Biotechnology Bulletin

Research Progress in the Functions of Key Enzymes of Ubiquitination Modification in Plant Stress Responses

GUO Hui-yan, DONG Xue, AN Meng-nan, XIA Zi-hao, WU Yuan-hua

2024, 40(4): 1-11. doi:10.13560/j.cnki.biotech.bull.1985.2023-1095

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Ubiquitination is one of post-translational modifications of plant proteins, which is involved in the regulation of plant growth, development and various stress responses by selective degradation of proteins. The reaction of ubiquitination modification is carried out by the synergistic action of three key enzymes. Ubiquitin molecules are activated by linking to thiol ester bonds of ubiquitin-activating enzymes, and the activated ubiquitin molecules form a complex with ubiquitin-conjugating enzymes, and finally bind to the target protein under the action of ubiquitin-ligase enzymes. With the development of proteomics sequencing technology, the research on ubiquitination modification has become more widespread and deeper. A large number of ubiquitination modified proteins and their modification sites have been identified, which is conducive to further understanding the regulatory mechanism of proteins and further analyzing the function of proteins. In this paper, we mainly introduced the reaction process of the ubiquitin-proteasome system, the structure, quantity and classification of key enzymes of ubiquitination modification, and focused on the functions of ubiquitin-activating enzymes, ubiquitin-binding enzymes and ubiquitin-ligases in plant response to abiotic and biotic stresses. Concurrently, we summarized the issues in studying functions of the key enzymes in plant ubiquitination modification, and discussed the crosstalk between ubiquitination and other modifications, which is of great significance for further research on ubiquitination modification in the field of plant responses to various environmental stresses.

Recent Advances in 14-3-3 Proteins and Their Functions in Plant

CHEN Ying-ying, WU Ding-jie, LIU Yuan, ZHANG Hang, LIU Yan-jiao, WANG Jing-yu, LI Rui-li

2024, 40(4): 12-22. doi:10.13560/j.cnki.biotech.bull.1985.2023-1091

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14-3-3 proteins are a highly conserved family of acidic proteins encoded by different genes in eukaryotic organisms. After interacting with the target proteins through the phosphorylation sites, 14-3-3 proteins influence the stability, subcellular localization, or interactions with other proteins of the target protein, thereby regulating its function. Various isoforms of 14-3-3 proteins have been found in different species, and these isoforms influence the processes such as plant growth and development. In this review, we summarize the type, subcellular localization, and expression patterns of 14-3-3 proteins in plants, with a specific focus on the functions of 14-3-3 proteins in plant hormone signaling, growth and development, and stress response. The aim of this review is to provide a theoretical foundation for further systematic research on 14-3-3 proteins.

Development of Hypocotyls and Apical Hooks in Dicotyledons and Their Regulatory Mechanisms for Seedling Emergence

HUA Zi-qing, ZHOU Jing-yuan, DONG He-zhong

2024, 40(4): 23-32. doi:10.13560/j.cnki.biotech.bull.1985.2023-1084

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Seedling emergence is a crucial stage in plant production as it greatly impacts or even determines the final yield. For dicotyledonous plants, the growth of the hypocotyl and the development of the apical hook play a significant role in seedling emergence. Hypocotyl growth and apical hook development are a continuously dynamic process and are important guarantees for seedling emergence. The elongation of the hypocotyls provides the necessary force for seedlings to penetrate the soil, while the apical hook protects the delicate apical meristems and cotyledons from the damages during the soil topping process. Focusing on the development of the hypocotyl and apical hook, this paper summarized the molecular mechanisms regulating hypocotyl elongation and thickening by arranging themselves through the cytoskeleton in dicotyledonous plants, as well as the growth mechanisms of cellular differentiation involved in apical hook formation, maintenance, and unfolding. This paper examined the synergistic effects and mechanisms of major environmental factors such as light and temperature, as well as plant endogenous hormones, on hypocotyl and apical hook development. Furthermore, the paper explored agricultural practices, including optimizing environmental factors through sowing methods and seedling thinning to enhance hypocotyl growth and apical hook development, which in turn improves seedling emergence rate and stand establishment rate. Lastly, the paper provided suggestions for further research on the molecular mechanisms regulating hypocotyl and apical hook development, along with insights into the adaptation mechanism of apical hook and hypocotyl development to adversity, as well as for achieving effective control of seedling emergence and stand establishment in dicotyledonous plants. This paper serves as a valuable reference for a deeper understanding of the seedling emergence mechanism in dicotyledonous plants and innovative plant seeding and seedling preservation techniques.

Review on the Regulation of Caleosin on Plant Lipid Droplet

PENG Feng, YU Hai-xia, ZHANG Kun, LIU Ying-ying, TAN Gui-yu

2024, 40(4): 33-39. doi:10.13560/j.cnki.biotech.bull.1985.2023-1051

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Plants frequently include subcellular organelles called lipid droplets, which encapsulate a range of lipids and store them in the cytoplasm. Lipid droplets involved in the control of lipid metabolism are crucial for maintaining plant lipid homeostasis. As the most important outer membrane protein of lipid droplets, caleosin plays a critical role in the formation and stabilization of lipid droplets as well as the accumulation of secondary metabolites and lipids. A thorough knowledge of caleosin regulating lipid droplet may provide as a foundation for further investigating lipid homeostasis and secondary metabolic processes related to lipid droplets. This paper presents the structure of plant caleosin, and highlights the significant elements and potential function in their protein sequences. We reviewed the species-specificity and tissue-specificity of the caleosin gene family, showing that not only the gene function of caleosin varies throughout the terrestrial plant evolutionary process, also its specific biological function varies among different tissues. Subsequently we summarized recent research on caleosin's influence on lipid droplet biosynthesis and clarified its function in triggering lipid droplet biosynthesis in response to environmental stimuli. This review indicates that caleosin’s ability to regulate lipid droplet metabolism is associated with the stabilizing influence of the protein's N-terminal sequence on lipid droplet structure. Furthermore, using aflatoxin, paclitaxel, and rutin as examples, we discussed the impact of caleosin on the accumulation of secondary metabolites encapsulated in lipid droplets. Concluding by discussing the prospects of gene editing technology and the application of artificial oil bodies, we aim to provide references for the scientific investigation of the mechanism of lipid quality formation and lipid homeostasis.

Research Progress in the Evolution Mechanisms for Insect Resistance to Insecticides and Bt-transgenic Plants

PENG Yu-jia, LI Wen-cui, LIU Yong-bo

2024, 40(4): 40-51. doi:10.13560/j.cnki.biotech.bull.1985.2023-1052

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To reduce the damage to crop from insects, some agricultural practices have been used such as spraying insecticides and planting Bt-transgenic insect-resistant plants. However, since insecticides are continuously sprayed and Bt-transgenic insect-resistant plants are continuously cultivated at large scales, target insects tend to evolve the resistance to insecticides and insect-resistant plants. This insect-resistance evolution of target insects not only decrease the insect-control effectiveness of insecticides and transgenic plants but also affect the functional services in agricultural ecosystems. Here, we reviewed the molecular mechanisms of insect resistance to chemical and microbial insecticides and Bt-transgenic plants. Insects evolve resistance to chemical insecticides via decreasing the sensitivity of target sites and enhancing the activities of detoxifying enzymes in insects, resistance to microbial insecticides via activating immune systems and changing symbiotic flora in insects, and resistance to Bt-transgenic plants via downregulating midgut binding receptors and decreasing midgut protease activities in insects. To delay the resistance evolution of insects and increase the insect-control efficiency of insecticides, it is urgent to systematically control agricultural insects through reducing the use of chemical insecticides and to increasing the integrative use of broad-spectrum and high-activity microbial insecticides as well as insect-resistant plants.

Application of Single-virus Tracking Technique in Animal and Plant Cells

YUAN Xiao-yan, ZHANG Yu-ge, YAO Li-juan, TANG Chen, LI Xiao-juan

2024, 40(4): 52-66. doi:10.13560/j.cnki.biotech.bull.1985.2023-1044

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Virus is a kind of simple microorganism specializing in intracellular parasitics, which is one of the main pathogens causing animal and plant diseases. The development of single-virus tracking（SVT）technology makes it possible to reveal the fine process of viral life activity. SVT is a new method to study the mechanism of viral infection based on fluorescence labeling and high spatio-temporal resolution imaging, which allows the observation of the life activity of a single or multiple viruses. In recent years, the SVT technology has made great progress in the study of virus entry, replication, and intercellular transmission, providing new insights into the mechanism of virus infection. In this paper, the principle and strategy types of virus labeling are briefly introduced, and the research progress of virus fluorescence labeling in single virus tracing is summarized, especially the labeling and imaging methods of different structures of viruses, aiming to provide new ideas for optimizing labeling strategies and solving the problem of efficient labeling of different types and structures of viruses.

Dynamic Changes of Arabidopsis Endoplasmic Reticulum Based on Enhanced Super-resolution Images

ZHANG Yi-heng, LIU Jia-zheng, WANG Xue-chen, SUN Zheng-zhe, XUE Ya-jun, WANG Pei, HAN Hua, ZHENG Hong-wei, LI Xiao-juan

2024, 40(4): 67-76. doi:10.13560/j.cnki.biotech.bull.1985.2023-1152

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【Objective】This work is to solve the bottleneck of accurately identifying fine structures, and dynamic changes cannot be concurrently met by imaging speed and imaging resolution in the study of plant cell endoplasmic reticulum.【Method】This study employed structured illumination microscopy techniques to achieve super-resolution real-time imaging of the ER in live Arabidopsis materials. Additionally, a self-supervised denoising framework（Blind2Unblind）was optimized to further enhance the signal-to-noise ratio of rapid microscopic imaging.【Result】Based on the images with high quality, a method for quantitative analysis of ER structures using time-lapse images was established. Moreover, detections of changes in ER structures under environmental stress were conducted to verify the effectiveness of the method. Moreover, correlation analyses of various parameters indicated a significantly positive correlation between the area,length of tubular ER and the number of growth tips and three-way junctions, while the area of ER cisternae and bulk flow had a significantly negative correlation with the area and length of tubules.【Conclusion】The optimized self-supervised denoising framework in this study improves the signal-to-noise ratio of images with structure illumination microscopy in living plant cells, enabling the quantification of complex structures and dynamics, such as tubular ER, cisternae in ER, bulk flow, growth tips, and nodes, with complex correlations among the structures.

Detection of NK Cell Cytotoxicity: Real-time Dynamic Imaging-Based Analysis

SANG Sen-hua

2024, 40(4): 77-84. doi:10.13560/j.cnki.biotech.bull.1985.2023-0870

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【Objective】Currently cellular immunotherapy is a fast-growing and reliable means of treating cancer. This study aims to develop a new method of detecting the effects of cell therapy products.【Method】By constructing tumor cells expressing green fluorescent protein（GFP）, a method for evaluating the cytotoxicity of cell therapy products was constructed based on a real-time live-cell dynamic imaging system.【Result】The real-time live-cell dynamic imaging system was used to monitor K562-GFP cells, and there was a good linear relationship between the total fluorescence intensity and the number of cells. In co-culture with extremely low effector to target（E∶T）ratio, high accuracy was also achieved. The cytotoxicity of natural killer（NK）cells was accurately evaluated by calculating the half-maximum effector concentration（EC₅₀）of NK cells for K562-GFP cells. Flow cytometry（FCM）was used to detect the correlation between apoptosis of K562-GFP cells and the quenching of cell fluorescent protein, which proved the feasibility of the detection method.【Conclusion】This method can be used to evaluate killing ability of immune cells of different types or production processes at a low cost, without requiring the use of dyes or antibodies.

Construction and Transcriptomic Analysis of Rice Histone H1 Triple Mutant

YANG Qi, WEI Zi-di, SONG Juan, TONG Kun, YANG Liu, WANG Jia-han, LIU Hai-yan, LUAN Wei-jiang, MA Xuan

2024, 40(4): 85-96. doi:10.13560/j.cnki.biotech.bull.1985.2023-1066

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【Objective】Histone H1 plays an important role in the maintenance and stabilization of higher chromatin structure. Elucidating the effect of rice histone H1 on gene expressions will help to better understand the regulatory functions of H1 in rice.【Method】Semi-quantitative RT-PCR and real-time quantitative PCR were used to detect the expressions of four H1 genes in rice. CRISPR technology was used to generate h1 mutant plants; subsequently, phenotypic and transcriptomic analysis of the h1 mutant was performed. 【Result】The four rice H1 genes were broadly expressed, and are weakly expressed in root. In T₀ generation of CRISPR-based gene editing plants, multiple mutations in H1.1-H1.4 were identified. In T₁generation, one Osh1.1 Osh1.3 Osh1.4 homozygous triple mutant was identified, which had various developmental defects and became the material for further transcriptomic sequencing analysis. In T₂ generation, quadruple and triple mutants were acquired, approximate 25% of which were albino seedlings, slow in plant development and defect in drought responses. By transcriptome sequencing of the h1 mutant, 1 055 differentially expressed genes were identified, of which the significantly up-regulated genes were approximately 2.5 times of the down-regulated genes, implying that H1 may inhibit gene expressions at genome-wide scale. 【Conclusion】In the mutant, pathways of photosynthesis, stress responses, amino acid and RNA metabolism were disrupted. The ribosome biogenesis and photosynthesis pathway genes were significantly up-regulated; while the stress-associated dehydrogenase genes were down-regulated. It is presumed that overexpression of ribosome pathway genes resulted in the disorder of protein homostasis, which ultimately caused plant developmental defects.

Cloning and Functional Analysis of OsLCT3, a Low-affinity Cation Transporter Gene of Rice

LI Xing-rong, TAN Zhi-bing, ZHAO Yan, LI Yao-kui, ZHAO Bing-ran, TANG Li

2024, 40(4): 97-109. doi:10.13560/j.cnki.biotech.bull.1985.2023-1025

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【Objective】Excessive cadmium（Cd）in some rice grains seriously affect food safety in China. The aim of this study is to identify the novel gene that regulate accumulation of Cd in rice grains and provide the genetic resource for reducing Cd accumulation in rice grains. 【Method】OsLCT3, a member of the low- affinity cation transporter family in rice, was cloned by reverse transcription PCR and RACE techniques. Natural variations of OsLCT3 and physicochemical properties of its coding protein were analyzed by bioinformatics methods. Its expression profiles throughout the growth period and under Cd, Mn, Fe stress were analyzed by quantitative real-time PCR（RT-qPCR）. Its subcellular localization was explored by investigating localization of OsLCT3-GFP fusion protein in in rice protoplasts. The effect of OsLCT3 knockout on divalent cation transport in rice was analyzed by measuring cadmium and other divalent mineral metal elements in various parts of plants at seedling stage in cadmium-stressed hydroponics and at maturity stage in cadmium-polluted soil. In addition, the effect of OsLCT3 on the yeast tolerance to Cd was verified by heterologous functional complementarity in yeast cells. 【Result】OsLCT3 only existed in some indica and japonica rice varieties, with a total coding region of 1 263 bp. The amino acid sequences were classified into five haplotypes based on coding region variation, and the encoded proteins had 12 sub-transmembrane structural domains. OsLCT3 was similar to the LCT-like proteins in Triticum aestivum and Aegilops tauschii, whereas it shared only 52% sequence identity with OsLCT2, 49% and 47% sequence identity with OsLCT1 in indica and japonica rice subspecies, respectively. OsLCT3 was highly expressed in the roots at all stages of growth and development. Its expression levels in the roots were suppressed by Cd, excessive iron（Fe）and manganese（Mn）stress. OsLCT3 was localized to the plasma membrane. Compared with the wild type plants, the oslct3 knockout lines showed the reduced plant height, the lower concentrations of Cd, Fe, and Zn in the shoots, higher concentration of Fe in the roots, and the same concentrations of other bivalent mineral elements. Moreover, oslct3 knockout lines demonstrated the decreased root-to-shoot translocation rates of cadmium, iron and zinc. Under field conditions, Cd concentrations in straw and brown rice of oslct3 knockout lines at maturity were significantly lower than those of the wild type plants, and there were no significant differences in manganese, copper, iron and zinc concentrations in straw and brown rice compared with the wild type plants. Expression of OsLCT3 in yeast resulted in increased sensitivity of yeast to cadmium stress.【Conclusion】OsLCT3 is involved in the translocation of Cd, Fe, and Zn from roots to shoots, and positively regulated the accumulation of Cd in rice grains.

Identification and Expression Pattern Analysis of GmHMGS Gene in Soybean

LOU Yin, GAO Hao-jun, WANG Xi, NIU Jing-ping, WANG Min, DU Wei-jun, YUE Ai-qin

2024, 40(4): 110-121. doi:10.13560/j.cnki.biotech.bull.1985.2023-1102

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【Objective】3-hydroxy-3-methylglutaryl-CoA synthase（HMGS）is a key enzyme in the mevalonate pathway and plays an important role in the growth, development and synthesis of terpenoids. Bioinformatics study and expression pattern analysis of the GmHMGS gene can lay the foundation for exploring the function of GmHMGS gene. 【Method】The gene was identified and analysed using bioinformatics approaches, and then its expression pattern was examined using real-time fluorescence quantitative PCR（RT-qPCR）. 【Result】The GmHMGS gene had six family members in total, named GmHMGS1 to GmHMG6. The analysis of physicochemical properties and gene structure showed that GmHMGS3, GmHMG4, GmHMGS5, GmHMGS6 were stable hydrophilic proteins, and all GmHMGS possessed conserved functional domains of HMG-CoA synthase. Promoter sequence analysis revealed that GmHMGS has hormone and adversity stress related action elements. The analysis of tissue expression pattern revealed that GmHMGS was expressed in roots, stems, leaves, flowers, grains, pod skins, and root nodules, with leaves having the highest relative expression of GmHMGS1, GmHMG2, GmHMG3, GmHMG5, and GmHMG6, and roots having the highest relative expression of GmHMGS4. The GmHMGS genes were responsive to PEG6000, NaCl, and H₂O₂ stresses, as well as MeJA and ABA exogenous hormones, and the relative expression of GmHMGS1 and GmHMGS6 was higher under a wide range of adversity stresses, according to expression analysis under abiotic stress and different exogenous hormone-induced conditions. 【Conclusion】GmHMGS gene family may be involved in response to drought, oxidative and salt stresses in soybean.

Cloning，Expression and Functional Identification of BrMLP328 Gene in Brassica rapa subsp. pekinensis

DU Ze-guang, REN Shao-wen, ZHANG Feng-qin, LI Mei-lan, LI Gai-zhen, QI Xian-hui

2024, 40(4): 122-129. doi:10.13560/j.cnki.biotech.bull.1985.2023-1082

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【Objective】 The BrMLP328 gene of Chinese cabbage （Brassica rapa subsp. pekinensis）was cloned, its expression pattern was analysed, and its function in regulating flowering was verified. This may lay the basis for further investigation into the mechanism of the role of this gene in regulating flower formation in Chinese cabbage.【Method】BrMLP328 was cloned by RT-PCR and analysed by bioinformatics. RT-qPCR was used to determine the relative expression of the BrMLP328. An overexpression vector was constructed and transformed into wild-type Arabidopsis thaliana by flower-dipping method, and the difference in flowering time between the T₂ generation plants and the wild type was compared.【Result】The coding sequence（CDS）of BrMLP328 has a total length of 456 bp, encoding 151 amino acids. The relative molecular weight of the protein was 17 493.82 Da and was localized in the nucleus. The expression of BrMLP328 was the highest in the stems of Chinese cabbage, followed by roots and lowest in flower buds. The expression in the growing point of the stem tip showed an elevated level after vernalization, after which it declined rapidly at the stage of flower bud differentiation and was maintained at a very low level. Compared with the wild type, the flowering time of the BrMLP328 of T₂ generation of transgenic plants delayed by 1.46-3.09 d.【Conclusion】BrMLP328 was cloned from Chinese cabbage. The expression of BrMLP328 was different in different tissues and different flowering stages, and the gene could delay flowering.

Identification and Expression Analysis of Epidermal Patterning Factor (EPF) Genes in Cucumis melo

CHEN Chun-lin, LI Bai-xue, LI Jin-ling, DU Qing-jie, LI Meng, XIAO Huai-juan

2024, 40(4): 130-138. doi:10.13560/j.cnki.biotech.bull.1985.2023-0969

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【Objective】Epidermal patterning factor（EPF）is a unique secreted protein found in plants that plays a crucial role in plant growth and development, particularly in stomatal morphogenesis. The aims of this study was to gain insights into the characteristics and functions of the EPF gene in melon.【Method】We conducted a comprehensive analysis of the entire CmEPF family, including gene structure, phylogeny, and tissue expression, using bioinformatics tools. 【Result】Our findings revealed that the melon genome contained 11 members of the CmEPF family, which can be classified into three subfamilies. The gene structure of CmEPF was found to be relatively conserved, typically consisting of 1-3 introns, with most genes located in the melon chloroplast. Phylogenetic analysis further confirmed the division of CmEPF into three subfamilies. By performing RT-qPCR analysis on various organs, we observed widespread expression of the CmEPF gene in melon, albeit with different expression patterns among family members in different organs. Notably, the CmEPF gene had high expression in the second leaf, which was correlated with the highest stomatal density, photosynthetic rate, and transpiration rate. These results suggest that the expression level of CmEPF is closely associated with photosynthetic parameters and photosynthesis. 【Conclusion】Our findings provide evidence that the CmEPF gene can affect stomatal density in melon, thereby affecting photosynthesis and regulating stomatal response to the external environment in melon leaves.

Effects of Melatonin on the Fruit Softening and Ethylene Synthesis of Post-harvest Oriental Melon

CHEN Qiang, HUANG Xin-hui, ZHANG Zheng, ZHANG Chong, LIU Ye-fei

2024, 40(4): 139-147. doi:10.13560/j.cnki.biotech.bull.1985.2023-1099

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【Objective】Melatonin plays an important role in post-harvest fruit preservation. To explore the efects of melatonin on the fruit softening and ethylene synthesis of post-harvest oriental melon.【Method】The fruit（Cucumis melo cv. ‘Huaguniang’）were soaked at 0,100, 200, 300, 400 μmol/L melatonin solution, by measuring the firmness, weight loss, decay rate, and endogenous ethylene production, the optimal concentration was selected. At the optimal concentration, the ACC content and the activities of ACO and ACS were studied. Furthermore, the transcriptome sequecing and real-time PCR were used to compare the gene expression of ethylene synthesis, signal transduction pathway and fruit softening between melatonin treatment and control. 【Result】200 μmol/L treatment delayed the weight loss rate, slow down firmness, and prevent fruit decay and deterioration effectively. Melatonin treatment decreased the ethylene synthesis of fruit and ACC level, while did not delay the arrival of peak period. The transcriptome sequecing results indicated that there were differentially expressed genes in ethylene synthesis, signal transduction pathways and fruit softening between melatonin and control fruits. Melatonin treatment inhibited the expressions of CmACO1-2, CmACS1, CmERFs, CmEXPs and CmEFRs.【Conclusion】200 μmol/L melatonin delays the weight loss rate, slows down firmness, and decreases the ethylene synthesis, by inhibiting the expressions of genes in ethylene synthesis, signal transduction pathways and fruit softening and prolong the storaage life.

Expression Characteristics and Functions of CaPIF4 in Capsicum annuum Under Salt Stress

LI Hui, WEN Yu-fang, WANG Yue, JI Chao, SHI Guo-you, LUO Ying, ZHOU Yong, LI Zhi-min, WU Xiao-yu, YANG You-xin, LIU Jian-ping

2024, 40(4): 148-158. doi:10.13560/j.cnki.biotech.bull.1985.2023-1045

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【Objective】Studying the molecular mechanisms of the pepper photoreceptor pigment phytochrome-interacting factor 4（PIF4）in response to salt stress plays an important role in understanding the molecular mechanisms of pepper's response to salt stress and breeding salt-tolerant varieties. 【Method】Using ‘Zunla-1’ cDNA as a template, CaPIF4 was cloned. Then the physical and chemical properties of CaPIF4 were analyzed using bioinformatics software. And the expression pattern and salt stress-induced expression was studied by real-time fluorescence quantitative PCR（RT-qPCR）and virus-induced gene silencing technology（VIGS）. 【Result】CaPIF4 was expressed in various tissues of pepper, including roots, stems, leaves, flowers, and fruits, with the highest expression in the leaves. And the expression of CaPIF4 increased with the extension of 300 mmol/L NaCl treatment, and reached the highest at 2 h after treatment. CaPIF4 gene was silenced using VIGS technology. The control group（CK）and gene transient silencing group（pTRV-CaPIF4）were treated in 300 mmol/L NaCl. And the phenotype showed that pTRV-CaPIF4 plants after salt stress presented more obvious salty damage compared with CK. Using VIGS technology to silence CaPIF4, the CK and the pTRV-CaPIF4 were treated with 300 mmol/L NaCl. It was found that the plants in the pTRV-CaPIF4 group were more severely wilted compared to CK after gene silencing, indicating a decrease in salt tolerance in the pTRV-CaPIF4 group. Subcellular localization results showed that CaPIF4 was located in the nucleus. Yeast transcriptional activity analysis revealed that CaPIF4 had transcriptional activation activity. Under high salt stress, the hydrogen peroxide content and antioxidant enzyme activity in the pepper leaves increased. Staining results showed that silencing the CaPIF4 gene resulted in a significant increase in H₂O₂ and O^2- levels. 【Conclusion】CaPIF4, as a transcription factor, may be involved in regulating salt tolerance in peppers.

Cloning and Expression Analysis of CsERF025L Transcription Factor in Cucumber

XIAO Ya-ru, JIA Ting-ting, LUO Dan, WU Zhe, LI Li-xia

2024, 40(4): 159-166. doi:10.13560/j.cnki.biotech.bull.1985.2023-0962

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【Objective】ERF transcription factor is a subfamily of AP2/ERF gene family, which plays a key role in plant growth and development and resistance to external stress. Cloning CsERF025-like（CsERF025L）and analyzing its expression pattern may lay a foundation for studying the function of CsERF025L. 【Method】CsERF025L from cucumber（Cucumis sativus L.）stem tip was cloned by RT-PCR, and bioinformatics analysis of CsERF025L protein physicochemical properties, subcellular localization and interacting proteins was performed. Real-time fluorescence quantitative PCR was used to analyze the expression characteristics of CsERF025L in cucumber. 【Result】The encoding sequence of CsERF025L was 510 bp, encoding 169 amino acids, with an AP2/ERF domain, belonging to the ERF subfamily of the AP2/ERF family. CsERF025L protein was an unstable hydrophobic protein located in the nucleus. CsERF025L protein interacts with members of P450 gene family, BEE3 transcription factor, DIR1 protein and MATE family members. CsERF025L promoter contained light response, hormone response, stress response and other elements. The expressions of CsERF025L in the cucumber seeds was the highest, which was significantly higher than that in leaves, stems and roots. The expression patterns of CsERF025L in the cucumber with different phyllotaxis types were compared, and the expressions of CsERF025L in the stem tip of opposite phyllotaxis cop1 were significantly higher than those of cucumber alternate phyllotaxis R1. 【Conclusion】The findings of this study suggest that CsERF025L is a nuclear-localized ERF transcription factor, potentially involved in the formation and regulation of cucumber alternate phyllotaxis.

Genome Wide Identification and Expression Analysis of the bZIP Gene Family in Panax quinquefolius

GUO Chun, SONG Gui-mei, YAN Yan, DI Peng, WANG Ying-ping

2024, 40(4): 167-178. doi:10.13560/j.cnki.biotech.bull.1985.2023-0970

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【Objective】The basic leucine zipper (bZIP) is one of the largest transcription factor families in plants, involved in multiple processes such as plant growth and hormone regulation. This study aims to identify the bZIP gene family in Panax quinquefolius and analyze its structure and expression patterns, providing a theoretical basis for further research on the function of the bZIP gene family in P. quinquefolius.【Method】Bioinformatics was used to analyze the physicochemical properties, phylogenetic relationships, gene structure, conserved motifs, cis acting elements, chromosomal localization, evolutionary selection pressure,and tissue-specific expression of the bZIP gene in P. quinquefolius. Simultaneously, exogenous hormone ABA was used to treat P. quinquefolius for transcriptome data analysis and RT-qPCR.【Result】A total of 121 bZIPs genes were identified in P. quinquefolius. According to the phylogenetic tree, these genes were divided into 12 subgroups. Gene structure and conserved motif analysis showed that proteins in the same subgroup had similar conserved motifs and domains, with conserved motif 1 being the most widely distributed and highly conserved in the bZIP protein of P. quinquefolius. The analysis of cis-acting elements showed that there were hormone responsive elements, defense and stress responsive elements in the upstream promoter element of bZIP, with ABA hormone responsive elements accounting for the largest proportion. Chromosome localization analysis showed that bZIP genes were unevenly distributed on 24 chromosomes. Conducting evolutionary selection pressure analysis, it was found that purification selection played an important role in the evolution of the bZIP gene family in P. quinquefolius; organizational specific expression analysis showed that the expression levels of PqbZIPs genes were relatively high in flowers and roots; transcriptome data revealed that bZIP transcription factors actively responded to ABA hormone treatment. Subsequently, RT-qPCR experiments were conducted to verify that the trend of gene expression changes was basically consistent. 【Conclusion】Members of the PqbZIP gene family have tissue-specific expression patterns and most respond to ABA hormone treatment.

Cloning and Functional Analysis of LtMYB305 in Liriodendron tulipifera

LIU Huan-huan, YANG Li-chun, LI Huo-gen

2024, 40(4): 179-188. doi:10.13560/j.cnki.biotech.bull.1985.2023-1069

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【Objective】MYB305 as a member of the MYB R2R3 subfamily, plays an important role in plant nectary development, nectar protein expression, starch accumulation and hydrolysis, and flavonoid synthesis. Therefore, it is of great significance to study the expression profile and function of MYB305 for the molecular mechanism study of Liriodendron tulipifera floral nectary. 【Method】Having floral nectary of nectariferous plant L. tulipifera as materials, the anthocyanidin content in five stages of floral nectary was detected by spectrophotometry, LtMYB305 gene was cloned via RACE（rapid-amplification of cDNA ends）technology, and relative expressions among different tissues were analyzed by RT-qPCR assay. Then, subcellular localization of LtMYB305 protein was verified using tobacco leaves as materials, and the function of LtMYB305 gene was studied by genetic transformation approaches of wild type Arabidopsis thaliana（col）. 【Result】The anthocyanidin in the nectaries of L. tulipifera was accumulated from the late expanding stage, increased sharply at the early flowering stage, and reached the highest of 27.14 μg/g at the withering stage, which was consistent with the process of nectar secretion and coloring. The full-length of LtMYB305 was 931 bp encoding 198 amino acids and its protein was hydrophilic and non-transmembrane. LtMYB305 was expressed highly only in the nectary of L. tulipifera at anthesis stage, and hardly in other tissues. LtMYB305 protein was localized in the nucleus. After the overexpression of LtMYB305, the nectary groove of the lateral nectary disappeared and deepened, the expressions of AtMYB30, AtPIN, AtSWEET9 genes related to flower nectary were up-regulated, and the expressions of AtCRC, AtBOP1/2, AtMYB21, AtSWEET3/4/7 genes were down-regulated. 【Conclusion】The LtMYB305 has typical characteristics of transcription factor and is involved to the nectary development in L. tulipifera.

Effects of Endophytic Fungal Inoculation on the Seedling Growth of Silage Maize

WANG Jia-wei, LI Chen, LIU Jian-li, ZHOU Shi-jie, YI Jia-min, YANG Jin-yuan, KANG Peng

2024, 40(4): 189-202. doi:10.13560/j.cnki.biotech.bull.1985.2023-1081

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【Objective】Endophytic fungi represent significant microbial assets that exert a beneficial influence on plant growth. The method of inoculation plays a pivotal role in determining the growth-promoting efficacy of endophytic fungi.This study aims to investigate the impact of various inoculation methods on the growth-promoting potential of endophytic fungi, thereby offering valuable insights for the practical utilization of endophytic fungi. 【Method】Four strains of endophytic fungi and Piriformospora indica of different species that could promote the growth of silage maize were screened from the endophytic fungi of desert plant roots in the early stage of the research group. Five strains of endophytic fungi were inoculated into the potted seedlings of silage maize by means of root irrigation with mycelial fragment suspension and root wrapping with mycelial mass. After 30 d, the biological characteristics of the seedlings inoculated with silage maize were determined, and the effects of different inoculation methods on the growth of silage maize were compared.【Result】The inoculation method had no significant effect on the mycelial infection rate and basal stem diameter of the five endophytic fungi in the roots of silage maize seedlings, but the other 17 traits related to the growth of silage maize were significantly affected by the inoculation method. The results of principal component analysis showed that the growth-promoting effect of the five endophytic fungi using mycelial fragment suspension root irrigation was significantly better than that of mycelial pellet root, and the difference between the two inoculation methods of P. indica and strain Tm36 was the largest. Ten traits of silage maize seedlings inoculated with P. indica and strain Tm36 were significantly different between the two inoculation methods, they were plant height, aboveground fresh weight, aboveground dry weight, average leaf area, leaf relative water content, root fresh weight, root surface area, root volume, whole plant fresh weight and whole plant dry weight. Among them, the five indexes of plant height, aboveground fresh weight, average leaf area, root fresh weight and whole plant fresh weight were the most different. The growth rate of P. indica inoculated with mycelial fragment suspension was 7.78, 3.74, 15.97, 7.93 and 5.36 times that of mycelial mass inoculation, and was 8.65 times, 4.33 times, 11.18 times, 16.58 times and 7.53 times that of inoculation strain Tm36.【Conclusion】The inoculation method significantly affects the growth-promoting effect of endophytic fungi. The inoculation method of mycelial fragment suspension root irrigation has better effect on the growth of silage maize seedlings than that of mycelial pellet root inoculation. It is recommended to use mycelial fragment suspension root irrigation method.

Responses of Sorghum Rhizosphere Soil Bacterial Communities to Salt Stress

GAO Yu-kun, ZHANG Jian-dong, YANG Pu-yuan, CHEN Dong-ming, WANG Zhi-bo, TIAN Yi-jin, Zakey Eldinn. E. A. Khlid, CUI Jiang-hui, CHANG Jin-hua

2024, 40(4): 203-216. doi:10.13560/j.cnki.biotech.bull.1985.2023-0685

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【Objective】This study aims to investigate the changes in the bacterial community structure of sorghum roots under salt stress, as well as the characteristics of the rhizosphere bacterial co-occurrence network structure.【Method】Two sorghum varieties, HN16（salt-sensitive）and GLZ（salt-tolerant）, with varying levels of salt tolerance were cultivated in pots and subjected to salt stress. The root microbiome of these plants was then analyzed using 16S amplicon sequencing technology.【Result】With the aggravation of salt stress, the contents of total phenols and flavonoids in the sorghum roots gradually increased, and the appropriate salt stress induced the synthesis of phenolic acids to improve the salt tolerance of sorghum. The results of the microbial community structure analysis showed that Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes and Chloroflexi were the dominant phyla in sorghum rhizosphere soils. Comparative analysis of samples showed that the bacterial composition of sorghum rhizosphere under salt stress was less affected by growth stage, and the bacterial community structure of sorghum rhizosphere changed with the aggravation of salt stress. Through WGCNA analysis of all filters OTUs, a total of 12 gene co-expression module were identified, pink module had significantly positive correlation with salinity and greenyellow module had significantly positive correlation with period and the contents of total phenols and flavonoids after salt treatment. The bacterial co-occurrence network was more complex in low salt stress soils than in higher salt stress soils, reflected by more nodes and more edges. The 13 network key OTUs were identified, OTU8480, OTU6866, OTU3247 and OTU3499 was the keyOTUs in S0 network; OTU6895, OTU4206, OTU6470, OTU1810 and OTU4916 in S3 network; OTU4217, OTU8426, OTU4847, and OTU6066 were the key OTUs in S7 network. 【Conclusion】The composition of rhizosphere bacteria in both varieties of sorghum has been modified in response to salt stress. After salt stress, with more complex co-occurrence network and tighter connections between OTUs of rhizosphere soil bacterial communities.

Identification of Salt-tolerant Plant Growth-promoting Bacterium W-1 and Its Effect on the Salt-tolerance of Sainfoin（Onobrychis viciaefolia）

GAO Zhi-wei, WEI Ming, YU Zu-long, WU Guo-qiang, WEI Jun-long

2024, 40(4): 217-227. doi:10.13560/j.cnki.biotech.bull.1985.2023-0905

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【Objective】To obtain salt-tolerant plant growth-promoting bacteria, several strains of salt-tolerant bacteria were isolated from plant roots in a saline-alkali soil in Jingtai, Gansu province, and some of them had obvious plant growth-promoting characteristics.【Method】Based on the previous work, a strain of salt-tolerant bacterium W-1 and a salt-sensitive plant sainfoin（Onobrychis viciaefolia）were selected as experimental materials, and the species classification, salt tolerance, acid and alkali tolerance and plant growth-promoting characteristics of strain W-1 were tested. The effects of strain W-1 on the physiological status of sainfoin treated with different concentrations of NaCl were investigated. 【Result】The salt-tolerant bacterium W-1 was Gram-positive, spore-producing, without capsule and flagellated. The 16S rDNA sequencing and comparison results showed that the bacterium W-1 was Bacillus siamensis. The maximum NaCl tolerance of strain W-1 was 13%, and the pH tolerance range was 4.5-8.5. W-1 has various plant growth-promoting functions, such as phosphate and potassium solubilization, nitrogen fixation, siderophores production, indole-3-acetic acid（IAA）production and 1-aminocyclopropane-1-carboxylic acid（ACC）deaminase activity. Strain W-1 significantly increased dry weight and fresh weight and promoted root growth of sainfoin seedlings. Under 100 and 150 mmol/L NaCl stresses, strain W-1 significantly increased the contents of soluble sugar, soluble protein, proline, chlorophyll, and catalase activity, decreased the content of hydrogen peroxide, leaf yellowing rate and mortality, and alleviated the adverse effects of salt stress on the growth of sainfoin seedlings.【Conclusion】The W-1 strain was an excellent plant growth- promoting bacterium with salt tolerance, acid and alkali tolerance, and enhanced the salt tolerance of legume forage.

Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim

XU Wei-fang, LI He-yu, ZHANG Hui, HE Zi-ang, GAO Wen-heng, XIE Zi-yang, WANG Chuan-wen, YIN Deng-ke

2024, 40(4): 228-241. doi:10.13560/j.cnki.biotech.bull.1985.2023-1172

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【Objective】The biocontrol ability of bacterium HX0037 against anthracnose disease of Trichosanthes kirilowii Maxim and its biocontrol mechanism were studied. 【Method】The antagonistic activities of HX0037 against four pathogens of fruit anthracnose disease were determined in petri dishes experiments. The biocontrol efficiencies of HX0037 against Trichosanthes leaf anthracnose and Trichosanthes fruit anthracnose were evaluated on detached leaves and fruits in vitro. In addition, HX0037 was further identified based on the analyses on morphological features, physiological and biochemical characteristics and molecular biological identification results of the whole genome, and the secondary metabolites of biocontrol bacteria HX0037 were also predicted. The lipopeptide compounds produced by strain HX0037 were extracted by acid precipitation and analyzed by high-performance liquid chromatography tandem mass spectrometry（LC-MS）. Finally, the possible biocontrol mechanism was explored through two experiments, observing the effects of lipopeptides produced by strain HX0037 on the hyphal growth and morphology of Colletotrichum gloeosporioides, and the abilities of strain HX0037 to produce enzyme and siderophore. 【Result】Strain HX0037 showed varied antimicrobial activity against four pathogens of fruit anthracnose disease, especially the best against C. gloeosporioides, a pathogen of Trichosanthes anthracnose disease, and it obviously changed the cell membrane permeability of the pathogenic fungi. Compared with plant tissues inoculated with the pathogen alone, inoculation with strain HX0037 significantly reduced the incidence of leaf and fruit anthracnose diseases of T. kirilowii Maxim, and the biocontrol efficacy was 61.50% and 52.51%, respectively. Strain HX0037 was further identified as Bacillus amyloliquefaciens, and its genome contained 12 secondary metabolite gene clusters, and it decomposed the protein, cellulose and starch, and also produced siderophore. The hyphae of C. gloeosporioides were distorted, intumescent, broken and more transparent after treatment with the antifungal lipopeptide produced by strain HX0037. Surfactin and fengycin were detected from the lipopeptide extracts of strain HX0037 by LC-MS. 【Conclusion】The B. amyloliquefaciens HX0037 has strong antagonistic activity on C. gloeosporioides, and is expected to be further developed as a biological agent for the biocontrol of Trichosanthes anthracnose diseases.

Analysis of Microbial Community Changes and Stress-resistant Enzyme Activities of Flue-cured Tobacco Three-stage Seedling Raising in Different Habitats

WANG Hao-jie, CHANG Dong, LI Jun-ying, MENG Hao-guang, JIANG Shi-jun, ZHOU Shuo-ye, CUI Jiang-kuan

2024, 40(4): 242-254. doi:10.13560/j.cnki.biotech.bull.1985.2023-1071

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【Objective】It is to analyze the effects of three-stage seedling raising on rhizosphere microbial diversity and anti-stress enzyme activity of tobacco seedlings.【Method】Zhongyan 100 was chosen as the test variety, with conventional floating seedling raising as the control, and the three-stage seedling raising was set up. By monitoring the growth and development of tobacco seedlings in the quadruple sports seedling greenhouse, the effects of three-stage seedling raising method on seedling environment, microbial diversity, agronomic traits and anti-stress enzyme activities of tobacco seedlings were systematically analyzed. Concurrently, its effect on the microbial diversity of tobacco rhizosphere soil in the field was traced and analyzed.【Result】In the seedling stage, the three-stage seedling raising method improved the physical and chemical properties of the pool water and reduced the temperature difference and humidity level in the seedling greenhouse compared with the floating seedling raising control. The daily average temperature difference decreased from 19.23℃ to 17.95℃, and the daily average humidity decreased from 75.17%RH to 69.53%RH. At the seedling stage, the three-stage seedling raising increased the abundance of Brevundimonas and Lysinibacillus in the seedling pool water, which were 13.90 times and 4.66 times that of the floating seedling raising, respectively. The fresh weight, root length, number of lateral roots, CAT enzyme activity, SOD enzyme activity, POD enzyme activity, root activity and seedling rate of tobacco seedlings increased by 34.33%, 34.37%, 36.41%, 31.91%, 25.34%, 58.07%, 29.08% and 5.91%, respectively, compared with the floating seedling raising control, while the MDA content decreased by 22.31%. At the same time, MDA content decreased by 22.31%. In the field period, the abundance of Ascomycota and Penicillium in tobacco rhizosphere soil increased, and a more favorable environment for tobacco growth was created.【Conclusion】The three-stage seedling raising method increased the abundance of beneficial bacteria in the rhizosphere of tobacco seedlings by improving the seedling raising environment, and significantly improved the agronomic traits and anti-stress enzyme activity of tobacco seedlings at the seedling stage, thus improving the quality of tobacco seedlings.

Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes

ZHONG Yun, LIN Chun, LIU Zheng-jie, DONG Chen-wen-hua, MAO Zi-chao, LI Xing-yu

2024, 40(4): 255-263. doi:10.13560/j.cnki.biotech.bull.1985.2023-1074

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【Objective】This study aims to clone the sterol glycosyltransferase gene AoSGT1 from Asparagus officinalis, to assess its catalytic properties, and to elucidate its role in the biosynthesis of asparagus saponins and their metabolic regulation.【Method】We designed specific primers based on asparagus transcriptome data to amplify the complete open reading frame（ORF）of AoSGT1. The gene was sequenced and analyzed through bioinformatics, and its expression profile was investigated via quantitative real-time PCR（RT-qPCR）. We also constructed a pGEX-4T-3-AoSGT1 expression vector, expressed it in Escherichia coli BL21（DE3）, and induced the recombinant protein production.【Result】AoSGT1 has a length of 1 800 base pairs, encoding 599 amino acids, with a relative molecular weight of 66.72 kD. It is a hydrophilic protein with no transmembrane domains or signal peptides. The phylogenetic analysis revealed a close homology between AoSGT1 and Dioscorea zingiberensis Dz3GT2, indicating their membership in the UGT80B1 subfamily. Multiple sequence alignment revealed that the protein sequence contained conserved domains “PSBD Box” and “PSPG Box” of sterol glycosyltransferase, indicating its potential glycosylation activity at the 3β-OH position of steroidal compounds. RT-qPCR results showed that AoSGT1 was highly expressed in the roots, while expression in stems and flowers was relatively low. Additionally, SDS-PAGE results revealed that the target protein was efficiently expressed in a soluble form within E. coli, matching the predicted size.【Conclusion】The AoSGT1 gene was successfully cloned and demonstrated to exhibit tissue-specific expression in asparagus, suggesting its potential involvement in the biosynthesis of steroidal saponins in asparagus. Additionally, heterologous expression of the target protein in E. coli was successfully achieved.

Biological Characteristics, Domestication and Cultivation of Wild Gymnopilus subpurpuratus

RAO Yong-bin, ZHANG Jun-li

2024, 40(4): 264-270. doi:10.13560/j.cnki.biotech.bull.1985.2023-1040

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【Objective】This work is to realize the efficient development and utilization of Xizang’s wild fungi resources and promote the development and growth of edible and medicinal fungi industry.【Method】A wild fungus collected fromYigong township, Bomi county, Linzhi city, Xizang autonomous region was isolated and purified, and the strain was identified according to morphological characteristics and its sequence.【Result】Combined with morphological characteristics and ITS phylogenetic tree results, the strain was identified as Gymnopilus subpurpuratus. The results show that the best carbon source for mycelium growth is glucose, the best nitrogen source is yeast powder, the optimal pH is 6.0, and the optimal temperature is 30℃. The results of domestication show that the filling-bag time in the culture medium with sawdust as the main cultivation material is 39 d, the formation time of primordium is 18 d, the harvest time is 8 dafter primordium was out, and the fresh weight of single fungus is 45.99 g.【Conclusion】To sum up, the artificial domesticated fruiting body of G. subpurpuratus is successfully obtained by using sawdust as the main cultivation material, which increases the new species of artificial domestication of medicinal fungi.

Mechanism of CMV Intervention in Peach Aphid Population Growth by Influencing the Expression of Effector MpC002

MAO Li-jie, LIANG Xiao, LIU Ying, WU Chun-ling, HAN Xiao-yan, CHEN Qing

2024, 40(4): 271-277. doi:10.13560/j.cnki.biotech.bull.1985.2023-1057

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【Objective】Peach aphid（Myzus persicae）is an important vector of cucumber mosaic virus（CMV）, and the effector MpC002 plays a key role in the feeding process. CMV and MpC002 are both present in aphid saliva, but little is known about their interaction and how MpC002 facilitate virus transmission. Therefore, this study aims to preliminarily investigate the mechanism by which CMV influences the expression of MpC002 for interfering the population growth of GPA. 【Method】The CMV copy number was calculated by the standard curve y=-3.163 1x+39.763, via applying the absolute quantitative polymerase chain reaction（qPCR）. 【Result】The CMV content in the peach aphid fed with CMV-infected pepper reached the highest at 10 s, then the CMV content decreased gradually along with the feeding time, and its virulence acquisition process conformed to the characteristics of non-persistent transmission. In addition, the expressions of MpC002 in the peach aphids fed on CMV-infested peppers within 5 min-24 h significantly decreased to 37%-58%, compared with those before feeding. While feeding on CMV-infested peppers, the average developmental duration of third, fourth instar and the entire nymph stages were 1.58, 2.07 and 6.47 d, which were significantly longer than those fed on the untreated peppers（1.25 d, 1.47 d and 5.33 d）; in contrast, the developmental duration of the first and second instars did not show significantly difference between CMV-infested and untreated peppers. The average laying period, the total peach aphid number and the average peach aphid in a single day were 10.03 d, 16.87 and 1.67, which were significantly shorter than those of untreated peppers（14.27 d, 39.73 and 2.82）. In addition, analysis of GPA life table parameters revealed that the net growth rate, intrinsic rate of increase, and finite rate of increase were 16.87, 0.21, and 1.21, respectively, which were significantly lower than those of peach aphids feeding on untreated peppers（39.73, 0.31, and 1.36, respectively）, whereas the mean generation time（13.02 d）and the population doubling time（3.26 d）were both significantly longer than those of peach aphids feeding untreated peppers（12.00 d and 2.30 d）. 【Conclusion】CMV significantly inhibited the expressions of MpC002, and then prolongated the developmental duration and decreased the reproduction, which in turn adversely affected its population growth.

Cloning and Expression of Protease SpP1 Gene and Characterization of Enzymatic Properties

YIN Liang, WANG Dai-wei, LIU Yue-ying, LIU Hai-yan, LUO Guang-hong

2024, 40(4): 278-286. doi:10.13560/j.cnki.biotech.bull.1985.2023-0813

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【Objective】The protease gene of Spirulina was cloned and expressed, and the enzymatic properties of recombinant enzyme were investigated, which lays a foundation for the further research of algal protease.【Method】The protease gene SpP1 was amplified from the genome of Spirulina platensis, and the pET28a-SpP1 recombinant plasmid was constructed, which was transfected into Escherichia coli BL21（DE3）to achieve heterologous expression, and the recombinant protease was isolated and purified by using a nickel column to study its enzymatic properties.【Result】The protease SpP1 was a member of the serine protease family, with a molecular weight of 47.04 kD, and its optimal temperature and pH values were 50℃ and 8.0, respectively; its thermal stability was poor, and it had a good acid-base stability in the pH=8.0-9.0 range. When casein was used as the substrate, the maximum reaction velocity V_max=8.237 U/mL, and the Michaelis constant K_m=16.369 μg/mL. The activity increased by 18-fold with the addition of 0.1 mol/L Mn ²⁺. Also 0.1 mol/L Fe³⁺, Zn²⁺, Ca²⁺, ethylenediaminetetraacetic acid（EDTA）had a significant promotion effect on the enzyme activity.【Conclusion】The protease SpP1 has the typical structure and property characteristics of serine protease family members, with good acid-base stability, and the addition of manganese ions can effectively enhance its catalytic activity.

Effect of Forsythia suspensa Leaves Tea on HCC Proliferation and Migration Function and Its Mechanism of Action

TENG Wen-long, WU Yong-na, WANG De-fu, NIU Yan-bing

2024, 40(4): 287-296. doi:10.13560/j.cnki.biotech.bull.1985.2023-0935

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【Objective】This work aims to investigate the effects and mechanisms of crude extract of F. suspensa leaves tea and its main components, phillyrin and Forsythia glycoside A, on the proliferation and migration function of hepatocellular carcinoma LM3. 【Method】 Water extraction method was used to extract green tea and black tea from Forsythia suspensa leaves tea, HPLC was used to detect the content of effective components such as phillyrin and Forsythia glycoside A in F. suspensa green tea and F. suspensa black tea. CCK8 experiment was used to explore the effects of crude extracts of two types of F. suspensa leaves tea, phillyrin and Forsythia glycoside A on the proliferation of hepatocellular carcinoma LM3. Two crude extracts of Forsythia suspensa leaves tea, phillyrin and Forsythia glycoside A, were used to culture hepatocellular carcinoma LM3. The migration of cells was observed at 24, 48, 72, and 96 h, and the effects of the two crude extracts of F. suspensa leaves tea, phillyrin and Forsythia glycoside A on the migration of hepatocellular carcinoma LM3 were detected through the cell scratch test. To reveal the molecular mechanism of crude extract of F. suspensa leaves tea and its main components phillyrin and Forsythia glycoside A affect the proliferation and migration of hepatocellular carcinoma. The expressions of proliferation and migration-related gene Ki-67, Erk, PI3K and mTOR were detected using RT-PCR technology.【Result】Both the green tea and black tea extracts of F. suspensa leaves tea, as well as phillyrin and Forsythia glycoside A, significantly inhibited the proliferation and migration of hepatocellular carcinoma LM3, while Forsythia glycoside A significantly inhibited the migration of hepatocellular carcinoma line LM3. Among them, the crude extract of F. suspensa green tea significantly inhibited the proliferation and migration of HCC at low, medium, and high concentrations when 24, 48, and 72 h. The inhibitory effect of the crude extract of F. suspensa black tea on the proliferation and migration of LM3 cells continued to increase with increasing concentration, and the best inhibitory effect was achieved at high concentration for 72 h. The analysis of RT-PCR results suggests that its main mechanism of action may be achieved by reducing the expression levels of proliferation and migration related genes such as Ki-67.【Conclusion】In summary, the crude extracts of F. suspensa green tea and F. suspensa black tea, as well as phillyrin and Forsythia glycoside A, can inhibit the proliferation or migration of HCC LM3 by reducing the expression of Ki-67.

Identification and Functional Study of T6SS Effector Protein PA0423 of Pseudomonas aeruginosa

WANG Cai-hong, JIANG Meng-yuan, SHAO Yu-han

2024, 40(4): 297-305. doi:10.13560/j.cnki.biotech.bull.1985.2023-1068

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【Objective】The objective of this study is to investigate the novel effector proteins and their functions in the type VI secretion system（T6SS）.【Method】 The secretion mode and function of PA0423 were explored by gene knockout, immunoblotting, co-immunoprecipitation, intraspecific and interspecific competition, Escherichia coli toxicity assays and crystal violet biofilm assays.【Result】Western blotting and co-immunoprecipitation experiments showed that PA0423 interacted with PA0262, and the secretion of PA0423 depended on H2-T6SS. PA0423 is a H2-T6SS-dependent antimicrobial effector protein with interspecific competitive advantages. PA0423 is toxic to Escherichia coli and PA0422 is its immune protein. PA0423 affected the formation of bacterial biofilms.【Conclusion】PA0423 is a H2-T6SS-dependent antimicrobial effector protein, which has bactericidal function and affects the formation of bacterial biofilms.

Identification and Transcriptional Regulation Analysis of Core Promoter in Bovine TARDBP Gene

ZHANG Qing-lan, ZHANG Ya-ran, JU Zhi-hua, WANG Xiu-ge, XIAO Yao, WANG Jin-peng, WEI Xiao-chao, GAO Ya-ping, BAI Fu-heng, WANG Hong-cheng

2024, 40(4): 306-318. doi:10.13560/j.cnki.biotech.bull.1985.2023-1048

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【Objective】 The aim of this study is to analyze the transcriptional regulation mechanism of bovine TARDBP gene, as well as its structure, function and promoter active region. 【Method】The complete coding region of bovine TARDBP gene was cloned by PCR. Bioinformatics software was applied to analyze the properties and the structure of bovine TARDBP protein. The expression of TARDBP gene was profiled in different tissues of lactating Holstein cows by real-time fluorescence quantitative PCR（RT-qPCR）. The transcriptional initiation site of TARDBP gene was determined by 5' RACE technique, and the promoter region and 5' terminal deletion fragment of this gene were cloned by PCR. The CpG island and potential transcription factor binding site within the promoter region were predicted by bioinformatics software. The core promoter region was then confirmed by the dual-luciferase reporter system.【Result】Bovine TARDBP gene was expressed in multiple tested tissues including mammary gland. TARDBP protein was water-soluble, and it contained RNA recognition motifs and DNA binding sites. There were two CpG islands and a large number of transcription factor binding sites including SP1, PPARA, PPARD, PPARG, SREBF1 and so on in the promoter region. The results of dual-luciferase activity analysis showed that the core promoter region of bovine TARDBP was between -476 and -149, and Sp1, PPARG, PPARD and SREBF1 binding sites were found in the core promoter, but there was no TATA-box. 【Conclusion】 Bovine TARDBP gene is distributed in various tissues of dairy cows. The core promoter region of bovine TARDBP gene is positioned at -476 to -149, and there are transcription factor binding sites related to milk fat synthesis and secretion in this region.

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2024, 40(4): 311-311.

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