16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进

【背景】在过去的十几年里,基于核糖体RNA基因的扩增子测序技术被广泛用于各种生态系统中微生物群落的多样性检测。扩增子测序的使用极大地促进了土壤、水体、空气等环境中微生物生态的相关研究。【目的】随着高通量测序技术的不断发展和参考数据库的不断更新,针对不同的环境样本的引物选择和改进仍然需要更深入的校验。【方法】本文收集了目前在微生物群落研究中被广泛采用的标记基因扩增通用引物,包括16S rRNA基因扩增常用的8对通用引物和2对古菌引物、9对真菌转录间隔区(internal transcribed spacer,ITS)基因扩增引物,以及18S rRNA基因扩增的4对真核微生物通用引物和1对真菌特异性引物。这些引物中包括了地球微生物组计划(Earth Microbiome Project,EMP)推荐的2对16S rRNA基因扩增引物、1对ITS1基因扩增引物和1对18S rRNA基因扩增引物。采用最近更新的标准数据库对这些引物进行了覆盖度和特异性评价。【结果】EMP推荐的引物依然具有较高的覆盖度,而其他引物在覆盖度及对特定环境或类群的特异性上也各有特点。此外,最近有研究对这些通用引物进行了一些改进,而我们也发现,一个碱基的变化都可能会导致评价结果或扩增产物发生明显变化,简并碱基的引入既可以覆盖更多的物种,但同时也会在一定程度上降低关注物种的特异性。【结论】研究结果为微生态研究中标记基因的引物选择提供了一个广泛的指导,但在关注具体科学问题时,引物的选择仍需数据指导与实验尝试。     

三七环二肽成分和人参内酰胺成分

From the roots of Panax notoginseng fourteen cyclodipeptides 1-14 were isolated including one new compound (1),seven new natural compounds (4-10) and six known compounds (2-3,11-14) together with one known other compound 15.The chemical structure of 1 was elucidated as cyclo-(Leu-Thr) based on spectral methods.From the roots of Panax ginseng five known lactams (16-20) includingpyrng lutamic acid were isolated together with butyric diacid,daucosterol and sucrose.The primary binactivity test showed that pyroglutamic acid and its n-butyl derivative have weak Ca^2 antagonistic activity.     

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

Observation of bias associated with re-amplification of DNA isolated from denaturing gradient gels

DNA from environmental PCR products separated by denaturing gradient gel electrophoresis (DGGE) was isolated from the background smear rather than from discrete bands of the DGGE gel. The interband region was considered as a potential source of less dominant members of natural microbial communities. Surprisingly, instead of detecting new bands from the re-amplified PCR products, patterns very similar to the original ones were obtained regardless of the position of the interband region. The results suggest that the separation of amplicons by DGGE may not be perfect and band re-amplification based sequence analyses need careful interpretation.     

微波法快速提取放线菌基因组DNA

原有的放线菌基因组DNA提取方法费时、费力、费用高,且对极端环境放线菌成功率较低。利用微波热振惊提取固体培养基表面放线菌菌落基因组DNA,具有快速、简便、费用低廉等优点。所得的基因组DNA可作为PCR反应的模板进行16S rRNA基因有效扩增。这为大量放线菌菌株的快速鉴别和系统分类创造了条件。     

Explant preparation affects culture initiation and regeneration of Panax ginseng and Panax quinquefolius

The influence of explant preparation on culture initiation and regeneration was investigated. Explant preparation was defined as the application of surface disinfection and homogenization to ginseng roots and embryos. Surface disinfection significantly affected culture initiation and subsequent embryogenesis. A high success rate (80–97%) in culture initiation was associated with explants from non-surface disinfected root tissue. The cumulative contamination rate after 8 months was below 4%; in contrast, a contamination rate of 85% was observed in disinfected root explants. For non-disinfected explants, visible callus was induced in 1 week, adventitious root and somatic embryos were formed in 2 and 6 months, respectively. Explants from disinfected roots required 1 month to induce visible callus, 5–7 months for adventitious roots and 10 months for somatic embryogenesis. For germinating embryos, disinfected embryos required double the time for embryogenesis than non-disinfected ones. It was considered that surface disinfection imposed a stress and subsequently a carry-over effect on explants.     

SSU rDNA phylogeny of cladoniiform lichens

To examine phylogenetic relationships among the "cladoniiform" lichenized fungi, i.e., the families Cladoniaceae, Baeomycetaceae, Icmadophilaceae, Stereocaulaceae, and Siphulaceae, and to provide evidence for the anticipated independent origins of podetia and pseudopodetia, we conducted phylogenetic analyses of SSU (small subunit) rDNA sequences from 39 lichen-forming fungi. These fungi represent all of the major growth forms of lichen associations, fruticose (including "cladoniiform"), foliose, and crustose. Our analysis suggests that lichen-forming fungi with a "cladoniiform" morphology arose multiple times within the ascomycetes. Additionally, each of the other thallus growth forms, crustose, foliose, and fruticose, have originated multiple times. It also seems to be clear that neither all podetiate nor all pseudopodetiate taxa form a monophyletic group. Therefore the term "podetium" should be restricted to homologous structures that are most probably limited to the genera Cladonia, Cladina, Pycnothelia, and allies. The "pseudopodetia" of Stereocaulon (Stereocaulaceae) and Cladia (Cladiaceae) may represent different states of the same homologous character. Our phylogenetic hypothesis supports the monophyletic origin of the order Lecanorales sensu stricto, including representatives of five suborders Cladoniineae, Lecanorineae, Teloschistineae, Agyriineae and Peltigerineae, but excluding representatives of the suborders Acarosporineae (Acarospora schleicheri and Megaspora verrucosa), Pertusariineae (Pertusaria trachythallina), and Umbilicarineae. The suborder Cladoniineae and the family Cladoniaceae both appear to be polyphyletic assemblages.     

Molecular heterogeneity in the 18s DNA gene of Alternaria sp. and Fusarium sp. producing mycotoxins in rice and maize grains

Background

Food contaminated with fungi and their toxins is a problem that threatens many developing countries. Kingdom of Saudi Arabia depends on the exported grain and legume seeds.

Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis

The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the Savignium group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.