伪狂犬病病毒广西B、W株gE基因的扩增、克隆及序列分析

在已鉴定了的伪狂犬病病毒（Pseudorabies Virus，PRV）11种糖蛋白中，gE是其中十分重要的一种非必需糖蛋白，是伪狂犬病病毒重要的毒力基因，它在介导感染细胞的融合，病毒在细胞间的扩散，病毒粒子的释放等方面均起着十分重要的作用，尤其是在影响伪狂犬病病毒神经嗜性和毒力方面的作用。利用PRV gE基因缺失疫苗，结合血清学方法可以区分疫苗接种猪和野毒感染猪。     

鹌鹑新城疫病毒分离株F基因的克隆和序列分析

研究以新城疫鹌鹑分离株(LA005)的基因组RNA为模板，应用RT-PCR技术扩增出其F基因的主要功能区片段，并进行了克隆和序列分析。结果表明：所扩增的目的片段长度为813bp，裂解位点氨基酸序列为112R-T-Q-R-R-Fll7，和F48E9标准强毒株的核苷酸和氨基酸的同源率分别为98．9％和98．5％，Cys残基位点和糖基化位点的数目和位置完全一致，说明该分离毒是一株和F48E9标准强毒株亲缘关系很近的强毒株。     

鸡组织细胞端粒相关序列的克隆及序列分析

根据真核细胞端粒DNA序列的共同特点，设计了一条引物（TELO02）（24nt)。按照TaKala克隆试剂盒的介绍方法，染色体基因组DNA经HaeⅢ和HinfⅠ两种限制性内切酶酶切后，先进行初级PCR（C1和TELO02）扩增，得到的产物为模板再经过次级PCR（C2和TELO2）扩增，得到的产物用试剂盒回收，并连接到PMD-18-T载体上，转化到JM109受体菌中，从阳性克隆中提取质粒DNA进行PCR和酶切鉴定，确认后进行序列测定，获得了一段243sbp的鸡端粒相关序列。此序列与人第7条染色体基因组末端序列的同源性为63.5%；与鼠端粒旁序列同源性为63.2%；而与MDV基因组序列的BamHⅠ酶切片段76.6%的同源。     

鹅细小病毒VP1与VP3非重叠序列的克隆与原核表达

根据鹅细小病毒GPVH1株基因组核苷酸序列设计了一对引物，对其结构蛋白VP1与VP3非重叠核苷酸序列进行PCR扩增，并克隆到表达性载体pGEX-6p-1，经酶切鉴定筛选出阳性克隆，并转化到大肠杆菌BL21(ED3)plysS进行原核表达，并用SDS—PAGE鉴定，结果表明融合蛋白在表达性细菌BL21中重获得了高效表达。     

鸡传染性支气管炎病毒(IBV)H120疫苗株S1基因的克隆与序列测定

根据国外已发表的鸡传染性支气管炎病毒(IBV)S1基因序列设计了一对引物，通过：RT-PCR特异性扩增出IBV H120疫苗株的S1基因，产物大小为1．62kb，与设计相符。同源性比较结果显示，H120株与H52、M41和BEAU株的S1基因核苷酸序列的同源性分别为97．1％、96．9％和96．8％，表明IBV H120疫苗株与标准毒株的S1基因具有高度的同源性。     

基于GIS的中国2大草地分类系统类的兼容性分析

以植被-生境学分类(vegetation-habitat classification system of grassland,VHCS)方法为基础的定性的中国草地分类系统,以及定量的气候-土地-植被综合顺序分类系统(comprehensive and sequential classification system of grassland,CSCS)是我国常用的2大草地分类系统。在实际应用中,因“草原”和“草地”概念的重叠、混淆和交叉,以及定性分类和定量分类的差异,造成了操作上的困扰,研究结果亦不便相互交流。本研究通过2大系统一级单位-“类”的分类指标、名称和属性的对比,分析了2个系统“类”的兼容性,建立了两者间的对应关系,并利用基本同期的CSCS分类图和数字化的中国草地资源图(VHCS),以内蒙古自治区和甘肃省为例,在ArcGIS平台上进行了验证分析。研究结果表明:1)以广义草原或草地概念为基础的CSCS是一个大系统,兼容了以中国为例的VHCS分类系统;2)兼容VHCS的CSCS的类,两者在分类指标、名称和属性方面均能达到统一;3)空间叠置分析表明,若不考虑森林和非地带性类,内蒙古和甘肃区域2个分类系统分类结果的兼容性分别达到61.4%和61.1%;有差异的区域,基本上表现出草地实际调查结果(VHCS)是比原生潜在草地(CSCS)在更恶劣气候条件下的低分类级别的草地类,说明人为干扰已超过了原生草地生态系统的生存阈限,导致草地逆行演替;4)对CSCS与VHCS分类结果的对比分析研究,可科学地揭示人为干扰下草地的演替状态,并明确草地恢复和重建的目标。     

进口白羽肉种鸡J亚群禽白血病病毒的全基因组序列分析

为研究引起我国部分地区进口白羽肉种鸡发生禽白血病疫情的病毒来源及其变异趋势,本研究对从进口白羽肉种鸡分离的8株J亚群禽白血病病毒(ALV-J)的全基因组序列进行测序分析。8株ALV-Jgp85氨基酸遗传演化分析显示,其与ALV-J肉鸡分离株(包括ALV-J原型病毒株HPRS-103、美国肉鸡ALV-J分离株、国内肉鸡分离株)及蛋鸡分离株氨基酸序列同源性均低于92.5%,而与国内2014年父母代白羽肉种鸡ALV-J分离株GD14J2同源性较高,为93.5%~95.1%,处于同一分支。8株ALV-J的3'非编码区(3'UTR)在rTM区缺失203bp,DR-1区缺失7bp,E元件位置缺失125bp,仅保留了22个碱基,这一缺失特征与GD14J2一致。8株ALV-J的3'LTRU3区序列与ALV-J肉鸡分离株及蛋鸡分离株的相应基因序列同源性均低于91.5%,而与GD14J2病毒株的同源性较高,为94.3%~96.3%。序列分析发现U3区存在连续11bp缺失和164位碱基突变(C/T),该突变形成2个新的转录调控元件AIBREP1、AIBREP2。综合gp85、3'UTR和U3区序列特征,推测引起本次进口白羽肉种鸡禽白血病的ALV-J与国内2014年父母代白羽肉种鸡ALV-J分离株GD14J2为同一来源。但本次分离到的ALV-J病毒株也有新的变异趋势,这些变异是否与ALV-J致病性相关,还需进一步研究。本研究为ALV-J有效防控和净化提供数据支持。     

Complete Nucleotide Sequence of Ryegrass Mottle Virus : A New Species of the Genus Sobemovirus

The genome of Ryegrass mottle virus (RGMoV) comprises 4210 nucleotides. The genomic RNA contains four open reading frames (ORFs). The largest ORF 2 encodes a polyprotein of 947 amino acids (103.6 kDa), which codes for a serine protease and an RNA-dependent RNA polymerase. The viral coat protein is encoded on ORF 4 present at the 3′-proximal region. Other ORFs 1 and 3 encode the predicted 14.6 kDa and 19.8 kDa proteins of unknown function. The consensus signal for frameshifting, heptanucleotide UUUAAAC and a stem-loop structure just downstream is in front of the AUG codon of ORF 3. Analysis of the in vitro translation products of RGMoV RNA suggests that the 68 kDa protein may represent a fusion protein of ORF 2-ORF 3 produced by frameshifting. The protease region of the polyprotein and coat protein have a low similarity with that of the sobemoviruses (approximately 25% amino acid identity), while the RNA-dependent RNA polymerase region has particularly strong similarity (54 to 60% of more than 350 amino acid residues). The sequence similarities of RGMoV to the sobemoviruses, together with the characteristic genome organization indicate that RGMoV is a new species of the genus Sobemovirus. Received 28 June 2000/ Accepted in revised form 14 November 2000     

A carlavirus-specific PCR primer and partial nucleotide sequence provides further evidence for the recognition of cowpea mild mottle virus as a whitefly-transmitted carlavirus

Cowpea mild mottle virus (CMMV) has physicochemical properties typical of carlaviruses, but has remained unclassified due to a number of unusual properties, including no serological cross-reaction with 18 carlaviruses; production of brush-like inclusion bodiesin vivo; and the ability to be transmitted by whiteflies (Bermisia tabaci). In this paper we report the use of a carlavirus specific PCR primer to identify CMMV as a member of the carlavirus group. This is confirmed by nucleotide sequence (958 nucleotides) from the 3 terminal region of CMMV RNA which contains a partial open reading frame (ORF) having high similarity with the coat proteins of other carlaviruses. The sequence also contains an 11.7K ORF at the 3 terminus, containing a zinc-finger motif which is unique to carlaviruses.     

A comparison of methods to estimate genomic relationships using pedigree and markers in livestock populations

Accurate prediction of breeding values depends on capturing the variability in genome sharing of relatives with the same pedigree relationship. Here, we compare two approaches to set up genomic relationship matrices for precision of genomic relationships (GR) and accuracy of estimated breeding values (GEBV). Real and simulated data (pigs, 60k SNP) were analysed, and GR were estimated using two approaches: (i) identity by state, corrected with either the observed ( G _{VR ‐O} ) or the base population ( G _{VR ‐B} ) allele frequencies and (ii) identity by descent using linkage analysis ( G _{IBD ‐L} ). Estimators were evaluated for precision and empirical bias with respect to true pedigree IBD GR. All three estimators had very low bias. G _{IBD ‐L} displayed the lowest sampling error and the highest correlation with true genome‐shared values. G _{VR ‐B} approximated G _{IBD ‐L} 's correlation and had lower error than G _{VR ‐O} . Accuracy of GEBV for selection candidates was significantly higher when G _{IBD ‐L} was used and identical between G _{VR ‐O} and G _{VR ‐B} . In real data, G _{IBD ‐L} 's sampling standard deviation was the closest to the theoretical value for each pedigree relationship. Use of pedigree to calculate GR improved the precision of estimates and the accuracy of GEBV.