Unlike a single genome, a pan-genome generally refers to a data set that contains all genomic information in a species or population. Over the past decade, pan-genomics has gradually become a research hotspot in rice, and relevant pan-genomes and tools have been widely applied in many downstream research fields such as population genetics, evolutionary biology and biological breeding practices. This paper focuses on the development process and application prospect of rice pan-genomics, reviews the development of connotation of rice pan-genomics and the timeline of its research results, summarizes the existing representative and important achievement tools of rice pan-genomics and their main applications in different fields, and looks forward to the challenges and development prospects for rice pan-genomics.

Potatoes are the most important non-grain food crop in the world. Hybrid potato breeding, that is, transforming potato from tetraploid asexual crop to diploid seed crop, has become a research hotspot in potato field. In the past decade, the rapid development of potato genomics with diploid as the main research object has further promoted the change of potato breeding technology. This paper systematically reviews the development process of potato genomics, and how genomics research can help solve the two difficult issues of hybrid potato breeding, self-incompatibility and inbreeding depression, and promote the construction of potato genome design breeding system, so that potato breeding will move towards the 4.0 era.

Small RNA（sRNA）-mediated RNA silencing（or RNA interference）is a conserved mechanism for regulating gene expression in eukaryotes. Endogenous or exogenous double-stranded RNAs（dsRNAs）are processed into sRNAs, which recognize complementary mRNAs or DNAs in a sequence-specific manner, and regulate gene expression at either the posttranscriptional level or the transcriptional level by degrading mRNAs, inhibiting translation and DNA methylation. Owing to the target-specific actions of dsRNAs, RNAi-based strategies have become ubiquitous tools in the study of gene function, the development of RNA drugs, the design of molecular breeding methods, and the exploitation of biopesticides. The discovery of the bidirectional transfer among species and function of sRNAs provides a theoretical basis for exploiting RNAi-based technologies. Previous studies have shown that several factors affect the efficiency of dsRNA-induced RNAi, such as the size and dose of dsRNAs, as well as the application method. The complex structure of RNA leads to its functional diversity in vivo. Here, we described the principles of RNAi-based strategies for crop protection, including host-induced gene silencing（HIGS）, spray-induced gene silencing（SIGS）and microbe-induced gene silencing（MIGS）. We summarized the experimental evidence that the structures of target mRNAs and sRNAs affect RNAi efficiency, aiming to deepen the understanding of the RNA structure affecting RNAi efficiency, to provide experience for target screening and dsRNA design, and provide reference for the development of efficient RNAi technology. We also reviewed the representative methods and computational tools for detecting and predicting RNA structures, thereby providing approaches in establishing efficient RNAi-based techniques.

Wheat（Triticum aestivum, AABBDD）is one of the most important food crops in the world, and is also the second staple food crop in China. The wide adaptability and ecological diversity of wheat varieties is partly determined by their flowering time. Furthermore, flowering time is an important agronomic trait that partly determines the yield and quality of wheat grain. Prolonged cold exposure in winter（winter cold）and increasing daylengths in spring and early summer are two key seasonal factors that ensure winter wheat flowering and bearing fruit at the proper time. The vernalization and photoperiodic pathways have been evolved in winter wheat to integrate the signals of winter cold and daylength changes with developmental state, leading to the floral transition at a proper season for seed production. In this review, we summarize molecular epigenetic progresses on wheat flowering-time regulation, discuss unsolved issues as well as prospects future directions in this field.

Mitochondria, the semi-autonomous organelles within eukaryotic cells, harbor specific genomes（mtDNA）and play pivotal roles in cellular life processes. mtDNA mutations in human are linked to a spectrum of genetic disorders, while in plants, recombination in mtDNA often leads to ORF genes associated with male sterility. The advent of gene editing technologies has revolutionized the study and treatment of mitochondrial diseases. With a plethora of mtDNA editing tools available, these technologies have also been instrumental in exploring functional genes and unknown sequences within plant mitochondrial genomes. Compared to nuclear genome editing, mtDNA editing still encounters certain challenges. This paper provides an overview of the development and current state of mtDNA editing technologies, examines their application in plant research, and contemplates future optimization strategies and the potential impact of these technologies on plant mitochondrial studies.

To meet the growing demand for food from human being, the study on editing important gene-coding regions to produce beneficial traits is countless. Transcriptional regulation is crucial for controlling gene expression, including the control of important agronomic traits in crops. The interaction between cis-acting elements and trans-acting factors determines the spatiotemporal expression patterns and levels of genes, and transcription factor binding sites and enhancers etc. play an important role in those pathways. What's more, the promoter, as the largest and most important cis-acting element, can be modified to alter the expression patterns of target genes, and create beneficial traits in crops, which is an effective strategy different from coding region editing breeding. There are two applications of promoter editing strategies. One is directed modification to generate specific beneficial traits, and the other is to randomly generate new alleles within specific promoter regions, followed by phenotypic selection, also producing new genetic variation resources. In this review, we mainly discuss the application of promoter editing in crops, including increasing yield, improving quality, and enhancing tolerance to biotic and abiotic stresses. It aims to focus on the cutting-edge precision breeding of crops, providing research ideas and theoretical references for the development and application of promoter gene editing technology.

Plant glutamate-like receptors（GLRs）are homologous to the inotropic glutamate receptors（iGluRs）in animals. Flowering plants often possess multiple GLR members that redundantly regulate plant growth and development, as well as responses to environmental stimuli. Plant GLRs share common action mechanisms as their animal homologues, but also display plant-specific features. In mammalian cells, iGluRs play vital roles in the central nervous system. During the process of neurotransmission mediated by iGluRs, presynapse-released neurotransmitters recognize and bind to postsynaptic iGluRs, leading to cation fluxes across the postsynaptic membrane and eventually depolarized the membrane. This depolarization is also known as action potential, the formation of which is fundamental to neurotransmission. During the past twenty years, plant GLRs have also been shown functioning as ion channels that mediate ion fluxes across various membranes. In this review, we comprehensively summarized the protein structural and evolutionary features of plant GLRs and discussed the residual sites that were reported functionally important for GLRs. Following this, we reviewed the latest research progress on GLR roles during different stages of plant growth and development, as well as during the responses to various biotic and abiotic stresses. Similarities and differences in the action mechanism of GLRs were compared to their animal homologues. We then pointed out what remains important to be investigated in the field. Finally, we prospected the important applicable potential and values of this protein family, aiming at providing clues for designing stress resilient crops.

Research on secondary metabolites is of great significance for plant growth and development, environmental adaptation, as well as human health and drug development. Liquid chromatography-mass spectrometry（LC-MS）has become the preferred strategy for secondary metabolism research. However, the annotation of metabolite structures is still hindered by the insufficient coverage of standard spectral libraries. Given that the coverage of metabolite structure databases far exceeds that of standard spectral libraries, establishing the association between metabolite structures and mass spectra through artificial intelligence methods to search molecular structure databases based on mass spectrometry data is an effective approach to address this issue. This paper reviews three strategies for establishing the association between metabolite structures and mass spectra using deep learning techniques and bioinformatics methods, including structure-to-spectrum, spectrum-to-structure, and known-to-unknown strategies. It also introduces the rationale and representative methods for each strategy. For each strategy, the paper discusses the advantages and limitations of its algorithms, as well as the challenges that may be encountered in practical applications. Additionally, the paper explores that factors should be considered when developing new algorithms and conducting benchmark tests, and how these factors may affect the evaluation of algorithms. Finally, the paper points out that integrating more orthogonal information is a future direction for achieving more accurate metabolite annotation.

Agricultural environmental pollution has posed a great challenge to crop production in China, and it is urgent to take effective control of it. Microbe-based environmental remediation technologies, without secondary pollution, are characterized by high efficiency and low operation cost, which has attracted increasing interest. Moreover, the combination of functional microbe and biochar can further improve the efficiency in eliminating environmental pollutants, and positive results have been achieved. In this context, we first summarized the role of functional microorganisms in the remediation of environmental pollutants and the current application status of biochar-loaded microorganisms in the environmental remediation. We then discussed the advantages and challenges of biochar-loaded microorganism technology in the remediation efficiency and resource utilization. Based on the development of current technology, we proposed the direction of further research on the biochar-loaded microorganism remediation technology, providing a basis for in-depth understanding of the effectiveness and safety of relevant techniques.

Microbiomes are crucial resources in life science, playing vital roles in advancing scientific knowledge, promoting human health, and improving environmental quality. The rapid evolution of second- and third-generation metagenomic technologies has greatly enhanced our understanding of microbial world. With the proliferation of microbiome data, the need to select appropriate analytical methods for efficiently extracting information has become increasingly essential. Here, we provide a comprehensive review of recent progress in microbiome studies, focusing on updated analytical tools for short-read second-generation sequencing data, including amplicon, culture-based methods, and metagenomic data. In addition, we offer the processing strategies for long-read third-generation sequencing data. We highlight the necessity for standardized data analysis workflows and present case studies that demonstrate the application of these methods in exploring microbiome interactions, particularly in plant and root-associated microbial systems. We discuss the strengths and limitations of various methods in analyzing microbiome composition, structure, and functionality, highlighting the potential of metagenomic data mining for practical applications. Finally, we address current limitations and challenges in microbiome study, and we discuss future trends toward the standardization and streamlining of microbiome study methodologies to accelerate progress in understanding microbiome functions and applications.

RNA modification is an important type of epigenetic modification, which refers to the addition of chemical modifying groups to the bases or ribose of RNA molecules, and can occur in a variety of RNAs, such as messenger RNAs（mRNAs）, transfer RNAs（tRNAs）, ribosomal RNAs（rRNAs）, and cyclic RNAs（circRNAs）, etc. RNA methylation and acetylation are two of the key types of modifications that perform important biological functions by regulating the genetic information of organisms. Both modifications are dynamically and reversibly regulated by three types of regulators, namely writers, erasers and readers, which have a crucial impact on different RNA metabolic processes such as RNA splicing, translocation, translation, transport and degradation. In recent years, with the development of RNA modification detection technologies, numerous studies have identified new RNA modification sites in plants and confirmed that methylation and acetylation modifications of RNAs play key roles in plant growth and development as well as in response to biotic and abiotic stresses. This paper firstly outlines the types of RNA modifications and their biological properties, and on this basis focuses on reviewing the research progress on RNA methylation and acetylation regulators in recent years, and finally summarizes the functions of RNA methylation and acetylation in plant growth and development and in response to stresses, and looks forward to some new research directions for RNA modifications in plants, with a view to providing theoretical basis and new ideas for further research related to RNA methylation and acetylation modification in plants.

Soil salinization is one of a major environmental factor constraining global agricultural development. Breeding saline-alkali tolerant crops is a fundamental strategy to address soil salinization. However, currently there are challenges such as limited saline-alkali-tolerant genes and germplasm resources. Exploring new genes for saline-alkali tolerance and elucidating the molecular mechanisms of plant responses to saline-alkali stress are crucial for developing saline-alkali-tolerant crops. Saline stress includes neutral salt stress and alkaline salt stress. Neutral salt stress induces osmotic stress, and excessive accumulation of Na⁺ and Cl^- may lead to ion toxicity, causing oxidative stress and a series of secondary stresses. Compared to neutral salt stress, alkaline salt stress also induces high pH stress. This review outlines the impact of saline-alkali stress on plant growth and development, as well as the molecular mechanisms underlying plant adaptation to saline-alkali stress and summarizes the significant research progress in plant saline-alkali stress responses over the past decade. It covers plant perception and signal transduction of saline-alkali stress and the molecular mechanisms of plant responses to osmotic stress, ion toxicity, oxidative stress, bicarbonate and carbonate stress, and high pH stress caused by neutral and alkaline salt stress. On this basis, the application of saline-alkali-tolerant genes in crop breeding is also discussed, and key scientific issues that require further research to improve plant saline-alkali tolerance are proposed. The aim of this work is to deepen the understanding of the molecular mechanisms of plant adaptation to saline-alkali stress, providing a theoretical basis for breeding high-yielding and saline-alkali-tolerant crops, and improving the development and utilization rate of salt-affected land.

Post-translational modifications（PTMs）of proteins refer to a series of chemical modifications that occur after protein synthesis, significantly affecting the structure, localization, stability, and function of proteins. In cell biology, PTMs play a crucial role in nearly all cellular signaling pathways and regulatory networks, particularly in vesicle trafficking, a critical process for intracellular material transport. Vesicle trafficking involves multiple steps, including vesicle formation, transport, and fusion. In this process, the correct modification and localization of proteins are essential for vesicle formation and transport direction. We first introduce the roles and classifications of several important protein PTMs. Subsequently, we systematically summarize the research progress of protein PTMs in vesicle trafficking, providing important references for revealing the molecular mechanisms of intracellular vesicle trafficking and deepening the study of membrane protein biological functions. In the future, we should further explore the specific mechanisms of protein PTMs in plant vesicle trafficking, and how these mechanisms affect plant growth, development, and environmental adaptability. This study will provide new strategies and methods for plant genetic improvement and stress-resistant breeding, is conducive to increasing crop yield and quality, and enhancing plants’ adaptability to environmental changes. In summary, through in-depth study in this field, it not only helps us reveal the basic principles of specific protein modifications in plant cells but also may provide new strategies for improving crop traits and enhancing plant stress resistance.

Grafting technology is one of the most cost-effective means to increase yield, improve quality and enhance stress tolerance in horticultural crops, and its application is still expanding. In addition to the requirement of continuous improvement of grafting technology, the mechanisms of graft affinity and scion-rootstock interactions need to be further explored. Graft survival includes a series of physiological and biochemical processes, and graft activation, hormonal pathways, or genes involved in vascular bundle formation may be involved in the regeneration and reconstruction of grafted tissues, and it is important to clarify their regulatory mechanisms. Grafting is a process of scion-rootstock mutual recognition and interaction, and long-distance signaling between rootstocks and scions is the basis for understanding the physiology of grafting. Grafted plants with scion-rootstock grafting combinations of different genotypes behave differently in terms of growth and development, phenotype, yield and stress resistance. In this paper, we review the research progress on the molecular mechanisms of scion-rootstock interactions in vegetable grafting, such as grafting survival, genetic signaling between rootstocks, grafting and epigenetic changes, and grafting and gene expression, with a view to providing theoretical references for in-depth studies on the molecular mechanisms of vegetable grafting, and providing guidance for rootstock selection and innovation of vegetable germplasm materials.

Synthetic biology, an emerging interdisciplinary field, involves molecular biology, bioengineering, microbiology, and system biology, aiming to create entirely new biological systems and products using principles of biology and engineering methods. The conceptualization of the “cell factory” has propelled synthetic biology towards industrial applications, allowing the bioengineering technology having a big step towards industrial application. However, challenges such as low production efficiency, genetic instability, and intricate regulatory processes persist, hindering the creation of highly efficient and robust “cell factories” for transformation. In recent years, the fields of cell engineering and genetic engineering have developed rapidly, with the maturation of technologies such as new cell elements, cell chassis, and gene circuit construction methods. Through precise gene editing and regulation, these technologies can enable the programming of specific functions in cells, such as enhancing cell metabolism, altering cell differentiation pathways, and designing new cell functional modules, with broad application prospects.This review provides a comprehensive overview of rapidly evolving cell programming technologies. These technologies, situated within the realms of cell engineering and genetic engineering, encompass novel cellular components, cellular chassis concepts, and gene circuit construction methods. The strategic integration of these advancements aims to address the existing challenges in synthetic biology. The utilization of these technologies is poised to empower engineered bacteria with enhanced working capacities, thus paving the way for the development of more efficient and resilient “cell factories.”

【Objective】 mitoTALENs plant mitochondrial gene editing technology can effectively achieve the knockout of mitochondrial genes, and then effectively conduct the study of mitochondrial gene function. However, the vector construction process of mitoTALENs is very complicated and there is still no relatively systematic and complete vector construction method as a reference. To solve this issue, we described in detail the complete process of mitoTALENs vector construction in combination with the methods published by predecessors and explored by ourselves, providing an important reference for researchers who use mitoTALENs technology to study plant mitochondrial gene function. 【Method】 With rice mitochondrial WA352 gene as the target gene, a target TAL was designed using its sequence-specific region. Firstly, the TALEN-left and TALEN-right vectors of mitoTALENs were constructed using the two-step assembly technology of Platinum gate TALEN assembly, respectively. Then, multisite LR reaction was used to react TALEN-left, TALEN-right, the entry vector containing other functional elements, and the destination vector to generate the final expression vector.【Result】 Ten vectors were assembled in the first step, two vectors were assembled in the second step, and one final expression vector was constructed by multisite LR reaction.【Conclusion】 The detailed introduction of mitoTALENs vector construction process provides an important reference for users and promotes the development of plant mitochondrial gene editing research.

【Objective】 To develope R-loop targeted editing technology for mammalian cells and explore its applications in drug resistance of tumor cells. 【Method】 An expression vector was constructed to express a dCas9-RNaseH1 chimaera that combines catalytically dead Cas9 lacking endonuclease activity with RNase H1 possessing R-loop hydrolysis activity for R-loop targeted editing. This dCas9-RNaseH1 chimaera was transfected to HeLa cells and stably expressed to construct a model cell line. To create a cell library for R-loop screening, a genome-wide gRNA library covering transcription start sites was transfected to the model cell line, through which R-loop functional sites affecting resistance to paclitaxel and cisplatin were identified. 【Result】 Total 744 R-loop functional sites affecting HeLa cell drug resistance were identified, which cover key biological pathways such as cell cycle, apoptosis, and signal transduction. Among them, 26 sites confered resistance to two drugs, while 8 sites rendered sensitivity to both drugs, suggesting potential shared biological pathways. Functional validation revealed that certain R-loop sites modulated the expressions of relevant genes（e.g., ZBTB20, SPON2, ACTRT1）, significantly impacting HeLa cell sensitivity to anticancer drugs. 【Conclusion】 A R-loop targeted editing system is successfully developed and a high-throughput screening platform is established for mammalian cells.

【Objective】 The emergence of high-fidelity editors in recent years is expected to widely reduce the off-target effect that occurs during gene editing in poultry with the conventional CRISPR/Cas9 system. However, there is still a lack of data support for the application of these high-fidelity editors in poultry. This study aims to further evaluate their on-target editing effects and off-target characteristics in poultry cells, so as to provide an effective reference for molecular genetic breeding and germplasm resource utilization in poultry.【Method】 Five high-fidelity Cas9 variants, which have been reported with high accuracy, were selected and co-transfected with the designed sgRNAs in chicken DF-1 cells, and the positive cells were collected for gene sequencing, and the editing efficiency and off-target effect of different high-fidelity editors were compared in parallel.【Result】 eCas9 maintained high editing activity while demonstrating low off-target effects in multiple sgRNAs tested against the avian influenza resistance-related gene ANP32B and the reproduction-related gene DAZL. Meanwhile, SuperFiCas9 had the lowest off-target effect among multiple tested high-fidelity variants, but a significant decrease in editing efficiency occurred in gene editing of poultry DF-1 cells.【Conclusion】 A variety of high-fidelity editors have shown editing activity in poultry cells, among which the eCas9 editor has high efficiency and low off-target properties, and can be used as a preferred high-fidelity editing tool for the genetic improvement of economically important traits and molecular breeding work in chickens.

As an important renewable resource, lignin is mainly deposited in the secondary cell wall of vascular plants and plays an important role in physiological processes such as nutrient transport, mechanical support, and plant pathogen defense. The formation of lignin can be divided into three processes: Intracellular synthesis, transmembrane transport and extracellular polymerization of monolignols. Exploring the transmembrane transport mechanism of monolignols is of great significance for revealing the mechanism of cell wall formation and wood improvement.. In this review, we sorted and summarized the relevant progress and latest technologies of monolignols transmembrane transport from the perspectives of transcriptomics, genetic engineering, click chemistry, fluorescence microscopy, and molecular simulation. Then we described the new technologies and existing technical bottlenecks in the research on monolignol transmembrane transport. In addition, we compared and analyzed the advantages and disadvantages of different technologies. Finally, we proposed the issues and challenges in the study of the mechanism of monolignols transmembrane transport. It is believed that the rapid development of imaging technology, structural biology, artificial intelligence and other technologies will provide new tools for further investigating the transmembrane transport mechanism of monolignols. This review is expected to provide new methods for the study of monolignols.

Bacillus is one of the most widely used biocontrol microbial resources in the world. In the era when genomics has become a universal basic discipline, the combined application of transcriptomics, proteomics, metabonomics and other multi-omics technologies have become an important means to deeply reveal the biological characteristics and biocontrol mechanisms of Bacillus. Currently, the NCBI database has published a total of 10 813 genome sequences of Bacillus, of which 1 842 have complete assembly annotations, accounting for 17.04% of the total number. The secondary metabolite gene clusters are the most conserved and specific part of the Bacillus genome. They are not only an important source of natural products, but also play important roles in the interactions and life cycle between bacteria and the environment. Up to now, research on Bacillus has mostly adopted integrated omics models and bioinformatics calculation methods, focusing on the mechanisms of Bacillus resistance formation, biofilm formation, colonization, growth promotion, and induction of plant stress resistance. With the continuous deepening of research, the gene regulatory mechanisms, protein expression, and metabolic pathways in the biocontrol process of Bacillus will be further elucidated, which will help discover new biocontrol active substances and optimize biocontrol strategies. This article reviews the research progress of multi-omics techniques such as genomics, transcriptomics, proteomics, and metabonomics on Bacillus. It aims to provide reference for the analysis of the biocontrol mechanism of Bacillus and the in-depth study of biocontrol strain improvement.

Environmental biosafety is closely related to social stability, human health, and even national defense security. Due to increasing pressure from climate change and anthropogenic activity, there is a risk of pathogenic ‘spillover’ and ‘spillback’. Current technologies are primarily designed for known pathogens. The rapid development and application of metagenomics-based bioinformatics may provide new opportunities and solutions for identifying unknown environmental pathogens and early warning of potential environmental health risks. The development of these technologies has not only promoted an understanding of the interactions between microbes and animals, humans, and the environment, but also is an important part of the concept of the One Health framework. This review briefly discusses the pathogenic ‘spillover’ and the increasing environmental health risks under global change. The focus is on bioinformatics technology in environmental biosafety study, covering data storage, processing, and mining. Finally, the review provide perspectives on big data-driven pathogen environmental risk assessment and new methods for pathogen control in the metagenomics era.

【Objective】 The objective of this study is to develop CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice and to expand the CRISPR/Cas gene-editing toolkit. 【Method】 The codon of LanCas9 from Lactobacillus subtilis KCTC 3501 was optimized, and further a chimeric SLanCas9 by fusing the active domains of LanCas9 and SpCas9 was generated. Thus CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice were constructed respectively. Using OsWRKY45, OsCPK4, OsCPK6, OsCPK7, OsMPK8, OsGSK3, and OsGSK4 as target genes, the 20 nt or 24 nt sgRNAs in NGG PAM or NAG PAM were designed, and their editing efficiency was analyzed through rice genetic transformation.【Result】 The LanCas9 efficiently identified NGG PAM and efficiency was higher when combined with a 20-nt sgRNA editor, resulting in an editing efficiency of 25.00% for the OsWRKY45 gene. Furthermore, the fused SLanCas9 not only showed the recognition of the NGG PAM and also demonstrated a certain level of editing efficiency at the NAG PAM site, achieving editing efficiencies of 100% and 39.58% respectively for different PAM sites. Additionally, SLanCas9 demonstrated multiple-gene editing capabilities with an impressive efficiency of up to 74.07% at NGG PAM sites. 【Conclusion】 This study has successfully developed novel CRISPR/LanCas9 and CRISPR/SLanCas9 gene editing technologies with independent intellectual property rights.

【Objective】 Soybean GmERD15c is one of the members of the ERD15 transcription factor family, and the gene expression and substance metabolism of soybean lines transgenic with GmERD15c gene under salt stress were explored, and the relationship between GmERD15c and soybean's tolerance to salt was revealed.【Method】 Using the transgenic soybean line and its recipient soybean ‘Dongnong 50'as the materials, and the roots after the salt stress treatment were to have analysis of transcriptomics and metabolomics.【Result】 There were significant changes in the metabolites of flavonoid synthesis pathway before and after salt stress, with significant variations in the content of dihydroquercetin, catechin, and fisetin after stress. And the content of quercetin was higher in the transgenic line of GmERD15c. Meanwhile, the combined analysis of transcriptomics and metabolomics revealed that the GmERD15c gene was closely related to the flavonoid biosynthesis-related enzymes.【Conclusion】 It is hypothesized that the soybean GmERD15c gene may affect the biosynthesis of flavonoids by regulating the enzymes related to flavonoid synthesis under salt stress, and flavonoids may reduce the oxidative stress caused by salt, which provides new clues for subsequently revealing the salt tolerance mechanism of the GmERD15c gene.

【Objective】 Quinoa, known as the “golden grain” for its rich beneficial secondary metabolites and nutritional components for human benefit, is explored in this study through the analysis of a color mutant isolated from quinoa natural population. By examining the metabolic composition of this mutant, analyzing the changes and transformation routes between metabolic pathways, and constructing a basic model of metabolic changes in the mutant, this study is aimed to provide a material basis for further identification and cloning of key genetic loci affecting important metabolic pathways in quinoa.【Method】 Using forward genetics, a natural mutant exhibiting reduced red pigmentation and intensified green coloration（green quinoa 1, gq1）was screened from the progeny of the quinoa cultivar, ‘Red Quinoa 1’（RQ1）. This mutant was compared with its original parent, RQ1, to identify differential metabolic components in the young panicle of grain filling stage using untargeted metabolomics. KEGG metabolic pathway analysis and correlation analysis of differential metabolites were employed to reveal the overall differences in metabolic pathways and some key nutritional components in the mutant gq1.【Result】 Genetic analysis conducted over four successive generations indicates that the color variation in the gq1 mutant quinoa plants was stably inherited and was controlled by a single genetic locus. Compared to its original parent RQ1, 409 differential metabolites were detected in the quinoa gq1 mutant, with the concentration of 110 metabolites increased and that of 299 metabolites decreased. Metabolomics analysis revealed a comprehensive decrease in tyrosine and its derivative metabolites in the quinoa gq1 mutant, which are crucial for plant secondary metabolism. Moreover, a significant reduction was observed in various amino acids, including six essential amino acids for humans, and components of the TCA cycle in the gq1 mutant. 【Conclusion】 The enrichment analysis of these differential metabolites through KEGG metabolic pathways indicates that the gq1 gene mutation leads to a general reduction in both primary and secondary metabolic components centered around tyrosine. This suggests that the gene could serve as a key genetic locus for the synergistic optimization of primary and secondary metabolism in quinoa.

【Objective】 To investigate the variation rule of carotenoids in the leaves of Anoectochilus roxburghii at different growth stages, and to analyze the molecular mechanism of carotenoid accumulation.【Method】 The carotenoids contents in the leaves of A. roxburghii at different growth stages（S3, in three months old; S6, in six months old; and S10, in ten months old）were investigated by liquid chromatograph mass spectrometer（LC-MS）and high-throughput transcriptome sequencing（RNA-seq）.【Result】 Combining the transcriptome and metabolome to analyze the leaves of A. roxburghii at different growth periods, the metabolomics results showed that there were 23 differential metabolites involved in carotenoid synthesis in the leaves, including neoxanthin, graphemexanthin, luteolin, and caroxanthin, etc., and the transcriptome results showed that there were 21 differential metabolism enzyme genes, including CYP97C1, CCD8, NCED, ZEP, and PDS, etc., which predicted that 14 transcription factors, including ERF, MYB, C2H2, WRKY and bZIP, were involved in regulating the differential carotenoid synthesis and accumulation in A. roxburghii leaves. and real-time quantitative PCR（RT-qPCR）experiments were performed to show that the expression trends of eight selected enzyme genes involved in the synthesis of carotenoids in the different phases of the process were consistent with the results of the sequencing of transcriptomics.【Conclusion】 Five transcription factors, including ERF, MYB, and C2H2, were involved in the regulation of carotenoid synthesis during different growth periods of Anoectochilus roxburghii, and were significantly associated with five key differential enzyme genes, including CYP97C1, CCD, and NCED, which together regulate six key differential metabolites, including Abscisic aldehyde, Zeaxanthin, and Lutein.

【Objective】 Due to the negative impact of long-term synthetic fertilizer application on the agricultural functions of rhizosphere microorganisms, we aim to develop an effective approach to enhance these functions in a wild-type Bacillus pumilus isolated from plant roots.【Method】 A novel CRISPR/Cas multi-gene editing system, UgRNA/Cas9, was used to disrupt the genome-scale cis-acting catabolite-responsive element（CRE）, aiming to alter the carbon source selectivity of B. pumilus. 【Result】 DNA sequencing results revealed that CRE-like sites in the partial genes of the carbon and secondary metabolic pathways underwent mutations including deletions, insertions, transitions, and transversions. Comparative metabolomic and transcriptomic analyses suggest a potential for the biosynthesis of pigments, surfactin, and bacilysin through the pentose phosphate and amino acid pathways. The introduction of UgRNA/Cas9-mediated edits into the CREs enhanced the ability of the strain to better adhere to the root, thereby promoting plant growth and strengthening resistance to pathogens. 【Conclusion】 Mutations in the genome-wide CRE sequence of LG3145 altered the direction of carbon metabolism flow in the bacterium, resulting in the establishment of a mutually beneficial relationship between the bacterium and plants.

【Objective】 A-to-I RNA editing is a co-transcriptional/post-transcriptional modification mediated by the ADAR enzyme family. The systematic mapping of the RNA editome in pigs provided candidate targets for molecular breeding in pigs. 【Method】 We collected 3 461 RNA-seq datasets from 16 types of pig tissues and detected RNA editing sites in pigs using bioinformatics methods.【Result】 A total of 130 989 RNA editing sites were detected, of which 124 208 were A-to-I RNA editing sites. Analysis of the characteristics of the A-to-I RNA editing sites revealed that 98.2% were located in repetitive regions, primarily within the PRE-1/Pre0_SS elements of pig-specific SINE retrotransposons. Only 12.3% of A-to-I RNA editing sites had coding potential. Finally, the study analyzed tissue-specific A-to-I RNA editing sites in seven tissues（adipose, brain, large intestine, small intestine, skeletal muscle, liver, and lung）. Functional enrichment analysis of the host genes in adipose tissue-specific sites showed that these genes were enriched in pathways related to cellular lipid metabolic processes, lipid metabolic processes, and thioester metabolic processes. Genes related to fat deposition within these pathways included PDK1, ACSL1 and PDE3B.【Conclusion】 This study comprehensively mapped the RNA editing sites in pigs, providing candidate targets for molecular breeding.

【Objective】 This work is aimed at establishing a library of endogenous expression elements of Streptococcus epizooticus，and investigating the application of it in increasing hyaluronic acid yields.【Method】 The 32 kinds of high, medium and low strength promoter and RBS combination（expression element, PR）were preliminarily screened, through transcriptomics analysis of S. zooepidemicus in the exponential or stationary stage of fermentation. Then, the strength of the 32 PRs was verified via detecting the transcriptional level of gfp gene and fluorescence intensity of GFP. Eventually, the key hyaluronic acid synthesis gene hasA, hasB, hasC, hasD, and hasE was overexpressed respectively with the selected strong expression element PR31, and the impact of the strong PR on enhancing hyaluronic acid production was assessed through a 2 L fermentation.【Result】 The relative transcription of the aforementioned genes were elevated to 8.17, 7.32, 3.72, 39.48 and 9-fold. Among them, the overexpression of hasA and hasD increased the hyaluronic acid yield by 43% and 31%, respectively, compared to the wild type, reaching 5.654 g/L and 5.283 g/L.【Conclusion】 A library of endogenous expression elements of S. zooepidemicus had been constructed successfully, which can be employed for metabolic engineering, such as reinforcing the hyaluronic acid synthesis pathway or weakening competitive pathways.

【Objective】 The rumen fungus Neocallimastix patriciarum GH11 family xylanase CDBFV has good application prospects in feed, food and related industries. Improving its thermal stability is crucial for optimizing its production and utilization. 【Method】 Potential thermostable mutants of CDBFV were designed using strategies such as molecular dynamics simulation and machine learning. And then heterologous expression and purification were carried out in Escherichia coli and Pichia pastoris. The optimal reaction conditions, specific enzyme activity and the relative residual activity after incubation at 85℃ for 3 min were determined, and the mechanisms for improving thermal stability were clarified through structural analysis. 【Result】 The ³⁶GNNS³⁹ motif at the N-terminus of CDBFV was highly flexible. Single mutant N37P and N38V created by modifying this motif showed the relative activites of 70.3% and 55.1% after incubation at 85℃ for 3 min, representing increases of 21.6% and 6.5% respectively compared to the wild-type（48.7%）. Based on the significant increase in relative activity observed in N37P, combined with the previously reported beneficial mutant N88G, the double mutant N37P/N88G was constructed. This double mutant presented a relative activity of 73.4%, and 24.7% improvement over the wild-type. In addition, when N37P/N88G was expressed in Pichia pastoris, it had a relative activity of 88.8% after treatment at 85℃ for 3 min. Structural analysis indicated that the N37P mutation introduced new hydrogen bonds in CDBFV, decreased the flexibility of the active site and disrupted glycosylation, thereby improving its thermal stability. 【Conclusion】 This study successfully generated the high-temperature-resistance double mutant N37P/N88G, offering new insights and approaches for improving the thermal stability of GH11 family xylanases. This advancement is anticipated to facilitate the extensive utilization of CDBFV in high-temperature settings like the feed industry.

【Objective】 A stable cell line expressing the MPXV（Mpox virus）surface proteins A35R or E8L was established and utilized for qualitative or quantitative analysis of the binding activities of antibodies or molecules associated with A35R or E8L on the cell surface.【Method】 Lentivirus vectors coding for A35R or E8L were constructed using gene recombination technology, and A35R and E8L were expressed and localized on the cell surface of engineered HEK-293T cell lines that were transduced by lentivirus carrying A35R or E8L. The expressions and localizations of A35R and E8L in the engineered cells were detected using Western Blot and immunofluorescence, and the binding affinity of the engineered cells to polyclonal antibodies was analyzed by flow cytometry. Finally, monoclonal cell lines were screened from the engineered cell lines and applied in the quantitative analysis of the binding activity of commercial anti-A35R or anti-E8L monoclonal antibodies.【Result】 Stable HEK-293T cell lines expressing A35R or E8L antigens on the surface were successfully established. Four monoclonal cell lines were selected through monoclonal screening, among which two stably expressed A35R（A35-2C10 and A35-4E3）and two stably expressed E8L（E8-6 and E8-8）. These monoclonal cell lines showed good fit in quantitative analysis of the binding activity of anti-A35R or E8L mAb.【Conclusion】 Cell lines with stable expressions of MPXV A35R or E8L may serve as tools and can be applied to assess the binding activities of neutralizing antibodies, nucleic acid aptamers, or other molecules associated with A35R or E8L in qualitative or quantitative analysis.