De novo assembly of Idesia polycarpa transcriptome and unsaturated fatty acid biosynthesis candidate genes Mining and functional Identification.

Unsaturated fatty acids (UFA) in lipids are the key to nutraceutical oil applications, with various potential applications in nutraceutical functional foods and pharmaceutical industries. In Idesia polycarpa (Salicaceae), more than 80 % of UFA have been found in the fruits; yet, the underlying genetic mechanism remains poorly understood. Due to the lack of theoretical research on the genes related to lipid biosynthesis and the complete genetic transformation system of I. polycarpa fruit, the selection and breeding of I. polycarpa, an excellent oil tree, has been severely restricted. In-depth understanding of the molecular mechanism and gene function of lipid biosynthesis of I. polycarpa fruit is therefore of great significance for the development of I. polycarpa resources. This is not only conducive to the genetic improvement of I. polycarpa by molecular breeding technologies but can also provide a reference for the study of the gene functions of other oil plants. In this study, the FA accumulation patterns of I. polycarpa fruits during 8 growth periods were analysed. Fruit from two developmental periods with different UFA levels were analysed for RNA sequencing by an Illumina NovaSeq 6000 HiSeq platform. De novo transcriptome assembly presented 115,350 unigenes and 4382 differentially expressed genes (DEGs). Functional annotation in the KEGG pathway and combined with DEG data revealed candidate genes potentially involved in UFA biosynthesis. Expression analysis of q-PCR of IpDGAT2, IpGPAT, IpKASII, IpSAD, IpFAD2, IpFAD3 and IpFAD8 suggested that these genes are highly involved in UFA biosynthesis. Full-length candidate genes were cloned and analysed by bioinformatic tools, and function analysis of IpSAD and IpFAD3 showed that these genes regulated the products of linoleic acid metabolism. This study provides a foundation for UFA biosynthesis in Idesia polycarpa, facilitating its genetic breeding in the future.

A Facile, Transfection-Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.

A genome-scale deep learning model to predict gene expression changes of genetic perturbations from multiplex biological networks.

Systematic characterization of biological effects to genetic perturbation is essential to the application of molecular biology and biomedicine. However, the experimental exhaustion of genetic perturbations on the genome-wide scale is challenging. Here, we show TranscriptionNet, a deep learning model that integrates multiple biological networks to systematically predict transcriptional profiles to three types of genetic perturbations based on transcriptional profiles induced by genetic perturbations in the L1000 project: RNA interference, clustered regularly interspaced short palindromic repeat, and overexpression. TranscriptionNet performs better than existing approaches in predicting inducible gene expression changes for all three types of genetic perturbations. TranscriptionNet can predict transcriptional profiles for all genes in existing biological networks and increases perturbational gene expression changes for each type of genetic perturbation from a few thousand to 26 945 genes. TranscriptionNet demonstrates strong generalization ability when comparing predicted and true gene expression changes on different external tasks. Overall, TranscriptionNet can systemically predict transcriptional consequences induced by perturbing genes on a genome-wide scale and thus holds promise to systemically detect gene function and enhance drug development and target discovery.

Variant graph craft (VGC): a comprehensive tool for analyzing genetic variation and identifying disease-causing variants.

BACKGROUND: The variant call format (VCF) file is a structured and comprehensive text file crucial for researchers and clinicians in interpreting and understanding genomic variation data. It contains essential information about variant positions in the genome, along with alleles, genotype calls, and quality scores. Analyzing and visualizing these files, however, poses significant challenges due to the need for diverse resources and robust features for in-depth exploration. RESULTS: To address these challenges, we introduce variant graph craft (VGC), a VCF file visualization and analysis tool. VGC offers a wide range of features for exploring genetic variations, including extraction of variant data, intuitive visualization, and graphical representation of samples with genotype information. VGC is designed primarily for the analysis of patient cohorts, but it can also be adapted for use with individual probands or families. It integrates seamlessly with external resources, providing insights into gene function and variant frequencies in sample data. VGC includes gene function and pathway information from Molecular Signatures Database (MSigDB) for GO terms, KEGG, Biocarta, Pathway Interaction Database, and Reactome. Additionally, it dynamically links to gnomAD for variant information and incorporates ClinVar data for pathogenic variant information. VGC supports the Human Genome Assembly Hg37 and Hg38, ensuring compatibility with a wide range of data sets, and accommodates various approaches to exploring genetic variation data. It can be tailored to specific user needs with optional phenotype input data. CONCLUSIONS: In summary, VGC provides a comprehensive set of features tailored to researchers working with genomic variation data. Its intuitive interface, rapid filtering capabilities, and the flexibility to perform queries using custom groups make it an effective tool in identifying variants potentially associated with diseases. VGC operates locally, ensuring data security and privacy by eliminating the need for cloud-based VCF uploads, making it a secure and user-friendly tool. It is freely available at https://github.com/alperuzun/VGC .

Efficient gene editing of a model fern species through gametophyte-based transformation.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P<0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications.

Genome-Wide Characterization of Alfin-like Genes in Brassica napus and Functional Analyses of BnaAL02 and BnaAL28 in Response to Nitrogen and Phosphorus Deficiency.

Alfin-like proteins (ALs) form a plant-specific transcription factor (TF) gene family involved in the regulation of plant growth and development, and abiotic stress response. In this study, 30 ALs were identified in Brassica napus ecotype 'Zhongshuang 11' genome (BnaALs), and unevenly distributed on 15 chromosomes. Structural characteristic analysis showed that all of the BnaALs contained two highly conserved domains: the N terminal DUF3594 domain and the C-terminal PHD-finger domain. The BnaALs were classified into four groups (Group I-IV), supported by conserved intron-exon and protein motif structures in each group. The allopolyploid event between B. oleracea and B. rapa ancestors and the small-scale duplication events in B. napus both contributed to the large BnaALs expansion. The promoter regions of BnaALs contained multiple abiotic stress cis-elements. The BnaALs in I-IV groups were mainly expressed in cotyledon, petal, root, silique, and seed tissues, and the duplicated gene pairs shared highly similar expression patterns. RNA-seq and RT-qPCR analysis showed that BnaALs were obviously induced by low nitrogen (LN) and low phosphorus (LP) treatments in roots. Overexpressing BnaAL02 and BnaAL28 in Arabidopsis demonstrated their functions in response to LN and LP stresses. BnaAL28 enhanced primary roots' (PRs) length and lateral roots' (LRs) number under LP and LN conditions, where BnaAL02 can inhibit LR numbers under the two conditions. They can promote root hair (RH) elongation under LP conditions; however, they suppressed RH elongation under LN conditions. Our result provides new insight into the functional dissection of this family in response to nutrient stresses in plants.

Genome-wide analysis of the WOX gene family and function exploration of RhWOX331 in rose (R. 'The Fairy').

WOXs are a class of plant-specific transcription factors that play key roles in plant growth and stress responses. However, the mechanism by which WOXs influence adventitious root development in Rosa hybrida remains unclear. In this study, RcWOX gene family in rose was identified and phylogenetically analyzed using bioinformatics analysis. A total of 381 RcWOX gene members were localized on seven chromosomes except of nine members. The main cis-acting elements involved in hormonal, light, developmental, and abiotic stress responses were identified in the promoters of RcWOX genes, suggesting their regulation by these signals. Nine RhWOX genes had significant different expression during rooting process of rose. RhWOX331, RhWOX308, RhWOX318 were positive with the formation of rose roots. RhWOX331 was positively involved in the formation of adventitious root primordia, which gene coding a transcription factor localized in the nucleus. The HOX conserved domain in the protein contributed to the self-activating activity of RhWOX331. We obtained genetically modified Arabidopsis to validate the function of RhWOX331. Overexpression of RhWOX331 gene alleviated the inhibition of root length of A. thaliana primary roots by high concentration of IBA and NPA, and significantly increased the number of lateral roots on the primary roots, as well as the height of A. thaliana plants. Additionally, RhWOX331 promoted adventitious root formation in A. thaliana and mitigated hormonal inhibition by exogenous 6-BA, NPA, and GA3. The RhWOX331 promoter contained cis-acting elements such as ABRE, Box 4 and CGTCA-motif et.al. GUS activity analysis showed that the gene acted at the cotyledon attachment site. Taken together, these studies identified a significant expansion of the RcWOX gene family, inferred roles of certain branch members in adventitious root formation, elucidated the function of RhWOX331 in adventitious root initiation, and laid the foundation for further research on the function of WOX gene family in roses.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in cotton.

BACKGROUND: In research to improve the quality of transgenic crops, it is often necessary to introduce multiple functionally related genes into recipient plants simultaneously to improve crop genetic traits effectively. Compared with unidirectional promoters, bidirectional promoters simultaneously regulate the expression of multiple genes and improve the efficiency of biotechnology. Therefore, in this study, bidirectional gene pairs were systematically analyzed in Gossypium hirsutum TM-1, and the structure, function and evolutionary relationships of the bidirectional genes were analyzed. The endogenous bidirectional promoters of cotton were mined, and their specific regulatory elements and biological functions were explored to provide useful promoter resources and a theoretical basis for cultivating new cotton germplasms with excellent fiber quality. RESULTS: Using an improved search model, a total of 1,383 bidirectional transcript pairs were identified in the Gossypium hirsutum TM-1 genome, and their gene structure and functional annotations were systematically analyzed. Thirty bidirectional intergenic sequences were randomly screened for promoter activity analysis via a transient expression system, and 25 intergenic sequences were found to have bidirectional promoter activity. Comparative analysis of the bidirectional gene profiles of the four cotton subspecies revealed that these subspecies presented abundant bidirectional gene pairs with high homology and that the bidirectional genes in the cotton subspecies were more similar in terms of their molecular functions, cellular components and biological processes. In addition, parallel analysis of bidirectional genes in dicotyledons and monocotyledons revealed that abundant bidirectional gene pairs exist in different species. Although the total number of orthologous bidirectional genes was similar, there was a significant difference in the number of orthologous bidirectional gene pairs between dicotyledons and monocotyledons. This evolutionary analysis of the function and structure of homologous bidirectional gene pairs in different varieties and different subspecies of the same species revealed potential pathways by which these gene pairs originated, which may be necessary for the evolution of a new species. CONCLUSION: In this study, many bidirectional gene pairs in Gossypium hirsutum TM-1 were identified using computer programming, and systematic analysis was conducted to explore their functions and evolutionary relationships. In addition, the promoter activity of the bidirectional intergenic sequences was verified. The combination of computer programming screening, experimental validation and other methods is expected to provide preferred bidirectional promoters for transgenic breeding work via multigene cotransformation methods, and this information is valuable for genetic engineering research and applications.

Functional Identification and Regulatory Active Site Screening of the DfDXS Gene of Dryopteris fragrans.

Dryopteris fragrans (L.) Schott has anti-inflammatory and antioxidant properties, and terpenoids are important components of its active constituents. The methyl-D-erythritol 4-phosphate (MEP) pathway is one of the major pathways for the synthesis of terpene precursors in plants, and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in this pathway. DXS has been shown to be associated with increased stress tolerance in plants. In this experiment, two DXS genes were extracted from the D. fragrans transcriptome and named DfDXS1 and DfDXS2. Based on phylogenetic tree and conserved motif analyses, DXS was shown to be highly conserved evolutionarily and its localization to chloroplasts was determined by subcellular localization. Prokaryotic expression results showed that the number and growth status of recombinant colonies were better than the control under 400 mM NaCl salt stress and 800 mM mannitol-simulated drought stress. In addition, the DfDXS1 and DfDXS2 transgenic tobacco plants showed improved resistance to drought and salt stress. DfDXS1 and DfDXS2 responded strongly to methyl jasmonate (MeJA) and PEG-mimicked drought stress following exogenous hormone and abiotic stress treatments of D. fragrans. The transcriptional active sites were investigated by dual luciferase and GUS staining assays, and the results showed that the STRE element (AGGGG), the ABRE element (ACGTGGC), and the MYC element (CATTTG) were the important transcriptional active sites in the promoters of the two DXS genes, which were closely associated with hormone response and abiotic stress. These results suggest that the DfDXS gene of D. fragrans plays an important role in hormone signaling and response to stress. This study provides a reference for analyzing the molecular mechanisms of stress tolerance in D. fragrans.

Knockout, Knockdown, and the Schrödinger Paradox: Genetic Immunity to Phenotypic Recapitulation in Zebrafish.

Previous research has highlighted significant phenotypic discrepancies between knockout and knockdown approaches in zebrafish, raising concerns about the reliability of these methods. However, our study suggests that these differences are not as pronounced as was once believed. By carefully examining the roles of maternal and zygotic gene contributions, we demonstrate that these factors significantly influence phenotypic outcomes, often accounting for the observed discrepancies. Our findings emphasize that morpholinos, despite their potential off-target effects, can be effective tools when used with rigorous controls. We introduce the concept of graded maternal contribution, which explains how the uneven distribution of maternal mRNA and proteins during gametogenesis impacts phenotypic variability. Our research categorizes genes into three types-susceptible, immune, and "Schrödinger" (conditional)-based on their phenotypic expression and interaction with genetic compensation mechanisms. This distinction provides new insights into the paradoxical outcomes observed in genetic studies. Ultimately, our work underscores the importance of considering both maternal and zygotic contributions, alongside rigorous experimental controls, to accurately interpret gene function and the mechanisms underlying disease. This study advocates for the continued use of morpholinos in conjunction with advanced genetic tools like CRISPR/Cas9, stressing the need for a meticulous experimental design to optimize the utility of zebrafish in genetic research and therapeutic development.

Functional analysis of AgJHAMT gene related to developmental period in Aphis gossypii Glover.

Aphis gossypii is one of the most economically important agricultural pests that cause serious crop losses worldwide, and the indiscriminate chemical application causes resistance development in A. gossypii, a major obstacle to successful control. In this study, we selected the up-regulated expression gene AgJHAMT, which was enriched into juvenile hormone pathway though transcriptome sequencing analysis of the cotton aphids that fed on transgenic cotton lines expressing dsAgCYP6CY3 (the TG cotton). The AgJHAMT gene was overexpressed in cotton aphids which fed on the TG cotton, and its expression profile during the nymphs was clarified. Then, silencing AgJHAMT could advance the developmental period of cotton aphids by 0.5 days compared with control groups. The T and t values of cotton aphids in the dsJHAMT treatment group (6.88 ± 0.15, 1.65 ± 0.06) were significantly shorter than that of the sprayed H2O control group (7.6 ± 0.14, 1.97 ± 0.09) (P < 0.05), respectively. The fast growth caused by AgJHAMT silencing was rescued by applying the JH analogue, methoprene. Overall, these findings clarified the function of AgJHAMT in the developmental period of A. gossypii. This study contributes to further clarify the molecular mechanisms of delaying the growth and development of cotton aphids by the transgenic cotton lines expressing dsAgCYP6CY3.

Low-dose 60Co-γ-ray irradiation promotes the growth of cucumber seedlings by inducing CsSAUR37 expression.

Cucumber (Cucumis sativus L.) is a major vegetable crop grown globally, with a cultivation history of more than 3000 years. The limited genetic diversity, low rate of intraspecific variation, and extended periods of traditional breeding have resulted in slow progress in their genetic research and the development of new varieties. Gamma (γ)-ray irradiation potentially accelerates the breeding progress; however, the biological and molecular effects of γ-ray irradiation on cucumbers are unknown. Exposing cucumber seeds to 0, 50, 100, 150, 200, and 250 Gy doses of 60Co-γ-ray irradiation, this study aimed to investigate the resulting phenotype and physiological characteristics of seedling treatment to determine the optimal irradiation dose. The results showed that low irradiation doses (50-100 Gy) enhanced root growth, hypocotyl elongation, and lateral root numbers, promoting seedling growth. However, high irradiation doses (150-250 Gy) significantly inhibited seed germination and growth, decreasing the survival rate of seedlings. More than 100 Gy irradiation significantly decreased the total chlorophyll content while increasing the malondialdehyde (MDA) and H2O2 content in cucumber. Transcriptome sequencing analysis at 0, 50, 100, 150, 200, and 250 Gy doses showed that gene expression significantly differed between low and high irradiation doses. Gene Ontology enrichment and functional pathway enrichment analyses revealed that the auxin response pathway played a crucial role in seedling growth under low irradiation doses. Further, gene function analysis revealed that small auxin up-regulated gene CsSAUR37 was a key gene that was overexpressed in response to low irradiation doses, promoting primary root elongation and enhancing lateral root numbers by regulating the expression of protein phosphatase 2Cs (PP2Cs) and auxin synthesis genes.

CRISPR/Cas9-Based Genome Editing of Fall Armyworm (Spodoptera frugiperda): Progress and Prospects.

The fall armyworm (Spodoptera frugiperda) poses a substantial threat to many important crops worldwide, emphasizing the need to develop and implement advanced technologies for effective pest control. CRISPR/Cas9, derived from the bacterial adaptive immune system, is a prominent tool used for genome editing in living organisms. Due to its high specificity and adaptability, the CRISPR/Cas9 system has been used in various functional gene studies through gene knockout and applied in research to engineer phenotypes that may cause economical losses. The practical application of CRISPR/Cas9 in diverse insect orders has also provided opportunities for developing strategies for genetic pest control, such as gene drive and the precision-guided sterile insect technique (pgSIT). In this review, a comprehensive overview of the recent progress in the application of the CRISPR/Cas9 system for functional gene studies in S. frugiperda is presented. We outline the fundamental principles of applying CRISPR/Cas9 in S. frugiperda through embryonic microinjection and highlight the application of CRISPR/Cas9 in the study of genes associated with diverse biological aspects, including body color, insecticide resistance, olfactory behavior, sex determination, development, and RNAi. The ability of CRISPR/Cas9 technology to induce sterility, disrupt developmental stages, and influence mating behaviors illustrates its comprehensive roles in pest management strategies. Furthermore, this review addresses the limitations of the CRISPR/Cas9 system in studying gene function in S. frugiperda and explores its future potential as a promising tool for controlling this insect pest.

Comparative Analysis of Transcriptomes Reveals Pathways and Verifies Candidate Genes for Clubroot Resistance in Brassica oleracea.

Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both resistant (R) and susceptible (S) B. oleracea plants infected by P. brassicae. Then the comparative analysis was carried out between the R and S samples at different time points during the infection stages to reveal clubroot resistance related pathways and candidate genes. Compared with 0 days after inoculation, a total of 4991 differential expressed genes were detected from the S pool, while only 2133 were found from the R pool. Gene function enrichment analysis found that the effector-triggered immunity played a major role in the R pool, while the pathogen-associated molecular pattern triggered immune response was stronger in the S pool. Simultaneously, candidate genes were identified through weighted gene co-expression network analysis, with Bol010786 (CNGC13) and Bol017921 (SD2-5) showing potential for conferring resistance to clubroot. The findings of this research provide valuable insights into the molecular mechanisms underlying clubroot resistance and present new avenues for further research aimed at enhancing the clubroot resistance of B. oleracea through breeding.

Assembly and functional profile of rhizosphere microbial community during the Salix viminalis-AMF remediation of polycyclic aromatic hydrocarbon polluted soils.

Arbuscular mycorrhizal fungi (AMF) are positive to the phytoremediation by improving plant biomass and soil properties. However, the role of AM plants to the remediation of polycyclic aromatic hydrocarbons (PAHs) is yet to be widely recognized, and the impact of AM plants to indigenous microbial communities during remediation remains unclear. In this work, a 90-day study was conducted to assess the effect of AMF-Salix viminalis on the removal of PAHs, and explore the impact to the microbial community composition, abundance, and function. Results showed that AMF-Salix viminalis effectively enhanced the removal of benzo[a]pyrene, and enriched more PAH-degrading bacteria, consisting of Actinobacteria, Chloroflexi, Sphingomonas, and Stenotrophobacter, as well as fungi including Basidiomycota, Pseudogymnoascus, and Tomentella. For gene function, AM willow enhanced the enrichment of genes involved in amino acid synthesis, aminoacyl-tRNA biosynthesis, and cysteine and methionine metabolism pathways. F. mosseae inoculation had a greater effect on alpha- and beta-diversity of microbial genes at 90 d. Additionally, AMF inoculation significantly increased the soil microbial biomass carbon and organic matter concentration. All together, the microbial community assembly and function shaped by AM willow promoted the dissipation of PAHs. Our results support the effectiveness of AM remediation and contribute to reveal the enhancing-remediation mechanism to PAHs using multi-omics data.

Efficient Broad-Spectrum Cyanophage Function Module Mining.

Cyanobacterial harmful algal blooms (CyanoHABs) cause health and environmental effects worldwide. Cyanophage is a virus that exclusively infects cyanobacteria. Using cyanophages to control blooms is the latest biological control method. However, little research on the genomics of cyanophages and the presence of numerous proteins with unidentified functions in cyanophage genomes pose challenges for their practical application and comprehensive investigation. We selected the broad-spectrum and efficient cyanophage YongM for our study. On the one hand, through rational analysis, we analyze essential genes, establish the minimal cyanophage genome and single essential gene modules, and examine the impact of essential modules on growth. Additionally, we conducted ultraviolet mutagenesis on YongM to generate more efficient cyanophages' critical modules through random mutagenesis. Then, we sequenced and analyzed the functionality of the mutational gene modules. These findings highlight several gene modules that contribute to a deeper understanding of the functional components within cyanophage genomes.

Rapid function analysis of OsiWAK1 using a Dual-Luciferase assay in rice.

In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study.

OsWNK9 regulates cadmium concentration in brown rice by restraining cadmium transport from straw to brown rice.

Selecting and breeding rice cultivars that enable strong cadmium (Cd) accumulation in rice straw but low accumulation in brown rice is a promising way to achieve Cd phytoremediation as well as to ensure the food safety of rice. Herein, we isolated a gene OsWNK9 from the quantitative trait locus associated with reducing Cd translocation from rice straw to brown rice and decreasing the Cd concentration in brown rice (BRCdC). Continuous strong expression of OsWNK9 was observed in nodes and internode and was induced after Cd supply. OsWNK9 was localized in the rice cell nucleus and participated in the regulation of Cd transport in yeast. Two independent oswnk9 rice mutants were generated via CRISPR/Cas9 gene-editing and showed significantly higher BRCdC than that of the wild type (WT). The BRCdC of knockout oswnk9 mutants was 0.227 mg kg-1and 0.238 mg kg-1, increased by 14 % and 19 % compared with that of the WT due to the lower Cd allocation in the basal stem, internode, and node III, which was unrelated to Cd uptake. Interestingly, OsWNK9 could promote iron (Fe) accumulation in rice under Cd-contaminated conditions, suggesting that OsWNK9 is an ideal gene for Cd phytoremediation and Fe biofortification in rice to support safe food production.

LEP Gene Promotes Milk Fat Synthesis via the JAK2-STAT3 and mTOR Signaling Pathways in Buffalo Mammary Epithelial Cells.

Leptin (LEP), a protein hormone well-known for its role in metabolic regulation, has recently been linked to lipid metabolism in cattle. However, its function in buffalo mammary glands remains unclear. To address this issue, we isolated and identified the LEP gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of LEP, designated as LEP_X1 and LEP_X2, were identified. The coding sequences (CDS) of LEP_X1 and LEP_X2 are 504 bp and 579 bp in length, encoding 167 and 192 amino acid residues, respectively. Bioinformatics analysis revealed that LEP_X2 is a hydrophobic protein with an isoelectric point below 7 and contains a signal peptide, while LEP_X1 is hydrophilic and lacks a signal peptide. Our study found that LEP gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with LEP_X2 expression remarkably higher than LEP_X1 in lactating BuMECs. Overexpression of both LEP_X1 and LEP_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including STAT3, PI3K, mTOR, SCD, and SREBF1, accompanied by an increase in cellular triglycerides (TG). Interestingly, LEP_X2 overexpression significantly suppressed LEP_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to LEP_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing LEP_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo LEP CDS, including a nonsynonymous substitution c.148C>T (p.Arg50Cys), which was predicted to decrease the stability of the LEP protein without affecting its function. These results collectively underscore the significant role of LEP in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.

O-Glycosyltransferase Gene BnaC09.OGT Involved in Regulation of Unsaturated Fatty Acid Biosynthesis for Enhancing Osmotic Stress Tolerance in Brassica napus L.

Osmotic stress is a major threaten to the growth and yield stability of Brassica napus. Post-translational modification with O-linked ß-N-acetylglucosamine (O-GlcNAc) is ubiquitous in plants, and participates in a variety of signal transduction and metabolic regulation. However, studies on the role of O-GlcNAc transferase (OGT) in osmotic stress tolerance of plants are limited. In previous study, a O-glycosyltransferase, named BnaC09.OGT, was identified from the B. napus variety 'Zhongshuang 11' by yeast one hybrid with promoter of BnaA01.GPAT9. It was found that BnaC09.OGT localized in both nucleus and cytoplasm. The spatiotemporal expression pattern of BnaC09.OGT exhibited tissue specificity in developmental seed, especially in 15 days after pollination. In view of osmotic stress inducing, the BnaC09.OGT overexpression and knockout transgenic lines were constructed for biological function study. Phenotypic analysis of BnaC09.OGT overexpression seedlings demonstrated that BnaC09.OGT could enhance osmotic stress tolerance than WT and knockout lines in euphylla stage under 15% PEG6000 treatment after 7 days. In addition, compared with WT and knockout lines, overexpression of BnaC09.OGT had significantly higher activities of antioxidant enzymes (SOD and POD), higher content of soluble saccharide, and while significantly less content of malondialdehyde, proline and anthocyanidin under 15% PEG6000 treatment after 7 days. On the other hand, the unsaturated fatty acid content of BnaC09.OGT overexpression was significantly higher than that of WT and knockout lines, so it is speculated that the BnaC09.OGT could increase unsaturated fatty acid biosynthesis for osmotic stress tolerance by promoting the expression of BnaA01.GPAT9 in glycerolipid biosynthesis. In summary, the above results revealed that the function of BnaC09.OGT provides new insight for the analysis of the pathway of O-glycosylation in regulating osmotic stress tolerance in B. napus.