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    26 September 2025, Volume 41 Issue 9
    Advances in RNA Interference Technology for Plant Functional Genomics and Crop Improvement
    HUANG Wen-jing, REN Si-chao, LIN Li, WANG You-ping, WU Jian
    2025, 41(9):  1-21.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0645
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    RNA interference (RNAi) is a sequence-specific gene silencing mechanism mediated by double-stranded RNA (dsRNA) and conserved in eukaryotes. Its molecular mechanism primarily involves the processing of dsRNA, the generation of small interfering RNA (siRNA) or microRNA (miRNA), and the sequence-specific degradation or translational inhibition of target mRNA mediated by the RNA-induced silencing complex (RISC). Based on this mechanism, RNAi technology has become a powerful tool for plant functional genomics research and demonstrates immense potential in the genetic improvement of crops for resistance to diseases and pests, as well as in green and sustainable pest control. This review systematically summarizes the discovery history and molecular mechanism of RNAi and focuses on its three core applications: Virus-induced gene silencing (VIGS) utilizes engineered viral vectors to achieve transient silencing of host genes, providing an efficient approach for plant gene functional studies. Host-induced gene silencing (HIGS) aims to breed transgenic crops with durable resistance to fungi, viruses, and pests by expressing RNAi molecules within the plant to target and silence key genes in pathogens or pests. Spray-induced gene silencing (SIGS), as a non-transgenic strategy, involves spraying designed target-specific dsRNA. Upon uptake by the target pathogen or pest, this dsRNA inhibits the expressions of their key genes, thereby interfering with their infection/infestation process, offering a novel, environmentally friendly strategy for disease and pest control. In the future, further advancements in RNAi technology are expected to benefit from the deep integration of synthetic biology, artificial intelligence, and nano-delivery systems to optimize target design, enhance silencing efficiency, and reduce off-target risks. With breakthroughs in key technologies and the overcoming of application bottlenecks, RNAi holds exceptionally broad application prospects for advancing plant science research and achieving sustainable agriculture.

    Advances in Spatial Metabolomics in Medicinal Plants
    LIU Yu-shi, LI Zhen, ZOU Yu-chen, TANG Wei-wei, LI Bin
    2025, 41(9):  22-31.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0276
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    Mass spectrometry imaging (MSI) is an innovative molecular imaging technique offering significant advantages, including label-free analysis, high molecular coverage, and exceptional sensitivity. MSI has been widely used in studies on the spatial distribution of metabolites in tissues. The continuous advancement of this technique has brought spatial metabolomics, a methodology that integrates MSI with metabolomics. This approach allows for simultaneous localization and imaging analysis of metabolites in tissues across diverse biological samples, encompassing animals, plants, and microorganisms. Recently, spatial metabolomics has demonstrated great potential in medicinal plant research as a crucial technique for precise spatial localization of metabolites in tissues. This review first presents the basic principles and experimental procedures of spatial metabolomics, followed by comparisons of several major MSI techniques, including matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), desorption electrospray ionization mass spectrometry imaging (DESI-MSI), and secondary ion mass spectrometry imaging (SIMS-MSI), where distinctive advantages and limitations of each approach are delineated, as well as key aspects in sample preparation and data processing are addressed. The review subsequently focuses on the applications of spatial metabolomics in medicinal plants, including visualizing the spatial distribution and accumulation pattern of metabolites, clarifying the biosynthesis and transport sites of metabolites, and discovering functional genes associated with biosynthetic pathways in medicinal plants. Finally, this review discusses the current challenges and prospects of spatial metabolomics, aiming to provide a new perspective for investigations in medicinal plant research.

    Research Progress in Genetic Transformation Technologies of Maize and Sorghum
    HU Lu, WANG Kai, XU Jing-yi, YE Li-hui, WANG Yong-fei, WANG Li-hua, LI Jie-qin
    2025, 41(9):  32-43.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0397
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    Plant genetic transformation technology, as a key method for crop genetic improvement, enables the stable integration of foreign functional genes to effectively regulate important traits such as plant growth and development, stress resistance, and environmental adaptability. The research on plant genetic transformation technology not only provides a key technical platform for gene function validation but also significantly promotes the establishment and development of precision crop breeding systems through the directed improvement of target traits. To achieve efficient and stable genetic transformation, researchers have been continuously optimizing and developing diverse technological approaches, primarily focusing on the effective delivery of exogenous genes and the high-efficiency regeneration of transformed plants. This review summarized the two primary methods for genetic transformation in maize (Zea mays L.) and sorghum (Sorghum bicolor (L.) Moench), the biolistic method and Agrobacterium-mediated transformation. The review highlighted critical factors affecting the efficiency of Agrobacterium-mediated transformation. And it also summarized key genes that enhance genetic transformation efficiency and their applications in plant genetic transformation. Additionally, the review summarized challenges and proposed strategies to optimize transformation systems using key regulatory genes. It also outlined the future research directions to provide insights for enhancing transformation efficiency and their applications in high-throughput molecular breeding.

    Establishment of TurboID Proximity Labeling Technology in Plants and Bacteria
    WANG Fang, SHAO Hui-ru, LYU Lin-long, ZHAO Dian, HU Zhen, LYU Jian-zhen, JIANG Liang
    2025, 41(9):  44-53.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0222
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    Objective TurboID-mediated proximity labeling technology is a novel protein-protein interaction research tool based on engineered biotin ligase, offering advantages including rapid labeling kinetics, high spatio-temporal resolution, and minimal cellular toxicity. However, its application remains limited to a few model species during current preliminary implementation stages. In this study, we developed TurboID expression systems in multiple representative species - monocots, dicots and prokaryotes - to systematically evaluate their labeling efficiency and applicability across evolutionary distant organisms. Method Recombinant vectors expressing “epitope tag-TurboID” fusion proteins were constructed for monocots (Oryza sativa), dicots (Arabidopsis thaliana, Solanum lycopersicum, and Nicotiana benthamiana), and bacteria (Escherichia coli), followed by transformation into respective species. Total proteins were extracted after biotin incubation, and fusion protein expression was verified via epitope tag-specific Western blot. Biotinylation of endogenous proteins was detected using streptavidin-HRP-based immunoblotting. Result Using ubiquitin promoters in plants and a T7 promoter in bacteria, we successfully achieved high-level expression of a “tag protein + TurboID” fusion protein in A. thaliana, S. lycopersicum, N. benthamiana, rice (Oryza sativa) callus, and E. coli. Western blot analysis confirmed robust expression of the fusion protein across all five systems. Following biotin treatment, multiple endogenous proteins were efficiently biotinylated in each system, indicating that the constructed TurboID system functioned effectively across monocots, dicots, and prokaryotes, demonstrating broad applicability and strong potential for further expansion. Conclusion A TurboID-based proximity labeling system is successfully established in monocotyledonous and dicotyledonous plants, as well as in bacteria, providing an efficient and reliable platform for analyzing complex protein interaction networks.

    Gene Mapping of Plant Height in Papaya Based on BSA-seq
    WU Xia-ming, ZHOU Chen-ping, YANG Min, XU Ze, KUANG Rui-bin, LIU Chuan-he, HE Han, WEI Yue-rong
    2025, 41(9):  54-61.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0351
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    Objective Plant height is one of the most important traits affecting the yield and quality of papaya (Carica papaya), and mapping the major candidate genes for papaya plant height provides genetic resources for the dwarf breeding of papaya. Method We used YY (taller plant) and GM (shorter plant) as the parents to construct a mapping population. The two parents and the offspring pools GY-H (taller pool) and GY-S (shorter pool) were then sequenced using BSA-seq technology. After the data were quality-controlled, the gene frequency gap between the pools was calculated by using the ED and SNP-index methods to locate the candidate intervals of the papaya plant height. Result The BSA-seq generated high-quality data, resulting in 337 980 SNP loci for gene mapping. The significant differences in genotype frequencies between the mixed pools were calculated using the ED and SNP-index methods, leading to the localization of candidate intervals with a size of 0.93 Mb on chromosome 3, 6, and 8. The interval was compared to the ‘Zihui’ papaya reference genome and found to contain 46 coding genes, 37 of which were annotated to various databases. Conclusion Cp_zihui12764, Cp_zihui06963, Cp_zihui07024, and Cp_zihui12732 are likely to be candidate genes to regulate the heights of the papaya plants.

    Elimination Technology of Sugarcane Streak Mosaic Virus in Chewing Cane
    HU Xin, LUO Zheng-ying, LIU Xin-long, WU Cai-wen, WU Zhuan-di
    2025, 41(9):  62-70.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0268
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    Objective This study is aimed to investigate efficient virus elimination technology for Sugarcane Streak Mosaic Virus (SCSMV) in chewing cane ‘Badila’, providing technical support for improving sugarcane yield. Method Using SCSMV-infected ‘Badila’ as experimental material, six hot water treatment groups were established: Untreated control, 50 ℃ for 2 h, 50 ℃ for 3 h, 52 ℃ for 3 h, 54 ℃ for 3 h, and 58 ℃ for 1 h. Virus-free plantlets were produced through shoot-tip culture and bud-tip culture. The virus-free plantlets were subjected to molecular detection of SCSMV, and their agronomic traits were investigated after pot cultivation to comprehensively analyze the virus elimination effects. Result Hot water treatment significantly reduced bud germination rates, with temperature and treatment duration showing a negative correlation with germination rate. No sprouting was observed at 54 ℃ and 58 ℃, making it impossible to obtain shoot-tip explants for tissue culture. However, the treatment had minimal impact on the survival rate of bud-tip culture. Even at 54 ℃ and 58 ℃, the bud apical meristems still maintained relatively high survival rates (83.3% and 76.9%, respectively). In terms of virus elimination, hot water treatment at 50 ℃ for 2 h, 50 ℃ for 3 h, and 52 ℃ for 3 h combined with shoot-tip culture only reduced the SCSMV detection rate to 80%. Under the same conditions, bud-tip culture reduced the rates to 70%, 60%, and 30%, respectively. Notably, under 58 ℃ treatment for 1 h, bud-tip culture achieved complete virus elimination (100%). Field trials demonstrated that virus-free sugarcane plants exhibited significantly better growth and agronomic traits compared to the control group. Plant height, internode length, single stem weight, brix and sugar content increased by 43.5%, 53.5%, 58.3%, 12.4% and 30.0%, respectively. Conclusion This study successfully established a virus elimination system combining hot water treatment and bud-tip culture, characterized by high plantlet regeneration and virus elimination efficiency.

    Genome-wide Identification and Expression Analysis of the ZIP Gene Family in Soybean
    HUANG Guo-dong, DENG Yu-xing, CHENG Hong-wei, DAN Yan-nan, ZHOU Hui-wen, WU Lan-hua
    2025, 41(9):  71-81.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0318
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    Objective Identifying and analyzing the expressions of GmZIP family members under aluminum stress in soybean lays the foundation for elucidating the biological function of GmZIP family. Method The members of GmZIP gene family were identified by bioinformatics method, which was used to establish the phylogenetic tree, predict protein network map and analyze the collinearity relationships, gene structure, and promoter cis-acting elements. Based on transcriptome sequencing, the expression patterns of GmZIP gene family in different tissues and under aluminum (Al) stress were analyzed. Result A total of 26 GmZIP were identified in the whole soybean genome, which were unevenly distributed on 14 chromosomes and divided into Ⅰ-Ⅴ subfamilies by phylogenetic analysis. The gene structure of GmZIP members were relatively conservative. All of GmZIP members contained ZIP conserved domain, multiple exons, and significantly different introns. Transcriptome analysis of different soybean tissues showed that most of GmZIP members had high expressions in the soybean root and nodule tissues. Three members of GmZIP (GmZIP6, GmZIP12, and GmZIP25) were up-regulated in Al-tolerant soybean germplasm Nannong 99-6, two members (GmZIP20 and GmZIP25) were up-regulated while one was down-regulated (GmZIP23) in Al-sensitive soybean germplasm soybean Zhongdou 32. The protein prediction results show that 16 of 26 GmZIP have interaction relationships, among which GmZIP23 and GmZIP25 were differentially expressed under Al toxicity stress. Based on fluorescence quantitative PCR detection, the expression of GmZIP25 in Al-resistant germplasm reached the highest at 72 h under aluminum stress treatment, while that in Al-sensitive germplasm reached the highest at 12 h. Conclusion These results suggest that GmZIP25 may play an important role in response to aluminum stress of soybean.

    Identification of Laccase (LAC) Gene Family in Potato (Solanum tuberosum L.) and Its Expression Analysis under Salt Stresses
    GONG Hui-ling, XING Yu-jie, MA Jun-xian, CAI Xia, FENG Zai-ping
    2025, 41(9):  82-93.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0337
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    Objective Laccases (LACs) are a class of copper-containing polyphenol oxidases that play significant roles in plant growth, development, and stress responses. The aim of this study is to identify the members of the StLAC gene family in potato (Solanum tuberosum L.) and analyze their expression characteristics, providing a reference for the functional study of StLACs. Method Based on the potato genome data, the StLAC gene family in potato was identified using bioinformatics methods. The expression patterns of StLAC genes in different tissues and under abiotic stress were analyzed using transcriptome data, and the expression patterns of StLAC genes under salt stress were verified by RT-qPCR. Result A total of 77 StLAC genes were identified in potato. Among them, 56 StLAC proteins had an isoelectric point >7, and only StLAC69 was a hydrophobic protein, while the other 76 members were hydrophilic proteins. Subcellular localization predicted results indicated that 20 StLAC proteins were located in the chloroplast, 19 in the vacuole, and the remaining StLAC proteins were located in the extracellular space, cytoplasm, nucleus, and plasma membrane. Analysis of cis-acting elements showed that the promoter regions of the StLAC gene family contained elements related to light response, growth and development, hormones, and stress response. Transcriptome data analysis revealed that StLAC genes were specifically expressed in different tissues and were induced under salt, drought, and hormone stress. The expression patterns of 12 StLAC family members (StLAC4/12/17/26/36/40/43/45/55/56/66/68) in the roots and leaves under salt stress were verified by RT-qPCR. The results showed that the expressions of StLAC4 and StLAC56 in the roots were upregulated by 3.8 to 15 times after stress, and the expressions of StLAC12, StLAC40, and StLAC56 in the leaves were upregulated by 17 to 34 times. Conclusion The StLAC gene family in potato is large and diverse, and may play an important role in the response of potato to salt stress.

    Genetic Diversity Analysis and Fingerprinting of Wild Potato and Landraces Based on SSR Markers
    LU Yao, YUAN Ping-ping, JIN Xin, MAO Xiang-hong, FAN Xiang-bin, BAI Xiao-dong
    2025, 41(9):  94-104.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0142
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    Objective Analyze genetic diversity of wild and landrace potato species at the molecular level and establish a fingerprint database for potato germplasm resources. Method SSR (Simple Sequence Repeat) molecular markers was used to analyze the genetic diversity of 73 wild potato and landrace potato species preserved at the CIP-China Center for Asia Pacific. Result The genetic diversity analysis revealed an average number of alleles (Na) of 1.843 7, Nei’s gene diversity index of 0.065 9, Shannon’s information index of 0.128 6, and an average polymorphism information content (PIC) value of 0.848 4, indicating high polymorphism but low genetic differentiation among the 73 germplasm resources. The germplasms were classified into seven subpopulations by Structure analysis, whereas they were divided into four groups via UPGMA clustering. Conclusion Five pairs of core primers are selected from 14 SSR primers, enabling complete differentiation of all 73 accessions. These primers were used to construct unique DNA fingerprints for the 73 wild and landrace potato accessions. This work provides a theoretical foundation and technical framework for future germplasm identification and screening systems in potato breeding.

    Regulatory Effect of Methyl Jasmonate on Postharvest Chilling Injury in Oriental Melon ‘Emerald’
    CHEN Qiang, YU Ying-fei, ZHANG Ying, ZHANG Chong
    2025, 41(9):  105-114.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0201
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    Objective Methyl jasmonate (MeJA), as an important plant hormone, plays a significant regulatory role in postharvest chilling injury of fruits. This study compared the differences in chilling injury index, weight loss rate, firmness, and soluble solids content between MeJA-treated and control fruits, analyzed the differentially expressed genes primarily affected by MeJA, and provided a theoretical basis for understanding the regulatory mechanism of MeJA on postharvest chilling injury in oriental melon ‘Emerald’. Method Oriental melon ‘Emerald’ fruits were fumigated with 50 μmol/L MeJA. The chilling injury index, firmness, weight loss rate, and soluble solids content were measured to clarify the regulatory effect of MeJA on fruit chilling injury. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (APX) were compared between MeJA-treated and control fruits. Transcriptome sequencing was used to screen differentially expressed genes and transcription factors related to chilling tolerance, and real-time quantitative PCR was performed for validation. Result During low-temperature storage, MeJA significantly reduced the chilling injury index of the oriental melon ‘Emerald’ fruits and delayed the decline in fruit firmness. MeJA treatment increased the activities of SOD, POD, and CAT enzymes but did not significantly alter APX enzyme activity. Transcriptome sequencing revealed that MeJA treatment and control fruits presented differentially expressed genes in the α-linolenic acid metabolism pathway, plant hormone signal transduction pathway, and antioxidant system. Conclusion Exogenous methyl jasmonate treatment effectively alleviates chilling injury in ‘Emerald’ oriental melon fruits and enhances their antioxidant capacity. The core transcription factor CmMYC2 of methyl jasmonate is suppressed, potentially through the negative regulation of downstream genes such as CmPOD, thereby achieving the regulatory effect of methyl jasmonate on chilling injury in oriental melon fruits.

    Bioinformatics Analysis and Flowering Regulation Function of FveBBX20 Gene in Woodland Strawberry
    DONG Xiang-xiang, MIAO Bai-ling, XU He-juan, CHEN Juan-juan, LI Liang-jie, GONG Shou-fu, ZHU Qing-song
    2025, 41(9):  115-123.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0346
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    Objective Exploring the function of FveBBX20 gene in flowering regulation provides foundation for further revealing the molecular mechanism, and a reference for molecular breeding of flowering in strawberry. Method Takingwoodland strawberry (Fragaria vesca 'Ruegen') as the experiment materials, the FveBBX20 gene was cloned by RT-PCR, and the gene sequences, protein structures and evolutionary relationships were analyzed by using bioinformatics websites and software. The protein localization was observed by tobacco transient expression. Transgenic Arabidopsisthaliana was obtained by flower dipping method, observing the flowering phenotype of transgenic materials. The expression of tissue and flowering-related genes was analyzed by real-time fluorescence quantitative PCR. Result The CDS length of FveBBX20 was 639 bp, encoding 212 amino acids and containing 2 B-box conserved domains. The CDS sequence of the woodland strawberry FveBBX20 was 639 bp in length, encoding 212 amino acids, and the relative molecular weight of protein was 23 958.62 Da, which was hydrophilic and contained two B-box conserved domains, belonging to the subgroup Ⅳ of the BBX family. Evolutionary analysis showed that FveBBX20 was closely related to its homologs from Argentina anserina and Rosa chinensis. Subcellular localization analysis showed that the FveBBX20 protein was localized in the nucleus, and FveBBX20 was expressed in different tissues of the woodland strawberry, with the highest expression level in flowers. Heterologous overexpression of the FveBBX20 gene in A. thaliana resulted in transgenic lines exhibiting delayed flowering phenotype, while the expressions of flowering-related genes AtCO, AtFT, AtSOC1, and AtFUL were significantly reduced. Conclusion FveBBX20 gene in the woodland strawberry delays flowering by inhibiting the expressions of flowering-related genes.

    Identification of the R2R3-MYB Gene and Expression Analysis of Flavonoid Regulatory Genes in Blueberry
    LIU Jia-li, SONG Jing-rong, ZHAO Wen-yu, ZHANG Xin-yuan, ZHAO Zi-yang, CAO Yi-bo, ZHANG Ling-yun
    2025, 41(9):  124-138.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0103
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    Objective Elucidating the expression patterns of the R2R3-MYB transcription factor family in blueberry during flavonoid regulation, thereby providing a theoretical foundation for exploring the regulatory mechanisms underlying flavonoid biosynthesis in blueberry. Method Bioinformatics was used to analyze the characteristics of R2R3-MYB genes in blueberry, such as physicochemical properties and subcellular localization, and RT-qPCR was to detect the expression patterns of VcMYBs in different tissues and different fruit development stages. Result A total of 278 R2R3-MYB genes were identified in blueberry, with amino acid sequence lengths ranging from 160 to 970 aa, and most of them were located in the nucleus. The blueberry VcMYBs was divided into 23 subfamilies by phylogenetic tree analysis, and the genes belonging to the same subfamily had similar structural characteristics. Cis-acting element analysis showed that VcMYBs may be involved in biological stress, abiotic stress, hormone induction, plant growth and development, and light response. Fourteen genes (SG4, SG5, SG6 and SG7) that may be involved in the regulation of flavonoid synthesis were randomly selected for further analysis. RT-qPCR analysis showed that the most of the VcMYBs had high expressions in fruits. Among them, VcMYB91(SG6) and VcMYB56(SG5) genes were lowly expressed in the green fruit stage and highly expressed in the mature stage, suggesting that they may be involved in fruit coloring. However, VcMYB88(SG4) and VcMYB96(SG4) showed the opposite expression pattern, suggesting that they may inhibit fruit coloring. In addition, VcMYB236(SG7) and VcMYB44(SG7) genes may be involved in flavonoid synthesis. Conclusion The 278 blueberry R2R3-MYB genes were identified via genome-wide analysis. Functional dissection revealed that four genes (VcMYB56, VcMYB91, etc.) from the SG5/6 subgroups regulate anthocyanin biosynthesis, six genes (VcMYB88, VcMYB96, etc.) from the SG4/5 subgroups participate in proanthocyanidin synthesis, and two genes (VcMYB236, VcMYB44) from the SG7 subgroup may govern flavonol metabolism.

    Gene Cloning and Functional Analysis of the Anthocyanin-related VcGSTF19 Gene in Blueberry (Vaccinium corymbosum L.)
    ZHANG Yong-yan, GUO Si-jian, LI Jing, HAO Si-yi, LI Rui-de, LIU Jia-peng, CHENG Chun-zhen
    2025, 41(9):  139-146.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0190
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    Objective Glutathione S-transferase Phi (F) subfamily members (GSTFs) play key roles in anthocyanin accumulation and transportation in many plants. To study the function of blueberry (Vaccinium corymbosum L.) GSTF, the blueberry anthocyanin-related GSTF19 gene was cloned and its function was studied. Method Reverse transcription PCR was used to clone the coding sequence (CDS) of VcGSTF19 related toanthocyanin from blueberry and construct overexpressing vector. Based on the blueberry fruit transient overexpression and Arabidopsis tt19 complementary transgenic transformation assays, the functions of VcGSTF19 were further studied. Result Transient overexpression of VcGSTF19 significantly promoted the accumulation of anthocyanins in blueberry peel. The transient overexpression of VcGSTF19 improved significantly the anthocyanin accumulation by 6.61-fold in blueberry fruit peel, compared to the empty vector control. Moreover, its transient overexpression significantly upregulated the expressions of anthocyanin biosynthesis related structural genes, including VcCHS, VcCHI, VcF3H, VcDFR, VcANS and VcUFGT. Heterologous transformation of Arabidopsis tt19 mutant with VcGSTF19 restored the anthocyanin accumulation in the mutant, with anthocyanin contents in rosette leaves accounting for approximately 6.21-fold of the tt19 mutant. Conclusion Blueberry VcGSTF19 gene plays an important role in anthocyanin accumulation and transport.

    Identification and Expression Analysis of SKP1 Gene Family in Grapevine (Vitis vinifera L.)
    LI Shan, MA Deng-hui, MA Hong-yi, YAO Wen-kong, YIN Xiao
    2025, 41(9):  147-158.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0210
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    Objective The characteristics and expression analysis of the S-phase kinase-associated protein 1 (SKP1) gene family (VvSKP1) in Vitis vinifera were studied, providing a theoretical basis for further research on the biological function of the VvSKP1 gene family. Method Based on the grape genome data, the SKP1 gene family members were screened and identified using bioinformatics methods. The characteristics of the encoded proteins, phylogenetic relationships, gene structure, chromosome localization, cis-acting elements, and collinearity were analyzed. The expression patterns of the SKP1 gene family under Botrytis cinerea and Peronospora variabilis infections were analyzed using real-time quantitative PCR (RT-qPCR). Result A total of 11 SKP1 family genes (VvSKP1-1 to VvSKP1-11) were identified from the grape genome, distributed on 7 chromosomes and 1 unknown chromosome, encoding 119 (VvSKP1-3) to 438 (VvSKP1-8) amino acids. Phylogenetic analysis revealed that the VvSKP1 family proteins could be classified into three subfamilies. The 11 SKP1 proteins of grape were found only in the first and second subfamilies, while the third subfamily consisted of all members of the wheat SKP1 gene family. VvSKP1 members of the same group or subfamily showed some similarity in gene structure and conserved motifs. The cis-acting elements in the gene promoter were mainly associated with light response, hormone response, and stress response. Synteny analysis showed that VvSKP1 had 8 pairs of homologous genes. Selection pressure analysis showed that the VvSKP1 gene was under purifying selection. The RT-qPCR analysis showed that 11 and 10 VvSKP1 genes responded positively to B. cinerea and P. variabilis infection, respectively. Conclusion Eleven members of the VvSKP1 gene family were identified from the grape genome. The expression patterns of the family members under the stress of B. cinerea and P. variabilis were different, indicating that the grape SKP1 gene family may be involved in the stress response processes of B. cinerea and P. variabilis.

    Cloning and Functional Analysis of LcTFL1 Gene in Litchi chinensis Sonn.
    SHI Fa-chao, JIANG Yong-hua, LIU Hai-lun, WEN Ying-jie, YAN Qian
    2025, 41(9):  159-167.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0147
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    Objective The TERMINAL FLOWER 1 (TFL1) gene is a crucial regulator in the plant flowering pathway. Our study aims to clone the LcTFL1 gene of litchi (Litchi chinensis Sonn.) and analyze its expression pattern and function, with the goal of identifying a marker gene for litchi flower formation regulation. This helps unlock the genetic mechanisms governing litchi floral development. Method Using previously obtained transcriptome data, we designed specific primers and successfully cloned the LcTFL1 gene from 'Feizixiao' litchi leaves by using the PCR technology. To verify its function, we used bioinformatics for protein structure and phylogenetic analysis. Through quantitative PCR technology, different tissues of litchi were selected to clarify the tissue expression pattern of this gene. Subcellular localization was determined by fusing LcTFL1 with a fluorescent tag and observing with confocal microscopy. By constructing a plant dual-source expression vector, we also overexpressed LcTFL1 in Arabidopsis thaliana to study its impact on flowering. Result The full-length coding region of LcTFL1 is 513 bp, which encodes 170 amino acids. It is a hydrophilic protein with a conserved PEBP domain. The promoter has cis-acting elements responsive to light, hormones, and stress. LcTFL1 localizes to the cytoplasm. Phylogenetically, it is the closest to JRO89 of Xanthoceras sorbifolium. Its expression is the highest in seeds, followed by apical buds and fruits. When the LcTFL1 gene was overexpressed in A. thaliana, the overexpressed A. thaliana plants showed that the number of rosette leaves at the initial flowering stage significantly increased, the length and width of leaves significantly reduced, and the plants demonstrated a late-flowering phenotype. Conclusion High expression of LcTFL1 delays A. thaliana flowering, indicating it acts as an inhibitory factor in flowering regulation. This discovery has implications for understanding plant development and for crop improvement in litchi.

    Integrate Transcriptomic and Metabolomic Analysis of Fruits Quality Differences between ‘Guiyou No. 1’ and ‘Shatianyou’ Pomelo (Citrus maxima
    LIU Jian-guo, LIU Ge-er, GUO Ying-xin, WANG Bin, WANG Yu-kun, LU Jin-feng, HUANG Wen-ting, ZHU Yun-na
    2025, 41(9):  168-181.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0279
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    Objective This study aims to elucidate the differences of fruit qualities and metabolic regulation between the self-pollinated cultivar pomelo ‘Guiyou No. 1’ and its female parent ‘Shatianyou’, it will provide a theoretical basis for future comprehensive research on the mechanisms of fruit quality formation and the metabolic regulation in pomelo. Method Based on physicochemical parameter measurements, transcriptomics, and widely targeted metabolomics, the fruit qualities, gene expression levels, and metabolite profiles were systematically analyzed in mature fruits of ‘Guiyou No.1’ and ‘Shatianyou’ pomelo. Result There were no significant differences in fruit weight, soluble solids content, titratable acidity, and vitamin C content between pomelo ‘Guiyou No.1’ and ‘Shatianyou’. Fruit shape index in ‘Guiyou No.1’ was significantly greater than that in ‘Shatianyou’, while the total amino acid content was significantly less than that in ‘Shatianyou’. Compared to ‘Shatianyou’, the 285 differentially expressed genes (DEGs) were screened, including 190 up-regulated genes and 95 down-regulated genes, respectively. These DEGs were primarily enriched in metabolic pathways, such as arginine and proline metabolism, fatty acid metabolism, and phenylpropanoid biosynthesis. Metabolome profiling suggested that a total of 90 differential metabolites were detected in ‘Guiyou No.1’, with 17 up-regulated and 30 down-regulated ones, compared with pomelo ‘Shatianyou’. Upregulated metabolites were mainly alkaloids and coumarins, while downregulated ones included amino acids, flavonoids, and phenolic acids. The differences of fruit quality between ‘Guiyou No.1’ and ‘Shatianyou’ were primarily influenced by the pathways of amino acids, alkaloids, and phenylpropanoids. Key genes such as GOT2, COMT, UGT73C, CYP71A and PAIC were identified as potentially playing crucial roles in the quality formation of pomelo fruits. Conclusion No significant differences in general qualities, are detected between mature fruits of ‘Guiyou No.1’ and ‘Shatianyou’ pomelo; however, there are obvious differences in terms of amino acids, flavonoids, phenolic acids, and alkaloids. The differential genes and metabolites between the two cultivars are mainly enriched in the metabolic pathways of amino acid and phenylpropanoid.

    Functional Study of RmEXPB2 Genein Rosa multiflora Based on the Identification of the Expansin Gene Family in Rosa sp.
    LI Yu-zhen, LI Meng-dan, ZHANG Wei, PENG Ting
    2025, 41(9):  182-194.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0216
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    Objective This study is aimed to systematically identify the expansin gene family in Rosa chinensis ‘Old Blush’, dissect their sequence characteristics and expression patterns, screen genes associated with prickle development. Based on this result, the biological function of RmEXPB2 gene in Rosa multiflora was further explored in order to lay the foundation for in-depth identification of the function of expansin genes in prickle formation in Rosa sp. plants. Method Bioinformatics tools were used to identify RcEXP genes in R. chinensis ‘Old Blush’ genome. The amino acid residue range of their encoded proteins and chromosomal distribution were analyzed. A phylogenetic tree was constructed to analyze the evolutionary relationships among expansins from Arabidopsis thaliana (AtEXPs), R. chinensis ‘Old Blush’(RcEXPs), and Fragaria vesca (FvEXPs). RcEXP expression patterns in different tissues were detected, and its expression in the prickle transcriptome was analyzed. R. multiflora, a model for prickle research, served as the experimental material, the candidate gene RmEXPB2 was isolated, and its expression pattern during prickle development were studied. The biological function of RmEXPB2 was preliminarily explored by heterologous expression in Arabidopsis. Result A total of 29 RcEXP genes were identified in the genome of R. chinensis ‘Old Blush’. The encoded proteins ranged from 243 to 313 amino acids, and they were unevenly distributed on 7 chromosomes. Phylogenetic tree analysis divided the expansins AtEXPs, RcEXPs, and FvEXPs into 4 subgroups. Among different tissues and organs, 15 RcEXP genes showed diverse expression patterns, with RcEXPB2 and RcEXPA4 predominantly expressed in prickles. The 13 RcEXP genes were screened from the transcriptome data of prickles, which were significantly expressed at specific stages of prickle development. The gene RmEXPB2, preferentially expressed in the prickles, was isolated from R. multiflora, and its expression increased during prickle development, peaking at the late stage. Heterologous expression of RmEXPB2 significantly promoted leaf expansion and root hair growth in transgenic Arabidopsis. Conclusion A total of 29 RcEXPs were identified in the genome of R. chinensis ‘Old Blush’, of which 13 members were significantly expressed at specific stages of prickle development. RmEXPB2 is a prickle preferentially expressed gene in R. multiflora, which may play an active role in the process of prickle elongation.

    Cloning of the LoAPS1 and Its Function Analysis during the Process of Dormancy Release in Lilium
    XU Xiao-ping, YANG Cheng-long, HE Xing, GUO Wen-jie, WU Jian, FANG Shao-zhong
    2025, 41(9):  195-206.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0231
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    Objective ATP sulfurylase (APS1) is a key enzyme in the first step of the sulfate assimilation process. Studying the function of APS1 during dormancy release in Oriental Lilium ‘Siberia’ provides important theoretical guidance for understanding the function of sulphur metabolic pathways in dormancy release in lily bulbs. Method The full-length sequence of LoAPS1 gene was obtained from different stages of dormancy relase by TA cloning, and its bioinformatics analysis was performed. Combined with the Lanzhou lily genome, the cis-acting elements of APS1 promoter were analyzed. Using the transcriptome database of different phases of dormancy release in lily and the riboflavin promotion of dormancy release in lily, the expression patterns of APS1 genes and related genes of sulphur metabolism pathway were analyzed. The subcellular localization of LoAPS1 protein was verified by tobacco subcellular localization transient transformation technique. The effect of silencing LoAPS1 on the process of dormancy release in lily bulbs was verified by virus induced gene silencing (VIGS) technique. Result The LoAPS1 gene had an ORF length of 1 413 bp, GenBank accession number was WMQ58782.1, and encoded 470 amino acids. The LoAPS1 protein contained two conserved structural domains, ATP-sulfurylase and PUA-like, with subcellular localization in chloroplasts, and might interact with sulphur metabolism pathways, such as APR, APK and SIR. The amino acid sequence of LoAPS1 had a high affinity with Musa acuminata and Zingiber officinale. The lily APS1 promoter contained four transcriptional start sites, including light-responsive elements, MYB-binding sites, and signal-responsive elements for methyl jasmonate (MeJA), gibberellin (GA3), and abscisic acid (ABA). The expression of LoAPS1 and related genes of sulfur metabolism pathway were down-regulated during the process of dormancy release of lily. The results of the VIGS and RT-qPCR showed that inhibition of LoAPS1 expression promoted the dormancy release in lily bulbs. Conclusion LoAPS1 is localized in chloroplasts and may respond to hormone signaling such as MeJA, GA3 and ABA, and inhibition of LoAPS1 expression promotes the dormancy release in lily bulbs.

    Cloning of bHLH Transcription Factor UNE10 and Its Regulatory Roles in the Biosynthesis of Volatile Compounds in Clove Basil
    WANG Bin, LIN Chong, YUAN Xiao, JIANG Yuan-yuan, WANG Yu-kun, XIAO Yan-hui
    2025, 41(9):  207-218.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0218
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    Objective bHLH transcription factors are key regulators in the biosynthesis of secondary metabolites in aromatic plants. The investigation into the regulatory roles of the bHLH transcription factor UNE10 in the biosynthesis of volatile compounds in clove basil (Ocimum gratissimum) provides critical regulatory factors for fine-tuning the essential oil components of this plant. Method The bHLH family gene OgUNE10, which has significantly high expression in the leaves of clove basil, was identified. The coding sequence of OgUNE10 was amplified using PCR, followed by its overexpression in the leaves of clove basil through Agrobacterium-mediated transformation to assess its impacts on the biosynthesis of volatile compounds. Result OgUNE10 expression in clove basil demonstrated notable tissue specificity, with particularly high levels in the leaves. The OgUNE10 gene was successfully cloned from the leaves and encoded a protein consisting of 161 amino acid residues, featuring a conserved bHLH domain. This protein was localized in the nucleus and presented low homology with bHLH amino acid sequences from model plants. The overexpression of OgUNE10 in the leaves significantly affected the contents of major components in the essential oil of clove basil, resulting in an increase in eugenol content while decreasing the contents of estragole and methyl cinnamate. Furthermore, OgUNE10 overexpression led to significant upregulations on three PAL (phenylalanine ammonia lyase) and two MES (methylesterase) genes, but downregulations on one MTA (methyltransferases) gene, while also affecting the expression of six terpene synthase-related genes. Conclusion OgUNE10 regulates the biosynthesis of volatile compounds by integrating the phenylpropanoid pathway, MEP and MVA pathways in clove basil.

    Analysis of Wax Components and Screening of Wax-deficient Gene Ggl-1 in Garlic (Allium sativum L.
    LIU Ze-zhou, DUAN Nai-bin, YUE Li-xin, WANG Qing-hua, YAO Xing-hao, GAO Li-min, KONG Su-ping
    2025, 41(9):  219-231.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0220
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    Objective This study is aimed to analyze the wax-deficient components in the leaves of the garlic mutant 8684-gl and identify the wax-deficient gene Ggl-1. The findings may lay foundation for exploring the molecular mechanisms underlying wax formation on garlic leaf surfaces and provide a theoretical basis for garlic pest resistance breeding and its application in agricultural production. Method The garlic wax-deficient mutant 8684-gl and its wild-type counterpart 8684 were used as research materials. Gas Chromatography-Mass Spectrometry (GC-MS) was employed to analyze the wax composition on leaf surfaces. Transcriptome sequencing was conducted to identify the Ggl-1 gene, and its expression was validated using real-time quantitative PCR (RT-qPCR). Result By GC-MS analysis the 39 wax components on the surface of garlic leaves were found, with 16-hentriacontanone (C₃₁H₆₂O) as the primary missing component in 8684-gl. Transcriptome analysis revealed that differentially expressed genes (DEGs) between 8684-gl and wild-type 8684 were mainly involved in lipid transport and metabolism, fatty acid biosynthesis and degradation, and the transport and catabolism of secondary metabolites. Two candidate genes for Ggl-1, Asa8G04000 and Asa4G02100 were identified. RT-qPCR analysis confirmed that these candidate genes presented significant different expressions between 8684-gl and its wild-type counterpart. Conclusion The primary wax-deficient component in 8684-gl was 16-hentriacontanone. The gene Asa8G04000 and Asa4G02100 are identified as candidate genes for the wax-deficient gene Ggl-1, providing insights into the genetic basis of wax biosynthesis in garlic.

    Identification and Functional Verification of Key Genes in Riboflavin Synthesis Pathway in Chinese Chestnut
    YU Wen-jie, FAN Si-ran, GAO Wen-li, XING Yu, QIN Ling
    2025, 41(9):  232-241.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0064
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    Objective Lumazine synthase (LS) and riboflavin synthase (RS) are key enzymes in riboflavin biosynthesis in Chinese chestnut. This work aims to explore the effects of LS and RS on the synthesis of Chinese chestnut riboflavin, and to provide a molecular basis for the synthesis of Chinese chestnut riboflavin. Method Having the protein sequences of riboflavin synthesis-related genes in the model plant Arabidopsis thaliana as bait, BLAST method was used to identify the riboflavin synthesis-related enzyme genes in the Chinese chestnut genome database. Transcriptome data was used to analyze the expression patterns of related genes in three stages of Chinese chestnut fruit development. Ultra-performance liquid chromatography (UHPLC) was applied to determine the riboflavin contents in eight Chinese chestnut varieties. Combined with inter-group difference and correlation analysis based on gene expression levels. Bioinformatics methods were adapted to analyze the structure, protein physicochemical properties, systematic evolution and promoter cis-acting elements of key genes. A chestnut callus transient transformation system was for verifying the functions of key genes. Result The Chinese chestnut contained GTP cyclohydrolase Ⅱ (GCHⅡ) , along with two pyrimidine deaminases (PYRD), one pyrimidine reductase (PYRR), one pyrimidine phosphatase (PYRP), two 3,4-dihydroxy-2-butanone 4-phosphate synthases (DHBPS), two lumazine synthases (LS), and one riboflavin synthase (RS) . The expressions of LS and RS were the highest in the kernel of Chinese chestnut. The riboflavin content in Chinese chestnut was between 0.104 and 0.054 mg/100 g. The expression of CmLS1 was significantly and positively correlated with the riboflavin content, the expression of CmRS was extremely significantly and positively correlated with the riboflavin content. CmLS1 was 693 bp in length and encoded 231 amino acids. CmRS was 843 bp in length and encoded 281 amino acids. CmLS1 had the highest homology with Corylus avellana. CmRS had the highest homology with Quercus suber,Quercus lobata and Corylus avellana. The promoters of CmLS1 and CmRS mainly contained hormone-responsive, light-responsive, low-temperature responsive and anaerobic induction cis-acting elements. After silencing CmLS1 and CmRS, riboflavin content in chestnut callus decreased by 33.1% and 49.1% respectively. Conclusion The riboflavin synthetases CmLS1 and CmRS of Chinese chestnut positively regulate riboflavin synthesis in the kernel and play an important role in riboflavin synthesis.

    Genome-wide Identification of the Chalcone Isomerase Gene Family in Eucommia ulmoides and Analysis of Their Expression Patterns
    CHENG Ting-ting, LIU Jun, WANG Li-li, LIAN Cong-long, WEI Wen-jun, GUO Hui, WU Yao-lin, YANG Jing-fan, LAN Jin-xu, CHEN Sui-qing
    2025, 41(9):  242-255.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0202
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    Objective Chalcone isomerase (CHI) is a key rate-limiting enzyme in the flavonoid biosynthesis pathway. The aim of this study is to conduct a genome-wide identification of the CHI gene family members in Eucommia ulmoides, systematically analyze their expression characteristics, and explore their biological functions in the growth, development, and stress response of E. ulmoides. Method Based on the genome data of E. ulmoides, bioinformatics methods were used to systematically identify the members of the CHI gene family, and comprehensively analyze their physicochemical properties, chromosomal localization, cis-acting elements in promoters, evolutionary relationships, conserved motifs, and gene structures. Meanwhile, transcriptome data and RT-qPCR technology were combined to investigate the expression patterns of EuCHIs in different tissues and under abiotic stress treatments. Result A total of 7 EuCHI gene family members (EuCHI-1 to EuCHI-7) were identified in the genome of E. ulmoides. The amino acid lengths of their encoded proteins ranged from 84 to 388 aa, the molecular weights ranged from 9 473.24 to 43 248.30 Da, the isoelectric points ranged from 4.81 to 9.42, the instability indices ranged from 23.09 to 46.43, and the hydrophilicity and hydrophobicity values ranged from -0.381 to 0.206. They were unevenly distributed on 5 chromosomes. Subcellular localization prediction showed that except for EuCHI-2, which was localized in the nucleus; the other members were all localized in the chloroplast, additionally, EuCHI-1 was also distributed in the cell membrane, EuCHI-4 was also distributed in both the cytoplasm and mitochondria. In the phylogenetic analysis, EuCHIs were classified into three subfamilies: Group Ⅰ, Group Ⅱ, and Group Ⅵ, and each member contained 1 to 10 exons. Promoter analysis indicated that EuCHIs might be involved in biological processes such as growth, development, and hormone response. Transcriptome data showed that most EuCHIs genes were involved in various biological processes of E. ulmoides, such as leaf color formation and floral development. The RT-qPCR results showed that EuCHIs had tissue expression specificity. Except for EuCHI-3, the other genes were highly expressed in the roots. Compared with the treatments of ABA and GA3 stress, the drought stress treatment could more significantly induce the expression of EuCHI genes. Conclusion Seven EuCHIs genes are identified and divided into three subfamilies. The expression patterns of EuCHIs differ among different tissues and under different stress conditions in E. ulmoides, indicating that they play important roles in the growth, development, and abiotic stress responses of E. ulmoides.

    Cloning and Functional Analysis of PtoYABBY2 and PtoYABBY12 in Populus tomentosa
    ZHANG Chao-chao, HAN Kai-yuan, WANG Tong, CHEN Zhong
    2025, 41(9):  256-264.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0122
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    Objective To explore the role of PtoYABBY2 and PtoYABBY12 genes in the flowering process and leaf development of Populus tomentosa, providing valuable reference genes for molecular design and breeding of P. tomentosa. Method PtoYABBY2 and PtoYABBY12 were cloned by homologous cloning, and bioinformatics, phylogeny, expression pattern and subcellular localization of them were analyzed. The function of these two genes was investigated by allogeneic transformation of Arabidopsis. Result PtoYABBY2 and PtoYABBY12 had complete YABBY and C2C2 domains, both belong to the YAB2 subclass of the YABBY gene family, and their proteins are located in the nucleus. PtoYABBY2 and PtoYABBY12 showed different expression patterns during the development of male and female flower buds in P. tomentosa. Two genes were expressed in the roots, stems, young leaves and mature leaves of P. tomentosa, with the high expression in mature leaves. Compared with wild-type Arabidopsis, the transgenic plants of PtoYABBY2 and PtoYABBY12 both showed obvious early-flowering phenotypes, but no significant changes in leaf morphology, inflorescence structure and floral organ morphology. In the transgenic lines, the relative expressions of flowering-promoting genes AtFT, AtSOC1, AtFUL and AtLFY were higher than those in wild-type Arabidopsis, while the relative expression of flowering-repressing gene AtFLC was significantly lower than that in wild-type Arabidopsis. Conclusion PtoYABBY2 and PtoYABBY12 play a key role in the flowering process, which can positively promote early flowering of plants.

    Identification of Surfactin-producing Bacillus Velezensis C5A-1 and Evaluation of the Plant Growth-promoting Effects of Its Surfactin
    LYU Zhen, GAN Tian, HUO Si-yu, ZHAO Chen-di, ZHAO Meng-yao, LI Ya-tao, MA Yu-chao, GENG Yu-qing
    2025, 41(9):  265-276.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0364
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    Objective This research is aimed to screen the high surfactin-producing strains, understand the genetic background and surfactin biosynthesis of the strains from the whole genome, and explore the functions of surfactin produced by the strains in plant growth-promoting. Method Surfactin-producing strains were isolated from plant rhizosphere soil by selective separation technique. The quantification and structural characterization of surfactin were performed using high-performance liquid chromatography and MALDI-TOF MS, respectively. The whole-genome was sequenced using Illumina + PacBio third-generation sequencing platform; and species identification, functional gene annotation, secondary metabolic gene clusters and surfactin biosynthesis were analyzed by bioinformatics method. Pot experiments were performed to detect the plant growth-promoting effect by surfactin. Result Strain C5A-1 with strong oil-displacing and antifungal activity was screened from poplar rhizosphere soil collected from Datong, Shanxi province. Surfactin homologues from C5A-1 were identified as C14- and C15-surfactin A, with the yield of 1 208.16 mg/L. The genomic average nucleotide identity (ANI) and digital DNA-DNA hybridization values between C5A-1 and B. velezensis FZB42 were 98.35% and 85.4%, respectively. C5A-1 genome size was 3 929 585 bp with 46.5% guanine-cytosine content, encoding 3 747 genes and harboring 12 secondary metabolite biosynthetic gene clusters. The surfactin biosynthetic gene cluster includes the core genes srfAA, srfAB, and srfAC. Surfactin produced by C5A-1 significantly enhanced the plant height, stem diameter, and aboveground and underground dry biomass of Populus tomentosa, Zea mays and Glycine max. Conclusion C5A-1 with high-producing surfactin was a novel strain belonging to B. velezensis, and the C14- and C15-surfactin A isoforms produced by C5A-1 demonstrated significant plant growth-promoting activity, which provides an optimized chassis cell factory for improving the surfactin yield by metabolic engineering strategies, and established a theoretical foundation for the practical application of surfactin in agricultural and forestry systems.

    Isolation, Identification, Biological Characteristics and Biocontrol Effects of Trichoderma harzianum M408 against Tomato Early Blight
    SU Xiu-min, HAN Wen-qing, WANG Jiao, LI Peng, WANG Qiu-lan, LI Wan-xing, CAO Jin-jun
    2025, 41(9):  277-288.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0306
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    Objective Tomato early blight caused by Alternaria solani is a main disease of tomatoes, a biocontrol strain with good antagonistic effect against tomato early blight was isolated and identified from the rhizosphere soil of zucchini after tomato-zucchini rotation. Method Antagonistic strains were identified using morphological observation and molecular biology techniques. Its optimal biological indicators such as carbon source, temperature, light, and pH value were conducted. Its biocontrol effects were evaluated through indoor confrontation and field control experiments. Result A superior strain M408 was screened to antagonize tomato early blight, and was identified as Trichoderma harzianum through morphological observation and ITS sequence analysis, the optimal carbon source for both nutritional growth and spore production was glucose; the optimal growth temperature was 27 ℃; the mycelial growth rate was the fastest in total darkness, and the spore production was the highest under full light, and the optimal pH was 6. In the vitro plate confrontation culture test, the inhibition rate of strain 408 against Alternaria solani was 100%, and the antagonistic index of strain M408 against A. solani reached level I. The inhibition rates of M408 strain original spore powder agent with spore concentration of 108, 107, 106, and 105 CFU/mL against A. solani W5 were 100%, 100%, 98.51%, and 93.43%, respectively, which were all higher than the inhibition rate of 89.55% of the chemical fungicide 325 g/L difenoconazole azoxystrobin against A. solani W5. In the field efficacy test, 7 d after the second spray, the biocontrol effect of T. harzianum M408 on early blight of tomato was obvious. The control effects of T. harzianum M408 strain original spore powder agent with spore concentration of 108, 107, 106 and 105 CFU/mL on tomato early blight were 89.44%, 87.07%, 84.56% and 81.01%, respectively, which were all higher than the relative control effect of 76.37% of 325 g/L difenoconazole azoxystrobin. Conclusion The strain of T. harzianum M408 is isolated from the rhizosphere soil of zucchini after tomato-zucchini rotation, it has a good biocontrol effect on early blight of tomato. It is a fungal resource with good biocontrol potential and has the value of further development and utilization.

    Whole Genome Analysis of Bradyrhizobium sp. Bd1 and the Negative Regulating Function of TetR3 during Cell Growth and Nodulation
    LI Ya-tao, ZHANG Zhi-peng, ZHAO Meng-yao, LYU Zhen, GAN Tian, WEI Hao, WU Shu-feng, MA Yu-chao
    2025, 41(9):  289-301.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0215
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    Objective This research is aimed to understand the genetic background of Bradyrhizobium sp. Bd1 at the genome level, and explore effective ways to promote the growth and nodules of Bd1. Method Illumina + PacBio third-generation sequencing platform was used to sequence the whole genome of Bd1. Bioinformatics method was employed to analyze the species identification, functional gene annotation, nitrogen fixation-related genes and TetR family transcription factors. TetR3 gene knockout mutant was constructed by homologous recombination double-exchange method to investigate the function of TetR3 in the growth and nodulation of Bd1. Result The genomic average nucleotide consistency (ANI) and digital DNA-DNA hybridization values between Bd1 and Bradyrhizobium diazoefficiens USDA110 were 99.98% and 98.7%, respectively. Bd1 genome size was 9 009 469 bp with 64.1% guanine-cytosine content, 8 403 coding sequences, within them 51 tRNA coding genes and 3 sets of ribosomal RNA genes. The number of nod, nif and fix genes were 9, 12 and 10, respectively. There were 58 genes encoding TetR family transcription factors with diverse structure. Compared with the wild Bd1, the growth and nodulation capacity were significantly enhanced, and glutathione S-transferase activity increased by 41.0% in TetR3-inactive mutant (Bd1ΔTetR3). Conclusion Bd1 is a novel strain belonging to B. diazoefficiens, which has the ability of nitrogen fixation in symbiotic nodulation with soybean. TetR3 negatively regulates the growth and nodulation in Bd1 by balancing cell REDOX capacity.

    Isolation, Identification, Optimization of Fermentation Conditions of High-yield Tremella fuciformis Polysaccharides Enzyme-producing Strain and Its Enzyme Characteristics Analysis
    LIAN Shao-jie, TANG Sheng-shuo, KANG Chuan-li, LIU Lei, ZHENG De-qiang, DU Shuai, TANG Li-wei, ZHANG Mei-xia, LIU Qiang
    2025, 41(9):  302-313.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0271
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    Objective Tremella fuciformis polysaccharides’ (TFPs) high molecular weight, high viscosity and other characteristics limit its biological activity and application. The purpose of this study is to screen strains with high yield of TFPs enzyme and optimize its fermentation conditions, so as to achieve the mass production of enzyme, thus effectively reduce the molecular weights of TFPs by enzymatic degradation method, and expand its application potential. Method Based on the characteristics of microbial enzymatic decomposition of TFPs, strains with TFPs as the sole carbon source were isolated from decomposed Tremella fuciformis samples. Combined the determination of TFPs degradation rate with 3,5-dinitrosalicylic acid (DNS) enzyme activity, strains with high yield of TFPs enzyme were screened. Single factor experiments and response surface methodology were used to optimize the fermentation medium (carbon source, nitrogen source, inorganic salts, etc.) and fermentation conditions (temperature, pH, rotation speed, etc.) of high-yield TFPs enzyme strains, and the catalytic characteristics (optimal pH, temperature, and degradation efficiency) of the TFPs enzyme were analyzed. Result The 40 strains isolated from the rotten T. fuciformis samples could use TFPs as sole carbon source, and 10 strains of them had high ability to degrade TFPs. The strain Y3522 with the highest enzyme activity was identified as Mesobacillus by 16S rRNA gene sequencing. The optimized medium components of Mesobacillus sp. Y3522 were as follows: TFPs (molecular weight of 1 250 kD) 8.07 g/L, casein peptone 25.47 g/L, K2HPO4 7.11 g/LNaCl 2.0 g/L,MgSO4·7H2O 1.0 g/L. The optimal fermentation conditions were a temperature of 35 ℃, pH of 7.5, inoculation volume of 3%, rotation speed of 250 r/min, and fermentation time of 18-24 h. After optimization, the enzyme activity increased by 60.1%. The optimal catalytic conditions for TFPs enzyme derived from Mesobacillus sp. Y3522 were pH 7.5 and temperature 35 ℃. In addition, 20% (V/V) crude enzyme solution degraded TFPs with a molecular weight of 3 000 kD (0.5%, m/V) to 922, 308, 85, and 18 kD at 30, 60, 90, and 120 min, respectively, demonstrating efficient molecular weight regulation ability. Conclusion The strain Mesobacillus sp. Y3522 producing high yield of TFPS are isolated and identified, its enzyme production fermentation system is optimized. The produced enzyme demonstrates efficient degradation ability on the molecular weights of TFPs.

    Screening of Amoxicillin-degrading Bacteria and Study on Its Degradation Mechanisms
    YAN Meng-yang, LIANG Xiao-yang, DAI Jun-ang, ZHANG Yan, GUAN Tuan, ZHANG Hui, LIU Liang-bo, SUN Zhi-hua
    2025, 41(9):  314-325.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0287
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    Objective This study is aimed to screen highly efficient amoxicillin (AMX) -degrading bacteria from pig manure sewage, optimize their degradation conditions, identify the degradation products and pathways of AMX, which may provide a basis and reference for removing antibiotic residues in the environment. Method Antibiotic domestication method and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were used to screen highly efficient AMX-degrading bacteria from pig manure. The highly efficient degrading bacteria were identified by morphological observation and 16S rDNA sequencing. The single-factor experiment method was adopted to optimize the culture conditions of AMX-degrading bacteria. The effects of culture temperature, pH value, inoculum size, and initial antibiotic concentration on the degradation of AMX by the strain were investigated. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to identify the products during the degradation of amoxicillin, so as to speculate the degradation pathway of amoxicillin. Through transcriptome sequencing, combined with genomics and bioinformatics methods, functional annotation of differentially expressed genes (DEGs) of strain AMX-1 was carried out. Result A highly efficient AMX-degrading bacterium was isolated and screened, named strain as AMX-1. Via morphological observation and molecular biological identification, strain AMX 1 was identified as Escherichia coli. The optimal conditions for strain AMX-1 to degrade AMX were as follows: Temperature was 35 ℃, pH was 7.0, the initial concentration of AMX was 50 mg/L, and the inoculum size was 6%. Under these conditions, the degradation rate of AMX reached 100% within 48 h. Eight intermediate products were identified during the degradation of AMX, and the degradation pathway of AMX was proposed. Transcriptome sequencing results showed that 786 essential degradation genes were up-regulated by the hydrolysis of ceftiofur sodium. Gene annotation results indicated that the degradation of AMX was related to a variety of biological processes. Conclusion In this study, a strain of E. coli AMX-1 with highly-efficient amoxicillin degradation is successfully screened and identified from pig manure. Under optimized conditions, the degradation rate of AMX by this strain can reach 100% within 48 h. The highly-efficient degradation ability and clear degradation mechanism demonstrated by strain AMX-1 provide important microbial resources and theoretical basis for the removal of amoxicillin residues in the environment.

    Effects of Shh Signal on the Proliferation and Differentiation of Follicular Granulosa Cells in Dairy Cows
    SUN Han-bing, XIANG Nian, FANG Xin-xin, YUE Jun-xiu, XU Qiu-shi, MA Li
    2025, 41(9):  326-334.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0333
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    Objective To investigate the effect of sonic hedgehog (Shh) signal on the proliferation and differentiation of bovine follicular granulosa cells. Method Ovaries from estrous dairy cows were collected and the expression characteristics of Shh in ovarian tissues were detected by IHC. Bovine follicular granulosa cells were isolated and cultured, and divided into: control group, Ad-Shh group (transfected with Shh overexpression adenovirus, MOI = 50 for 48 h), and Ad-Shh + Cyc group (treated with Shh pathway inhibitor cyclopamine (Cyc, 5 μmol/L) for 24 h on the basis of overexpression of Shh). The proliferation activities of cells was analyzed by immunofluorescence, MTS, and EdU methods; the cell cycle variations were analyzed by flow cytometry; and the expressions of Shh, Ki67, and proliferation and differentiation-related proteins were detected by Western blot. Result IHC results showed that Shh was highly expressed in the bovine ovarian tissues; immunofluorescence results indicated that the overexpression of Shh promoted the expression of Ki67 in granulosa cells, and Cyc reversed this change (P<0.01); MTS and EdU results demonstrated that Cyc reversed the enhancing effect of overexpression of Shh on cell proliferation activity (P<0.01). Flow cytometry results showed that the overexpression of Shh promoted the transition of granulosa cells from G1 phase to S phase (the proportion of G1 phase cells decreased from 91.42% to 71.58%, a decrease of 19.84%, and the proportion of S phase cells increased from 4.88% to 22.22%, an increase of 17.34%), and Cyc slowed down the cell cycle process (P<0.01). Western blot results showed that the overexpression of Shh promoted the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.01), and Cyc reversed the promoting effect of overexpression of Shh on the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.05). Conclusion Shh signal positively regulates the proliferation and differentiation of bovine follicular granulosa cells.

    Metabolite Early Warning Model for the Risk of Early Progression in Esophageal Squamous Cell Carcinoma
    ZHANG Ya-qi, WANG Qin-qin, SHEN Xia, LI Xu-miao, GAO Min, LI Jun, LI Chen, WANG Hui
    2025, 41(9):  335-344.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0418
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    Objective An early warning model for the risk of esophageal squamous cell carcinoma (ESCC) was constructed based on metabolomics to identify high-risk populations accurately. Method Eighty-four patients with low-grade intraepithelial neoplasia (LGIN) were included, and serum was collected at baseline. They were divided into a progress group (N=28) and a non-progress group (N=56) according to whether they progressed to high-grade intraepithelial neoplasia (HGIN) or ESCC during follow-up. Untargeted metabolomics analysis was performed using reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with high-resolution mass spectrometry. Differences in metabolic profiles between groups were assessed by combining univariate and multivariate analyses, and pathway enrichment analyses were performed for differential metabolites. The samples were divided into training and test sets in the ratio of 7:3. In the training set, univariate logistic regression combined with least absolute shrinkage and selection operator (LASSO) regression was used to screen key metabolites associated with disease progression, and a risk warning model was constructed based on the regression coefficients. Model performance was assessed by the receiver operating characteristic curve and area under the curve (AUC). Result A total of 1 431 metabolites from 10 classes were identified. Differential metabolites were significantly enriched in steroid hormone biosynthesis, primary bile acid synthesis, and linoleic acid metabolic pathways. Finally, 18 key metabolites closely related to the progression of the disease were selected, including sn-glycero-3-phosphocholine, hexadecanoic acid, xanthurenic acid, and N-amidino-aspartate. The risk warning model showed good predictive ability in the test set (AUC=0.812). Conclusion Based on the prospective follow-up cohort, multiple key metabolites and metabolic pathways are identified, and metabolite early warning models for the risk of early progression of ESCC are constructed. The model has good prediction robustness and generalization ability and may provide theoretical support for early risk assessment and intervention strategy optimization in people at high risk of ESCC.

    Computational Literature-based Knowledge Discovery for Soybean Coupling Traits
    GUAN Zhi-hao, SHAN Zhi-yi, XIONG He, ZHAO Rui-xue
    2025, 41(9):  345-356.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0082
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    Objective This study aims to develop an automated soybean trait discovery model using computational literature. The goal is to accurately identify soybean coupling traits before field trials and analyze their genetic networks. This approach addresses the limitations of traditional laboratory methods and offers a more efficient way to discover knowledge for soybean breeding research. Method Firstly, the annotation strategy of soybean corpus was constructed according to the authoritative domain ontology, and the soybean coupling trait knowledge was represented in the form of Subject-Predication-Object (SPO) semantic triples. Secondly, a semantic triplet extraction model was constructed based on the domain dictionary. Adapted positive-unlabeled learning (AdaPU) algorithm and R-BERT (pre-trained Transformer encoder for relation extraction) algorithm were used to automatically extract the knowledge entities and their regulatory relationships in soybean breeding literature, and the genetic regulatory networks of soybean traits were obtained. Finally, the coupling trait connected subnetworks and the reachable paths between trait nodes in the network were mined, and the literature review method was used to verify the results and analyze the genetic mechanism. Result Experimental results show that the knowledge extraction model achieved an accuracy of 79.41%, a recall rate of 88.52%, and an F1 score of 83.72%. A total of 776 unique soybean trait knowledge triples were identified, encompassing 33 gene concepts, 119 protein concepts, and 96 trait concepts. Among these, 478 triples represented “associated with” relationships, 264 “up-regulation” relationships, and 34 “down-regulation” relationships. Within the soybean trait knowledge network, six coupling traits connected subgraphs were discovered, and 139 trait coupling paths within the largest connected subnetwork. Conclusion This study identified the feasibility of trait knowledge discovery based on large-scale literature. By deeply mining knowledge units, it uncovers the underlying coupling traits and their associated molecular mechanisms, providing plant breeding researchers with potential pleiotropic genes and coupling traits for experimental design, thereby enhancing the efficiency of hypothesis generation in scientific research. The structured trait knowledge generated by this study contributes to the development of knowledge graphs in the field of soybean breeding and serves as a reliable knowledge foundation for domain-specific large language models, facilitating the development and application of AI agent.

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    2025, 41(9):  357. 
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    2025, 41(9):  358. 
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    2025, 41(9):  359. 
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