CRISPR/Cas9在放线菌合成生物学研究中的应用和发展

放线菌是活性天然产物和抗生素药物的重要来源。利用合成生物学高效地开发其中丰富的天然产物资源,将为加速新药开发奠定坚实的基础。CRISPR/Cas9作为一种多功能基因编辑系统,因其便捷高效而被广泛应用于真核生物的遗传操作。但在原核生物尤其是放线菌中的应用仍处于起步阶段,机遇和挑战并存。本综述总结了目前CRISPR/Cas9系统在放线菌基因编辑和调控,以及活性天然产物的产量提升、生物合成机制解析和资源开发等方面的研究进展。同时,也对该系统在应用中面临的包括重组修复效率低,以及靶向切割效率不足等关键挑战进行了分析,并提出了相应的优化解决方法。随着CRISPR/Cas9在放线菌应用中的不断完善和发展,将极大地推动放线菌的合成生物学研究,促进其中天然产物资源的有效挖掘和应用开发。     

Population biology of a selfish sex ratio distorting element in a booklouse (Psocodea: Liposcelis)

Arthropods harbour a variety of selfish genetic elements that manipulate reproduction to be preferentially transmitted to future generations. A major ongoing question is to understand how these elements persist in nature. In this study, we examine the population dynamics of an unusual selfish sex ratio distorter in a recently discovered species of booklouse, Liposcelis sp. (Psocodea: Liposcelididae) to gain a better understanding of some of the factors that may affect the persistence of this element. Females that carry the selfish genetic element only ever produce daughters, although they are obligately sexual. These females also only transmit the maternal half of their genome. We performed a replicated population cage experiment, varying the initial frequency of females that harbour the selfish element, and following female frequencies for 20 months. The selfish genetic element persisted in all cages, often reaching very high (and thus severely female‐biased) frequencies. Surprisingly, we also found that females that carry the selfish genetic element had much lower fitness than their nondistorter counterparts, with lower lifetime fecundity, slower development and a shorter egg‐laying period. We suggest that differential fitness plays a role in the maintenance of the selfish genetic element in this species. We believe that the genetic system in this species, paternal genome elimination, which allows maternal control of offspring sex ratio, may also be important in the persistence of the selfish genetic element, highlighting the need to consider species with diverse ecologies and genetic systems when investigating the effects of sex ratio manipulators on host populations.     

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.     

Migratory patterns and connectivity of two North American grassland bird species

Effective management and conservation of migratory bird populations require knowledge and incorporation of their movement patterns and space use throughout the annual cycle. To investigate the little‐known migratory patterns of two grassland bird species, we deployed 180 light‐level geolocators on Grasshopper Sparrows (Ammodramus savannarum) and 29 Argos‐GPS tags on Eastern Meadowlarks (Sturnella magna) at Konza Prairie, Kansas, USA, and six US Department of Defense (DoD) installations distributed across the species' breeding ranges. We analyzed location data from 34 light‐level geolocators and five Argos‐GPS tags attached for 1 year to Grasshopper Sparrows and Eastern Meadowlarks, respectively. Grasshopper Sparrows were present on the breeding grounds from mid‐April through early October, substantially longer than previously estimated, and migrated on average ~2,500 km over ~30 days. Grasshopper Sparrows exhibited strong migratory connectivity only at a continental scale. The North American Great Lakes region likely serves as a migratory divide for Midwest and East Coast Grasshopper Sparrows; Midwest populations (Kansas, Wisconsin, and North Dakota; n = 13) largely wintered in Texas or Mexico, whereas East Coast populations (Maryland and Massachusetts, n = 20) wintered in the northern Caribbean or Florida. Our data from Eastern Meadowlarks provided evidence for a diversity of stationary and short‐ and long‐distance migration strategies. By providing the most extensive examination of the nonbreeding movement ecology for these two North American grassland bird species to date, we refine information gaps and provide key insight for their management and conservation.     

Evidence for contemporary and historical gene flow between guppy populations in different watersheds,with a test for associations with adaptive traits

In dendritic river systems, gene flow is expected to occur primarily within watersheds. Yet, rare cross‐watershed transfers can also occur, whether mediated by (often historical) geological events or (often contemporary) human activities. We explored these events and their potential evolutionary consequences by analyzing patterns of neutral genetic variation (microsatellites) and adaptive phenotypic variation (male color) in wild guppies (Poecilia reticulata) distributed across two watersheds in northern Trinidad. We found the expected signatures of within‐watershed gene flow; yet we also inferred at least two instances of cross‐watershed gene flow—one in the upstream reaches and one further downstream. The upstream cross‐watershed event appears to be very recent (41 ± 13 years), suggesting dispersal via recent flooding or undocumented human‐mediated transport. The downstream cross‐watershed event appears to be considerably older (577 ± 265 years), suggesting a role for rare geological or climatological events. Alongside these strong signatures of both contemporary and historical gene flow, we found little evidence of impacts on presumably adaptive phenotypic differentiation, except perhaps in the one instance of very recent cross‐watershed gene flow. Selection in this system seems to overpower gene flow—at least on the spatiotemporal scales investigated here.     

The effects of food level and social density on reproduction in the Least Killifish,Heterandria formosa

The feedbacks from population density to demographic parameters, which drive population regulation, are the accumulated results of several ecological processes. The compensatory feedback from increased population density to fertility includes at least two distinct factors, the effects of decreases in per capita food level and increases in the social density (the number of interacting individuals). Because these effects have been studied separately, their relative importance is unknown. It is also unclear whether food limitation and social density combine additively to influence fertility. We investigated these questions with two factorial experiments on reproduction in the Least Killifish, Heterandria formosa. In one experiment, we crossed two levels of density with two levels of a total food ration that was distributed to all individuals. In the other experiment, we crossed two levels of density with two levels of per capita food. Whereas the first experiment suggested that the effects of variation in food level and density were synergistic, the second experiment indicated that they were not. The apparent synergism—the statistical interaction of food and density levels—was the result of confounding per capita food with social density in that design. In the second experiment, the effects of social density on reproductive rate were stronger than the effects of food level, whereas the effects of food level were stronger on offspring size at parturition than those of social density. The results suggest that the social stresses that emerge at higher densities play an important role in the compensatory response of fertility to density, a role, that is, at least as important as that of decreased per capita food levels.     

Global genetic diversity,lineage distribution,and Wolbachia infection of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil (Hypera postica) is a well‐known example of a worldwide‐distributed pest with high genetic variation. Based on the mitochondrial genes, the alfalfa weevil clusters into two main mitochondrial lineages. However, there is no clear picture of the global diversity and distribution of these lineages; neither the drivers of its diversification are known. However, it appears likely that historic demographic events including founder effects played a role. In addition, Wolbachia, a widespread intracellular parasite/symbiont, likely played an important role in the evolution of the species. Wolbachia infection so far was only detected in the Western lineage of H. postica with no information on the infecting strain, its frequency, and its consequences on the genetic diversity of the host. We here used a combination of mitochondrial and nuclear sequences of the host and sequence information on Wolbachia to document the distribution of strains and the degree of infection. The Eastern lineage has a higher genetic diversity and is found in the Mediterranean, the Middle East, Eastern Europe, and eastern America, whereas the less diverse Western lineage is found in Central Europe and the western America. Both lineages are infected with the same common strain of Wolbachia belonging to Supergroup B. Based on neutrality tests, selection tests, and the current distribution and diversification of Wolbachia in H. postica, we suggested the Wolbachia infection did not shape genetic diversity of the host. The introduced populations in the United States are generally genetically less diverse, which is in line with founder effects.     

Temporal stability and assignment power of adaptively divergent genomic regions between herring (Clupea harengus) seasonal spawning aggregations

Atlantic herring (Clupea harengus), a vital ecosystem component and target of the largest Northwest Atlantic pelagic fishery, undergo seasonal spawning migrations that result in elusive sympatric population structure. Herring spawn mostly in fall or spring, and genomic differentiation was recently detected between these groups. Here we used a subset of this differentiation, 66 single nucleotide polymorphisms (SNPs) to analyze the temporal dynamics of this local adaptation and the applicability of SNP subsets in stock assessment. We showed remarkable temporal stability of genomic differentiation corresponding to spawning season, between samples taken a decade apart (2005 N = 90 vs. 2014 N = 71) in the Gulf of St. Lawrence, and new evidence of limited interbreeding between spawning components. We also examined an understudied and overexploited herring population in Bras d'Or lake (N = 97); using highly reduced SNP panels (N_SNPs > 6), we verified little‐known sympatric spawning populations within this unique inland sea. These results describe consistent local adaptation, arising from asynchronous reproduction in a migratory and dynamic marine species. Our research demonstrates the efficiency and precision of SNP‐based assessments of sympatric subpopulations; and indeed, this temporally stable local adaptation underlines the importance of such fine‐scale management practices.     

Environmental variation shapes genetic variation in Bouteloua gracilis: Implications for restoration management of natural populations and cultivated varieties in the southwestern United States

With the increasing frequency of large‐scale restoration efforts, the need to understand the adaptive genetic structure of natural plant populations and their relation to heavily utilized cultivars is critical. Bouteloua gracilis (blue grama) is a wind‐dispersed, perennial grass consisting of several cytotypes (2n = 2×–6×) with a widespread distribution in western North America. The species is locally dominant and used regularly in restoration treatments. Using amplified fragment length polymorphism (AFLP) and cpDNA analyses, we assessed the genetic variability and adaptive genetic structure of blue grama within and among 44 sampling sites that are representative of the species’ environmental and habitat diversity in the southwestern United States. Five cultivars were also included to investigate genetic diversity and differentiation in natural versus cultivated populations. Three main findings resulted from this study: (a) Ninety‐four polymorphic AFLP markers distinguished two population clusters defined largely by samples on and off the Colorado Plateau; (b) substructure of samples on the Colorado Plateau was indicated by genetic divergence between boundary and interior regions, and was supported by cytotype distribution and cpDNA analysis; and (c) six AFLP markers were identified as “outliers,” consistent with being under selection. These loci were significantly correlated to mean annual temperature, mean annual precipitation, precipitation of driest quarter, and precipitation of wettest quarter in natural populations, but not in cultivated samples. Marker × environment relationships were found to be largely influenced by cytotype and cultivar development. Our results demonstrate that blue grama is genetically variable, and exhibits genetic structure, which is shaped, in part, by environmental variability across the Colorado Plateau. Information from our study can be used to guide the selection of seed source populations for commercial development and long‐term conservation management of B. gracilis, which could include genetic assessments of diversity and the adaptive potential of both natural and cultivated populations for wildland restoration.