一株禽流感病毒全基因的序列分析

通过反转录-聚合酶链式反应(RT-PCR)技术对禽流感病毒A/Turkey/Wisconsin/1/66(H9N2)的8个基因片段即PA、PB1、PB2、NS、NP、M、HA、NA分别进行扩增,然后将其克隆到PMD18-T载体后进行序列测定和拼接;并将克隆到的8个基因片段与以下毒株各个基因的相应序列进行比较分析:Duck/HongKong/Y280/97(DHKY280/97)、Duck/HongKong/YT439/97(DHKY439/97)、Quail/HongKong/G1/97(QHKG1/97)、A/Chicken/Beijing/1/94(CBJ1/94)、Chicken/HongKong/G9/97(CHKG9/97)、A/Turkey/California1/66(TC/1/66).结果表明:我们克隆到的TW1/66株的8个基因片段均含有相应病毒基因的完整开放阅读框架:TW1/66的各基因与TC/1/66株相应各基因同源性最高(NS基因除外,同源性只有(67.4%).与其它各毒株各基因同源性均较低,但与DHKY439/97各基因同源性高于与DHKY280/97、QHKG1/97、CBJ1/94、CHKG9/97各基因同源性.     

Detection of papillomavirus-DNA in mesenchymal tumour cells and not in the hyperplastic epithelium of feline sarcoids

Abstract We examined 12 formalin-fixed paraffin-embedded feline skin tumours which had the histopathological features of fibropapillomas for the presence of papillomavirus (PV) DNA using touchdown polymerase chain recation (PCR), DNA sequencing and nonradioactive in situ hybridization. Nine of the tumours contained a 102-bp PCR product demonstrated using consensus PV primers that amplify a portion of the L1 gene. The nucleotide sequences are closely related, but not identical to that of ovine PV type 2, rabbit oral PV and reindeer PV. The deduced amino acid sequences had strong homologies with the major capsid protein L1 of deer PV, bovine papillomavirus (BPV) 1 and BPV 2, and European elk PV. Although PV antigens were not detected in any of the tumours by immunohistochemistry, PV DNA was demonstrated in individual mesenchymal cells or cell nests of 4/12 tumours by in situ hybridization. A nonproductive infection of mesenchymal fibroblast-like tumour cells with a papillomavirus would explain the lack of PV antigen expression and the absence of PV DNA in the hyperplastic epithelium. Because these tumours and their pathogenesis are similar to equine sarcoids, we suggest that they should be reclassified as 'feline sarcoids' instead of fibropapillomas.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

北极狐多巴胺受体D1基因的克隆测序

为了获得北极狐多巴胺受体D1基因序列,给北极狐自咬行为提供理论依据。采用聚合酶链式反应方法,从北极狐耳组织扩增出多巴胺受体D1基因的部分外显子序列,并对其进行克隆测序,将该序列提交到Genebank上。Genebank中的Blast分析表明,北极狐多巴胺D1受体基因与家狗(Canis familiaris)的同源性为99%,与牛(Bos taurus)的同源性为93%,与人(Homo sapiens)的同源性为92%。     

新扬州鸡早期增重的OPAY02-SCAR分子标记研究

OPAY02型2条多态性条带经克隆、测序和引物设计后,转换成SCAR标记,并对86个新扬州鸡随机交配后代基因组DNA进行了PCR扩增.2条带DNA序列与红色原鸡基因组序列比对结果表明,大分子量条带与位于红色原鸡第3号染色体上序列有98%的同源性,共检测到8个SNPS,其中195位的碱基T→G,316位的A→T,538位的G→A,731位的T→A,1 147位的G→A,1 329位的T→C,1 927位的C→T,2 081位的C→T,小分子量条带与红色原鸡没有同源序列,推测新扬州鸡野祖除红色原鸡外,还有其它来源.SCAR标记分析表明,经条件优化随机扩增的OPAY02型标记稳定、可靠,可用于遗传分析.2条带所在座位群体基因型平衡性测验结果表明,所测新扬州鸡群体处于平衡状态,选择可以打破平衡,有利于动物育种.     

Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv . poae . Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains.     

猪链球菌2型分泌性核酸酶A(SsnA)活性检测及其基因序列的克隆与测序分析

对8株猪链球菌2型重庆分离株的核酸酶A全基因进行克隆测序,结果表明该基因长度为3 126 bp,与Gen-Bank发表的唯一的该基因的序列相比,核苷酸同源性高于98%,推导的氨基酸同源性高于94%。根据核酸酶A基因的测序结果建立扩增片段长度为301 bp的PCR检测方法,并用甲苯胺蓝DNA酶琼脂对猪链球菌分泌性核酸酶A的活性进行检测。检测了从病猪内脏分离的猪链球菌致病株35株,33株核酸酶A基因PCR检测阳性(阳性比例为94.3%),其中有24株分泌活性检测为阳性(阳性比例68.6%);正常猪扁桃体分离株14株,PCR检测为阳性的10株(阳性比例71.4%),其中有核酸酶A分泌活性为8株(阳性比例57.1%),检测菌株中有2型猪链球菌44株,38株扩增出核酸酶A基因的片段PCR检测阳性并且分泌活性为阳性的有32株,猪链球菌1型、7型、9型、13型、1/2型各1株,均能扩增出核酸酶A基因的片段,但只有13型猪链球菌有核酸酶A分泌活性。对猪链球菌在不同生长时期核酸酶活性的检测显示,猪链球菌在培养4、8、16、32、48 h均有核酸酶A的分泌,并且C a2 和M g2 为分泌性核酸酶A的活性必需因子。     

6株猪O型口蹄疫病毒VP1基因的克隆与序列分析

根据口蹄疫病毒（FMDV）VP1基因的序列，设计并合成了2对用于扩增VP1基因的引物。从组织中提取总RNA，首先用P1、P2引物对6株猪。型口蹄疫病毒进行RT—PCR扩增，获得1000bp的片段；再用P3、P4引物进行巢式PCR扩增，结果获得850bp的片段。将850bp的片段克隆到pMD18—-T载体中，通过PCR鉴定，将阳性重组质粒进行测序并分析。结果发现6株FMDV的核苷酸同源性为80．2％～99．4％，其推导的氨基酸序列同源性为86．9％～99．5％；构建遗传发生树，发现6株FMDV属于两个不同的基因型，其中的Shunde00、Sihui01、Shenzhen99、Fushan01株属一个基因型（与Hongkong93、广东86分离株属同一基因型）；Guangzhou99、Shenzhen00株属另一个基因型（与UKG-12—2001株、JPN2000株属同一基因型）。通过对口蹄疫病毒VP1基因的测序与分析，了解其变异情况，为科学地防控FMD提供分子水平的依据。