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    26 June 2024, Volume 40 Issue 6
    Harmony in Diversity: Auxin and Redox Signaling During Root Hair Development
    XIE Yan-jie
    2024, 40(6):  1-4.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0477
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    Reactive oxygen species and auxin play important roles in plant growth and development, and their signal transduction and regulatory mechanisms have always been an important field of plant science research. The classic pathway TIR1/AFB-Aux/IAA-ARF mediates nucleic auxin signaling through transcriptional regulation. However, how auxin signal is relayed from cytoplasm to nucleus is still unclear. Recently, Prof Li Chao's team from East China Normal University discovered that redox signals are involved in the auxin-regulated root hairing process. The auxin receptor protein TIR1/AFB2 undergoes Oxi-PTMs before migrating to the nucleus, which determines the initiation of the auxin transcriptional signal for root hair development, and this step is controlled by the upstream FER/LG1-RAC/ROP-RBOHC module. The results of this research have discovered the crosstalk mechanism between auxin and redox signals, and provide a paradigm for regulating biological processes through protein Oxi-PTMs.

    Roles of MYB Transcription Factor in Regulating the Responses of Plants to Stress
    HU Ya-dan, WU Guo-qiang, LIU Chen, WEI Ming
    2024, 40(6):  5-22.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1186
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    MYB is one of the biggest multifunctional transcription factors(TFs)families in plants. It is widely involved in regulating multiple process at the transcription level, including plant growth and development, signal transduction of various phyhormones and response to abiotic and biotic stresses. The N-terminal of MYB contained a typical MYB domain, which is divided into different subgroups according to the number of R repeats in the domain. However, the C-terminal domain is very different, thus it is functionally diverse. Many studies have been shown that MYB can bind to the downstream target gene promoter cis-acting elements MYBCORE and AC-box alone or through interaction with other proteins after being activated by environmental signals, and participate in regulating the expressions of downstream target genes, thereby regulating plant tolerances to stresses. Additionally, MYB responded to abiotic and biotic stresses by regulating signaling pathways such as abscisic acid(ABA), brassinolide(BR), jasmonic acid(JA)and reactive oxygen species(ROS). In this review, the structure, classification, and action type of the MYB families in plants were summarized, and the regulatory mechanisms of MYB in the response to abiotic and biotic stresses such as salt, drought, extreme temperature, nutrient deficiency, heavy metals, and pathogenic bacteria, were mainly reviewed. The key direction of future studies were also prospected. This paper may provide genetic resources and theoretical support for genetic improvement of stress resistance of crops and biological breeding in the future.

    Advances in the Molecular Mechanisms of Plant Tissue Culture and Regeneration Regulated by Totipotency-related Transcription Factors
    WANG Di ZHANG Xiao-yu SONG Yu-xin ZHENG Dong-ran TIAN Jing LI Yu-hua WANG Yu WU Hao
    2024, 40(6):  23-33.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1164
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    Plant cells demonstrate the potential to develop into completely new organisms under suitable culture conditions, a phenomenon known as plant cell totipotency. Tissue culture techniques based on this totipotency have been extensively applied in the fields of asexual reproduction and genetic improvement through genetic transformation in plants. Abiotic stress, plant hormones and transcription factors synergistically regulate plant regeneration in vitro. Among these, transcription factors that play a pivotal role in plant regeneration are referred to as totipotency-related transcription factors. Recent advancements have shed light on the molecular mechanisms by which these totipotency-related transcription factors regulate plant in vitro regeneration. Numerous transcription factors have been identified and preliminarily applied in studies aimed at enhancing the efficiency of plant genetic transformation. According to the function of plant cell totipotent transcription factors in plant regeneration, this paper summarizes the signal transduction mechanism in plant regeneration in vitro in recent years, summarizes the role of plant cell totipotent transcription factors in improving the efficiency of plant genetic transformation, and looks forward to its application prospect, so as to provide scientific basis for establishing an effective asexual propagation system.

    Regulation of Maize Anthocyanin Biosynthesis Metabolism
    HU Jin-jin, LI Su-zhen, MA Xu-hui, LIU Xiao-qing, XIE Shan-shan, JIANG Hai-yang, CHEN Ru-mei
    2024, 40(6):  34-44.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0075
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    Anthocyanidins exist in the form of glycosides bound to glucose or fibre disaccharide molecules, and are a class of water-soluble natural pigments that are widely found in fruits, vegetables and flowers in nature. Anthocyanins have physiological activities such as antioxidant, anti-inflammatory, anti-tumour, and virus growth inhibition, thus they have broad application prospects in the food and pharmaceutical fields. Purple corn is an anthocyanin-rich crop, and the study of its anthocyanin biosynthesis pathway is of high practical significance. With the in-depth study of anthocyanin synthesis pathway in plants, the anthocyanin biosynthesis pathway has been gradually clarified, but the anthocyanin regulatory genes and the mechanism still have a lot of room for exploration. In recent years, researchers have made great progress in the study of anthocyanin-rich maize, but the precision breeding of anthocyanin-rich maize needs further research. In this paper, we reviewed the anthocyanin biosynthesis pathway, key enzyme genes and regulatory genes of maize, systematically analysed the effects of structural genes in the anthocyanin synthesis pathway of maize as well as regulatory genes, such as MYB, bHLH, WD40, etc., and elaborated on the regulation of anthocyanins by the MBW ternary complex. We also discussed the unresolved issues in breeding anthocyanin-rich maize and the future research direction, with a view to providing reference for future research on the genetic improvement of anthocyanins in maize and the selection of maize varieties with high anthocyanin content.

    Effects of Arbuscular Mycorrhizal Fungi on Plant Stress Resistance and Secondary Metabolite Synthesis
    YUAN Hai-peng YE Yun-shu SI Hao JI Qiu-yan ZHANG Yu-hong
    2024, 40(6):  45-56.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1117
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    Arbuscular mycorrhizal(AM)is a symbiotic system composed of the fungi of the phylum Glomeromycota and plant roots. Seventy-one percent of vascular plants have AM. Glomeromycota is now under a single class, four orders, eleven families and twenty-seven genera, with about 300 species of arbuscular mycorrhizal fungi(AMF). AMF improves plant resistance to abiotic stresses such as drought, salinity, heavy metals, and biotic stresses induced by other organisms as well as regulates the synthesis of plant secondary metabolites.This article reviews the research progress on the effects of AMF on plant adversity stress and secondary metabolites. The effects of AMF on life activities such as plant morphology and structure, physiology and biochemistry, gene expression and secondary metabolism are described. The mechanism of action of AMF in enhancing plant resistance to adversity stress and regulating the synthesis of plant secondary metabolites is summarized. It aims to provide a reference for the in-depth study of the action mechanism of AMF in response to plant adversity stress and regulation of plant secondary metabolite synthesis.

    Banana Rhizosphere Microecology and Its Relationship with Fusarium Wilt Control
    QI Yan-xiang, XIE Yi-xian, PENG Jun, ZENG Fan-yun, ZHANG Xin
    2024, 40(6):  57-67.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1222
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    Fusarium wilt seriously endangers banana production, it causes yield loss and even no yield. It is spread by soil and transportation of seedlings, and chlamydospores of its pathogen survive in soil for 30 years. It is very difficult for the disease to be controlled, and it is always the focus of the government, academic field and banana industry. The experts, scholars, and related practitioners of domestic and abroad have done extensive and deep related research for solving this hard issue. This paper reviewed the previous research progress from the occurrence and harm of banana Fusarium wilt, the relationship between plant rhizosphere microecology and soil-borne diseases, banana rhizosphere microecology and their effects and contributions in Fusarium wilt control. The paper proposed the research trends of the interaction mechanism between Fusarium wilt and banana rhizosphere microecology, strain screening scope and isolating incubation methods, development and application of biocontrol agents with high efficiency and strong stability, aiming to provide references for subsequent in-depth study of banana rhizosphere microecology, the excavation and innovation utilization of excellent biological control resources and the efficient control of banana Fusarium wilt.

    Research Progress in Carbon Sequestration by High-valued Isochrysis Strain
    CAI Nan, FANG Jing-ping, CHEN Bi-lian, HE Yong-jin
    2024, 40(6):  68-80.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1196
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    CO2 is one of the main greenhouse gases. A large amount of CO2 is released into the atmosphere through industrial production and human activities, leading to a significant increase in global CO2 concentration. This has resulted in the increasing global temperatures, climate warming, and frequent occurrences of extreme weather events, posing a threat to global human security. In order to mitigate the increase of CO2 and address global warming, countries around the world have set carbon peak targets and are committed to carbon capture and utilization. Marine Isochrysis galbana characterizes of a fast growth rate and high carbon-fixing efficiency; moreover, biological carbon sequestration mediated with Isochrysis strain and wastewater synthesized various high-value biologically active substances such as protein, polyunsaturated fatty acids; which is regarded to be a promising way of fixing CO2. In this review, we firstly summarize the prevalent CO2 capture technologies and compares their advantages and disadvantages. Furthermore, we elaborate the carbon metabolism mechanism of Isochrysis strain for the carbon fixing process, and explain the relationship among the Calvin cycle, TCA cycle, and the metabolism of various biologically active substances during photosynthetic carbon fixation. Based on the photosynthetic carbon-fixing mechanism of Isochrysis strain, we propose some methods about cultivation conditions, photobioreactors and modified microalgal cells by genetic engineering and synthetic biology to improve carbon-fixing efficiency for Isochrysis strain. Lastly, we illustrate the correlation between carbon fixation of Isochrysis strain and the synthesis of bioactive components, offering essential details for high valued Isochrysis-derived bioproducts by deep processes. This review will provide essential perspectives to address carbon neutrality by microalgal carbon sequestration and exploit high-value microalgal bioproducts by Isochrysis strain.

    Research Progress in the Production of Functional Fatty Acid DHA by Microalga Thraustochytrids
    ZHANG Mei-yu, ZHAO Yu-bin, WANG Ling-yun, SONG Yuan-da, ZHAO Xin-he, REN Xiao-jie
    2024, 40(6):  81-94.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0011
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    Docosahexaenoic acid(DHA), an essential polyunsaturated fatty acid in human body, plays a crucial role in maintaining cardiovascular health, combating cancer, and supporting vision and brain function. Traditional methods of extracting DHA from deep-sea fish oil suffer from strong fish odor and complex processing, driving researchers to explore more efficient and environmentally friendly alternatives. Thraustochytrids, with their rapid growth, low heavy metal contamination, and high DHA content, have emerged as potential microorganisms for industrial DHA production. However, several key issues persist in DHA production through Thraustochytrids fermentation, including enhancing fermentation production and reducing costs. This paper aims to comprehensively review the research status of DHA production through Thraustochytrids fermentation, covering strain screening and improvement, DHA biosynthesis pathways, genetic transformation, metabolic engineering, and fermentation control strategies. The paper first summarizes methods for natural screening and mutagenic improvement of wild-type strains to enhance DHA oil production in Thraustochytrids. It then delves into the research progress of DHA biosynthesis pathways in Thraustochytrids, emphasizing the role of key cofactors in DHA production. Additionally, it outlines the current application status of genetic transformation technology for introducing exogenous DNA into Thraustochytrids cells, offering valuable insights for enhancing genetic transformation efficiency and stability. Regarding DHA metabolism regulation, the paper discusses the promoting effect of nitrogen limitation on DHA synthesis and the impact of temperature and oxygen supply on production efficiency. Finally, it summarizes the main challenges and future development trends in leveraging Thraustochytrids for DHA production, aiming to facilitate their widespread application in pharmaceuticals, nutraceuticals, and food industries, and achieve efficient large-scale production.

    Research Progress of Cold Plasma Activated Solution in Antibacteria and Removing Biofilm
    CAI Zhi-cheng, WANG Yuan-yuan, SANG Xiao-han, ZENG Li-xian, DENG Wen-tao, WANG Jia-mei
    2024, 40(6):  95-104.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1199
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    Bacteria utilize quorum sensing(QS)to secrete signaling molecules and extracellular polymeric substances(EPS), enabling bacterial aggregation and adhesion to both biotic and abiotic surfaces, ultimately forming resilient and potentially harmful biofilms under certain conditions. Currently, the detrimental impact of biofilms in various fields such as medical, food, and agricultural industries is escalating. Particularly, certain pathogenic bacteria not only pose threats to human health but also incur significant socio-economic costs. Traditional disinfection and sterilization methods often struggle to entirely eradicate biofilms, while also presenting risks of secondary contamination. The strong adhesion of biofilms to different surfaces across various media constitutes a significant challenge in their removal. Therefore, immersion and cleaning with highly active solutions represent effective strategies for biofilm elimination. The reactive oxygen species(ROS)and reactive nitrogen species(RNS)activated in water by cold plasma technology disrupted bacterial cell walls and peptidoglycan structures, effectively inhibiting microbial adhesion and aggregation. As a potent antimicrobial solution, cold plasma-activated water has found extensive application in biomedical, food decontamination, and seed germination fields. However, the active agents in cold plasma-activated water are prone to dissipation, challenging long-term storage and leading to diminished antimicrobial efficacy. Recent advancements have seen the development of cold plasma-activated solutions, either by incorporating various media or combining other technologies, which effectively prolong the lifespan of active agents, marking a new direction in disinfection and antimicrobial research. This review delineates the mechanisms of antimicrobial action of cold plasma-activated water and solutions, with a focus on their efficacy in biofilm eradication, highlighting the biofilm formation process. It aims to provide a theoretical reference for biofilm removal and control in environmental and medical fields.

    Progress in the Development and Application of RNA Reference Materials
    WANG Dan, ZHAO Wen-jiang, QI Xin, LI Xin-ran, LI Kai, FAN Li, LI Liang
    2024, 40(6):  105-113.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1202
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    RNA reference materials is an important physical standard to ensure the accuracy and consistency of nucleic acid in vitro diagnostic data. It is widely used in the detection of viral diseases. It can also be used in calibration instruments, evaluation kits and proficiency testing, which purpose is to ensure the accuracy and consistency of the measurement process and measurement results. Since 1997, WHO has developed the first hepatitis C nucleic acid international reference material for nucleic acid amplification technology, which makes the results of different laboratories and different methods in the world comparable. By 2023, there are nearly 100 kinds of RNA certified reference materials released by China on the National Sharing Platform for Reference Materials. The research and application of RNA reference materials have been paid more and more attention by metrology institutions, relevant international academic organizations and enterprises all over the world. Especially after the outbreak of the novel coronavirus epidemic, the development of RNA reference materials has become a hot spot in the development of biological reference materials. With the continuous development of the reference materials development technology, the accuracy of the reference materials has also been improved, which provides a guarantee for the accuracy and consistency of the measurement results. The preparation methods, sub-package, fixed value technology, uncertainty evaluation and application fields of RNA reference materials are briefly reviewed, which provides a reference for the development of RNA reference materials and their future applications in various fields.

    Application of Transcription Factor-based Biosensors in Environmental Analysis
    ZHANG Di, JU Rui, LI Li-mei, WANG Yu-qian, CHEN Rui, WANG Xin-yi
    2024, 40(6):  114-125.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1184
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    Transcription factors are a class of proteins that regulate gene expression in cells by directing RNA polymerases to bind to specific DNA sequences. Transcription factors, as important regulatory proteins for prokaryotes to adapt to the natural environment, can specifically sense ligand molecules in environmental media. By coupling with different signal transduction and amplification systems, biosensing systems with transcription factors as recognition elements can construct flexible sensing strategies in whole-cell system, cell-free system and in vitro reaction systems. The transcription factor-based biosensors(TFBBs)have the advantages of high sensitivity, small size, low-cost, and on-site monitoring, showing great potential in the analysis of environmental pollutants. This paper introduces the reaction system types, core component compositions and working principle of TFBBs, as well as lists their application progress in the detection of environmental pollutants including heavy metal ions, aromatic compounds and antibiotics. On this basis, the opportunities and challenges for TFBBs in the detection of environmental pollutants are discussed, and the development trend of TFBBs is prospected. It points out that the rapid development of interdisciplinary emerging technologies such as synthetic biology and artificial intelligence will promote the artificial design and sensing performance improvement of TFBBs, making them applicable in a wider range of fields.

    Establishment and Application of Real-time PCR for Sugarcane Striate Virus
    WANG Chao-min, HE Mei-dan, WANG Wen-zhi, YUAN Qian-hua, ZHANG Shu-zhen, SHEN Lin-bo
    2024, 40(6):  126-133.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0129
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    【Objective】 It is to establish a rapid, sensitive and specific SYBR Green I quantitative PCR method for the detection of sugarcane striate virus(SStrV).【Method】 A specific amplification primer was designed from the conserved sequence of the SStrV genome sequence, the recombinant plasmid pMD19-T-SStrV-qN containing SStrV gene was constructed as a positive plasmid standard. Using it as template, the SStrV fluorescence quantitative PCR assay was established. And the sensitivity, specificity, stability, and subsequently of this method were tested, then the SStrV loads in different tissue sites of sugarcane were detected.【Result】 The recombinant plasmid containing SStrV genome sequence was diluted into a standard at a 10-fold ratio, and they were used as a template for real-time PCR, the standard curve y = -3.337 x + 38.197 was obtained, and the correlation coefficient r2 = 0.999 was obtained, indicating that the Cq value was linearly related to the logarithm of the copy number of the standard concentration. With this established real-time PCR, the least detection limit was 13 copies of the recombinant plasmid/μL, which was 100 times more sensitive than ordinary PCR. The method specifically detected SStrV with high specificity, and the coefficient of variation within and between groups was n 0.13%-0.94%, indicating that the method was of good repeatability. The load of SStrV was significantly different among different tissue sites of the sugarcane, and the load of SStrV was the highest in +4 leaves, which was significantly different from other tissue sites.【Conclusion】 The SYBR Green I quantitative PCR method is established to provide an efficient quantitative detection method for the diagnosis of SStrV, and it is determined that +4 leaves are the best sampling site for the detection of SStrV in sugarcane.

    Transcriptome Analysis of Fertility Transformation in Weakly Restoring Hybrid F1 of Soybean Cytoplasmic Male Sterility
    BAI Zhi-yuan, XU Fei, YANG Wu, WANG Ming-gui, YANG Yu-hua, ZHANG Hai-ping, ZHANG Rui-jun
    2024, 40(6):  134-142.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0066
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    【Objective】 It is to explore the molecular mechanism of fertility transformation in weakly restoring hybrid F1 of soybean cytoplasmic male sterility at the RNA level, then to provide valuable information on the molecular mechanism of male sterility in soybean from the perspective of fertility transition.【Method】 Using weakly restoring hybrid F1(H3A× SXTH3)as the research object, and short light(plant fertility is sterile)and normal light(plant fertility is fertile)treatments were set up during the seedling stage. Mixed flower buds of different sizes were collected during the peak flowering period for transcriptome sequencing and quantitative real time PCR(RT-qPCR)analysis.【Result】 Total 3 917 differentially expressed genes were screened out. After short light treatment during the seedling stage, 2 134 genes were downregulated and 1 783 genes were upregulated. Bioinformatics analysis was conducted on differentially expressed genes, and gene ontology(GO)significant enrichment analysis showed that carbohydrate metabolic process, transmembrane transporter activity, and cell periphery functions played the main biological functions during fertility transition. The significant enrichment analysis of the kyoto encyclopedia of genes and genomes(KEGG)pathway showed that the main metabolic pathways for fertility transformation were pentose and glucuronate interconversions, starch and sucrose metabolism, and plant circadian rhythm. RT-qPCR analysis of 11 genes revealed that genes related to soybean cytoplasmic male sterility and pentatricopeptide repeats protein(PPR)were involved during fertility transformation of weakly restoring cytoplasmic male sterile hybrid F1 in soybean.【Conclusion】 It was speculated that the fertility transformation of weakly restoring hybrid F1 was related to abnormal expressions of genes related to plant circadian rhythm, PPR, and soybean cytoplasmic male sterility, such as mitochondrial, pollen wall development, carbohydrate metabolism, sugar transport, and reactive oxygen species metabolism. Key genes in the circadian rhythm pathway change cause changes in the expression levels of PPR genes and soybean cytoplasmic male sterility related genes, then fertility transformation will occur.

    Barrier Effect of Zinc and Selenium Combined Application on Mercury Accumulation in Rice
    HUANG Qiu, LIU Jing, QIN Fan-xin, LUO Bang-lin, LUO Lin, LI Wan-yu, XU An-qi
    2024, 40(6):  143-151.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0064
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    【Objective】 To study the effects of combined application of zinc and selenium on the accumulation and transport of mercury in rice in a soil-rice system, and to investigate the barrier effect of combined zinc and selenium application on mercury in rice, and provide a reference for the safe utilization of mercury-contaminated soil. 【Method】 Selecting mercury-contaminated soil in the mining area of Bijiang District, Tongren, Guizhou, having the rice variety Chuankangyou 727 as the research subject, and using pot experiment, we measured the growth and development characteristics of the rice plant, as well as the available mercury in the soil and the mercury content in various organs of rice.【Result】 The simultaneous application of zinc and selenium reduced the available mercury content in the soil by 45% to 75.2%. Alternatively, when low concentrations of selenium and zinc were applied together, the available mercury content in the soil reduced by 75.2%. The application of zinc and selenium in combination resulted in a reduction of mercury levels in different organs of rice plant by 15.2% to 92.3%. The most effective reduction effect was in the mercury content of the grain, with a decrease of 87.6% to 92.3%. Except for the high concentration of selenium and the combination of high and low concentrations of zinc, which promoted the transportation of mercury from the roots to the grains in rice plant, the blocking effect of a zinc-selenium combination on the accumulation and transportation of mercury in the rice plant was better than that of zinc or selenium applied individually. The combination of zinc and selenium reduced the enrichment coefficient of mercury from roots to grains in rice by 88% to 92%. Similarly, when low concentration selenium was combined with both high and low concentrations of zinc, the transportation coefficient of mercury from the roots to the grains in rice plant reduced by 69.2% and 61.5% respectively.【Conclusion】 The combined application of zinc and selenium has a significant blocking effect on mercury in rice, effectively alleviating the toxic effects of mercury on rice growth and development.

    Cloning and Expression Analysis of BrCYP83B1 Gene in Chinese Cabbage
    WANG Yu-shu, ZHAO Lin-lin, ZHAO Shuang, HU Qi, BAI Hui-xia, WANG Huan, CAO Ye-ping, FAN Zhen-yu
    2024, 40(6):  152-160.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1127
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    【Objective】 The cytochrome P450 family is an important enzyme system involved in glucosinolate synthesis in cruciferous plants, where in the CYP83 subfamily plays a predominant role in core structure formation, this work aims to investigate the Chinese cabbage (Brassica rapa ssp. Pekinensis) CYP83B1 gene functional role.【Method】 RT-PCR was used to clone the CYP83B1 gene. Bioinformatics analysis software was utilized to predict the encoded protein's physicochemical properties, homology of the encoded protein, and promoter cis-acting elements. The expression pattern of BrCYP83B1 was analyzed by RT-qPCR, and a plant overexpression vector was constructed for further experimentation. 【Result】 The cDNA length of BrCYP83B1 was 1 500 bp, encoding a total of 499 amino acids. The protein belonged to cytochrome P450 superfamily and predominantly located in the cytoplasm. Its secondary structure primarily comprised of α-helixes and irregular coil. Homologous comparison illustrated that BrCYP83B1 had close relationship with Brassica napus L and Brassica oleracea L. var. italica. The BrCYP83B1 promoter contained cis-acting elements that were involved in the response to salicylic acid(SA), abscisic acid(ABA), and methyl jasmonate(MeJA), suggesting that the expression of BrCYP83B1 gene may be regulated by hormones. The expression of BrCYP83B1 was detected in various plant organs, including the roots, stems, leaves, flowers and fruits from RT-qPCR results. Notably, the highest expression was observed in the leaves. Moreover, BrCYP83B1 significantly presented induction upon treatment with MeJA, while its expression was repressed by SA. Additionally, ABA treatment initially up-regulated and subsequently down-regulated the gene. 【Conclusion】 BrCYP83B1 may be involved in the response regulation of Chinese cabbage to hormones.

    Identification and Induced-expression Analysis of REVEILLE Family in Brassica napus L.
    LIU Rong, TIAN Min-yu, LI Guang-ze, TAN Cheng-fang, RUAN Ying, LIU Chun-lin
    2024, 40(6):  161-171.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1153
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    【Objective】 REVEILLE family genes have important biological functions, but there are few studies on this family gene in Brassica napus L. This project was conducted to further understand the function of RVE family genes in B. napus and their relationship with abiotic stress. 【Method】 Bioinformatics methods were used to identify33, 16 and 16 RVE genes in B. napus, B. rapa and B. oleracea, and to analyze their phylogenetic development, chromosome localization, gene structure, physical and chemical properties, and promoter cis-acting elements. 【Result】 All RVE proteins were classified into two subfamilies. The 33 BnaRVE were located on 18 chromosomes of the A sub-genome and 15 chromosomes of the C sub-genome. Most members belonged to relatively stable hydrophobic proteins. Most of the Ka/Ks values were < 1, indicating that the family was subjected to strong purification selection. The gene structure varied greatly, and the number of introns ranged from 4 to 11. Collinearity analysis showed that there were large number of homologous genes in B. napus and Arabidopsis, B. rapa and B. oleracea. Most RVE family members of B. napus expressed RVE in the cotyledon and leaves, and the expression was the highest. BnaRVE promoter contained a large number of cis-acting elements related to hormone and biological stress. Through quantitative PCR analysis, the expressions of 4 BnaRVE genes were significantly upregulated under ABA and MeJA induction as well as low temperature stress. 【Conclusion】 Thirty-three members of the BnaRVE family were identified in the genome of B. napus. Different genes showed different expression patterns in different developmental stages and different tissues. Different genes have different expression patterns in response to various stresses. RVE family members have positive response function to ABA, MeJA and cold stress.

    Effects of Different Treatments on the Nutrients, Fiber Structure, and Glucosinolate Contents in Rapeseed Straw
    YANG Dai-yi, FAN Yang, TU Yan, XU Zhi-yu, XUE Ying-hao, SUN Yuan-feng, WANG Jin, HAO Xiao-yan, MA Tao
    2024, 40(6):  172-179.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0077
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    【Objective】 Physical(extrusion)and microbial(Aspergillus niger and Bacillus subtilis)treatments were applied to analyze the changes in the nutrient content, fiber structure and glucosinolates of the rapeseed straw after fermentation, aiming to improve the feeding value of rape straw as roughage for ruminants.【Method】 The experiment included 6 treatments: Rape straw group(M group, that is, control group), control plus Aspergillus niger group(MA group), control plus Bacillus subtilis group(MB group), control puffed group(PM group), control puffed plus A. niger group(PMA group), and control puffed plus B. subtilis group(PMB group).【Result】 Compared with the M group(2.60%), the crude protein content significantly increased 3.16%(MA group), 3.24%(MB group), 3.31%(PM group), 4.02%(PMA group), 3.73%(PMB group)and the NDF content significantly reduced in other groups, 81.6%(MA group), 80.3%(MB group), 80.4%(PM group), 77.3%(PMA group)and 78.3%(PMB group)(P<0.05). The crystallinity(27.0% vs 25.3%)and specific surface area(1.22 m2/g vs 1.19 m2/g)in MA group was higher that than in M group(P<0.05), the hydrogen bonding force was weakened, and there was no significant difference in the degree of polymerization in MA compared with that in the M group(P>0.05). Compared with the M group(0.49 μmol/g), the glucosinolate content of the other groups was significantly lower(P<0.05). The difference in the glucosinolate contents in PMA(0.24 μmol/g)and PMBs(0.22 μmol/g)groups was not significant(P<0.05).【Conclusion】 The use of microbial and physical treatments of rape straw can reduce the NDF level by breaking fiber structure and reducing the content of glucosinolates, thus improving its feeding value in ruminants.

    Phylogenetic Evolution and Expression Analysis of SPL Gene Family in Juglans regia
    ALIYA Waili, CHEN Yong-kun, KELAREMU Kelimujiang, WANG Bao-qing, CHEN Ling-na
    2024, 40(6):  180-189.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1171
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    【Objective】 The SQUAMOSA promoter binding protein like(SPL)transcription factor is a crucial regulatory element in the induction of plant flowering. This study aims to identify the members of the SPLJrSPL)gene family within the walnut genome and analyze their expression characteristics. The findings of this study will serve as a valuable reference for further investigations into the functional aspects of SPL in walnut flowering induction. 【Method】 The present study employed bioinformatics techniques to identify members of the JrSPL gene family, subsequently to predict their fundamental physicochemical properties, conserved domains, evolutionary relationships, and cis acting elements of promoters. Additionally, transcriptome sequencing and RT-qPCR technology were employed to investigate the expressions of JrSPL family members in various tissues of walnut, as well as following grafting-induced flowering. 【Result】 The JrSPL family consisted of 28 members, showing significant conservation in their gene and protein structures, which were dispersed across 14 distinct chromosomes within the walnut genome. Notably, there were 17 collinear relationships with Arabidopsis and 24 with Populus tomentosa, respectively. Through phylogenetic analysis, these members were categorized into 8 distinct groups. Additionally, the JrSPL promoter region encompassed a substantial quantity of light responsive elements, hormone responsive elements, stress responsive elements, and other similar regulatory motifs. The transcriptome analysis revealed that the JrSPL genes demonstrated varying expressions across different tissues of walnut, including male flowers, female flowers, apical buds, and leaves. Notably, the expressions of most genes were relatively high in female flowers, with 6 genes having significant expression, suggesting their potential involvement in the induction of flowering. In the context of grafting-induced walnut flowering materials, with the except of JrSPL8, the expressions of 12 JrSPLs observed in the flowering materials were found to be greater than those in the control group. Specifically, JrSPL2 and JrSPL25 showed significant expressions in the male flowers of mixed inflorescences, whereas JrSPL8 had high expression in the apical buds. 【Conclusion】 The JrSPL gene exerts a significant influence on the process of walnut flowering, with its high expression serving as the primary determinant of early flowering.

    Evolution, Expression, and Functional Analysis of the HSP20 Gene Family from Simmondisa chinensis
    LI Bo-jing, ZHENG La-mei, WU Wu-yun, GAO Fei, ZHOU Yi-jun
    2024, 40(6):  190-202.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0012
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    【Objective】 Clarifying the structure and function of members of the jojoba HSP20 family(ScHSP20)will provide support for subsequent study.【Method】 A systematic analysis was conducted on the ScHSP20 family members in jojoba from structural, phylogenetic, and expression perspectives, and functional analysis of ScHSP20-17 was performed.【Result】 The genome of jojoba encompassed 35 HSP20 gene loci, with 8 genes involved in tandem duplications and 5 in segmental duplications. Phylogenetic analysis showed that 35 ScHSP20 clusters into 12 subfamilies, with the highest number of nuclear cytoplasmic subfamilies. Promoter analysis revealed that the majority of ScHSP20 family members possessed cis-acting elements in their promoter regions associated with the responses to hormone and abiotic stress. Transcriptome analysis indicated that the expression levels of some ScHSP20 genes, such as ScHSP20-35 and ScHSP20-17, underwent significant changes during flower and seed development, and genes like ScHSP20-17 and ScHSP20-28 were induced by high temperatures. Subcellular localization analysis showed that the protein encoded by ScHSP20-17 was located in the nucleus, and the overexpression of ScHSP20-17 in Escherichia coli, Saccharomyces cerevisiae, and Nicotiana benthamiana markedly improved their tolerance to high temperature stress.【Conclusion】 Some members of the ScHSP20 family are involved in the reproductive development and response of jojoba to abiotic stress.

    Genome-wide Identification and Expression Analysis of bHLH Transcription Factor Family in Dendrobium nobile
    WANG Jian, YANG Sha, SUN Qing-wen, CHEN Hong-yu, YANG Tao, HUANG Yuan
    2024, 40(6):  203-218.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0907
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    【Objective】 bHLH transcription factors exist widely in plants and are one of the important transcription factor families that regulate plant growth and development and respond to stress. The DnbHLHs transcription factor family was identified from the whole genome of Dendrobium nobile and its expression characteristics were analyzed, which laid the foundation for further study on the biological function of DnbHLHs transcription factor. 【Method】 The bHLH transcription factor family members were identified from the whole genome of D. nobile by bioinformatics method, and their physical and chemical characteristics, phylogenetic relationship, gene structure, chromosome localization and promoter cis-acting elements were analyzed. Transcriptome data and RT-qPCR were used to analyze the tissue expression patterns of DnbHLHs transcription factors and their expression patterns under different abiotic stresses. 【Result】 A total of 137 bHLH transcription factors were identified in D. nobile. The amino acid number of DnbHLHs protein was 85-1 260, the molecular weight was 9 855.23-140 024.19 Da, the isoelectric point was 4.52-10.66, the instability coefficient was 31.94-87.42, the fat solubility index was 56.66-106.06, and the hydrophilic index was -0.815-0.185. DnbHLHs were distributed on 19 chromosomes of D. nobile, all of which had a bHLH domain and were homologous to the bHLH of Arabidopsis thaliana, and there are collinear relationships between groups and species. Analysis of gene expression patterns showed that DnbHLHs family members had tissue specificity, and DnbHLH26 and DnbHLH33 were responsive to various stresses. 【Conclusion】 A total of 137 DnbHLHs transcription factor family members are identified from the whole genome of D. nobile. The physicochemical properties and conserved mods of the family members showe specificity and diversity. There are tissue expression differences among different DnbHLHs transcription factors, and DnbHLH26 is mainly induced by high salt and high temperature stress. DnbHLH33 is mainly induced by high salt, high temperature and low temperature stress.

    Genome-wide Identification and Expression Pattern Analysis of the Bcl-2-related Anti-apoptotic Family in Cucumis sativus L. and Cucurbita moschata Duch.
    HU Yong-bo, LEI Yu-tian, YANG Yong-sen, CHEN Xin, LIN Huang-fang, LIN Bi-ying, LIU Shuang, BI Ge, SHEN Bao-ying
    2024, 40(6):  219-237.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1195
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    【Objective】 In order to provide favorable genes for the analysis of the grafting healing mechanism of Cucumis sativus L. or Cucurbita moschata Duch. grafted seedlings and the molecular breeding of resistance of the cucumber and pumpkin vegetables, we conducted the analysis of the expression patterns of Bcl-2 associated athanogene(BAG)family proteins in the cucumber and pumpkin in response to abiotic stress and in response to light during cucumber or pumpkin grafting healing. 【Method】 Based on the genome information of the cucumber and pumpkin, the BAG gene family in the cucumber and pumpkin was identified through bioinformatics methods, and its physicochemical characteristics, chromosome mapping, gene structure, phylogeny and collinearity were analyzed. The expression patterns of BAG family proteins in the cucumber and pumpkin in response to abiotic stress and in response to light during cucumber or pumpkin grafting healing were analyzed. These data were based on the public database and the transcriptome sequencing data of the cucumber or pumpkin grafted seedlings in the process of grafting and healing. 【Result】 A total of 12 and 18 members of the BAG family were identified in the cucumber and pumpkin, respectively and they were all divided into two subfamilies and the gene members were highly conserved. The BAG of subfamily I was mainly involved in gene regulation and stress response, while the BAG of subfamily II was mainly involved in plant development. The BAG genes of cucumber and pumpkin had multiple linear relationships with Arabidopsis, Oryza sativa L. and Solanum lycopersicum L., respectively, but the CsaV3_1G017210 was relatively conservative and there was no collinearity with the BAG genes in Arabidopsis, rice, tomato and pumpkin. Different BAG genes possessed tissue-specific expression patterns. CsaV3_6G000970 and CmoCh08G008520 belonged to the BAG family molecular chaperone regulator 6. In both cucumber and pumpkin, they were upregulated during the grafting healing process and in response to abiotic stress. 【Conclusion】 BAG family genes respond differently to abiotic stress and light during the grafting healing process of the cucumber or pumpkin. These genes synergistically regulate the growth, development and grafting healing of the cucumber and pumpkin. BAGs play an important role in the process of the cucumber and pumpkin abiotic stress and cucumber or pumpkin grafting healing.

    Identification and Analysis of E2 Gene Family in Pepper(Capsicum annuum L.)
    CHANG Xue-rui, WANG Tian-tian, WANG Jing
    2024, 40(6):  238-250.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0044
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    【Objective】 Ubiquitin-binding enzyme E2(UBC)is an important transit enzyme in the ubiquitin-proteasomal pathway that transfers ubiquitin to target proteins, which is involved in plant growth and development and many stresses. The E2 gene family members were identified from the whole genome of pepper, and the expression patterns of related genes were analyzed, which provided a theoretical basis for subsequent research.【Method】 Based on the genome-wide information of pepper, the E2 family was identified by bioinformatics. The basic physio-chemical properties, subcellular localization, chromosomal localization, gene structure and phylogenetic analysis, cis-acting elements, abiotic stress response, tissue and organ expression patterns, and protein-protein interaction network of the family members were systematically analyzed. 【Result】 There were 48 members in the E2 gene family, which were unevenly distributed on 11 chromosomes, and its encoded proteins were mainly localized in the extracellular and nucleus. After comparative evolutionary analysis of pepper, tomato and Arabidopsis thaliana, they were divided into the Group I-Group XIII subfamily, which had similar conserved protein motifs and gene structures. A total of 26 proteins were found to interact. The cis-element analysis showed that the promoter region of CaUBC gene contained a large number of light-responsive and hormone-responsive elements, and there were a variety of cis-acting elements related to growth, development and stress resistance in the upstream 2 000 bp region. CaUBC was involved in the stress, under different abiotic stresses and hormone treatments, they were divided into three categories, among which the third type of genes were highly expressed in high temperature environment; different expressions showed in tissues and organs, among which the expression of CaUBC38 in fruits changed significantly. RT-qPCR analysis showed that the relative expression was the highest at 50 d and the lowest at 10 d. 【Conclusion】 The 48 members of E2CaUBC)gene family are systematically identified from the pepper genome. The expression characteristics of CaUBC family members during its growth, development, and abiotic stress responses are revealed.

    Cloning and Functional Analysis of Flavonoid 3'-hydroxylase(F3'H)Gene in Rhododendron hybridum Hort
    WU Ze-hang, YANG Zhong-yi, YAN Yi-cheng, JIA Yong-hong, WU Yue-yan, XIE Xiao-hong
    2024, 40(6):  251-259.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0034
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    【Objective】 The flavonoid 3'-hydroxylase(F3'H)is a key enzyme in the synthesis of anthocyanins in plants, and the function and expression characteristics of F3'H gene in Rhododendron hybridum Hort were investigated.【Method】 The petals and roots, stems and leaves of R. hybridum Hort at different developmental stages were used as experimental materials. The flavonoid 3'-hydroxylase gene sequence information of flavonoid biosynthesis pathway was screened from the R. hybridum Hort transcript database and bioinformatics analysis was performed, and the RhF3'H gene was cloned by reverse transcription(RT-PCR)technology. The anthocyanin content in the petals of R. hybridum Hort at different stages was determined by ultraviolet spectrophotometer, and the expression of RhF3'H gene was analyzed by RT-qPCR technology on different tissues of petals at different development stages and maturity stages. The overexpression vector 35S:RhF3'H-GFP recombinant vector was constructed by Gateway technology for subcellular localization verification, and p1302-RhF3'H overexpression vector was constructed to infect the petals of R. hybridum Hort.【Result】 The RhF3'H gene was 1 557 bp in length and encoded 518 amino acids, and the protein encoded by RhF3'H belonged to the P450 superfamily with a conserved F3'H domain. Phylogenetic tree analysis showed that RhF3'H was the most closely related to Dimocarpus longan and Litchi chinensis F3'H proteins. The results of RT-qPCR showed that RhF3'H was expressed in different petal stages and roots, stems, and leaf tissues of R. hybridum Hort. The expressions of RhF3'H gene were higher in the blooming and decaying stages of different petal development stages, which was consistent with the anthocyanin content, and subcellular localization analysis showed that RhF3'H mainly existed on the cell membrane. Compared with CK and p1302, p1302-RhF3'H was significantly higher in the petals of R. hybridum Hort and its anthocyanin content significantly increased.【Conclusion】 The RhF3'H gene was expressed in the petal cell membrane, and the expression pattern was consistent with the accumulation trend of anthocyanins. Overexpression of the RhF3'H gene promotes the synthesis of anthocyanins.

    Metabolome Difference Analysis of Fermented Cigar Tobacco Leaves in Summer and Winter
    YU Xin-lei, HE Jie-wang, LIN Guo-ping, LI Jin-hai, WANG Da-ai, YUAN Yue-bin, LIU Sheng-gao, LI Zhi-hao, TAO De-xin
    2024, 40(6):  260-270.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1101
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    【Objective】 The differences of metabolites in cigar tobacco fermented in summer and winter were analyzed. 【Method】 Widely targeted metabolomics detection(UPLC-ESI-MS/MS)was used in combination with multivariate statistical analysis(PCA and OPLS-DA). 【Result】 During the fermentation period(lasting for 34 d), the average temperature of the fermentation room in summer and winter were 27.2℃ and 14.7℃, respectively, with a difference of 12.5℃, and the average humidity was 77.6% and 66.7%, respectively, with a difference of 10.9%, thus summer was more in line with the environmental requirements of relatively high temperature and high humidity for tobacco fermentation. A total of 905 metabolites were detected in cigar leaf samples, including alkaloids, phenolic acids, flavonoids, lipids, amino acids, organic acids, terpenes, sugars, nucleotides, and lignin. The top three metabolites were alkaloids, organic acids, and lipids, accounting for 24.5%, 23.7%, and 15.0%, respectively. After the fermentation, the contents of organic acids, lipids, amino acids and phenolic acids significantly increased, and the contents of alkaloids, terpenes, flavonoids, sugars and lignin decreased to varying degrees. In summer fermented tobacco leaves, the metabolic transformation of alkaloids and terpenoids was more intense, the contents of organic acids, amino acids, flavonoids, lipids and sugars were higher, the chemical composition was more coordinated, the aroma quality was richer, the flue gas was soft, the aftertaste was sweet, and the sensory quality was better.【Conclusion】 The natural high temperature and high humidity environment in summer in southwest Hubei is more suitable for the ideal state of cigar tobacco agricultural fermentation. The cigar leaves produced in the current year should be naturally aged in winter and then fermented in the next summer, which may be used as a reference scheme to explore and promote the quality and efficiency of the fermentation process of cigar.

    Effects of Acid Mine Drainage on Physicochemical Factors and Nitrogen-fixing Microorganisms in the Root Zone of Mature Rice
    TIAN Sheng-ni, ZHANG Qin, DONG Yu-fei, DING Zhou, YE Ai-hua, ZHANG Ming-zhu
    2024, 40(6):  271-280.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1146
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    【Objective】 Acid mine drainage(AMD)is a kind of wastewater with low pH, high sulfate concentration and heavy metal enrichment. This study is aimed to investigate the abundance and composition of azotobacter community in the root zone of rice at mature stage and its response to AMD, and to elucidate the main driving factors of the change of soil azotobacter community structure. 【Method】 Rice pot experiment was carried out on paddy soil contaminated and uncontaminated by AMD from Tongling mining area of Anhui province, and three different treatments were set up(A: AMD irrigated contaminated soil; B: clean water irrigated contaminated soil; CK: clean water irrigated uncontaminated soil), nifH gene high-throughput sequencing technology was used to analyze the characteristics of azotobacter community in rice root zone soil under different treatments. 【Result】 The contents of SO42-, NO3- and heavy metals in rice root zone soil increased significantly, the soil acidified, and the diversity of azotobacter community decreased. The dominant nitrogen-fixing bacteria in rice root zone soil included Anaeromyxobacter, Geobacter, etc. The number of nitrogen-fixing bacteria enriched in CK treatment was significantly higher than that in A and B treatments, and Verrucomicrobia were mainly enriched in B treatment, while Proteobacteria were mainly enriched in CK treatment. pH、Cu、Pb and Zn were the main factors driving the community structure of azotobacter in rice root zone. Desulfovibrio and Desulfurivibrio with sulfur reduction function have significantly contributed to the change in nitrogen. 【Conclusion】 AMD had significant effects on soil chemistry and azotobacter in the root zone of rice, and restoring clean water irrigation promoted the recovery of azotobacter. The above findings provide new insights and ideas for biological remediation of AMD-contaminated soil.

    Construction of cDNA Library of Setosphaeria turcica and Screening of Transcription Factor StMR1 Interacting Proteins
    WANG Qiu-yue, DUAN Peng-liang, LI Hai-xiao, LIU Ning, CAO Zhi-yan, DONG Jin-gao
    2024, 40(6):  281-289.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0121
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    【Objective】 To screen the interaction proteins of transcription factors of Setosphaeria turcica, and to analyze the molecular mechanism of melanin-regulated transcription factor StMR1 in regulating the pathogenicity of S. turcica. This study provides a reference for elucidating the regulatory network of transcription factors in the infection process of S. turcica and elucidating the pathogenic mechanism of the pathogen.【Method】 Different germination stages of hyphae and spores of S. turcica were collected as test materials, and the cDNA library of S. turcica was constructed by gateway method, The bait vector of transcription factor StMR1 was constructed by homologous recombination, and its interacting proteins were screened by yeast two-hybrid technology and verified one-to-one.【Result】 The average fragment length of the constructed library was greater than 1 000 bp, the library capacity of the primary library and the secondary library were 1.2×107 and 1.04×107 CFU, respectively, and the recombination rate was 100%, which could be used for yeast two-hybrid screening. The bait vector pGBKT7-StMR1 that could be used for screen library was successfully constructed, and three interacting proteins were obtained through primary screening and re-screening, and one-to-one verification of the interaction between short-chain dehydrogenase, glycosyltransferase and leucine-rich repeat protein and transcription factor StMR1 were verified.【Conclusion】 A high quality and abundant cDNA library was successfully constructed and the protein interacting with StMR1 was screened

    Heterologous Biosynthesis of Cordycepin in Aspergillus oryzae
    YAN Huan-huan, SHANG Yi-tong, WANG Li-hong, TIAN Xue-qin, LIAO Hai-yan, ZENG Bin, HU Zhi-hong
    2024, 40(6):  290-298.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0006
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    【Objective】 This study aims to synthesize cordycepin by constructing engineered Aspergillus oryzae strains that heterologously express key genes for cordycepin synthesis. 【Method】 The uidine/uracil and histidine diauxotrophic strain of Aspergillus oryzae AoΔpyrGΔHisB served as the background, and overexpressed the key gene CmCns1-CmCns3 for cordycepin synthesis using the Agrobacterium-mediated transformation technique. The subcellular localization of CmCns1 and CmCns2 within A. oryzae was visualized through fluorescence microscopy. High-performance liquid chromatography(HPLC)was used to determine the content of cordycepin in transgenic A. oryzae. Additionally, glycine and adenine, both precursors in the synthesis of cordycepin, were introduced into the fermentation broth to investigate their impact on cordycepin synthesis in A. oryzae. 【Result】 The CmCns1 and CmCns2 from Cordyceps militaris were localized in lipid droplets in A. oryzae. Cordycepin synthesis achieved through the overexpression of CmCns1 alone, the co-expression of CmCns1 and CmCns2, and the co-expression of CmCns1-CmCns3 in A. oryzae, and the highest extracellular production of cordycepin reached 37.74 μg/mL after 48 h of fermentation. The addition of glycine and adenine to the fermentation broth of A. oryzae could not effectively increase the content of cordycepin.【Conclusion】 This study successfully achieved heterologous expression of synthetic cordycepin genes in A. oryzae.

    Identification of a Streptomyces Strain and Study on the Fermentation Process of Geldanamycin Production
    YANG Lu, YUAN Yuan, FANG Zhi-kai, LIN Ru, JIANG Hong, ZHOU Jian
    2024, 40(6):  299-309.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1215
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    【Objective】 Streptomyces sp. FIM18-0592 was identified and its fermentation process was optimized for extracellular geldanamycin yield with the aim of increasing the yield and reducing the fermentation cost.【Method】 Strain identification was carried out by morphological features, culture characteristics and physiological and biochemical properties, combined with 16S rDNA sequence analysis to construct a phylogenetic tree; a one-factor test was used to optimize the culture conditions and media formulations, and the contents of the media formulations were further optimized by the steepest-climbing test and the response surface test. 【Result】 Through preliminary culture observation of the morphological characteristics, culture characteristics and physiological and biochemical characteristics of Streptomyces geldanamycininus FIM18-0592, we found that it grew well on ISP2 and other media, with vigorous aerial mycelium and melanin production. Combined with 16S rDNA molecular identification, the Streptomyces sp. was identified as Streptomyces geldanamycininus. The fermentation conditions and medium formulation were optimized by one-factor test, and the most optimal culture conditions for the fermentation of the strain were speed of 140 r/min, liquid content of 12%, inoculum concentration of 7%(V/V), and fermentation time of 144 h. The optimal carbon source, nitrogen source, and mineral salt were glucose, soybean cake powder and NH4HPO4, respectively. The optimal fermentation medium was determined by steepest-climbing test and response surface optimization test as follows: Glucose 10.42%, soybean cake powder 1.68%,(NH4)2SO4 0.3%, lactic acid 0.3%, glycerol 4%, magnesium sulphate 0.1%, and calcium carbonate 0.4%. Under these conditions, the fermentation titer of geldanamycin reached 2 887 μg/mL, which was 66% higher than the original fermentation process. 【Conclusion】 Streptomyces sp. FIM18-0592 belongs to Streptomycete geldanamycininus, whose fermentation process is optimized to significantly increase the yield of geldanamycin, laying the foundation for the development and utilization of geldanamycin and its derivatives.

    Expression of Lithocarols Biosynthesis Gene litI and Functional Analysis of Its Promoter
    LI Meng-ran, YE Wei, LI Sai-ni, ZHANG Wei-yang, LI Jian-jun, ZHANG Wei-min
    2024, 40(6):  310-318.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1225
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    【Objective】 Lithocarols are a novel class of polyisoprenyl diphenylketone compounds, which have a good antitumor activity. The lithocarols biosynthesis gene litI was cloned, expressed and purified, the promoter of the gene litI was functionally identified, which will lay a molecular foundation for the biosynthesis and transcriptional regulation of lithocarols. 【Method】 The biosynthetic gene litI was cloned and expressed in Escherichia coli, and the expressed protein LitI was preliminarily purified by nickel affinity chromatography column. The properties and structure of the protein were analyzed by bioinformatics method. The litI gene promoter fragment was amplified, the luciferase expression system was constructed to analyze the transcriptional activity of the promoter, and its functional components were predicted by the PlantCARE promoter analysis website. 【Result】 LitI protein is a hydrophilic protein, which has a relative molecular weight of 51 kD and a secondary structure including 51.32% α-helix, 8.99% extended chain, 3.95% β-angle, and 35.75% irregular curling. The gene litI promoter shows strong transcription activity, and it could initiate the transcription of ampicillin resistance gene in E. coli, and litI promoter contained TATA box and CAAT box. 【Conclusion】 LitI protein is obtained by heterologous expression, its properties and structure are analyzed, and the promoter fragment of litI gene with strong transcription activity is identified.

    Screening of CTX-M-14-type Ultra-broad-spectrum β-lactamase Inhibitors Based on Pharmacophore Modelling
    WANG Meng-fan, ZHAO Zi-yu, WANG Chun-guang, LIU Ting-yu, CHEN Xi, ZHANG Tie
    2024, 40(6):  319-329.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1228
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    【Objective】 Development of inhibitors against cefotaxime-hydrolase-14(CTX-M-14)-type ultra-broad-spectrum β-lactamases(ESBLs)to mitigate the serious harm caused by bacterial drug resistance. 【Method】 DiscoveryStudio Visualizer(DS Visualizer)was used to construct a receptor structure-based pharmacophore(SBP)and a ligand common feature-based qualitative pharmacophore model(HIPHOP), and the validated models were used as query conditions for virtual screening of the ZINC database targeting CTX-M-14 protein, and glycyrrhizic acid(GL), a monomer component of traditional Chinese medicine(TCM)with good fitting scores, was obtained. The related force analysis, molecular dynamics simulation and binding free energy calculation were carried out to analyse the binding mode, binding capacity and stability of glycyrrhizic acid and CTX-M-14 protein. Finally, the antibacterial sensitizing activity, enzyme inhibition and enzyme inhibition of glycyrrhizic acid were investigated by the combined bacterial inhibition assay and enzyme kinetic assay. 【Result】 Glycyrrhizic acid, a single component of traditional Chinese medicine, mainly formed hydrogen bonds and van der Waals forces with multiple amino acid residues in the active centre of CTX-M-14 protein, and the docking fraction and binding free energy of the two were -10 kcal/mol and -22.06 kcal/mol, respectively. Glycyrrhizic acid synergistically interacted with cefotaxime sodium(FICI ≤ 0.5); glycyrrhizic acid competitively inhibited the hydrolysis of the substrate antibiotic by β-lactamase, and the enzyme inhibition and protection rate of cefotaxime sodium reached 58.53%, which was close to that of clavulanic acid(60.98%). 【Conclusion】 The Chinese medicine monomer glycyrrhizic acid can bind stably to CTX-M-14 protein and increase the sensitivity of multi-drug-resistant Escherichia coli E320 and recombinant protein-positive bacterium BL-21 to cefotaxime sodium by competitively inhibiting the hydrolysis of the substrate antibiotic by β-lactamase, so as to achieve a reduction in the quantity and increase in the effectiveness of the antibiotic.

    Change of Single-cell Transcription Profile and Analysis of Intercellular Communication in Myogenic Cell Differentiation
    QIN Jian, LI Zhen-yue, HE Lang, LI Jun-ling, ZHANG Hao, DU Rong
    2024, 40(6):  330-342.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1005
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    【Objective】 Based on single-cell RNA sequencing(scRNA-seq), the changes of gene expression profile during bovine myogenic cell differentiation were revealed, and the mechanisms of ligand-receptor interaction mediating intercellular communication were explored, which lays the foundation for the construction of dynamic regulatory network in myogenic differentiation.【Method】 The scRNA-seq raw data of bovine myogenic cells in NCBI-GEO public database were deeply analyzed, including the identification of cell cluster and the analyses of differential gene expression profile, correlation, GO enrichment, PPI, intercellular communication, and pseudotime, by using databases or softwares such as Seurat, ClusterProfiler, STRING, Cytoscape, CellChatDB, and Monocle2.【Result】 Four cell clusters with unique transcriptional characteristics, including Myoblasts, Myocytes, Fibroblasts, and FAPs, were identified based on the gene expression correlations and marker genes. By comparing the gene expression profiles and analyzing the pseudotime differentiation trajectory of myoblast subclusters, it was found that there was strong heterogeneity among subclusters, of which Myoblasts_1 was the starting point of differentiation, Myoblasts_0 and 3 were in the early stage of differentiation, while Myoblasts_2 was the most obvious subcluster with muscle characteristics and they might be the late-stage Myoblasts adjacent to forming Myocytes. There were differences in muscle-related GO terms enriched by differential genes between Myoblasts_2 and Myocytes, and there were complex protein interactions among these genes. The communication mechanisms between homotypic or heterotypic cells of Myoblasts_0-5, Myocytes, Fibroblasts, and FAPs involved various ligand-receptor interactions, such as PTN-NCL, IGF2-IGF2R, and ANGPTL2-(ITGA5 + ITGB1).【Conclusion】 There are gradually changing of gene expression profiles and intercellular communication during the differentiation of myogenic cells, reflecting the complex dynamic heterogeneity and molecular regulatory mechanisms.

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