity of this important region it is essential to work with local ejidos on grazing management.     

Advances in the value of eggs and egg components for human health

The avian egg is an important source of nutrients, containing all of the proteins, lipids, vitamins, minerals, and growth factors required by the developing embryo, as well as a number of defense factors to protect against bacterial and viral infection. Moreover, eggs are now understood to contain substances with biological functions beyond basic nutrition, and extensive research has been undertaken to identify and characterize these biologically active components. This review mainly focused on biological activities of proteins and peptides derived from egg components. Several biological activities have now been associated with egg components, including novel antimicrobial activities, antiadhesive properties, immunomodulatory, anticancer, and antihypertensive activities, antioxidant properties, protease inhibitors, nutrient bioavailability, and functional lipids, highlighting the importance of egg and egg components in human health and in disease prevention and treatment. Continued research to identify new and existing biological functions of hen egg components will help to define new methods to further improve the value of eggs as a source of numerous biologically active compounds with specific benefits for human and animal health and secure their role in the therapy and prevention of chronic and infectious disease.     

Structural basis for antioxidant activity of trans-resveratrol: ab initio calculations and crystal and molecular structure

From the experimental crystal structure and ab initio calculations on resveratrol and its derivatives, structural features of mechanistic importance are described. The molecular structure reveals the relative coplanarity of the trans-stilbene skeleton, and the molecular packing in the solid state shows an extensive hydrogen bond network that elucidates the flip-flop motion of the three hydroxyl groups that alternately form and break H bonds with each of the neighboring phenolic oxygens. The dynamic behavior provoked by the alternation of hydrogen bond formation and breaking can result in the ready mobility of up to three hydrogen atoms per resveratrol molecule that can be transferred to reactive oxidants that are rich in electron density. In addition, theoretical studies confirm the planarity of resveratrol as well as for half of the molecule of a condensation dimeric derivative of resveratrol, trans-sigma-viniferin. Furthermore, these studies show the p-4'-OH group to be more acidic compared to the other two m-OH groups. These features correlate with the biological activity of resveratrol as an antioxidant and support earlier studies showing H-atom transfer to be the dominant mechanism by which phenolic antioxidants intercept free radicals.     

突变型抑癌基因p53在蛋鸡J亚群禽白血病中的表达

摘要: 蛋鸡J亚群禽白血病是病毒引起的肿瘤性传染病。为了探讨J亚群禽白血病病毒的致癌分子机理, 观察蛋用型鸡发生J亚群禽白血病时p53抑癌基因在各脏器中的表达, 应用链霉亲和素-生物素-过氧化物酶复合物免疫组化（SABC）法，研究了蛋用型鸡J亚群禽白血病自然病例的肝脏、肾脏、腺胃、肿瘤、输卵管、心脏、胰脏、骨髓、脾脏、肺脏、十二指肠、胸腺和法氏囊。结果表明：突变型p53在肝脏、肾脏、腺胃、肿瘤、输卵管中表达量最高，心脏、胰脏、骨髓、脾脏、肺脏、十二指肠、胸腺及法氏囊均可检出中等量的表达。这一结果提示突变型抑癌基因p53的表达参与蛋鸡J亚群禽白血病发生、发展过程。     

Trends associated with residential development in riparian breeding bird habitat along the Snake River in Jackson Hole, WY, USA: Implications for conservation planning

Throughout North America, bird population declines may be attributable to loss of habitat on the breeding grounds. Human land uses such as residential development, greatly impact landscapes and biota, particularly breeding birds. Riparian areas, which are essential to breeding birds, are particularly vulnerable to development pressures. In North America, most studies have focused on eastern birds and it is unclear how western bird assemblages respond to the effects of residential development. We sampled bird community parameters and habitat variables at three spatial scales (microhabitat, macrohabitat, and landscape) along a residential development gradient within the Snake River riparian corridor in Jackson Hole, WY, USA. Principal component analysis and multiple linear regression statistical tests were used to determine the effects of housing densities on avian community parameters, guilds, individual species distributions, and environmental variables. Landscape-level features were most affected by residential development and trends associated with increasing housing densities, such as anthropogenic habitat fragmentation primarily structured local bird communities. Overall species richness and diversity declined with increasing residential development. Neotropical migrant species were most negatively impacted and consistently declined in proportional representation on forested plots as residential development densities increased. Food generalists, ground gleaners, and avian nest predators all increased with increasing residential development. Brood parasites, on the other hand, did not increase with increasing housing densities and their distribution may reflect the availability of nest host species. These results suggest that residential development within riparian habitats may be exerting a strong negative influence on western bird communities and at high densities may lead to a depauperate avian biota. The results of our study indicate that preserving forest structure may benefit certain species but will not likely conserve as diverse and rich an avian assemblage as minimizing fragmentation associated with residential development.     

应用RT-PCR及RFLP技术对鸡传染性支气管炎病毒进行诊断与分型的研究进展

本文概述了近年来国内外应用ＲＴ－ＰＣＲ及ＲＦＬＰ技术对对外开放传染性支气管炎病毒（ＩＢＶ）进行诊断与分型的研究进展。介绍以ＩＢＶ通用引物的ＲＴ－ＰＣＲ进行诊断及其应用、型特异引物的ＲＴ－ＰＣＲ及其应用以及ＲＴ－ＰＣＲ结合ＲＦＬＰ分析技术进行分型的应用,而且还展望了这上结技术在临床诊断及分子流行病学研究上的应用前景。     

“十四五”土壤生物学分支学科发展战略

土壤生物是地球生物多样性的重要组成部分。土壤生物驱动着土壤中有机质降解、元素循环、污染物转化与降解以及温室气体的产生与消耗,在全球粮食安全、环境保护以及应对气候变化等方面发挥着重要作用。土壤生物学是研究土壤生物多样性与分布、土壤生物的过程与功能以及土壤生物的调控与应用的科学。21世纪以来,分子生物学技术的突破与生态学理论的广泛应用极大地推进了土壤生物学研究的发展。本文回顾了土壤生物学科的发展历程,详细介绍了土壤生物学科的发展现状,提出了土壤生物学科在理论与应用上的发展趋势,并对未来土壤生物学科的发展方向进行了展望。随着多学科交叉融合以及研究手段的进步,土壤生物学迎来了一个新的发展时期。土壤生物学研究在生物资源挖掘、时空分布格局、生态服务功能和生物调控等方面取得的重要成果,将更好地服务于土壤健康、植物健康、人类健康以及我们的星球健康。     

Basic strategies of molecular biological research in plant nutrition - a review

A review is given on basic methodical strategies of molecular biological research in plant nutrition. Membrane transport protein identification and analysis by the use of cDNA libraries and functional expression in yeast mutants and Xenopus oocytes is demonstrated. The various methods of gene isolation and analysis, including molecular data of model plants, are pointed out. The significance of research on transgenic plants permitting overexpression of introduced genes, antisense technique and analysis of promoter/reporter gene constructs is emphasized. The numerous examples of recent research simultanously indicate contemporary and future topics for molecular biological research in plant nutrition.