濒危植物太行菊与长裂太行菊的ITS序列分析

对太行菊属（Opisthopappus）的太行菊（O. taihangensis）和长裂太行菊（O. longilobus）13个种群的核糖体DNA ITS进行测序,分析不同种、不同种群间的ITS序列差异。结果表明：排序后的ITS序列总长度为682 bp,含有15个简约信息位点;根据ITS序列差异共确定出18种单倍型,太行菊和长裂太行菊种群均表现出高的单倍型多样性和核苷酸多样性;两个种均有独有的单倍型,又有共有单倍型;聚类分析表明18种单倍型形成明显两支,Hn1和Hn8分别位于两支的中心为祖先单倍型。ITS序列将太行菊和长裂太行菊13个种群聚类成一个单源支系,但长裂太行菊中两个种群（林虑山LLS和石板岩SBY）与太行菊种群聚为一支,显示出两种之间存在着基因交流或杂交。在进化过程中,长裂太行菊可能经历了长距离侵殖,太行菊在太行山隆升之前经历了种群的扩张,随着太行山的隆升逐渐形成现今的分布格局。     

乙型脑炎病毒NS1’蛋白移码序列的鉴定

本文通过对猪乙型脑炎病毒(Japanese encephalitis virus,JEV)基因组中移码序列分析,设计合成三条引物,通过PCR扩增出假定的NS1’移码片段,并将移码片段三倍重复克隆入pET-28a表达载体中。重组质粒转化E.coli BL-21后,经IPTG诱导在37℃下实现目的片段的可溶性表达。纯化表达产物,并免疫小鼠制备了多抗血清,以多抗血清为一抗,对JEV感染的Vero细胞进行免疫荧光分析,结果发现抗移码片段表达蛋白的血清可识别JEV蛋白。以JEV感染Vero细胞,提取感染细胞总蛋白,分别以NS1单抗和以上制备的多抗血清为一抗,进行Western blot分析。结果显示,NS1单抗为一抗,可染出48 kDa的NS1条带和56 kDa的NS1’条带;而以多抗血清为一抗,仅染出56 kDa的条带。上述结果显示,多抗血清识别的NS1’蛋白为NS1’移码后片段表达的蛋白。     

Saccharomyces Cerevisae and Arabidopsis Thaliana: Useful Model Systems for the Identification of Molecular Mechanisms Involved in Resistance of Plants to Toxins

Secondary metabolites produced by pathogens during the infection process are thought to play a role as pathogenicity or virulence determinants in many plant diseases. Baker's yeast and the plant Arabidopsis thaliana are attractive models for elucidating molecular mechanisms of resistance to toxic substances. For the Fusarium mycotoxin deoxynivalenol, the following resistance mechanisms were identified in yeast: (1) reduced toxin uptake due to the ABC transporter protein Pdr5p (molecular efflux pump), (2) detoxification by the acetyltransferase Ayt1p, and (3) modification of the ribosomal target by amino acid changes in the ribosomal protein L3 (Rpl3p). PDR5-like genes exist in plant genomes as large gene families and could play an important role as a first line of defence against a broad range of toxic metabolites. Amino acid alterations in the highly conserved RPL3 genes could likewise play a role in trichothecene resistance in plants. The knowledge obtained using model systems should be valuable in biotechnological approaches to disease control and marker-assisted resistance breeding.     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

Detection of nucleolar organizer regions on chromosomes by flourescence in situ hybridization with human 28S rRNA gene and cloning of 28S rRNA gene in Sika deer

The number and loci of nucleolar organizer regions (NOR) on chromosomes in Sika deer (Cervus nippon centralis) were determined by fluorescence in situ hybridization with a human 28S ribosomal RNA (rRNA) gene as a probe. Sika deer that live in Nikko National Park and its neighboring areas (Asio and Seta) in Japan were used. All of the analyzed metaphases had three or four NOR at the end of the first and second longest telocentric autosomes. Nucleolar organizer region association, which is associated specifically on parts of NOR between chromosomes, was also observed clearly. A Sika deer 28S rRNA gene was produced by a polymerase chain reaction method. The nucleotide sequence of a Sika deer 28S rRNA gene determined by an automatic sequencer was 97 bp, and showed homogeneity of 88% for the human sequence.     

Direct detection of <Emphasis Type="Italic">Taphrina deformans</Emphasis> on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.     

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.     

Emerging Technologies in Microbial Ecology Aid in Understanding the Effect of Monensin in the Diets of Broilers in Regard to the Complex Disease Necrotic Enteritis

Necrotic enteritis is a complex disease condition of broiler chickens, commercial layer pullets, and turkeys and requires the presence of a toxigenic strain of Clostridium perfringens, alteration of bird diets, and damage to the intestinal epithelium. All of these alone, but especially in combination, result in significant alterations of the intestinal microflora. The intestinal microflora is part of a complex ecosystem that is involved in augmenting intestinal development, immune surveillance, and competitive exclusion against pathogenic organisms. However, when the microflora fails to protect the mucosa from pathogens, antimicrobials are used to reduce the numbers of pathogenic organisms or to prevent their colonization of the intestine. We were interested in studying the effects of monensin on the bacterial community within the ileum of chickens because ionophore antimicrobials are commonly used to prevent Eimeria infections in broilers. We used two 16S ribosomal DNA community analysis protocols, terminal restriction fragment length polymorphism analysis combined with 16S rDNA clone libraries. These methods showed that monensin caused significant alterations in the microbial community structure of the ileum. Whereas the ileal bacterial community of control birds primarily consisted of lactobacilli, monensin-treated birds had communities rich in clostridia. None of the birds exhibited signs of intestinal disease or mucosal lesions, which suggested that the clostridia were avirulent. Although the therapeutic benefits of monensin may largely result from its anticoccidial effects, its ability to foster a competitively exclusive bacterial community may contribute to the intestinal health of birds, thus preventing the development of necrotic enteritis.     

大花蕙兰授粉后子房cDNA差异表达片段序列分析

 应用mRNA差异显示(DDRT-PCR) 方法比较分析了大花蕙兰(Cymbidium hybridium ) 授粉后2 d与未授粉子房的基因表达差异。结果分离到了7个在授粉后子房中特异表达的cDNA片段, 分别命名为CDD-313, CDD-272, CDD-265, CDD-243, CDD-193, CDD-218,CDD-470。在GenBank中进行同源性比较发现前4个没有同源序列与之匹配; 后3个片段推导的氨基酸序列分别与ABC型transporter, GTPase和40S核糖体蛋白质S3 (RPS3) 有高度同源性。反向Northern杂交进一步证实它们存在, 并在授粉子房中高度表达。其中, 最为有意义的是CDD-470, 其推定的氨基酸与参与细胞生长分化的植物源多功能蛋白RPS3高度同源性, 进而推测其与RPS3有相似的功能, 可能与兰花子房发育有关, 这为解释花发育分子机制提供了新资料。     

Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

A polymerase chain reaction-restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus. __________ Translated from Journal of Nanjing Forestry University, 2005, 30(4): 5–9 [译自: 南京林业大学学报]