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    26 July 2023, Volume 39 Issue 7
    Research Progress in lncRNA and Their Responses to Low Temperature Stress in Plant
    CHEN Xiao, YU Ming-lan, WU Long-kun, ZHENG Xiao-ming, PANG Hong-bo
    2023, 39(7):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0051
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    Low temperature is a major limiting factor for plant growth and production, affecting planting range, crop yield, quality, and even causing death. To cope with low-temperature stress, plants have evolved complex defense mechanisms. lncRNAs are a group of transcripts generated by genomic transcription and found in both the cytoplasm and nucleus of cells. In recent years, researches have shown that lncRNAs respond to low-temperature stress in plants through mechanisms such as polyadenylation, synergistic effects with specific proteases, and competitive binding with miRNAs. This review provides a summary of the definition, origin, and classification of lncRNAs, with a focus on their roles in low-temperature stress response in the model plant Arabidopsis thaliana and agro-economic crops. The findings presented in this review will serve as a valuable reference for those studying plant low-temperature tolerance mechanisms and molecular breeding for cold-tolerance.

    Advances in the Regulation of Plant Growth, Development and Stress Physiology by Small Peptide Hormones
    HU Hai-lin, XU Li, LI Xiao-xu, WANG Chen-can, MEI Man, DING Wen-jing, ZHAO Yuan-yuan
    2023, 39(7):  13-25.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1468
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    Small peptide hormones are usually referred to as peptides containing 5 to 100 amino acids in length. In plants, the contents of small peptide hormones are very low, the molecular weight is small, the quantity is large, and the source and processing mechanism is complex. This gives small peptides a variety of biological functions. They can bind to receptors at very low concentrations, regulate physiological processes such as cell division and growth, tissue and organ differentiation, flowering and fruiting, maturation and senescence, and coordinate plant responses to various stress environments. As an important medium of signal transduction among cells, the molecular mechanism of small peptide hormones in regulating growth and development is the hotspot and front topic in Botany. First the research progress of the structure, synthesis, classification and function of small peptide hormones were systematically reviewed. Furthermore, the research progress of CIF, CLE, RALFs, PSK, SYS in regulating plant growth and development and stress physiology was emphatically summarized, and the application prospect of plant small peptide hormones was outlined, which may provide an important theoretical basis for the in-depth research, development and application of plant small peptide hormones.

    Advances in the Regulation of Iron Homeostasis by bHLH Transcription Factors in Plant
    LI Yu, LI Su-zhen, CHEN Ru-mei, LU Hai-qiang
    2023, 39(7):  26-36.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1474
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    Iron is an essential micronutrient for plant growth and development. Iron deficiency is widely existing in plants growing in neutral or alkaline soils. However, the normal growth and development of plants need to maintain the balance of iron,which is tightly regulated at both transcriptional and post-transcriptional levels. Many transcription factors are involved in the regulatory network of iron homeostasis in plants, among which members of the basic helix-loop helix(bHLH)family are essential for iron homeostasis. In this review, key bHLH transcription factors that regulate iron homeostasis are summarized, and the mechanisms by which these transcription factors regulate iron homeostasis in plant growth and development are reviewed, aiming to provide a theoretical basis to uncover the regulation of iron homeostasis in plants.

    Advances in the Mechanism of Pepper in the Response to Heat Stress
    ZHANG Bei, REN Fu-sen, ZHAO Yang, GUO Zhi-wei, SUN Qiang, LIU He-juan, ZHEN Jun-qi, WANG Tong-tong, CHENG Xiang-jie
    2023, 39(7):  37-47.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0132
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    Pepper is an important vegetable crop and condiment, rich in nutrients. However, high temperature restricts the growth and development of pepper, seriously affects its yield and quality. Therefore, it is of great significance to study the response mechanism of heat stress to cultivate pepper varieties with strong heat resistance. Based on the physiological and biochemical, molecular, and omics mechanism, this review summarizes the recent advances in heat-response regulation mechanisms in pepper. Furthermore, the existing problems in current researches are pointed out and the key points in future studies are also proposed, aiming to promote the analysis of pepper heat+resistant mechanism and heat-resistant variety breeding process.

    Application of DNA Methylation in Interpreting Natural Variation in Moso Bamboo
    LI Ying, YUE Xiang-hua
    2023, 39(7):  48-55.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1555
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    DNA methylation can change gene expressions and cause variable splicing without any alterations in the DNA sequence, thus changing cell phenotype and even function; moreover, this modification can be transmitted to progeny DNA during the process of DNA replication and plays an important regulatory role in plant somatic mutation. Moso bamboo(Phyllostachys edulis)is mainly propagated by vegetative process, however, it has abundant natural mutation types, and the underlying genetic regulation mechanism is not clear so far. In this paper, we reviewed the advances of natural variation in Moso bamboo, DNA methylation and its transcriptional regulation in plant development, and prospected the potential application of DNA methylation in bamboo natural variation, aiming to provide theoretical guidance and technical support for accurate breeding of bamboo plants.

    Advances in the Biosynthetic Pathways and Related Genes of Lotus Alkaloids
    WANG Ling, ZHUO Shen, FU Xue-sen, LIU Zi-xuan, LIU Xiao-rong, WANG Zhi-hui, ZHOU Ri-bao, LIU Xiang-dan
    2023, 39(7):  56-66.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1343
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    Many parts of lotus(Nelumbo nucifera Gaertn.)can be used as medicine, among which benzyl isoquinoline alkaloids are the main active components in lotus leaf and lotus plumule. The study found that lotus leaf contains nuciferine, roemerine and other aporphine alkaloids, with good lipid-lowering weight-reducing effect. Lotus plumule mainly contains bisbenzylisoquinoline alkaloids such as neferine and liensinine, which can resist arrhythmia. In recent years, the significant pharmacological activity of lotus alkaloids and the phenomenon of ‘homologous and different effects’between lotus leaf and lotus plumule have inspired more and more researchers to study the biosynthetic pathways and key enzymes of lotus alkaloids. Therefore, this paper reviews the research progress of alkaloid composition types, alkaloid synthesis pathways and key enzyme genes of lotus leaves and lotus plumule, aiming to provide reference for analyzing the alkaloid synthesis pathways of lotus and the molecular mechanisms of pharmacodynamic differentiation of lotus leaves and lotus plumule.

    Advances in Genomic Studies and Metabolic Engineering of Red Yeasts
    LI Yu-zhen, MEI Tian-xiu, LI Zhi-wen, WANG Qi, LI Jun, ZOU Yue, ZHAO Xin-qing
    2023, 39(7):  67-79.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1426
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    Red yeast is a general term for a class of red pigment-producing yeast, and they are widely distributed in various ecological environments such as soil, sea, rivers and lakes. Red yeasts have the ability to produce polysaccharides, oils, carotenoids and other active substances and thus own great potential for applications in agriculture, food, medicine and cosmetics. In recent years, studies on red yeast including their applications, genome sequencing and key metabolic enzymes have expanded the in-depth knowledge of this important group of yeast. In addition, progress has been made in genome editing and metabolic engineering modification of these yeasts, providing basis for developing more efficient red yeast strains for industrial applications. Here, we review the applications of red yeast and related research advances in genomic studies and metabolic engineering. This review would be helpful for better understanding the physiology and metabolic features of red yeast, as well as utilization of renewable biomass for sustainable bioproduction using red yeast.

    Research Progress in the Regulation of Isobutanol Synthesis Pathway in Saccharomyces cerevisiae
    CHENG Ting, YUAN Shuai, ZHANG Xiao-yuan, LIN Liang-cai, LI Xin, ZHANG Cui-ying
    2023, 39(7):  80-90.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1350
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    As one of the microorganism commonly used in industrial production, Saccharomyces cerevisiae is of low pH resistance and strong stress resistance. The use of microorganisms to produce bio-based products has become an important development direction of green biomanufacturing. Isobutanol is a kind of branched-chain alcohol, which has a good application prospect in the chemical industry, energy, and other fields. As a biofuel, the synthesized isobutanol from S. cerevisiae is a sustainable, renewable, economical and environment-friendly approach to meet the needs of human development. The construction of an efficient isobutanol synthesizing S. cerevisiae cell factory may enable the green manufacturing of isobutanol as a biofuel. However, the ability of S. cerevisiae to synthesize isobutanol has limited the industrial development of isobutanol, the rational metabolic regulation strategy combined with synthetic biology is one of the effective methods to improve the ability of microbial isobutanol synthesis. This review summarized the latest developments in the regulating strategies of isobutanol biosynthesis in S. cerevisiae, including promoting isobutanol synthesis pathways, blocking competitive pathways, balancing cofactors, relocating synthetic pathways, regulating transcription factors, and improving microbial isobutanol tolerance. These strategies have significantly improved the conversion and yield of isobutanol in engineering yeast, but there are still many deficiencies in the industrial production of isobutanol. In order to promote the industrial production of isobutanol, the current bottlenecks and their solutions for isobutanol production in S. cerevisiae were discussed, aiming to provide valuable information for the green manufacturing of isobutanol.

    Research Progress in the Extraction and Functional Activities of Bioactive Peptides from Fish Skin
    YOU Zi-juan, CHEN Han-lin, DENG Fu-cai
    2023, 39(7):  91-104.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1265
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    Deep processing of fish skin waste in aquatic products processing can turn waste into treasure in high-value-added way and reduce environmental pollution, and the bioactive peptides from fish skin possess antioxidant, antihypertensive and antibacterial effects, etc. The methods of extraction, separation and identification of bioactive peptides from fish skin waste and their functional activities were discussed in depth, which may provide theoretical basis for the development of food, health products, cosmetics, pharmaceuticals and chemical products. In this paper, the advantages and disadvantages of enzyme, chemical and fermentation methods for the extraction of bioactive peptides from fish skin are reviewed. Compared with chemical methods, enzyme method is widely used, by which the bioactive peptides have higher activity; the cost of fermentation method is low, and it is suitable for mass production. The separation, purification and identification methods of fish skin bioactive peptides, such as ultrafiltration, nanofiltration, gel filtration, ion exchange, high performance liquid chromatography and mass spectrometry are summarized. The combination of multiple separation and identification methods to obtain fish skin bioactive peptides with specific functional activities is the first choice, but to obtain high purity and high activity of the target products is still a difficult breakthrough. In addition, the functional activities of fish skin bioactive peptides, such as antioxidant properties, inhibitory activities of angiotensin converting enzyme(ACE), antibacterial properties, and other bioactivities are analyzed, and the structure-activity relationship between functional activities and molecular weight, sequence structure and location of the peptides is summarized. Finally, the deficiencies of functional activity research, the development of bioactive peptides, and the direction of further research are prospected, aiming to provide reference for the fish processing industry in making high-value functional products.

    Progress in Research and Application of Natural Bio-materials as Immobilized Carriers
    LI Huan-min, GAO Feng-tao, LI Wei-zhong, WANG Jin-qing, FENG Jia-li
    2023, 39(7):  105-112.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1475
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    Microorganism has shown great functional potential in practical application, but the defects of free microorganism causing high production cost, low efficiency, poor environmental tolerance and low recovery inhibit its use, while the immobilization technology can improve the above properties of microorganism and has been applied in many fields. Among various methods, the combined immobilization based on adsorption has achieved remarkable results, and it is of great significance to increase the adsorption capacity and adsorption capacity of carrier materials, thus the development of efficient, durable and low-cost carrier materials is the key to the popularization and application of microbial immobilization technology. In this paper, the application of natural biomass materials as the immobilized carrier of microorganisms for environmental treatment is reviewed. The research on the modification and optimization of carriers based on biomass materials and substitution of mycelium pellets are summarized and prospected. The strategy of combining chemical engineering and genetic engineering to strengthen the application efficiency of microbial immobilization is emphasized in the future.

    Advances in Plant Cell Wall Structure and Imaging Technology
    ZHANG Man, ZHANG Ye-zhuo, HE Qi-zou-hong, E Yi-lan, LI Ye
    2023, 39(7):  113-122.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1347
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    Plant cell wall is a complex cross-linking network that provide mechanical support for plant cells during growth, development and adaptation to a changing environment. It has the functions of regulating plant morphology, resisting stress, transporting water, etc. In addition, most of the biomass accumulated by plant photosynthesis is stored in the cell wall. Therefore, it is of great significance to study the composition and structure of the cell wall for better utilization of plant energy. The nano-microstructure of plant cell wall is one of the frontier hotspots in plant research. With the development of new imaging technologies, a large number of studies had been conducted on the composition and structure of the cell wall. In this work, we summarized the composition, structure, imaging technology and mechanical property of the plant cell wall, in order to provide new ideas for studying plant cell wall.

    Establishment and Optimization of Virus-induced Gene Silencing System in Bougainvillea peruviana ‘Thimma’
    LIU Zhen-yin, DUAN Zhi-zhen, PENG Ting, WANG Tong-xin, WANG Jian
    2023, 39(7):  123-130.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1266
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    Bougainvillea peruviana ‘Thimma’, belonging to the family Nyctaginaceae, has high ornamental values in landscape. It is an ideal material to study the regulation of flower and leaf coloration. It is vital to establish a rapid and efficient gene verification system in B. peruviana. In this study, phytoene desaturase gene(PDS)was used as an indicator gene, we explored the effects of different inoculation sites and fragment length on the silencing effects of endogenous PDS mRNA in B. peruviana leaves induced by tobacco rattle virus(TRV)and established an optimized virus-induced gene silencing system(VIGS)for B. peruviana. The results showed that the silencing effect of leaf friction injection was more significant than that of tender stems friction injection. Additionally, PDS gene silencing was induced by both the inserted fragment of 336 bp and 457 bp, and the latter one was more significant. The VIGS system of B. peruviana is preliminarily constructed, which can be applied to the following gene function research.

    Functional Analysis of Soybean Gene GmHMGR Responding to Exogenous Hormones and Abiotic Stresses
    WANG Shuai, FENG Yu-mei, BAI Miao, DU Wei-jun, YUE Ai-qin
    2023, 39(7):  131-142.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0015
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    GmHMGR gene is one of the key enzyme genes in the mevalonate pathway of soybean(Glycine max L.), and plays an important role in the stress regulation. Exploring the function of the GmHMGR gene in soybean stress resistance will serve as a foundation for the analysis of soybean antioxidant, salt resistance mechanisms, and molecular breeding. The function of the GmHMGR gene under abiotic stress and hormone treatments was analyzed using real-time quantitative PCR, yeast transformation, and A. thaliana genetic transformation. The results showed that the GmHMGR gene responded to PEG6000, NaCl, H2O2, MeJA and ABA treatments. Overexpressing GmHMGR1, GmHMGR3, GmHMGR5, GmHMGR6 and GmHMGR7 genes improved yeast's salt resistance and antioxidant capacity. Overexpressing GmHMGR4 and GmHMGR6 genes in A. thaliana upregulated the expression levels of terpenoids metabolism-related enzymes genes(including AtDXR, AtSQS1, AtSQS2 and AtCAS), and improved its resistance capacity under oxidative and salt stress. GmHMGR gene may be involved in response to oxidative and salt stresses in soybean.

    Optimization and Application of Tobacco Rattle Virus-induced Gene Silencing System in Soybean
    LI Wen-chen, LIU Xin, KANG Yue, LI Wei, QI Ze-zheng, YU Lu, WANG Fang
    2023, 39(7):  143-150.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1449
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    Virus-induced gene silencing(VIGS)technology has been widely used in plant gene function research. The efficiency of gene silencing in soybean mediated by tobacco rattle virus(TRV)-based vector system remains to be clarified. The seamless cloning technique was used to construct a TRV-VIGS system to explore the silencing efficiency in soybean target genes in different tissues with different inoculation methods, providing a basis for gene function studies in soybean. Taken phytoene desaturase(GmPDS)and ubiquitin ligase(GmATL3)as target genes, pTRV1 and the recombinant vectors solution were inoculated into soybean(Glycine max)plants Zhonghuang13 by three methods: injection, agroinoculation and a combination of injection plus agroinoculation. The silencing phenotypes were observed at 28 d after inoculation, and the relative expressions of the genes in the roots and leaves were quantified by RT-qPCR to determine the silencing effectiveness of the different methods. Silencing of GmPDS resulted in yellowing and chlorosis on the edges and inside of the leaves, while chlorosis spots and fold chlorosis phenotypes in the leaves surface occurred. The results of RT-qPCR showed that silencing efficiency of GmPDS of all three inoculation methods was close to 100%. The silencing efficiency of GmATL3 by the injection was 80%-95% in the leaves and 40%-60% in the roots; the silencing efficiency of the root inoculation and injection plus root inoculation was 70%-90% in roots and 15%-50% in leaves. Different inoculation methods caused different levels of silencing phenotypes and silencing efficiencies for different endogenous genes. The silencing efficiency of an inoculation method on different tissues was different, and the highest silencing efficiency on the leaves and roots were injection method and injection plus agroinoculation, respectively.

    Cloning and Expression Analysis of CsTMFs Gene in Tea Plant
    SUN Ming-hui, WU Qiong, LIU Dan-dan, JIAO Xiao-yu, WANG Wen-jie
    2023, 39(7):  151-159.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1408
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    Based on the whole genome data of tea plant(Camellia sinensis), members of tea plant TATA element modulatory factor(CsTMF)gene family were screened and identified. The full-length coding sequence(CDS)of CsTMFs in the tea plant leaves was further cloned. The conserved domain, gene structure, physical and chemical properties, protein secondary structure and phylogenetic relationship of CsTMFs were analyzed by bioinformatics method. Tissue-specific expression was analyzed based on transcriptome data, and qPCR was used to detect the expressions of CsTMFs in the tea plant leaves under cold stress and drought stress. The result showed that there were two genes of CsTMF gene family in the tea plant. The lengths of CDS region in two CsTMF genes were 2 934 bp and 2 904 bp, respectively. There were complete conserved domains, TMF-DNA-bd and TMF-TATA-bd in both two CsTMF genes. CsTMF1 and CsTMF2 had tissue expression specificity. CsTMF1 was highly expressed in the flowers and stems, while CsTMF2 was highly expressed in the mature leaves and stems. The results of qPCR showed that CsTMF1 was induced by cold stress, while CsTMF2 was inhibited by cold stress and drought stress.

    Mechanism of Melatonin Seed Priming in Improving Salt Tolerance of Capsicum annuum
    WEI Xi-ya, QIN Zhong-wei, LIANG La-mei, LIN Xin-qi, LI Ying-zhi
    2023, 39(7):  160-172.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1253
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    Melatonin can enhance plant salt tolerance, but its effect and mechanism of improving plant salt tolerance through seed priming are rarely reported. In order to investigate the effects of melatonin(MT)seed priming on the growth and physiological characteristics of pepper seedlings under salt stress, its influences on the growth, photosynthetic characteristics, antioxidant metabolism and osmotic regulation of pepper seedlings under salt stress were studied using ‘Maoshu 360’ as material. The results showed that MT seed priming significantly alleviated the adverse effects of salt stress on the growth of pepper plants. Compared with seed without seed priming treatment, seed priming treatments with different concentrations of melatonin significantly improved the growth of pepper seedlings under salt stress, specifically, these indexes plant root length, above ground height, stem fresh weight, leaf fresh weight, root fresh weight, FV'/Fm', Qp, NPQ, Fo, FV/Fm and chlorophyll content increased. Physiological analysis showed that the contents of malondialdehyde, hydrogen peroxide and superoxide anion in the leaves of pepper under salt stress significantly reduced by seed priming with different concentrations of melatonin. The contents of soluble sugar, soluble protein, proline, AsA and DHA in the leaves significantly increased. The activities of POD, SOD, APX and GR significantly increased. In addition, melatonin seed priming treatment significantly increased the content of K+ in the leaves, stems and roots, and decreased the content of Na+ and Na+/ K+ in the leaves of seedlings under salt stress. The above results showed that 100 μmol/L melatonin seed priming treatment had the best effect, which significantly improved the tolerance to soil salt, reduced the absorption of Na+ by plants, and alleviated the damage of salt stress to plants.

    Effects of EMS Mutagenesis on the Seeding Survival and Major Agronomic Traits of Sorghum in M1 Generation
    FAN Xin-qi, WANG Hai-yan, CHEN Jing, ZHANG Xiao-juan, GUO Qi, LIANG Du, ZHOU Fu-ping, NIE Meng-en, ZHANG Yi-zhong, LIU Qing-shan
    2023, 39(7):  173-184.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1499
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    The current study mainly explored the effects of different EMS concentration on seedling survival and major agronomic traits in sorghum M1 generation, and found the optimal EMS concentration in different sorghum varieties. It can provide the basic varieties and theoretical guidance on the mutant construction, the germplasm resources innovation and the new varieties cultivation with high yield and quality in sorghum. Six sorghum restorer lines were used in this study. The seeds were firstly soaked by EMS with different concentrations(0, 0.20%, 0.25%, 0.30%, and 0.35%)for 12 h. The seedling survival and related agronomic traits were investigated after sowing in the field. With the EMS concentration increasing, the seedling survival rate decreased gradually, while the mutation rate of plant height, leaf, panicle type, fertility and maturity stage increased in mutagenesis plants. In sorghum, 0.25% EMS was the optimal concentration of 1121R, LgB7B Daligao, L zao 615·L17, and 28R·Ji16. The 0.35% EMS was the optimal concentration of L17, while the optimal concentration of Digan L17 was higher than that under 0.35% EMS. Thus, EMS treatment has a great effect on seedling survival and major agronomic traits in sorghum, and it has different effects on different varieties.

    QTL Mapping of Sorghum Plant Height and Internode Numbers Based on Super-GBS Technique
    XU Jian-xia, DING Yan-qing, FENG Zhou, CAO Ning, CHENG Bin, GAO Xu, ZOU Gui-hua, ZHANG Li-yi
    2023, 39(7):  185-194.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1510
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    Plant height and its relative traits are key factors that affect plant type and yield of sorghum. Mapping major QTL for these traits may provide a basis for genetic improvement in sorghum. In this study, a RI population including 205 lines obtained from a cross between American cultivar BTx623 and Guizhou liquor-brewing cultivar Hongyingzi was used to investigate plant height and internode numbers under five environments. The genetic map was constructed based on genotype data from Super-GBS technology, and major QTLs were identified by inclusive composite interval mapping(ICIM). Totally, eighteen major QTLs were detected on chromosome 1, 3, 4, 8, and 9, of which 7 and 11 QTLs were related to plant height and internode numbers, respectively. Ten major QTLs were repeatedly detected in multiple environments or two traits, of which qPH9.1 was consistent with the known plant high gene Dw1, while qPH1.2, qPH3.2, qIN3.2 and qIN8.1 were colocalized with four homologous genes involving in plant height or internode elongation in rice.

    Isolation and Functional Verification of Genes Responding to Iron and Cadmium Stresses in Lycium barbarum
    YU Hui, WANG Jing, LIANG Xin-xin, XIN Ya-ping, ZHOU Jun, ZHAO Hui-jun
    2023, 39(7):  195-205.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1304
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    In this paper, the effects of iron(Fe)and cadmium(Cd)stress on the expression patterns of genes of LbFRO(ferric reduction oxidase), LbZIP(zinc-regulated transporters and iron-regulated transporter-like protein)and LbNRAMP(natural resistance-associated macrophage protein)in Lycium barbarum L. were investigated for providing theoretical basis and technical support in preventing and controlling yellow leaf disease of wolfberry in Ningxia, cultivation of high-efficiency Fe-utilizing wolfberry varieties and low-cadmium accumulation varieties. The seedlings of L. barbarum L. No. 1 were used as experimental materials, and the genes related to iron(Fe)and cadmium(Cd)absorption and transport were isolated by transcriptome sequencing technology; the expression patterns of the target genes under Fe and Cd stress were verified by real-time fluorescence quantitative PCR; the full-length sequences of the target genes were cloned by conventional PCR method; and the yeast expression vector of those genes were constructed and transformed into Fe/Cd sensitive yeast strain ΔCCC1/YK44 for functional verification. The results showed that LbFRO, LbIRT1, LbIRT2 and LbNRAMP3 were significantly up-regulated under Fe and Cd stress, phylogenetic tree analysis showed that those genes of L. barbarum L. were closely related to rice and Arabidopsis thaliana, the functions of transgenic yeast further verified that LbFRO, LbIRT2 and LbNRAMP3 genes enhanced the sensitivity of yeast to iron(Fe)and cadmium(Cd), indicating that these genes may be involved in the absorption and transport of Fe and Cd in L. barbarum L.

    Effect of Premature Bolting on the Rhizosphere Soil Microenvironment of Angelica sinensis
    XIE Tian-peng, ZHANG Jia-ning, DONG Yong-jun, ZHANG Jian, JING Ming
    2023, 39(7):  206-218.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1446
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    The changes of rhizosphere soil microenvironment during Angelica sinensis bolting were explored to provide new ideas for solving the problem of premature bolting. A. sinensis was taken as the research object, the soil properties, bacterial community and metabolite in the rhizosphere soil were determined by 16S rDNA and GC-MC techniques in bolting and unbolting A. sinensis, and the correlation among them was studied. The results show that nitrate nitrogen content in the rhizosphere soil of bolting groups were significantly higher than those of unbolting ones. There was no difference between bolting and unbolting groups in the diversity of rhizosphere soil bacterial community and the structure of dominant bacteria, thus the bacterial community was alike. However, there were significantly differences among 10 non-dominant genera. There were 66 metabolites in the rhizosphere soil of bolting groups, which were different from the unbolting ones, 52 of which were significantly up and 14 significantly down. The differential metabolites were mainly concentrated in 7 related pathways of amino acid metabolism, heterogeneous biodegradation and metabolism, and lipid metabolism. The nitrate nitrogen content, Phaselicystis, Rubellimicrobium were significantly negatively correlated to the differential metabolites of bolting groups(P<0.05). The nitrate nitrogen was mainly negatively related to the biosynthesis of unsaturated fatty acids, while Phaselicystis and Rubellimicrobium were mainly negatively related to the degradation of aminobenzoate, arginine and proline metabolism. The premature bolting of A. sinensis was related to the increase of nitrate nitrogen content in the rhizosphere soil, and the premature bolting will cause significant changes in the relative abundance of non-dominant bacteria and metabolites in the rhizosphere soil. The increase of nitrate nitrogen content may be related to the change of the relative abundance of Nitrosomonas, but further research is still needed.

    Effects of Applying Biochar on the Rhizosphere Fungal Community and Survival Rate of Panax notoginseng Under Continuous Cropping
    ZHAO Lin-yan, XU Wu-mei, WANG Hao-ji, WANG Kun-yan, WEI Fu-gang, YANG Shao-zhou, GUAN Hui-lin
    2023, 39(7):  219-227.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1358
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    Fungal community composition and diversity are important indicators for soil health. In order to explore the effects of biochar on the soil fungal community of Panax notoginseng under continuous cropping, biochar was applied at 0, 12, and 15 t/ha(T0, T1, and T2)to the soil which has cultivated P. notoginseng for 10 years. After transplanting for 18 months, soil fungal diversity and the abundance of pathogenic Fusarium oxysporum in the rhizosphere of P. notoginseng under different treatments were analyzed using high-throughput sequencing and qPCR technology. Moreover, the correlations between soil physicochemical factors and changes in fungal community, and the effects of biochar on the survival rate of P. notoginseng were explored. The results showed that soil pH, NO3--N, and available potassium(AK)contents in the rhizosphere soil increased by 6.5%, 13.6%, and 40.3% respectively under T2 treatment, however, soil NH4+-N content decreased by 21.2%. Biochar application significantly increased fungal α diversity and altered fungal community composition. At the phylum level, the relative abundance of Mortierellomycota significantly increased under biochar treatment. At the genus level, while the relative abundance of Fusarium decreased, the relative abundance of Mortierella increased significantly(P<0.05), qPCR analysis showed that the abundance of pathogenic F. oxysporum significantly decreased with biochar application(P<0.05). The survival rate of P. notoginseng increased by 24.0% under T2 treatment, which was positively correlated with soil pH, fungal α diversity, and relative abundance of Mortierella, but negatively correlated with NH4+-N content and F. oxysporum abundance(P<0.05). Therefore, the application of biochar improved soil physicochemical properties, changed soil fungal community, increased the abundance of beneficial fungi, and decreased the abundance of pathogenic F. oxysporum, which effectively increased the survival rate of P. notoginseng under continuous cropping. Our data indicated that biochar was effective to reduce the replant problem of P. notoginseng.

    Screening and Identification of Broad-spectrum Antagonistic Bacterial Strains Against Vegetable Soft Rot Pathogen and Its Control Effects
    MA Jun-xiu, WU Hao-qiong, JIANG Wei, YAN Geng-xuan, HU Ji-hua, ZHANG Shu-mei
    2023, 39(7):  228-240.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1349
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    To obtain a broad-spectrum biocontrol strain with high efficiency against vegetable soft rot bacteria, in this study, a biocontrol strain DJ1 with good inhibition effect on three pathogens was screened from vegetable rhizosphere soil by the method of bacteriostasis circle, using soft rot pathogen of Chinese cabbage Pectobacterium carotovorum BC2, onion Burkholderia gladioli YC1 and baby cabbage Pseudomonas sp. WWC2 as the targets. Antagonistic strains were isolated and screened from vegetable rhizosphere soil by gradient dilution and bacteriostatic zone method. Through morphological, physiological and biochemical analysis and 16S rDNA sequence analysis, the species of the biocontrol strains were identified, and their the growth characteristics were studied. The Oxford Cup method was used to determine the bacteriostasis of the biocontrol strains on 3 strains of soft rot and 3 strains of human pathogenic bacteria, and the plate confrontation method to determine the bacteriostasis on 8 strains of plant pathogenic fungi. Needling inoculation method was applied to determine the control effect on vegetables leaves in vitro and field, and X-gal chromogenic method to determine the degradation activities of quorum-sensing signal factors against Chinese cabbage soft rot pathogen. The growth characteristics of the strain, its ability to degrade quorum sensing signal factors, its bacteriostatic spectrum and its field control effect on soft rot of Chinese cabbage were studied. The results showed that 18 antagonistic strains were selected from 1 012 bacteria isolated from 20 soil samples, among which 1 biocontrol strain DJ1 had antibacterial activity against 3 soft rot pathogens YC1, BC2 and WWC2. The antibacterial zone diameters were(10.60±0.20),(6.92±0.56)and(3.92±0.16)mm, respectively. The strain DJ1 was identified as Bacillus velezensis. The optimal growth temperature of strain DJ1 was 30℃, and it had good salt tolerance and grew under 1%-5% NaCl. It had the ability to degrade the quorum-sensing signaling factors of Chinese cabbage, and inhibited the growth of Escherichia coli, Staphylococcus aureus and 8 plant pathogenic fungi. The 1×108 CFU/mL of bacterial solution was 84.30%, 60.21% and 69.96% in vitro against soft rot of Chinese cabbage, onion and baby cabbage, respectively, and 79.91% in field. Therefore, B. velezensis DJ1 presents potential application in the control of vegetable soft rot.

    Exploration, Identification and Phosphorus-solubilizing Condition Optimization of a Multifunctional Strain
    XIE Dong, WANG Liu-wei, LI Ning-jian, LI Ze-lin, XU Zi-hang, ZHANG Qing-hua
    2023, 39(7):  241-253.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1450
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    In order to explore multifunctional microorganism resources to achieve the effective utilization of agricultural waste, some efficient phosphate-solubilizing bacteria were isolated from the compost of mushroom chaff and chicken manure, and their phosphate-solubilizing abilities were subsequently optimized. Efficient phosphorus-solubilizing bacteria were screened from the compost by inorganic phosphorus selective medium. The selected strain was identified through the combination of morphological observation, physiological and biochemical test and molecular biology identification. Single factor and response surface experiments were used to optimize the inorganic phosphorus-solubilizing conditions of this strain. In addition, the compost potential of this strain was evaluated by determining the ability of releasing organophosphorus and potassium, and indole acetic acid production. An efficient phosphate-solubilizing strain Pb1 was isolated and identified as belonging to Bacillus smithii strain. The highest phosphorus-solubilizing capacity of strain B. smithii Pb1 was achieved when the culture condition was kept as: glucose 15.0 g /L, ammonium sulfate 3 g/L, tricalcium phosphate 7.0 g/L, inorganic salt 0.48 g/L, temperature 55.0℃, rotating speed 225 r/min, initial pH 7.0, loaded liquid 60.0 mL/250 mL, and inoculum volume 3.0%(V/V). Further response surface optimization showed that under the conditions of tricalcium phosphate 6.4 g/L, rotating speed 207 r/min and loaded liquid 65.0 mL/250 mL, the solubilized phosphorus content in the fermentation broth of strain Pb1 cultured for 3 dreached 534.68 mg/L, which was 1.58 times higher than that before optimization. Moreover, B. smithii Pb1 was found to be capable of detoxifying organophosphates, detoxifying potassium and producing indole-3-acetic acid with the content of 238.99 mg/L, 81.06 mg/L and 23.26 mg/L, respectively. B. smithii Pb1 demonstrated the ability of releasing inorganic phosphorus, organophosphates and potassium, and producing indoleacetic acid at high temperature(55℃), laying the foundation for the development of B. smithii Pb1 as an aerobic composting bacterial agent.

    Ecological Monitoring of Pit Mud in Yibin Strong-fragrance Baijiu-producing Region
    YOU Ling, JIAN Xiao-ping, FAN Fang-yong, YANG Zhi, WANG Tao
    2023, 39(7):  254-265.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1406
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    In order to explore the reasonable index system of pit mud ecological monitoring in Yibin strong-fragrance Baijiu region, 32 samples were gathered from 6 Baijiu factories with similar process and raw materials. The significant difference in the sensory quality of Baijiu from these pits was distinguished, and the physicochemical indexes, the contents of main organic acids and bacterial flora of these pit muds were detected and the relationship between these indexes and flavor substances or flavor characteristics of Baijiu were analyzed. It was found that the pit mud in Yibin Baijiu region showed following characteristics: pH was about(4.66±1.43), the titrated acid was about(1.04±0.75)mmol NaOH/10 g, the conductivity was about(711±321)μs/cm, the calcium ion content was about(32.6±67.0)mg/kg, the iron ion content was about(20.7±24.8)mg/kg, the available nitrogen content was about(97.1±42.4)mg/kg, the available phosphorus content was about (137±60)mg/kg, and the available potassium content was about(275±122)mg/kg, the contents of caproic acid, acetic acid, ethyl caproate, propionic acid, hexyl caproate and butyric acid calculated by 2-ethylbutyric acid were about(6.49±2.84),(0.04±0.05),(1.15±1.07),(0.04±0.05),(0.97±0.98)and(0.20±0.13)g/100 g respectively, and Caproiciproducens, Syntrophaceticus, Syntrophomonas, Clostridium, Lactobacillus, and Sedimentibacter were dominant bacterial genera. The pit muds with good sensory quality of Baijiu had the following common characteristics: the pH was moderately high to about(6.17±1.57), and the acidity was moderately low to about(1.14±0.92)mmol NaOH/10 g; the contents of hexyl caproate in the pit mud that produces high-quality Baijiu tended to be higher, and the content of butyric acid tended to be lower; the abundance of Caproiciproducens, Caloramatoraceae, Peptostreptococcales-Tissierellales tended to be higher, the abundance of Sporosarcina tended to be lower. The results showed that the indexes of pit mud, such as pH, acidity, conductivity, calcium ion content, iron ion content, available nitrogen, phosphorus and potassium content, main organic acids and caproate ester content, and the abundance of dominant bacteria, can reflect the ecological status of pit mud from different aspects, and suitable for ecological monitoring indicators of pit mud in Yibin strong-fragrance Baijiu region.

    Screening of an Efficient Denitrifying Phosphorus-accumulating Bacterium and Its Denitrification and Phosphorus Removal
    YUAN Ye, ZHOU Jia, QU Jian-hang, ZHANG Bo-yuan, LUO Yu, LI Hai-feng
    2023, 39(7):  266-276.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1522
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    The objective is to explore the denitrification and phosphorus removal conditions and performance of a efficient denitrifying phosphorus-accumulating bacterium, and its treatment effect on simulated wastewater. Denitrifying phosphorus-accumulating strain D4 was isolated by pure culture technology. Preliminary identification of the strain was completed based on 16S rRNA gene sequence analysis and physiological and biochemical characteristics, and the denitrification and dephosphorization conditions were optimized by single factor and response surface method, so as to achieve efficient biological denitrification and dephosphorization of mimic food wastewater. As results,denitrifying phosphorus-accumulating bacterium D4 was Gordonia sp.. The optimal conditions for nitrogen and phosphorus removal were sodium acetate(3.32 g/L)as carbon source, 5% inoculum, initial phosphorus content of 18.61 mg/L, 31.3℃, and pH 7.9. Under these conditions, the removed amounts of total phosphorus, nitrate nitrogen and ammonia nitrogen by strain D4 were 14.18, 39.67 and 69.71 mg/L, respectively; and the corresponding removal rates were 84.50%, 97.67% and 96.22%. Results of treatment to different simulated wastewater showed that strain D4 had the best nitrogen and phosphorus removal effects in soybean product wastewater, with the removed amount of nitrogen and phosphorus being 101.58 mg/L and 10.40 mg/L, respectively. In conclusion, strain D4 has promising denitrification and phosphorus-accumulating ability and is a good strain resource for biological denitrification and phosphorus removal of food processing wastewater with high nitrogen and phosphorus such as soybean product wastewater.

    Study on the Biosynthesis of l-SLR by Efficient Prokaryotic Expression of Berberine Bridge Enzyme
    MEI Huan, LI Yue, LIU Ke-meng, LIU Ji-hua
    2023, 39(7):  277-287.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1517
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    l-Scoulerine(l-SLR)is a key intermediate of benzylisoquinoline alkaloids(BIAs), which are synthesized by berberine bridge enzyme(BBE). Based on the previous successful prokaryotic expression of Eschscholzia californcia berberine bridge enzyme(EcBBE, EcBO), an engineered E. coli strain with efficient expression of EcBO, which efficiently transformed l-Reticularine(l-RL)into l-scoulerine(l-SLR), was constructed by replacing the prokaryotic expression vector and co-expressing with chaperone protein. The results showed that the engineered strain A co-expressed by EcBO and chaperone pGro7 increased the activity of EcBO to 194.14 U/L, 9.23 times compared with the original strain. Further optimization of the culture conditions for strain A biosynthesis l-SLR showed that in TB medium and at the conditions of 0.04 mmol/L isopropylthio-β-D-galactoside(IPTG), 4 mg/mL L-arabinose, induced for 18 h at 16℃ then 0.2 mg/mL l-RL conversion for 18 h at 37℃, the l-SLR yield reached 144.19 mg/L, 4.72 times higher than that by the initial strain. In conclusion, the highly active expression of EcBO was achieved by co-expressing the chaperone pGro7 and EcBO in the prokaryotic system, which significantly improved the biosynthesis efficiency of l-SLR, and provides a new strategy for the efficient biosynthesis of isoquinoline alkaloids such as l-SLR.

    High-throughput Profiling and Analysis of Shared Antibiotic Resistance Genes in Human Feces, Skin and Water Environments
    ZHOU Zhen-chao, ZHENG Ji, SHUAI Xin-yi, LIN Ze-jun, CHEN Hong
    2023, 39(7):  288-297.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1430
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    Antibiotic resistance genes(ARGs)are emerging contaminants, whose spread in the environment exacerbates antibiotic resistance issues. Little is known about the critical and shared antibiotic resistance genes(ARGs)and their potential hosts in human and water environments. Here, we characterized the ARG profile and bacterial community in human and water environments from a Chinese rural village using high-throughput quantitative polymerase chain reaction and 16S rRNA gene sequencing. A total of 70 shared ARGs, 7 shared mobile genetic elements(MGEs), and 58 shared bacteria between human and water environments were identified. The dominant shared ARGs in feces were both tetracycline and MLSB resistance genes. Further, we identified 20 bacterial biomarkers by linear discriminant analysis effect size(LEfSe). The network revealed a significant association among shared ARGs, MGEs, and bacteria(P < 0.05), suggesting the potential transfer of ARGs between humans and environments via these bacteria. This study revealed the shared ARGs in drinking water, feces, skin, wastewater, and river samples. Our results may provide a better understanding of the co-occurrence of these genetic elements and identify the potential hosts of ARGs between humans and the environment in peri-urban areas. In conclusion, this study demonstrated the co-occurrence and transmission of shared ARGs and bacteria in humans and water environments.

    Whole Genome Sequencing and Analysis of Listeria monocytogenes Strain LMXJ15
    DU Dong-dong, QIAN Jing, LI Si-qi, LIU Wen-fei, WEI Xiang-li, LIU Chang-yong, LUO Rui-feng, KANG Li-chao
    2023, 39(7):  298-306.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1093
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    In order to understand the genomic characteristic and evolutionary relationship of Listeria monocytogenes LMXJ15 from Xinjiang, PacBio and Illumina HiSeq platforms were used to have the whole genome sequencing of LMXJ15, then the relevant software were applied to predict and annotate the sequenced data. Multilocus sequence typing(MLST)and collinearity analysis were conducted to analyze the homologies among the LM strains. The results showed that the length of LMXJ15 genome was 3 017 732 bp with a GC value of 37.81%. It was predicted to contain 3154 protein-coding genes with a total length of 2 734 315bp. The genome of LMXJ15 contained 6 rRNA operons and 57 tRNA, 88 virulence genes being predicted from the genome. Through the CRT prediction, a CRISPR sequence was found in the LMXJ15 genome sequence. A total 2 241 genes of 3 GO contents were obtained by comparing GO database. Total 88 possible virulence genes were predicted. MLST analysis showed that LMXJ15 was sequence type(ST)8 and belonged to CC8(clonal complex 8). Evolutionary analysis showed that the LMXJ15 had a close evolutionary relationship with two clinical IZSAM_Lm_15_17439_A144 isolates from Italy, LmN1546 isolates from Switzerland, R479a isolates from smoked salmon, and had the same ST type and serotype(1/2a), and comparative genomics among 4 LM strains showed scant collinearity. In summary, we preliminarily analyzed the genomic characteristics and virulence genes situation of LMXJ15 isolate, which may provide basic data for analysis of the evolution relationship and the virulence of L. monocytogenes in Xinjiang province.

    Establishment of MDH2 Knockout Cell Line Using CRISPR/Cas9 Technology and Study of Anti-deoxynivalenol Effect
    SHI Wei-tao, YAO Chun-peng, WEI Wen-Kang, WANG Lei, FANG Yuan-jie, TONG Yu-jie, MA Xiao-jiao, JIANG Wen, ZHANG Xiao-ai, SHAO Wei
    2023, 39(7):  307-315.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1429
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    The aim of this study is to establish the IPEC-J2 cell line with MDH2 gene knockout by CRISPR/Cas9 genome editing technology and investigate whether MDH2 gene knockout confers resistance to deoxynivalenol. sgRNA sequences targeting MDH2 gene were designed and cloned into PX459 vector; the recombinant plasmid PX459-sgRNA-MDH2 was introduced into the IPEC-J2 cells by electroporation,and puromycin was added to screen KO cell colony generations. The MDH2 KO single cell colony generations were isolated by limited dilution method and validated by genotype sequencing, quantitative PCR and Western blotting to obtain MDH2-KO monoclonal cell colony generation. Finally, the cell viability was detected by CCK8 kit and apoptosis and necrosis assay kit to determine the resistance of MDH2 knockout cell colony generation to deoxynivalenol. The sequencing results showed that the MDH2 knockout vector was successfully constructed. The obtained cell colony generations were validated as MDH2 knockout monoclonal cell colony generations by RT-qPCR and immunoblotting. The CCK8 cell viability assay showed that knockout of MDH2 increased the viability of IPEC-J2 cells by 18.67%, 19.59%, 26.36%, and 27.01% at different concentrations of deoxynivalenol(4, 2, 1, and 0.5 μg/mL)for 5 d compared with wild-type cells. Flow cytometry assays showed that knockout of MDH2 reduced the mortality rate of IPEC-J2 cells by 30.33%, 15.81%, 16.00%, and 14.70% at different concentrations of deoxynivalenol respectively compared with wild-type cells for 5 d. We obtained the MDH2-KO monoclonal cell colony generation by editing the MDH2 gene in IPEC-J2 cells using the CRISPR/Cas9 system. The resistance of MDH2-KO cell colony generation to deoxynivalenol was demonstrated by cell viability and cell death assays, which provids insight into the toxic mechanism of deoxynivalenol-induced cell death and offers a new strategy for the prevention and treatment of deoxynivalenol.

    Stellera chamaejasme L. Inhibiting Cell Proliferation by Reducing YAP1 Expression in Hepatocellular Carcinoma
    ZHOU Wen-han, ZHENG Kang-ning, LI Yong-min
    2023, 39(7):  316-324.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1418
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    This study is to investigate the effect and mechanism of Stellera chamaejasme L.(SCL)on inhibiting proliferation of hepatocellular carcinoma cells, providing basis for the pathological mechanism study and clinical treatment of primary hepatocellular carcinoma. Firstly, CCK-8 assay was performed to illustrate the ability of tumor growth in the HepG2215 cells treated with SCL serum containing blood. Western blot was used to detect the expression of YAP1(yes-associated protein 1)protein in the HepG2215 cells. Secondly, Yap1flox/flox and Yap1LKO mice were induced to form liver tumors in situ by DEN(diethylnirtosamine)/TCPOBOP(3,5-dichloro-2-[4-(3,5-dichloropyridin-2-yl)oxyphenoxy]pyridine). SCL decoction was given to the stomach of the mice for 7 d, the liver was taken out, and the general shape of the liver tumor was observed. H&E was performed to observe the pathological morphology in tumor tissue. Western blot was to detect the expressions of YAP1 in the liver tumor tissues. The results from cell experiment demonstrated that SCL inhibited the proliferation of HepG2215 cells and decreased YAP1 expression. Moreover, animal experiment revealed that SCL reduced tumor volume and YAP1 expression both in Yap1flox/flox and Yap1LKO mice with tumors in liver. The hepatocellular carcinoma tumor volume of Yap1LKO mice in NS group reduced when compared with Yap1flox/flox mice. Yap1 knockdown reduced the effect of SCL inhibiting tumor growing in SCL group when compared with Yap1flox/flox mice. LC-MS was used to detect the active components of SCL drug-containing serum and water decoction, and molecular docking was carried out with YAP1 protein. LC-MS results showed that the active ingredients were caffeic acid 3-sulfate, 5-benzyloxolan-2-one, 3,4,5-trihydroxy-6-[(2-oxo-2h-chromen-5-yl)oxy]oxane-2-carboxylic acid and isoscopoletin in SCL. Among them, caffeic acid 3-sulfate, 3,4,5-trihydroxy-6-[(2-oxo-2h-chromen-5-yl)oxy]oxane-2-carboxylic acid and isoscopoletin directly formed stable docking with YAP1 protein. These results suggest that SCL inhibits liver tumor cell proliferation by reducingYAP1 expression.

    Study on the Heterogeneity of Human Gingival Mesenchymal Stem Cells at Single Cell Level
    WU Hao, LIU Zi-wei, ZHENG Ying, DAI Ya-wen, SHI Quan
    2023, 39(7):  325-332.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0253
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    Gingival mesenchymal stem cells(GMSCs)cell clusters and functional heterogeneity were analyzed through single cell RNA sequencing technology. Human GMSCs were isolated and cultured in vitro, GMSCs were sequenced by 10× scRNA-Seq platform. The cells were grouped according to the gene expression differences, and analyze the expression of surface markers and cell cycle differences in each cluster. Differences in gene expression and gene expression profile of osteogenic, lipogenic and chondrogenic differentiation were compared,and the interactions between different clusters of cells were also analyzed. Human GMSCs were successfully extracted and grew in a vortex like manner. The GMSCs were composed of 9 cell clusters. ENG, NT5E, THY1, ITGB1 and CD44 were highly expressed, and CD34, CD14, PECAM1 and PTPRC were negatively expressed. The proportion of G2/M phase of GMSCs was 41.59%. The G2/M phase ratio of cell cluster 6 was the lowest(16.44%), while the cell cluster 2 was the highest with 99.08%. The expressions of adipogenic, chondrogenic and osteoblastic differentiation related genes were not identical in the 9 cell clusters. Nine clusters had different gene expression profiles, and 2/4 cell clusters showed high similarity, with relatively high expressions of TOP2A, UBE2C, KPNA2, CENPF genes. DKK1, KRT7 and ID1 genes were highly expressed in the cell cluster 5. MALAT1, FN1 and NEAT1 genes were highly expressed in the cell cluster 9. Moreover, GO analysis showed that each cell cluster enriched different pathways. The communication between cell cluster 9 and other cell clusters were the most active. The single-cell level study suggests that there are different functional clusters of GMSCs, and the proliferation capacity and genomic expression profile of each cluster were heterogeneous.

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