Identification of QTLs and candidate genes for water-soluble protein content in soybean seeds.

Soybean represents a vital source of premium plant-based proteins for human nutrition. Importantly, the level of water-soluble protein (WSP) is crucial for determining the overall quality and nutritional value of such crops. Enhancing WSP levels in soybean plants is a high-priority goal in crop improvement. This study aimed to elucidate the genetic basis of WSP content in soybean seeds by identifying quantitative trait loci (QTLs) and set the foundation for subsequent gene cloning and functional analysis. Using 180 F10 recombinant inbred lines generated by crossing the high-protein soybean cultivar JiDou 12 with the wild variety Ye 9, our researcher team mapped the QTLs influencing protein levels, integrating Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and gene expression profiling to identify candidate genes. During the 2020 and 2022 growing seasons, a standard bell-shaped distribution of protein content trait data was observed in these soybean lines. Eight QTLs affecting protein content were found across eight chromosomes, with LOD scores ranging from 2.59 to 7.30, explaining 4.15-11.74% of the phenotypic variance. Notably, two QTLs were newly discovered, one with a elite allele at qWSPC-15 from Ye 9. The major QTL, qWSPC-19, on chromosome 19 was stable across conditions and contained genes involved in nitrogen metabolism, amino acid biosynthesis, and signaling. Two genes from this QTL, Glyma.19G185700 and Glyma.19G186000, exhibited distinct expression patterns at maturity, highlighting the influence of these genes on protein content. This research revealed eight QTLs for WSP content in soybean seeds and proposed a gene for the key QTL qWSPC-19, laying groundwork for gene isolation and enhanced soybean breeding through the use of molecular markers. These insights are instrumental for developing protein-rich soybean cultivars.

Comparative Analysis of the Mitochondrial Genome of Eggplant (Solanum melongena L.) to Identify Cytoplasmic Male Sterility Candidate Genes.

Cytoplasmic male sterility (CMS) is important for commercial hybrid seed production. However, it is still not used in eggplant (Solanum melongena L.), and corresponding regulatory genes and mechanisms of action have not been reported. We report CMS line 327A, which was derived from the hybridization between cultivated and wild eggplants. By looking at different stages of anther development under a microscope, we saw that the 327A anther's tapetum layer vacuolized during meiosis, which caused abortion. To investigate the 327A CMS regulatory genes, the mitochondrial genomes of 327A and its maintainer line 327B were assembled de novo. It was found that 15 unique ORFs (Open Reading Frame) were identified in 327A. RT-PCR and RT-QPCAR tests confirmed that orf312a and orf172a, 327A-specific ORFs with a transmembrane domain, were strongly expressed in sterile anthers of 327A. In addition, orf312a has a chimeric structure with the ribosomal protein subunit rpl16. Therefore, orf312a and orf172a can be considered strong candidate genes for CMS. Concurrently, we analyzed the characteristics of CMS to develop a functional molecular marker, CMS312, targeting a future theoretical basis for eggplant CMS three-line molecular breeding.

Optimizing Trilobatin Production via Screening and Modification of Glycosyltransferases.

Trilobatin (TBL) is a key sweet compound from the traditional Chinese sweet tea plant (Rubus suavissimus S. Lee). Because of its intense sweetness, superior taste profile, and minimal caloric value, it serves as an exemplary natural dihydrochalcone sweetener. It also has various health benefits, including anti-inflammatory and glucose-lowering effects. It is primarily produced through botanical extraction, which impedes its scalability and cost-effectiveness. In a novel biotechnological approach, phloretin is used as a precursor that is transformed into TBL by the glycosyltransferase enzyme ph-4'-OGT. However, this enzyme's low catalytic efficiency and by-product formation limit the large-scale synthesis of TBL. In our study, the enzyme Mdph-4'-OGT was used to screen 17 sequences across species for TBL synthesis, of which seven exhibited catalytic activity. Notably, PT577 exhibited an unparalleled 97.3% conversion yield within 3 h. We then optimized the reaction conditions of PT577, attaining a peak TBL bioproduction of 163.3 mg/L. By employing virtual screening, we identified 25 mutation sites for PT577, thereby creating mutant strains that reduced by-products by up to 50%. This research enhances the enzymatic precision for TBL biosynthesis and offers a robust foundation for its industrial-scale production, with broader implications for the engineering and in silico analysis of glycosyltransferases.

Review of glucose oxidase as a feed additive: production, engineering, applications, growth-promoting mechanisms, and outlook.

The regulation and prohibition of antibiotics used as growth promoters (AGP) in the feed field are increasing because they cause antimicrobial resistance and drug residue issues and threaten community health. Recently, glucose oxidase (GOx) has attracted increasing interest in the feed industry as an alternative to antibiotics. GOx specifically catalyzes the production of gluconic acid (GA) and hydrogen peroxide (H2O2) by consuming molecular oxygen, and plays an important role in relieving oxidative stress, preserving health, and promoting animal growth. To expand the application of GOx in the feed field, considerable efforts have been made to mine new genetic resources. Efforts have also been made to heterologously overexpress relevant genes to reduce production costs and to engineer proteins by modifying enzyme properties, both of which are bottleneck problems that limit industrial feed applications. Herein, the: different sources, diverse biochemical properties, distinct structural features, and various strategies of GOx engineering and heterologous overexpression are summarized. The mechanism through which GOx promotes growth in animal production, including the improvement of antioxidant capacity, maintenance of intestinal microbiota homeostasis, and enhancement of gut function, are also systematically addressed. Finally, a new perspective is provided for the future development of GOx applications in the feed field.

Mining of a novel reductase and its application for asymmetric reduction of p-methoxyacetophenone.

(R)-1-(4-methoxyphenyl) ethanol [(R)-1b] is an essential precursor for the synthesis of aryl propanoic acids' anti-inflammatatory drugs. Biocatalysts for (R)-1b preparation are limited and reductase has problems of low substrate concentration and low conversion rate. As a result, there is a constant need for discovering novel biocatalysts with excellent catalytic performances. In this study, a novel reductase LpSDR from Lacisediminihabitans profunda for the biocatalytic reduction of p-methoxyacetophenone (1a) to (R)-1b was obtained based on gene-mining technology, and some key reaction parameters were also investigated to improve the conversion rate of 1a using whole cells of recombinant Escherichia coli expressing reductase LpSDR as biocatalysts. It was found that the optimal concentration of isopropanol, ZnSO4·7H2O solution, 1a, and recombinant E. coli resting cells, the optimal reaction temperature, buffer pH, and reaction time were 1.95 mol l-1, 0.75 mmol l-1, 75 mmol l-1, 250 g (wet weight) l-1, 28°C, 7.0, and 21 h, respectively. Under the above conditions, a conversion rate of 99.5% and an enantiomeric excess of 99.6% were obtained, which were superior to the corresponding values previously reported. This study provides a novel reductase LpSDR, which is helpful in reducing 1a to (R)-1b.

Alternative Splicing Variation: Accessing and Exploiting in Crop Improvement Programs.

Alternative splicing (AS) is a gene regulatory mechanism modulating gene expression in multiple ways. AS is prevalent in all eukaryotes including plants. AS generates two or more mRNAs from the precursor mRNA (pre-mRNA) to regulate transcriptome complexity and proteome diversity. Advances in next-generation sequencing, omics technology, bioinformatics tools, and computational methods provide new opportunities to quantify and visualize AS-based quantitative trait variation associated with plant growth, development, reproduction, and stress tolerance. Domestication, polyploidization, and environmental perturbation may evolve novel splicing variants associated with agronomically beneficial traits. To date, pre-mRNAs from many genes are spliced into multiple transcripts that cause phenotypic variation for complex traits, both in model plant Arabidopsis and field crops. Cataloguing and exploiting such variation may provide new paths to enhance climate resilience, resource-use efficiency, productivity, and nutritional quality of staple food crops. This review provides insights into AS variation alongside a gene expression analysis to select for novel phenotypic diversity for use in breeding programs. AS contributes to heterosis, enhances plant symbiosis (mycorrhiza and rhizobium), and provides a mechanistic link between the core clock genes and diverse environmental clues.

Discovery of enzymes to biotransform ginsenoside Rd into ginsenosides F2 and CK using metagenomics and genomic mining.

Ginsenosides are the main active components of ginseng, including many types and different contents. Among them, minor ginsenosides have better biological functions and pharmacological activities than those of the major ginsenosides. However, minor ginsenosides cannot be obtained in large quantities, but by means of enzymatic transformation technology, some major ginsenosides can be de-glycosylated at a specific position to generate minor ginsenosides. In this study, we report two glycosidase genes associated with the conversion of ginsenoside Rd to ginsenosides F2 or CK. SWMU-CK-1 was identified among the total genes extracted from the feces of plum deer by local Blast screening for putative ginsenoside conversion function, which could cause the conversion of ginsenoside Rd → F2 → CK. The other gene was found in the Bifidobacterium breve 689b SGAir 0764 chromosome genome, which might have the same function as the ß-glucosidase gene testified by the gene matching, named SWMU-F2-2, and can achieve the Rd → F2 transformation. This study reports two genes that enable achieving the biotransformation of rare ginsenosides, while it provides a new insight and a promising approach to explore new genes and develop new functions of existing genes.

Characterization of a new L-carnosine synthase mined from deep-sea sediment metagenome.

L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. ß-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM ß-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.

Comprehensive Genomic Analysis of Marine Strain Streptomyces sp. 891, an Excellent Producer of Chrysomycin A with Therapeutic Potential.

Chrysomycin A is one of the most promising therapeutic candidates for treating infections caused by multidrug-resistant Gram-positive bacteria. By hybridizing next-step generation (Illumina) and third-generation (PacBio) sequencing technologies, a high-quality chromosome-level genome together with a plasmid was firstly assembled for chrysomycin A-producing marine strain 891. Phylogenetic analysis of the 16S rRNA gene and genome sequences revealed that this strain unambiguously belonged to the genus Streptomyces, and its genomic features and functional genes were comprehensively analyzed and annotated. AntiSMASH analysis of this strain unveiled one key biosynthetic gene cluster, T2PKS, responsible for the biosynthesis of chrysomycin, the biosynthesis pathway of which was putatively proposed. These findings definitely shed light on further investigation for construction of a robust industrial strain with high-yield chrysomycin A production using genetic engineering techniques and combinatorial biology approaches.

Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a piggyBac Transposon Promoter-Trapping Vector System.

Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 106 seeded HeLaS3 cells. 5' RACE analyses identified the well-known CYP24A1 gene as a calcitriol-responsive gene, as well as two new bexarotene- or calcitriol-responsive genes, BDKRB2 and TSKU, respectively. TSKU, interestingly, also responded to bexarotene. Endogenous levels of the TSKU and BDKRB2 transcripts displayed only slight changes and were not detected in the comprehensive analyses performed to date. Dose-response analyses of BDKRB2 and TSKU reporter cells in parallel revealed a differential profile in response to each vitamin A agonist, suggesting a bioanalyzer. The present study demonstrates that producing multiple reporter cells by a type of random screening can efficiently identify novel genes with unusual characteristics and be used for the profiling of the properties of vitamin compounds. Similar approaches to the method shown here may be useful for identifying new markers and for the analysis or diagnosis of nutrients, toxins, metabolites, etc.

Mapping QTL/QTN and mining candidate genes for plant height and its response to planting densities in soybean [Glycine max (L.) Merr.] through a FW-RIL population.

Plant height (PH) determines the morphology and seed yield of soybean, so it is an important breeding target, which is controlled by multiple genes and affected by plant density. In this research, it was used about a four-way recombinant inbred lines (FW-RIL) with 144 families constructed by double cross (Kenfeng 14 × Kenfeng 15) × (Heinong 48 × Kenfeng 19) as experimental materials, with the purpose to map QTL/QTN associated with PH under densities of 2.2×105 plant/ha (D1) and 3×105 plant/ha (D2) in five environments. The results showed that response of PH to densities varied in accordance to genotypes among environments. A total of 26 QTLs and 13 QTNs were identified specifically in D1; 20 QTLs and 21 QTNs were identified specifically in D2. Nine QTLs and one QTN were discovered commonly in two densities. Fifteen QTLs and 9 QTNs were repeatedly detected by multiple statistical methods, densities, or environments, which could be considered stable. Eighteen QTLs were detected, as well as 7 QTNs underlying responses of PH to density increment. Six QTNs, co-located in the interval of QTL, were detected in more than two environments or methods with a longer genome length over 3000 kb. Based on gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, five genes were predicted as candidates, which were likely to be involved in growth and development of PH. The results will help elucidate the genetic basis and improve molecular assistant selection of PH. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01209-0.

Identification, characterization and expression analysis of lineage-specific genes within Triticeae.

Lineage-specific genes (LSGs) are a set of genes in a given taxon without significant sequence similarity to genes and intergenic sequences of other taxa and are functional. The tribe Triticeae mainly includes species of different ploidy levels, such as staple food crops wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). This study is aimed at mining and characterizing the Triticeae-specific genes (TSGs) using expressed sequence data of wheat. A total of 3812 TSGs was identified and they were generally characterized by smaller size, fewer exons, shorter open reading frames and lower expression levels. Most TSGs were expressed with tissue preference and many of them were predominantly expressed in reproduction related tissues, especially in young stamen. Nearly one third of the TSGs were stress-responsive and inducible under abiotic and/or biotic stresses. A co-expression-based annotation supported the relevance of some TSGs with reproduction and stress responses, indicating their potential economic importance.

Exploitation of Drought Tolerance-Related Genes for Crop Improvement.

Drought has become a major threat to food security, because it affects crop growth and development. Drought tolerance is an important quantitative trait, which is regulated by hundreds of genes in crop plants. In recent decades, scientists have made considerable progress to uncover the genetic and molecular mechanisms of drought tolerance, especially in model plants. This review summarizes the evaluation criteria for drought tolerance, methods for gene mining, characterization of genes related to drought tolerance, and explores the approaches to enhance crop drought tolerance. Collectively, this review illustrates the application prospect of these genes in improving the drought tolerance breeding of crop plants.

An embedded gene selection method using knockoffs optimizing neural network.

BACKGROUND: Gene selection refers to find a small subset of discriminant genes from the gene expression profiles. How to select genes that affect specific phenotypic traits effectively is an important research work in the field of biology. The neural network has better fitting ability when dealing with nonlinear data, and it can capture features automatically and flexibly. In this work, we propose an embedded gene selection method using neural network. The important genes can be obtained by calculating the weight coefficient after the training is completed. In order to solve the problem of black box of neural network and further make the training results interpretable in neural network, we use the idea of knockoffs to construct the knockoff feature genes of the original feature genes. This method not only make each feature gene to compete with each other, but also make each feature gene compete with its knockoff feature gene. This approach can help to select the key genes that affect the decision-making of neural networks. RESULTS: We use maize carotenoids, tocopherol methyltransferase, raffinose family oligosaccharides and human breast cancer dataset to do verification and analysis. CONCLUSIONS: The experiment results demonstrate that the knockoffs optimizing neural network method has better detection effect than the other existing algorithms, and specially for processing the nonlinear gene expression and phenotype data.

Strategies for Discovering New Antibiotics from Bacteria in the Post-Genomic Era.

New antibiotics are urgently required in clinical treatment and agriculture with the development of antimicrobial resistance. However, products discovered by repeating previous strategies are either not antibiotics or already known antibiotics. There is a growing demand for efficient strategies to discover new antibiotics. With the continuous improvement of gene sequencing technology and genomic data, some mining strategies have emerged. These strategies are expected to alleviate the current dilemma of antibiotics. In this review, we discuss the recent advances in discovery of bacterial antibiotics from the following aspects: activation of silent gene clusters, genome mining and metagenome mining. In the future, we envision the discovery of natural antibiotic will be accelerated by the combination of these strategies.

GeM-Pro: a tool for genome functional mining and microbial profiling.

Gem-Pro is a new tool for gene mining and functional profiling of bacteria. It initially identifies homologous genes using BLAST and then applies three filtering steps to select orthologous gene pairs. The first one uses BLAST score values to identify trivial paralogs. The second filter uses the shared identity percentages of found trivial paralogs as internal witnesses of non-orthology to set orthology cutoff values. The third filtering step uses conditional probabilities of orthology and non-orthology to define new cutoffs and generate supportive information of orthology assignations. Additionally, a subsidiary tool, called q-GeM, was also developed to mine traits of interest using logistic regression (LR) or linear discriminant analysis (LDA) classifiers. q-GeM is more efficient in the use of computing resources than Gem-Pro but needs an initial classified set of homologous genes in order to train LR and LDA classifiers. Hence, q-GeM could be used to analyze new set of strains with available genome sequences, without the need to rerun a complete Gem-Pro analysis. Finally, Gem-Pro and q-GeM perform a synteny analysis to evaluate the integrity and genomic arrangement of specific pathways of interest to infer their presence. The tools were applied to more than 2 million homologous pairs encoded by Bacillus strains generating statistical supported predictions of trait contents. The different patterns of encoded traits of interest were successfully used to perform a descriptive bacterial profiling.

Mining human cancer datasets for kallikrein expression in cancer: the 'KLK-CANMAP' Shiny web tool.

The dysregulation of the serine-protease family kallikreins (KLKs), comprising 15 genes, has been reportedly associated with cancer. Their expression in several tissues and physiological fluids makes them potential candidates as biomarkers and therapeutic targets. There are several databases available to mine gene expression in cancer, which often include clinical and pathological data. However, these platforms present some limitations when comparing a specific set of genes and can generate considerable unwanted data. Here, several datasets that showed significant differential expression (p<0.01) in cancer vs. normal (n=118), metastasis vs. primary (n=15) and association with cancer survival (n=21) have been compiled in a user-friendly format from two open and/or publicly available databases Oncomine and OncoLnc for the 15 KLKs. The data have been included in a free web application tool: the KLK-CANMAP https://cancerbioinformatics.shinyapps.io/klk-canmap/. This tool integrates, analyses and visualises data and it was developed with the R Shiny framework. Using KLK-CANMAP box-plots, heatmaps and Kaplan-Meier graphs can be generated for the KLKs of interest. We believe this new cancer KLK focused web tool will benefit the KLK community by narrowing the data visualisation to only the genes of interest.

Pool deconvolution approach for high-throughput gene mining from Bacillus thuringiensis.

Novel genes from Bacillus thuringiensis (Bt) are required for effective deployment in agriculture, human health, and forestry. In an improvement over conventional PCR-based screening, next generation sequencing (NGS) has been used for identification of new genes of potential interest from Bt strains, but cost becomes a constraint when several isolates are to be sequenced. We demonstrate the potential of a DNA pooling strategy known as pool deconvolution to identify commercially important toxin genes from 36 native Bt isolates. This strategy is divided into three steps: (a) DNA pooling, (b) short read sequence assembly followed by gene mining, and (c) host isolate identification. With this approach, we have identified insecticidal protein (ip) genes including nine three-domain (3D) cry genes, three cyt-type genes, three mtx genes (mosquitocidal toxin), and one bin and vip-type gene each. Three cry-type and three cyt-type genes were cloned, out of which, two cry-type genes, ip11 and ip13, were named as cry4Ca2 and cry52Ca1, respectively by the Bacillus thuringiensis nomenclature committee ( http://www.biols.susx.ac.uk/Home/Neil_Crickmore/BT/ ). Our results show that the pool deconvolution approach is well suited for high-throughput gene mining in bacteria.

Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach.

Optimizing the production of the high-value renewables such as OMEGAs through pathway engineering requires an in-depth understanding of the structure-function relationship of genes involved in the OMEGA biosynthetic pathways. In this preliminary study, our rationale is to identify and characterize the ∼221 putative genes involved in production of OMEGAs using bioinformatic analysis from the Streptophyte (plants), Chlorophyte (green algae), Rhodophyta (red algae), and Bacillariophyta (diatoms) lineages based on their phylogenomic profiling, conserved motif/domain organization and physico-chemical properties. The MEME suite predicted 12 distinct protein domains, which are conserved among these putative genes. The phylogenomic analysis of the putative candidate genes [such as FAD2 (delta-12 desaturase); ECR (enoyl-CoA reductase); FAD2 (delta-12 desaturase); ACOT (acyl CoA thioesterase); ECH (enoyl-CoA hydratase); and ACAT (acetyl-CoA acyltransferase)] with similar domains and motif patterns were remarkably well conserved. Furthermore, the subcellular network prediction of OMEGA biosynthetic pathway genes revealed a unique interaction between the light-dependent chlorophyll biosynthesis and glycerol-3-phosphate dehydrogenase, which predicts a major cross-talk between the key essential pathways. Such bioinformatic analysis will provide insights in finding the key regulatory genes to optimize the productivity of OMEGAs in microalgal cell factories.

The Road from Host-Defense Peptides to a New Generation of Antimicrobial Drugs.

Host-defense peptides, also called antimicrobial peptides (AMPs), whose protective action has been used by animals for millions of years, fulfill many requirements of the pharmaceutical industry, such as: (1) broad spectrum of activity; (2) unlike classic antibiotics, they induce very little resistance; (3) they act synergically with conventional antibiotics; (4) they neutralize endotoxins and are active in animal models. However, it is considered that many natural peptides are not suitable for drug development due to stability and biodisponibility problems, or high production costs. This review describes the efforts to overcome these problems and develop new antimicrobial drugs from these peptides or inspired by them. The discovery process of natural AMPs is discussed, as well as the development of synthetic analogs with improved pharmacological properties. The production of these compounds at acceptable costs, using different chemical and biotechnological methods, is also commented. Once these challenges are overcome, a new generation of versatile, potent and long-lasting antimicrobial drugs is expected.