New severe strains of Melon necrotic spot virus: symptomatology and sequencing

New strains of Melon necrotic spot virus (MNSV), designated MNSV-YS and MNSV-KS, caused much more severe growth retardation on melon plants than MNSV-NH, which was previously reported as the most severe strain of MNSV in Japan. MNSV-YS spread much more quickly than MNSV-NH in infected plants, and induced more severe growth retardation, even though the appearance of necrotic lesions on inoculated cotyledons was much slower. MNSV-KS had properties intermediate between those of the other two strains. The results suggest that faster-spreading strains can multiply more rapidly as a result of lower levels of activity in inducing necrotic lesions in melon plants. The complete sequences of MNSV-YS and MNSV-KS were determined, and an RT–PCR–RFLP method based on these sequences was successfully developed to detect and discriminate between the three strains.     

马铃薯晚疫病菌分子遗传及与寄主互作研究

近年来马铃薯晚疫病的重新暴发再次引起世界各国极大关注,特别对马铃薯晚疫病菌的分子遗传学的研究,包括病菌基因组遗传、转录和物理图谱的构建,病菌致病的分子机制以及马铃薯-马铃薯晚疫病菌互作分子机制等。本文就近几十年来对马铃薯晚疫病菌在生物学和病理学,分子遗传学等研究方面作简要综述,并对其研究趋势进行了展望。     

禾谷镰刀菌和稻瘟病菌基因组中的微卫星序列比较

利用禾谷镰刀菌Fusarium graminearum和稻瘟病菌Magnaporthe grisea基因组测序结果,对这两种植物病原真菌基因组中的微卫星(SSR)序列进行了系统地分析和比较.结果表明,在禾谷镰刀菌基因组中,共发现4679个SSR序列,总长度为96.2kb,占基因组全长的0.27%.平均7.7kb碱基中有一个大于15 bp的SSR序列.在稻瘟病菌基因组中共发现16398个SSR系列,其总长度达到330kb,约占整个基因全长的0.85%,平均2.36kb碱基中就分布有1个SSR序列.在禾谷镰刀菌基因组中,数量最多的是五碱基重复序列,其次是六碱基重复序列;稻瘟病菌基因组中数量最多的是单碱基重复序列,其次为三碱基重复序列和五碱基重复序列.两基因组中数量最少的都是二碱基重复序列.尽管这两种植物病原真菌都属子囊菌,基因组大小也十分接近,但无论是在SSR的总体数量上,还是在各类SSR的分布上,两种植物病原真菌都存在十分显著的差别.     

Interaction of benzimidazole-N-sulfonamides with the cytochrome b/c1 complex of Pythium aphanidermatum

Experiments with intact cells and submitochondrial fractions of Pythium aphanidermatum (Edson) Fitz. indicated an interference of benzimidazole-N-sulfonamides with the NADH- or succinate-driven electron transport system between cytochromes b and c. Comparison with Ustilago maydis (DC) Corda and Botrytis cinerea Pers. ex Fr. revealed that this effect is Oomycetes specific. The molecular interaction between benzimidazole-N-sulfonamides and the mitochondrial cytochrome b/c₁ complex from P. aphanidermatum has been investigated. Binding assays with [¹⁴C]52232 RP (dimefluazole) indicated a time- and dose-dependent labelling of two proteins. The molecular mass of one labelled protein and the competition of the binding with antimycin A suggest that benzimidazole-N-sulfonamides interact with the Q₁-centre of cytochrome b. Furthermore, experiments with doubly labelled [³H][¹⁴C]CGA 323103 revealed a possible irreversible inactivation of the b/c₁ complex leading to covalent linkage of the dimethylsulfonamoyl moiety to the target site.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

羊驼Cytb基因序列的测定及其研究

测定了羊驼线粒体DNA细胞色素b基因部分序列，并与骆驼科其它物种细胞色素b基因进行同源序列比较，分析了碱基组成及变异情况，并用邻接法和最大简约法构建了分子系统树，在分子水平上探讨了羊驼在骆驼科动物中的分类地位。结果表明，羊驼与骆马和美洲驼存在的遗传差异相对比羊驼与原驼大，为传统分类学中的羊驼是原驼驯化以后的一种家养驼的提法提供了分子学依据。     

禽腺联病毒全基因组的克隆及感染性病毒的拯救

为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序。序列分析表明,AAAV YZ-1株的基因组为4684bp,两端具有141bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%。将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建。     

A lost Sorraia maternal lineage found in the Lusitano horse breed

A common female founder individual of the Portuguese horse breeds Sorraia and Lusitano was found while conducting research on the variation of the Lusitano mitochondrial DNA lineages in relation to studbook information. We obtained 416‐bp control region sequences from 16 descendents of a female Sorraia founder (Pomba) still represented in the living population of the Lusitano, according to the most recent edition of this breed's studbook. The same haplotype was found for all analysed samples and belongs to the haplogroup described by several authors as having predominantly Iberian, South American and North African haplotypes bringing new insights on the relationship between the Sorraia and the other Iberian breeds. This work illustrates how weak the boundary of breed establishment can be, especially at the same geographical region. Using the same founders in different breeds is surely one of the explanations to frequently shared haplotypes among recent breeds, resulting in a lack of consistency between mtDNA sequences and breeds and/or geographical regions.     

口蹄疫病毒WFL株基因组全长cDNA的克隆及序列分析

根据口蹄疫病毒基因组的结构特点以及GenBank上公布的全序列，用DNAMAN分别设计了涵盖整个基因组序列的3对引物，从接种口蹄疫病毒WFL株的细胞培养液中提取了病毒基因组RNA，采用RT-PCR方法和RACE法分别扩增了3条基因片段，并将扩增片段分别与T载体连接，在体外分别进行了5-半分子和3’半分子的构建。最后将5’半分子和3’半分子连接成基因组全长cDNA分子。经PCR鉴定、酶切鉴定及全长cDNA测序，证实成功构建了口蹄疫病毒wFL株基因组全长eDNA分子。序列分析结果表明，供试口蹄疫病毒基因组全长为8155nt，5＇UTR长1059nt；具有一个大的读码框，其核苷酸长度为6969nt。包括201aa的前导蛋白基因和2122aa的聚合蛋白基因；3＇UTR长127nt，包括34nt的polv（A）。