Table of Content

26 December 2024, Volume 40 Issue 12

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Research Progress in the Effects of Plant Abscisic Acid and Its Receptor Gene PYL9

CHEN Ying-e, LIANG Qiao-lan

2024, 40(12):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0494

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Abscisic acid（ABA）is an essential plant phytohormone for plant growth and development. It is involved in important processes such as the regulation of seed germination and dormancy, root growth, stomatal closure, leaf senescence, and abscission. Its function is mainly achieved through a series of signal transductions, and the first step of its signal transduction depends on the binding of its receptor. PYR1-like protein 9（PYL9）serves as a key receptor in the ABA signaling pathway, positioned upstream. Upon the detection of ABA signals, it suppresses the activity of protein phosphatase 2C（PP2C）, thereby activating SNF2-related protein kinase（SnRK2）and facilitating downstream gene's expression to initiate the ABA signaling cascade. This pivotal role contributes significantly to the regulation of plant stress resistance, as well as growth and development, by modulating abscisic acid signal transduction, cellular metabolism, and physiological and biochemical responses within plants. This paper provides a comprehensive review of the regulatory role of the ABA in plant growth, interactions with different hormone signals, synthesis and metabolic pathways, signal transduction composition and function, as well as the structure, functional characteristics, and important role and existing issues of ABA receptor PYL9 in stress response. The paper also prospects its application in future. It is also anticipated to offer a theoretical basis for systematic research on the function of ABA receptor gene PYL9 and mechanisms of plant antiviral defense reactions.

Database-aided Design of Antimicrobial Peptides

SHAO Chang-xuan, ZHANG Shao-hua, DENG Hao-ran, YU Wei-kang, ZHU Yong-jie, SHAN An-shan

2024, 40(12):  12-19.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0277

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Antimicrobial peptides（AMPs）are a diverse class of peptides ubiquitously present in nature, showcasing a broad spectrum of antibacterial activity. Due to their distinctive antibacterial mechanisms, they are considered a novel alternative to conventional antibiotics. In recent decades, substantial research has been conducted on AMPs. However, challenges persist in the development and application of these peptides, including difficulties extraction in vivo, high production costs, systemic cytotoxicity, and instability in physiological conditions. Consequently, scientists employ natural AMPs as a model to generate derived peptides with the aim of creating novel peptide drugs that possess enhanced potency and reduced toxicity. The process of experimental peptide design is deep concerned by its time-consuming, labor-intensive, and costly nature. However, with the continuous advancement of computational methods, publicly accessible databases now offer substantial amounts of AMPs data. These databases serve as a valuable resource for the development and construction of novel AMPs. In this paper, the existing AMP databases and database-aided design methods are reviewed with the aim to provide reference for drug development of AMPs.

Construction Strategies of Allosteric Transcription Factor Biosensors and Their Application Advances in Food Safety

ZHOU Zi-ying, SONG Xiao-dong, LIU Yang-er, WU Yi-fan, ZHU Long-jiao, GU Dong-yue, HE Guo-qing, LI Xiang-yang, XU Wen-tao

2024, 40(12):  20-33.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0392

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With the rise of synthetic biology, transcription factor-based biosensors are gradually transitioning from in vivo to in vivo sensing. These sensors play a role in various detection fields, especially in the field of food safety, due to their high safety, strong stability, and fast response. Currently, most of the reviews on allosteric transcription factor（aTF）biosensors focus on construction of whole-cell biosensors in vivo. However, drawing on previous studies, this paper focuses on exploring the in vivo construction of aTF biosensors, such as using cell-free transcription-translation systems and compatible buffer systems as reaction vectors. In this paper, we provide a detailed review of the construction strategies of aTF-based in vivo biosensors and the progress of their application in food safety detection. Firstly, the construction of aTF biosensors is systematically described, including the molecular recognition mechanism of aTF, two signal amplification strategies of isothermal amplification and CRISPR-Cas, two signal output modes of optics and electrochemistry, as well as the use of two sensing systems, namely, the compatibility buffer and cell-free. Secondly, the progress of the application of aTF biosensors in the detection of food contaminants such as heavy metal ions, pesticide and veterinary drug residues, food additives, and foodborne pathogens is highlighted. Finally, the challenges faced by aTF biosensors are discussed in depth, and their future development trends are envisioned with a view to further expanding their potential applications in new fields.

Establishment of a Universal PCR Detection System for Different Maize Events

WANG Jing, ZHANG Xiao-lei, BAI Yu, SHENG Yu-xin, GUAN Hai-tao, WEN Hong-tao

2024, 40(12):  34-44.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0361

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【Objective】Establishing a universal PCR detection system would address the issue of inconsistent amplification systems and reaction conditions of different events, thereby enhancing the efficiency of event detection. 【Method】 In this study, we analyzed and compared the differences in amplification systems and reaction conditions of qualitative and quantitative PCR detection methods for the specificity of transgenic material events. The most widely used PCR systems and conditions parameters were selected as unified parameters to validate the PCR qualitative detection methods for different maize events in the laboratory. 【Result】 General PCR amplification system: Total volume 25.0 μL, 25 mmol/L MgCl₂ solution 1.5 μL, 2.5 mmol/L dNTPs mixed solution 2.0 μL, final concentration of upstream and downstream primer 0.4 μmol/L, Taq DNA polymerase 0.025 U/μL, 25 mg/L DNA template 2.0 μL, LOD 0.1%. Reaction conditions: Denaturation at 94℃ for 5 min, 35 cycles of denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, and final extension at 72℃ for 7 min. Universal real-time fluorescent qualitative PCR amplification system: Total volume 20.0 μL, 25 mmol/L MgCl₂ solution 2.0 μL, dNTPs mixed solution（2.5 mmol/L each）1.6 μL, final concentration of upstream and downstream primers and probe 0.4 μmol/L, Taq DNA polymerase 0.04 U/μL, 25 mg/L DNA template 2.5 μL, LOD 0.1%. Reaction conditions: denaturation at 95℃ for 5 min; 95℃ denaturation 15 s, 60℃ annealing extension 60 s, 40 cycles.【Conclusion】This system and its associated amplification system and reaction conditions can be used to detect different event materials.

Specific Qualitative PCR Detection Method for Transgenic Maize Zheda Ruifeng 8

TIAN Jin, ZHANG Yue-qiu, ZHANG Hua, CHEN Zi-yan, TIAN Lu, WANG Hao-qian, GAO Fang-rui, LIANG Jin-gang, CHEN Hong

2024, 40(12):  45-52.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0324

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【Objective】Zheda Ruifeng 8 is an insect-resistant transgenic maize developed by Hangzhou Ruifeng Biotechnology Co. Ltd., the safety certificate for production and application was obtained in December 2021. The specific PCR detection method for Zheda Ruifeng 8 was investigated to provide technical support for market regulation and industrialization. 【Method】In this study, the flanking sequence of the insertion end of exogenous gene of Zheda Ruifeng 8 event was used as the target sequence, specific primers were designed at the 5'-end（right flanking）and the 3'-end（left flanking）respectively. Twenty pairs of primers for each of the left flanking and the right flanking were screened by the standard reaction system and the reaction procedure that was consistent with that of the standard gene zSSIIb in maize, and the primers with high specificity and fine stability were obtained. A preliminary method for the detection of PCR of the Zheda Ruifeng 8 event was established by changing the two key parameters of the annealing temperature and the concentration of the primer. 【Result】The screened Zheda Ruifeng 8 PCR amplification primer RF 8-RB-R4/F1 may specifically amplify the target fragment of 265 bp, and a standard reaction system and reaction procedure consistent with the standard gene zSSIIb in maize was established, and it achieved high throughput for batch detection. 【Conclusion】The established specific qualitative PCR detection method demonstrated excellent stability and adaptability. It could consistently detect a copy number fraction as low as 0.1%, equivalent to approximately 20 copies. This method proved to be highly accurate and specific, effectively detecting the transformants of Zheda Ruifeng 8.

Identification of Banana Hybrid and Selfing Offspring Based on Internal Transcribed Spacer（ITS）Sanger Sequencing

ZENG Hong-yun, XU Lin-bing, WU Yuan-li, HUANG Bing-zhi

2024, 40(12):  53-60.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0501

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【Objective】The aim of this study is to utilize the Internal Transcribed Spacer（ITS）region as a molecular marker in identifying newly developed hybrid and selfing offspring banana progeny, thereby laying the foundation for advancements in banana breeding and genetics research.【Method】DNA of parents and their suspected offspring was extracted, then PCR amplifification and Sanger sequencing were conducted, and sequence alignment was performed using software “DNAMAN” and “SnapGene” to analyze the polymorphism of ITS, thereby determining the hybrid and selfing offspring. The plant was classified as a positive plant when the ITS sequence of the suspected offspring plant showed differences at one or more base sites compared with the parent; conversely, if no such differences were found, the plant was determined to be a false positive plant.【Result】The study encompasses three sets of putative progeny derived from cultivated banana crossed with wild banana（‘Guang Fen No. 1’ × ‘Tian Ye BB No. 1’）, selfed offspring of a cultivated variety（‘Fen Za No.1’）, and a cross between two wild bananas（‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’）. Applying the established banana ITS Sanger sequencing protocol, the notable disparities linked to the ITS sequences of these candidate offspring compared to their parents were identified. Phenotypic observations further confirmed variances in the offspring plants, suggesting they were positive plants. In the hybrid offspring of ‘Guang Fen No.1’ and ‘Tian Ye BB No.1’, polymorphisms in the “410 bp region” were observed in the ITS Sanger sequencing profiles, accompanied by distinct changes in bunch characteristics. Among four selfed offspring of ‘Fen Za No.1’, polymorphic peaks in the “50 bp” and “410 bp regions” were detected, with two lines exhibiting distinctive leaf traits. For the ten seedlings resulting from the ‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’ cross, significant differences in the sequencing profiles were noted at “115 bp” “431 bp” and “452 bp” relative to their parents.【Conclusion】The ITS Sanger sequencing approach emerges as a complementary tool to phenotypic assessments and SSR-based identification methods, facilitating rapid, flexible, and convenient material authenticity confirmation in distinguishing banana hybrid or selfed offspring. This methodology aids breeders and researchers in efficiently confirming their materials accuracy in breeding and genetic studies.

Rational Design of Recombinant Adeno-associated Virus Transient Expressing System and Establishment of Its Efficient Production Process

LI Rui-rui, NA Dao-yuan, WANG Hong-lin, ZHAO Liang, TAN Wen-Song, YE Qian

2024, 40(12):  61-71.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0547

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【Objective】This study focuses on understanding the impact of crucial viral and helper genes within the triple plasmid system on rAAV production to guide rational design of viral gene cassette and process optimization, thereby enhancing rAAV production efficiency.【Method】First, we clarified the composition and ratio of the rep and cap gene, as well as the critical sequences of helper viral genes that influenced the production of rAAV. Based on these insights, we rationally designed the viral gene cassettes on the plasmids and constructed a novel triple system. Finally, the critical process parameters of this new system were optimized to establish a high-efficiency rAAV production process.【Result】The proper ratio of rep and cap gene expression was crucial for rAAV production. Retaining only the essential helper protein sequences E4orf6（adenovirus early region 4 opening reading frame 6）and DBP（DNA binding protein）in the helper viral gene expression cassette achieved the same rAAV production efficiency. Herein, we developed a novel triple plasmid system, which led to a 2.6 fold increase in genome titer compared to the traditional triple plasmid system. Further the optimization of transfection parameters resulted, the genome titer reached 7.8×10¹¹ vg/mL, and the CSVY（cell specific virus yield）reached 1.5×10⁵ vg/cell, which were 3.7 fold and 2.4 fold compared to the traditional triple plasmid system, respectively.【Conclusion】By exploring the effects of the composition and ratio of rAAV viral genes and helper viral genes on the production of rAAV, and we re-designed the triple plasmid system. Finally, we established a new rAAV manufacturing process with a novel triple plasmid system, which were elevated 16-fold in genome titer and 10-fold in CSVY, respectively, compared with that of the traditional triple plasmid system. This study lays the foundation for efficient industrial production of rAAV.

Research Progress in Nucleic Acid Molecular Diagnostic Technology for Mycoplasma pneumoniae

YU Yong-xia, ZHU Ning, LIU Guang-min, ZHU Long-jiao, XU Wen-tao

2024, 40(12):  72-83.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0119

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Mycoplasma pneumoniae is the smallest known cell organism, and is one of major pathogens causing community-acquired pneumonia. The symptoms of infection can differ widely in the early stages and can affect different organs in a body. Early diagnosis is challenging because early clinical symptoms and radiological examinations lack specificity and are prone to misdiagnosis and missed diagnosis, which can threaten physical health. At present, both domestic and international M. pneumoniae detection mainly relies on laboratory diagnostic methods. This article begins by examining the intricate pathogenesis of M. pneumoniae, which includes adhesion damage, membrane fusion damage, invasion damage, toxicity damage, immune damage, and inflammatory damage. It also briefly explores commonly used molecular diagnostic techniques in the laboratory, such as nucleic acid isothermal amplification technology and variable temperature amplification technology. The isothermal amplification techniques include loop-mediated isothermal amplification（LAMP）, chain displacement reaction, sequence-dependent nucleic acid amplification（NASBA）, and recombinase-aided amplification（RAA）. Additionally, variable temperature amplification techniques refer to traditional polymerase chain reaction（PCR）, broad-range PCR, nested PCR, real-time PCR, and multiplex PCR. The text also includes a comprehensive discussion on biosensing platforms for M. pneumoniae detection, which encompasses lateral flow assay, electrochemical biosensors, fluorescence biosensors, and more. This manuscript aims to summarize the advantages and disadvantages of current M. pneumoniae detection techniques, providing a reference for early diagnosis. It also anticipates future non-extraction, integration, and rapid detection methods. Along with reduced costs, these advancements could enable patients to perform self-examinations at home, avoid unnecessary use of anti-infective drugs, and usher in a precision era for clinical diagnosis and treatment.

Identification and Expression Analysis of High Mobility Gene Families in Spinach

ZOU Ting-ting, YANG Li, QU Xi-tong, CHEN Zi-hang, PAN Xi, SHAO Hong-xu, WANG Xiao-li

2024, 40(12):  84-92.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0420

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【Objective】High mobility group（HMG）proteins participate in the regulation of DNA structure and chromatin, which play an important role in gene transcription regulation. Identifying the family of HMG genes in spinach（Spinacia oleracea L.）may provide a theoretical basis for studying the biological functions of spinach HMG proteins.【Method】The members of the spinach SoHMG family were identified at the whole genome level, and their physicochemical properties, gene structure, conserved motifs, promoter elements, and salicylic acid-responsive expression profiles were analyzed. 【Result】The spinach genome contained 16 members of the SoHMG gene family, with a coding region length of 240-4 029 bp and a coding amino acid number between 79-1 342. The 16 SoHMG genes were scattered across six chromosomes. By evolutionary tree analysis, SoHMG members were divided into two subfamilies, including three members in A subfamily and 13 in B subfamily. The members of the same subfamily have similar conserved domains. Some hormone-responsive elements, such as salicylic acid（SA）, were found in the promoter of the SoHMGB subfamily. The SoHMG genes were expressed in the roots, stems, and leaves of spinach. Most of genes had higher expressions in the leaves. SA treatment induced the expressions of most of the SoHMG genes. Among them, the expressions of SoHMGB1（SOV1g000250.1）, SoHMGB7（SOV5g026260.1）, and SoHMGB2（SOV1g027200.1）were more than 10 times higher than those in control. 【Conclusion】There are 16 members of HMG gene family in spinach genome, which is divided into 2 subfamilies. Three genes（（SoHMGB1, SoHMGB7, and SoHMGB2））are significantly upregulated under SA treatment, which may play an important role in the SA signal response.

Cloning and Expression Analysis of CabZIP42 Gene in Pepper

LI Bai-xue, LI Jin-ling, CHEN Chun-lin, DU Qing-jie, LI Meng, WANG Ji-qing, MA Yong-bin, XIAO Huai-juan

2024, 40(12):  93-101.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0465

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【Objective】To explore the role of bZIP gene in response to abiotic stress would provide genetic resources for improving the stress resistance of peppers. 【Method】A candidate gene CabZIP42 was obtained and cloned based on transcriptome sequencing in pepper ‘G7’, the molecular characteristics of the encoded protein were analyzed by bioinformatics methods, and its expression patterns were analyzed by RT-qPCR in different tissues and under different stress treatments.【Result】The coding region of CabZIP42 was 1 233 bp in length, encoding 410 amino acids, with a predicted molecular weight of 44.85 kD and a theoretical isoelectric point of 9.47. The CabZIP42 protein contained two conserved domains, the basic amino acid region N-x9-R and the leucine zipper region x6-L-x6-L-x6-L. The secondary structure of the protein was dominated by random coils, and contained a small amount of α-helix. Phylogenetic analysis showed that CabZIP42 had the most recent protein evolutionary relationship with tomato and potato. Promoter prediction analysis showed that there were multiple hormonal and stress-related cis-acting elements on the CabZIP42 promoter.【Conclusion】The expression of CabZIP42 gene was the highest in pepper leaves, and it was involved in ABA signal transduction in response to drought, high temperature and high salt stress.

Identification of the HXK Gene Family in Cucumis melo and Their Expression Analysis under Abiotic Stresses

ZHANG Shuai-bo, YIN Jin-peng, WANG Ji-qing, XIAO Huai-juan, LI Meng

2024, 40(12):  102-112.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0418

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【Objective】 To clarify the genetic characteristics and potential functions of the hexokinase family in melon（Cucumis melo）. 【Method】 The melon HXK gene family was identified at the whole genome level, and its protein's physicochemical properties, subcellular localization, systematic evolution, gene structure, chromosome localization, conserved motifs, protein structure, promoter cis acting elements, etc., were analyzed. Based on transcriptome data, the expression patterns of CmHXK gene in male flowers, female flowers, roots, leaves, fruits, and fruit development processes were analyzed. In addition, the expression patterns of CmHXK gene under abscisic acid, drought, salt, and low temperature stress were measured by RT-qPCR. 【Result】A total of six CmHXK gene family members were identified on chromosomes 2, 4, 5 and 8, containing 6-9 exons, 5-8 introns, with an average amino acid number of 462, an average molecular weight of 50.2 kD and an isoelectric point of 4.99-6.34. Systematic evolutionary analysis showed that the melon HXK members had the closest homology with cucumber. The main cis-acting elements in the melon CmHXK promoter were light responsive elements, hormone responsive elements, and stress responsive elements. The expression of CmHXKs was specific in different organs. Among them, CmHXK1, CmHXK2, and CmHXK5 expressed at high level in male flowers, female flowers, roots, leaves, and fruits during the development and maturation of fruits. However, the expressions of CmHXK3, CmHXK4, and CmHXK6 decreased with the development and maturation of fruits. The responses of CmHXKs to the four abiotic（abscisic acid, drought, salt, and cold）stresses differed. Among them, CmHXK1, CmHXK3, and CmHXK4 were strongly induced by ABA. CmHXK1, CmHXK3, and CmHXK6 were strongly induced by salt stress, and CmHXK1, CmHXK2, and CmHXK3 were strongly induced by drought stress. CmHXK1 and CmHXK6 were strongly induced by cold stress. 【Conclusion】 The CmHXK family are highly conserved and play important roles in the growth, development, and abiotic stress in melon. Among them, CmHXK1 is the most sensitive to four abiotic stresses.

Study on the Regulation of Anthocyanin Metabolism by miR156-PhSPL3 in Pericallis hybrida

SUN Dan-ni, LI Hao, CUI Yu-meng, HUANG He

2024, 40(12):  113-123.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0325

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【Objective】Pericallis hybrida is an important potted ornamental flower with a variety of colors and anthocyanin metabolic branches, which is an ideal material to study the molecular mechanism of flower color regulation. miR156-PhSPL3 module is differently expressed in different varieties of P. hybrida, exploring the mechanism of this module in the formation of flower color would provide a theoretical basis for the regulation mechanism of flower anthocyanin metabolism.【Method】PhSPL3 and two Ph-miR156 precursor genes（Ph-MIR156a and Ph-MIR156b）were cloned from P. hybrida for bioinformatics analysis and tissue expression analysis. Experimental studies using VIGS, yeast one-hybrid（Y1H）and two-hybrid（Y2H）system, and dual-luciferase assays elucidated the function and regulatory role of miR156-PhSPL3 in anthocyanin metabolism.【Result】The open reading frame of PhSPL3 gene was 1 119 bp long, encoded a protein of 372 amino acids and belonged to Clade VII branch of the SPL family. Ph-MIR156a and Ph-MIR156b precursor stem-loop sequences were 108 bp and 104 bp long respectively, both containing mature miR156 sequences. Expression analysis revealed the high expression of PhSPL3 in carmine and blue varieties of P. hybrida ray florets, inversely correlated with Ph-miR156 expression. Dual-luciferase assays demonstrated that Ph-miR156 targeted to PhSPL3. Virus-induced gene silencing（VIGS）of PhSPL3 reduced anthocyanin content and downregulated expressions of anthocyanin metabolic genes PhCHS2, PhF3H1, PhANS, and regulatory gene PhbHLH17. Y2H and Y1H revealed PhSPL3 interacted with PhMYB5 and PhMYB7 and specifically bound to the promoters of PhCHS2 and PhbHLH17, thus regulating their expressions. 【Conclusion】The miR156-PhSPL3 pathway in P. hybrida may play a role in the biosynthesis of anthocyanin substances by directly regulating structural genes on the anthocyanin metabolism branch or indirectly regulating genes regulating anthocyanin metabolism.

Cloning SmERF B3-45 from Salix matsudana and Functional Analysis on Its Tolerance to Salt

HUA Xuan, TIAN Bo-wen, ZHOU Xin-tong, JIANG Zi-han, WANG Shi-qi, HUANG Qian-hui, ZHANG Jian, CHEN Yan-hong

2024, 40(12):  124-135.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0385

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【Objective】To validate whether SmERF B3-45 plays a positive regulatory role in plant response to salt stress may lay the foundation for revealing the role of AP2/ERF transcription factors in regulating the salt tolerance of Salix matsudana.【Method】The cis-acting elements in the promoter region of SmERF B3-45 from the AP2/ERF superfamily of Populus euphratica were analyzed. The full-length CDS sequence of SmERF B3-45 was cloned using specific primers, and bioinformatics and subcellular localization analysis were conducted. The function was elucidated by constructing overexpression vectors and transforming Arabidopsis thaliana mutants ERF-OE1 and ERF-OE2, and by using virus-induced gene silencing（VIGS）.【Result】Cis-acting element analysis suggested that SmERF B3-45 may be involved in the expression regulation pathways responding to stress. RT-qPCR results showed that the expression of SmERF B3-45 was induced by NaCl treatment and was widely expressed in different tissues of Populus euphratica. Subcellular localization indicated that the SmERF B3-45 protein was localized in the nucleus. In transgenic Arabidopsis, the expression of SmERF B3-45 significantly increased. Under salt stress, compared with the wild type, the root length of overexpressing SmERF B3-45 Arabidopsis significantly increased, while the total protein content, Na⁺ content, MDA content, and Na⁺/K⁺ ratio significantly decreased, and the CAT content and K⁺ content significantly increased. Gene-silenced plants showed a significant downregulation of SmERF B3-45 expressions. Compared with the control, the total protein content of the gene-silenced plants significantly reduced, while MDA and proline content were significantly higher than that of the negative control plants. Additionally, the silenced plants demonstrated wilting leaves, indicating that the silencing of SmERF B3-45 reduced the tolerance of Salix matsudana to salt. 【Conclusion】SmERF B3-45 is confirmed as a positive regulatory transcription factor in the plant response to salt stress.

Expression of HbTRXh5 Gene of Hevea brasiliensis in Yeast and Analysis on Its Resistance to Stress

WU Shuang, LU Rui-lin, FENG Cheng-tian, YUAN Kun, WANG Zhen-hui, LIU Jin-ping, LIU Hui

2024, 40(12):  136-144.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0488

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【Objective】Cloning and analysis of expression characteristics of the thioredoxin gene HbTRXh5 from rubber tree（Hevea bsinensis）, exploring its function in abiotic stress, which aims to provide genetic resources for genetic improvement of stress tolerance in rubber tree.【Method】RT-PCR method was used to clone HbTRXh5 gene from rubber tree, and bioinformatics methods were employed to analyze its sequence characteristics and phylogenetic relationship. Quantitative real-time PCR was applied to analyze the expressions of HbTRXh5 in various tissues of rubber trees and under abiotic stress. The yeast expression vector of HbTRXh5 was constructed and transformed into yeast, and then the survival differences between transgenic yeast and control yeast after cold, salt, and oxidative stress treatments were compared. 【Result】The coding region of HbTRXh5 gene was 354 bp in length and encoded 117 amino acids. HbTRXh5 contained a conserved thioredoxin domain and a CGPC active site, belonging to the subgroup I of h-type thioredoxin. HbTRXh5 was expressed in various tissues of rubber tree, with the highest expression in latex. Low temperature, salt, and oxidative stress-induced by H₂O₂ and methyl violet up-regulated the expression of HbTRXh5. HbTRXh5 gene was successfully transformed into the yeast strain INVSc1 and induced to have expression. Compared with the control yeast transformed with the pYES2 empty vector, the HbTRXh5 gene transgenic yeast had a higher survival rate after H₂O₂ treatment, but showed a lower survival rate after cold and salt stress treatments. 【Conclusion】The expression of HbTRXh5 is regulated by low temperature, salt, and oxidative stress in rubber tree. The overexpression of HbTRXh5 in yeast enhances the tolerance to oxidative stress, but reduces the tolerance to low temperature and salt stresses.

Identification and Expression Analysis of MYB Gene Family in Dendrobium catenatum Under the Combined Stress of High Temperature and Drought

TIAN Shan-shan, HUANG Shi-yu, YANG Tian-wei, GAO Man-rong, ZHANG Shang-wen, HE Long-fei, ZHANG Xiang-jun, LI Ting, SHI Qian

2024, 40(12):  145-159.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0151

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【Objective】To study the structure and function of the transcription factor（TF）family in the Dendrobium catenatum MYB（DcMYB）gene, And identify the prime members and their expression under high-drought stress, which may provide aiding in elucidating D. catenatum's stress adaptation mechanisms.【Method】Utilizing whole-genome transcriptomic data from the combined stress of high temperature & drought, key MYB TFs responsive to this stress were screened, then analyzed, identified via informatics, and their expression patterns under various stresses were validated via real-time PCR.【Result】The 38 DcMYBs genes were identified, they were divided into three types: 1R-MYB, R2R3-MYB, and 3R-MYB, and R2R3-MYB accounted for 89%. The DcMYBs proteins’ lengths were 203-581 aa, they were of good thermal stability, and most of them were of hydrophilic properties. Prediction of subcellular localization revealed that all were present in the nucleus, and some of them coexisted in other organelles. The MEME analysis uncovered that the MYB genes in the same subgroup had similar structural characteristics. DcMYBs was divided into 19 subgroups（D1-D19）after evolution. The prediction for cis-acting element showed that DcMYBs were involved in bio/abiotic stress & hormonal induction, DcMYB6, DcMYB15, DcMYB16, DcMYB21, DcMYB23, and DcMYB35 contained drought-responsive MBS. DcMYB6 and DcMYB35 also harbored 4 ABA-responsive ABREs. Under the combined stress of high temperature and drought, the R2R3-MYB subfamily's genes were significantly induced.The the key genes were consistent with the transcriptome data by RT-qPCR validation. DcMYB6 and DcMYB35 were upregulated under stress, DcMYB15, DcMYB16, DcMYB21, and DcMYB23 were downregulated. Treatment with exogenous abscisic acid, 20% PEG6000, and drought-responsive MBS gene showed an initial rising followed by a declining, suggesting the involvement of six genes in ABA signaling during drought drought. 【Conclusion】Under stress, 38 DcMYBs genes were identified in D. catenatum, dominated by R2R3-MYB type. The encoded proteins are of thermal stability, hydrophilicity, and are predicted to be in nucleus. DcMYBs genes have high homology with Arabidopsis MYB. The specific genes are significantly induced by drought and ABA, and they might be likely involved in biological, abiotic stress, hormone induction, and play important roles in the growth development of D. catenatum under stress environment.

Cloning and Expression Analysis of DkTPS7 in Dendrobium kingianum

KONG Lan, YE Xiu-xian, LIN Rong-yan, LIN Bing, ZHONG Huai-qin

2024, 40(12):  160-169.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0403

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【Objective】 Terpene synthase（TPS）is a key enzyme in the biosynthesis of terpenoids, which possesses an important role in the formation of plant flower fragrance. Exploring the role of TPS gene in formation of Dendrobium flower fragrance may provide references for the dynamic changes and related formation mechanism of Dendrobium flower fragrance at different flowering stages. 【Method】 Headspace solid phase microextraction-gas chromatograohy-mass spectrometry（HS-SPME-GC-MS）was used to compare the difference in the fragrance components during flowering stages of Dendrobium kingianum. RT-PCR was applied to clone a DkTPS7 gene, and its bioinformatics analysis was conducted. The agrobacterium-mediated transformation was to detect their subcellular localizations. RT-qPCR technology was to detect the expression profiles in different varieties, during flowering stages, and on diurnal variations. 【Result】 A total of 523 frangrance components were identified. Terpenoids was the most abundant volatiles constituting the fragrance of this variety. DkTPS7 included a 1 797 bp open reading frame for 598 amino acids. The DkTPS7 contained three conserved domains and belonged to TPS-b subfamily. Subcellular localization analysis revealed that DkTPS7 was localized in the plastid. The result of RT-qPCR analysis showed that DkTPS7 was highly expressed in Dendrobium kingianum, while it has almost no expression in Dendrobium ‘Zajiaozihua’ and Dendrobium parishii. The expression of DkTPS7 was the highest at the half flowering stage of Dendrobium kingianum, followed by the bud stage and the lowest at the bud stage. The expression of DkTPS7 also showed a trend of raising first and then decreasing. 【Conclusion】 DkTPS7 is cloned from D. kingianum. The expression of DkTPS7 has variety and temporospatial characters, and the expression pattern is consistent with the accumulation trend of monoterpenoid.

Function Verification of Genes Involved in 22 (R)-hydroxycholesterol Biosynthesis in Veratrum nigrum and Their Heterologous Synthesis

XU Yan-jiao, HONG Kai-yun, LU Yi-wang, WANG Chang-qing, LIANG Yan-li, HE Si-mei

2024, 40(12):  170-181.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0412

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【Objective】 22 (R)-hydroxycholesterol is an important precursor of veratrol alkaloid biosynthesis. The function of cholesterol C-22 hydroxylase in Veratrum nigrum was verified by heteroexpression, and the yeast chassis was constructed to produce 22 (R)-hydroxycholesterol, which lays a foundation for deciphering the biosynthetic pathway of veratrol alkaloid.【Method】 Based on the transcriptome data of V. nigrum, three full-length CYP90B subfamily gene sequences were cloned from the root of V. nigrum, the yeast Y33 expression vector was constructed and transferred into the cholesterol yeast chassis for functional verification. The yeast fermentaed products in shake flask were detected by high performance liquid chromatography and liquid chromatography-mass spectrometry. The enzyme VnCYP90B27-1 with cholesterol C-22 hydroxylase catalytic function was screened. VnCYP90B27-1 was integrated into the yeast chromosome by multi-fragment assembly, homologous recombination and lithium acetate conversion to construct a yeast chassis for 22 (R)-hydroxycholesterol. 【Result】 RT-qPCR showed that the expressions of the three candidate genes in the roots and leaves was consistent with the trend of transcriptome expression. The expression of VnCYP90B27-1 in the roots was significantly higher than that in the leaves. The results of phylogenetic tree showed that VnCYP90B27-1 had high homology with VcCYP90B27 of Veratrum californicum and VnCYP90B27 of V. nigrum, and belonged to CYP90B subfamily. In addition, the results of heterologous expression in the yeast showed that VnCYP90B27-1 had the function of cholesterol C-22 hydroxylase, and successfully achieved the heterologous synthesis of 22 (R)-hydroxycholesterol in S. cerevisiae, with the yield of (5.37±0.37) mg / L in shake flask. 【Conclusion】The function of 22 (R)-hydroxycholesterol synthesis gene VnCYP90B27-1 from V. nigrum is successfully cloned and verified. The 22 (R)-hydroxycholesterol yeast chassis is constructed. It is proved that the catalytic activity of VnCYP90B27-1 in S. cerevisiae is higher than that of VcCYP90B27 from V. californicum, which provides genetic resources for heterologous synthesis of steroid alkaloids.

Identification of Aux/IAA Gene Family in Corydalis hendersonii Hemsl. and Analysis on Their Expression Pattern under UVB Treatment

LIANG Jia-lin, ZHAO Shuang, LI Xing-er, ZHAO Cheng-zhou, LI Ping

2024, 40(12):  182-192.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0401

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【Objective】 To identify the Aux/IAA gene family in Corydalis hendersonii, and to provide a theoretical basis for the study of the function of C. hendersonii Aux/IAA genes and their resistances to adverse stresses.【Method】Based on the full-length transcriptome data of C. hendersonii, the members of the Aux/IAA（ChAux-IAA）gene family were identified and analyzed, and their proteins'physicochemical properties, phylogenetic tree, gene structure, protein tertiary structure, and protein interaction relationships were analyzed. In addition, C. Hendersonii was treated with UV-B at different times（0.5 h and 48 h）to detect the expression patterns of the ChAux/IAA gene family. 【Result】The ChAux/IAA gene family of C. hendersonii comprises a total of 20 members, most of which are located in the cell nucleus, with a minority found in chloroplasts, mitochondria, and cell walls. The proteins encoded by the ChAux/IAA gene family are hydrophilic. The presence of light-responsive elements in the cis-acting elements of the ChAux/IAA gene family suggests that this gene family may be involved in plant growth and development and in resisting ultraviolet stress. Analysis via RT-qPCR revealed that under UVB treatment, the expressions of all 20 ChAux/IAA members increased compared to the control group, with ChIAA2, ChIAA3, ChIAA4, ChIAA10, ChIAA13 and ChIAA21 showing a more significant increase in expression. This indicates that the ChAux/IAA gene family plays an important role in the plant's resistance to ultraviolet stress. 【Conclusion】A total of 20 ChAux/IAA gene family members are identified, and the ChAux/IAA gene family plays an important role in UV-B stress.

Study on the Effect of Rhodococcus rhodochrous NB1 on the Tolerance to Salt and Growth-promoting of Maize and Its Whole Genome

YIN Zi-wei, HONG Yu

2024, 40(12):  193-207.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0503

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【Objective】To study the salt tolerance and growth-promoting characteristics of Rhodococcus rhodochrous NB1, analyze its whole genome information, and explore the salt tolerance promoting genes of NB1 strains.【Method】NB1 strain were identified using morphological observations and 16S rRNA gene sequence analysis. The nitrogen fixation, phosphorus lysis and ACC deaminase production were identified by modified medium, Pikovaskaia's medium, DF liquid medium and ADF liquid medium. The NB1 strain was inoculated to NB solid concentrations of 0, 5%, 10% and 15% on the trophic base, the tolerable concentration of the strain was determined after 48 h of incubation. Maize seeds treated with and without the NB1 strain were inoculated onto 1/2 MS medium, and the plant height, root length, fresh weight and root weight were determined after 15 d of continuous culture. Growth indicators of maize seedlings watered and untreated with NB 1 broth were measured in the form of pot experiments under the same salt concentration stress. Whole gene analysis of NB1 strain was conducted using Illumina second generation sequencing and PacBio third generation sequencing. 【Result】The NB1 strain belongs to Rhodococcus rhodochrous, it may produce ACC deaminase, has the ability of fixing nitrogen and dissolving phosphorus, and may tolerate the salt concentration of 5%. Under soil-free culture conditions, the plant height, root length, fresh weight and root weight of maize seedlings treated with NB1 strain increased significantly. After pot planting, the plant height and fresh weight were significantly higher than that of CK. The NB1 strain has 5 259 coding genes, the total length of coding genes is 5 230 674 bp, and the average GC content is 68.30%. They are annotated to 5 235, 4 379, 4 195, 3 758, 3 263, and 2 449 genes in NR, Pfam, COG, Swiss-Prot, GO, and KEGG databases, respectively. The protein structures encoded from 178 genes in NB1 strain belongs to the CAZy family and containing genes of chitinase,sucrase and other enzymes.Meanwhile, it is predicted that there are 14 secondary metabolite gene clusters, 457 virulence factors and 324 resistance genes in NB 1 strains, and related genes such as tetrahydropyrimidine and tetracycline antibiotics with salt resistance and promoting properties were found from the NB1 genome. 【Conclusion】Rhodococcus rhodochrous NB1 has salt resistance and growth promoting effect on maize, which provides new strain resources for plant salt resistance.

Composition of Root-associated Bacteria of Polygonum cuspidatum and Their Relationship with the Bioactive Ingredients

DU Jie, HUANG Xuan-yi, ZHANG Yan, JIANG Qing-chun, YU Zhi-he, WANG Yun, LIU Zhong-yu

2024, 40(12):  208-217.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0375

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【Objective】This work aims to explore the effect of root-associated bacterial microbiomes on the quality of Polygonum cuspidatum by analyzing the correlation between the bioactive compounds and microbiomes. 【Method】The bulk soil, rhizosphere soil and roots of P. cuspidatum in the first, second and third years were collected, high-throughput sequencing technology was used to analyze bacterial microbiome compositions, and the contents of bioactive compounds were determined, including polydatin, resveratrol, emodin and physcion. The relationship between the root-associated bacteria and the bioactive components was explored through Pearon correlation analysis.【Result】Bioactive ingredients were distinct across different cultivation years（P < 0.05）. Pseudomonas and Sphingomonas were the most abundant genus of bulk soil and rhizosphere microbiome, while Pseudomonas, Chloroplast and Actinoplanes were the most dominant in the root endophyte. The abundance of Sphingomonas in the rhizosphere soil was decreasing year by year, the abundance of Pseudomonas in the root was decreasing year by year, while the abundance of Chloroplast in root was increasing year by year. The alpha diversity significantly varied along with the soil and root sample with different growth years, the abundance and diversity of bacteria in the bulk soil and rhizosphere were higher than that in root（P < 0.05）. The microbiome composition among the bulk soil, rhizosphere soil and roots extremely significantly differed（P < 0.001）, it also significantly differed across 1-year, 2-year, and 3 -year ones（P < 0.05）. The microbiome composition among the bulk soil and rhizosphere soil was significantly affected by cultivation years（P < 0.05）, while the root endosphere microbiome was not significantly affected by the growth years（P > 0.05）. The content of polydatin was significantly positively correlated with the relative abundance of Microvirga and Gaiella in rhizosphere, significantly positively correlated with the relative abundance of Pseudomonas in the root, and significantly negatively correlated with the relative abundance of Actinoplanes and Nocardioides in the root. The content of resveratrol was significantly negatively correlated with the relative abundance of Actinoplanes and Nocardioides in the root. The content of emodin was significantly positively correlated with the relative abundance of Arthrobacter in the rhizosphere, significantly positively correlated with the relative abundance of Mycobacterium in root, and significantly negatively correlated with the relative abundance of Pseudomonas in root. The content of physcion was significantly positively correlated with the relative abundance of Arthrobacter in the rhizosphere, significantly positively correlated with the relative abundance of Mycobacterium in the root, as well as significantly negatively correlated with the relative abundance of Actinoplanes in the root. 【Conclusion】This study provides insights into the interaction networks between P. cuspidatum root-associated bacteria and bioactive compounds, giving us a new opportunity to manipulate the production of bioactive compounds in future.

Isolation, Screening and the Optimization of Fermentation Conditions of Biocontrol Bacteria against Eleutherococcus senticosus Black Spot

DING Yan-zhe, YAO Xin-xin, SUN Zhuo, YANG Li-min, HAN Zhong-ming, WANG Yun-he

2024, 40(12):  218-226.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0400

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【Objective】The biocontrol bacteria against Eleutherococcus senticosus black spot were isolated, screened and identified, and the fermentation conditions of bacteria were optimized. This study may provide a good source of biocontrol bacteria against E. senticosus black spot.【Method】The serial dilution method was used to isolate bacteria from healthy E. senticosus rhizosphere soil, and the plate confrontation method was used to screen strains with strong antagonistic effects on A. tenuissima, and the inhibitory effects on other 5 pathogenic fungi were measured. The taxonomic status of this strain was clarified based on morphological characteristics, physiological and biochemical characteristics and 16S rDNA gene sequencing. The culture medium components and fermentation conditions were optimized using single-factor experiments and orthogonal experiments. The effect of fermentation broth of this strain on the biocontrol efficiency against E. senticosus black spot was also studied.【Result】Total 419 strains of bacteria were isolated, among which the strain YZ-228 had the highest inhibitory rate of 67.38% against A. tenuissima, the inhibition rate of sterile filtrate against A. tenuissima was 64.32%. And strain YZ-228 had the inhibitory effects against Rhizoctonia solani, Fusarium solani, Alternaria panax, Cylindrocarpon destructans, and Fusarium oxysporum, with an inhibition rate of 62.80%-67.89%. Strain YZ-228 was identified as Bacillus cereus. Soluble starch and beef extract were the best carbon and nitrogen sources for YZ-228. Under the condition that the pH, temperature, inoculum, liquid volume and speed were 7.0, 28℃, 3%, 50 mL/250 mL and 200 r/min, respectively, the bacterial solution fermented for 1 d had a good antagonistic effect on A. tenuissima, with an inhibition rate of 74.19%. It was 11.99% higher than that in beef extract-peptone-yeast extract medium（BPY）. In addition, inoculation of the fermentation broth of strain YZ-228 significantly reduced the disease index of E. senticosus black spot, and its control effect was 59.15%. 【Conclusion】B. cereus YZ-228 has good control effect on E. senticosus black spot and has the potential to be used as a biological control agent for E. senticosus black spot.

Differential Analysis of Key Genes Involved in the Accumulation of L-malic Acid by Aspergillus niger Fermentation

LIU Shu-tong, WU Sheng, TAN Yi-yang, WANG De-pei, XUE Xian-li

2024, 40(12):  227-238.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0512

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【Objective】 Microbial production of L-malic acid is currently an extremely efficient producing method. In order to reveal the biosynthetic mechanism of L-malic acid production by Aspergillus niger, the differential changes of key genes in the metabolic regulation process were explored with the help of transcriptomic analysis. 【Method】The acid production level of MA-1 strain reached 88.27 g/L at 144 h of fermentation, and three time points（48, 72, and 120 h）with different acid production rates were selected for transcriptome analysis. 【Result】GO analysis showed that the regulation of biological processes, RNA binding and ribosome-related secondary entries under the significant genes were the most differentiated and the largest number. Transcriptome analysis revealed that transcriptional regulators such as HacA, Ace1, and Rpn4 were consistently at high levels during fermentation from 48 to 120 h, with FPKM values reaching 20 181.64-94 573.00, and that glucose transporter protein（Rco-3）, and ketoglutarate/malate transporter protein Yhm1 showed relatively high transcript levels, with FPKM values reaching 4 971.83-6 575.46; in the glycolytic EMP pathway, its important rate-limiting enzyme pyruvate kinase（FPKM values of 13 109.15-25 649.30）and ANI_1_1984024 among the six hexokinase enzymes（FPKM values up to 4 111.68-7 325.43）showed relatively high transcript levels, and then the pathway for the conversion from glucose to pyruvate. The transcript levels of other enzymes in the glucose-to-pyruvate pathway showed a decreasing trend at 120 h. The transcript levels of the whole genes in the TCA cycle were maintained at a high level to provide sufficient energy for cellular metabolism, and the highest level of citrate synthase in the rTCA pathway, followed by malate dehydrogenase, and the level of the glyoxylate cycle was low.【Conclusion】The main pathways of L-malate synthesis in A. niger MA-1 strain were presumed to be rTCA and pyruvate carboxylation pathway.

Four Strains of Gram-negative Bacteria Labeled with Red and Green Fluorescent Proteins and Their Biological Characterization

WU Qi-ye, LU Li-na, LIU Ying-long, PING Yuan, HE Peng-bo, HE Yue-qiu, WU Yi-xin, HE Peng-fei

2024, 40(12):  239-247.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0550

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【Objective】To construct broad host range replication type fluorescent protein plasmid and to evaluate the applicability of fluorescent protein plasmid by labeling four strains of Gram-negative bacteria. 【Method】gfp, mCherry and the constitutive promoter PpsbA were cloned into the plasmid pBBR1MCS2' in order to construct the expression vector pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry, and the expression vectors were introduced into Ralstonia solanacearum EP1, Pectobactererium carotovorum subsp. carotovorum WB, Klebsiella Pneumoniae JF and Burkholderia cepacia 1-2 by conjugative transfer. Their fluorescence performance of colonies and cells was observed, growth curves and plasmid retention rates were detected, and relevant functions were verified. 【Result】The fluorescent protein expression vectors pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry were successfully constructed, and the four Gram-negative bacteria were successfully labeled with green and red fluorescent proteins. The colonies and individual cells of the fluorescent protein-labeled bacteria emitted corresponding fluorescence, and the growth rate was consistent with that of the wild-type strains. However, the stability of the fluorescent protein plasmids varied greatly among the four strains. The green and red fluorescent protein plasmids were the most stable in the strain JF, with plasmid retention rates of 90.33% and 94.67%, respectively after 40 h of incubation without selective pressure; followed by strain WB and EP1, and the worst in strain 1-2, with fluorescence not detected after 5 and 25 h of incubation, respectively. In addition, there was no significant difference in the pathogenicity between fluorescently labeled WB and wild-type strain WB. 【Conclusion】 Two fluorescent protein expression vectors are successfully constructed and introduced into four different Gram-negative bacterial strains, including R. solanacearum EP1. In the plasmid retention rate, green and red fluorescent proteins are less stable in strain 1-2, and are stably expressed in the remaining three strains. These vectors provide important research materials for the subsequent study of labelling Gram-negative bacteria and investigating their related functions.

Identification of the Promoter for the Biosynthesis Gene of Polyketide Meroterpenoids in Phomopsis tersa FS441

LIU Yu-ping, ZHANG Wei-yang, ZHANG Wei-min, YE Wei, LI Dong-li

2024, 40(12):  248-255.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0347

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【Objective】 Phomeroids are novel skeleton polyketide meroterpenoids isolated from the marine fungus Phomopsis tersa FS441, demonstrating promising anti-tumor activity. To facilitate the transcriptional regulation of the crucial genes for the biosynthesis of phomeroids, including the terpene cyclase gene ctg1509 and the P450 monooxygenase gene ctg1511, the functions for the promoters of these two genes were identified in this study.【Method】 This study cloned the promoter p1509 and p1511 of ctg1509 and ctg1511 genes in marine fungus P. tersa FS441. The transcriptional activities of the p1509 and p1511 promoters were validated using luciferase reporter gene vectors, the plate experiment and the growth curve were employed to identify the function of the two promoters. Moreover, PlantCARE database was used to analyze the regulatory elements of the promoter. 【Result】 The promoter of p1509 has the strongest transcriptional activity, which is stronger than the positive promoter PgpdA. p1509 and p1511 promoters can initiate the transcription of ampicillin resistant gene AmpR in Escherichia coli. p1509 and p1511 promoters’ regions not only contain core components such as CAAT box and TATA box, but also include cis-acting regulatory elements involved in low temperature response and light response. 【Conclusion】 This study confirmed the function of p1509 and p1511 promoters, and excavated a novel strong promoter, p1509, which had stronger transcriptional activity than the positive control and contained abundant regulatory elements.

Cloning and Expression Analysis of FvALT from Fusarium verticillioides

HAO Nan, GENG Shan, ZHAO Yu-wei, HOU Zhi-han, ZHAO Bin, LIU Ying-chao

2024, 40(12):  256-263.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0432

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【Objective】 Fusarium verticillioides is the main pathogen of maize tassel rot, thus exploring the biological function of FvALT will provide a theoretical basis for the development of novel targeted fungicides. 【Method】Physiological and biochemical characteristics and homology of FvALT were analyzed using online tools such as ProtParam, ProtScale, WoLF PSORT and Clustal X. The protein structure was obtained using SOMPA and SWISS-MODEL, and the binding ability of FvALT to substrates was analyzed using AutoDock. The target proteins were then obtained by prokaryotic expression and affinity nickel column chromatography, and the binding ability of FvALT to the substrate was verified by fluorescence titration technique.【Result】FvALT is a hydrophilic protein and localized in the cytoplasm, its protein secondary structure is dominated by α-helix and irregular coiling, and is highly conserved in Fusarium, with a typical PTZ00377 superfamily structural domain. The optimal conditions for the induction of FvALT are 0.4 mol/L IPTG, 16℃ for 16 h. FvALT has strong binding effects with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰ and ASP²⁵⁸. FvALT has a strong binding effect with α-ketoglutarate, and the binding sites are ARG³¹⁰, SER¹⁴⁹, SER²⁹⁸, SER³⁰⁰, and ASP²⁵⁸.【Conclusion】FvALT has a typical alanine aminotransferase profile and binds specifically to the substrate alpha-ketoglutarate.

Induction and Circular Structure Analysis of Prophage in Carbapenem-resistant Hypermucoviscous Klebsiella pneumonia K2-ST375

LIU He-lan, CHEN Ze-hui, LI Ming-zhe, ZHOU Yong-wen, LI Rui, WANG Yong-xiang

2024, 40(12):  264-274.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0346

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【Objective】 To analyze and study the distribution of prophages, genetic structure of inducible prophages, and the characteristics of their encoded genes in clinical carbapenem-resistant hypermucoviscous Klebsiella pneumoniae（CR-HMKP）K2-ST375 strain, this may lay an important foundation for further research on the biological functions of prophage in CR-HMKP.【Method】The distribution of prophages in CR-HMKP K2-ST375 strain was predicted using Prophage Hunter. Mitomycin C was used to induce the prophages and their circular structure was verified through circularization primers. Comprehensive bioinformatics software such as RAST were utilized to annotate the genes of the inducible prophage, Prophage-4, and to analyze the physicochemical properties, structural characteristics, and genetic information of the endolysin encoded by Prophage-4.【Results】The predicted results of Prophage Hunter indicate that the chromosome of CR-HMKP K2-ST375 strain carries a total of four active prophages, named Prophage-1 - Prophage-4. Upon induction with mitomycin C, Prophage-4 can be excised from the host chromosome to form a circular structure. Sequence analysis shows that there is a 40 bp homologous repeats sequence between Prophage-4 and the host chromosome. Gene annotation results suggest that the genes encoded by Prophage-4 are mainly involved in phage structure and assembly, DNA metabolism, lysogenic cycle, and host lysis. The endolysin encoded by Prophage-4 shares the same conserved structural domains with the endolysin R21-like protein of the Enterobacteriaceae bacteriophage P21. Genetically, they are closely related in terms of evolutionary lineage. Phylogenetic tree analysis results show that Prophage-4 and the lytic Klebsiella phage BUCT541 are located on the same evolutionary branch, indicating that Prophage-4 possesses potential lytic capabilities. 【Conclusion】The Prophage-4 carried in clinical CR-HMKP K2-ST375 strain can be induced by mitomycin C and excised from the host's chromosome to form a circular structure mediated by 40 bp homologous repeat sequences. The endolysin is closely related to the endolysin protein encoded by phage P21.

Transfer of Antibiotic-resistance Gene AmpR by Escherichia coli DH5α Through Outer Membrane Vesicles

ZHUANG Ke, LIANG Zhi-xuan, HE Ying-ting, XIE Qiu-ling

2024, 40(12):  275-281.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0335

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【Objective】 To investigate the ability of Escherichia coli DH5α outer membrane vesicles（OMVs）transferring the antibiotic resistance gene AmpR among bacteria. 【Method】 The OMVs were extracted by ultracentrifugation; then the morphology, particle size and concentration of OMVs were detected and characterised by transmission electron microscopy, nanoparticle tracking analysis（NTA）and Western blot. Meanwhile, PCR was performed to identify AmpR, an ampicillin resistance gene in OMVs. OMVs were co-incubated and cultured with blank Escherichia coli DH5α, and the bacterial growth on medium with ampicillin was observed, and single colonies were picked to analysize the presence of AmpR by PCR. 【Result】 The average particle size of the extracted vesicles was 125 nm, which was within the range of OMVs particle size, and the OMVs marker protein OmpC was positive, indicating that the extracted vesicles were bacterial OMVs. It was also identified that the ampicillin resistance gene AmpR was in these OMVs. After co-incubation with OMVs, blank E. coli DH5α grew on LB solid plates, and AmpR gene was identified by colony PCR in these bacterial cells. 【Conclusion】 Escherichia coli DH5α with resistant genes can transfer resistance by passing AmpR to surrounding E. coli via outer membrane vesicles OMVs.

Whole Genome Sequencing and Genome Evolution Analysis of Capsular Serotype A and D Pasteurella multocida of Bovine

WANG Zi, SHI Jin-chuan, WANG Yong-qiang, SUN Miao, MENG Ling-hao, GENG Chao, LIU Kai

2024, 40(12):  282-290.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0371

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【Objective】The capsular serotype of the Pasteurella multocida isolated from bovine in the Wulagai management area was identified. Whole genome sequencing and genome evolution analysis were performed for the strains with different serotypes. 【Method】Taking the lung tissue samples from bovine for pathogen isolation and purification, the 16S rRNA identification, capsular serotype typing, drug sensitivity experiments, and mouse pathogenicity experiments were performed. Whole-genome sequencing and genomic evolution analysis were performed for the strains with different serotypes. 【Result】Two strains of bovine Pasteurella multocida were successfully isolated, designated as Pm-YQ and Pm-SM. The Pm-YQ strain was capsular serotype A and the Pm-SM strain was capsular serotype D. Both strains presented relatively high virulence but low resistance to antibiotics. The whole genome lengths of the isolates were 2 274 102 bp and 2 244 957 bp, respectively, encoding 2 070 and 2 007 genes. The Pm-YQ strain belonged to sequence type ST 179, while Pm-SM strain belonged to ST 1. Through the construction of whole-genome phylogenetic tree, Pm-YQ strain was found to be closely related to three strains registered in GenBank from Germany, Denmark, and the United States, Pm-SM strain was found in the same evolutionary branch with a capsular serotype F of P. multocida strain isolated from Chongqing, China. 【Conclusion】The isolation, identification and whole genome sequencing of two strains with different capsular serotypes of bovine P. multocida were completed. The Pm-YQ strain was closely related to the German isolate, and the Pm-SM strain was relatively far related to the currently registered bovine P. multocida strains.

Roles of miR-3604 on the Receptivity, Proliferation, and Apoptosis of Bovine Endometrial Epithelial Cells

JI Zhong-xiang, LUORENG Zhuo-ma, LI Yu-hang, WANG Yu-mei, HU Xi-min, LI Yan-qing, WANG Xing-ping

2024, 40(12):  291-298.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0245

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【Objective】 To investigate the role of miR-3604 in the receptivity of bovine endometrial epithelial cells（bEECs）.【Method】Progesterone（P4）and interferon tau（IFN-τ）were used to induce bEECs to construct a tolerant cell model. On this basis and after the overexpression and interference of bovine miR-3604 in the tolerant bEECs, the RT-qPCR, flow cytometry, EdU and CCK8 techniques were used to detect the receptivity of bovine miR-3604 in bEECs. Role in cell proliferation and apoptosis. 【Result】The expressions of receptive marker gene HOXA10, IL-6 and VEGF significantly reduced in the miR-3604 overexpression group of receptive bEECs（P < 0.05）. The expressions of apoptosis marker gene CASP3 and BAX were significantly up-regulated（P < 0.05）. The apoptosis rate of acceptive bEECs significantly decreased（P < 0.01）, while the cell proliferation rate significantly increased（P < 0.05）. At the same time, the results of the miR-3604 interference experiment were contrary to the results of the overexpression experiment.【Conclusion】The expression of miR-3604 in receptive bEECs increased, which may inhibit the receptivity and apoptosis of bEECs, and promote the proliferation of bEECs.

Alleviating Roles and Mechanisms of Ethanol Extract from Inonotus obliquus to Intestinal Injury Induced by Lipopolysaccharide

MA Wen-ao, YANG Wei, LI Ying-chun, ZHU Yan-bin, CHEN Zhi-bao, LIU Na

2024, 40(12):  299-308.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0422

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【Objective】 To investigate the protective effect and molecular mechanism of ethanol extract of Inonotus obliquus（EEIO）on lipopolysaccharide（LPS）-induced intestinal injury. 【Method】 Using LPS to induce mouse small intestinal epithelial cells and C57BL/6 mice, LDH and NO levels were measured after EEIO treatment. RT qPCR and ELISA methods were to detect the effects of EEIO on the expressions of inflammatory factor IL-1β and IL-18. RT-qPCR and WB methods were used to detect the effects of EEIO on MAPK and pyroptosis signaling pathways. 【Result】 Both in vivo and in vitro results showed that EEIO effectively alleviated the increase in LDH and NO levels induced by LPS. After EEIO processing, the expressions of IL-1β and IL-18 were significantly reduced. EEIO inhibited the MAPK（ERK, P38 and JNK）signaling pathway, significantly reducing the levels of p-ERK/ERK, p-p38/p38 and p-JNK/JNK. Meanwhile, the pyroptosis pathway was inhibited, and the levels of ASC, Caspase-1, GSDMD and NLRP3 significantly reduced. The positive drug resveratrol（RES）had a similar effect on various detection indicators as EEIO, but its effect was not as good as EEIO. 【Conclusion】 EEIO regulates the MAPK signaling pathway, inhibits the pyroptosis pathway, and reduces the expression of inflammatory factors, thus alleviating LPS-induced intestinal injury.

Content

2024, 40(12):  309. 

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Copyright

2024, 40(12):  310. 

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Cover

2024, 40(12):  311. 

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