Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

WUSCHEL (WUS)-related homeobox (WOX) family genes are important regulatory factors in plant developmental processes such as stem cell maintenance, embryogenesis, and organogenesis. In this study, the WOXs in sugarcane were identified and the number of gene family members, sequence characteristics, and evolutionary relationships have been elucidated. Additionally, the ScWOXs capable of regulating sugarcane callus proliferation and shoot regeneration were mined, aiming to provide valuable genetic resources to enhance the efficiency of genetic transformation and gene editing in sugarcane. Firstly, the conserved domain file of WOX protein (Pfam ID: PF00046) and the Hidden Markov Model (HMM) file were downloaded from Pfam. Using PF00046 as the template sequence, the candidate sequences of ScWOX family proteins were obtained by aligning the amino acid sequences of the sugarcane cultivar 'R570' with HMM3.0. The members of the ScWOX gene family were identified based on the conserved domain analysis. Subsequently, a series of analyses were performed on ScWOXs, including the analysis of physicochemical properties, prediction of subcellular localization, analysis of gene structures, protein motifs, and cis-elements in promoters, as well as analysis of chromosome localization and collinearity, and presented the relevant results via TBtools. Finally, qRT-PCR was employed to analyze the expression patterns of ScWOXs in the sugarcane cultivar 'YZ08-1609' during callus proliferation and shoot regeneration, aiming to identify ScWOXs responsive to callus proliferation and differentiation. A total of 82 ScWOXs were identified from the genome of 'R570' and classified into three major clades (ancient, intermediate, and the WUS/modern clade) and 12 subclades (ScWOX1‒ScWOX12). The deduced ScWOXs had the lengths of 204‒956 aa, relative molecular weights of 22.04‒103.86 kDa, and theoretical isoelectric points of 6.00‒10.79. They were all hydrophilic proteins, and most ScWOXs were localized in the nucleus. ScWOXs contained 1‒3 exons and 3‒10 conserved motifs. The promoters of ScWOXs were enriched with cis-acting elements responsive to plant growth and development, meristem activation and expression, phytohormone regulation, abiotic stress, and light. ScWOXs presented uneven distribution on the 44 chromosomes and 5 scaffolds of 'R570'. Among ScWOXs, there were 272 collinear gene pairs. Additionally, 87 and 81 WOX collinear gene pairs were identified for sugarcane with sorghum and rice, respectively. Gene expression analysis showed that ScWOX4, ScWOX9, ScWOX10, and ScWOX12 were up-regulated during callus proliferation and shoot regeneration in 'YZ08-1609'. These genes are promising candidates for promoting sugarcane callus proliferation and shoot regeneration. The results provide abundant genetic resources for optimizing the genetic transformation and gene editing systems in sugarcane.

The homologous regions (hrs) of Bombyx mori nucleopolyhedrovirus (BmNPV) have been demonstrated to function as transcriptional enhancers and initiation sites of DNA replication. This study aimed to elucidate the protein-binding characteristics of hrs in the BmNPV genome and their regulatory mechanisms in viral infection. Using DNA pull-down coupled with LC-MS/MS, we systematically analyzed four highly interactive hrs (hr1, hr2L, hr3, and hr5), successfully identifying 215‒612 specific binding proteins for each region. Our findings revealed that these hrs not only bind to with numerous host proteins but also with multiple viral proteins. Notably, 15 proteins exhibited binding affinity to all four hrs, which suggested that these core interacting proteins may play pivotal roles in hrs-mediated regulation. Further analysis demonstrated that 67.3% of the binding proteins possessed multivalent binding properties, indicating that hrs may coordinate viral genome regulation through shared protein interaction networks. These results provide significant insights into the crucial regulatory functions of hrs in BmNPV infection, offer potential targets for developing antiviral strategies in silkworms, and contribute to a deeper understanding of baculovirus-host interactions at the molecular level.

Abscisic acid, stress and ripening-induced (ASR) proteins play crucial roles in plant ripening induction and stress responses. Although ASR proteins have been identified in various plant species, systematic studies in sugarcane remain limited, and their structural and functional characteristics are poorly understood. To this end, this study aimed to systematically identify members of the sugarcane ASR gene family at the genome-wide level, elucidate their molecular characteristics and evolutionary relationships, and investigate their expression patterns during growth, development, and stress responses. The results showed that a total of 40 ShASR genes from the Saccharum spp. cultivar 'R570' and 15 SsASR genes from the wild species Saccharum spontaneum were identified. All the predicted ASR proteins contained the characteristic ABA/WDS domain. Phylogenetic analysis, using rice ASR proteins as a reference, classified the sugarcane ASRs into three distinct subfamilies. Physicochemical property predictions indicated that sugarcane ASR proteins were stable and hydrophilic, mainly localized to the nucleus. Further analyses of gene structures, duplication patterns, and chromosomal distribution revealed that ASR genes mainly expanded through whole-genome or segmental duplication events and exhibited subfamily-specific clustering on chromosomes. Promoter analysis showed enrichment of cis-acting elements related to stress and plant hormone responses, as well as growth and development. Transcriptomic data revealed that most ASR genes were expressed in sugarcane leaf, leaf sheath, pith, skin, and bud samples and were responsive to smut pathogen infection and drought stress. RT-qPCR results confirmed that six ShASR genes (ShASR1, ShASR6, ShASR14, ShASR20, ShASR25, and ShASR40) showed differential expression patterns in response to exogenous plant hormone treatments and smut pathogen challenge. These findings suggested that ShASR genes might play important roles in regulating stress responses, disease resistance, and development in sugarcane. This study provides a comprehensive understanding of the sugarcane ASR gene family and offers valuable genetic resources for molecular breeding of stress-tolerant sugarcane cultivars.

Parainfluenza virus type 5 (PIV5) is a negative-sense single-stranded RNA virus belonging to the genus Rubulavirus of the family Paramyxoviridae. The virus has a broad host range and can infect various animals, including ticks, horses, dogs, pigs, hamsters, guinea pigs, cattle, and giant pandas. With extremely low pathogenicity in humans and a favorable safety profile, PIV5 has garnered significant attention as a promising novel vaccine vector platform. This article outlines the pathogenic biological characteristics of PIV5, summarizes the advances in reverse genetics technologies for PIV5 and other common paramyxoviruses, and analyzes the advantages and challenges of PIV5 as a vaccine vector. The aim is to provide insights for the development of novel PIV5-based vector vaccines.

Single-domain antibodies (sdAbs) have garnered increasing attention in the biomedical field due to their low molecular weights and exceptional tissue penetration capacity. Compared with mammalian cell and Escherichia coli expression systems, the Pichia pastoris expression system offers advantages such as low costs, short production cycles, and robust support for protein folding. To enhance the expression efficiency of sdAbs in yeast, we constructed a co-expression system in P. pastoris involving six molecular chaperones (HDPI, YPDI, BZIP, TO, RPPO, and Sec) and the target sdAbs. The effects of these chaperones on the structural and functional properties of sdAb were systematically evaluated through circular dichroism (CD) spectroscopy for secondary structure analysis, measurements of thermal melting temperature (T_m) and concentration of urea when 50% protein unfolded (CUU), turbidity assays, and trypsin digestion experiments. The results demonstrated that: Co-expression of Sec significantly increased the expression level of sdAbs by 1.68 times; None of the chaperones significantly altered the secondary structure or antigen-binding activity of the sdAbs; Only the BZIP co-expression group showed enhanced thermal stability, with a Tm increase of 2.4 ℃; The YPDI group exhibited markedly improved resistance to urea-induced denaturation, with a 1.2 mol/L increase in CUU; In trypsin digestion assays, the YPDI group displayed the highest stability, retaining 58.3% of the enzyme activity after 3 h; Turbidity assays indicated that YPDI and BZIP effectively suppressed antibody aggregation, whereas HPDI, TO, and RPPO promoted aggregation. In conclusion, this study reveals distinct regulatory effects of different molecular chaperones on the physicochemical properties of sdAbs, providing a theoretical foundation and practical insights for optimizing the production processes of sdAbs.

To explore the mechanisms underlying the therapeutic efficacy of cell transplantation in mouse liver, liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics was employed to screen the differential metabolites in the liver tissue of mice undergoing cell transplantation. We then explored the biological significance of differential metabolites through pathway analysis, correlation analysis, and cluster analysis, aiming to decipher the potential mechanisms affecting the therapeutic outcomes of cell transplantation. The augmenter of liver regeneration (ALR) has been reported to regulate stem/progenitor cell fate. An acute liver failure (ALF) model was induced in mice with carbon tetrachloride (CCl₄), and the mice were allocated into three groups (n=5) and transplanted with liver epithelial progenitor cells (LEPCs) with normal expression, overexpression, and knockdown of ALR via splenic injection. Liver tissue samples were collected for LC-MS-based untargeted metabolomics analysis. Differential metabolites were screened and subjected to Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis to identify potential targets influencing the efficacy of cell transplantation. A total of 4 861 metabolites were detected in both positive and negative ion modes. Comparisons were conducted among the three groups with the threshold of fold change (FC)≥2 or≤0.5. The overexpression group and the normal expression group showed minor differences in metabolomic profiles, demonstrating high metabolic similarity. Therefore, we focused on comparing the overexpression group with the knockdown group. Twenty-four differential metabolites were identified between the two groups, with the overexpression group showing 16 upregulated metabolites and 8 downregulated metabolites. Among them, geniposidic acid (GPA) exhibited the most significant upregulation. KEGG pathway enrichment analysis revealed that the overexpression group was primarily associated with six metabolic pathways. Importantly, GPA has been repeatedly reported to participate in the regulation of the bile acid metabolism pathway. ALR overexpression in LEPCs may enhance the therapeutic efficacy by upregulating GPA expression and modulating the bile acid metabolism pathway, thereby creating a favorable liver microenvironment for transplanted cells.

This study aims to investigate the feasibility of an ATP bioluminescence assay for rapid detection of microbial contamination in cell-based products. Firstly, we evaluated the applicability of a sample pre-treatment method for its ability to eliminate background ATP interference derived from animal cells. The effectiveness of the ATP bioluminescence assay for rapidly detecting microbial contamination was concurrently assessed. On this basis, the ATP bioluminescence assay combined with the pre-treatment method and the pharmacopeial sterility test were employed to analyze the animal cell samples spiked with microorganisms at low levels. The detection limits of both methods were determined, and the non-inferiority of the ATP bioluminescence assay relative to the pharmacopeial method was evaluated. The results showed that the pre-treatment method effectively eliminated the interference caused by background ATP signals from animal cells. The ATP bioluminescence assay detected microbial contamination rapidly. Compared with the pharmacopeial sterility test method, the ATP bioluminescence assay showed no statistically significant difference in detection limit and demonstrated non-inferiority in terms of decision equivalence. Therefore, within a risk-based strategy, the ATP bioluminescence assay is suitable for the rapid detection of microbial contamination in cell-based products. It provides rapid and effective contamination monitoring methods for the risk release of cell therapy drugs and the process control of aseptic processes for other biological products based on cell fermentation.

As an essential aspect of structural biology, RNA secondary structures play a crucial role in maintaining molecular stability and regulating various biological functions. Since experimental approaches for resolving RNA secondary structures are often complex and costly, developing efficient and accurate prediction methods has become a key direction in RNA structural research. Computational biology, an important analytical tool, provides structural biology with theoretical foundations and algorithmic support. Due to its great potential in RNA secondary structure prediction, it has attracted extensive attention in biomedical research. This article first outlined the main computational methods for RNA secondary structure prediction, including energy-based methods, multiple-sequence methods, traditional machine learning methods, deep learning methods, and tertiary structure-based RNA secondary structure prediction methods and compared the advantages and disadvantages of various algorithms. Then, this article discussed the applications of related techniques in biomedical fields, with a particular focus on the identification of RNA-binding protein sites. Finally, it provided an outlook on the future development of RNA secondary structure prediction methods. This review is expected to provide important references for relevant research.

Peanut (Arachis hypogaea L.) is an important oilseed crop widely cultivated in tropical and subtropical regions. The growth-regulating factors (GRFs) are key transcription factors that regulate plant growth and responses to stress. To improve the peanut yield and stress tolerance, it is crucial to investigate the roles of GRFs in growth, development, and stress responses. In this study, we analyzed the physicochemical properties, evolutionary relationships, chromosomal localization, and sequence variations of the AhGRF gene family by bioinformatics methods. Using qRT-PCR, we revealed the expression patterns of AhGRF genes under drought and cold stress conditions. Subcellular localization expression vectors were constructed to determine the cellular distribution of AhGRF2b and AhGRF3b. Finally, yeast two-hybrid (Y2H) assays were performed to identify interacting proteins of AhGRF3b. The results revealed that twenty-four AhGRF genes were identified in peanut, which were unevenly distributed across 16 chromosomes. The deduced proteins ranged from 268 to 630 aa in length, with molecular weights spanning 29 842.27 to 67 980.83 Da. Most AhGRFs were acidic and predicted to be localized in the nucleus. Phylogenetic analysis classified the AhGRF family members into six distinct clades. Multiple sequence alignment demonstrated that the majority of AhGRF genes contained conserved QLQ and WRC domains. Under drought and cold stress conditions, several AhGRF genes, particularly AhGRF2b and AhGRF3b, exhibited significantly upregulated expression, which indicated their responsiveness to abiotic stresses. Transient expression in tobacco showed that AhGRF2b was localized in both the nucleus and cytoplasm, while AhGRF3b was localized in the nucleus. Furthermore, Y2H assays revealed that AhGRF3b may interact with AhCAT3 (catalase), suggesting that AhGRF genes may enhance stress tolerance by regulating reactive oxygen species scavenging. These findings provide a theoretical basis for improving the stress tolerance in peanut breeding programs.

This study aimed to explore novel β-glucosidases with unique environmental adaptability and investigate their potential application in hydrolyzing ginsenoside Rb1. A GH3 family β-glucosidase gene TsBgl3 was successfully cloned from the marine-derived intestinal bacterium Tamlana sp. I1, and a recombinant enzyme with good solubility was obtained through an optimized Escherichia coli heterologous expression system. It was identified that the molecular weight of the recombinant enzyme TsBgl3 was 80.8 kDa, and the optimal reaction conditions were pH 6.0 and 37 ℃. This enzyme exhibited remarkable low-temperature catalytic properties and maintained a relative activity of 16.56% at 0 ℃. Kinetic analysis indicated that TsBgl3 exhibited high substrate affinity and catalytic efficiency for the substrate 4-nitrophenyl-beta-D-glucopyranoside (pNPG), with the K_m, V_max, and k_cat/K_m values of 3.65 mmol/L, 578.04 μmol/(mg·min), and 213.01 L/(mmol·s), respectively. It is worth noting that TsBgl3 exhibited excellent salt tolerance, with its enzymatic activity increasing by 57.47% in a 2 mol/L NaCl solution. In addition, the saponin hydrolysis experiment demonstrated that TsBgl3 could specifically hydrolyze the β-(1, 6)-glucosidic bond at the C-20 position in the ginsenoside Rb1 molecule, showing high specificity. Moreover, the substrate could be completely converted to ginsenoside Rd within 11 h (HPLC detected conversion rate > 99%). In conclusion, we successfully obtained a novel β-glucosidase, TsBgl3, which possessed both cold adaptability and high salt tolerance. This enzyme not only provides an efficient biocatalyst for the green preparation of rare ginsenosides but also offers a new path for the development and utilization of marine microbial resources.