Table of Content

26 March 2025, Volume 41 Issue 3

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Research Progress in Starch Biosynthesis and Regulatory Factors in Maize Kernel

WANG Tao, HU She-wei, ZHANG Yu, DENG Wen-wen, SHANG Chun-yuan, WANG Wan-yi

2025, 41(3):  1-13.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0922

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Maize, as a widely cultivated crop worldwide, is not only an indispensable food source in the human diet, but also plays an important role in the feed industry and clean energy. The starch rich in its kernels constitutes the main body of maize biomass and directly determines the yield level of maize. It is an indispensable raw material in agricultural production and food processing. However, with the continuous growth of the global population, the acceleration of urbanization, and the reduction of agricultural land and frequent natural disasters caused by climate change, the global food supply system is facing unprecedented challenges. As important crops in the world,it is very crtical to cultivate high-yield varieties by analyzing its starch biosynthesis pathway. This paper summarizes the key enzymes and transporters involved in the maize starch biosynthesis pathway and discusses the specific roles of these factors in the process of starch synthesis. Concurrently, this paper reviews the grain phenotypes of mutants of key enzymes in the synthesis pathway, and analyze the effects of gene mutations on starch yield and quality. In addition, this paper also summarizes the various factors regulating starch biosynthesis and discusses how to improve the yield and quality of maize by optimizing these factors. Finally, this paper looks forward to the future research direction, aiming to provide a solid theoretical basis and practical guidance for molecular design breeding of high quality and high yield maize.

Advances on Molecular Modifications of Formate Dehydrogenase for CO₂ Reduction

LU Feng, HUANG Yu-hong, LIN Yan-na, MA Fu-qiang

2025, 41(3):  14-24.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1038

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With the urgent global demand for sustainable energy transformation and greenhouse gas emission reduction, the efficient green conversion of CO₂ has become a research hotspot in various fields such as energy, environmental science, and chemical engineering. Especially in the context of combating climate change, CO₂ capture and utilization is seen as one of the key strategies to achieve carbon neutrality. Formate dehydrogenase (FDH) as an important biocatalyst for reducing CO₂ to formate, has shown significant potential in green chemistry and bioenergy conversion. However, it still has certain limitations regarding catalytic activity, thermal stability, and coenzyme specificity. In recent years, with the continuous development of protein engineering technology and molecular biology, researchers have proposed a variety of strategies to modify the performance of FDH, significantly enhancing its application prospects. For example, enzyme engineering means such as directed mutation and structure optimization can improve the substrate affinity of the enzyme, enhance the rigidity and conformational stability of the enzyme, and change the coenzyme binding site to broaden its application prospects in the process of green transformation. In this paper, the research progress of FDH catalytic CO₂ reduction in recent years will be comprehensively summarized, focusing on the improvement measures of FDH in terms of catalytic efficiency, thermal stability, coenzyme specificity, etc., the specific strategies adopted in the molecular transformation process will be discussed, and the rules of these strategies will be summarized, in order to provide new ideas and methods for the molecular transformation of FDH in the future and promote the development of its application in CO₂ reduction reaction. In addition, with the development of artificial intelligence, machine learning, gene editing and other technologies, the molecular modification of FDH will be more efficient and precise in the future, and these emerging technologies are expected to screen out FDH mutants with excellent performance in a short time, providing a feasible green solution in solving the global climate change and energy crisis.

Theoretical Proof， International Comparison and Chinese Approach for Gene Edited Food Labeling System

ZHANG Wen-fei, YANG Fei, LIU Xu-xia

2025, 41(3):  25-34.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0655

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Based on the characteristics of gene editing technology and the actual needs of expanding and expediting biological breeding industrialization, it is necessary to establish an independent gene edited food labeling system based on the detectability standard. There are many approaches of gene edited food labeling system internationally, the EU categorizes the objects of gene edited food labeling; the US reflects the transparency of the labeling system from the aspects of legal basis, system content and public participation; and the Canada is open to introducing gene edited food into a voluntary transparency initiative. In the process of industrializing gene edited crops in China, the top-level design of gene edited food labeling system should be strengthened by formulating special legal documents, improving the domestic rules system, strengthening international communication and cooperation, and docking international rules benignly. The content of gene edited food labeling system should be optimized by identifying the objects of labeling, refinement of the contents of labeling and unification of the types of labeling. The guarantee measures of gene edited food labeling system should be improved by perfecting the traceability system, establishing the public database and improving consumer awareness, in order to maintain consumers’ rights to choose and know, promote the high-quality and sustainable development of the biological breeding industry, ensure national biosecurity and improve the competitiveness of application and supervision on emerging biotechnology of China in the agricultural field.

Creation of Rice Mutant by Gamma-ray and Its Molecular Identification

LI Xin-peng, ZHANG Wu-han, ZHANG Li, SHU Fu, HE Qiang, GUO Yang, DENG Hua-feng, WANG Yue, SUN Ping-yong

2025, 41(3):  35-43.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0667

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Objective Gamma-ray mutagenesis has been widely used in rice breeding because of its high efficiency and simple method. OsGRF4 is an important and rare pleiotropic gene in rice, which can significantly improve nitrogen use efficiency, yield and cold tolerance, while reduce seed shattering, this work aims to obtain different types of mutant resources and mine the regulatory genes of OsGRF4. Method The rice material NIL-GRF4 was mutated by γ rays in this study, the authenticity and genetic background of the mutants were identified by functional marker and SSR markers. Result By traits investigating, 15 mutants of grain shape, 6 mutants of zebra leaf, 4 mutants of tiller and 3 mutants of sterile were obtained. Among them, an important grain type mutant was obtained. Its grain length reached 14.10 mm, and its grain length and 1 000-grain weight increased by 1.50 mm and 8.85 g respectively compared with the wild type, with an increase of 11.90% and 23.95% respectively. The authenticity and genetic background of the mutants were identified by using the functional markers of OsGRF4 and 48 pairs of SSR markers. The results showed that the mutants were all derived from wild-type NIL-GRF4, and the genetic background of the mutants was very similar to that of wild-type NIL-GRF4. Conclusion The F₂ populations have been constructed by crossbreeding the mutants of grain shape, zebra leaf and sterile with the wild type, providing important gene resources for rice breeding.

Establishment and Optimization of a Tobacco Chromosome Preparation System

HAN Meng-qiao, WU Jiang, LI Li-hua, WANG Zhao-yi, DENG Xi, WEI Feng-jie, REN Min, SUN Yang-yang, LI Fu-xin

2025, 41(3):  44-50.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0915

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Objective To develop a specialized chromosome preparation system for tobacco, laying the groundwork for advancements in tobacco chromosome engineering and the systematic exploration of distant hybridization research. Method The chromosome preparation system was established using the enzyme digestion method, with common tobacco as the test material. The experiment was designed to sample the root tip tissues at eight time intervals. Pre-treatment included four methods: A control group (without any pre-treatment), treatment with 20% wind medicated oil at room temperature for 2 h, exposure to an ice-water mixture at 0℃ for 48 h, and N₂O treatment, in which the N₂O treatment was further divided into samples collected at 10-min intervals from 20 to 70 min; the root tip samples were enzymatically digest for different durations, including 35, 45, and 55 min. Chromosome preparation was performed and observed for each treatment and duration, with the optimal method for tobacco chromosome preparation identified based on chromosome length, clarity, and dispersion. Merging improved processing methods and durations to establish a tobacco chromosome preparation system. The validation applicable to the method was further confirmed by preparing chromosome samples from various tobacco varieties. Result It is the optimal time between 10‍‍∶‍30 and 12‍‍∶‍‍30 for tobacco root tip sampling, 30‒40 min for N₂O pretreatment and 45 min for enzymatic digestion were suitable for preparing chromosomes. High-quality chromosome samples were successfully obtained by applying this optimization system to three different germplasm resources: the ancestral species for common tobacco, common tobacco, and progeny derived from distant hybridization. Conclusion A preparation system for tobacco chromosomes is established, capable of preparing high-quality chromosome samples from tobacco root tips within 3 h. This system provides crucial technical support for research in distant hybridization and chromosome engineering.

The High-yield Bafilomycin A1 Strain Obtained from UV Mutagenesis and Its Medium Optimization

CHEN Hai-min, SUN Fei, YUAN Yuan, WU Jia-wen, JIANG Hong, ZHOU Jian

2025, 41(3):  51-61.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0898

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Objective Bafilomycins are a class of macrolide compounds produced by the fermentation of Streptomyces griseobrunneus, which have antagonistic effect on a variety of plant fungi, and its representative compound is bafilomycin A1 (Baf A1). In order to further enhance the yield of Baf A1 in Streptomyces sp. FIM-B0711 and reduce the production cost, the high-yielding strain of Baf A1 was selected and its fermentation process was optimized. Method High-yield mutant strains were generated through ultraviolet mutagenesis, followed by optimization of the fermentation process using single factor experiment, the steepest ascent experiment, and response surface methodology. Result The Baf A1 yield of the UV-21 strain reached 397.99 mg/L, representing a 23.60% increase compared to the original strain. Single factor experiment identified that maltodextrin, soybean peptone, and calcium carbonate were as the optimal carbon source, nitrogen source, and inorganic salt for strain UV-21, respectively. The optimal culture conditions were determined to be pH 7.0, an inoculum concentration of 3% (volume fraction), a liquid volume of 30 mL/250 mL, and a fermentation time of 96 h. The steepest ascent experiment revealed that the highest relative titer of 147.18% was achieved with a carbon source concentration of 45 g/L, nitrogen source concentration of 15 g/L, and inorganic salt concentration of 0.5 g/L. The response surface optimization demonstrated that the optimal fermentation process for the strain involved maltodextrin at 45.98 g/L, soybean peptone at 14.96 g/L, calcium carbonate at 0.48 g/L, valine at 3 g/L, an initial pH of 7.0, an inoculum concentration of 3% (volume fraction), a liquid volume of 30 mL/250 mL, and a fermentation time of 96 h. Under these optimized conditions, the fermentation titer reached 627.58 mg/L, a 57.69% increase compared to the initial process. Conclusion A high-yield Baf A1 strain with 627.58 mg/L is obtained based on ultraviolet mutagenesis and medium response surface optimization experiment, which increases the yield by 57.69% compared to the original strain FIM-B0711.

Improving the Efficiency of Gene Editing by Optimizing sgRNA in Aspergillus tubingensis

LIANG Li-cun, WANG Ke-fen, SONG Zu-huan, LIU Meng-ting, LI Jia-yu, LUO Hui-ying, YAO Bin, YANG Hao-meng

2025, 41(3):  62-70.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0784

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Objective In the CRISPR/Cas9 gene editing system, sgRNA is one of the important gene editing elements. It binds to the Cas9 protein and complements the genomic DNA through the spacer sequence to guide the Cas9 protein to accurately cut the genome. In order to improve the gene editing efficiency of Aspergillus tubingensis, sgRNA optimization is a feasible strategy. Method The promoter and hairpin structure of sgRNA were optimized, and the gene editing efficiency was verified in A. tubingensis. Result The gene editing efficiency of sgRNA with “lock” structure was 9.37% higher than that of the control group when gene editing was performed by RNP method. When sgRNA was expressed in vivo, the gene editing efficiency of tRNA^Gly15 and tRNA^Gly17 promoters was 14%-16% higher than that of 5S rRNA promoter, respectively. Using the tRNA^Gly15 promoter to express sgRNA with a “lock” structure increased the white spore rate and increased the gene editing efficiency of A. tubingensis to 96%. Conclusion The “lock” structure and two promotors of tRNA^Gly15 and tRNA^Gly17 may improve the gene editing efficiency of A. tubingensis.

Design and Application of Guide RNAs for Non-coding RNAs

QIN Yu-ting, PAN Sen-tao, CHEN Yu-ping

2025, 41(3):  71-82.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0750

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Non-coding RNA(ncRNA) refers to functional RNA molecules that typically do not encode proteins but play important physiological roles by regulating the expression and function of coding genes. microRNA (miRNA), circular RNA (circRNA), and long non-coding RNA (lncRNA) are currently known as the three major types of regulatory ncRNAs with important physiological and pathological significance. The loss, rearrangement, and dysregulation of their genes are closely related to the occurrence and development of various diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) are types of immune-defense systems widely existing in bacteria and archaea, consisting of CRISPR-associated proteins (Cas) and guide RNAs (gRNAs). Cas nucleases shear and remove exogenous genes, while gRNAs determine their precise recognition and effective cleavage of target DNA/RNAs. CRISPR/Cas technology has excellent capabilities of site-specific genome editing and cleavage of RNA transcripts and can precisely and efficiently edit, intervene, and detect abnormalities in these ncRNAs and thus contribute to successful diagnosis and treatment of diseases. This paper summarizes the composition and design principles of Cas9 and Cas13 gRNA and the unique molecular characteristics of miRNA, circRNA, and lncRNA genes and their respective molecular features produced by the RNA itself or during RNA formation, emphasizes the design strategies and target selection of their gRNAs, as well as how Cas9 and Cas13 nucleases can accurately detect and intervene in abnormal miRNA, circRNA, and lncRNA under the guidance of gRNA with high specificity and efficiency. The article further discusses the similarities and differences in gRNA design among three types of ncRNAs, as well as the challenges faced by CRISPR/Cas applications in ncRNAs. It also gives prospects for future gRNA designs for more accurate targeting at ncRNAs, aiming to provide some reference for the employment of CRISPR/Cas technology in managing ncRNAs-associated diseases.

Reducing the Expression of GBSSI Gene in Barley via the Editing in the 5′UTR Region

XUE Rui-ying, LIU Yong-ju, JIANG Yan-yan, PENG Xin-ya, CAO Dong, LI Yun, LIU Bao-long, BAO Xue-mei

2025, 41(3):  83-89.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0813

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Objective Barley is the fourth largest grain crop. The total starch content and the ratio of amylose and amylopectin in the grain are the key factors to determine the quality and characteristics of barley. The fine control of amylose content is of great significance for the improvement of barley flour quality. Method In this study, barley Cas9 overexpression lines were used as receptors, and the 5′non-coding region (5′ UTR) of GBSSI gene was targeted by BSMV-SG-mediated gene editing system to achieve the fine regulation of GBSSI gene expression. Result Our results indicated that somatic editing occurred in the leaves of the fifth tiller from 14 M0 generation 5′UTR plants, with an editing efficiency ranging from 1.22% to 84.49%. Seeds from the fifth tiller exhibiting peak editing efficiency were harvested, leading to identification of six edited lines among twenty-three M1 generation plants, resulting in an edited plant proportion of 26.08%. RT-qPCR analysis revealed a reduction in GBSSI gene expression by 40%–82% compared to controls. Conclusion These findings demonstrate that GBSSI gene expression can be effectively regulated through modification of its 5′UTR region, providing novel insights into precise regulation mechanisms for amylose synthesis.

Functional Analysis of Soybean GmPM31 Gene Promoter Involvement in Response to High Temperature and Humidity Stress

LIN Zi-yi, WU Yi-zhou, YE Fang-xian, ZHU Shu-ying, LIU Yan-min, LIU Su-shuang

2025, 41(3):  90-97.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0435

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Objective To study the function of GmPM31 promoter on seed vigor formation in response to high temperature and high humidity stress, and to lay a foundation for comprehensively unravelling the function of soybean small heat shock proteins involved in the response to high temperature and high humidity stress. Method Homozygous T3 transgenic Arabidopsis thaliana with GmPM31 promoter was treated with high temperature and high humidity stress (40℃/100% relative humidity), and wild-type A. thaliana was used as control to detect its tolerance and stomatal opening. Analysis of GUS gene expression driven by the soybean GmPM31 promoter by GUS histochemical staining and quantitative real-time PCR. The seed vigor was detected by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Result Compared with the control group, the tolerance of GmPM31-transgenicpromotor A. thaliana to high temperature and high humidity increased. The activity of GUS increased, and they were expressed in the leaves, roots and flowers, and germination rate and vigor increased. Conclusion GmPM31 promoter improve the resistance of seeds to high temperature and high humidity stress and the ability to resist deterioration.

Mechanism of Tolerance of Protein Phosphatase AhPDCP37 in Peanut to Drought

QIAN Qi, WANG Zeng-hui, SUN Rong-hua, LUO Ying-zhi, SU Liang-chen

2025, 41(3):  98-103.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0721

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Objective To investigate the mechanism of tolerance of the peanut protein phosphatase AhPDCP37 to drought under dehydration stress, and to provide a research basis for biotechnological means of modifying new plant varieties. Method The phenotype and leaf stomatal opening of transgenic Arabidopsis plants overexpressing AhPDCP37 were observed under drought conditions, and physiological and biochemical characteristics as well as changes in the expression of genes related to the response to drought stress and those related to the ABA signaling pathway were measured. Result The malondialdehyde content in Arabidopsis overexpressing AhPDCP37 was less up-regulated under drought stress compared with the wild type, whereas superoxide dismutase and peroxidase activities showed a significant up-regulation of expression under drought treatment. Meanwhile, leaf stomatal openings showed a decreasing trend in all groups under drought stress, but the decrease in the overexpression of AhPDCP37 treatment group was significantly lower than that in the wild-type treatment group. This indicates that AhPDCP37 enhanced the survival ability of plants under drought conditions by decreasing stomatal opening and increasing the content of cellular antioxidant enzymes. Under drought conditions, the expressions of positively regulated genes (WDR55, APA1) and genes related to ABA signaling pathway (NCED3, ABF3, and RD29A) were significantly up-regulated in the Arabidopsis treatment group overexpressing AhPDCP37 compared with the wild-type treatment group, and the expressions of negatively regulated genes of drought stress response WRKY70 significantly decreased compared with the wild-type treatment group. Conclusion AhPDCP37 improves tolerance in plants to drought under drought stress conditions mainly through its involvement in the regulation of stomatal movement and antioxidant enzyme activities.

Functional Studies of the Gossypium hirsutumGhNFD4 Gene in Response to Drought in Cotton

MA Li-hua, HOU Meng-juan, ZHU Xin-xia

2025, 41(3):  104-111.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0839

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Objective To analyze the biological function of GhNFD4 in response to drought stress in terrestrial cotton, and to provide genetic resources and theoretical basis for molecular breeding of terrestrial cotton for stress tolerance. Method Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression pattern of GhNFD4 in cotton seedlings under PEG-mimicked natural drought stress; tobacco crisped virus (TRV)-induced gene silencing (VIGS) was utilized to explore the regulatory role of GhNFD4 in drought stress, and the interfering sequences of GhNFD4 were cloned and the gene silencing vectors were constructed. The GhNFD4 silencing plants were further obtained by infiltrating cotton seedlings through transient transfection technology, observing the phenotypic differences between silencing plants and control plants at 15 d of natural water loss, detecting the accumulation of ROS products by using NBT and DAB staining, and detecting the water loss rate and drought-related physiological indexes of isolated leaves. Result The expression of GhNFD4 was induced by natural drought stress simulated by PEG and peaked at the 12th h of stress. After 15 d of natural drought, the leaves of GhNFD4-silenced plants showed more severe wilting and darker coloring after NBT and DAB staining compared with the negative control plants, and the water loss rate of the isolated leaves and the relative electrical conductivity (REL), malondialdehyde (MDA), and hydrogen peroxide (H₂O₂) contents of the leaves after drought stress were significantly higher compared with the control, and the total antioxidant enzyme (T-AOC) activity was significantly lower compared with the control activity was significantly decreased compared with the control group. Conclusion GhNFD4 is induced by drought stress and actively participates in the positive regulation of drought stress in cotton by regulating the activity of antioxidant enzymes under drought conditions, which improves the resistance of cotton to drought stress.

Identification of SEC1 Complex Components and Functional Validation of the GhSCY1 Gene in Cotton

FENG Xiao-kang, LIANG Qian, WANG Xue-feng, SUN Jie, XUE Fei

2025, 41(3):  112-122.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0923

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Objective The SEC1 complex is a protein transport system located on the thylakoid membrane, composed of SCY1, SECE1, and SECA1. The SEC1 system is involved in the transport and integration of thylakoid proteins, playing a very important role in maintaining the stability of chloroplast structure. Identifying SEC1 complex genes from the whole genome of upland cotton using bioinformatics aims to provide new gene resources for photosynthesis mechanism research and high-yield, stress-resistant cotton breeding. Method Based on the protein sequences of the Arabidopsis SEC1 system component genes, cotton SEC1 system components were identified from the upland cotton genome, and then the physicochemical properties, transmembrane structures, protein interactions, promoter elements, and expression patterns of these system components were analyzed. The effects of the main channel protein component GhSCY1 on chlorophyll synthesis, chloroplast structure, and morphology were studied using VIGS and transmission electron microscopy techniques. Result Components of the SEC1 transport system in upland cotton were identified, GhSCY1A/D, GhSECE1A/D, and GhSECA1A/D. GhSCY1A/D contains one SecY conserved domain and seven transmembrane domains, while GhSECE1D only contains one transmembrane domain. It is predicted that GhSCY1A/D and GhSECE1D together form the chloroplast transmembrane channel protein in the SEC1 transport system. GhSECA1A/D possesses a SecA domain and lacks transmembrane domains, mainly providing energy for the protein transport process. The expressions of the SEC1 system genes were high in the leaves and could participate in the response to temperature stress. After silencing the GhSCY1 genes (GhSCY1A and GhSCY1D), the cotton leaves demonstrated a mottled pale yellow phenotype, a significant decrease in chlorophyll content, abnormal chloroplast structure and morphology, and no starch granule accumulation. Conclusion The cotton SEC1 system is constituted by GhSCY1A/D, GhSECE1A/D, and GhSECA1A/D, among which the GhSCY1A/D genes play an important role in maintaining chloroplast structure and morphology and in the biosynthesis of chlorophyll.

Genome-wide Identification and Expression Profiles of DIR Gene Family in Potato

ZHANG Yi-xuan, MA Yu, WANG Tong-tong, SHENG Su-ao, SONG Jia-feng, LYU Zhao-yan, ZHU Xiao-biao, HOU Hua-lan

2025, 41(3):  123-136.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0825

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Objective All members of the DIR gene family in potato (Solanum tuberosum) were identified by the latest potato genome information. Bioinformatic techniques and transcriptome data were used to predict and analyze the phylogenetic relationship, gene structures, promoter elements and expression profiles of DIR gene family members. These results will lay a foundation for further study on the function of DIR genes in potate growth and development as well as their responses to stresses. Method Based on the protein sequences of Arabidopsis DIR gene and the model file of Dirigent domain, the whole genome identification of potato DIR family members was carried out. The physicochemical characteristic, chromosome localization, collinearity analysis, conserved motif, gene structure and cis-acting elements of potato DIR genes were analyzed by bioinformatics tools such as ExPASy, MEME, MCScanX, GSDS, PlantCARE, and TBtools, respectively. Transcriptome data and RT-qPCR were based to understand the expression patterns of DIR genes in different tissues and under stress treatment, and to explore the candidate DIR genes related to growth and development and stress responses. Result A total of 34 DIR genes were identified from the potato genome and unevenly distributed on 11 chromosomes. Phylogenetic analysis showed that 34 StDIR genes were divided into three subgroups. The conserved motifs and gene structures of StDIR members in the same subgroup were relatively consistent. Most of the StDIR genes contained only one exon. Analysis of cis-acting elements indicated that the StDIR genes had diverse hormone-related elements and abiotic stress response elements. Synteny analysis revealed that 5, 13 and 18 DIR genes in potato were collinear with 3, 15 and 16 DIR genes in rice, Arabidopsis and eggplant, respectively. The expression analysis of StDIR gene showed that the StDIR genes had relatively high expressions in the leaves, roots, stolons, and callus. However, the transcript abundance of the StDIR genes was lower in sepals, carpels, stamens, and petals. Furthermore, StDIR genes had different expressions under salt, drought, heat, and cold stresses, indicating that StDIR genes play important roles in abiotic stress responses. Conclusion A total of 34 DIR genes are identified in potato and divided into three subgroups. The conserved motif and gene structure in the same subgroup are basically consistent. The expression pattern of StDIR genes vary in different tissues and significantly respond to drought and high temperature stresses.

Screening and Interaction Verification of Transcription Factors Stpatatin 05 Gene in Potato

YU Ting, HUANG Dan-dan, ZHU Yan-hui, YANG Mei-hong, AI Ju, GAO Dong-li

2025, 41(3):  137-145.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0991

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Objective The aim of this study is to identify the transcription factors binding to patatin promoters and provide new gene resources for patatin protein accumulation. Method We selected Stpatatin 05 gene as a candidate gene. Its promoter was cloned and integrated into the pAbAi vector to obtain the pAbAi-Stpatatin 05 bait vector. The resultant vector was then transformed into Y1H yeast cells to yield the yeast bait strain. Yeast one-hybrid (Y1H) screening was conducted based on the cDNA library of young potato tuber tissues. The interaction of candidate proteins was verified and the interaction motif was analyzed. The effect on the expression of the target gene was investigated using dual-luciferase reporter system. Result A candidate protein StMYB86-like was obtatined by Y1H. StMYB86-like belongs to the MYB-like transcription factors family and encodes a protein of 320 amino acids. It is located in the nucleus. The full length sequence of StMYB86-like was cloned to verify the interaction. The Y1H results showed that the yeast cells in the control group did not grow normally under the condition of 30 mmol/L 3-amino-1, 2, 4-triazole (3-AT), while the yeast cells in the experimental group grew normally, supporting the interaction between StMYB86-like and the promoter of Stpatatin 05 gene. Y1H results indicated that StMYB86-like bound to the CAACTG motif in the promoter, and the dual-luciferase reporter system showed that StMYB86-like negatively regulates the expression of its downstream target gene. Conclusion StMYB86-like interacts with Stpatatin 05 promoter; which may play a role in the accumulation of patatin protein in potato tubers.

Identification and Comparative Analysis of the StGAox Genes in Diploid and Tetraploid Potatoes

QIN Yue, YANG Yan, ZHANG Lei, LU Li-li, LI Xian-ping, JIANG Wei

2025, 41(3):  146-160.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1018

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Objective Gibberellin oxidases (GAoxs), including GA20 oxidase (GA20ox), GA2 oxidase (GA2ox), and GA3 oxidase (GA3ox), are key regulatory enzymes in the biosynthesis of gibberellin (GA) in plants, playing crucial roles in plant growth, development, and stress responses. This study is aimed to identify members of the the StGAox gene family from the whole genomes of diploid and tetraploid potatoes, and to conduct a compareative analysis of the evolutionary patterns of this gene family in potatoes with different ploidy levels, providing insights into the potential functions of StGAox in potatoes. Method Based on the whole genome information of three diploid and three tetraploid potatoes, bioinformatics methods were used to identify and the StGAox gene family and comparatively analyze the phylogenetic relationships, gene structure, chromosome localization, collinearity, gene replication patterns, subcellular localization, cis-acting elements, and expression patterns in stolons for members of this family in potatoes with different ploidy levels. Result A total of 35, 26, and 56 GAox homologous genes were identified in the diploid potatoe Solanum tuberosum group Phureja DM1-3 516 R44 (DM), S. chacoense M6 (M6), and S. tuberosum group Tuberosum RH89-039-16 (RH), respectively. In tetraploid potato varieties, S. tuberosum cv. Atlantic (Atl), S. tuberosum cv. Cooperation-88 (C88), and S. tuberosum cv. Otava (Ot), 99, 133, and 105 homologous genes were identified, respectively. These genes were unevenly distributed on 12, 24, or 48 chromosomes, with their encoded proteins mainly located in the nucleus, cytoskeleton, and chloroplasts. By comparative evolutionary analysis between diploid and tetraploid potatoes, as well as tomatoes and Arabidopsis, the genes were classified into three subfamilies: GA20ox, GA3ox,and GA2ox, with similar conserved protein motifs and gene structures. Collinearity analysis revealed varying degrees of translocation in the StGAox among the six potatoes. In terms of gene replication, compared with diploid potato DM, 21 and 51 collinear genes were found in the diploid potatoes M6 and RH, respectively, while 72, 117, and 95 StGAox genes in the tetraploid potatoes Atl, C88, and Ot, respectively, were identified as collinear genes. Cis-regulatory element analysis showed that stress-related CAAT-box, growth-related TATA-box, hormone-related CGTCA-motif, ERE and TGACG-motif, and light-response elements such as Box 4 and G-boxes were the most abundant. Expression patterns analysis indicated that differential expression of StGAox in stolons, with most having TPM values below 20. Additionaly, 31 StGAox genes were exclusively expressed in swollen stolons, while 12 genes had significantly higher expressions in swollen stolons compared to the apical hooked stolons. Conclusion A total of 454 GAox genes are identified in diploid and tetraploid potatoes, revealing the gene structure, motif composition, chromosome distribution, gene replication patterns, and expression profiles of the GAox family in stolons with different ploidy levels.

Genome-wide Identification and Expression Analysis of CCDs Family in Brassia rapa L.

WANG Chen, LIU Guo-mei, CHEN Chang, ZHANG Jin-long, YAO Lin, SUN Xuan, DU Chun-fang

2025, 41(3):  161-170.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0722

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Objective The members of the carotenoid cleavage dioxygenase gene family in Brassia rapa L. were identified to gain insight into the function and tissue expression characteristics of the BrCCD family genes. Method Bioinformatics methods were used to identify the CCDs gene family in B.rapa L., construct a phylogenetic tree, and analyze the chromosome distribution, gene structure, conserved motif, intraspecific collinearity and promoter cis elements. Combined with transcriptome data and RT-qPCR technology, the expression of CCDs genes in different tissues was analyzed. Result Total 16 BrCCD genes were identified in B.rapa L., which were divided into six subgroups and distributed unevenly on eight chromosomes. Colinearity analysis showed that there was a collinearity between seven pairs of CCDs genes. Cis element analysis showed that CCDs family members may respond to various regulatory processes such as growth and development, hormone regulation, and abiotic stress. Transcriptome and quantitative PCR data analysis showed that the expression of BrCCD was tissue-specific, and the expression of BrCCD-L gene was higher in flowers, leaves and seeds, BrCCD1b was higher in flowers, and the rest was higher in seeds and leaves. Result Total 16 CCDs genes were identified in the genome of B.rapa L., which showed different expression patterns in different tissues. The BrCCD-L gene was highly similar to the CCD2 gene with saffron genus, and was significantly expressed in different tissues.

Identification of β‍-amylase Gene Family in Melon and Their Response to Abiotic Stresses

HAN Jiang-tao, ZHANG Shuai-bo, QIN Ya-rui, HAN Shuo-yang, ZHANG Ya-kang, WANG Ji-qing, DU Qing-jie, XIAO Huai-juan, LI Meng

2025, 41(3):  171-180.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0926

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Objective β‍-amylase (BAM) catalyzes the hydrolysis of starch and plays a crucial role in plant growth and stress response. Identifying the BAM gene family members from the whole melon genome may provide genetic resources for function study of BAM gene and resistance breeding in melon. Method This work analyzed the physicochemical properties, phylogenetic evolution, gene structure, conserved motifs, promoter cis-acting regulatory elements of the BAM family's protein. RT-qPCR was used to analyze their expression patterns under drought, ABA, low temperature, and salt stress. Result Nine CmBAMs were identified from the whole-genome of melon (Cucumis melo), and they were located on chromosome 1, 4, 5, 6, 7, and 11, with an average length of 543 amino acids. The average molecular weight was approximately 58.7 kD, and the isoelectric point ranged from 5.47 to 8.89. Phylogenetic analysis demonstrated that BAMs in melon were highly homologous with cucumbers (Cucumis sativus) with over 94.5% homology and one-to-one correspondence. CmBAMs promoter in melon mainly harbored hormone-responsive elements, photo-responsive elements, abiotic stress elements, and protein binding elements. Expression pattern analysis of CmBAMs under drought, ABA, low temperature, and salt stresses revealed that CmBAMs responded to these four abiotic stresses at varied level. Notably, CmBAM1, CmBAM3, and CmBAM9 were dramatically induced by drought, while CmBAM3, CmBAM5, and CmBAM9 showed strong induction by ABA. CmBAM1, CmBAM5, and CmBAM9 were obviously induced by cold stress whereas CmBAM3 showed the highest induction under salt stress. Conclusion Nine CmBAMs are identified from the whole genome of melon, and CmBAM1, CmBAM3, CmBAM5, and CmBAM9 were the most sensitive to the four abiotic stresses, suggesting that they might play a key role in abiotic stress and thus can be selected as candidate genes for further research.

Effects of Different Concentrations of Melatonin on the Physiological Characteristics of Strawberry Seedlings under High-temperature Stress

WU Xia-ming, YANG Min, ZHOU Chen-ping, KUANG Rui-bin, LIU Chuan-he, HE Han, XU Ze, WEI Yue-rong

2025, 41(3):  181-189.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0635

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Objective The impacts of different concentrations of melatonin treatments on the growth and physiological characteristics of strawberry seedlings under high-temperature stress were investigated to establish a theoretical foundation for the advancement of heat-resistant cultivation technology for strawberries. Method ‘Hongyan’ strawberry seedlings were subjected to different concentrations of melatonin via root irrigation and foliar spraying to examine the effects of exogenous melatonin treatments on the growth and physiological characteristics of strawberry seedlings under high-temperature stress. Result The growth of strawberry seedlings was constrained under high temperature stress. However, melatonin treatment alleviated the growth inhibition of strawberry seedlings under high-temperature stress compared to the control. Key parameters such as net photosynthesis rate, transpiration rate, and stomatal conductance of strawberry seedlings showed significant increases. Additionally, the activities of antioxidant enzymes (SOD, POD, and CAT) and proline content were significantly elevated, while hydrogen peroxide content, superoxide anion radical production, relative conductivity and MDA content were notably reduced, with the most pronounced effects observed with a 400 μmoL/L^{treatment. Conclusion Under high-temperature stress, a 400 μmoL/L melatonin treatment improved the ability of strawberry seedlings to withstand heat-induced adversity by enhancing photosynthetic efficiency, boosting antioxidant enzyme activities, maintaining cell membrane stability, and reducing the accumulation of harmful substances.}

Identification of the TCP Gene Family in Actinidia chinensis var. Hongyang and Their Expression Analysis in Fruit

SONG Shu-yi, JIANG Kai-xiu, LIU Huan-yan, HUANG Ya-cheng, LIU Lin-ya

2025, 41(3):  190-201.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0820

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Objective The members of TEOSINTE-BRANCHED1/CYCLOIDEA/PCF in kiwifruit ‘Hongyang’ (Actinidia chinensis var. Hongyang) (AcTCPs) gene family were identified based on whole-genome. Taxonomic and evolutionary relationships of the members were analyzed, and the expression profiles of the AcTCP genes in tissue-specific expression, fruit development process, and hormones treatments were explored. These findings provide a basis for further exploring the function of the AcTCP gene in kiwifruit. Method Bioinformatics methods were used to analyze the physicochemical properties, gene structure, conserved motifs, cis-acting elements and inter-species covariance of TCP transcription factors in kiwifruit ‘Hongyang’ were analyzed. RNA-seq and RT-qPCR were used to investigate the expression profiles of AcTCPs in different tissues, fruit development and in response to hormonal stimuli. Result A total of 40 AcTCP genes with complete TCP domains were identified on19 chromosomes in kiwifruit ‘Hongyang’ and named AcTCP1-AcTCP40. Using phylogenetic analyses, conserved motif, and gene structure analyses, the family members were divided into PCF (21), CIN (12) and CYC/TB1 (7). Synteny analysis showed that genome-wide or segment-based replication played an important role in the expansion of the kiwifruit ‘Hongyang’ TCP gene family. Analysis of tissue-specificity showed that the six candidate genes expressed significantly high in male flowers and fruits. The results of RT-qPCR experiments at different stages of fruit development found that AcTCP1 showed elevated expressions at later stages of fruit development, while AcTCP11, AcTCP35, AcTCP10, and AcTCP32 demonstrated high expression during the initial stages of fruit development, and the expression pattern was consistent with the transcriptomic results. In fruit, abscisic acid (ABA) and ethylene (ET) treatments to the fruits down-regulated six candidate AcTCPs, gibberellin (GA₃) treatment up-regulated the expressions of AcTCP1, AcTCP35 and AcTCP32, and the forchlorfenuron (CPPU) treatment up-regulated the expressions of AcTCP1, AcTCP35 and AcTCP32 in initial stages, whereas GA₃ and CPPU decreased the expressions of the others AcTCPs. Conclusion A total of 40 TCP members from kiwifruit ‘Hongyang’ were systematically identified. The expression patterns of six candidate genes highly express in the fruit and male flowers. The expressions of AcTCP35, AcTCP32, AcTCP1, AcTCP10, AcTCP11 and AcTCP20 in the fruit are involved in kiwifruit development and are regulated by various exogenous hormones (ABA, ET, GA₃, and CPPU).

Transcriptome Analysis of Axillary Bud Formation and Development in Sugarcane

LI Xu-juan, LI Chun-jia, LIU Hong-bo, XU Chao-hua, LIN Xiu-qin, LU Xin, LIU Xin-long

2025, 41(3):  202-218.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0755

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Objective The key metabolic pathways and regulatory genes of sugarcane axillary bud formation and development were explored to provide valuable genetic resources for the breeding of sugarcane varieties and the formulation of genetic improvement strategies. Method The tender, semi-large, young and mature axillary buds of ‘XTT22’ were selected as the material, and the growth points of stem apex were used as the control. First, transcriptome libraries of the four stages of axillary bud formation and development were obtained by RNA-seq. The DESeq2 software was used to screen differentially expressed genes (DEGs), as well as GO and KEGG functional enrichment analysis was carried out. Then real-time fluorescent quantitative PCR (RT-qPCR) was used to verify the reliability of transcriptome sequencing results. Finally, the core genes related to the formation and development of sugarcane axillary buds were excavated by weighted gene co-expression network analysis (WGCNA). Result A total of 62 630 DEGs were obtained during the formation and development of sugarcane axillary buds. KEGG enrichment analysis mainly concentrated in mannose-type O-glycan biosynthesis, indole alkaloid biosynthesis, brassinolide biosynthesis and other pathways. KEGG enrichment pathway network analysis showed that hormone signal transduction, cyanamide metabolism and phenylpropanoid biosynthesis are the core pathways of axillary bud formation and development, while β‍-alanine metabolism and nitrogen metabolism are the specific core pathways in the development of tender axillary buds. C-5 branched-chain dibasic acid metabolism, tryptophan metabolism and fatty acid progression pathway are the core enrichment pathways of young and mature axillary buds respectively. The RT-qPCR results of 9 DEGs showed similar expression patterns to those of RNA-seq results. The 20 core genes related to the formation and development of sugarcane axillary buds, including UBR7, IRK and POLD WGCNA analysis was identified. Conclusion During the formation and development of sugarcane axillary buds, mannose-type O-glycan, sucrose, starch and nitrogen are needed as the material and energy basis, and plant hormones and secondary metabolites are also needed to regulate the related growth and development process. UBR7, IRK and POLD genes may play a positive role in the formation and development of sugarcane axillary buds, while DGK1, PCaP1 and LSD1 may negatively regulate the formation and development of sugarcane axillary buds.

Morphological Characterization， Karyotype and ISSR Marker of Lilium longiflorum and L. longiflorum ‘Snow Queen’

CHENG Shi-yuan, HE Jia-li, HAN Fu-bin, YANG Na-na, YANG Li-ping, FU Yong-yao

2025, 41(3):  219-229.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0798

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Objective Lilium longiflorum ‘Snow Queen’ belongs to the Lilium longiflorum hybrid lily, and clarifying differences between L. longiflorum and L. longiflorum ‘Snow Queen’ will lay the foundation for their identification and development. Method Using classical measurements, chromosome tableting and ISSR marker methods were used to systematically analyze the morphological characterization, karyotype and genetic variation respectively. Result Morphological observations showed that the leaves of L. longiflorum were much narrower and the capsule length slightly increased than those in L.longiflorum ‘Snow Queen’, while there were no significant differences in plant height, flowering diameter, flowering time, bulb weight and the outer scale weight. The projected area of pollen grains in L. longiflorum ‘Snow Queen’ was much larger than that of L. longiflorum, of which the highest number was distributed between 8 700-8 900 μm², accounting for 14.4%. Leaf epidermal cells and stomatas of L. longiflorum ‘Snow Queen’ were significantly wider than those in L. longiflorum. Karyotype analysis showed that L. longiflorum and L. longiflorum ‘Snow Queen’ were both diploids, with the chromosome formula 2n=2x=24. The karyotype formula of L. longiflorum was 2n=2x=24=2m+6sm+10st+6t, and that of L. longiflorum ‘Snow Queen’ was 2n=2x=24=2m+4sm+16st (2SAT) +2t, and karyotype asymmetry coefficient was much greater in the latter. ISSR marker analysis indicated that L. longiflorum ‘Snow Queen’ experienced a genetic variation at a rate of 20.00% compared to L. longiflorum control. Conclusion There were significant differences in leaf morphology, pollen grains, karyotype and molecular genetic levels between L. longiflorum and L. longiflorum ‘Snow Queen’, providing basic information for their identification and development in future.

Cloning and Expression Analysis of AwANS Genes in Allium wallichii

PENG Ting, LIN Ying, TAN Yuan-yuan, RAO Ying, HUANG Qin, ZHANG Wen-e, WANG Bo, TIAN Rui-feng, LIU Guo-feng

2025, 41(3):  230-239.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1004

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Objective Allium wallichii is a characteristic alpine flower. Anthocyanidin synthase (ANS) is a key enzyme in the anthocyanin biosynthesis, which lies downstream of flavonoid biosynthesis. This study is to investigate the role of ANS in the flower color formation of purple and white A. wallichii. Method pH-differential method was used to determine the total anthocyanin contents at different developmental stage of flowers. The coding sequences of AwANS genes were cloned by RT-PCR. The sequence alignments and expression patterns of AwANSs were also analyzed. Result The total anthocyanin contents of purple flowers increased gradually with flower development and peaked at S4, while those of white flowers could barely be measured. Two sequences of AwANS (AwANS^pa and AwANS^pb ) were cloned from white A. wallichii flowers and both of them had the CDS sequence length of 1 074 bp CDS with 357 amino acids. However, there were five sequences of AwANS (AwANS^wa, AwANS^wb, AwANS^wc, AwANS^wd, and AwANS^we ) cloned from purple A. wallichii flowers. Among them, AwANS^wa and AwANS^wd were 1 074 bp and 1 077 bp in CDS length, encoding two proteins with 357 and 358 amino acids, respectively. The protein translations of the remaining three sequences were terminated prematurely because of large fragment deletion. Phylogenetic analysis indicated that AwANSs were the closest to A. cepa. qRT-PCR analyses indicated that AwANSs in purple A. wallichii flowers had the highest expressions in blooming flowers and had the lowest expressions in the roots, pistils and fruits. The expression of AwANSs enhanced gradually with flower development and reached the peak at S5 in purple A. wallichii flowers. However, AwANSs expression were almost undetectable in white flowers. Conclusion The expressions of AwANSs have obvious spatial and temporal specificity and are the highest in flowers. Compared with purple A. wallichii, anthocyanins could not be detected and AwANSs are barely expressed throughout flower development in white flowers, suggesting AwANSs might play a key role in the flower color formation of purple A. wallichii.

Chloroplast Genome Characteristics and Phylogenetic Analysis of Scrophulariaceae

LI Bin, SU Xiang-ping, LIU Chang, WANG Yu-bing, ZHANG Yong-hong, ZHOU Chao, XU Qing

2025, 41(3):  240-254.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0753

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Objective To explore the characteristics and phylogenetic relationships of chloroplast genome of the Scrophulariaceae. Method The chloroplast genomes of Hubei Scrophularia ningpoensis were sequenced, assembled, and annotated. Additionally, a total of 46 complete chloroplast genomes of Scrophulariaceae were downloaded from GenBank database for comparative analysis. Result The full length of the chloroplast genome in Scrophulariaceae ranges from 142 336 to 154 710 bp, with a GC content between 37.7% and 38.1%. The genome shows a typical quadripartite structure, comprising a large single copy region of 83 531 to 97 103 bp and a small single copy region of 17 375 to 18 600 bp, as well as an inverted repeat region spanning 13 497 to 25 695 bp. Comparative analysis of chloroplast gene composition within the Scrophulariaceae reveals evolutionary patterns of gene gain and loss. Collinearity analysis indicates that the genomic arrangement of the Scrophulariaceae chloroplast genome is relatively conserved, although instances of genome rearrangement events have also been observed. Analysis of long repeat sequences shows that the majority of the Scrophulariaceae chloroplast genome consists of forward repeats and palindromic repeats. Furthermore, simple repeat sequence analysis identified 117 to 156 SSR sites within the Scrophulariaceae chloroplast genome, with single nucleotides composed of A/T being the most frequent, accounting for 85.50% to 91.50% of the total. Relative synonymous codon usage (RSCU) analysis identified 25 optimal codons, the majority of which ended with A/U. Divergence time analysis suggests that the common ancestor of Scrophulariaceae and closely related species diverged approximately 70.5 million years ago (MYA) and formed a monophyletic branch around 52.4 MYA, with most species of Scrophulariaceae appearing nearly 50 MYA. Conclusion Although the chloroplast genomes of the Scrophulariaceae demonstrate similar structural characteristics, they have also experienced gene acquisition and loss as well as genome rearrangement events during their evolutionary history. A more refined phylogenetic tree of the Scrophulariaceae has been constructed based on the chloroplast genomes. Furthermore, the divergence time analysis indicates that the rapid diversification of Scrophulariaceae species mainly occurre around 50 million years ago.

Comparative Transcriptomic Analysis of Clove Basil （Ocimum gratissimum） Leaves in Response to Cadmium Stress

WANG Bin, WANG Yu-kun, XIAO Yan-hui

2025, 41(3):  255-270.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0618

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Objective Clove basil (Ocimum gratissimum) has the potential for both remediation and safe utilization of cadmium (Cd)-contaminated soil. Elucidating the molecular mechanisms of clove basil in response to Cd stress and identifying key regulatory factors closely associated with Cd tolerance may provide candidate gene resources for the innovation of germplasm resources of clove basil tolerant to Cd stress. Method This study employed comparative transcriptome to analyze the transcriptomic changes in clove basil leaves during 72 h of Cd treatment (35 μmol/L). Result Cd stress significantly inhibited the growth of clove basil. The leaves, stems, and roots of clove basil accumulated high levels of Cd, with the highest content in the roots. Additionally, Cd stress significantly affected the composition and content of essential oils, inducing the biosynthesis and accumulation of methyl ester cinnamic acid in the leaves. The transcriptome analysis results showed that differentially expressed genes (DEGs) were significantly enriched in pathways such as ribosome, plant-pathogen interaction, and the MAPK signaling pathway in the 24 h comparative group. Whereas DEGs were significantly enriched in pathways including photosynthesis, photosynthesis-antenna proteins, and porphyrin and chlorophyll metabolism in the 72 h comparative group. Genes within the light cyan1 module were significantly correlated with the Cd stress phenotype. These genes were primarily enriched in defense pathways such as plant hormone signal transduction, plant-pathogen interaction, and the MAPK signaling pathway. Furthermore, the upregulated DEGs in response to Cd stress were also significantly enriched in these three pathways, suggesting that the activation of these pathways was the main mechanism by which clove basil leaves responded to Cd stress. Cd stress treatment induced the expressions of 13 transcription factor genes, in which 5 genes from the bHLH family, suggesting crucial roles for bHLH transcription factors in regulating Cd stress tolerance in clove basil. Conclusion The molecular mechanisms by which clove basil responds to Cd stress closely resemble those of other plant species, and the bHLH transcription factor appears to act as key regulators of Cd tolerance in clove basil.

Screening， Indentification and Promotion Performance Investigation of Tobacco Growth-promoting Rhizobacteria

MA Yao-wu, ZHANG Qi-yu, YANG Miao, JIANG Cheng, ZHANG Zhen-yu, ZHANG Yi-lin, LI Meng-sha, XU Jia-yang, ZHANG Bin, CUI Guang-zhou, JIANG Ying

2025, 41(3):  271-281.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0774

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Objective In order to provide important microbial fertilizer germplasm resources for promoting plant growth and improving farmland soil environment, the growth promoting bacteria with multiple functions were screened out in the rhizosphere of tobacco plants, and their effects on promoting plant growth and development were studied. Method The strains were screened from the rhizosphere of tobacco plants. The IAA production of these strains was determined by Salkowski colorimetry,and their phosphorus- and potassium-dissolving abilities were determined by molybdenum antimony resistance colorimetry and flame photometry. The dominant growth-promoting strains were screened by morphological, physiological and biochemical identification and 16S rDNA molecular identification. The optimal growth-promoting conditions were studied by orthogonal test, and the growth-promoting effect was studied by pot experiment. Result An efficient and multifunctional growth-promoting strain Arthrobacter woluwensis DY8 was screened. Its IAA-producing ability reached 36.7 mg/L, potassium-dissolving and inorganic phosphorus-dissolving ability reached 72.3 mg/L and 148.6 mg/L, respectively, and it antagonized pathogenic bacteria. Among these, the IAA production capacity reached its highest when the liquid volumewas 30 mL/250 mL, the pHwas 8, peptone was used as the nitrogen source, and glucose was used as the carbon source. In the tobacco potted experiment, after inoculating with strain DY8, in comparison with the control group, the contents of IAA, available phosphorus and potassium in tobacco soil were significantly augmented and the biomass and photosynthesis of tobacco also increased. Principal Component Analysis (PCA) results indicate that DY8 enhanced tobacco biomass by increasing the IAA content in the soil. In pot experiments with bok choy and wheat, DY8 treatment improved root structure, promoted root development, and increased above-ground biomass. Conclusion DY8 is a multifunctional growth-promoting bacterium that generates IAA, solubilizes phosphorus and potassium. It has a fine growth-promoting effect on economic crops, vegetable crops.

Analysis of Genetic Diversity for Wild Germplasm of Pleurotus pulmonarius in China Based on Whole-genome SNP Loci and Trait Characteristics

YUE Yuan-yuan, HU Zhe-yuan, HE Qi, HUANG Chen-yang, ZHENG Su-yue, ZHAO Meng-ran

2025, 41(3):  282-293.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0969

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Objective The genetic variation of the wild resources in Pleurotus pulmonarius was analyzed, aiming to provide the excellent parent strains and data support for the breeding of new varieties. Method A total of 37 wild strains were used to analyze the genetic diversity based on SNP loci obtained by whole genome resequencing, and to observe the phenotypic traits such as cultural characteristics and fruiting characteristics. Result The number of SNPs detected in the wild strains ranged from 71 521 to 114 390, and the average genetic distance between strains was 0.180. Cluster analysis showed that the closer genetic relationship among most strains from the same location, and those was closer among the strains from Shaanxi, Sichuan and Xinjiang regions in comparison with the strains from Yunnan. Abundant phenotypic diversity in culture characteristics such as favorable temperature, temperature sensitivity, colony recovery, and fruiting characteristics such as primordium formation and differentiation, fruiting body maturation, and cap color were observed in wild resources of P. pulmonarius. The cluster analysis based on phenotypic characteristics showed that all strains were divided into two groups according to primordium differentiation at the similarity coefficient of 0.52, which was highly consistent with the classification results of the principal component analysis. Conclusion The genetic variation in the wild germplasm of P. pulmonarius is abundant,which have fine potential for domestication and utilization. The degree of genetic differentiation at the genome level among wild strains might be related to their geographical distributions rather than their phenotypic characteristics.

Screening， Identification and Whole Genome Analysis of a Cellulase Producing Strain

CHE Jian-mei, ZHENG Xue-fang, WANG Jie-ping, CHEN Yan-ping, CHEN Bing-xing, LIU Bo

2025, 41(3):  294-307.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0448

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Objective To screen out the microbial strains with strong cellulose degrading enzyme activity for efficient fermentation of silage. Method The cellulase-producing strains were screened by carboxymethyl cellulose sodium plate from existing strain banks and their cellulase activity was determined. Morphological observation, physiological and biochemical analysis and 16S rRNA were used to identify this strain. The growth curve, growth temperature, pH and salt tolerance of strain FJAT-25102 were measured to study its growth characteristics. In further, the whole genome data of this strain was analyzed by bioinformatics to explore the cellulase genes. Result According to the ratio D/d between transparent circle D and colony diameter d, the strain with the highest D/d ratio was FJAT-25102. Further determination of its enzyme activity showed that its cellulase activity was 221.81 U/mL at 48 h. It was found that this strain had the ability to produce protease and amylase. Strain FJAT-25102 was identified as Micrococcus luteus by morphological, physiological, biochemical and phylogenetic analysis. Strain FJAT-25102 was in the growth initiation stage after 2 h of culture, and the OD₆₀₀ value reached the maximum at 26 h of culture. It grew between 20-50℃, pH 5-11 and 0%-5% NaCl concentration, and had certain high temperature resistance and saline-alkaline resistance. The genome of strain FJAT-25102 consisted of a ring chromosome with a size of about 2 570 649 bp and a GC content of 72.98%. The genome contained 2 378 predicted protein-coding sequences. All possible genes involved in cellulose degradation in strain FJAT-25102 included possible alpha-glucosidase GH13, endoglucanase GH74, and acetylxylanesterase CE1, CE7, and CE3. Among them, GH13 and GH74 regulated enzymes in the glycoside hydrolase family of FJAT-25102 may play an important role in the process of cellulose degradation. The contents of crude fiber, acid detergent fiber and neutral detergent fiber in the silage treated by strain FJAT-25102 significantly reduced. Conclusion The selected Micrococcus luteus FJAT-250102 may provide microbial strain resources for silage fermentation. The determination of its whole genome and investigation of cellulase genes may provide the basis for the subsequent research on the cellulase production mechanism of this strain.

Screening and Degrading Characteristics of Cellulose-degrading Strains in Chinese Herb Residues

ZHANG Yu, ZHOU Chun-fu, PENG Yi-xuan, ZHANG Jia-hao, YIN Xin-ru, QIAO Ce-ce, XIE Yue, WANG Jian-fei, WANG Xiang

2025, 41(3):  308-318.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0482

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Objective To isolate and screen efficient cellulose degrading strains from Chinese herb residues, and they were used to improve resource utilization. Method CMC-Na plate was used to primarily screen cellulose-degrading bacteria, combined Congo red staining and disintegration test of filter paper strips to have re-screening. They were identified by physiology, biochemistry, and 16S rDNA molecular biology. Then the cellulase activities of strains with different growth and antibiotic culture conditions were investigated, and the degrading abilities of strains were determined using Chinese herb residues as the only carbon source. Result Three strains with cellulose-degrading ability were isolated and screened from Chinese herb residues. H-1 and Z-1 were identified as Bacillus sp., and Z-2 as Niallia sp. The optimal enzyme production conditions for strain H-1 and Z-1 were 10 mL/50 mL, pH 6, and 30℃.The optimal enzyme production conditions for strain Z-2 10 mL/50 mL, pH 7, 37℃, and adding oxytetracycline and enrofloxacin did not have a significant effect on the enzyme activities of strain H-1, Z-1 and Z-2. The degradation rates of Chinese herb residues by strain H-1, Z-1 and Z-2 were 56.37%, 54.76% and 41.53%, and the rates of degrading cellulose were 22.12%, 20.01% and 9.78%, respectively. Conclusion The three screened strains have robust cellulase activity, are not affected by antibiotics, and show an effective degradation capability for Chinese herb residues.

Enzymatic Characterization and Directed Evolution of Agrobacterium tumefaciens O-demethylase Atu1420

WANG Hao, CAO An-ni, GAO Xin-yi, GUO Min-liang

2025, 41(3):  319-329.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0993

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Objective Protocatechuic acid (PCA) is a widely used phenolic compound. Biosynthesis method of PCA is a potential alternative to the highly-polluting chemical synthesis. Finding and improving enzymes for synthesizing PCA is the key to biosynthesis. Agrobacterium tumefaciens O-demethylase Atu1420 is an enzyme that can convert vanillic acid (VA) to PCA. This work aims to characterize the enzymatic properties of Atu1420 and improve it. Method Atu1420 was expressed in Escherichia coli, and the enzymatic properties of Atu1420 were determined by methods such as high-performance liquid chromatography (HPLC). Based on the predicted structure and catalytic mechanism of Atu1420, 19 sites were selected to conduct site-directed mutagenesis on Atu1420. A visual method for detecting the catalytic activity of Atu1420 was developed by using 4-aminoantipyrine to screen variants of Atu1420. The structure of Atu1420 variants was predicted by alphaFold, and the potential mechanism for the improvement of catalytic efficiency of variants was analyzed. Result The V_max of Atu1420 was determined to be 33.5±1.6 nmol/(L·s), the K_m is 82.7±3.5 μmol/L, the k_cat is (6.7±0.3)×10^-1 s^-1, k_cat/K_m is 8.1×10^-3 L/(μmol·s), the optimal pH is between 7 and 8, and the optimal temperature is 30°C. Five variants with enhanced enzyme activity were screened out from the variants obtained by site-directed mutagenesis. The variant with the strongest activity is G35S, and its catalytic activity is 66.0% higher than that of the wild type. Combined mutations at these five sites did not produce a significant additive effect on enzyme activity. Comparative analysis of the structures indicates that the deflection of the arginine residue at position 121 may be the reason for the improvement of the catalytic efficiency of the variants. Conclusion The enzymatic properties of Atu1420 have been characterized, and five Atu1420 variants with improved catalytic efficiency have been obtained. The increase in the catalytic efficiency of the variants may be caused by the deflection of the arginine residue at position 121.

Impact of Low-temperature Fermentation with Mixed Microbial Agent on the Quality of Corn Straw Yellow Silage Feeds

REN Xin-ru, ZHAO Hong-lu, LI Ya-jing, LIU Rong-jun, ZENG Fan-li, WANG Qin-hong, WANG Zhen

2025, 41(3):  330-342.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0801

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Objective To produce high-quality corn straw yellow silage feed with high protein content and high palatability, a low-temperature fermentation process of corn straw yellow silage was developed using agricultural resource waste corn straw as the primary raw material and cold-resistant low-temperature Saccharomyces cerevisiae, lactic acid bacteria, and Bacillus subtilis as fermentation strains. Method Firstly, crude protein content was used as the primary index to investigate the compounding ratio of fermentation agents. The effects of inoculation amount, fermentation time, and ammonium sulfate addition amount on fermented feed were then explored using the single-factor test in conjunction with the response surface optimization test. Finally, the quality of low-temperature (10℃) fermented feed was thoroughly assessed. Result The crude protein content of the feed was the highest at 20.05%,while the compound ratio of cold-resistant S. cerevisiae, lactic acid bacteria, and Bacillus subtilis was 3∶1∶3, the inoculation amount of compound microbial agent was 18.50%, the fermentation time was 20.50 d, and the addition amount of ammonium sulfate was 2.53%. When compared to standard temperature (30℃) fermentation, low-temperature fermentation produced a greater scent in straw feed, with 20 aroma components showing significant variations. Phenylethyl alcohol had the greatest amount (20.22 μg/g), giving the feed a rose fragrance. Concurrently, characteristic aromatic odor compounds such as ethyl methoxyacetate (fragrance), dicyclobutyl oxalate (aromatic), and ethyl methyl carbonate (fruity) were found in low-temperature fermented feed. In addition, low-temperature fermentation reduced fungal contamination. Compared with fermentation without and with added microbial agents at room temperature, the fungal toxin content in low-temperature fermented feed reduced by 46.44% and 22.9%, respectively. Conclusion Low-temperature fermentation with mixed microorganisms can effectively improve the quality of corn straw fermented yellow silage feed, especially in terms of increasing the protein content of corn straw feed and enhancing its aroma and appetite.

Content

2025, 41(3):  343. 

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Copyright

2025, 41(3):  344. 

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Cover

2025, 41(3):  345. 

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