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    26 December 2023, Volume 39 Issue 12
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    Research Progress in Immune Receptor Functions of Pattern-Recognition Receptor in Plants
    YE Hong, WANG Yu-kun
    2023, 39(12):  1-15.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0765
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    In the process of long-term adaptation to environment, plants have evolved a large number of cell surface and intracellular immune receptors to sense various biological and abiotic stimuli, and then trigger receptor-dependent immune responses. Cell surface Pattern-Recognition Receptor（PRR）is able to recognize pathogenic microbial model molecules and activate basic immunity, thereby enabling plants to acquire corresponding tolerance. At present, with the help of efficient research methods, researchers have made some progress in PRR-mediated plant disease resistance and environmental stress tolerance. Here, we reviewed the recent achievements of plant PRR on types and structures, ligand recognition and binding mechanisms, characteristics and mechanism of innate immunity mediated by PRRs, and identification of novel PRRs. Meanwhile, this review also focused on PRR-mediated salt resistance. The results will help us further understand the immune basis of plant-environment interaction, and will provide theoretical basis and guidance for the use of genetic engineering to cultivate excellent disease resistant and stress resistant plant varieties.

    Roles of Plasma Membrane Na+/H+ Antiporter SOS1 in Maintaining Ionic Homeostasis of Plants
    ZHU Ye-sheng, WU Guo-qiang, WEI Ming
    2023, 39(12):  16-32.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0793
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    Plants regulate ion homeostasis to adapt to saline environment through a series of complex transport systems. SOS（salt over sensitive）signal pathway is the major signal pathway for plants to respond to abiotic stress, which is mainly composed of plasma membrane Na+/H+ antiporter SOS1, serine/threonine protein kinase SOS2, and calcium sensor SOS3. As one of the main members of SOS signaling pathway, SOS1 widely exists in higher plants. Due to the early evolutionary differences, the structural, physical, and chemical properties of SOS1 from different species had certain specificity. The SOS1 protein is a homodimer, and each monomer is composed of transmembrane and intracellular domains, which provides a stable docking platform for integrating signals from different pathways and regulating Na+ transport. Transcription level of the SOS1 gene was regulated by different stress conditions. SOS1 activity was inhibited or activated through Ca2+ signal regulation, phosphorylation, self-inhibition, and synergistic regulation with other ion transporters. SOS1 has shown to regulate circadian rhythm and pH, and maintain ion homeostasis in plants, which plays an important role in the response of plant to abiotic stress. In this review, the structure, function, regulation mechanism of SOS1 and its role in maintaining plant ion homeostasis are reviewed, and its future research direction is also prospected. The information in this review provide a theoretical support and excellent genetic resources for the genetic improvement of crops to produce new, stress-resistant varieties.

    Research Progress in the Mechanism of Plant Photosystem II（PSII）Responsing to Abiotic Stress
    CHENG Shuang, ULAANDUU Namuun, LI Zhuo-lin, HU Hai-ling, DENG Xiao-xia, LI Yue-ming, WANG Jing-hong, LIN Ji-xiang
    2023, 39(12):  33-42.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0658
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    Plant growth process is influenced by a series of abiotic stresses represented by environmental factors（temperature, drought, salinity, and heavy metals, etc.）, which hinder the photosynthetic process mainly characterized by substance conversion and energy metabolism. Photosystem II（PSII）is a multi-subunit pigment-protein complex located on the thylakoid membrane, which is particularly important for plant growth and development by capturing light energy to complete the photolysis of water and plastoquinone reduction. Generally, abiotic stress may inhibit the activity of PSII, affect its structure, and hinder electron transport and energy conversion. As the most vulnerable part of photosynthesis, PSII has attracted much attention in recent years due to its relationship with environmental factors. Based on this, this article summarized the physiological responses of plant PSII under major abiotic stresses such as temperature, drought, salt and heavy metals, and reviewed the structure and function of PSII, electron transfer process, photoinhibition and photoprotection based on chlorophyll fluorescence technology. It also provides a theoretical basis for further research on the physiological and molecular mechanisms of plant PSII response to abiotic stress.

    Application and Biosynthesis Strategies of Unnatural Amino Acids
    LI Hai-ning, ZHANG Hong-bing, GENG Ge-xia, LI Ran, JIA Zhen-hua
    2023, 39(12):  43-55.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0648
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    Unnatural amino acids（UAAs）are a special class of amino acids that are not constrained by genetic codons. They have unique spatial configurations and chemical properties, which provide new resources and opportunities in several fields. Currently, UAAs can be synthesized by both chemical synthesis and biosynthesis. Compared with chemical synthesis, biosynthesis is a promising synthesis with higher efficiency and stereoselectivity, lower cost and pollution. This paper reviews the research progress and application cases of UAAs in protein probes, enzyme engineering, antibody-drug conjugates, and antimicrobial peptides, etc. It shows how UAAs break through the limitations of natural amino acids and expand the structural and functional diversity of proteins. It also introduces the biosynthesis strategies of UAAs, including static and dynamic regulatory strategies in metabolic engineering and fermentation optimization strategies. Finally, it looks forward to the future development trends and challenges in the biosynthesis and application of unnatural amino acids, which will provide a reference and inspiration for the UAAs synthesis and development.

    Research Progress in S-adenosyl-L-methionine Dependent 3-amino-3-carboxypropyl Utilizing Enzymes
    HE Jia-le, DONG Min
    2023, 39(12):  56-70.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0220
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    S-adenosyl-L-methionine（SAM）is a very important cofactor in biology. It possesses three unstable C-S bonds, which can be selectively cleaved by various SAM-dependent enzymes, catalyzing a wide range of biochemical reactions. Methyltransferases and radical SAM enzymes, which cleave C-S bonds and use methyl and 5'-dexoyadenosine of SAM respectively, have been intensively studied. Over 1.8 million protein sequences of methyltransferases have been deposited in the UniProt database. Radical SAM enzymes have grown into one of the largest enzyme superfamilies, consisting of over 700 000 members. However, enzymes utilizing the 3-amino-3-carboxypropyl（ACP）group of SAM are relatively rarely reported. This paper aims to classify and introduce these enzymes while also provide a prospective research direction for the future.

    A Quantitative Detection Approach for Glucose Uptake in Aspergillus niger: A Case Study of Glucose Transporter MstC
    GAO Kai-yue, GUO Yu-ting, DU Yi-mou, ZHENG Xiao-mei, MA Xin-rong, ZHAO Wei, ZHENG Ping, SUN Ji-bin
    2023, 39(12):  71-80.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0759
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    Aspergillus niger is an important strain for industrial production of organic acids and enzymes, possessing a powerful hydrolytic enzyme system and glucose transport system, which enable to copy with extracellular carbon sources variance and uptake glucose to support cell growth and industrial production. As an important carbon source and an essential signaling molecule, glucose and its uptake has considerable influence on cell growth and fermentation performance. Regarding to issue of lacking simple methods for quantitative characterization of glucose absorptive capacity in filamentous fungi, we developed a quantitative detection pipeline for glucose uptake capacity assay for filamentous fungi, using a non-metabolizable, fluorescent glucose analog, 2-（N-（7-nitrobenz-2-oxa-1,3-diazol-4-yl）amino）-2-deoxyglucose（2-NBDG）as a fluorescent probe, then incubating pre-cultured A. niger spores with 2-NBDG, and using with fluorescence microscopy and flow cytometry. It showed that the optimized 2-NBDG work concentration and incubation time were 150 μmol/L and 4 h, respectively. Using this quantitative analysis method, it was found that the overexpression of low affinity glucose transporter MstC increased glucose uptake by 1.44 times. Meanwhile, on the basis of previous studies, mutant R188K of MstC was designed, and the point mutation directly caused the loss of glucose transport activity of MstC, which indicates that Arg188 is a key amino acid site affecting the glucose transport capacity of MstC. The establishment of this quantitative glucose uptake detection method and its application to the study of MstC function not only deepens the quantitative understanding of glucose absorption in filamentous fungi, but also provides technical support for the transformation and optimization of glucose transport system. This study provides a feasible quantitative approach to investigate the glucose uptake of filamentous fungi, and also revealed the physiological function of key glucose transporter MstC, which paves the way for glucose uptake engineering in this industrially important fungal cell factory.

    Detection of Escherichia coli O157: H7 Based on Antibody Aptamer Sandwich Biosensor
    HOU Wei-chen, YE Ke, LI Jie, ZHANG Yang-zi, XU Wen-tao, ZHU Long-jiao, LI Xiang-yang
    2023, 39(12):  81-89.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0698
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    Escherichia coli O157:H7 is an important food borne pathogen related to public health. Antibody molecules are the most important effector molecules in the humoral immune response. They have a variety of biological activities. The main biological function is to specifically bind to the corresponding antigen. Aptamers are shorter DNA sequences synthesized in vitro, which can bind specifically by recognizing specific regions and targets. Based on the specific recognition function of antibody and aptamer, the capture effect and magnetic separation of immunomagnetic beads, and the construction of biosensor by polyclonal antibody and tailoring aptamer, real-time fluorescence quantitative polymerase chain reaction（qPCR）may achieve the detection of target at (8×103)-(8×106) CFU/mL. The limit of detection is 800 CFU/mL for quantitative detection.

    Establishment of a Luciferase-assisted Quantitative Method for Measuring Ultrasonic Disruption of Escherichia coli Cells
    LI Yi-ya, WU Yi-fan, DING Neng-shui, FAN Xiao-ping, CHEN Fan
    2023, 39(12):  90-98.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0129
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    In this study, we present a novel and effective method for measuring ultrasonic disruption of Escherichia coli cells based on firefly luciferase. This quantitative approach provides high sensitivity and rapid detection of cell disruption, by which the disruption was characterized. To evaluate this method, we employed an E. coli strain expressing firefly luciferase as an internal reference, which was mixed with the target bacterial suspension at a specific ratio before sonication. The release of both luciferase and target protein activities into the extracellular solution under different ultrasonic conditions was then investigated, and the assisted quantitative effect was evaluated. The results demonstrated that: A ratio of 1∶500（volume ratio）E. coli strain expressing firefly luciferase added to the target bacterial suspension was sufficient for sonication quantification. The effect of sonication was calculated by the change of luciferase activity within the supernatant. Importantly, luciferase activity also provided clues for maintaining target protein activity during sonication. The luciferase protein incorporated into the final supernatant showed no effect on the purification of the target protein by nickel beads. Bacteria containing firefly luciferase can be stored at -80℃ for at least 90 d without significant loss of luciferase activity. Moreover, the disruption resistance was not changed during the long-term cryo-storage process. Therefore, freezing bacterial suspension can be thawed and directly mixed with target cells for assisting quantification without losing accuracy. In conclusion, the luciferase-assisted quantification of E. coli ultrasonic disruption is convenient, robust, and efficient.

    Research Progress in the Application of RNAi Technology in Parasitoid Wasps
    ZHANG Long-xi, LYU Lin, ZHANG Huan-huan, ZHOU Jin-cheng, CHE Wu-nan, DONG Hui
    2023, 39(12):  99-108.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0580
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    RNA interference technology has been widely used for investigating functions of genes for more than 20 years since its inception. In Hymenoptera, parasitoid wasps are a group of important natural enemy against pests in agriculture and forestry. The inhibition of target gene expression relies on the delivery of dsRNA. However, the parasitoids develop on host insects, and interact with their hosts. This biological characteristic makes it challenging to conduct RNAi technology. The RNAi technology has already been conducted in the studies involved the molecular mechanisms of at least 13 species of parasitoid wasps, e.g. Nasonia vitripennis, Microplitis mediator, Cotesia vestalis, Pteromalus puparum, and Meteorus pulchbernis. This study reviewed the studies of the molecular mechanisms by using RNAi method, and summarized the factors influencing RNAi efficiency in parasitoid wasps, aiming to provide the reference for application of RNAi technology in parasitoid wasps.

    Single Cell Assay for Transposase Accessible Chromatin with High-throughput Sequencing Technology and Its Applications
    ZHAO Ruo-han, ZHAO Jing-ying, BAI Yi-cheng, ZHANG Rui-fang, JIA Jun-jing, DOU Teng-fei
    2023, 39(12):  109-117.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0518
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    Single-cell assay for transposase-accessible chromatin with high-throughput sequencing（scATAC-seq）is a high-throughput sequencing technique that utilizes transposase to investigate the chromatin accessibility at the single-cell level, which is of great significance for investigating the epigenetic regulation of the whole genome. It can reveal important information about chromatin organization from multiple perspectives, mapping the binding regions of transcription factors and the positioning of nucleosomes in cells. Currently, scATAC-seq has been widely applied in the fields of biology and medicine, primarily for generating open chromatin profiles, studying cell differentiation and development, elucidating disease pathogenesis, and exploring the tumor microenvironment. This article provides an overview of the development, data analysis, and relevant applications of scATAC-seq technology in studying the open chromatin regions at the single-cell level, aiming to provide insights for the investigation of chromatin accessibility at the single-cell whole-genome level, identification of cis-regulatory elements, and deciphering genetic regulatory networks, thereby contributing to advancements in life science research.

    Application of Cell-specific Nucleic Acid Aptamers in Disease Treatment
    ZHONG Zhao-bin, ZHU Long-jiao, ZHANG Bo-yang, ZHANG Yang-zi, CHEN Ke-ren, XU Wen-tao
    2023, 39(12):  118-127.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0289
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    Nucleic acid aptamers are single-stranded deoxyribonucleic acid（ssDNA）or ribonucleic acid（RNA）composed of 20 to 100 nucleotides, and are generally screened by systematic evolution of ligands by exponential enrichment（SELEX）. Aptamers can specifically bind to a variety of target substances, including toxins, metal ions, biological macromolecules, microorganisms, etc., and have been widely used in food detection, medical diagnosis, and other fields. As the most basic unit of the human body, cells are the focus of research on the pathological mechanism and treatment strategy of most diseases. In recent years, nucleic acid therapy has become the focus of research in the field of disease treatment with the advantages of safer and more efficient one. Researchers realized that the targeted binding ability of nucleic acid aptamers can be applied to cell targeted therapy. Cell-specific aptamers are different from traditional aptamers in that their specific targets are cellular components. They can directly bind to substances related to cell growth and metabolism to affect cell physiological activities, and can also be used as targeted delivery tools to deliver drugs, which has a breakthrough effect in the precision treatment of diseases. Cell-specific nucleic acid aptamers have attracted much attention. In this article, we review the research progress of cell-specific nucleic acid aptamers in the treatment of a variety of diseases in order to provide reference for their application in the treatment of human diseases.

    Character Identification and Genetic Analysis of Progeny from Distant Hybrid Between Gossypium hirsutum and G. thurberi
    HOU Lin-hui, ZHENG Yun, RONG Er-hua, WU Yu-xiang
    2023, 39(12):  128-135.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0539
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    Distant hybridization and polyploidization are fundamental processes for the development of new species and germplasm innovation. In this study, we obtained fertile offsprings by interspecific distant hybridization and chromosome doubling between Gossypium hirsutum and Gossypium thurberi. The objective is to penetrate beneficial agronomic traits of G. thurberi into G. hirsutum and expand the genetic diversity of G. hirsutum. In this study, we obtained hybrid F1 by synthesizing the female parent G. hirsutum with the male parent G. thurberi, and had allohexaploid cotton by chromosome doubling. And then we examined the morphology, cytology, physiology, biochemistry and SSR molecular markers of the F1 and allohexaploid. The results of morphological identification showed that the allohexaploid plant was similar to the heterotriploid, with mid-parents'characters. The allohexaploid had more stamens than the parent and F1 had, with reddish spots, which were different from the parents and F1. Cytological identification showed that stomatal density was in a decreasing trend with the increase of ploidy; while stomatal length, chloroplast number and pollen grain diameter all showed an increasing trend. The meiotic behavior of heterotriploids and allohexaploids revealed that both normal and abnormal tendencies, with more polyploids in triploids and significantly more normal tetrads in hexaploids. In triploids, the percentage of normal tetrads was 24.33%, while the percentage of normal tetrads was 82.17% in the polyploid hexaploids, indicating the recovery of hexaploid fertility. Physiological and biochemical results showed that the activities of enzymes increased with the increase of ploidy in different generations. The SSR molecular marker identification results revealed that distant hybridization led to chromosomal recombination in triploid hybrids and heterohexaploids, resulting in the amplification of both parental bands and new specific bands. The comprehensive results show that hybrids are successfully synthesized by distant hybridization and fertile heterohexaploids are successfully obtained by chromosome doubling, which provides valuable materials for cotton germplasm innovation and further genetic research.

    Genetic Diversity Analysis and Molecular Identity Card Construction by SSR Markers of 52 Solanum tuberosum L. Varieties（Lines）
    AN Miao, WANG Tong-tong, FU Yi-ting, XIA Jun-jun, PENG Suo-tang, DUAN Yong-hong
    2023, 39(12):  136-147.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0512
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    The evaluation on the genetic diversity of potato germplasm resources（Solanum tuberosum L.）is the prerequisite and basis for genetic breeding, which may provide breeding materials for exploring the genetic background of potato germplasm resources and breeding new varieties. The genetic relationship and diversity of 52 potato varieties（lines）was evaluated by SSR markers, and the fingerprint was constructed. Sixteen primers with abundant polymorphism and good stability were selected from 25 pairs of SSR primers, and 147 polymorphic bands were amplified by 16 pairs of SSR primers from 52 potato varieties（lines）, and the average polymorphic band ratio was 64.71%. The average Nei's genetic diversity（H）and Shannon's index（I）were 0.14 and 0.24, respectively. At the similar genetic coefficient of 0.23, the 52 potato materials were divided into three categories by cluster analysis. Group I included 49 varieties（lines）, 46 varieties（lines）from North China, and 3 imported varieties（lines）from abroad, all of them were suitable for planting in North Shanxi province. Group II included two varieties, ‘Beifang 016’ and ‘Jinshu 16’, characterized by yellow potato skin and high dry matter content. Group III only included ‘Jinshu 15’, and was not clustered according to geographical origin. At the genetic similarity coefficient of 0.36, group I can be divided into 7 subclasses. Based on the principle of identifying the most varieties with the least primers, C59, S25, C33 and S151 were used to distinguish all varieties tested, and the digital fingerprints and molecular identity cards based on SSR markers were constructed. The genetic diversity of potato germplasm resources can be evaluated by using SSR markers, and different potato varieties（lines）can be accurately identified and traced by molecular identification cards.

    Transcriptome Analysis of Cucumber Seeds Early and Late Germination Under PEG Drought Simulation
    XIE Yang, ZHOU Guo-yan, SU Hang, XING Yu-meng, YAN Li-ying
    2023, 39(12):  148-157.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0819
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    Drought is one of the most common abiotic stress factors that affect the growth and development of cucumber and reduce the yield. To analyze the molecular mechanism of cucumber drought stress response, the seeds of drought resistant cucumber ‘KS33’and drought sensitive cucumber ‘KS30’ were treated with PEG drought simulation, and transcriptomic sequencing was performed at early and late germination stages. The results showed that early germination was an important database for drought resistance gene mining, among which 727 up-regulated and 345 down-regulated differential genes were identified in PEG drought simulation induction of drought-resistant materials in early germination compared with the control（DT1_TvsDT1_CK）. Compared with the control（DS1_TvsDS1_CK）, 1 226 up-regulated and 111 down-regulated differential genes were identified by PEG drought simulation induction during early germination of sensitive materials.Via KEGG enrichment analysis the 20 key drought resistance candidate genes were identified to be enriched in linoleic acid metabolism（csv00591）, pentose, glucuronic acid conversion（csv00040）, phenylpropanoid biosynthesis（csv00940）and plant hormone signal transduction（csv04075）pathways. The drought-resistant gene CsaV3_4G023820, CsaV3_2G006420 and two related SNP molecular markers were selected by associated SNP mutation site and RT-qPCR analysis.

    Effects of Cucumber Endocarp Juice on Seed Germination
    YANG Yan, MO Yu-xing, ZHOU Yi, CHEN Hui-ming, XIAO Lang-tao, WANG Ruo-zhong
    2023, 39(12):  158-168.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0390
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    The roles, chemical composition and mechanisms of endocarp juice on seed germination were studied, which may provide a theoretical basis for the research of cucumber seed germination inhibitors from cucumber source. Viviparous cucumber（C-L）seed was used as materials and cultivated by not viviparous（M-H）endocarp juice（mass fractions was 100% and 50%）and its various extracts（mass fractions was 3%）. The chemical composition of juice was analyzed. During seed germination, the physiological and biochemical factors were analyzed when the culture medium was a mass fraction of 100% juice and distilled water（control）. Cucumber endocarp juice with a mass fraction of 100% and 50% effectively inhibited cucumber seed germination, with inhibition rates of 100% and 73%, respectively, at 20 h. Throughout the process, the EtOH and n-BuOH layers inhibited the seed germination, at 26 h, the inhibition rates were 12.22% and 47.77% higher than those of the control, respectively. Myristic acid, palmitic acid, citric acid, and histidine were identified in the juice that affected seed germination. The soluble sugar content of the seeds was insignificant change after the 100% juice treatment process. From 0 to 20 h, ABA content was always higher than that of the control, and ABA/GA3 was significantly higher than that of the control. The expressions of the ABA synthase genes CsNCED1 and CsNCED2 rose from 10 to 15 h and from 15 to 25 h, respectively. After 10 h, the expressions of ABA signal transduction genes CsPYL2, CsPP2C2, CsSnRK2, and catabolic gene CsCYP707A1 increased significantly, with the highest expression of four genes detected at 20 h. Cucumber endocarp juice contains myristic acid, palmitic acid, citric acid, and histidine, which may decrease seed germination by increasing the expressions of ABA synthesizing CsNCED2 and signal transduction genes CsPYL2, CsPP2C2, and CsSnRK2, as well as inhibiting soluble sugar metabolism.

    Identification and Expression Analysis of Spinach PSY Gene Family
    REN Li, QIAO Shu-ting, GE Chen-hui, WEI Zi-tong, XU Chen-xi
    2023, 39(12):  169-178.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0392
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    PSY（phytoene synthase, PSY）is the first rate-limiting enzyme in the plant carotenoid synthesis pathway and plays an important role in plant growth and development. In order to understand the function of PSY gene family in spinach, the number, structure, promoter elements, amino acid characteristics and gene evolution of PSY gene family members in spinach were identified by bioinformatics method, and the expression pattern of PSY genes in spinach tissue and in response to the changes in the ratio of red to blue light was analyzed by real-time fluorescence quantitative PCR（RT-qPCR）. Meanwhile, the expressions of R1B3（red∶blue light = 1∶3）and R3B1（red light∶blue light = 3∶1）and the correlation between SoPSYs gene expression and total carotenoid content were analyzed. The results showed that there were 4 PSY genes in spinach, containing SQS_PSY conserved domain, and the SoPSY protein was a hydrophilic protein. PSY1 and PSY2 of spinach were closely related to beet in Amaranthaceae, and PSY3 and PSY4 were closely related to PSY3 in tomato. There were various light-responsive and phytohormone-related elements in the promoter sequence of spinach PSY genes. SoPSY genes had tissue expression specificity, SoPSY1, SoPSY2, and SoPSY3 were only expressed in the leaves, and SoPSY4 was mainly expressed in the roots. Spinach carotene content decreased after treatment with R1B3 and R3B1. Among spinach genotypes, the expression patterns of each SoPSY gene in response to the ratio of red to blue light were different. The expression of SoPSY1 was not significantly changed in cultivated spinach, the expression patterns of SoPSY2 were reversed in both spinach genotypes, and the relative expression patterns of SoPSY3 were consistent in the wild and cultivated spinach, and the relative expressions of these three genes was closely related to the change of total carotenoid content. This study lays the foundation for an in-depth understanding of the biological function of the PSY genes in spinach and their regulatory mechanism in response to changes in the ratio of red to blue light.

    Cloning and Prokaryotic Expression Analysis of MaMC6 in Banana
    TENG Meng-xin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI Jing-yang, LI Xin-guo
    2023, 39(12):  179-186.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0694
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    Metacaspases（MCs）is a regulatory gene of programmed cell death in plants, and it participates in the response of plants to stress. MaMC6, a member of MaMCs family, was cloned from Brazilian banana（Musa AAA Cavendishcv. Brazil）cells by PCR, and its bioinformatics analysis, expression pattern analysis and functional verification were carried out. The results showed that MaMC6 was composed of 320 amino acids, was a stable protein, hydrophilic, of no transmembrane structure, contained a caspase-like domain, belonged to type II metacaspase protein, and had a defense and stress response. The results of RT-qPCR analysis showed that the relative expression of MaMC6 after 100 mmol/L NaCl treatment was 7.70 times higher than that of the control at 2 h, and the gene expression at 2 h after CaCl2 treatment under salt stress was 3.99 times higher than that of the control, while the use of calcium chelating agent EGTA and calcium channel blocker LaCl3 increased the expression of MaMC6 under salt stress, reaching 9.92 and 11.20 times of the control at 2 h. The results of subcellular localization showed that MaMC6 was located in the cytoplasm and nucleus. The results of E. coli transformation showed that the growth of recombinant strain pET28a-MaMC6 was better than that of control strain pET28a under NaCl, mannitol, and high-temperature stress. To sum up, MaMC6 may be involved in the response of banana to abiotic stress. This study provides a reference for follow-up study on the role of MaMCs in banana stress resistance.

    Genetic Diversity Analysis and Molecular Identity Establishment of Clausena lansium Germplasm Resources from Guangdong Province Based on SSR Markers
    LU Yu-sheng, PENG Cheng, CHANG Xiao-xiao, QIU Ji-shui, CHEN Zhe, CHEN Hui-qiong
    2023, 39(12):  187-199.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0349
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    This work aims to reveal the genetic diversity and relationship among Guangdong wampee germplasms, and provide reference for the effective protection and efficient utilization of wampee germplasm resources. The wampee germplasms from Guangdong province were examined using 12 pairs of SSR primers to determine their genetic diversity. UPGMA was utilized for cluster analysis, while numerical and letter codes were assigned to create molecular identity cards. The results showed that the 12 pairs of primers generated a total of 43 alleles（Na）, with an average of 3.583 alleles per locus in the 84 analyzed materials. The expected heterozygosity（He）ranged from 0.294 to 0.665, with a mean of 0.524. The polymorphism information content（PIC）ranged from 0. 291 to 0.642, with a mean of 0.484. The Shannon information index（I）ranged from 0.567 to 1.096, with a mean of 0.913. The 84 germplasms were divided into six groups based on UPGMA clustering, and molecular identity was established for each examined germplasm resource. Guangdong wampee germplasm resources have high genetic diversity, while their clustering was not strictly conducted by their geographical origins. Based on the SSR genotyping, a digital and letter-based coding method was used to construct the molecular identity card for wampee resources.

    Heterologous Expression of Paeoniflorin Converting Enzyme G6046 and the Identification of Its Enzymatic Activities
    HAN Ying-yi, LI Zhang-han, CAO Xue-li, PEI Hai-run
    2023, 39(12):  200-208.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0653
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    Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that shows various protective effects on the cardio-cerebral vascular system and always used in traditional herbal medicine. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites and mainly metabolized in intestinal bacteria. In this paper，the induction expression and purification of paeoniflorin converting enzyme G6046 in Escherichia coli BL21 DE3 were studied. The induction reaction conditions were optimized to improve the enzyme yield and the optimal conditions for induction reaction were found to be 24 h induction reaction at 16℃. Enzyme production of bacterium reached 5.34 mg/g, representing a 51.69% increase in enzyme production. Based on the conditions of induction expression, the conditions（pH and temperature）for the reaction of G6046 with paeoniflorin were optimized, pH 9.0 and 45℃ were proved to be the optimized conditions, resulting in a specific enzyme activity of 14.56 U/mg. The reaction system was enlarged under this optimized condition, and the reaction scale was from G6046-substrate（1∶1; mg/mg）to G6046-substrate（1∶20; mg/mg）, the yield of the converted product significantly increased, which lays a foundation for the isolation of peonidin conversion product.

    Control Ability and Identification of Biocontrol Agent HK11-9 Against Corynespora Leaf Spot of Cucumber
    ZHANG Lin-lin, SHEN Hu-sheng, YANG Bing, HE Meng-han, PIAO Feng-zhi, SHEN Shun-shan
    2023, 39(12):  209-218.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0407
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    In order to explore the biocontrol ability of biocontrol agent HK11-9 against Corynespora leaf spot of cucumber, the antifungal activity of HK11-9 against Corynespora cassiicola were detected by in petri dishes experiments, the control effect of HK11-9 against Corynespora leaf spot of cucumber were detected by in vitro and in vivo inoculation experiments, the colonization ability of HK11-9 on cucumber leaves and its effect on the activities of defense enzymes were conducted by pot experiment, at the same time, the biological functions of HK11-9 was determined. The results showed that, HK11-9 significantly inhibited the mycelia growth and conidia germination of Corynespora cassiicola in the petri dishes experiment, HK11-9 presented significant control effect against Corynespora leaf spot of cucumber at the in vitro and in vivo inoculation experiment, the control efficiencies were 62.80% and 67.73% respectively. HK11-9 colonized stably in cucumber leaves, maintained a density of 104 CFU/g in cucumber leaves, and HK11-9 significantly improved the activities of defense enzymes in cucumber leaves. In addition, HK11-9 decomposed protein, cellulose and starch, produced siderophore, and HK11-9 demonstrated antifungal activity against a variety plant pathogenic fungi. Based on the morphological, physiological characteristics and 16S rRNA sequence analysis, the strain HK11-9 was identified as Paenibacillus polymyxa. In summary, the biocontrol agent HK11-9 has great potential to control Corynespora leaf spot of cucumber.

    Physiological Response and Drought Resistance Evaluation of Endophytic Bacteria to Sugarcane Under Drought Stress
    LUO Yan-ju, XIE Lin-yan, ZOU Qing-lin, LI Si-jie, LIU Han, LIU Lu-feng, HE Li-lian, LI Fu-sheng
    2023, 39(12):  219-228.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0818
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    This work aims to explore the physiological response of inoculated endophytic bacteria to sugarcane under drought stress and the effect of alleviating drought-tolerant, and to provide research basis for the development and utilization of sugarcane drought-tolerant functional strains and the promotion of drought resistance cultivation techniques in sugarcane. Four sugarcane endophytes with strong in vitro drought tolerance were selected as the target strains. Two soil water contents were set up in pot experiment, namely normal watering（CK: soil water content 25%-30%）and moderate drought stress（soil water content 10%-12.5%）, and a total of 8 treatments were inoculated with single endophyte, combined endophyte and no endophyte, respectively. Physiological and biochemical indexes of sugarcane seedling stage were determined, and drought resistance of each treatment was evaluated comprehensively. Compared with CK, drought stress promoted the accumulation of proline（Pro）, soluble protein（SP）, soluble sugar（SS）and malondialdehyde（MDA）in sugarcane plants. The activity of superoxide dismutase（SOD）and peroxidase（POD）increased. The contents of leaf relative water content（RWC）, water loss rate in vitro（RWL）and chlorophyll（ChI）decreased. Compared with the treatment without inoculation, the inoculation of endophytic bacteria effectively alleviated drought stress under drought stress, among which ZM strain inoculated alone（ZD）had the highest comprehensive evaluation value of drought resistance D, and the gray correlation analysis showed that SOD, ChI, RWC and Pro were closely related to the D values of each treatment. ZM strain inoculation has the best effect on alleviating drought stress of sugarcane seedling, and can improve the drought resistance of sugarcane seedling by increasing the contents of ChI, RWC, Pro and SOD activity.

    Isolation and Identification of Bacillus velezensis ZHX-7 and Its Antibacterial and Growth-promoting Effects
    LI Ying, SONG Xin-ying, HE Kang, GUO Zhi-qing, YU Jing, ZHANG Xia
    2023, 39(12):  229-236.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0152
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    In order to obtain highly effective biocontrol microorganism for controlling peanut diseases, using Aspergillus niger as the indicator, a highly effective biocontrol strain ZHX-7 was screened from soil samples by agar disk dilution and dual-culture assay. The soil samples were collected from the rhizosphere of diseased and healthy peanut plants in continuous cropping field in Laixi, Shandong province. The strain ZHX-7 was identified as Bacillus velezensis based on the morphological characteristics, 16S rDNA and gyrB sequence analysis. The bacterial suspension, sterile fermentation broth and volatile gas of ZHX-7 significantly inhibited the growth of Sclerotium rolfsii, Fusarium neocosmosporiellum, Phoma arachidicola and Alternaria alternata. The antagonistic experiment results showed that the inhibition rates of ZHX-7 suspension against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 64.40%, 70.37%, 77.43% and 65.32%, respectively. In the sterile fermentation broth of ZHX-7 diluted 10 times with PDA medium, the inhibition rates of ZHX-7 against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 67.81%, 52.16%, 23.52% and 69.93%, respectively. The double dish test showed that the inhibition rates of ZHX-7 volatile gas against the growth of S. rolfsii, F. neocosmosporiellum, P. arachidicola and A. alternata were 86.82%, 35.40%, 81.70% and 44.19%, respectively. ZHX-7 produced secondary metabolites such as protease, cellulose and amylase. In addition, under the condition of pot culture in greenhouse, ZHX-7 fermentation broth significantly promoted the growth of peanut. Compared with the control, the fresh mass and dry mass increased by 31.24% and 26.47%, respectively. ZHX-7 fermentation broth improved the resistance of peanut to S. rolfsii, and the control effect reached 51.76%. In conclusion, a strain of B. velezensis ZHX-7 screened in this study may inhibit a variety of peanut pathogenic fungi and promote peanut growth at the same time. It is a multifunctional strain with broad antagonistic spectrum against peanut diseases, and high potential for biocontrol application.

    Isolation and Identification of a Psychrotolerant Heterotrophic Nitrification-aerobic Denitrification Bacterium and Its Nitrogen-removing Characteristics
    DONG Yi-hua, WANG Ling-xiao, REN Han-xue, CHEN Feng
    2023, 39(12):  237-249.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0452
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    In order to improve the biological nitrogen removal efficiency of low-temperature wastewater, a psychrotolerant heterotrophic nitrification and aerobic denitrification bacterium was isolated from soil and river sediment in cold regions, and its nitrogen removal characteristics and pathway were investigated. The isolated bacterium was identified based on its colony and cell morphological observation as well as 16S rRNA gene sequence analysis. The nitrification, denitrification, and simultaneous nitrification and denitrification performance of the strain were investigated with NH4+-N, NO3--N, and NO2--N as the sole nitrogen source and NH4+-N and NO3--N as mixed nitrogen sources. The nitrogen-removing functional enzyme genes of the strain were amplified by polymerase chain reaction（PCR）to speculate the nitrogen-removing pathway at low-temperature. A heterotrophic nitrification and aerobic denitrification bacterium was screened from the river sediment and identified as Pseudomonas veronii, named P. veronii DH-3. When strain P. veronii DH-3 was cultured aerobically for 48 h at 10℃ with NH4+-N, NO3--N, and NO2--N as the sole nitrogen sources（N 105 mg/L）, the corresponding nitrogen removal efficiencies were 99.07%, 96.89%, and 90.29%, respectively, without accumulation of nitrite. When NH4+-N and NO3--N were used as mixed nitrogen sources, NH4+-N was completely removed in 48 h and the NO3--N removal rate was 87.09%, indicating that strain P. veronii DH-3 had excellent simultaneous nitrification and denitrification performance at 10℃. The nitrogen balance analysis results showed that when NO3--N and NO2--N were the sole nitrogen sources respectively, the conversion rates of nitrogenous gas and intracellular nitrogen were lower than those of NH4+-N, indicating that the heterotrophic nitrification ability of the strain DH-3 was stronger than that of aerobic denitrification. Meanwhile, the successful expressions of denitrification functional genes hao, napA, nirS, nirK, cnorB, and nosZ further confirmed that the heterotrophic nitrification and aerobic denitrification ability of strain DH-3. Based on the above results, it was speculated that the main nitrogen-removing pathway of the strain was heterotrophic nitrification, aerobic denitrification, and assimilation. These findings indicate that the psychrotolerant P. veronii DH-3 possesses excellent heterotrophic nitrification and aerobic denitrification capability, which provides the theoretical support for biological purification of low-temperature nitrogen wastewater.

    Screening of a Heterotrophic Nitrifying-aerobic Denitrifying Bacterium and Its Nitrogen Transforming Characterization
    JIANG Hui-hui, WANG Qiang, FU Wei-lai, RAO Zhi-ming, ZHANG Xian
    2023, 39(12):  250-260.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0614
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    The excessive nitrogen in aquaculture easily causes the deterioration of water quality and threatens the growth and reproduction of aquatic animals, thus a safe and efficient nitrogen removal method is urgently needed. An ammonia-degrading bacterium was selected from the sludge at the bottom of an aquaculture pond in Fujian province. After the strain identification, the adaptive domestication was carried out. The effects of different conditions on the growth of the strain and its ammonia-nitrogen degradation were studied. Subsequently, nitrite was used as the only nitrogen source to detect the aerobic denitrification characteristics of the strains. The strain was identified as Bacillus aryabhattai by 16S rRNA and named as JN01. After acclimation, the growth of the strain and removal rate of ammonia nitrogen increased at varied degrees, and the removal rate of nitrogen was up to 87.29% with the initial NH4+-N concentration was 200 mg/L. The results of single factor experiment showed that the degradation rate of ammonia nitrogen of the strain reached 92.78% when NH4+-N concentration was 200 mg/L, sodium succinate as carbon source, carbon to nitrogen ratio was 15∶1, pH 7.5 and culture temperature was 30℃. Under the condition that nitrite nitrogen was the only nitrogen source, strain JN01 also performed aerobic denitrification, and the degradation rate of NO2--N was 82.30%. In conclusion, B. aryabhattai JN01 has promising heterotrophic nitrification and aerobic denitrification characteristics, and has the potential to concurrently solve the excessive ammonia nitrogen and nitrite in aquaculture wastewater.

    Studies on Anti-inflammatory Activity and Chemical Diversity of Secondary Metabolites from Symbiotic Fungi in Stony Corals
    LIAO Qing-nan, ZHOU Long-jian, YANG Zhi-you, FENG Yun-kai, HUANG Yi-jun, HU Xue-qiong, ZHANG Yi, LIU Ya-yue
    2023, 39(12):  261-275.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0501
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    Stony coral symbiotic fungi were used to investigate the effects of different culture conditions on the anti-inflammatory activities and chemical diversities of their secondary metabolites. Based on the OSMAC（one stain many compounds）strategy, 31 strains of stony coral-derived symbiotic fungi were cultured in small-scale fermentation with potato dextrose broth（PDB）and brown rice medium at three salinities（0.3%, 3.0%, and 10.0%）, respectively. Lipopolysaccharide（LPS）-induced BV-2 microglia were used as the inflammation model, from which the strains with anti-inflammatory activity were selected. Via thin-layer chromatography（TLC）,ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry（UPLC-QTOF-MS）and feature-based molecular networking（FBMN）, the chemical diversity of the secondary metabolites from the anti-inflammatory active strains was further analyzed. The results showed that five strains demonstrated significant anti-inflammatory activity under the brown rice medium conditions and showed moderate or strong inhibitory activity against LPS-induced nitric oxide（NO）release in BV-2 cells, with a significant concentration-dose dependence. While the crude extract of strain C3-9 also showed the significant inhibition to NO production at low concentrations of 10 and 20 µg/mL, further TLC and FBMN analyses revealed that the secondary metabolites of strain C3-9 were rich in anti-inflammatory compounds and highly diverse. The secondary metabolites of the stony coral co-epiphytic fungus C3-9 have significant anti-inflammatory activity and diversity, providing a basis for the subsequent discovery of anti-inflammatory active compounds.

    Extraction of Polysaccharide from Hericium corallinum and Analysis on Its in vitro Antioxidant Activity
    TU Xiao-yuan, CHU Lu-lu, WANG Mian, CHEN Bing-zhi, JIANG Yu-ji
    2023, 39(12):  276-286.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0710
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    Hericium corallinum, a newly cultivated and domesticated species, has a high content of polysaccharides with physiologically active properties such as antioxidation. Using the polysaccharide extraction rate as a benchmark, the response surface methodology was utilized to optimize the hot water extraction process of H. corallinum polysaccharides（HCP）. UV, FT-IR and X-ray diffraction were used to preliminarily characterize HCP, and its antioxidative activity in vitro was measured. The results showed that the optimal process parameters for extracting HCP were an extraction temperature of 93℃, an extraction time of 2.0 h, a material-to-liquid ratio of 1∶33（g/mL）, and a particle size of 80 mesh. The characterization results showed that HCP contained a small amount of proteins and nucleic acids, with typical absorption peaks of polysaccharide compounds and pyranose ring structure, and showed the presence of crystalline and non-crystalline structures. In vitro antioxidant experiments showed that HCP had good scavenging effects on DPPH, ABTS+ and ·OH radicals, and their EC50 values were 0.663 mg/mL, 0.767 mg/mL and 0.952 mg/mL, respectively. HCP has good antioxidant activity, and the results lay a research foundation for further exploring the antioxidant activity and other effects of coralline HCP in vivo.

    Genome Comparison and Virulence Factor Analysis of Pathogenic Escherichia coli from Porcine
    WU Li-dan, RAN Xue-qin, NIU Xi, HUANG Shi-hui, LI Sheng, WANG Jia-fu
    2023, 39(12):  287-299.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0578
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    The pathogenicity of different strains of porcine-derived Escherichia coli varies greatly. By focusing on the types and quantities of virulence factors carried in the genomes of the strains, we can analyze the molecular mechanism of porcine-derived E. coli causing disease. Next-generation high-throughput DNA sequencing technology was used to determine the genomic DNA sequence of porcine-derived pathogenic E. coli, and bioinformatics methods were adapted to analyze the genome structural characteristics, evolutionary types, virulence factor genes and base variation of different virulent strains. PCR method was to verify the candidate virulence factor genes. The full lengths of the genomes from different virulent strains of porcine pathogenic E. coli ranged from 4.62 Mb to 5.3 Mb, containing 3 364 to 3 557 coding genes. Most phylogenetic groups and multi-locus sequence typing of strains belonged to group A and ST10 clonal complex groups. Six virulent strains carried 112 to 280 virulence factor genes. Virulence factor genes, especially toxin genes, in strong virulent strains were more than that in the weak virulent strains, and the number of genes in the secretion system of each strain varied greatly. In addition, there were high-impact variations（e.g., insertion deletion, frameshift mutation, and nonsense mutation）in virulence factor genes. And representative virulence factor genes and their mutations were confirmed by PCR method. The virulence of porcine-derived pathogenic E. coli was correlated with genome size and number and variation in coding genes, and was not necessarily related to the GC content, prophage, and CRISPR sequence in genome.

    Comparative Mitochondrial Genome and Phylogenetic Analysis of Atteva charopis Turner, 1903
    LIN Xing-yu, SONG Nan
    2023, 39(12):  300-310.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0171
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    Mitochondrial genomes（mitogenomes）advance our understanding of molecular evolution and phylogenetic relationships in Yponomeutoidea. In this study, we sequenced and analyzed the mitochondrial genome of Atteva charopis Turner, 1903. We reconstructed the phylogenetic relationships of Yponomeutoidea based on the nucleotide and amino acid data of the mitochondrial genome. In phylogenetic analyses, we selected 10 exemplars of Yponomeutoidea as ingroups，and two species of Tineoidea（Phyllocnistis citrella and Caloptilia theivora）as outgroups. The phylogenetic analyses were conducted using Maximum likelihood and Bayesian inference methods. The mitochondrial genome of Atteva charopis was a circular molecule of 15 163 bp in length（GenBank accession No. OQ130005）, containing 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. Compared to the putative insect trn gene（trnI-trnQ-trnM）, there has been a trn gene rearrangement（trnI-trnM-trnQ）in this mitochondrial genome. Both phylogenetic inference methods produced a similar tree topological structure:Family Lyonetiidae, Praydidae and Attevidae were the monophyletic groups. However, the family Plutellidae was the non-monophyletic group. The family Attevidae was closely related to Praydidae. In addition，A. charopis was the sister to A. aurea. Our results provide a mitochondrial genome data for the intensive study of the phylogeny of Yponomeutoidea.

    Study on the Function of Phosphomevalonate Kinase in Aspergillus oryzae
    SHANG Yi-tong, YAN Huan-huan, WANG Li-hong, TIAN Xue-qin, XUE Ping-hong, LUO Tao, HU Zhi-hong
    2023, 39(12):  311-319.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0702
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    Phosphomevalonate kinase（PMK）is a key enzyme in the mevalonate（MVA）pathway. In fungi, the MVA pathway is the upstream of ergosterol biosynthesis and therefore PMK is also known as Erg8. In order to study the role of phosphomevalonate kinase in ergosterol synthesis pathway of Aspergillus oryzae, the function of AoErg8 gene in A. oryzae was preliminarily studied. Bioinformatics method was used to identify AoErg8. Phylogenetic tree and yeast allogeneic complementation were used to analyze whether this gene was conserved. Then the subcellular location was determined via fluorescence protein labelling and gene expression pattern were analyzed via RT-qPCR. Finally, the effects of overexpression of this gene on the growth and ergosterol content of A. oryzae were determined. AoErg8 was evolutionarily conservative, and its expression was different under different growth times and different abiotic stress. AoErg8 restored the temperature sensitive phenotype of Saccharomyces cerevisiae erg8 mutant. AoErg8 was located in the cytoplasm. The overexpression of AoErg8 led to the decrease of ergosterol content, and affected growth and spore formation. Therefore, the function of AoErg8 in A. oryzae was conservative and its overexpression reduceed the ergosterol content, also affected the growth of colonies and sporulation. This study further reveals the biosynthesis and regulatory mechanisms of ergosterol in this important filamentous fungus A. oryzae and lays the foundation for genetic engineering for lipid metabolism in A. oryzae or other fungi.

    Heterologous Expression, Enzymatic Characterization of Laccase BmLac and Degradation of Gossypol by It
    WEI Ting-liu, MIAO Hua-biao, WU Qian, HUANG Zun-xi
    2023, 39(12):  320-328.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1469
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    To develop enzymatic proteins for the efficient degradation of gossypol, a strain of B. amyloliquefaciens XP-13 was screened from soil, the gene BmLac was amplified from its genome, then the recombinant plasmid Ppic9k-BmLac was constructed and introduced into Pichia pastoris GS115 for heterologous expression. The characteristics of the laccase BmLac and the degradation effect of gossypol were studied. The results showed that the enzyme activity was 101.56 U/mL at pH 4 and 55℃ under the optimal reaction conditions with ABTS as the substrate; the relative enzyme activity was retained over 70% after incubation at 90℃ for 1 h; the relative enzyme activity was retained over 96% after incubation at pH 3-7 for 1 h. The enzyme activity increased 1.93 times by adding 1 mmol/L Cu2+, but high concentrations（10 mmol/L）of Fe2+, Fe3+ and Al3+ completely inhibited the enzyme activity. After reaction at 55℃ for 2 h, the degradation rate of gossyrol by this enzyme reached 94.57%（without mediator）and 98.73%（with ABTS）. In conclusion, the laccase BmLac has good thermal stability and wide pH adaptability and can effectively degrade gossypol, and this enzyme provides the basic support for the effective degradation of free gossypol in the cottonseed meal.