Table of Content

26 December 2025, Volume 41 Issue 12

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Recent Advances in Plant SUMO E3 Ligases

FANG Yu-jie, LIU Kuan, CUI Han-bing, WANG You-ping

2025, 41(12):  1-15.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0637

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As a crucial reversible post-translational modification form of proteins in eukaryotes, sumoylation participates in various life processes in plants by regulating the stability, subcellular localization, and activity of proteins. Sumoylation modification is mediated by a serial of enzymatic cascade reactions, among which the SUMO E3 ligases plays a decisive role in the selection of target proteins. Recent studies have revealed that SUMO E3 ligases play pivotal regulatory roles in growth, development, and stress adaptation of plants by mediating the sumoylation modification of multiple types of target proteins. This review systematically summarizes the structural characteristics, functional differentiation of SUMO E3 ligases in plants and their regulatory networks in various physiological processes. It focuses on their roles in the molecular mechanisms and signaling integration pathways involved in abiotic stress responses, developmental regulation, and immune response, aiming to provide a theoretical basis for elucidating the regulatory networks and biological functions of plant sumoylation modification, and offer potential targets for crop stress resistance improvement and high-yield breeding.

Advance on Seed Dormancy and Its Regulation Mechanism

DUAN Ruo-xin, CHEN Ying-ying, LIN Jin-xing, LI Rui-li

2025, 41(12):  16-26.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0467

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Seed dormancy is defined as the inability of seeds to germinate in favorable conditions, which can prevent pre-harvest germination of crops. Studying seed dormancy in forestry is beneficial to forest cultivation. Studies showed that the plant hormone abscisic acid (ABA) induces and maintains seed dormancy, while gibberellin (GA) breaks dormancy. The two hormones regulate seed dormancy and germination in an antagonistic manner. Although some studies have investigated the hormonal regulation of seed dormancy and germination, there is a lack of a systematic summary of the molecular mechanisms underlying seed dormancy. Therefore, this review provides an overview of the types of seed dormancy and the effects of light and temperature signals on seed dormancy. It focuses on the regulatory mechanisms of seed dormancy by ABA, GA, and the interactions between these hormones. Additionally, it summarizes the research on dormancy-specific regulatory factors (such as DOG1, DOG18, Sdr4) and epigenetic regulation mechanisms. The aim is to deepen the understanding of the complex regulatory network of seed dormancy and provide theoretical references for plant trait improvement and selective molecular breeding.

Cytochrome P450 Involved in Secondary Metabolites Biosynthesis in Response to Biotic Stresses

WANG Jia, GAO Ming, ZHAO Yun-xiao, CHEN Yi-cun, WANG Yang-dong

2025, 41(12):  27-39.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0510

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Plant cytochrome P450 (CYP450) enzymes constitute a critical class of heme-binding proteins, distinguished by the characteristic 450 nm absorption peak in their reduced carbon monoxide-bound form. As one of the largest single oxygenase superfamilies in plants, CYP450 enzymes are widely involved in various biological processes such as growth and development, secondary metabolism synthesis, and stress defense by catalyzing multiple oxidation reactions including hydroxylation, epoxidation, and dealkylation. They are thus hailed as “universal biological catalysts”. Under biotic stress conditions (such as pathogen invasion and pest feeding), plantactivate CYP450-mediated secondary metabolic pathways such as phenylpropanoids, terpenoids, fatty acids, and glucosinolates to synthesize compounds with pesticidal or antimicrobial properties, thereby enhancing the plant’s ability to resist pests and diseases. In recent years, with the development of high-throughput sequencing technology and molecular biology, multiple CYP450 enzymes associated with these biological processes have been isolated, screened, and functionally characterized in diverse plant species. In view of this, this review provides a comprehensive overview of the nomenclature, structural characteristics, classification and distribution of CYP450 enzymes in plants. Concurrently, it focuses on introducing the research progress of CYP450 family genes in secondary metabolite biosynthesis and biotic stress responses. It provides a reference for the mining and functional study of CYP450 family genes in plants, as well as provides a basis for the role of secondary metabolite biosynthesis and biotic stress response based on this.

Research Advances in Microbial Remediation of Soil Pesticide Residues

ZHU Shan-shan, XU Jun, PAN Xing-lu, DONG Feng-shou, ZHENG Yong-quan, WU Xiao-hu

2025, 41(12):  40-49.  doi:10.13560/j.cnki.biotech.bull.1985.2025-1011

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Pesticide residues pose a major challenge to soil ecological security and agricultural product safety. Microbial degradation has emerged as a promising strategy for the remediation to farmland pollution due to its high efficiency and environmental compatibility, demonstrating broad application prospects. This review systematically elucidates the study and application advances in pesticide-degrading microorganisms, covering the following key aspects: In terms of degrading strain screening, various strategies—from traditional enrichment culture to microfluidic single-cell screening—have significantly enhanced both the efficiency and diversity of obtaining efficient degraders. Regarding degradation mechanisms, this review provides an in-depth analysis of key degradation gene functions, catalytic mechanisms of intracellular and extracellular enzymes, and synergistic enhancement of co-metabolic pathways. Concerning ecological processes, this review focuses on the colonization mechanisms of degrading bacteria in the rhizosphere and phyllosphere through chemotaxis and biofilm formation, along with their subsequent functional responses. It further examines both active migration mediated by flagellar motility and passive migration facilitated by environmental factors including hydraulic transport. In application technologies, this review examines the preparation processes of both liquid and solid formulations along with their respective advantages and limitations, and assesses the challenges in practical implementation, such as environmental adaptability, colonization efficiency, and ecological interactions. Finally, the review emphasizes that future efforts should focus on formulation innovations and plant-microbe interactions to enhance field stability and degradation efficacy of inoculants, thereby facilitating large-scale application of this technology and promoting agricultural green development.

Research Advances in AI-driven Enzyme Modifying and Design

GUO Fa-xu, FENG Quan, ZHANG Jian-hua, ZHOU Huan-bin, YANG Sen, WANG Jian, ZHOU Guo-min

2025, 41(12):  50-65.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0627

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Enzymes play a crucial role in both biological systems and industrial applications. Due to their unique catalytic properties, they are among the key choices for catalytic processes. However, traditional enzyme engineering and design approaches face significant challenges, such as the vastness of sequence space and the complexities associated with multi-objective optimization. In recent years, artificial intelligence (AI) technologies, particularly deep learning and generative AI methods, have provided novel perspectives and solutions for enzyme modification and design, enabling breakthroughs in overcoming these limitations with large-scale data support. AI-driven strategies have facilitated efficient exploration of sequence space, accurate prediction of structure-function relationships, and the coordinated multi-objective optimization using reinforcement learning frameworks. These methods have not only significantly accelerated the enzyme engineering process but also led to groundbreaking advancements in the enhancement of catalytic efficiency, thermal stability, and substrate specificity. This review systematically summarizes the latest research on AI-driven enzyme modification and design, providing an in-depth analysis of foundational database construction, intelligent modification strategies, and design methodologies. Furthermore, it discusses current challenges related to data, models, and engineering applications, as well as future directions. These innovations open up vast possibilities for the design of high-performance, multifunctional enzymes and are poised to propel fields such as biomanufacturing, environmental remediation, and agricultural biotechnology toward more efficient, intelligent, and sustainable development.

Construction and Application Study of a General Vector pCamRUBY for Visual Screening of Transgenes

DOU Shuo, DING Ruo-xi, SUN Xing, GUO Wen-jing, KONG Wen-hui, YUAN Jing-xian, ZHANG Dong-mei, WANG Xing-fen, MA Zhi-ying, WU jin-hua, WU Li-zhu

2025, 41(12):  66-73.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1133

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Objective To construct and verify an universal vector for visual screening transgenic plants and improving the efficiency of plant genetic transformation. Method Having gene RUBY as the reporter gene, a universal expression vector pCamRUBY was constructed using 2A technology. Validation vectors pCamSpeRUBY and pCamKanRUBY were constructed using Spe and Kan resistance genes, respectively. Arabidopsis and cotton were used as the test materials for genetic transformation and pigment content identification. Result This vector has the characteristics of high visualization, small vector sequence, MCS sites composed of Sal I, Apa I, Xba I, Sac Iand Spe I, and T2A (optimized) sequences. The red positive transgenic Arabidopsis and cotton plats and tissues were visually screened with the naked eye respectively, and stable genetic inheritance was obtained. Conclusion The universal expression vector pCamRUBY can directly be linked to the normal exogenous genes, and the positive transgenic plants show red character. The transgenic plants can be directly observed and screened with naked eye, and not affect other phenotypic traits including chlorophyll content of positive plants.

Modifying the Probiotic Escherichia coli Nissle 1917 for the Biosynthesis of Indirubin via Metabolic Engineering

MAO Li-jing, JIN Xiao-xuan, SHI Wan-ting, HU Fei-yang, ZHANG Yuan-rong, XIONG Liang-bin, REN Lu

2025, 41(12):  74-81.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0718

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Objective Indirubin, a bi-indolic alkaloid derivative isolated from the traditional Chinese herbal medicine Indigo Naturalis, represents a potent therapeutic compound having broad-spectrum antibacterial, anti-inflammatory, and anti-tumor properties. Currently, it is clinically utilized primarily in combination therapy regimens for the treatment of chronic myeloid leukemia (CML), psoriasis, and related pathologies. This study investigates the potential of the safe Escherichia coli Nissle 1917 (EcN) as a chassis for constructing a green supply system for the active components of the traditional Chinese medicine indirubin. Method Based on the engineering modification of the natural endogenous plasmid of EcN, the product output efficiency of the indirubin biosynthesis pathway constructed using the flavin monooxygenase fmo and its mutants (fmo^K223R and fmo^K223R/D317S )) from Methylophaga sp. SK1 was systematically evaluated. Combined with metabolic engineering, key genes in the competitive branch of the tryptophan biosynthesis pathway were knocked out, including pheA, a key gene for phenylalanine biosynthesis, and tyrA, a key gene for tyrosine biosynthesis. Further optimization was achieved by inactivating the upstream genes of the tricarboxylic acid cycle, including pykA (pyruvate kinase) and ppc (phosphoenolpyruvate carboxylase). Finally, the product synthesis level was further improved by deleting the repressor protein gene trpR in the tryptophan pathway and strengthening the anti-feedback inhibition gene trpE^S40F in the tryptophan pathway. Result After 48 h of shaking culture in a 250 mL flask (50 mL culture), the indirubin titer reached (176.9 ± 4.5) mg/L, representing a 3.5-fold increase compared to the parent strain harboring only the wild-type fmo gene. The production in the 5 L bioreactor was (379.3 ± 12.3) mg/L. Conclusion Employing the endogenous cryptic plasmid of the probiotic EcN to express the heterologous flavin monooxygenase mutant gene fmo^K223R, and integrating this with systematic metabolic engineering of endogenous tryptophan biosynthesis pathways, the production of indirubin reached over 300 mg/L, demonstrating the excellent potential of EcN as a cell factory for biosynthesis of active ingredients in traditional Chinese medicines.

CRISPR-based High-throughput Screening Technology and Its Applications in Livestock Research

TIAN Yun, KONG Chen, YANG Chong, LIU Tong-gao, GAO Hong-rui, LI Jia-hui, MA Yun, CAI Bei

2025, 41(12):  82-94.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0500

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Animal husbandry is an important part of agriculture, and its economic value is closely related to the phenotypic traits of livestock and poultry (such as meat production, egg production rate, etc.). These traits are regulated by complex genetic networks and are affected by environmental factors, causing it difficult to mine functional genes that determine these traits. The emergence of genetic screening technology makes the discovery of genotype-phenotype relationship simple, but the early genetic screening technology has low throughput and is difficult to analyze the large amount of data generated, resulting in low efficiency. With the iterative update of biotechnology, genetic screening technology has entered the era of high-throughput screening, which provides an efficient means for analyzing genotype-phenotype relationship. Traditional high-throughput screening methods mainly include RNA interference (RNAi) and cDNA overexpression. However, RNAi is prone to have off-target and cannot completely inhibit gene expression. The construction of cDNA library is costly and may cause cytotoxicity. The development of CRISPR/Cas system provides a new strategy for genetic screening. Based on the adaptive immune mechanism of bacteria, the system targets DNA sequences by guide RNA and accurately edits genes by Cas protein, which has the advantages of simple operation, high specificity and wide application range. CRISPR high-throughput screening technology mainly includes multiple modes such as loss-of-function (CRISPRko), gain-of-function (CRISPRa/CRISPRi), and base editing, which can directly perturb at the DNA level and overcome the shortcomings of traditional methods. This paper briefly introduces the types and characteristics of genetic screening technology and its application in animal husbandry, and looks forward to the great potential of CRISPR high-throughput screening technology in animal husbandry breeding.

Establishment and Functional Validation of a Reverse Genetics System for the Chinese Influenza D Virus D/JY3002

HU Wan-ke, CHEN Yun-xia, LUO Di-zhou, WU Si-yu, LI Jian-bo, ZHAI Shao-lun, JU Xiang-hong, LIAO Ming, WEI Wen-kang, YU Jie-shi

2025, 41(12):  95-105.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0496

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Objective The current prevalent influenza D virus in Chinese cattle herds primarily belongs to the D/Yama2019 genetic lineage. The development of a reverse genetics system for the D/Yama2019 genetic lineage influenza D virus offers a crucial research tool for the in-depth investigation of its replication and pathogenic mechanisms. Method The Chinese influenza D virus strain D/bovine/CHN/JY3002/2022 (Abbreviated as D/JY3002), belonging to the D/Yama2019 genetic lineage, was utilized as the research material. The DNA fragments corresponding to the genomic segments that encode the primary antigen of the virus, Hemagglutinin-Esterase-Fusion (HEF), were successfully cloned into the engineered bidirectional expression vector pCC1-DualPro. The DNA fragments corresponding to the remaining genomic segments were seamlessly cloned into the widely utilized bidirectional expression vector pHW2000. Subsequently, the recombinant influenza D virus was successfully rescued through improved and optimized operational procedures. Result The rescued influenza D virus strain (rD/JY3002) demonstrated a replication ability comparable to that of the naturally isolated Chinese influenza D virus strain (D/JY3002), and the replication kinetics of the two viruses are similar. Moreover, the rD/JY3002 virus can be stably propagated for at least five generations. Using the established reverse genetics system, recombinant influenza D viruses containing the green fluorescence reporter gene (GFP) and the HEF genes from other genetic lineages (D/OK and D/660) were successfully generated. They were respectively designated as rD/JY3002-GFP, rD/JY3002-D/OK-HEF, and rD/JY3002-D/660-HEF. These results confirmed that reassortment occurred among influenza D viruses from different genetic lineages. Conclusion An efficient and stable reverse genetics system for the influenza D virus Chinese strain has been established. This system can be utilized to further advance exogenous gene presentation technology, employing the influenza D virus as a vector.

Boosting Rice Yield in Saline-alkali Lands Using Enterobacter hormaechei QT2： A Wild Rice Endophytic Bacterium

LI Jun-chen, ZHAO Xiang-yuan, WANG Fu-dong, WANG Yi-fan, ZHANG Jian-feng, WANG Shu-sheng

2025, 41(12):  106-113.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0654

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Objective Screening wild rice-derived endophytic bacteria with high potential for mitigating salinity-alkalinity stress, and evaluating the effects of Enterobacter hormaechei QT2 on the growth, physiology, and yield of cultivated rice under such stress conditions. This study aims to provide a theoretical basis for stabilizing rice yields and enhancing efficiency in saline-alkali lands, as well as for developing stress-resistant microbial agents. Method The endophytic bacterium E.hormaechei QT2 was isolated from the leaves of wild rice, and its growth-promoting characteristics and yield-enhancing effects were tested in a pot experiment (middle-heavy saline-alkaline soil) and a field validation (pH 9.3, conductivity 1 262 μS/cm). Result Under saline-alkali stress, inoculation of QT2 significantly improved the growth phenotype of cultivated rice (P<0.05), demonstrated the increase of plant height, root length, fresh weight and dry weight increased by 101.0%, 46.1%, 137.4% and 447.0%, respectively. Meanwhile, the osmotically regulated substances were significantly accumulated in the leaves of cultivated rice, and the contents of soluble sugars, soluble proteins and proline increased by 135.7%, 94.0% and 205.2%, respectively. The content of malondialdehyde (MDA) reduced by 59.6%, and the membrane stability index (MSI) increased by 42.3%. The activity of antioxidant system in the leaves of the cultivated rice was significantly enhanced, and the activities of superoxide dismutase (SOD), peroxidase (POD) and the content of glutathione (GSH) increased by 57.5%, 73.3% and 39.4% respectively. The photosynthesis of cultivated rice leaves was enhanced, and the contents of chlorophyll a, chlorophyll b and carotenoids increased by 30.69%, 78.57% and 75.62%, respectively. In addition,strain QT2 also showed a variety of plant growth-promoting properties (nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA production, ACC deaminase production and ammonia production). Field experiments further confirmed that inoculation with QT2 increased the yield of saline cultivated rice by 29.5%, and the input-output ratio of strain QT2 reached 1:46. Conclusion The endophytic bacterium E.hormaechei QT2 effectively alleviates the inhibitory effects of saline-alkali stress on cultivated rice by synergistically regulating osmotic balance, enhancing antioxidant defense, and improving photosynthetic efficiency, thereby significantly promoting growth and increasing yield.

Cloning and Low-phosphorus Tolerance Analysis of SiSPX9 Gene in Foxtail Millet

GUO Hao-jie, WANG Cheng, YANG Fu-rong, DU Bing, MENG Chao-min

2025, 41(12):  114-123.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0404

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Objective SPX gene family plays an important role in signal perception, absorption and transport of phosphorus in plants. To explore the function of SiSPX9 in response to low-phosphorus stress would provide a theoretical basis for analyzing the mechanism of low-phosphorus tolerance in foxtail millet (Setaria italica). Method SiSPX9 was cloned from foxtail millet root and analyzed by bioinformatics. Real-time quantitative PCR (RT-qPCR) was used to analyze the expressions of SiSPX9 in different tissues and the expression patterns of different low-phosphorus stress durations. SiSPX9 was heterologously expressed in Arabidopsis thaliana by Agrobacterium-mediated method, and the germination rate, root length and root surface area of transgenic plants were detected under low-phosphorus treatment. Result The CDS of SiSPX9 gene was 759 bp in length, encoding 253 amino acids, with the conserved domain of SPX family. The results of protein structure prediction showed that the secondary structure of the encoded protein was composed of 68.38% α-helix, 1.19% β-sheet and 30.43% extended chain, and the tertiary structure was consistent with the secondary structure. Through homologous evolution analysis, it was found that millet SiSPX9 had the highest similarity with sorghum and corn, and belonged to the same branch in evolution. The results of RT-qPCR analysis showed that the expression characteristics of SiSPX9 gene in different tissues were different, with the highest expression in the roots and the lowest expression in the leaves. Under different low-phosphorus stress duration treatments, the expression of SiSPX9 increased first and then decreased with the increase of low-phosphorus stress time. The expression reached the highest value at 12 h of low-phosphorus stress, which was 15.3 times that of suitable phosphorus treatment. Functional analysis showed that under low-phosphorus stress, the germination rate, root length and root surface area of wild type (Col-0) and SiSPX9 overexpression Arabidopsis lines were inhibited to a certain extent, but SiSPX9-overexpressing lines were significantly better than Col-0. Conclusion The overexpression of SiSPX9 markedly augments the germination rate and root development of Arabidopsis in conditions of low-phosphorus stress. This suggests that SiSPX9 enhances the tolerance to phosphorus deficiency by modulating the phosphorus signaling pathway.

Genome-wide Identification of WRKY Gene Family in Gossypium hirsutum and Expression Analysis of WRKY44 in Fiber Development

XU Jia-yu, ZHAO Ge, TANG Ye, LIU Wen-wen, PENG Qing-zhong, WU Jia-he

2025, 41(12):  124-138.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0390

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Objective To investigate the characteristics of the WRKY gene family in upland cotton (Gossypium hirsutum L.) and elucidate the roles of WRKY transcription factors in fiber growth and development, thereby providing insights into their underlying molecular mechanisms. Method Genome-wide identification of the GhWRKY genefamily was performed, including analyses of chromosomal localization, physicochemical properties, subcellular localization, gene structure, and promoter cis-elements. Moreover, real-time quantitative PCR (qPCR) was employed to analyze the expression patterns of the GhWRKY gene family during fiber development, and a virus-induced gene silencing approach was to analyze the function of GhWRKY44. Result A total of 225 WRKY family members were identified in the G. hirsutum genome, and classified into three subfamilies. These genes were unevenly distributed across 26 chromosomes. The promoter cis-elements analysis showed that the promoters of GhWRKYs contained numerous regulatory elements related to plant hormones, growth and development, stress responses, and light response. Eight GhWRKYs (GhWRKY11/14/31/48/133/149/160/224) had preferential expressions at different stages of fiber development. Among them, the gene GhWRKY14 and GhWRKY133 shared similar temporal expression patterns and three-dimensional structures and were identified as orthologs (copies) of Arabidopsis thalianaWRKY44 in G. hirsutum. Accordingly, both were collectively designated as GhWRKY44. Silencing of GhWRKY44 significantly inhibited the growth and development of trichomes. Conclusion This study provides a wide analysis of the WRKY gene family in G. hirsutum and screens the key gene GhWRKY44 in fiber growth and development, thus laying the foundation for further exploring the function of WRKY44 genes in fiber growth and development.

Comparative Transcriptomics Reveals Synergistic Control of Cucumber Sex Determination by Ethylene Signaling and Epigenetic Regulation

YANG Zong-hui, LI Li-bin, MENG Zhao-juan, GAO Tian, ZHU Li-xia, DU Hai-mei, DONG Wei-wei, CAO Qi-wei

2025, 41(12):  139-155.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0571

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Objective To identify the key genes and regulatory pathways that control sex determination in cucumber (Cucumis sativus L.), with the aim of providing a theoretical basis for breeding high-yield cucumber varieties and laying a foundation for understanding the mechanisms of sex determination in dicotyledonous plants. Method Near-isogenic lines of cucumber, the gynoecious line X8g and the monoecious line X8, were used as materials. Transcriptome sequencing (RNA-Seq) analysis was conducted on the apical meristems of flower buds at the four-leaf-one-heart stage. An integrated strategy combining differential expression analysis, principal component analysis (PCA), and variance decomposition analysis was employed to screen for key genes. OrthoFinder was used to identify orthologous genes between cucumber and Arabidopsis thaliana to perform a cross-species comparative transcriptomic analysis, investigating the conservation and specificity of the regulatory networks. The function of the core candidate gene, CsACO2, was validated using Virus-Induced Gene Silencing (VIGS), and its silencing efficiency and specificity were assessed by qPCR. The role of epigenetic regulation was verified by treating the gynoecious line with the DNA methylation inhibitor 5-azacytidine. This study investigated key genes and pathways in cucumber sex determination using RNA-Seq analysis of gynoecious and monoecious lines. Result A total of 197 differentially expressed genes were identified between the gynoecious and monoecious lines, with 43 being upregulated and 154 downregulated. These genes were significantly enriched in pathways such as floral organ morphogenesis and α-linolenic acid (a precursor for jasmonic acid synthesis) metabolism. The multi-dimensional analysis identified a set of core candidate genes, including the known key ethylene synthesis gene CsACO2 and newly discovered MADS-box transcription factors like AGL6 and MADS4. The cross-species comparative analysis showed a weak overall correlation in expression patterns of orthologous genes but revealed the potential importance of target gene sets of the ethylene response factor EIN3, the NAC053 transcription factor, and H3K27me3 epigenetic modification in regulating cucumber sex. Functional validation experiments confirmed that the specific silencing of CsACO2 induced the formation of male and bisexual flowers in the gynoecious line. Similarly, treatment with 5-azacytidine also caused the gynoecious line to produce male flowers. Conclusion This study systematically reveals that cucumber sex determination is governed by a complex molecular network dominated by the ethylene signaling pathway, potentially involving jasmonic acid metabolism, various transcription factors, and epigenetic modifications.

Identification and Expression Characteristics Analysis of CmRGLG Gene Family in Melon

WANG Ya-ping, JIN Lan, HAO Jin-feng, CHANG Ming, WANG Yan-dan, GAO Feng

2025, 41(12):  156-167.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0307

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Objective RGLG proteins belong to the RING (Really Interesting New Gene, RING) type E3 ubiquitin ligases, which participate in plant growth, development, and abiotic stress responses by ubiquitinating and degrading other proteins. By identifying the members of the CmRGLGs gene family (CmRGLGs) in melon and analyzing their expression patterns, this study lays a theoretical foundation for further investigation into their potential functions. Method Bioinformatics methods were employed to analyze the chromosomal localization, gene structure, physicochemical properties of encoded proteins, phylogenetic evolution, syntenic relationships, and protein-protein interactions of CmRGLGs. The expression levels of these genes in different organs, under varying phytohormone concentrations, and after abiotic stress treatments were analyzed using RT-qPCR in young melon leaves. Result A total of six CmRGLGs members (CmRGLG1-CmRGLG6) were identified in the melon genome, named based on their chromosomal positions. The encoded proteins were hydrophilic, with amino acid lengths ranging from 364 to 596 aa. Phylogenetic analysis classified CmRGLGs into three distinct branches. Intraspecific and interspecific collinearity analysis revealed no gene duplication events among CmRGLGs. CmRGLG1, CmRGLG2, CmRGLG4, and CmRGLG5 showed collinear genes in Arabidopsis thaliana, Cucumis sativus, and Solanum lycopersicum. The promoter regions contained cis-acting elements associated with phytohormones and abiotic stress responses. Protein-protein interaction network prediction revealed that CmRGLGs-interacting proteins were primarily enriched in ubiquitin-protein transferase activity, protein metabolism, biosynthesis, and organic cyclic compound binding pathways. Expression analysis showed that CmRGLG5 and CmRGLG6 were expressed in the stems at the lowest level, while CmRGLG1, CmRGLG2, CmRGLG3, and CmRGLG4 were more abundant in the flowers. Additionally, each gene presented varying expression under different phytohormone treatments and abiotic stress conditions. Conclusion Under different phytohormone treatments, most CmRGLG members in melon showed downregulated expression trends. Under abiotic stress treatments, the majority of members demonstrated an initial upregulation followed by downregulation in their expressions.

Identification of VvOMTs Gene Families and Functional Analysis of Promoters in Wine Grapes

XUE Xiao-bin, NING Lin, ZHOU Yu, LIU Hong-jun, GAO Zhao-zu, WANG Zhen-ping, LI Dong-mei

2025, 41(12):  168-176.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0459

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Objective To characterize the response of grape (Vitis vinifera L.) VvOMTs gene family promoters to hormone and drought signals, to elucidate the promoters functions of the VvOMTs gene family, and to provide a theoretical basis for the regulation of the synthesis of 3-alkyl-2-methoxypyrazine aroma substances by VvOMTs under drought conditions. Method Bioinformatics methods were used to analyze the physicochemical properties of the protein, chromosomal localization, gene structure, and promoters cis-acting elements; and the promoters of the VvOMT2/3 gene was verified by an Agrobacterium-mediated transient transformation system in tobacco in response to abscisic acid (ABA), methyl jasmonate (MeJA), and mannitol. Result The CDS region of the coding sequence of the grape VvOMTs gene family ranged from 1 062‒1 110 bp, and the number of protein amino acids ranged in353-369 aa; the four VvOMTs were evenly distributed on the two grape chromosome 3 and 12; subcellular localization predictions indicated that the VvOMTs proteins existed in the cytoplasm; the promoters cis-acting element analysis indicated that the VvOMTs genes might be subjected to induction by light, multiple plant hormones and drought stress. The results of GUS staining, gene quantification and enzyme activity assay of transiently transformed tobacco showed that under the induction of ABA, MeJA and mannitol, the tobacco leaves transformed with VvOMT2/3 promoters showed a blue color reaction, and the expression of the GUS gene of VvOMT2/3 significantly increased and showed a tendency of increasing and then decreasing, and the GUS enzyme activity of VvOMT2/3 significantly increased. The GUS enzyme activity of VvOMT2/3 significantly increased and showed a trend of increasing and then decreasing under hormone treatment, and a bimodal trend under drought treatment. Conclusion A total of four methoxytransferase genes (VvOMT1-4) regulating methoxypyrazine synthesis are identified from the grape genome, and the VvOMT2 and VvOMT3 gene promoters are involved in and regulated by drought, ABA and MeJA responses.

Screening and Identification of Salt-tolerant Hub Genes in Karelinia caspia Using WGCNA

XU Cong-cong, ZHENG Mei, LI Cui, ZHAO Chun-qiao, HE Wei, HOU Xin-cun, GUO Qiang

2025, 41(12):  177-189.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0451

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Objective Salt-tolerance-specific gene modules were explored by investigating RNA-seq, which elucidated the molecular mechanisms underlying salt adaptation in Karelinia caspia. Method Leaves of K. caspia subjected to different NaCl concentrations (200, 300, and 400 mmol/L) were analyzed using RNA-seq and weighted gene co-expression network analysis (WGCNA). Key salt-responsive modules and genes were identified by functional annotation and network topology. Result RNA-seq generated 103.66 GB of data, yielding 66 823 unigenes, of which 45.15% (30 171) were annotated across six public protein databases. A total of 10 243 differentially expressed genes (DEGs) were identified, with 10 co-expression modules extracted via WGCNA. Notably, the brown, pink, and yellow modules demonstrated strong positive correlations with the 400, 300, and 200 mmol/L NaCl treatments, respectively. KEGG enrichment analysis revealed that genes within these modules were significantly involved in secondary metabolite biosynthesis, plant hormone signal transduction, and MAPK signaling pathways. Hub genes, such as PILS6, REM4.1, DOF21, MAPKKK18, GATA8-like, SAUR76, ABH, CIPK6, DIR22, and 4CL2, were screened based on connectivity and functional annotation. These suggested that they might play a pivotal role in adaptation of K. caspia to salt. Conclusion This study identified key salt-tolerance-specific gene modules and hub genes in K. caspia via integrated transcriptome and WGCNA analyses, thereby providing a theoretical foundation for exploration and utilization of salt-tolerant genetic resources in K. caspia.

Potential Regulatory Differences of Endogenous Hormones on Flavonoids in Anoectochilus roxburghii at Different Growth Stages Based on Multi-omics Analysis

ZENG Jing-jing, LUO Pan-lan, YAN Shu-jun, ZHENG Tao, YANG Jun-jie, CAI Kun-xiu, CAO Jia-yu, ZHANG Tian-xiang, LI Luan, CHEN Ying

2025, 41(12):  190-200.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0861

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Objective To investigate the influences of endogenous hormones at different growth stages of Anoectochilus roxburghii on flavonoid metabolism and to reveal the molecular basis for the differences in flavonoid accumulation. Method Using A. roxburghii as the experimental material, leaves from different growth stages (S3: 3 months, S6: 6 months, S10: 10 months) were selected. High-throughput RNA sequencing (RNA-Seq) and liquid chromatography-mass spectrometry (LC-MS/MS) were employed to obtain the transcriptomic and metabolomic data of A. roxburghii. Bioinformatics analysis was used to predict the differential enzyme genes related to flavonoids, metabolites, and differential hormone metabolites, and to explore the relationships among them. Result A total of 17 differential enzyme genes related to flavonoids, 16 differential metabolites, and 19 differential hormone metabolites were predicted. F3'H and FLS were identified as the key genes affecting flavonoid metabolism in the leaves of A. roxburghii, while cyanidin 5-O-β-D-glucoside 3-O-β-D-primeveroside and cyanidin 3-O-β-D-primeveroside might be the key metabolites causing the color differences in the leaves. Correlation analysis showed that 11 differential hormone metabolites were significantly related to 13 differential enzyme genes related to flavonoids, such as CHS, PGT1 and F3H. Moreover, 19 transcription factors were predicted to regulate eight differential enzyme genes, including F3H, F3'H, FLS, CHS, PGT1, and CCoAOMT. Among these transcription factors, 16 contained cis-elements responding to plant hormones (salicylic acid, abscisic acid, methyl jasmonate, gibberellin, ethylene, and auxin). The expressions of six genes (three transcription factors and three enzyme genes) were verified by qRT-PCR, and the results were consistent with the transcriptomic analysis. Conclusion At different growth stages, 16 transcription factors in A. roxburghii respond to five types of hormone elements, thereby regulating the expressions of flavonoid metabolism-related enzyme genes F3'H and FLS, and ultimately affecting the accumulation of flavonoid metabolites, delphinidin 5-O-β-D-glucoside 3-O-β-D-sambubioside and cyanidin 3-O-β-D-sambubioside.

Identification and Expression Analysis of Anthocyanin-associated R2R3-MYB Genes in Canna indica

YANG Chen-xin, LI Meng-xiu, JIANG Tang, ZHANG Wen-e, PAN Xue-jun

2025, 41(12):  201-213.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0507

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Objective To identify the R2R3-MYB gene family in Canna indica, analyze its expression patterns in different flower colors, and provide a theoretical reference for understanding the molecular regulatory mechanism of flower color and breeding new varieties of C. indica. Method Using R2R3-MYB protein sequences from Arabidopsis thaliana as reference, the members of the R2R3-MYB gene family were identified from the C. indica genome through HMM modeling and BLAST searches, and their physicochemical properties, subcellular localization, phylogenetic relationships, chromosomal distribution, conserved motifs, and promoter cis-acting elements were analyzed. Based on pre-existing transcriptome data from eight C. indica varieties within the research group, TBtools was used to visualize their expression patterns in fully open petals. Petals from four varieties with distinct flower colors (orange 'Yinli', pink 'Grand Due', red 'Anwang', yellow 'Sajin') were selected, and the expression patterns of 11 candidate CiMYB genes were detected via quantitative real-time PCR (RT-qPCR), and the relationship between gene expression and petal anthocyanin/flavonoid content was analyzed. Protein-protein interactions (PPI) in C. indica were redicted using Arabidopsis protein. Result A total of 185 R2R3-MYB genes were identified in C. indica. The encoded proteins are hydrophilic and nuclear-localized, distributed across 9 chromosomes. Approximately 94% of members contained the core conserved motifs (Motif1, Motif2, Motif3), and promoter regions are enriched with light-responsive elements. Phylogenetic analysis grouped them with Arabidopsis homologs into 30 subgroups, with 11 genes homologous to MYB proteins regulating anthocyanin synthesis in Arabidopsis. RNA-seq results showed differential expression of these 11 candidate genes across canna varieties with different flower colors. PPI network prediction indicated that CiMYB65 significantly co-expressed with DFR-A (a key anthocyanin synthesis gene) and directly regulated the anthocyanin synthesis RT-qPCR analysis revealed that CiMYB21, CiMYB39, CiMYB65, and CiMYB20 had the highest expressions in 'Grand Due', 'Sajin', 'Anwang', and 'Yinli', respectively. Gene expressions were significantly positively correlated with both anthocyanin and flavonol content. Conclusion The C. indica'sgenome contains 185 R2R3-MYB genes. Among them, genes such as CiMYB20, CiMYB39, and CiMYB65 participate in regulating C. indica flower color formation by modulating anthocyanin synthesis.

Genetic Diversity of Taxus chinensis var. mairei in Taihang Mountains Based on SSR Molecular Markers

DU Yu-qing, JIANG Lu-yuan, WANG Yi-heng, WU Chen-wei, YANG Meng-lu, LIU Xu-sheng, WANG Xiao-jun, QIU De-you, FAN Wei, YANG Yan-fang

2025, 41(12):  214-224.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0490

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Objective Taxus spp. are Class I protected endangered plants in China. Elucidating the genetic diversity and variation within T. chinensis var. mairei populations in the Taihang Mountain region will provide valuable insights for both conservation of genetic resources and development of improved cultivars. Method In this study, the genetic diversity and genetic variation of nine T. chinensis var. mairei populations in the Taihang Mountains and three populations from Zhejiang, Hunan and Fujian provinces (total of 106 individuals) were analyzed based on 8 pairs of SSR primers with good stability and polymorphism. Result Across the twelve T.chinensis var. mairei populations, genetic diversity analysis revealed the following averages: observed alleles (Na) = 2.781, effective alleles (Ne) = 2.074, Shannon information index (I) = 0.679, observed heterozygosity (Ho) = 0.390, and expected heterozygosity (He) = 0.381. Analysis of the genetic differentiation index (Fst) and gene flow intensity (Nm) revealed predominantly low to moderate levels of genetic differentiation (Fst<0.15) and frequent gene flow (Nm>>1) among the T. chinensis var. mairei populations within the Taihang Mountains region. In contrast, higher genetic differentiation (Fst>0.25) and low gene flow (Nm<1) were observed between these Taihang Mountains populations and populations from Zhejiang, Hunan, and Fujian provinces. Furthermore, Analysis of Molecular Variance (AMOVA) revealed that the genetic variation in T. chinensis var. mairei populations occurred primarily within individuals, while relatively low proportions of variation were found among populations and among individuals. Based on STRUCTURE and cluster analyses, the 106 samples could be divided into two major groups. Accessions from Fujian, Hunan, and Zhejiang provinces clustered together, while the T. chinensis var. mairei populations from the Taihang Mountains formed a distinct cluster, and the individuals of Taihang mountain were mixed with each other and had rich genetic background. Conclusion This study reveals that the T. chinensis var. mairei populations collectively possess relatively high genetic diversity. However, populations within the Taihang Mountains region demonstrate lower levels of genetic diversity. Within this region, populations showed low to moderate genetic differentiation and relatively high levels of gene flow. In contrast, significant genetic differentiation was observed between the Taihang Mountains populations and those from lower-latitude regions.

Integrated Metabolomic and Transcriptomic Analysis Reveals the Mechanism of Prunus davidiana Response to Freezing Stress

ZHANG Xiao-dan, YIN Zheng, LIU Qing-chen, LI Xue-mei, LIU Xiao-hua, LIANG Mei-xia

2025, 41(12):  225-239.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0416

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Objective Freezing stress is one of the primary environmental factors limiting peach tree growth, fruit quality, and yield. However, the molecular mechanisms underlying peach tree responding to low-temperature stress remained poorly understood. This study investigated the dynamic reprogramming patterns of amino acid, carbohydrate, and lipid metabolism in peach trees under low-temperature stress, and explored their molecular associations with cold resistance, aiming to provide a theoretical basis for molecular breeding for cold tolerance and the optimization of cultivation techniques. Method Using Prunus davidiana as experimental material, we implemented gradient freezing temperature treatments (5, -5, -15 and -25 ℃). Physiological indices were measured and integrated with transcriptomic and metabolomic comparative analyses. Through systematic integration of physiological-biochemical measurements, metabolomics, and transcriptomics technologies, we comprehensively analyzed the multi-omics regulatory networks. Result Freezing stress triggered the accumulation of proline and soluble sugars in P. davidiana, accompanied by increased malondialdehyde content and elevated electrolyte leakage rates, indicating damage to cellular membrane systems. Metabolomic analysis revealed significant enrichment of carbohydrates and derivatives under low-temperature conditions, partial amino acid reduction, while the content of tricarboxylic acid cycle-related organic acids (e.g., 2-oxoglutarate and citrulline) increased. Flavonoid biosynthesis, arginine synthesis, and nitrogen metabolism pathways were significantly activated during stress. Transcriptomic analysis confirmed the substantial upregulation of glucuronic acid biosynthesis-related genes under cold stress, demonstrating coordinated responses with metabolite changes to freezing stress. These results elucidated the molecular mechanism through which P. davidiana enhanced cold resistance via metabolic-gene network regulation. Conclusion This study reveals that P. davidiana activated starch and sucrose metabolism, as well as phenylpropanoid and flavonoid biosynthesis pathways in response to freezing stress. A total of 21 key metabolites and 15 core genes are identified, elucidating the coordinated mechanisms of metabolic and genetic regulation underlying cold resistance.

Functional Study of DoDELLA2 in Dendrobium officinale Kimura et Migo

YANG Tao, LI Lin, MO Xiao-lian, CHEN Xiao-long, WANG Jian, HUANG Yuan, ZHAO Jie-hong, ZOU Jie

2025, 41(12):  240-253.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0534

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Objective To identify the function of DoDELLA2, the major gene of the DELLA gene family in Dendrobium officinale, in plant morphological development, and to investigate the downstream genes and signaling pathways regulated by the DoDELLA2 gene, so as to provide a theoretical basis for the molecular breeding of D. officinale. Method RT-qPCR was employed to analyze the tissue expressions of members of the DELLA gene family in D. officinale, and a potential major gene DoDELLA2 of the DELLA gene family in D. officinale was screened out. The full-length DoDELLA2 gene was synthesized, followed by the construction of the 35S-DoDELLA2 overexpressing vector for heterologous transformation into Arabidopsis thaliana. Functional studies were conducted by combining phenotypic observation with transcriptome data analysis. Result Compared with the wild type, A. thaliana overexpressing DoDELLA2 showed phenotypic differences in the growth period, including a 10-16 d delay in bolting time, a significant 40.04% reduction in plant height, elliptical leaf apices with deeper leaf margin serrations, and a significant increase in the number of siliques but a significant reduction in silique length. Transcriptome analysis revealed 696 differentially expressed genes (DEGs) between DoDELLA2-overexpressing A. thaliana and the wild type. Among these, the significantly enriched up-regulated genes mainly included protein-coding genes such as transporter proteins, gibberellin synthetases, and cysteine-rich secretory proteins. The significantly enriched down-regulated genes mainly included the peroxidase protein superfamily, cytochrome P450 protein superfamily, and plant pathogenesis-related protein (PR1). The DEGs were significantly enriched in biological processes such as lignin biosynthesis and the transition from vegetative to reproductive growth, as well as KEGG pathways including phenylpropane biosynthesis, flavonoid biosynthesis, and diterpenoid biosynthesis. Additionally, the expressions of key gibberellin biosynthesis enzyme genes AtGA₂₀OX2, AtGA₂₀OX3, and AtGA₃OX1 were upregulated, showing obvious feedback regulation. Conclusion The overexpression of the DoDELLA2 gene influenced the morphological development of A. thaliana by regulating downstream genes in the GA signaling pathway. Transcriptome differential analysis indicated that DoDELLA2 overexpression significantly affected the expressions of gibberellin synthase genes and certain resistance gene families, providing important targets and a theoretical foundation for molecular breeding practices aimed at improving the quality and traits of Dendrobium officinale.

Identification of the AsparagusCYP51 Gene Family and the Response to Abiotic Stress

ZENG Liang-qin, DONG Chen-wen-hua, LIN Chun, LIU Zheng-jie, MAO Zi-chao

2025, 41(12):  254-266.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0469

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Objective Sterol 14α-demethylase (CYP51), a key enzyme in the plant sterol biosynthesis pathway, plays an important role in plant responses to abiotic stress. This study is aimed to identify CYP51 gene family members genome-wide in Asparagus, analyze their molecular characteristics, and validate the function of AtaCYP51G2 from Asparagus taliensis in response to abiotic stress, thereby providing a basis for understanding the molecular mechanisms of stress resistance in Asparagus plants. Method Bioinformatic methods were used to identify CYP51 family members in A. taliensis, A. officinalis, and A. setaceus. Their physicochemical properties,chromosomal localization, gene duplication, phylogenetic relationships, gene structures, and cis-acting elements in promoter regions were analyzed. Transgenic Arabidopsis thaliana lines overexpressing AtaCYP51G2 were generated, and phenotypic observations and physiological/biochemical measurements were performed under drought, salt, and osmotic stress conditions. Real-time quantitative PCR was employed to detect the expression levels of the target gene in transgenic lines and the changes in AtaCYP51G2 expression under stress treatments. Result Four CYP51 gene family members were identified in Asparagus species. Promoter analysis revealed the presence of hormone- and stress-responsive cis-acting elements, which were particularly abundant in the AtaCYP51G2 promoter. Subcellular localization experiments indicated that the AtaCYP51G2 protein is localized to the endomembrane system. The overexpression of AtaCYP51G2 enhanced the tolerance of transgenic Arabidopsis to drought and osmotic stress and partially alleviated oxidative damage under salt stress. Conclusion The AsparagusCYP51 gene family has a small number of members and is highly conserved in terms of gene structure and protein sequence. The overexpression of AtaCYP51G2 enhances the tolerance of transgenic Arabidopsis to drought, salt, and osmotic stress, providing a theoretical basis and potential genetic resources for improving stress resistance in Asparagus plants.

Molecular Characterization， Expression Profiling， and Protein Interaction Analysis of the EuGIF1 Gene in Eucommia ulmoides

WANG Ruo-Ruo, QU Peng-Kun, ZHANG Xin, WANG Luo, ZHU Ying, TIAN Shuang-Yi, ZHAO De-Gang

2025, 41(12):  267-279.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0438

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Objective To systematically identify the GIF gene family in Eucommia ulmoides and analyze their expression patterns and protein interaction characteristics, aiming to decipher the regulatory network of organ development and optimize genetic transformation strategies. Method HMMER and BLASTPwere used to identify GIF genes in Eucommia ulmoides. Bioinformatics tools were employed to analyze protein physicochemical properties, 3D structures, and promoter cis-elements. RT-qPCR was adapted to validate the tissue-specific and GA₃-induced expression of EuGIF1. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted to verify the interaction between EuGIF1 and EuGRF5. Additionally, the overexpression vector carrying the EuGIF1 gene was to genetically complement the gif1 mutant of Arabidopsis thaliana. Result Three EuGIF genes EuGIF1/2/3 with conserved SSXT/SNH domains were identified in E. ulmoides. EuGIF1 and EuGIF2 were located on chromosome 7 of the E. ulmoides genome, and EuGIF3 was located on chromosome 17. The proteins encoded by EuGIF1,EuGIF2 and EuGIF3 all contained conserved SSXT/SNH domain.Promoter sequence analysis revealed that the promoter region of the EuGIF1 gene contained two gibberellin-response elements. EuGIF1 had high expression in apical meristems and rapid induction by GA₃. 3D structural prediction of the protein revealed potential interaction interfaces between the SSXT/SNH domains. Co-expression analysis suggested functional synergy between EuGIF1 and EuGRF1/3/5, but Y2H and BiFC assays showed no direct interaction between EuGIF1 and EuGRF5. The overexpression of EuGIF1 in A. thalianagif1 mutant failed to complement its narrow-leaf phenotype. Conclusion EuGIF1 may play a significant role in gibberellin-mediated organ development in E. ulmoides, whereas its interaction with GRFs shows species specificity. Furthermore, the functional conservation of this gene appears limited across divergent species.

Transcriptome Analysis of Different Parts of Ferula Sinkiangensis K. M. Shen and Exploration of Genes Related to Sesquiterpene Synthesis

SI Xu-peng, CUI Xiu-wen, OUYANG Li-zhi, FANG Dan-dan, PEI Long-ying, FANG Gui-ping, PU Xi-lei, MA Yi-mian, ZHANG Zheng

2025, 41(12):  280-293.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0452

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Objective By analyzing the transcriptome information of different parts of Ferula sinkiangensis, we aim to explore the key metabolic pathways and regulatory genes involved in the biosynthesis of sesquiterpenes, providing genetic resources for molecular breeding and resource development of F. sinkiangensis Method The Unigene sequences of different parts of the roots, stems and leaves of F. sinkiangensis were obtained by transcriptome sequencing, and bioinformatics analysis was carried out. Result A total of 116 517 transcripts were obtained by transcriptome sequencing, and a total of 44 896 Unigene sequences were annotated. F. sinkiangensis has the highest proportion of homologous sequence matching with Daucus carota subsp. sativus. A total of 19 290 Unigenes were annotated in biological processes, cell composition and molecular function. The metabolic pathways of terpenoids and polyketides (11 pathways, 204 Unigenes) and other secondary metabolite synthesis pathways (17 pathways, 233 Unigenes) were enriched by KEGG. Further studies showed that 57 Unigenes were related to sesquiterpene synthesis, involving 34 full-length transcripts of 9 key enzyme genes. The differentially expressed genes were mainly enriched in the MEP pathway, and the RT-qPCR results of six enzyme genes with obvious expression trends showed similar expression patterns with the RNA-seq results. The expression analysis showed that the relative expressions of P450 and TPS, the key genes downstream of sesquiterpene synthesis, were the highest in the roots, the expressions of DXS and HMGR were the highest in the leaves, and the expressions of AACT and DXR were the highest in the stems. Meanwhile, a total of 28 017 CDS sequences, 1 886 transcription factors, 14 277 SSR loci and 9 844 pairs of corresponding primers were predicted in the transcriptome. Conclusion Through transcriptome analysis, the biosynthetic pathway of sesquiterpenes in F. sinkiangensis is clarified, and it is speculated that P450 and TPS in the roots are the key downstream genes of sesquiterpene synthesis.

Biocontrol Effect of Burkholderia contaminans KRS634 on Cotton Red Rot Disease

WU Qiong, CHEN De-yong, ZHU He, WANG Dan, ZHANG Xiao-jun, CHEN Jie-yin

2025, 41(12):  294-303.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0497

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Objective To develop an environmentally-friendly, safe, and highly effective biocontrol agent against cotton red rot disease caused by Fusarium verticillioides. Method F. verticillioides was used as the target fungus, antagonistic strains were screened by the plate confrontation method to obtain a strong antagonistic activity strain. The strain was identified and analyzed through morphological observation, phylogenetic analysis of 16S rRNA sequences, and plant growth-promoting characteristics. Pot experiments were further carried out to determine the biocontrol and plant growth-promoting effects of the strain. The inhibitory effects of the strain on various fungi were tested by the confrontation culture method and the sealed fumigation method. Result The strain KRS634 exhibiting strong antagonistic activity against F. verticillioides was screened through plate confrontation assays, and was identified as Burkholderia contaminans based in 16S rRNA sequence analysis. KRS634 had the plant growth-promoting characteristics including phosphorus solubilization, potassium solubilization, nitrogen fixation, and siderophore production. The indoor experiments showed that KRS634 effectively reduced the occurrence of cotton red rot disease at the seedling stage. The disease plant rate of cotton red rot disease treated with KRS634 was 85.71%, and the disease index was 36.31, which was significantly lower than that of the control group. The fermentation broth of KRS634 also increased the emergence rate of cotton and promoted the growth of cotton. In addition, KRS634 had a broad-spectrum antagonistic effect on various pathogenic fungi such as Verticillium dahliae, Magnaporthe oryzae, and Colletotrichum gloeosporioides. Conclusion The strain B. contaminans KRS634 has a good application potential in the prevention and control of fungal diseases such as cotton red rot disease. It can be used as a candidate strain for the development of biocontrol agents against cotton red rot disease.

The Role of GSTs in the Detoxification Metabolism of Aphis craccivora to Afidopyropen

XUE Chao, DUAN Ai-ling, WANG Ai-yu, YANG Yuan-xue

2025, 41(12):  304-312.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0580

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Objective Aphis craccivora is a globally prominent agricultural pest that severely threatens the yield of peanuts and other crops. Afidopyropen, as a novel and highly efficient insecticide, demonstrates potent control efficacy against sap-sucking pests. Clarifying the metabolic resistance mechanisms of A. craccivora to afidopyropen is of critical importance for the development of scientifically robust field management strategies. Method The sensitivity of A. craccivora to afidopyropen was evaluated through leaf-dip bioassay. The role of glutathione S-transferases (GSTs) in detoxification metabolism was assessed through enzyme activity assays and synergist toxicity bioassays. Tissue expression pattern, developmental expression pattern, and insecticide-induced expression profiles of GSTs were analyzed via quantitative real-time PCR (RT-qPCR). RNA interference (RNAi) was employed to identify key GSTs associated with tolerance to afidopyropen. Result Afidopyropen treatment significantly increased GST activity in A. craccivora. Pretreatment with the synergist diethyl maleate (DEM) enhanced the susceptibility of A. craccivora to afidopyropen, indicating the involvement of GSTs in detoxification metabolism in A. craccivora. Tissue and temporal expression pattern analyses revealed that most GSTs were highly expressed in the midgut and fat body, with expressions rising markedly during developmental progression. Afidopyropen induced the upregulation of GSTT2 and GSTS2. RNAi-mediated silencing of GSTT2 significantly increased the sensitivity to the afidopyropen in A. craccivora. Conclusion GSTT2 plays an important role in response to afidopyropen stress by reducing its sensitivity to the insecticide. This finding lays the foundation for further investigation into the detoxification mechanisms of A. craccivora against afidopyropen.

Isolation and Identification of Biocontrol Bacteria for Potato Anthracnose in Three Strains and Optimization of Fermentation Parameters for Cultures with High Antibacterial Activity

LUO Ming-kai, ZHANG Hao-jie, SHI Hui-qin, LI Ya-nan, FENG Rui-chao, SHEN Shuo

2025, 41(12):  313-327.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0209

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Objective Potato anthracnose, caused by Colletotrichum coccodes, severely impacts both yield and quality. Bacillus spp. are commonly used as biocontrol agents, making the search for efficient and low-toxicity strains to manage this disease highly significant. This study focused on three Bacillus strains with strong antibacterial activity and their composite microbial community. By optimizing the fermentation parameters of the composite Bacillus, the aim is to enhance its inhibitory effect against the pathogen, providing a reference for the development of composite biocontrol agents. Method Endophytic bacteria were isolated from potato tubers by tissue isolation and scribing methods, strains with strong inhibitory effect on potato anthrax were screened by drug-sensitive paper slide method, the taxonomic status of the active strains was determined by combining morphological and molecular biological identification, and the optimal ratio of the three strains was determined by drug-sensitive paper slide method, and the composite group was optimized by one-factor test and response surface design. The fermentation medium and fermentation conditions of HQS6513 were optimized by one-way test and response surface design. Result Three strains QS10-6, QS2-5 and QS2-13 with obvious inhibitory effect on potato coccodes (Colletotrichum coccodes) were screened out from potato tubers and identified as Bacillus atrophaeus, Bacillus atrophaeus and Bacillus velezensis, response surface method analysis was optimized to obtain the optimal fermentation medium for the composite colony HQS6513 as bran 2.5%, yeast extract powder 2.6%, NaCl 1.0%, and the optimal fermentation conditions as incubation temperature of 32 ℃, pH 5, inoculum amount of 2.0%, and shaking table speed of 190 r/min, and the optimized bacterial inhibition rate of the composite colony HQS6513 reached 69.66%. Conclusion Three strains of Bacillus spp. were isolated, and the composite colony HQS6513 composed of these three strains of Bacillus spp. had high inhibitory activity against C. coccodes, and the optimization of fermentation formula and culture conditions for it resulted in the increase of cell production and inhibitory activity of the composite flora HQS6513.

Screening， Identification and Whole Genome Analysis of a Paenibacillus Strain Resistant to Root Rot of Rehmannia glutinosa

ZHAO Yi-fan, WANG Tong, YE Lan, ZHAO Le, ZHANG Bao, DU Peng-qiang, HE Hai-rong

2025, 41(12):  328-341.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0491

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Objective Strains resistant to Rehmannia glutinosa root rot were screened to provide a material basis and strain resources for the biological control of R. glutinosa root rot. Method The rhizosphere microorganism of ring rot-infected R. glutinosa were isolated, and strains with antagonistic activities against pathogenic fungi were screened by using plate confrontation assay. Besides, a pot experiment was carried out to explore the active bacteria in preventing and controlling the root rot disease of R. glutinosa. The strain was identified through colonial morphology observation, physiological and biochemical, and molecular biology analysis. The strain was also sequenced throughout its entire genome, its genomic information was analyzed, and its disease resistance mechanism was explored. Result Based on the plate confrontation results, a strain QH-1 with broad-spectrum antagonistic activity against plant pathogenic fungi was screened, showing an inhibition rate of nearly 90% against the pathogen of R. glutinosa root rot. Strain QH-1 was identified as Paenibacillus polymyxa, it was a Gram⁺ bacterium and produced peanut-like endospores that grew at 10-55 ℃, pH 6-8, the concentration of NaCl 0-5%. Carbon and nitrogen source utilization tests showed that it utilized multiple nitrogen sources but only two carbon sources that were maltose and sorbitol. Additionally, the strain produced catalase, amylase, lipoidase, gelatinase, and proteinase. The genome of this strain consisted of a circular chromosome and a circular plasmid, with a size of 5 692 874 bp and 37 590 bp, and a content of (G+C)% of 45.4%. Total 17 biosynthesis gene clusters were explored by using antiSMASH. Of these 17 biosynthesis gene clusters, 6 biosynthesis gene clusters had 100% similarities with known clusters including fusaricidin B, paenibacillin, paenilan, tridecaptin, polymyxin and paenicidin. Thus, strain QH-1 has the potential of producing these antibacterial compounds like mentioned above. Conclusion Strain QH-1 is a P. polymyxa strain resistant to R. glutinosa root rot. Its genomic information indicates the ability of producing multiple antimicrobial substances, providing a solid theoretical and material foundation for further development of microbial agents and natural antimicrobial compounds against R. glutinosa root rot.

Functional Study of bZIP Transcription Factor CsFcr3 in Regulating the Sensitivity of Colletotrichum siamense to DMI and QoI Fungicides

YANG Hong, ZHANG Chao, XUE Wen-xuan, HUANG Wei-yuan, LIN Chun-hua, WANG Li-feng

2025, 41(12):  342-350.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0543

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Objective The bZIP transcription factor CsFcr3 was cloned from Colletotrichum siamense, the predominant species responsible for Colletotrichum leaf disease (CLD) in rubber trees. This study characterized its sequence and structural features and elucidated its biological role in regulating the sensitivity of C. siamense to six different fungicides. These findings establish a foundational understanding of resistance regulatory mechanisms in pathogenic fungi and provide a theoretical framework for the scientific and efficient management of fungal diseases in rubber tree cultivation. Method The bZIP transcription factor CsFcr3, having high sequence homology with the fluconazole resistance protein Fcr3 from Candida albicans, was cloned from Colletotrichum siamense using homology-based cloning. Comprehensive sequence and structural analyses were conducted to characterize this gene. Real-time quantitative PCR (RT-qPCR) was employed to profile the expression patterns of CsFcr3 under fungicide stress. Additionally, the gene knockout of CsFcr3 was performed by homologous recombination and PEG-mediated protoplast transformation, and the changes in sensitivity of the knockout mutants to different fungicides were analyzed. Result The open reading frame (ORF) of CsFcr3 encompassed 1 101 base pairs, encoding a protein of 336 amino acids with a conserved bZIP_Yap domain. RT-qPCR results demonstrated significant upregulation of CsFcr3 expression under stress induced by demethylation inhibitor (DMI) fungicides (difenoconazole, tebuconazole, prochloraz) and quinone outside inhibitor (QoI) fungicides (pyraclostrobin). Three CsFcr3 knockout mutants (ΔCsFcr3-3, ΔCsFcr3-15, and ΔCsFcr3-16) were successfully generated. Compared to the wild-type strain, the ΔCsFcr3s presented significantly reduced sensitivity to fluconazole, tebuconazole, and prochloraz, increased sensitivity to pyraclostrobin, and no significant differences in sensitivity to difenoconazole and fludioxonil. Conclusion The bZIP transcription factor CsFcr3, which contains conserved motifs and structural domains typical of the bZIP family, is cloned from C. siamense. Phylogenetic analysis reveals its close homology to the Saccharomyces cerevisiae Yap3p protein. Functional characterization demonstrates that CsFcr3 primarily regulates C. siamense sensitivity to DMI and QoI fungicides, exhibiting no significant regulatory role in response to pyrrolnitrin-class fungicides.

Effects of γ-Aminobutyric Acid on Antioxidant Capacity and Cecal Microbial Structure of Broilers

MA Shun, ZHAO Xu, LIU Xue, CHEN Guang-kun, GAO Yue, DING Jin, ZHANG Jing-yi, ZHAO Yi-meng, GONG Li-yu, LI Hong-tao

2025, 41(12):  351-359.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0553

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Objective To explore the effects of γ-aminobutyric acid (GABA) on growth performance, meat quality, antioxidant capacity and cecal microbial structure of broilers. Method A total of 408 one-day-old Ross 308 broilers with similar body weights were randomly divided into 2 groups (6 replicates per group and 34 chickens per replicate): the control group (CON group) was fed with the basal diet, while the GABA group was fed with the experimental diet supplemented with GABA (50 mg/kg) based on the basal diet. The trial lasted for 42 d. Result Dietary supplementation with GABA significantly increased the ether extract content in the breast muscle, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the total antioxidant capacity (T-AOC) in the livers of broilers (P<0.05), while significantly decreased the shear force in breast muscle and the malondialdehyde (MDA) content in serum (P<0.05). Microbial analysis indicated that the cecal microbiota composition of broilers in the GABA group was significantly different from that in the CON group (P<0.05). Compared with the CON group, the relative abundances of Alistipes and Blautia significantly increased (P<0.05), while the relative abundances of Rikenella and Parabacteroides significantly decreased in the GABA group (P<0.05). Moreover, the relative abundances of Alistipes and Blautia were positively correlated with the ether extract content in breast muscle, the activities of SOD and GSH-Px, as well as the T-AOC in the liver (P<0.05), while negatively correlated with the shear force in breast muscle and the MDA content in serum of broilers (P<0.05). The relative abundance of Rikenella was negatively correlated with the activities of GSH-Px and T-AOC in the liver of broilers (P<0.05). Additionally, the PICRUSt2 functional predicting results showed that the cecal microbiota of broilers in the GABA group significantly enhanced functions in pentose and glucuronate interconversions, sphingolipid metabolism, nitrotoluene degradation, steroid hormone biosynthesis, and N-glycan biosynthesis (P<0.05), and significantly reduced functions in amoebiasis disease compared with the CON group (P<0.05). Conclusion Adding 50 mg/kg of GABA to the diet may promote the fat deposition in breast muscle, improve the tenderness of breast muscle, enhance the antioxidant capacity, and change the structure of cecal microbiota of broiler at 42 d of age.

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2025, 41(12):  360. 

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2025, 41(12):  361. 

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