aBIOTECH最新文献

Accurate taxonomic classification is essential to understanding microbial diversity and function through metagenomic sequencing. However, this task is complicated by the vast variety of microbial genomes and the computational limitations of bioinformatics tools. The aim of this study was to evaluate the impact of reference database selection and confidence score (CS) settings on the performance of Kraken2, a widely used k-mer-based metagenomic classifier. In this study, we generated simulated metagenomic datasets to systematically evaluate how the choice of reference databases, from the compact Minikraken v1 to the expansive nt- and GTDB r202, and different CS (from 0 to 1.0) affect the key performance metrics of Kraken2. These metrics include classification rate, precision, recall, F1 score, and accuracy of true versus calculated bacterial abundance estimation. Our results show that higher CS, which increases the rigor of taxonomic classification by requiring greater k-mer agreement, generally decreases the classification rate. This effect is particularly pronounced for smaller databases such as Minikraken and Standard-16, where no reads could be classified when the CS was above 0.4. In contrast, for larger databases such as Standard, nt and GTDB r202, precision and F1 scores improved significantly with increasing CS, highlighting their robustness to stringent conditions. Recovery rates were mostly stable, indicating consistent detection of species under different CS settings. Crucially, the results show that a comprehensive reference database combined with a moderate CS (0.2 or 0.4) significantly improves classification accuracy and sensitivity. This finding underscores the need for careful selection of database and CS parameters tailored to specific scientific questions and available computational resources to optimize the results of metagenomic analyses.

Mosses, particularly desiccation-tolerant (DT) species, are important model organisms for studying genes involved in plant development and stress resistance. The lack of a simple and efficient stable moss transformation system has hindered progress in deciphering the genetic mechanisms underlying traits of interest in these organisms. Here, we present an Agrobacterium tumefaciens-mediated transformation system for DT mosses that uses Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301-GUS. This system achieved transformation efficiencies of 74% and 81% in Physcomitrium patens and Bryum argenteum protonemata, respectively, without the need for culture and callus formation prior to regeneration. We detected GUS enzyme activity in the regenerated transgenic moss via histochemical staining. Southern blot, PCR, and RT-qPCR analyses confirmed the presence of the GUS gene. In addition, we successfully used this system to transform wild DT Syntrichia caninervis. Furthermore, P. patens and B. argenteum transformed using this system with the stress resistance gene EsDREB from the desert plant Eremosparton songoricum (Litv.) exhibited improved salt tolerance. We thus present an efficient tool for the genetic analysis of DT moss species, paving the way for the development of stress-resistant crop cultivars.

A regulon refers to a group of genes regulated by a transcription factor binding to regulatory motifs to achieve specific biological functions. To infer tissue-specific gene regulons in Arabidopsis, we developed a novel pipeline named InferReg. InferReg utilizes a gene expression matrix that includes 3400 Arabidopsis transcriptomes to make initial predictions about the regulatory relationships between transcription factors (TFs) and target genes (TGs) using co-expression patterns. It further improves these anticipated interactions by integrating TF binding site enrichment analysis to eliminate false positives that are only supported by expression data. InferReg further trained a graph convolutional network with 133 transcription factors, supported by ChIP-seq, as positive samples, to learn the regulatory logic between TFs and TGs to improve the accuracy of the regulatory network. To evaluate the functionality of InferReg, we utilized it to discover tissue-specific regulons in 5 Arabidopsis tissues: flower, leaf, root, seed, and seedling. We ranked the activities of regulons for each tissue based on reliability using Borda ranking and compared them with existing databases. The results demonstrated that InferReg not only identified known tissue-specific regulons but also discovered new ones. By applying InferReg to rice expression data, we were able to identify rice tissue-specific regulons, showing that our approach can be applied more broadly. We used InferReg to successfully identify important regulons in various tissues of Arabidopsis and Oryza, which has improved our understanding of tissue-specific regulations and the roles of regulons in tissue differentiation and development.

In the face of carbon, nitrogen, and phosphorus starvation, microorganisms have evolved adaptive mechanisms to maintain growth. In a previous study, we identified a protein predicted to contain acyl-CoA-binding domains in the plant pathogenic fungus Verticillium dahliae. The predicted protein, designated VdAcb1, possesses an atypical signal peptide. However, the functions of this acyl-CoA-binding protein in V. dahliae are not clear. In this research, in vivo or in vitro assays confirmed that VdAcb1 is secreted extracellularly from V. dahliae, although it does not have the typical signal peptide. Furthermore, the unconventional secretion of VdAcb1 was dependent on VdGRASP, a member of the compartment for unconventional protein secretion (CUPS). The deletion mutant strain of VdAcb1 (ΔVdAcb1) exhibited significant sensitivity to carbon starvation. RNA-seq revealed that the expression of genes related to filamentous growth (MSB2 pathway) and sugar transport were regulated by VdAcb1 under conditions of carbon starvation. Yeast one-hybrid experiments further showed that the expression of VdAcb1 was positively regulated by the transcription factor VdMsn4. The ΔVdAcb1 strain showed significantly reduced virulence on Gossypium hirsutum and Nicotiana benthamiana. We hypothesize that under conditions of carbon starvation, the expression of VdAcb1 is activated by VdMsn4 and VdAcb1 is secreted into the extracellular space. In turn, this activates the downstream MAPK pathway to enhance filamentous growth and virulence of V. dahliae.

Robust genome editing technologies are becoming part of the crop breeding toolbox. Currently, genome editing is usually conducted either at a single locus, or multiple loci, in a variety at one time. Massively parallel genomics platforms, multifaceted genome editing capabilities, and flexible transformation systems enable targeted variation at nearly any locus, across the spectrum of genotypes within a species. We demonstrate here the simultaneous transformation and editing of many genotypes, by targeting mixed seed embryo explants with genome editing machinery, followed by re-identification through genotyping after plant regeneration. Transformation and Editing of Mixed Lines (TREDMIL) produced transformed individuals representing 101 of 104 (97%) mixed elite genotypes in soybean; and 22 of 40 (55%) and 9 of 36 (25%) mixed maize female and male elite inbred genotypes, respectively. Characterization of edited genotypes for the regenerated individuals identified over 800 distinct edits at the Determinate1 (Dt1) locus in samples from 101 soybean genotypes and 95 distinct Brown midrib3 (Bm3) edits in samples from 17 maize genotypes. These results illustrate how TREDMIL can help accelerate the development and deployment of customized crop varieties for future precision breeding.

The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs.

Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture.

The diversity of plant natural products presents a rich resource for accelerating drug discovery and addressing pressing human health issues. However, the challenges in accessing and cultivating source species, as well as metabolite structural complexity, and general low abundance present considerable hurdles in developing plant-derived therapeutics. Advances in high-throughput sequencing, genome assembly, gene synthesis, analytical technologies, and synthetic biology approaches, now enable us to efficiently identify and engineer enzymes and metabolic pathways for producing natural and new-to-nature therapeutics and drug candidates. This review highlights challenges and progress in plant natural product discovery and engineering by example of recent breakthroughs in identifying the missing enzymes involved in the biosynthesis of the anti-cancer agent Taxol^®. These enzyme resources offer new avenues for the bio-manufacture and semi-synthesis of an old blockbuster drug.

Salicylic acid (SA) is a phytohormone required for plant growth and defense signaling. There are two major SA biosynthesis pathways in plants: the isochorismate synthase (ICS) pathway and the phenylalanine ammonia-lyase (PAL) pathway. It has been demonstrated in several plant species, including the model plant Arabidopsis, that SA is derived predominantly from the ICS pathway. Here, we employed the CRISPR/Cas9 system to generate ICS knockout mutants in rice (Oryza sativa L.). The Osics mutants display severe growth defects, and are completely devoid of phylloquinone, an isochorismate-derived product. The growth defects of Osics can be rescued through exogenous application of 1,4-dihydroxy-2-naphthoic acid (NA), a precursor of phylloquinone. Remarkably, the basal SA levels are not altered in the Osics mutants. Our findings support a role of OsICS in the biosynthesis of phylloquinone, and imply that SA biosynthesis in rice may occur through an alternative route other than the ICS pathway.