Frankia的REP-PCR、RAPD-PCR鉴定及遗传多样性研究

本文采用溶菌酶法、CTAB法、微波法及CTAB+微波法等4种方法进行Frankia菌Cp11基因组DNA的提取。结果表明四种方式均可行，但以CTAB法及微波法最为有效。提取方法与REP-PCR带型间关系的研究表明，方法上的差异不会影响到REP-PCR带型的变化。对14株Frankia菌株作REP-PCR分析，并对之进行比较分类，证明REP-PCR方法不但能实现菌株间的差异鉴别，还能有效地进行Frankia菌的分类。这一结果与DNA同源相关性所得的分类结果具有良好的相关性。选用20个随机引物，对分自2个分类接种群的8株Frankia纯培养的总DNA进行随机扩增。其中引物OPW15和OPW16能扩增得到较为稳定的RAPD图谱。扩增产物分子量大都分布在0.5-4Kb之间。从稳定的RAPD扩增图谱看，Frankia菌间存在有丰富的遗传多样性；在选定适当引物情况下，能依据共同带型将Frankia菌化归为同一分类接种群。

rep-PCR对赤杨根瘤内局Frankia菌基因多样性研究

本文应用重复基因外回纹序列rep-PCR方法直接扩增长日山三种不同赤杨和云南不同地区早冬瓜根瘤内的Frankia菌基因，所获得的REP指纹图谱揭示Frankia菌具有丰富的基因多样性。研究结果表明Frankia菌多样性随地域，海拔，坡问不同而变：多样性指数与自然选抒）玉力大小成正相关：自然选择压力是产生和保持Frankia菌基因多样性的重要因子。同时发现Frankia菌基因型与地域有紧密关系：所观察的云南各地区中，感染高黎贡山旱冬瓜的Frankia菌基因多样性最为丰富。推测与高黎贡山旱冬瓜共生的Frankia可能作为种储备库，为其它地区的寄主植物提供了祖先菌株。在对长白山三种赤杨（东北赤杨，西伯利亚赤杨和色赤杨）根瘤内几百游殆菌基因多样性考察过程中发现，Frankia菌基因类型与寄主赤杨种型关系密切。同时海拔对感染东北赤杨的Frankia菌基因多样性也有一定影响。感染西伯利亚赤杨和色赤杨的Frankia菌享有相同的基因型，可能有较近的亲缘关系。

Determination of SARS-coronavirus by a microfluidic chip system

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-Cov). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.

Frankia基因组DNA提取及其PCR带型

本文采用溶菌酶法、CTAB法、微波（MW）法及CTAB＋MW法等4种方法提取Frankia菌CpIl基因组DNA。结果表明四种方式均可满足试验要求，其中以CTAB法及微波法最为有效。在比较提取方法与PCR带型间的关系中，发现提取上的差异不影响PCR带型的变化。

长白山赤杨丛枝菌根真菌多样性的半巢式LP-PCR-SSCP检测分析

利用半巢式LP PCR SSCP技术 ,对中国吉林长白山不同海拔生境下 3种赤杨共生丛枝菌根真菌的多样性进行检测分析 .结果表明 ,该地区赤杨属东北赤杨、西伯利亚赤杨及色赤杨共生丛枝菌根真菌在科乃至种的水平上并未随宿主的变化表现出丰富的多样性 ;3个树种在自身属的水平上与共生的球囊霉科 (Glomaceae)至少 1种AMF ,即G .intraradix ,在种的水平上表现出不相关于宿主海拔高度的某种相互选择性 .

长白山四种赤杨丛枝菌根真菌侵染多样性的巢式PCR-RFLP分析

采集中国吉林长白山不同海拔的4种赤杨根须样本,利用巢式PCR-RFLP方法检测丛枝菌根真菌(AMF)对样品的侵染情况,PCR结果经限制性内切酶分析.结果表明,赤杨根内AMF存在丰富的基因多样性.AMF的侵染有从宿主混乱性向宿主专一性发展的趋势.东北赤杨AMF的宿主专一性水平最强,球囊霉属已成为东北赤杨的优势侵染类群;其余3种赤杨的AMF则出现宿主混乱现象.宿主因素比海拔因素对AMF侵染有更重要的影响.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. (C) 2003 Elsevier B.V. All rights reserved.

几种PCR方法在克隆鸭肠炎病毒未知基因中的应用

以DEV基因组DNA为模板,用简并PCR、改良Targeted gene walking PCR、改良的热不对称交错PCR和Long-PCR,获得了5350bp、11083bp和2905bp3段DEV未知基因片段,DNA序列分析发现包含9个开放阅读框,将这些序列提交GenBank分别获得的登录号为:EF554396~EF554403。结果表明,多种PCR方法联合使用可以高效的实现对鸭肠炎病毒未知基因的克隆。

利用rep-PCR研究云南尼泊尔桤木根瘤内Frankia基因多样性

利用rep-PCR指纹技术对云南5个地区的尼泊尔桤木根瘤内Frankia菌基因多样性进行研究,实验发现Frankia菌呈现丰富的基因多样性.所有71个样品被分为11种不同的基因类型.除高黎贡山外,不同地域都有某种基因型占优势,显示Frankia菌基因型与地域有紧密关系.所观察的5个地区中,高黎贡山Frankia菌基因型种类最多,多样性指数最高,基因多样性最为丰富.结合高黎贡山地理资料及其它生物多样性相关研究,推测与高黎贡山尼泊尔桤木共生的Frankia可能作为种储备库,为其它地区的寄主植物提供了祖先菌株.图8表1参13