PCR-SSCP技术在嗜盐放线菌链单孢菌属快速筛选中的应用

为提高嗜盐放线茵的研究效率,快速、准确的从大量分离菌株中去除重复菌株、筛选出目的菌株,在特异性引物快速定属的基础上,以嗜盐放线茵链单孢茵属的34株菌株为研究对象,采用与PCR相结合的单链构象多态性分析(PCR-SSCP),扩增出16S rRNA基因中的两个高变区,根据结果对34株菌株进行聚类分析,并将其16S rRNA基因片段测序予以验证.结果表明,聚类后34株菌株可大致分为3类,且与16S rRNA基因片段分析结果一致.从而可快速去除重复菌株并反映出菌株间的系统进化关系.同时实验数据可构建成库,使后续分离菌株的筛选工作只需比对数据即可完成,利于提高工作效率,降低实验成本.     

Can Preference for Oviposition Sites Initiate Reproductive Isolation in Callosobruchus maculatus?

Theory has identified a variety of evolutionary processes that may lead to speciation. Our study includes selection experiments using different host plants and test key predictions concerning models of speciation based on host plant choice, such as the evolution of host use (preference and performance) and assortative mating. This study shows that after only ten generations of selection on different resources/hosts in allopatry, strains of the seed beetle Callosobruchus maculatus develop new resource preferences and show resource-dependent assortative mating when given the possibility to choose mates and resources during secondary contact. The resulting reduced gene flow between the different strains remained for two generations after contact before being overrun by disassortative mating. We show that reduced gene flow can evolve in a population due to a link between host preference and assortative mating, although this result was not found in all lines. However, consistent with models of speciation, assortative mating alone is not sufficient to maintain reproductive isolation when individuals disperse freely between hosts. We conclude that the evolution of reproductive isolation in this system cannot proceed without selection against hybrids. Other possible factors facilitating the evolution of isolation would be longer periods of allopatry, the build up of local adaptation or reduced migration upon secondary contact.     

The genetic variation of compatibility in Biomphalaria glabrata and Schistosoma mansoni

Interactions between different Biomphalaria glabrata stocks and Schistosoma mansoni strains were studied. A series of inbred stocks of B. glabrata were characterized as to genetic variations in susceptibility at different ages to a series of different S. mansoni strains. A series of inbred strains of S. mansoni were characterized as to genetic variations in infectivity for B. glabrata stocks at different ages. Also described is a process of selection for substrains from a single S. mansoni isolate that differ genetically in snail infectivity.     

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFP^KI) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFP^KI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.     

A color-based stable multi-copy integrant selection system for Pichia pastoris using the attenuated ADE1 and ADE2 genes as auxotrophic markers

The methylotropic yeast Pichia pastoris has been used for more than two decades to successfully produce a large number of recombinant proteins. Currently, a wide variety of auxotrophic and drug based selection markers are employed to screen for clones expressing the protein of interest. For most proteins an increased copy number of the integrated plasmid results in higher levels of expression, but these multi-copy integrants can be unstable due to the propensity of P. pastoris for homologous recombination. Here we describe a multi-copy selection system based on ade1 and ade2 auxotrophic parent strains and the respective attenuated markers with truncated promoter regions. We show that for all four proteins we tested, the use of the attenuated markers leads to increased protein expression when compared with selection based on the full strength markers. The fact that the adenine auxotrophic strains grow more slowly than the complemented counterparts essentially ensures the stability of multi-copy integration. At the same time, the accumulation of a red dye in the auxotrophic strains also provides an easy, color-based selection for transformants with multiple copies.     

对92株茶树菇菌株的遗传多样性分析和农艺性状的评价

为了解茶树菇（Agrocybe aegerita）种质资源的遗传多样性和筛选优良的茶树菇新品种,采用菌株拮抗试验方法观察了92株茶树菇菌株间拮抗反应及其类型,ISSR-PCR（inter-simple sequence repeat-PCR）分子标记方法对92株茶树菇菌株的遗传多样性进行了综合分析。拮抗试验将92株茶树菇菌株分为27组;筛选出的20条ISSR引物共扩增出317条清晰条带,多态性条带平均比率为82.60%;在遗传相似系数为0.742时,ISSR分子标记分析可将92株茶树菇划分为6大类群,拮抗试验和ISSR分子标记分析的结果基本一致。通过对比农艺性状分析,初步筛选出滇农5、滇农14、茶5-800、白茶、闽农5及滇农8作为工厂化生产茶树菇菌种。结果表明茶树菇的遗传多样性丰富,结合栽培出菇试验可为茶树菇品种选育和杂交育种的亲本选择提供参考。     

Absence of clinal variation in virgin retention capacity in Australian Drosophila melanogaster

The ability of virgin Drosophila melanogaster adults to retain eggs is thought to be an adaptation to persisting in temperate areas, based on differences in this trait between European and African populations, and based on seasonal changes in this trait in France. By retaining eggs in the absence of males and under conditions of poorer nutrition (conditions common in temperate areas during colder months), females reduce the wastage of resources and increase their probability of surviving spring into summer, enabling them to initiate summer population expansions. To test for variation in virgin egg retention along a climatic gradient, we characterized clinal variation in strains collected from eastern Australia extending from temperate Tasmania to tropical northern Queensland. Despite testing a large number of strains and repeated testing of the cline ends, we did not detect any evidence for clinal variation in virgin egg retention. Therefore although D. melanogaster in temperate Australia overwinter at the adult stage, there is no evidence for selection on virgin retention capacity producing clinal patterns. This contrasts with other evidence for clinal variation in egg production patterns over winter.     

A systematic strain selection approach for halotolerant and halophilic bioprocess development: a review

Halotolerant and halophilic microorganisms have potential applications in a number of very relevant environmental and industrial bioprocesses, from wastewater treatment to production of value-added chemicals. While numerous microbial strains have been identified and studied in the literature, the number of those successfully used in industrial applications is comparatively small. Literature is abundant in terms of characterisation of specific strains under a microbiology perspective; however, there is a need for studies tackling the selection of strains for bioprocess applications. This review presents a database of over 200 halophilic and halotolerant prokaryote strains compiled from taxonomic microbiological resources and classified by trophic groups as well as by their salinity, pH and temperature tolerance and optimum ranges, all under a process development perspective. In addition to this database, complementary systematic approaches for the selection of suitable strains for a given trophic activity and environmental conditions are also presented. Both the database and the proposed selection approaches together constitute a general tool for process development that allows researchers to systematically search for strains capable of specific substrate degradations under specific conditions (pH, T, salinity). Many exiting established halotolerant and halophilic environmental and industrial bioprocesses appear to have been developed following strategies in line with the systematic approaches proposed here.     

Intrapopulation variability of antibiotic biosynthesis in streptomycetes

Spontaneous variants making up parallel series of hereditary variability inside the populations of antibiotic-producing actinomycetes differ in the level of their antibiotic activity. As a rule, spontaneous variants of the basic type possess the highest antibiotic activity. Other variants representing parallel series have a lower activity level. This raises the possibility to carry out a directed selection of previously known active colonies from populations on the basis of their easily discernible morphological properties. It enhances the efficiency of selection work both in the case of stabilization of the level of antibiotic activity and in the case of obtaining more productive commercial strains.     

Interplay of Darwinian and frequency-dependent selection in the host-associated microbial populations

In order to analyze the microevolutionary processes in host-associated microorganisms, we simulated the dynamics of rhizobia populations composed of a parental strain and its mutants possessing the altered fitness within "plant-soil" system. The population dynamics was presented as a series of cycles (each one involves "soil-->rhizosphere-->nodules-->soil" succession) described using recurrent equations. For representing the selection and mutation pressures, we used a universal approach based on calculating the shifts in the genetic ratios of competing bacterial genotypes within the particular habitats and across several habitats. Analysis of the model demonstrated that a balanced polymorphism may be established in rhizobia population: mutants with an improved fitness do not supplant completely the parental strain while mutants with a decreased fitness may be maintained stably. This polymorphism is caused by a rescue of low-fitted genotypes via negative frequency-dependent selection (FDS) that is implemented during inoculation of nodules and balances the Darwinian selection that occurs during multiplication or extinction of bacteria at different habitats. The most diverse populations are formed if the rhizobia are equally successful in soil and nodules, while a marked preference for any of these habitats results in the decrease of diversity. Our simulation suggests that FDS can maintain the mutualistic rhizobia-legume interactions under the stress conditions deleterious for surviving the bacterial strains capable for intensive N2 fixation. Genetic consequences of releasing the modified rhizobia strains may be addressed using the presented model.     

A differential drug screen for compounds that select against antibiotic resistance

Antibiotics increase the frequency of resistant bacteria by providing them a competitive advantage over sensitive strains. Here, we develop a versatile assay for differential chemical inhibition of competing microbial strains, and use it to identify compounds that preferentially inhibit tetracycline-resistant relative to sensitive bacteria, thus "inverting" selection for resistance. Our assay distinguishes compounds selecting directly against specific resistance mechanisms and compounds whose selection against resistance is based on their physiological interaction with tetracycline and is more general with respect to resistance mechanism. A pilot screen indicates that both types of selection-inverting compounds are secreted by soil microbes, suggesting that nature has evolved a repertoire of chemicals that counteracts antibiotic resistance. Finally, we show that our assay can more generally permit simple, direct screening for drugs based on their differential activity against different strains or targets.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Clonal Interference in the Evolution of Influenza

The seasonal influenza A virus undergoes rapid evolution to escape human immune response. Adaptive changes occur primarily in antigenic epitopes, the antibody-binding domains of the viral hemagglutinin. This process involves recurrent selective sweeps, in which clusters of simultaneous nucleotide fixations in the hemagglutinin coding sequence are observed about every 4 years. Here, we show that influenza A (H3N2) evolves by strong clonal interference. This mode of evolution is a red queen race between viral strains with different beneficial mutations. Clonal interference explains and quantifies the observed sweep pattern: we find an average of at least one strongly beneficial amino acid substitution per year, and a given selective sweep has three to four driving mutations on average. The inference of selection and clonal interference is based on frequency time series of single-nucleotide polymorphisms, which are obtained from a sample of influenza genome sequences over 39 years. Our results imply that mode and speed of influenza evolution are governed not only by positive selection within, but also by background selection outside antigenic epitopes: immune adaptation and conservation of other viral functions interfere with each other. Hence, adapting viral proteins are predicted to be particularly brittle. We conclude that a quantitative understanding of influenza’s evolutionary and epidemiological dynamics must be based on all genomic domains and functions coupled by clonal interference.     

The evolution of resource specialization through frequency-dependent and frequency-independent mechanisms

Levins's fitness set approach has shaped the intuition of many evolutionary ecologists about resource specialization: if the set of possible phenotypes is convex, a generalist is favored, while either of the two specialists is predicted for concave phenotype sets. An important aspect of Levins's approach is that it explicitly excludes frequency-dependent selection. Frequency dependence emerged in a series of models that studied the degree of character displacement of two consumers coexisting on two resources. Surprisingly, the evolutionary dynamics of a single consumer type under frequency dependence has not been studied in detail. We analyze a model of one evolving consumer feeding on two resources and show that, depending on the trait considered to be subject to evolutionary change, selection is either frequency independent or frequency dependent. This difference is explained by the effects different foraging traits have on the consumer-resource interactions. If selection is frequency dependent, then the population can become dimorphic through evolutionary branching at the trait value of the generalist. Those traits with frequency-independent selection, however, do indeed follow the predictions based on Levins's fitness set approach. This dichotomy in the evolutionary dynamics of traits involved in the same foraging process was not previously recognized.     

Comparative Growth of Natural Bacterial Isolates on Various Lignin-Related Compounds

Bacterial strains were isolated on the basis of their ability to proliferate in a minimal medium containing one of a series of lignin-related compounds as the sole carbon and energy source. These included the aromatic monomers guaiacol, vanillic and coumaric acids, a dimer and a trimer possessing the arylglycerol-β-aryl ether linkage, anisoin, and both the ether-soluble and -insoluble fractions of kraft lignin. The growth of the strains on each of these compounds was measured. The results showed that the metabolic properties of the strains varied according to the structure of the carbon sources used for their selection. Spectrophotometric tracings of the culture medium during the log phase of growth of one of the strains on the β-O-4 dimer revealed decomposition with the release of guaiacol.     

Linear selection indices for non-linear profit functions

Summary Two methods of deriving linear selection indices for non-linear profit functions have been proposed. One is by linear approximation of profit, and another is the graphical method of Moav and Hill (1966). When profit is defined as the function of population means, the graphical method is optimal. In this paper, profit is defined as the function of the phenotypic values of individual animals; it is then shown that the graphical method is not generally optimal. We propose new methods for constructing selection indices. First, a numerical method equivalent to the graphical method is proposed. Furthermore, we propose two other methods using quadratic approximation of profit: one is based on Taylor series about means before selection, and the other is based on Tayler series about means after selection. Among these different methods, it is shown that the method using quadratic approximation based on Taylor series about means after selection is the most efficient.     

设施化专用香菇种质资源挖掘

以香菇L808菌株基因组序列为参照,开发了300份插入/缺失(InDel)标记。通过5个菌株的初筛,选出均匀分布于基因组的82份多态标记对42份香菇种质资源进行遗传背景分析。同时在设施化栽培条件下考察了种质资源的生育期、菌棒转色、菌棒硬度、现蕾期和产量等表型。结果表明：供试香菇种质资源的遗传多样性丰富,栽培菌株与野生菌株的遗传距离较远,遗传相似性系数平均值为0.51。群体结构分析将种质资源分为6个亚群,与基于遗传距离的系统聚类结果较为一致。亚群间菌株具有较远的亲缘关系,遗传分化指数平均值为0.290,适合用于杂交育种。表型结果显示,设施化栽培条件下的种质资源农艺性状分化程度高,亚群间菌株的生育期、现蕾期和产量均有显著差异。种质资源多样性分析结果为香菇杂交育种工作奠定了基础。     

Ecological Similarity and Coexistence of Epiphytic Ice-Nucleating (Ice) Pseudomonas syringae Strains and a Non-Ice-Nucleating (Ice) Biological Control Agent

De Wit replacement series were used to study competitive interactions between epiphytic IcePseudomonas syringae strains and the biological frost control agents IceP. syringae TLP2del1 and Pseudomonas fluorescens A506. Mixtures containing two strains in different proportions but at a constant total population size were inoculated onto potato leaves. The population sizes of each strain and the total population size were determined when the community had reached equilibrium. A near-isogenic P. syringae strain pair exhibited an interaction similar to that expected for strains competing equally for limiting environmental resources. Replacement series with nonisogenic Ice and IceP. syringae strain pairs suggested that these strains competed for limiting resources according to their relative competitive abilities. There was no evidence of any niche differentiation between the IceP. syringae strains and the IceP. syringae strain. The growth responses of epiphytes following addition of nutrients to the phyllosphere indicated that the epiphytic P. syringae populations were nutrient limited and that, under growth chamber conditions, the populations were more limited by the availability of carbon than by the availability of nitrogen. Determination of in vitro carbon source utilization profiles provided further evidence for the lack of niche differentiation between the Ice and the IceP. syringae strains. Niche overlap indices calculated for the IceP. syringae strains with respect to IceP. syringae TLP2del1 were uniformly high, indicating ecological similarity, and were consistent with the observed low level of coexistence. The biological frost control agent P. fluorescens A506 replaced P. syringae. This was correlated with a high degree of niche overlap between these species.     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

俄罗斯远东及黑龙江省春小麦种质资源的遗传多样性

利用31个SSR引物分析56份俄罗斯远东地区春小麦(Triticum aestivum)及56份黑龙江省2010生(区)试品系的遗传变异和群体结构。结果表明,黑龙江省春小麦和俄罗斯远东春小麦明显分化为两大类群,聚类结果同地理来源的划分基本一致;居群间和个体间都存在显著性差异;群体的遗传分化程度较高,但群体间的基因交流有限,血缘相对比较单一。鉴于部分黑龙江省同一育种单位的品种(系)间的遗传距离非常相近,目前亟需拓宽和创制新的小麦种质资源。实验结果证明种质资源遗传多样性与地理来源和人为选择压力密切相关。