霍山石斛种内遗传稳定性的RAPD初探

目的 研究霍山石斛荚果内遗传稳定性及荚果间的差异大小。方法 利用从20个随机引物筛选出来的3个稳定性较好的10碱基引物对来自5个不同荚果的霍山石斛种群进行RAPD检测。结果 5个荚果内遗传相似系数较大,在92.5926%-100.00%,其中H1、H9、H10、H14存在一定程度的变异,H23是一个较为稳定的群体;5个荚果间H1、H9、H10、H23的遗传距离较小,H14与其他4个荚果的遗传距离则较大。结论 建立霍山石斛稳定的无性快繁体系是切实可行的,同时说明霍山石斛种内存在一定的变异,H14与其他荚果的差异最大。     

文山三七栽培群体变异类型的分子鉴定

目的 了解三七栽培群体的遗传背景及变异情况,为三七育种提供依据。方法 采用RAPD方法分析三七栽培群体的NDA变化。结果 从160个10bp的寡核苷酸引物中筛选出8个条带清晰、重复性好的特异性引物对三七栽培群体中的七个变异类型共34个样品进行了PCR扩增,共扩增出了49条带,其中多态性条带37条,变异类型之间的多态性差异达75.5％,同一类型不同个体间的多态性差异为75.2％,结论 三七栽培群体从遗传背景来看还是一个杂合群体,具有丰富的遗传多样性。     

药用菊花遗传多样性的RAPD分析

目的:分析不同药用菊花栽培类型的遗传差异。方法:采用2%CTAB法进行药用菊花的DNA提取,并用RAPD技术对22个类型药用菊花种质资源的DNA进行检测,采用NTSYS软件对扩增结果进行统计和聚类。结果:筛选得到的26个引物共得到了233条扩增DNA片断,其中89.7%的扩增条带表现出多态性,每个引物平均可扩增得到8.04条多态性片断。聚类分析结果表明利用RAPD技术可将全部供试材料区分开。结论:药用菊花种质资源在分子水平上确实存在较大差异;药用菊花栽培类型间的差异与环境因素有关,但更大程度上由其遗传因素决定。     

Choice of Methodology for Assessing Genetic Impacts of Environmental Stressors: Polymorphism and Reproducibility of RAPD and AFLP Fingerprints

PCR-based multi-locus DNA fingerprints represent one of the most informative and cost-effective measures of genetic diversity and are useful population-level biomarkers of toxicologic and other anthropogenic impacts. However, concerns about reproducibility of DNA fingerprints have limited their wider use in environmental biology. We assessed polymorphism and reproducibility of two common fingerprinting techniques, RAPD (randomly amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), in pedigreed populations of rainbow trout (Oncorhynchus mykiss) to derive general rules for selective removal of problematic fingerprint bands. We found that by excluding bands that comprised less than 1% of total intensity, and by excluding the largest and smallest 10% of the bands, we could achieve nearly 100% reproducibility of AFLP fingerprints. Similar application of band exclusion criteria to RAPD fingerprints did not significantly enhance their reproducibility, and at least 15% of RAPD bands were not fully repeatable, heritable, or transmittable. The RAPD technique produced more polymorphic fingerprints than AFLP; however, considering that a substantial proportion of RAPD markers did not demonstrate Mendelian inheritance patterns, the AFLP methodology is to be preferred for future research.     

Disruption of beta-catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice

BACKGROUND & AIMS: Human liver cancer can be divided into 2 categories that are characterized by activation of beta-catenin and genomic instability. Here we investigate whether similar categories exist among 5 transgenic models of liver cancer, including c-myc, transforming growth factor-alpha, E2F-1, c-myc/transforming growth factor-alpha, and c-myc/E2F-1 mice. METHODS: The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. beta-Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of beta-catenin and expression of alpha-fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry. RESULTS: Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor-alpha mouse) and activation of beta-catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor-alpha tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of beta-catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of alpha-fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of beta-catenin activation in the presence of a relatively stable genome and low alpha-fetoprotein levels. CONCLUSIONS: We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor-alpha and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and beta-catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.     

Direct Transmission of H. pylori from Challenged to Nonchallenged Mice in a Single Cage

To understand whether direct transmission of H. pylori occurs from infected mouse to noninfected mouse, the system using a mouse model we developed previously was tested. Six nude mice were challenged with H. pylori inocula; one group consisted of one challenged nude mouse 1 week after inoculation raised with four nonchallenged nude mice in a single cage. For the single cage, a polycarbonate cage or a mesh-floor cage was used. Then three groups were kept in a polycarbonate cage and the other three groups kept in a mesh-floor cage to avoid H. pylori transmission through stool. After coraising for 1, 2, or 3 weeks, all mice were sacrificed to determine the existence of H. pylori in the stomach, saliva, and stool by culture or PCR and H. pylori-associated gastritis. RAPD fingerprinting patterns using different primers of isolated strains from challenged and nonchallenged mice were compared to understand the origin of transmitted strains. During 3 weeks after coraising of H. pylori challenged and nonchallenged mice, H. pylori was detected in the stomachs in 3 of 12 nonchallenged mice in the polycarbonate cage and in 2 of 12 nonchallenged mice in the cage with a steel mesh floor. H. pylori was detected from saliva or stool in two nonchallenged, infected mice in the polycarbonate cage. Moreover, RAPD fingerprinting using different primers of the total five strains isolated from five nonchallenged, infected mice in both cages showed the same pattern and concordance with that of the challenged strain and the strains isolated from challenged mice. It is demonstrated that intimate interaction is the cause of H. pylori transmission via saliva and stool.     

Molecular epidemiology of cutaneous leishmaniasis and heterogeneity of Leishmania major strains in Iran

Objective To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran. Methods A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB/c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA‐PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique. Results Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica. In rural areas, the causative agent of CL was mainly L. major (95%L. major vs. 5%L. tropica), in urban areas it was L. tropica (65%L. tropica vs. 35%L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB/c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area. Conclusion Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country.     

不同地理株杂交培育的柔嫩艾美球虫及其免疫保护率

目的　培育 3个不同地理株柔嫩艾美球虫 (Eimeriatenella)的杂交株 ,以探讨研制球虫疫苗的可能性。　方法　通过免疫试验 ,从 5个不同地理株中选择 3株作为杂交亲本株 ,对此 3株分别进行两次杂交 ,获得的后代混合卵囊经单卵囊分离、扩增 ,分别提取卵囊DNA ,利用随机扩增多态性DNA(RAPD)进行分析 ( 3 0条引物 ) ,分离、培育出两代杂交株。再进行免疫试验 ,比较杂交株与亲本株的免疫保护性。　结果　选择了免疫原性较好、免疫保护率较高的广州株、保定株、长春株作为杂交亲本 ,分别进行保定株×长春株、广州株×F1株两次杂交 ,获得的后代卵囊 ,提取卵囊DNA ,RAPD分析后 ,得到了保定株×长春株的杂交株F1(F1Z7)和广州株×F1株的杂交株F2 (F2Z3 )。免疫试验结果 ,杂交株F1与F2的免疫保护率分别为 80 %和 84% ;亲本株广州株为 77% ,保定株为 69% ,长春株为 63 %。　结论　分离、培育出了F1、F2两代杂交虫株。其免疫保护率均高于各亲本株 ,提示杂交株获得了亲本株的部分保护性 ,其免疫的雏鸡对各虫株的攻击均有好的保护力。尤其是F2株 ,免疫保护率平均达到 84%。     

我国利什曼原虫RAPD分析

目的　我国不同疫区利什曼原虫分离株的RAPD分析。　方法　用 7种随机引物 ,扩增来自我国 3个疫区 (得自利什曼病患者、病犬及白蛉 )的利什曼原虫分离株和国际标准株 ,并将扩增产物进行聚类分析。　结果　①来自我国山丘疫区及平原疫区的L d .分离株分别聚为两类 ,两者遗传距离较远 ;②来自新疆荒漠疫区、近荒漠疫区及平原地区的L d .XJ771、L d .XJ90 1和L d .XJ80 1聚在一类 ,表明它们的遗传距离较近 ;③山丘疫区虫株中来自病人和病犬的分离株区别不明显 ,两者同源性高 ,表明犬在传播中起着重要作用 ;④印度平原型标准株L d .DD8与我国平原疫区虫株聚在一类 ;⑤L infantum与我国山丘疫区的L d .分离株分属于两类 ;⑥L dJed与平原疫区L d .分离株亲缘关系较近 ;⑦L infantum与L tropica最早聚合 ,遗传距离最近。　结论　我国不同疫区利什曼原虫分离株在基因水平上存有差异。     

伯氏疟原虫氯喹抗性株和敏感株基因组DNA比较研究

目的　比较伯氏疟原虫敏感株 (N株 )和抗性株 (RC株 )基因组DNA差异。　方法　应用基因组DNA随机多态性扩增 (RAPD)和锚定引物扩增DNA(APAD) ,分别对抽提的伯氏疟原虫N株和RC株基因组DNA进行PCR扩增 ,并对扩增的产物进行电泳 ,对结果进行统计分析。　结果　 3 7条随机引物RAPD方法扩增出 44 0条DNA条带 ,其中RC株和N株的共有条带是 196条 ,差异带 43条。 84条锚定多聚A引物APAD方法扩增出 95 2条DNA条带 ,其中RC株和N株的共有条带是 43 6条 ,差异带 5 3条。两种方法得到N株和RC株基因组DNA的同源性分别为 0 .89和0 .91;距离系数分别为 0 .197和 0 .15 5。　结论　伯氏疟原虫N株和RC株基因组DNA具有高度同源性。