TILLING技术在功能基因组学中的应用

TILLING(定向诱导基因组局部突变)技术是近年发展起来的一种高通量筛选化学诱变的点突变的技术,它利用专一识别点突变的核酸酶结合PCR来检测单核苷酸多态性(SNP)。TILLING技术起源于植物基因组研究,逐渐扩展到动物及人类功能基因组学的研究中。无论是筛选突变体还是研究特定基因的重要性,TILLING都具有高通量、自动化的优势。随着此项技术应用范围的扩展,从诱变剂和内切酶的选择到具体的操作方式,以及结果的识别和统计方法,都有了不少改进。在其他相关学科不断发展的大环境下,TILLING技术也在不断发展,其在功能基因组学研究中的作用也会更显著。     

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.     

An Internet‐based platform for the estimation of outcrossing potential between cultivated and Chilean vascular plants

A national‐scale study of outcrossing potential within Chilean vascular flora was conducted using an upgraded algorithm, which adds parameters such as pollinator agents, climate, and geographic conditions. Datasets were organized and linked in a Web platform ( www.flujogenico.cl ), in which the development of a total outcrossing potential (TOP) predictor was formulated. The TOP predictor is the engine in the Web platform, which models the effect of a type of agricultural practice on others (coexistence calculation mode) and on the environment (biodiversity calculation mode). The scale for TOP results uses quintiles in order to define outcrossing potential between species as “very low,” “low,” “medium,” “high,” or “very high.” In a coexistence analysis considering 256 species (207 genera), the 10 highest TOP values were for genera Citrus, Prunus, Trifolium, Brassica, Allium, Eucalyptus, Cucurbita, Solanum, Lollium, and Lotus. The highest TOP for species in this analysis fell at “high” potential, 4.9% of the determined values. In biodiversity mode, seven out of 256 cultivated species (2.7%) were native, and 249 (97.3%) corresponded to introduced species. The highest TOP was obtained in the genera Senecio, Calceolaria, Viola, Solanum, Poa, Alstroemeria, Valeriana, Vicia, Atriplex, and Campanula, showing “high” potential in 4.9% of the values. On the other hand, 137 genetically modified species, including the commercial and pre‐commercial developments, were included and represented 100 genera. Among these, 22 genera had relatives (i.e., members of the same genus) in the native/introduced group. The genera with the highest number of native/introduced relatives ranged from one (Ipomea, Limonium, Carica, Potentilla, Lotus, Castanea, and Daucus) to 66 species (Solanum). The highest TOP was obtained when the same species were coincident in both groups, such as for Carica chilensis, Prosopis tamarugo, and Solanum tuberosum. Results are discussed from the perspective of assessing the possible impact of cultivated species on Chilean flora biodiversity. The TOP predictor ( http://epc.agroinformatica.cl/ ) is useful in the context of environmental risk assessment.     

Lipid composition of tobacco cells cultivated at various temperatures

Tobacco cell suspensions were grown under controlled conditions to determine whether temperature aftected the fatty acid pattern of the cellular lipids. At any temperature ranging between 17° and 35°, the total fatty acid content and the levels of fatty acids or individual lipids varied during the growth period, The optimum temperature for lipid biosynthesis and polyunsaturated fatty acid accumulation was between 20° and 26°. Increase in the level of polyunsaturated fatty acids was associated with lower temperatures during the active cell division period.     

An efficient CRISPR/Cas9 platform for targeted genome editing in rose (Rosa hybrida)

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-related nuclease 9(Cas9) system enables precise, simple editing of genes in many animals and plants.However, this system has not been applied to rose(Rosa hybrida) due to the genomic complexity and lack of an efficient transformation technology for this plant. Here, we established a platform for screening single-guide RNAs(sgRNAs) with high editing efficiency for CRISPR/Cas9-mediated gene editing in rose using suspensio...     

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

Tomato TILLING technology: development of a reverse genetics tool for the efficient isolation of mutants from Micro-Tom mutant libraries

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1-SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.     

Copper nanoclusters: an efficient fluorescence sensing platform for quinoline yellow

Fluorescence quenching behavior of artificial food colorant quinoline yellow (QY), on interaction with l ‐cysteine stabilized copper nanoclusters (l ‐Cys‐CuNCs) is investigated in this work. For this purpose, l ‐cysteine stabilized CuNCs were synthesized and characterized using various analytical techniques. Results demonstrated that the synthesized probe (size ~2 nm) had very promising optical features such as bright blue fluorescence, significant quantum yield and excellent photostability. l ‐Cys‐CuNCs can function as a fluorescence sensor by selectively sensing QY among other yellow colorants, giving a detection limit as low as 0.11 μM. The developed sensor exhibited a linear concentration range from 5.50 to 0.20 μM. The developed fluorescence assay was successfully applied for testing commercial samples, thereby making this sensing strategy significant for quality control of food stuffs.     

Bacterial magnetosomes as an efficient gene delivery platform for cancer theranostics

Background

Gene therapy has gained an increasing interest in its anti-tumor efficiency. However, numerous efforts are required to promote them to clinics. In this study, a novel and efficient delivery platform based on bacterial magnetosomes (BMs) were developed, and the efficiency of BMs in delivering small interfering ribonucleic acid (siRNA) as well as antiproliferative effects in vitro were investigated.

Results

Initially, we optimized the nitrogen/phosphate ratio and the BMs/siRNA mass ratio as 20 and 1:2, respectively, to prepare the BMs–PEI–siRNA composites. Furthermore, the prepared nanoconjugates were systematically characterized. The dynamic light scattering measurements indicated that the particle size and the zeta potential of BMs–PEI–siRNA are 196.5 nm and 49.5 ± 3.77 mV, respectively, which are optimum for cell internalization. Moreover, the confocal laser scanning microscope observations showed that these composites were at a proximity to the nucleus and led to an effective silencing effect. BMs–PEI–siRNA composites efficiently inhibited the growth of HeLa cells in a dose-as well as time-dependent manner. Eventually, a dual stain assay using acridine orange/ethidium bromide, revealed that these nanocomposites induced late apoptosis in cancer cells.

Conclusions

A novel and efficient gene delivery system based on BMs was successfully produced for cancer therapy, and these innovative carriers will potentially find widespread applications in the pharmaceutical field.

     

Optimizing TILLING populations for reverse genetics in Medicago truncatula

Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project ( http://www.eugrainlegumes.org ), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula . Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase     

A diploid wheat TILLING resource for wheat functional genomics

Background

Cultivated peanut (Arachis hypogaea L.) is an important crop worldwide, valued for its edible oil and digestible protein. It has a very narrow genetic base that may well derive from a relatively recent single polyploidization event. Accordingly molecular markers have low levels of polymorphism and the number of polymorphic molecular markers available for cultivated peanut is still limiting.

Results

Here, we report a large set of BAC-end sequences (BES), use them for developing SSR (BES-SSR) markers, and apply them in genetic linkage mapping. The majority of BESs had no detectable homology to known genes (49.5%) followed by sequences with similarity to known genes (44.3%), and miscellaneous sequences (6.2%) such as transposable element, retroelement, and organelle sequences. A total of 1,424 SSRs were identified from 36,435 BESs. Among these identified SSRs, dinucleotide (47.4%) and trinucleotide (37.1%) SSRs were predominant. The new set of 1,152 SSRs as well as about 4,000 published or unpublished SSRs were screened against two parents of a mapping population, generating 385 polymorphic loci. A genetic linkage map was constructed, consisting of 318 loci onto 21 linkage groups and covering a total of 1,674.4 cM, with an average distance of 5.3 cM between adjacent loci. Two markers related to resistance gene homologs (RGH) were mapped to two different groups, thus anchoring 1 RGH-BAC contig and 1 singleton.

Conclusions

The SSRs mined from BESs will be of use in further molecular analysis of the peanut genome, providing a novel set of markers, genetically anchoring BAC clones, and incorporating gene sequences into a linkage map. This will aid in the identification of markers linked to genes of interest and map-based cloning.     

小麦TILLING分析中CELⅠ酶切及PCR反应体系的优化

在小麦中建立稳定的基于CELⅠ酶切的目的基因突变位点检测技术,有助于高通量鉴定目的基因片段的点突变及提高突变检测效率。本研究以冬小麦品种新麦18空间诱变SP2群体为材料,以小麦糯质基因Waxy为目标片段,通过优化基因组DNA提取方法、调整PCR反应体系中dNTP、Mg2+及引物浓度、改变目标片段CELⅠ酶切缓冲液成分,以及调整纯化过程中的空气相对湿度等方式,优化了小麦TILLING技术体系。在利用PVP-40法提取DNA过程中,研磨器振动频率提高到30/s,KAc溶液的反应时间延伸为20min时,基因组DNA质量和纯度最佳;在设定的浓度范围内dNTP和Mg2+浓度对产物影响差异不明显,均能高效扩增出目的条带。引物浓度对产物影响差异显著,最佳引物浓度为0.4μmol/L。20μl酶切体系中,最佳CEL I酶浓度为0.1U且利用超纯水代替CELⅠ缓冲液。最终在小麦中建立起了基于CELⅠ酶切的高通量TILLING筛选技术体系。     

A structurally informed autotransporter platform for efficient heterologous protein secretion and display

ABSTRACT: BACKGROUND: The self-sufficient Autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a beta-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a beta-helical core structure (beta-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated beta-stem appeared unstable after translocation, demonstrating the importance of an intact beta-stem. By interrupting the cleavage site between passenger and beta-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.     

Tailoring cyanobacteria as a new platform for highly efficient synthesis of astaxanthin

With the ability to recycle CO₂ into value-added chemicals, cyanobacteria have been considered as renewable microbial cell factories. Astaxanthin, a highly valued carotenoid with potent antioxidant activity, could be beneficial to human health. Astaxanthin biosynthesis in engineered chassis has been achieved previously, but it generated a relatively low yield. Here, we successfully constructed a highly efficient astaxanthin biosynthetic pathway in cyanobacterium Synechocystis sp. PCC 6803, and achieved more than a 500-fold increase in astaxanthin production via stepwise reconstruction of the biosynthetic pathway and rational rewiring of the endogenous metabolism. The engineered strain produced up to 29.6 mg/g of astaxanthin (dry cell weight), which is the highest yield reported in the engineered chassis to date. Moreover, multi-omics analyses revealed that establishing a high astaxanthin flux may enhance photosynthesis and central metabolism in the engineered strain to compensate for the depleted pigments, which could be valuable for astaxanthin overproduction. This study presents a novel alternative for high-efficiency biosynthesis of astaxanthin directly from CO₂.     

EUROCarbDB: An open-access platform for glycoinformatics

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.     

An experimental biomimetic platform for artificial olfaction

Artificial olfactory systems have been studied for the last two decades mainly from the point of view of the features of olfactory neuron receptor fields. Other fundamental olfaction properties have only been episodically considered in artificial systems. As a result, current artificial olfactory systems are mostly intended as instruments and are of poor benefit for biologists who may need tools to model and test olfactory models. Herewith, we show how a simple experimental approach can be used to account for several phenomena observed in olfaction. An artificial epithelium is formed as a disordered distributed layer of broadly selective color indicators dispersed in a transparent polymer layer. The whole epithelium is probed with colored light, imaged with a digital camera and the olfactory response upon exposure to an odor is the change of the multispectral image. The pixels are treated as olfactory receptor neurons, whose optical properties are used to build a convergence classifier into a number of mathematically defined artificial glomeruli. A non-homogenous exposure of the test structure to the odours gives rise to a time and spatial dependence of the response of the different glomeruli strikingly similar to patterns observed in the olfactory bulb. The model seems to mimic both the formation of glomeruli, the zonal nature of olfactory epithelium, and the spatio-temporal signal patterns at the glomeruli level. This platform is able to provide a readily available test vehicle for chemists developing optical indicators for chemical sensing purposes and for biologists to test models of olfactory system organization.