Table of Content

26 August 2025, Volume 41 Issue 8

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Artificial Intelligence Transforms Protein Engineering： From Structural Analysis to Synthetic Biology through Algorithmic Advancements

CAI Ru-feng, YANG Yu-xuan, YU Ji-zheng, LI Jia-nan

2025, 41(8):  1-10.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0300

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The intricate relationship between protein function and its three-dimensional structure has long been a fundamental guiding principle in life sciences research. While scientists have dedicated substantial effort to deciphering protein structures, the exponential growth in sequence data fueled by rapid advances in protein sequencing technologies has significantly outpaced progress in structural studies. Over the past decade, the burgeoning field of artificial intelligence (AI), underpinned by core algorithms such as deep learning and neural networks, has emerged as a transformative force in protein engineering, offering new avenues to address this disparity. Leveraging AI, next-generation methods for protein structure prediction and design have achieved remarkable breakthroughs. These advanced algorithm-based tools have dramatically enhanced both the accuracy and speed of protein structure modeling. They are not only accelerating progress in structural biology and drug discovery but also providing crucial foundations for protein synthesis. Furthermore, AI is catalyzing a paradigm shift in protein research, moving beyond ‘structure determination’ towards ‘inverse design’. By constructing multidimensional models that elucidate sequence-structure-function relationships, researchers can now reverse-engineer protein sequences with desired structural characteristics based on specific functional requirements. This capability for precise protein sequence design is paving new pathways for biosynthetic applications.This review focuses on the pivotal role of AI in protein engineering. Firstly, it outlines the current challenges in the protein engineering and the bottlenecks in traditional protein structure determination methods. Then it introduces the development of AI-based structure prediction tools, followed by an analysis of their application in protein synthesis. Finally, it then explores the algorithm-driven revolution facilitating the transition from structure determination to de novo protein synthesis, and discusses potential future directions, aiming to provide a reference framework for ongoing research in this field.

Research Advances in Targeted Mutagenesis-mediated Microbial Evolutionary Engineering

DIAO Chen-yang, CUI You-zhi, LI Bing-zhi

2025, 41(8):  11-21.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0191

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Natural mutations in organism's genomes confer diverse traits that facilitate evolutionary adaptation to environmental challenges. However, the slow evolutionary rates inherent to natural settings limit their practical utility. Strategies involving targeted base modifications at specific DNA sequences enable accelerated laboratory evolution, which has been widely applied in non-rational protein engineering and metabolic pathway optimization. Nevertheless, conventional mutagenesis methods remain labor-intensive, suffer from low efficiency, and lack targeting precision. In contrast, targeted mutagenesis technologies introduce mutations at precisely defined genomic loci, significantly improving efficiency and shortening evolution cycles. In recent years, targeted mutagenesis has emerged as a key area of research in microbial evolutionary engineering. These technologies enhance the optimization of microbial metabolic pathways, improve stress tolerance in chassis strains, and have found widespread applications in biocatalysis, environmental remediation,and industrial fermentation. This review summarizes recent advances in targeted mutagenesis systems developed for key microbial chassis organisms, focusing on the design principles and applications of error-prone replication systems, CRISPR-Cas-mediated mutagenesis systems, and the MutaT7 mutagenesis system. Additionally, it analyzes the strengths and limitations of current laboratory evolution systems and discusses future prospects for precision-targeted mutagenesis technologies.

Research Advances in Amylose Biosynthesis and Strategies for Enhancing Its Content in Plants

WANG Sai-di, ZHANG Gao-yang, LYU Huan-huan, SUN Zhong-ke, LI Cheng-wei

2025, 41(8):  22-33.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0255

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Amylose, primarily composed of D-glucopyranose units linked by α-1,4-glycosidic bonds, is a critical component of starch. Its unique molecular structure and physicochemical properties underpin its broad applications in food, industrial, and pharmaceutical sectors. Amylose content varies across plant species with distinct genetic traits, significantly influencing grain processing characteristics, starch quality, nutritional composition, and textural properties of derived products. Furthermore, amylose plays essential roles in plant growth, development, reproduction, and stress resistance to biotic/abiotic challenges. Consequently, enhancing amylose content in plants has emerged as a pivotal target for both crop quality improvement and industrial utilization. This review systematically summarizes the biosynthetic pathways of amylose, analyzes its genetic regulatory mechanisms, and explores strategies to elevate amylose content through temperature modulation, plant growth regulators, and cultivation practices. Additionally, current challenges and future prospects in amylose enhancement are critically evaluated. The synthesis of this knowledge aims to provide a theoretical foundation for advancing research on amylose optimization and expanding its practical applications.

Application and Research Progress of Gene Editing Technology in Bovine

YU Yong-xia, DU Zai-hui, ZHU Long-jiao, XU Wen-tao

2025, 41(8):  34-41.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1083

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Gene editing refers to the process of modifying specific targets in an organism’s genome through gene editing techniques, achieving efficient and precise gene knockout, insertion, replacement, and other processes. Modern gene editing techniques include zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9), and self-cleaving gene targeting technology in combination with programmable nucleases (SEGCPN). This paper reviews the breakthrough research progress of gene-edited cattle, including the famous gene-edited hornless cattle Buri, Spotigy and the US Food and Drug Administration (FDA) safety certified PRLR-SLICK, and summarizes the application of gene-edited cow in production, medicine and environment. The aim is to summarize the current application and research progress of gene editing technology in domestic cattle, in order to accelerate the industrialization process of gene editing technology in China and expand the application range of gene editing cattle.

Mechanisms and Applications of Bacillus in Controlling Plant Diseases

PENG Tian-tian, MA Yun-long, XU Pei-dong, CHEN Di, XIE Bing-yan, LI Yan

2025, 41(8):  42-52.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0171

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Plant diseases cause a serious threat to the economic benefits of crops, while the overuse of chemical pesticides exacerbates the risks to the environment and human health. Therefore, biological control strategies for plant diseases are considered the main trend for disease management in future. At present, Bacillus microbial biocontrol agents have been extensively utilized in plant disease management. These bacteria exert their disease control effects through multiple mechanisms, including targeting pathogens for competitive action and secreting antimicrobial substances to directly inhibit the growth of pathogens; targeting plants for colonizing the rhizosphere and form biofilms, inducing systemic resistance of plant, and promoting plant growth to enhance plant resistance to disease. Concurrently, they can also improve the soil environment of rhizosphere, creating favorable conditions for the healthy growth of plants. This article reviews the mechanisms of action of Bacillus strains in controlling plant diseases and their application status as microbial biocontrol agents and discusses its application prospects and challenges in modern agriculture, aiming to provide valuable reference for the development of subsequent biopesticide products based on these biocontrol microorganisms.

Research Progress in Molecular Defense Mechanisms of Chlamydomonas reinhardtii in Response to Heavy Metal Stress

LI Ya, JIANG Lin, XU Chuang, WANG Su-hui, MA Zhao, WANG Liang

2025, 41(8):  53-64.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0088

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With the rapid development of urbanization and industrialization, heavy metal pollution has posed an immeasurable threat to aquatic ecosystems. Through the bioaccumulation effect in food chains, it severely endangers human health and has become a globally concerning environmental issue. In this context, green environmental remediation solutions represented by bioremediation have garnered significant attention due to their eco-friendliness and sustainability. As crucial primary producers in aquatic ecosystems, microalgae demonstrate high diversity and ecological adaptability, employing various molecular mechanisms to respond to heavy metal stress. Chlamydomonas reinhardtii, a unicellular eukaryotic microalga, maintains intracellular metal ion homeostasis through multi-layered physiological structures and regulatory mechanisms. Its unique capabilities in metal biotransformation and biosorption have established it as an important model organism for studying bioremediation of heavy metal-contaminated water bodies. The deepening understanding of C. reinhardtii’s metal homeostasis mechanisms and rapid advancements in modern biotechnology have provided theoretical foundations for elucidating its defense strategies against heavy metal stress, while offering scientific guidance for its application in water remediation. This review summarizes key components and regulatory mechanisms involved in its responses to heavy metal stress, spanning from cell surface adaptations (e.g., cell walls and extracellular polymeric substances) to intracellular processes (e.g., metal transport proteins, heavy metal-binding factors, and metal-regulating organelles). Furthermore, it explores the potential applications of modern molecular biology techniques in enhancing C. reinhardtii’s bioremediation capabilities and discusses its prospects in aquatic environmental protection.

Efficient CRISPR Gene Editing in Rice Assisted by RUBY

DENG Mei-bi, YAN Lang, ZHAN Zhi-tian, ZHU Min, HE Yu-bing

2025, 41(8):  65-73.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0339

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Objective To develop a RUBY-assisted CRISPR/Cas9 gene-editing vector (GCR) enabling visual screening of transgenic plants and gene-editing events. Method The GCR vector was constructed by coupling RUBY with the Cas9/gRNA expression cassette. Using OsAGO2, OsAGO3, and OsAGO7 in rice (Oryza sativa) as target genes, two multi-gene editing vectors (GCR-237-1 and GCR-237-2) were designed. These vectors were introduced into rice (Zhonghua 11, ZH11) via Agrobacterium-mediated genetic transformation to generate gene-edited plants. Result After the transformation of GCR-237-1 and GCR-237-2 into rice callus, RUBY markers effectively indicated transgenic positive events, and the target genes of stable transformed red plants were efficiently edited. Conclusion The GCR vector enables effective gene editing in rice, and transgenic or gene-edited plants can be rapidly identified through direct visual observation.

Enhancing 5-Aminolevulinic Acid Biosynthesis through Tandem Rare Codon-mediated Attenuation of HemB Expression

LI Jia-yi, LI Jin-yi, BAI Xue, BAI Ying-guo, LIU Bo, ZHANG Zhi-wei

2025, 41(8):  74-81.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0095

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Objective To reduce 5-aminolevulinic acid degradation and enhance its accumulation, the expression of 5-aminolevulinic acid dehydratase (HemB), which is a key enzyme in the catabolic pathway of 5-aminolevulinic acid (5-ALA), was attenuated via a tandem rare codon engineering strategy. Method Corynebacterium glutamicum was used as the chassis cell for the synthesis of 5-ALA. To reduce the catabolism of 5-ALA, tandem rare codons according to codon usage bias of C. glutamicum were introduced at the 5' end of the gene encoding HemB to weaken its expression. Result The metabolically engineered C. glutamicum strain showed a reduced growth rate, simultaneously the content of 5-ALA degradation intermediates decreased and 5-ALA accumulation was significantly enhanced in the fermentation broth. Conclusion The rare codon engineering strategy effectively attenuates HemB expression to enhance 5-ALA accumulation, and this strategy can be applied to fine-tuning expression of other proteins and remodeling other metabolic pathways.

Research Progress in HEK293 Cell Suspension Culture Technology and Its Application

YUE Xin-yu, JI Wen-na, ZHOU Jin-yu, JIN Li-wu, YANG Di, LIU Zhen-bin, SUN Na, QIAO Zi-lin, MA Zhong-ren, WANG Jia-min

2025, 41(8):  82-91.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0029

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The production of biopharmaceutical products primarily relies on mammalian cells, especially in the manufacture of complex protein drugs, due to their ability to perform intricate post-translational modifications (PTMs), which are critical for the function and stability of proteins. Most biopharmaceutical products, such as recombinant therapeutic proteins, vaccines, anticancer drugs, and other clinically relevant medications, are produced using mammalian cell platforms. HEK293 cells (human embryonic kidney 293 cells) are one of the most commonly used host cells among all human cell lines; however, the high investment costs associated with adherent cells in production applications and the potential external risks posed by serum additives have initially limited their use in production. In contrast, HEK293 suspension cells offer advantages over adherent cells, such as significantly reduced production costs and enhanced safety in producing biological products due to the absence of serum. Currently, HEK293 suspension cells have been widely applied in various fields and have become a robust vehicle and new platform for the production of proteins, vaccines, and gene therapy drugs. This article reviews the origin of HEK293 cells and their derived cell lines, summarizes the current suspension adaptation techniques for adherent cells, and outlines serum-free media used for HEK293 cells. It also describes the advancements in the application of HEK293 cells in vaccine production, drug production for gene therapy, and fundamental laboratory research. The significance and existing problems of large-scale production of HEK293 cells are discussed, along with prospective research directions and the application prospects of large-scale production in the future.

Construction and Phenotypic Variation Analysis of Ningchun No. 4 Wheat EMS Mutant Library

DOU Fei-fei, REN Yu-zhao, WANG Shi-lei, LIU Chun-ying, WANG Xiao-dong, WANG Zhao-yi, LIU Cai-xia, LIU Feng-lou, WANG Zhang-jun, LI Qing-feng

2025, 41(8):  92-101.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0027

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Objective To accelerate the creation of high-quality germplasm resources for saline wheat and to enrich genetic diversity, we constructed Ningchun No. 4 EMS mutant library. Method In this study, 10 000 seeds of the wheat variety Ningchun No. 4 were treated with 0.5% ethyl methanesulfonate (EMS) solution, and a library of M₁ generation mutants was constructed and statistically analyzed for the mutation rates of their phenotypic traits. In addition, the M₁ generation material was subjected to targeted screening with 250 mmol/L NaCl solution to obtain salt-tolerant mutants, and the mutants were phenotyped as well as measured for morphological indices, substance accumulation, and physiological indices. Result The 3 709 plants survived in M₁ generation with a survival rate of 37.09%, of which 1 226 had phenotypic mutant traits with a mutation rate of 33.05%. The 1 226 plants were rich in mutation types, containing 151 mutations for leaf traits, 212 mutations for spike traits, 161 mutations for tillers, 133 mutations for plant height, 130 mutations for fertility and fecundity, and 75 mutations for other traits, with mutation rates of 13.89%, 5.72%, 4.34%, 3.59%, 3.50%, and 2.02%, respectively. After salt stress treatment, Ningchun No. 4 showed yellowing and wilting of leaves, significant differences in seedling height, maximum root length, number of primordial roots, aboveground fresh weight, root fresh weight, aboveground dry weight, and root-crown ratio compared to the mutant, while root dry weight showed no significant change. In terms of physiological indices, malondialdehyde (MDA) content and cell membrane permeability of the mutants were significantly lower than those of the wild type, proline content, peroxidase (POD) and catalase (CAT) activities were significantly higher than those of the wild type, and there was no significant difference in the soluble sugar content. Conclusion The types of mutants produced by EMS mutagenesis from Ningchun No. 4 are abundant, providing rich germplasm resources for rapid localization of wheat genes for excellent traits and study of their functions.

Identification of LHC Gene Family in Setaria italica and Expression Analysis under Abiotic Stresses

CHENG Xue, FU Ying, CHAI Xiao-jiao, WANG Hong-yan, DENG Xin

2025, 41(8):  102-114.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0187

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Objective ​​Light-harvesting complexes (LHCs)​​ serve as essential structural components of the photosynthetic apparatus and are also implicated in the responses to abiotic stress. In this study, bioinformatics analysis was carried out to identify the LHC gene family in foxtail millet (Setaria italica) at the whole-genome level, with further analysis of its expression profiles under diverse abiotic stress conditions. It would be helpful for the functional study of genes related to photosynthesis and provide theoretical support for molecular breeding in foxtail millet. Method The members of LHC gene family were obtained from the foxtail millet genome. Subsequently, a series of analysis were conducted, including phylogenetic analysis, physicochemical properties and subcellular localization, gene structure, conserved motif identification, and promoter cis-acting elements. Transcriptomic data and RT-qPCR were combined to investigate gene expression across different tissues and under abiotic stress conditions. Result A total of 30 LHC gene family members were identified and classified into four subfamilies of Lhc, Lil, PsbS and FCⅡ. These genes were unevenly distributed on nine chromosomes. Synteny analysis revealed one pair of segmental duplications and two pairs of tandemly duplicated genes. Cis-acting elements in the gene promoters of all members included light-responsive, hormone-responsive, abiotic stress-responsive and plant defense/stress-responsive elements. Protein structure prediction indicated that secondary structures were predominantly α-helices and random coils, while tertiary structures demonstrated subfamily-specific features. Strong protein-protein interactions were observed within the SiLhc subfamily. Expression analysis showed that 27 SiLHCs were highly expressed in the leaves. Members within the same subfamily presented functional divergence in response to identical stress conditions, while genetic background further drove differential abiotic stress responses. SiLhcb4, SiLhcb5 and SiFCⅡ were identified as abiotic stress-sensitive genes by RT-qPCR analysis, displaying significant downregulation under drought, salinity, and alkaline stress conditions. Conclusion The structural and functional similarities among the four subfamily members of SiLHC, combined with their divergent transcriptional responses to abiotic stresses, suggested their roles in the photosynthetic system and the regulation of responses to abiotic stress.

Heterologous Overexpression of Sorghum SbSnRK2.1 Enhances the Resistance to Salt Stress in Arabidopsis

LI Ya-qiong, GESANG La-mao, CHEN Qi-di, YANG Yu-huan, HE Hua-zhuan, ZHAO Yao-fei

2025, 41(8):  115-123.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0114

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Objective This study aims to identify SNF1-RELATED KINASE 2 (SnRK2) genes from the sorghum genome that regulate plant responses to salt stress, thereby providing a theoretical basis for investigating salt tolerance mechanisms in sorghum. Method Bioinformatics techniques were employed to identify and analyze the sequence characteristics of sorghum SnRK2 gene family members (SbSnRK2s). Quantitative PCR was used to examine the dynamic expression patterns of SbSnRK2s within 12 h of salt stress treatment. Genes showing significant upregulation under salt stress were heterologously overexpressed in Arabidopsis thaliana to evaluate changes in salt stress tolerance. Result Eleven SbSnRK2s genes were identified in the sorghum genome, unevenly distributed across seven chromosomes. Phylogenetic analysis of protein sequences revealed close evolutionary relationships between SbSnRK2s and SnRK2 genes from maize, another C₄ plant. Promoter analysis identified regulatory elements responsive to hormones and stress. All 11 SbSnRK2s exhibited upregulated expression under salt stress, with SbSnRK2.1 showing the most pronounced induction (>150-fold increase at 9 h post-treatment). Arabidopsis thaliana heterologously expressing SbSnRK2.1 demonstrated significantly higher survival rates, primary root lengths, and fresh weights compared to wild-type plants under salt stress conditions. Conclusion SbSnRK2.1 demonstrates remarkable upregulation in response to salt treatment, and its heterologous overexpression significantly enhances the tolerance of Arabidopsis thaliana to salt stress, indicating its crucial role in plant responses to salinity stress.

Mining the Core Genes Being Tolerant to Cadmium in Wild Soybean by WGCNA

ZHU Li-juan, ZHANG Kai, WEN Xiao-lei, CHU Jia-hao, SHI Feng-yu, WANG Yan-li

2025, 41(8):  124-136.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1113

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Objective Wild soybean has characteristics of tolerance to stress, gradually becoming a germplasm resource for improved cultivated soybeans. It is significant to clarify the molecular regulation mechanism of tolerance to cadmium in wild soybeans and provide a basis for cultivating tolerant soybean varieties. Method In this study, 200 wild soybeans in eastern Hebei were used as experimental materials. Wild soybean seedlings were treated with Hoagland nutrient solution containing 75 mol/L CdCl₂, and the dry weight of the seedlings was measured. Transcriptome sequencing was performed on Cd-tolerant wild soybean R and sensitive material S at 24 h and 48 h under Cd treatment, respectively. KEGG and GO enrichment analyses were performed on differentially expressed genes (DEGs), and weighted gene co-expression network analysis (WGCNA) was used to detect Cd-tolerant core genes. Result The 200 wild soybean seedlings have shown remarkable differences under stress. Compared with the control, the shoot dry weight and root dry weight of seedlings under cadmium treatment significantly reduced. Transcriptome analysis showed that 6 443 and 4 496 DEGs were identified in R and S materials, respectively. GO and KEGG analyses found that these DEGs were enriched in photosynthesis and stress response pathways. Combined with WGCNA, the key turquoise and blue modules were significantly correlated with cadmium tolerance in wild soybeans. According to the connectivity and functional annotation of the genes in the module, it was predicted that 8 genes, such as LOC114376469, LOC114412091, LOC114388638, and LOC114399512 may play a role in the process of cadmium stress in wild soybean. Conclusion Two specific modules related to the tolerance of wild soybean to cadmium are identified, and LOC114376469, LOC114412091, LOC114388638, and LOC114399512 are screened to be the core genes related to the tolerance of wild soybean to cadmium.

Heterologous Overexpression of Soybean GmNF-YB24 Improves the Resistance of Transgenic Tobacco to Drought

ZHAI Ying, JI Jun-jie, CHEN Jiong-xin, YU Hai-wei, LI Shan-shan, ZHAO Yan, MA Tian-yi

2025, 41(8):  137-145.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0046

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Objective Nuclear factor (NF-Y) genes are involved in the regulation of drought resistance in plants. The function of resistance to drought and mechanism of resistance to drought of soybean GmNF-YB24 were analyzed, which may provide theoretical basis for the subsequent application of NF-YB genes in the genetic breeding of soybean with high resistance. Method The expression of GmNF-YB24 under salt, drought and cold stresses was detected by RT-qPCR. GmNF-YB24 was cloned and its plant expression vector was constructed to transformed tobacco. The resistance of GmNF-YB24 transgenic tobacco to drought was evaluated. Result Salt, drought and cold stresses induced the expression of GmNF-YB24, and the expression of GmNF-YB24 was the most obvious in response to drought stress. The open reading frame of GmNF-YB24 was 516 bp, and the encoded protein contained 171 amino acid residues. The GmNF-YB24 protein contained a histone-fold motif (HFM). The plant expression vector pRI101-GmNF-YB24 was constructed and transformed into tobacco. Five GmNF-YB24 transgenic tobacco plants (OE1-OE5) were obtained. After drought and rewater treatment, the performance of GmNF-YB24 transgenic tobacco was superior to that of wild-type tobacco. Under drought stress, compared with wild-type tobacco, GmNF-YB24 transgenic tobacco had more proline and soluble carbohydrate, lower relative electrolyte leakage and malondialdehyde content, and antioxidant enzyme activity increased and accumulation of peroxide reduced. The expressions of stress-related genes NtOsmotin and NtERD10B in GmNF-YB24 transgenic tobacco were higher than those in wild-type tobacco. Conclusion Heterologous overexpression of GmNF-YB24 in tobacco improves the resistance of transgenic tobacco to drought.

Identification and Expression Analysis of GH17 Gene Family of Pyrenophora graminea

REN Rui-bin, SI Er-jing, WAN Guang-you, WANG Jun-cheng, YAO Li-rong, ZHANG Hong, MA Xiao-le, LI Bao-chun, WANG Hua-jun, MENG Ya-xiong

2025, 41(8):  146-154.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0104

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Objective Glycoside hydrolase 17 (GH17) plays a significant role in various physiological and pathogenic processes in plants, animals and fungi. The identification and preliminary analysis of the PgGH17 family members in the Pyrenophora graminea provide a scientific basis for further in-depth research on the function of GH17 genes, and concurrently lay a solid theoretical foundation for understanding the function of pathogenicity genes in the P. graminea and exploring the host-pathogen interaction mechanism. Method Using P. graminea as the research subject, we identified and analyzed gene families through bioinformatics approaches. Subsequently, real-time quantitative PCR (RT-qPCR) was employed to monitor the temporal expression patterns of these genes during the infection process. Result The PgGH17 gene family comprises six members, with amino acid sequence lengths ranging from 305 to 648 aa. All proteins are hydrophilic, exhibiting isoelectric points (pI) of 5.06–8.66 and aliphatic indices of 65.43–82.00. Signal peptide lengths vary from 0 to 22 aa, with most members localized to the extracellular matrix; only one gene harbors a transmembrane domain. Intron numbers range from 1 to 3 per gene. Phylogenetic analysis revealed that these family members cluster into seven distinct subclades. Quantitative reverse transcription PCR (RT-qPCR) results demonstrated that, with the exception of PgGH17-3, the expressions of other PgGH17 genes during infection generally present first an initial upregulation followed by subsequent downregulation. Conclusion Six GH17 family members are identified in P. graminea, and with the exception of GH17-3, all show upregulated expressions during infection, suggesting their functional involvement in the pathogenicity of this fungal pathogen.

Unfolded Protein Response Enhances Plant Resistance to Disease by Regulating Tryptophan Metabolism

CAO Yuan-yuan, ZHOU Shu-hao, ZHANG Hai-rong, CUI Xiao-na

2025, 41(8):  155-164.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0275

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Objective To investigate the relationship between endoplasmic reticulum stress (ERS) induced unfolded protein response (UPR), tryptophan metabolism, and plant disease resistance, and to clarify the regulatory mechanisms of the transcription factors bZIP28 and bZIP60 in tryptophan metabolism and their impact on plant disease resistance. Method RNA-seq and qRT-PCR were used to analyze the expressions of genes involved in the tryptophan metabolism pathway under ER stress conditions. The synthesis of tryptophan-derived metabolites, camalexin and IAA, was measured under ER stress. The expressions of bZIP28 and bZIP60 after pathogenic infection and the disease resistance of plants overexpressing these genes were assessed. Yeast one-hybrid assays and dual-luciferase reporter assays were conducted to examine the regulation of WRKY33 by bZIP28/60. Result ER stress triggered the UPR, leading to the up-regulation of tryptophan metabolic genes and increased levels of camalexin and IAA. Pathogenic infection up-regulated the expressions of bZIP28 and bZIP60. Plants overexpressing bZIP28/60 demonstrated enhanced resistance to disease. bZIP28/60 indirectly regulated the expression of WRKY33, with the core regulatory sequence located within the -364 to -566 bp region of the WRKY33 promoter. Conclusion ER stress modulates the expression of WRKY33 through the transcription factors bZIP28 and bZIP60, resulting in elevated levels of camalexin and IAA, thereby enhancing resistance to pathogen infection and alleviating ER stress.

Genetic Diversity of 220 Pepper Germplasm Resources Using SCoT， SRAP， and SSR Molecular Markers

PEI Hong-xia, WANG Lu-yao, LI Sheng-mei, GAO Jing-xia

2025, 41(8):  165-174.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0208

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Objective To comprehensively analyze the genetic diversity and phylogenetic relationships of pepper germplasm resources preserved at the Ningxia Academy of Agriculture and Forestry Sciences, providing a theoretical foundation for the conservation, introduction, and application of pepper germplasm resources. Method Genetic diversity analysis was conducted on 220 pepper germplasm resources from 15 regions across China using three molecular marker techniques: SSR, SRAP, and SCoT. Result Six SCoT markers amplified 1 to 4 bands, 14 SRAP markers amplified 2 to 4 bands, and 3 SSR markers amplified 2 to 5 bands, with all markers showing 100% polymorphic band percentages. Further analysis revealed that SSR markers had the highest average polymorphic information content (PIC) of 0.77, significantly higher than SCoT (0.55) and SRAP (0.55), indicating that SSR markers were more advantageous for detecting genetic diversity, with HpmsE088 being the most effective SSR marker. Cluster analysis, principal coordinate analysis, and population genetic structure analysis divided the germplasm from the 15 regions into two major groups (Group 1 and Group 2). Group 1 included germplasm from Sichuan, Shenyang, Xinjiang, Shandong, Ningxia, and Hunan in China, while Group 2 included germplasm from China (Yunnan, Jiangsu, Henan, Gansu, Beijing, and Anhui), Netherlands, Japan, and the United States. Additionally, 83.18% of the germplasm demonstrated high homozygosity. A correlation was observed between fruit shape characteristics and cluster grouping, with a significantly higher proportion of linear fruit pepper germplasm in Group 1 compared to Group 2, while all short horn-shaped fruit pepper germplasm were classified into Group 2. Conclusion The pepper germplasm resources preserved at the Ningxia Academy of Agriculture and Forestry Sciences show high genetic diversity and can be primarily divided into two groups based on genetic information. The findings provide a theoretical basis for future introduction strategies and parental selection.

Growth-promoting Mechanism of Trichoderma harzianum T9131 on Astragalus membranaceus Seedlings

BAI Yu-guo, LI Wan-di, LIANG Jian-ping, SHI Zhi-yong, LU Geng-long, LIU Hong-jun, NIU Jing-ping

2025, 41(8):  175-185.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0377

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Objective To explore the regulatory mechanism of Trichoderma harzianum T9131 on the growth promotion of Astragalus membranaceus seedlings and provide a theoretical basis for T9131 to increase the yield of A. membranaceus. Method The A. membranaceus seedlings grown for 30 d were treated by root irrigation with T. harzianum T9131 spores suspension. Samples were taken from the roots of the A. membranaceus seedlings at 0, 24 h, 48 h and 45 d after root irrigation, and the plant height, root length and fresh weight of the A. membranaceus seedlings at 45 d were determined. Transcriptome libraries were constructed at three time points of 0, 24 and 48 h by RNA-Seq. Differentially expressed genes (DEGs) in the three comparison groups (T_24 h vs T_0 h, T_48 h vs T_0 h, and T_48 h vs T_24 h) were obtained by using DESeq2 software. Venn analysis was conducted on the three groups of DEGs to clarify that the three groups had a total of DEGs. KEGG enrichment analysis was performed on the common DEGs to screen the growth-promoting related genes, and the expression of growth-promoting-related genes in A. membranaceus seedlings in the early stage after 45 d of induction with T9131 and induction with exogenous hormone ABA was analyzed by RT-qPCR. The contents of 15 hormones in the root samples at 0 h, 48 h and 45 d were determined by the LC-MS/MS method. Result T9131 could significantly promote the plant height and fresh weight of A. membranaceus seedlings.Transcriptome analysis of A. membranaceus seedlings induced by T9131 indicated that there were 149 differentially expressed genes in the three comparison groups. KEGG enrichment pathway analysis showed that the pathways related to growth promotion mainly included terpenoid backbone biosynthesis, carotenoid biosynthesis, diterpenoid biosynthesis, cyanoamino acid metabolism, zeatin biosynthesis, and plant hormone signal transduction. The gene types enriched in the pathway include HMGR, CYP707A2, GA2ox2, E3.2.1.21, UGT73C6, PYL4, GH3.1, and JAZ, totaling 14 genes. Except for the gene UGT73C6 (DN24891_c1_g1) down-regulated by T9131 induction, the remaining genes were all up-regulated in expression. Fourteen genes induced A. membranaceus seedlings in T9131 at 45 d, seven genes including HMGR (DN10463_c0_g2, DN989_c0_g1, DN113450_c0_g1, DN989_c2_g1), CYP707A2 and JAZ were significantly up-regulated, while the remaining genes were down-regulated. Analysis of the expressions of the 14 genes induced by ABA showed that the expressions of 6 genes, including HMGR (DN10463_c0_g2, DN989_c0_g1, DN113450_c0_g1, DN989_c2_g1), CYP707A2 (DN397_c0_g2), and E3.2.1.21 (DN2073_c0_g1), were significantly down-regulated by ABA induction, while the expressions of the remaining genes were up-regulated. Hormone determination indicated that T9131 significantly reduced the content of ABA. Conclusion T. harzianum T9131 may promote the growth of A. membranaceus by regulating the up-regulated expressions of genes related to the growth promotion of A. membranaceus, such as HMGR, CYP707A2 and JAZ, and by reducing the content of ABA. Exogenous ABA can reduce the expressions of HMGR and CYP707A2.

The Role of NbJAZ3 in the Infection of Nicotiana benthamiana by Alfalfa Mosaic Virus

LAI Shi-yu, LIANG Qiao-lan, WEI Lie-xin, NIU Er-bo, CHEN Ying-e, ZHOU Xin, YANG Si-zheng, WANG Bo

2025, 41(8):  186-196.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0084

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Objective To explore the effect of NbJAZ3, a gene encoding jasmonate ZIM-domain protein 3 (JAZ3), on the infection of Nicotiana benthamiana by alfalfa mosaic virus (AMV), and to provide a theoretical basis for further analyzing the mechanism of AMV infection. Method Using N. benthamiana and AMV as materials, AMV was inoculated into N. benthamiana by friction inoculation method. The leaves of N. benthamiana at 1, 7, 15 and 21 d after inoculation were used for transcriptome sequencing analysis. The differential genes in jasmonic acid (JA) signal transduction pathway were screened, and the expression of NbJAZ3 in AMV infection was detected by real-time fluorescence quantitative PCR (RT-qPCR). NbJAZ3 was cloned, biological information was analyzed, phylogenetic tree was constructed, and multiple sequence alignment was performed.The transient overexpression vector of pCAMBIA1300-NbJAZ3 was constructed. The subcellular localization of NbJAZ3 protein was observed by laser confocal microscopy, and the protein expression was detected by Western blot. NbJAZ3 was transiently overexpressed in N. benthamiana leaves and inoculated with AMV, and the relative expression of AMV CP at 5 d after inoculation was detected by RT-qPCR. The pTRV2-NbJAZ3 silencing vector was constructed by gene silencing technology induced by tobacco brittle fracture virus. After silencing NbJAZ3, AMV was inoculated by friction. The relative expression of AMV CP was detected by RT-qPCR. Result Transcriptome sequencing and RT-qPCR further verified that NbJAZ3 was down-regulated on different days after AMV inoculation, and the relative expression of NbJAZ3 was the lowest on the day15, which was 84.43% lower than that of CK. The NbJAZ3 CDS sequence was 1 143 bp in length, encoding 380 amino acids, containing two conserved domains of ZIM and Jas-motif. NbJAZ3 had the highest homology with NsJAZ3 of Nicotiana sylvestris, and the similarity was 96.58%. Microscopic observation showed that NbJAZ3 was localized in the nucleus, cytoplasm and cell membrane of N. benthamiana leaves. Compared with the control group, the relative expression of AMV CP decreased by 83.82% and increased by 78.58% in N. benthamiana with transient overexpression and transient silencing of NbJAZ3 on the day 5 of AMV inoculation, respectively. Conclusion AMV infection can cause the down-regulation of NbJAZ3 in N. benthamiana. Transient overexpression of NbJAZ3 can inhibit the infection of AMV, while silencing this gene can promote the infection of AMV, further indicating that NbJAZ3 can be used as a positive regulator of N. benthamiana resistance to AMV infection and play an important role in resisting virus infection.

Genome-wide Identification and Expression Analysis of bZIP Transcription Factor Family in Dendrobium officinale

ZENG Dan, HUANG Yuan, WANG Jian, ZHANG Yan, LIU Qing-xia, GU Rong-hui, SUN Qing-wen, CHEN Hong-yu

2025, 41(8):  197-210.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0094

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Objective To identify the bZIP gene family of Dendrobium officinale,and clarify the biological function of DobZIP transcription factors through bioinformatics and gene expression analysis, thus aiming to provide theoretical guidance for further exploration of the molecular mechanism of DobZIP transcription factors. Method Based on the genome data of Dendrobium officinale in the IMP database, we identified the members of DobZIP gene family, and performed multiple of bioinformatics analysis. Furthermore, we used transcriptome data and real-time fluorescent quantitative PCR technology to analyze the tissue expression pattern and abiotic stress expression of DobZIP transcription factors. Result A total of 54 DobZIP genes were identified. None of the DobZIP proteins had signal peptides, and only DobZIP54 had a transmembrane domain. The subcellular localization of the DobZIP proteins were mainly in the nucleus. Members of the DobZIP family were classified into 10 subfamilies, and members of the same subfamily contained similar structures and motifs. DobZIP genes were unevenly distributed on 18 chromosomes, with two pairs of genes showing tandem relationships. There were multiple collinear relationships between species, and DobZIP family had a high homology with the OsbZIP family in rice. Cis-acting elements analysis indicated that the DobZIP family members all contained multiple cis-acting elements involved in growth and development and response to abiotic stress. Gene expression pattern analysis showed that the DobZIP family members had tissue-specific expression, and DobZIP4、DobZIP10、DobZIP28、DobZIP32 and DobZIP37 were induced by cold, drought and NaCl stress to varying degrees. In addition to DobZIP32 and DobZIP37, DobZIP4, DobZIP10 and DobZIP28 were also induced by high temperature stress. Conclusion A total of 54 DobZIP genes are identified in the genome of Dendrobium officinale. DobZIP genes was in tissue-specific expression, and DobZIP4, DobZIP10, DobZIP28, DobZIP32 and DobZIP37 are induced to varying degrees under cold, drought, and NaCl stress.

Identification and Expression Patterns Analysis of ADH Gene Family in Salvia miltiorrhiza

HUA Wen-ping, LIU Fei, HAO Jia-xin, CHEN Chen

2025, 41(8):  211-219.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1233

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Objective Alcohol dehydrogenase (ADH) is one of the key enzymes in plant fermentation metabolism under anaerobic conditions, and plays important roles in plant resistance to waterlogging stress. This work is aimed to identify the SmADH genes from Salvia miltiorrhiza and provide a theoretical basis for the functional study of the SmADH gene family and the breeding of flood-resistant varieties. Method Bioinformatics methods were used to identify members of the gene family based on the S. miltiorrhiza genome data, and to analyze their physicochemical properties, sequence characteristics, evolutionary, and expression regulation. The RT-qPCR was then used to study the expression patterns of SmADHs under different stresses. Result A total of seven SmADH genes were identified, belonging to two subfamilies. SmADH proteins were mainly acidic proteins. The number of encoded amino acids ranged from 379 to 438, with a molecular weight of 40.79 to 46.89 kD. Except for SmADH5/6 localized in chloroplasts, other members were localized in the cytoplasm. Genetic feature analysis showed that members of the same subfamily of SmADHs possessed conserved motifs, similar intron numbers, and intron distributions. Chromosome localization revealed that SmADH1 was located on chromosome 1, SmADH2/3/4 on chromosome 2, SmADH5/6 on chromosome 6, and SmADH7 on contig030. Regulation by miRNA analysis showed that SmADH3/4/5/6 were regulated by miRNA family members such as smi-miR167, smi-miRN5 and smi-miR482. Promoter cis-acting element analysis showed that the promoter regions of SmADHs contained a large number of stress-responsive and hormone-responsive elements. Transcriptome data and qRT-PCR analysis indicated that SmADH2/5/6 had the highest expressions in S. miltiorrhiza roots. Under waterlogging stress, SmADH2/6 expression was rapidly upregulated. After salt stress, the expression of SmADH2/6 was significantly upregulated, while the expression of SmADH5 was inhibited. The expression of SmADH2/5/6 was significantly upregulated after PEG treatment, while the expression of SmADH6 was induced by GA3. Conclusion SmADH2/5/6, which are highly expressed in the roots of S. miltiorrhiza, play a role under various stresses, and SmADH2/6 play a particularly important role in resisting waterlogging stress.

Identification of Plasma Membrane H⁺-ATPase Gene Family in Safflower and Expression Analysis in Response to Low Nitrogen and Low Phosphorus Stress

LA Gui-xiao, ZHAO Yu-long, DAI Dan-dan, YU Yong-liang, GUO Hong-xia, SHI Gui-xia, JIA Hui, YANG Tie-gang

2025, 41(8):  220-233.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0057

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Objective Genome-wide identification and analysis of the plasma membrane H⁺-ATPase (PMA) gene family in safflower (Carthamus tinctorius L.) may provide the basis for investigating this gene family and its function in response to low nitrogen and low phosphorus stress. Method Bioinformatics was used to identify and systematically analyze the members of the CtPMA gene family. Real-time fluorescence quantitative PCR (RT-qPCR) was applied to analyze the tissue-specific expression pattens and the responses to low nitrogen and low phosphorus stress. Result A total of 10 CtPMAs (named CtPMA1-CtPMA10) were identified in safflower, encoding proteins ranging from 785 to 958 aa, with relative molecular weight of 85.23 to 105.52 kD, and isoelectric points of 5.26 to 7.91. All were predicted to be localized to the plasma membrane by the subcellular localization. All members contained conserved domains, including the Cation_ATPase_N domain, the E1_E2 ATPase domain, and the HAD-superfamily hydrolase subfamily ⅢA domain. Cis-acting element analysis revealed a lot of regulatory elements related to growth, development, hormones and stress responses. Gene collinearity indicated that whole-genome duplication and segmental duplication played key roles in the evolution of the CtPMA gene family. RT-qPCR results showed that the CtPMA1 and CtPMA7, which were highly expressed in the roots, were significantly induced by low nitrogen and low phosphorus stress. This finding suggests that CtPMA1 and CtPMA7 may play a crucial role in the process of nitrogen and phosphorus uptake in safflower roots. Conclusion A total of 10 CtPMAs members are identified in the safflower genome.These members demonstrate evolutionary conservation and tissue-specific expression patterns. The highly expressed CtPMA1 and CtPMA7 transcripts in the roots were significantly induced under low nitrogen and phosphorus stress. This suggests that safflower may improve the absorption and utilization efficiency of nitrogen and phosphorus by increasing its CtPMAs transcription level in order to resist nutritional stress.

Cloning of Gene CtbHLH128 in Safflower and Response Function Regulating Drought Stress

LI Kai-jie, WU Yao, LI Dan-dan

2025, 41(8):  234-241.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0059

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Objective Basic helix-loop-helix (bHLH) transcription factors represent one of the largest families of transcription factors in eukaryotes, and their members play crucial roles in plant growth, development, and stress responses. This study enriches the research on the function of the bHLH transcription factor family genes and lays an important foundation for analyzing the molecular regulatory mechanism of safflower (Carthamus tinctorius L.) seed development. Method Based on the complete CDS sequence of CtbHLH128 in the genome, primers were designed using Primer 6.0 software to clone and obtain the correct sequence of the gene, and bioinformatics was used to analyze the number of amino acids encoded by the gene, conserved domains, and phylogenetic tree. The CtbHLH128 fragment was constructed into a plant expression vector and transformed into Arabidopsis thaliana by Agrobacterium infection. Herbicide screening and PCR were used to identify positive strains. Drought stress was applied to the positive strains at the seedling and germination stages to explore the role of this gene in drought stress. Result The CtbHLH128 consists of 948 bp nucleotides, encoding 315 amino acids. The molecular weight of its encoded protein is 33 742.32 Da, and the theoretical isoelectric point is 9.02. CD-Search analysis results revealed that its C-terminus contained a typical HLH conserved domain located at amino acid residues 249-301. Phylogenetic tree revealed high similarity with bHLH128 gene in sunflower and lettuce. The CtbHLH128 gene was overexpressed in Arabidopsis, and three T3 positive strains were obtained through resistance screening and PCR identification. The positive strains were subjected to drought stress-related phenotypic analysis. The results revealed that transgenic Arabidopsis plants showed greater sensitivity to drought conditions at the seedling stage, but enhanced resistance to drought at the germination stage. Conclusion This suggests that the CtbHLH128 gene may possess potential functions in the responses to drought stress and seed germination.

Genome-wide Identification and Expression Pattern Analysis of WRI1 Gene Family in Erythropalum scandens

HUANG Shi-yu, TIAN Shan-shan, YANG Tian-wei, GAO Man-rong, ZHANG Shang-wen

2025, 41(8):  242-254.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1212

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Objective Identification of the WRI1 transcription factor in Erythropalum scandens provides a foundation for further investigation into its role in lipid biosynthesis. Method The whole genome data of E. scandens were used to identify the WRI1 transcription factors in E. scandens by bioinformatics methods. Based on transcriptome data, the expression patterns of WRI1 in different tissues of E. scandens were analyzed. Result A total of 17 WRI1proteins (EsWRI1-1-EsWRI1-17) containing two AP2 conserved domains were identified in E. scandens. The number of amino acids was 359-776, and the average molecular weight was 56 kD. Most of them were acidic and unstably hydrophilic proteins. Subcellular localization showed that it was mostly located in the nucleus, and 17 EsWRI1s members were unevenly distributed on 8 chromosomes. The results of protein sequence phylogenetic analysis divided 17 WRI1 proteins into nine subfamilies (Group 1-Group 9). Among them, Group 3 members were closely related to Arabidopsis thaliana and Glycine max, and EsWRI1-10 was closely related to A. thaliana. The promoter analysis of the EsWRI1s gene family showed that it contained more motifs related to biotic and abiotic stress, plant response to hormone,and plant growth and development, among which the growth and development-related elements were the most. The analysis of gene expression patterns showed that the expressions of EsWRI1-10, EsWRI1-14 and EsWRI1-16 genes in immature kernels were higher than those in other tissues, among which EsWRI1-10 gene played a major role in the formation of kernels, EsWRI1-11, EsWRI1-17 and EsWRI1-7 were highly expressed in different tissues of E. scandens. The RT-qPCR validation was consistent with the transcriptome data. Conclusion A total of 17 WRI1 genes are identified. Their encoded proteins possess favorable thermal stability and hydrophilicity and are predicted to be mainly located in the cell nucleus. The EsWRI1s gene family shares high homology with that of A. thaliana, G. max and M. oleifera. Specific genes are expressed specifically in immature seed kernels, implying their significant roles in kernel growth and development and potentially in lipid synthesis of E. scandens.

Cloning and Expression Analysis of the UV-B Receptor Gene McUVR8 in Mentha canadensis L.

KANG Qin, WANG Xia, SHEN Ming-yang, XU Jing-tian, CHEN Shi-lan, LIAO Ping-yang, XU Wen-zhi, WU Wei, XU Dong-bei

2025, 41(8):  255-266.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0013

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Objective The ultraviolet-B (UV-B) receptor UVR8 plays a key role in regulating plant growth and development in response to adversity stress. The aim is to explore the protein properties and expression characteristics of McUVR8, a UV-B receptor gene in mint (Mentha canadensis L.), and to predict its interacting proteins, thus to provide theoretical references for the subsequent probing of the function of McUVR8. Method The McUVR8 gene was obtained by RT-PCR, and its physicochemical properties, protein structure and subcellular localization, and interacting proteins were analyzed and predicted using a variety of bioinformatics tools. Tissue and leaf sequence expression patterns of McUVR8 and its expression changes under different hormones and stresses were analyzed using RT-qPCR. Result McUVR8 is 1 353 bp in length, encoding 450 amino acids, distributed in both nucleus and cytoplasm, with conserved helix-looped RCC1 core domain and C17 and C27 domains, and there is high homology with SsUVR8 of Salvia splendens, SdBUR8 of Salvia divinorum and ShUVR8 of Salvia hispanica, which are also in the Labiaceae family. McUVR8 was expressed in different tissues and leaf sequences of M. canadensis, with some differences, with the lowest expression in the roots, the highest expression in the flowers, and the highest expression in the first leaf in different leaf sequences. In addition, the McUVR8 gene responded to MeJA, ABA, and drought, NaCl, and two heavy metal and aluminum stress treatments in both leaves and roots. Finally, McUVR8 was screened and verified to interact with McCOP1. Conclusion McUVR8 may have important roles in regulating mint growth and development, responding to hormonal or adversity signals, and it may be involved in regulating mint secondary metabolism, adversity response, and other processes by binding to COP1, WRKYs, and other interacting proteins.

Expression and Enzyme Activity Identification of Cell Wall Invertase Gene SmCWIN6 from Red Pitaya

ZHENG Qian-ming, YAN Shuang, XIE Pu, WANG Hong-lin

2025, 41(8):  267-275.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0388

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Objective Cell wall invertase (CWIN) degrades sucrose into glucose and fructose, and acts as the key enzyme for sugar metabolism in higher plants. The physiological functions of CWINs in soluble sugar metabolism of red pitaya (Selenicereusmonacanthus) fruit were investigated, which provides a basis for regulating and improving fruit flavor and quality. Method Total protein from adult stem tissues and fruit pulp at various developmental stages was extracted to detect the CWIN enzyme activity; the sequence domains of SmCWIN family were analyzed; the expression patterns of SmCWINs in flower, stem, fruit peel and pulp tissues were detected by gene digital expression profiles and real-time fluorescent quantitative PCR. The subcellular localization of SmCWIN6 was detected by transiently transformed in tobacco mesophyll cells. By expressed in the baker’s yeast (Saccharomyces cerevisiae) and then total protein was extracted, the enzyme activity of SmCWIN6 was analyzed in vitro. Result The CWIN enzyme activity in adult stems of red pitaya was low, reached highest at 23 d after pollination in fruit, and then decreased significantly with fruit ripening. Of the red pitaya SmCWIN family, only SmCWIN6 contained domains and conserved amino acid residues related to sucrose degradation. SmCWIN6 was the only CWIN member that expressed in fruits and mainly expressed at 20-25 d after pollination; after then gradually down-regulated with fruit development, and weakly expressed at fruit ripening (30 d after pollination). Subcellular localization assay together with the plasmolysis experiment showed that SmCWIN6 protein was located into the apoplast space, possibly in the cell wall. After been expressed by yeast cells and detected in vitro, the SmCWIN6 protein degraded sucrose into glucose and fructose with the maximum enzyme activity at pH=3.0. Conclusion Red pitaya SmCWIN6 degrades sucrose to produce glucose and fructose in the apoplast, is expressed at 20-25 d after pollination in fruits, and involved in the extracellular soluble sugars metabolism during fruit development.

Phylogenetic Analysis of Lonicera Sect. Nintooa Based on Chloroplast Genomes Data

WEI Yao, ZHANG Jing-jing, CUI Yun-xiao, LIU Yu, LIU Hai-rui

2025, 41(8):  276-288.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0116

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Objective Evolutionary analysis of chloroplast genomes of Sect. Nintooa (Lonicera) species is significant for the scientific conservation and development of resources related to Sect. Nintooa. It also provides references for the subsequent species identification of the genus Lonicera. Method Using the leaves of Lonicera similis and Lonicera pampaninii as materials, the chloroplast genomes of the two plants were sequenced, assembled and annotated, and the whole chloroplast genomes were obtained. Result The sequence of whole chloroplast genome of L. similis was 155 312 bp and that of L. pampaninii was 155 360 bp. The chloroplast genome of L. similis and L. pampaninii contained the same type and number of genes, 130 genes were identified, and GC content was 38.5%. The chloroplast genome structure of the two species were highly similar to other Lonicera plants, there were no significant expansion or contraction observed. There was gene deletion in L. calcarata. A total of 102 SSR loci were detected, with the highest proportion of mono-nucleotide repeat SSRs dominated by (A/T). Three regions with high variation were identified from the chloroplast genomes, each for LSC, SSC and IRb region. There were 32 high-frequency codons in the two plants which preferred to end with A/U. Phylogenetic analysis indicates that the Lonicera showed a close relationship to the Leycesteria. Among the species within Sect. Nintooa, the Subsect. Volubilis wereclustered to be a branch, and L. similis and L. pampaninii had the closest relationship. L. calcarata in Subsect. Calcaratae had long distance to Subsect. Volubilis. The reconstruction of ancestral area revealed that Himalaya and Southwest China probably were the original location of Sect. Nintooa as well as the species mainly migrated to the Southeast China, and then region F becomes the major settlement place. Conclusion The chloroplast genome of Sect. Nintooa contains a conserved genomic structure and gene composition. However, there are differences in the non-coding areas of L. calcarata. The Sect. Nintooa shows higher preference for codons ending in A/U. The ancestral distribution area of the Sect. Nintooa species is the Himalayas and the southwestern region of China.

Screening， Identification and Preservation of Lactic Acid Bacteria Inhibiting the Growth of Rot-causing Fungus Lonicera caerulea L.

LI De-hai, YIN Li, ZHOU Cai-xue, WANG Ze-tong, SUN Chang-yan

2025, 41(8):  289-299.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0107

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Objective To screen and identify a strain of lactic acid bacteria that can inhibit rot-causing fungi in post-harvest Lonicera caerulea L., and to explore its effect on post-harvest preservation of L. caerulea L, this may provide new technical means to solve the problem of post-harvest rotting of L. caerulea L. Method After the isolation and purification of lactic acid bacteria from pickles and fermented berries, the P-2, which can inhibit the rot-causing fungi (Alternaria alternata and Rhizopus microsporus) in post-harvest L. caerulea L., were screened and identified by 16S rRNA gene sequence analysis. To evaluate the antibiotic sensitivity of P-2 and the inhibitory effect of cell-free supernatant (CFS) on rot-causing bacteria under different conditions. At last, P-2 CFS was used as a preservative to keep L. caerulea L fresh. Result Among the 22 strains of isolated and purified lactic acid bacteria, P-2 strain showed the best bacteriostatic effect and was identified as Pediococcus acidilactici. P-2 is resistant to kanamycin, gentamicin and streptomycin, sensitive or neutral to other antibiotics, its CFS has strong antifungal activity under acidic conditions, and is sensitive to protease. The MIC of P-2 CFS against A. alternata and R. microsporus were 12.8 and 25.6 mg/mL, respectively. Compared with the control group, the CFS of P-2 reduced the weight loss rate and decay rate of L. caerulea L, slowed down the decline of hardness, total phenol, flavonoid and vitamin C content of L. caerulea L during storage, and prolonged the shelf life of L. caerulea L. for at least 7 d. Conclusion The CFS of P. acidilactici P-2 can inhibit the post-harvest rot-causing bacteria of L. caerulea L., and can be used as a green preservative in the post-harvest preservation of berries.

Analysis of the Growth-promoting Effects and Active Components of Volatile Organic Compounds Produced by Bacillus velezensis NZ-4

SHI Yan-hua, LI Shuo, GAO Yu-zhu, ZHENG Bao-kun, ZHU Jie-hua, ZHANG Dai, YANG Zhi-hui

2025, 41(8):  300-310.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0135

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Objective In previous research, five Bacillus strains were identified to secret volatile substances with antifungal effects. This study aims to further elucidate the growth-promoting function of volatiles produced by biocontrol Bacillus strains and to identify their active volatile compounds. The goal is to obtain the strains that demonstrate both antifungal and plant-promoting properties, and to develop the volatile natural products with growth-promoting effects. Method The best growth-promoting strains were comprehensively evaluated using the pot fumigation method, selective medium assay and an entropy calculation method. The effect of volatile substances secreted by the strains on the contents of three endogenous hormones,cytokinin, gibberellin, and auxin in potato leaves,was determined by high-performance liquid chromatography (HPLC). The expressions of related genes (StCKX1, StLAX1, and StGA2ox1) were analyzed using real-time quantitative PCR. The specific components of VOCs produced by the strain were detected using two-dimensional gas chromatography-mass spectrometry (GC×GC-MS), and identified by the NIST/EPA/NIH database. Three pure compounds with the highest relative contents were then procured, and the plant growth-promoting effects of these substances on plants were tested in vitro. Result The heights and root lengths of plants increased by 40.01% and 15.15%, respectively, after fumigation with volatiles from Bacillus velezensis NZ-4. B. velezensis NZ-4 was also capable of solubilizing phosphorus, fixing nitrogen, and producing siderophore. It received the highest comprehensive score (0.947 6) in the evaluation using the entropy method and was identified as the optimal growth-promoting strain. The levels of cytokinin, gibberellin, and auxin represented increases of 1.09-fold, 0.87-fold, and 1.89-fold, compared to the control group.The expressions of the StGA20ox1 and StLAX1 genes increased by 3.61-fold and 4.71-fold, respectively. GC×GC-MS analysis revealed that 2-heptanone, 3-ethyltoluene, and 2-nonanone were the most abundant compounds. A concentration of 1 μg/μL of 2-heptanone and 100 ng/μL of 3-ethyltoluene significantly promoted plant growth, while 2-nonanone had no significant effect. Conclusion The VOCs produced by B. velezensis NZ-4 promote the growth of potato, with 2-heptanone and 3-ethyltoluene identified as the active volatile compounds. These VOCs have potential for development into novel natural products with growth-promoting properties.

Combined Transcriptomic and Metabolomic Analysis of Virulence Differences among Three Lecanicillium lecanii Strains

CHAI Jun-fa, HONG Bo, JIA Yan-xia

2025, 41(8):  311-321.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1281

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Objective To study the virulence differences among various strains of Lecanicillium lecanii, and to identify key differentially expressed genes (DEGs) and differential secondary metabolites (DEMs) associated with strain virulence through integrated transcriptomic and metabolomic data analyses. Method RNA-seq and LC-MS/MS techniques were used to detect the gene expression and secondary metabolites of three L. lecanii strains cultured for 8 d and liquid fermentation for 8 d, and the differential genes were verified by RT-qPCR. Result Indoor virulence results showed that J-1 secondary metabolites were the most virulent against Myzus persicae, with highly significant differences from J-2 and V-1. Transcriptome and metabolome results: 225 DEGs and 59 DEMs were obtained for J-1_vs_J-2, 2464 DEGs and 75 DEMs were obtained for J-1_vs_V-1. Differential genes and differential metabolites were enriched by KEGG analysis, mainly in phenylalanine metabolism, tyrosine metabolism and ABC transporter protein, 79 DEGs and 19 DEMs, salicylic acid, 3-hydroxyphenylacetic acid, phenyllactic acid, hippuric acid, and methyl beta-D-galactoside were significantly up-regulated and norepinephrine was highly significantly down-regulated between high and low virulence strains. c75905.graph_c0,c78027.graph_c0,c77968.graph_c0, c78586.graph_c1, c74779.graph_c0, and c78871.graph_c0 were significantly up-regulated. RT-qPCR results showed that the key differential genes were consistent with the expression trends in the transcriptome. Conclusion Six key genes and six significantly enriched differential metabolites that may be involved in regulating the virulence of L. lecanii strains are found by combined transcriptome and metabolome analysis, which may provide a basis for the construction of highly virulent strains.

Isolation and Identification of a High-yielding Magnolol and Honokiol Strain from Magnolia officinalis and Optimization of the “Sweating” Process

ZHANG Ru, LI Yi-ming, ZHANG Tong-xi, SUN Zhan-bin, REN Qing, PAN Han-xu

2025, 41(8):  322-334.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0422

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Objective To clarify the correlation between the microbial colonies and the quality of Magnolia officinalis during the process of “sweating”, and to investigate the regulatory mechanisms of key microbial species on the biosynthesis and accumulation of magnolol and honokiol in M. officinalis. Method Traditional culture separation and HPLC were systematically employed to isolate and screen functionally critical microbial strains during the “sweating” process of M. officinalis. The target strains were identified using molecular biological techniques, and phylogenetic trees were constructed to analyze their evolutionary status and genetic relationships. The process parameters of “sweating” of strain HP3 were optimized by using single-factor experiments combined with the Box-Behnken response surface method. Result Forty-eight strains were isolated and purified from the “sweating” M. officinalis samples. Subsequent screening revealed 9 strains that significantly increased the production of magnolol and honokiol. Among these, strain HP3 presented the most pronounced efficacy and was identified as Bacillus velezensis. The optimization of the “sweating” process parameters for strain HP3 revealed the following optimal conditions: Sweating temperature 31.84 ℃, sweating time 1.9 d, boiling time 6.37 min and drying temperature 80 ℃. Under these conditions, the total amount of magnolol and honokiol was as high as 5.149%, an increase of 62.67% compared with the non-sweated samples. Conclusion A strain HP3 was isolated and identified from the “sweating” process of M. officinalis, which demonstrated remarkable capability in enhancing the total content of magnolol and honokiol. Furthermore, the optimal sweating process parameters for strain HP3 were determined through response surface optimization experiments. The study has revealed the bacterial communities closely related to the quality of M. officinalis during its “sweating” process, providing an important basis for the development of microbial resources of M. officinalis medicinal materials.

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2025, 41(8):  335. 

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2025, 41(8):  336. 

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2025, 41(8):  337. 

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