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    26 July 2022, Volume 38 Issue 7
    Advances in Histone Variants in Plant Epigenetic Regulation
    XUE Man-de, ZHAO Feng-yue, LI Jie, JIANG Dan-hua
    2022, 38(7):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0071
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    The basic subunit of chromatin is nucleosome that consists of DNA and histone proteins,and it serves as a carrier of genetic and epigenetic information in regulating life activities. Histone variants are related isoforms in the same histone family. In addition to their sequence divergence,their chromatin assembly is done under specific molecular chaperones. The incorporation of histone variants into nucleosomes generates profound impacts on nucleosome property and chromatin activity. Growing studies in plants support a pivotal role of histone variants in chromatin regulation including chromatin structure and maintenance of the epigenetic information,DNA damage repair and transcription regulation,which subsequently impact plant development and environmental response. Here we summarize recent progress on the chromatin locations of plant histone variants,molecular chaperons and their biological functions,and we propose a few new directions for the study of plant histone variants,aiming to provide a reference for deeply studying the histone variant-mediated epigenetic regulation.

    Research Progress in Histone Demethylase in Plant
    TANG Qian-qian, LIN Chu-yu, TAO Zeng
    2022, 38(7):  13-22.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0165
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    Histone methylation plays an important role in regulating gene expression in eukaryotic cells. Histone methylation is dynamically regulated by histone methyltransferases(HMTs)and demethylases(HDMs),among which methylation on lysine residues is the most common. In this paper,the latest research progress of plant histone lysine demethylase is systematically summarized,and its important role and regulation mechanism in the growth and development process of plant flowering time regulation and circadian rhythm regulation,as well as the responses of plants to drought,temperature,pathogenic bacteria and other stresses are expounded. The paper provides reference for further utilization of plant histone lysine demethylase in crop genetic improvement.

    Research Progress in the Roles of DNA and Histone Methylations in Epigenetic Regulation
    ZHANG Miao, YANG Lu-lu, JIA Yan-long, WANG Tian-yun
    2022, 38(7):  23-30.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1054
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    In eukaryotes,as a common mechanism of epigenetic regulation,DNA and histone methylations mainly affect chromatin structure and gene transcription expression,especially in gene expression regulation of mammalian cells and maintaining genome stability. This paper reviews DNA methylation modification and its role in gene expression regulation,gene expression regulation mode,function and research progress of histone methylation modification,as well as the interaction between DNA and histone methylations in epigenetic regulation.

    Research Progress in the Structural and Functional Analysis of Mammalian DNA Methyltransferase DNMT1 and DNMT3
    WANG Chen-chen, ZHANG Fan-li, CHEN Pei-qi, WENG Si-yao, WANG Hui-fang, CUI Xiao-juan
    2022, 38(7):  31-39.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1589
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    The DNA methyltransferases are an important protein family involved in epigenetic modification,and DNMT1,DNMT2,DNMT3A,DNMT3B and DNMT3L are the main members in mammalian. Particularly,the methyl group are transferred to the fifth carbon atom of cytosine catalyzed by DNMT1,DNMT3A and DNMT3B,which are involved in numerous biological processes of early embryonic development,germ cell development,learning and memory,and disease occurrence. In recent years,with the development of the study on the DNA methylation,the mechanism of DNA methyltransferases has attracted extensive attention from scientists. In this paper,the structural characteristics of mammalian DNA methyltransferases,the dynamic regulation of mammalian DNA methylation,and the role of DNA methyltransferases in ontogeny and disease occurrence are reviewed.

    Research Progress in Chromatin Immunoprecipitation Followed by Sequencing
    CHEN Gui-fang, YANG Jia-yi, GAO Yun-hua, REN Ge
    2022, 38(7):  40-50.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0807
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    Chromatin immunoprecipitation followed by sequencing(ChIP-seq)is an important method to study the interaction between DNA and target proteins,it has been widely used to study transcription factor and the functions and distributions of post-translation histone modification. In recent years,researchers have been optimizing the key steps of this method,including cell separation,chromatin fragmentation,immunoprecipitation and sequencing library construction,allowing the ChIP-seq suitable for the analysis of small amount of cells. In cleavage under targets and release using nucleus(CUT&RUN)and cleavage under targets and tagmentation(CUT&Tag),the enzyme is “targeted” to the target protein through specific antibodies,and the chromatin near the target protein is digested by MNase or Tn5 transposase,which simplifies the operation process. The paper provided an overview of the principle behind ChIP-seq and its data analysis methods,compared the ChIP-based optimization methods and derived methods,and summarized the studies of transcription factors and histone modification in the regulation of biological clock,hormone signal transduction,signal transduction pathway and stress response in the process of plant growth and development,as well as the application of ChIP-seq technique.

    Expression Pattern Analysis of Histone Methyltransferase Under Drought Stress in Hybrid Wheat
    CHEN Jia-min, LIU Yong-jie, MA Jin-xiu, LI Dan, GONG Jie, ZHAO Chang-ping, GENG Hong-wei, GAO Shi-qing
    2022, 38(7):  51-61.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1524
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    The aim of this study is to screen the histone methyltransferases involved in the drought stress of wheat hybrids,and to provide functional markers for the improvement of stress resistance of two-line hybrid wheat. Screening of the total 175 TaHMT family genes in the wheat through the public databases on the website and the comparative genomics strategies,we obtained 8 candidate histone methyltransferases genes induced by drought stress(TaHMT21TaHMT24TaHMT31TaHMT42TaHMT49TaHMT105TaHMT143TaHMT157). The physical and chemical properties,protein structures,phylogenetic relationships,gene structures and conserved motifs of 8 TaHMTs were systematically analyzed via bioinformatics. Using drought treated hybrid wheat combinations(BS278*09YH91-5 and parental)as experimental materials,the candidate genes of 8 histone methyltransferase were verified via quantitative fluorescent PCR. It was found that their expressions were induced by drought stress,and the expression pattern presented mode of up-regulation first and then down-regulation with the drought stress time prolonged. The expressions of gene TaHMT21TaHMT24 and TaHMT42 accorded with the phenotypes of hybrid combinations,indicating that they may regulate the drought resistance of hybrids. These results provide important clues to deeply study the function of wheat TaHMT family genes in the drought resistance of hybrids as well as also provide excellent stress resistance gene resources for wheat molecular breeding.

    Optimization and Application of the Chromatin Immunoprecipitation Based on Rice Protoplast
    SHI Jia, ZHU Xiu-mei, XUE Meng-yu, YU Chao, WEI Yi-ming, YANG Feng-huan, CHEN Hua-min
    2022, 38(7):  62-69.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1592
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    Transcription factors(TFs)are important regulatory proteins that control gene expression and regulate various biological functions in plant. Chromatin immunoprecipitation(ChIP)is a very widely used technique to investigate the direct target genes of TFs. However,ChIP is always limited by antibody specificity or time-consuming construction of genetic materials. In this paper,the chromatin immunoprecipitation system based on rice protoplasts(ChIP-RP)was established by optimizing the transient transformation of rice protoplasts,formaldehyde fixation and ultrasonic fragmentation of immunoprecipitated nucleic acid. Moreover,the enrichment of target genes by OsNF-YA4 protein,a transcription factor of rice,were verified via ChIP-RP method. This technical system will greatly reduce the limitations of antibody specificity or constructing stable genetic materials,facilitate rapid screening and verification of direct target genes of rice transcription factors,and promote the deciphering of the molecular mechanism of rice transcription factor regulatory functions.

    Expression Analyses of m6A Methylase Genes in Bovine Adipogenesis
    YANG Xin-ran, WANG Jian-fang, MA Xin-hao, ZAN Lin-sen
    2022, 38(7):  70-79.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1231
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    The aim of this study is to investigate the mRNA expressions of relevant genes,including METTL3,METTL14,WTAP for m6A methyltransferase,and FTO and ALKBH5 for m6A demethylaes,in bovine various tissues and in the proliferation and differentiation of preadipocytes,aiming to provide a reference for clarifying the role and mechanism of m6A modification in adipogenesis. Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expressions of m6A methylases in the various tissues of newborn and adult Qingchuan beef cattle. Then,CCK-8 treatment and oil-red O staining were adapted to detect the growth curves of the preadipocytes cells and lipid droplet formation during adipogenic differentiation. The 5 genes expressed lowly in skeletal muscles,and highly in rest tissues,while the mRNA expressions of them in the adipose tissue of adult cattle was significantly higher than that of newborn cattle(P < 0.05). The isolated preadipocytes had a good growth state and adipogenic differentiation was accordance with the general pattern. The expressions of METTL3,METTL14 and WTAP decreased in the early stage of bovine preadipocytes proliferation,then increased and remained at low level. The expression of FTO showed a slow upward trend. The temporal expression of ALKBH5 increased in the early stage,decreased in the metaphase,and increased continuously after the day 4(P < 0.01). The expression patterns of METTL3,METTL14 and FTO were similar in the adipogenic differentiation of bovine preadipocytes,which decreased in the early stage of differentiation,and highly expressed in the metaphase,then decreased. The expressions of WTAP and ALKBH5 increased with the progress of differentiation. Our results indicate that m6A modification plays a potentially important role in fat development and deposition of Qinchuan cattle,and m6A methylases may regulate the lipogenesis of bovine preadipocytes in vitro.

    Sugar Signaling and Its Role in Plant Response to Environmental Stress
    CHEN Hong-yan, LI Xiao-er, LI Zhong-guang
    2022, 38(7):  80-89.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1289
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    Sugar is not only carbon and energy sources as well as structural material in plant cells,but also a signaling molecule,which plays a key role in plant growth,development,and response to environmental stresses. Abiotic stresses,such as high temperature,low temperature,drought,salinity,and heavy metal,are the major stress factors limiting crop yield. The exact mechanism of sugar,as signaling molecule,in plants response to these stresses still remains unclear. Based on the current advance in sugar signaling and its role in the formation of stress tolerance in plants,the hexose kinase(HXK)-,regulator of G protein signaling 1(RGS1)-,glycolysis(EMP)-,and pentose phosphate pathway(PPP)-dependent signaling pathways were separately summarized;and the role of sugar signaling in the developing of plant stress tolerance,including heat,cold,drought,salt,and heavy metal stress tolerance was discussed. In addition,the future research direction of H2S signaling in the field of plant biology was prospected.

    Research Progress of Sterol in Regulating Plant Growth and Development
    LI Ping, GUO Fa-ping, TIAN Min, SHUI Yang, XU Na-na, BAI Da-song, YU De-jin, ZHANG Jie, HU Yun-gao, PENG You-lin
    2022, 38(7):  90-98.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1181
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    Sterols are isoprene compounds and play an important role in the growth and development of organisms. Sterols are not only structural components of eukaryotic cell membrane,but also precursors of sterol hormone biosynthesis,which play a key role in plant cell division,embryogenesis and development,and participating in stress. In plants,the multiple functions of sterol derived brassinosteroids(BRs)in growth and development have been extensively studied. BRs as a class of plant hormone,cooperate with other hormones to play a variety of functions in plant growth and development. BRs play an important role in all aspects of plant life cycle from cell division,cell expansion,stomatal conductance and root development. In addition to these functions,BRs,as an important product of the phytosterol synthesis pathway,act as an important signal molecule responding to adversity stress and regulating plant morphogenesis. This article outlines the research progress of the related genes in the BRs synthesis pathway,and summarizes the biosynthesis of brassinolide and its research progress in regulating plant growth and development. Finally,the research prospects of brassinolide are discussed and prospected.

    Nutritional Interactions Between Symbiotic Microbiota and Insect Hosts
    WANG Zheng-yan, HU Hai-sheng, YONG Han-zi, LU Yu-jie
    2022, 38(7):  99-108.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1166
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    There are a wide range of nutritional symbiosis between symbiotic bacteria and insect hosts. Insect hosts provide nutrition and energy sources to symbiotic bacteria,and symbiotic bacteria can provide essential nutrients to insects,help insects digest food,and participate in nitrogen cycle and maintain normal physiological environment of insect hosts. The shared metabolic pathways between certain symbiotic bacteria and insect hosts provide impetus for host-symbiont coevolution. To provide deep insight into the cross-feeding between symbiotic bacteria and hosts,this paper reviewed nutritional function of symbiotic bacteria on insect hosts,the transshipment mode of nutrients between symbiotic bacteria and insects,and the regulation of insects on the production and transshipment of nutrients from symbiotic bacteria.

    Research Progress in Modern Taxonomy and Nomenclature of Aspergillus
    LIU Jing-ju, ZHANG Yu-sen, CHEN Juan, SUN Bing-da, ZHAO Guo-zhu
    2022, 38(7):  109-118.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0779
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    The discovery and use of Aspergillus fungi have been a long history,while its taxonomy constantly changes. According to articles of ‘International code of nomenclature for algae,fungi and plants’(Melbourne Code,July,2011),“one fungus,one name,1F1N” has been applied to fungi since January 1,2013. It changed the rule of that the fungi could have multiple names and teleomorph could represent the holomorph,which had a great impact on Aspergillus taxonomy. Since then,Aspergillus and its related genera had been systematically revised on a large scale in the world. The new revisions paid more attention to the results of molecular phylogeny,and the standard modern polyphasic methods recommended for Aspergillusclassification mainly included:I. Molecular biology by determining universal ITS rDNA molecular barcode and specific second barcode(calmodulin CaM,β-tubulin BenA and RNA polymerase II second subunit RPB2,etc.);II. morphological characteristics(substrate,standard medium,incubation conditions,colony,conidial head,ascospores,etc.);III. extrolite analysis(toxins,enzymes,organic acids,antibiotics and other secondary metabolites,etc.). The modern classification of Aspergillus formed by the new classification system retainsAspergillus as the only legal generic name,including 446 species,subdivided in 6 subgenera,27 sections and 75 series,Aspergillus glaucus was the type species. All related teleomorphic genera and species(names)were abolished,combined,or removed independently. In this paper,we briefly summarized the taxonomic history of Aspergillus,focusing on revision changes,modern taxonomy and genome-wide analysis techniques of Aspergillus in its study under the influence of new article of Melbourne Code,also including some comments and suggestions about present research status of Aspergillus in China. The purpose of this study is to provide reference for the standardization of classification,nomenclature,use of legal names,and the mining of Aspergillus fungi and gene resources.

    Biosynthesis of Natural Carotenoids:Progress and Perspective
    ZHOU Lin, LIANG Xuan-ming, ZHAO Lei
    2022, 38(7):  119-127.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1184
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    Carotenoids are the general term for a large class of natural pigment substances broadly in higher plants,microorganisms and algae,one of important natural pigments groups for food. Cell factories are widely used in the production of various chemicals,food and drugs,and are the central link of green biological manufacturing. It is economically feasible and environmentally friendly to synthesize source-scarce,difficult-to-be-obtained and high value-added natural carotenoids in cell factories. Carotenoids play important roles in anti-oxidation,eye-vision protection and anti-cancer. In the present review,the analysis of carotenoid biosynthesis pathway,the selection and modification of chassis cells,the module assembly and adaptation of pathway,and the strategy of synthetic biology during the construction of cell factory used for carotenoids biosynthesis are introduced in details. Future opportunities and challenges in the industrial production of carotenoids are discussed.

    Role of Gut-Liver Axis in Animal Nutritional Metabolism and Immunity
    JIANG Xian-zhe, ZHANG Bo-yan, LUO Hai-ling, ZHANG Xin-meng, WANG Bing
    2022, 38(7):  128-135.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1322
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    The gut-liver axis is a bidirectional relationship between intestinal microorganisms and the liver,which is essential for the intestinal microbe to affect the host. Growing studies have shown that the gut-liver axis plays a key regulatory role in animal nutrition metabolism and body immunity. Based on the bridge role of the gut-liver axis in the interaction between intestinal microbe and host,this review discusses two regulatory mechanisms of gut-liver axis in animal nutrition metabolism and body immunity:endocannabinoid system and gut microbiota-derived signals. Gut microbiota-derived signals include short-chain fatty acids,bile acids,choline metabolites,amino acid metabolites,and microbial-related molecular patterns. Finally,the paper discusses application prospect of gut-liver axis theory in animal husbandry production,aiming to provide support for healthy and efficient animal feeding without antibiotics.

    Research Status and Challenges of COVID-19 Vaccine
    WEN Ya-ya, SONG Li, WANG Qiao-ju, PAN Zhi-ming, JIAO Xin-an
    2022, 38(7):  136-145.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1042
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    The outbreak of COVID-19 at the end of 2019 has caused social and economic chaos around the world,which is a serious threat to human life and health. With the continuous efforts of countries around the world in the development of COVID-19 vaccine,a number of vaccines have been approved for emergency use or registered on the market. This article summarizes the clinical trial results,safety and effectiveness of 11 vaccines currently used at home and abroad under COVID-19 pandemic,as well as briefly analyzes the challenges faced in the development and application of the current vaccine.

    Establishment of Real-time Fluorescent Quantitative PCR Detection Method for Genetically Modified Herbicide-tolerant Soybean GE-J12
    CAO Ying-fang, ZHAO Xin, LIU Shuang, LI Rui-huan, LIU Na, XU Shi-yong, GAO Fang-rui, MA Hui, LAN Qing-kuo, TAN Jian-xin, WANG Yong
    2022, 38(7):  146-152.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1330
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    GE-J12 is a new herbicide-resistant soybean line with G2-EPSPS gene and GAT gene developed by the Chinese Academy of Agricultural Sciences. In this study,primers and probes were designed based on the specific sequence of the transformant at the 5' end of the insertion site of the exogenous gene in the herbicide-resistant soybean GE-J12 strain. A realtime fluorescent quantitative PCR detection method based on TaqMan hydrolysis probe was established,after primer probe screening,specificity testing,reaction system and program optimization,and standard curve construction,detection limit and quantitative limit test and other tests. This method presents strong specificity and high accuracy. When the RSD was < 25%,the detection limit was 0.032%,the quantification limit was 0.16%,and the linearity was > 0.992. After testing with 5%,3%,and 1% fixed-value sample,the average deviation of the true value was 2.87%-16.67%. This method can be used to accurately quantify the specific sequence content of genetically modified herbicide-tolerant soybean GE-J12 transformants,which provides a new method for the detection of new soybean lines of GE-J12.

    Development and Application of the Combinatorial Marker for the Rice Blast Resistance Gene Pigm
    LI Bai, CAI Zhi-jun, WANG Lei, CHEN Jie, CAO Kui-rong, LI Jun, CHONG Gao-jun
    2022, 38(7):  153-159.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1228
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    Pigm is one of the rice blast resistance genes,with broad-spectrum and lasting resistance. A variety of molecular markers have been developed for Pigm gene,but the detection specificity or recognition is still insufficient. Seven Pigm markers were analyzed by NCBI,including Pigm-4,T9E4,DG-2,M80410,R060939,GMR-3 and T-Pigm,and total 40 rice varieties species were detected and verified. According to the sequence analysis and detection results,a combinatorial marker was developed and applied to Zhennuo 19 × Kongyu 131(Pigm)molecular marker-assisted selection. Sequence analysis showed that M80410 marker had strong specificity,but could not distinguish heterozygous genotypes;Pigm-4,T-Pigm and T9E4 etc. markers distinguished heterozygous genotypes,but their specificity was slightly insufficient. Combined with the detection results of rice varieties,Pigm-4 and M80410 were selected as combinatorial marker and used in assisted breeding. The glutinous rice line NK29 was selected,which had good resistance to rice blast. Combinatorial marker can quickly and accurately identify the homozygous dominant,homozygous recessive and heterozygous genotypes of Pigm,which lays a foundation for Pigm molecular breeding.

    Structural and Functional Characterization of AOX Gene Family
    WANG Hui, MA Yi-wen, QIAO Zheng-hao, CHANG Yan-cai, ZHU Kun, DING Hai-ping, NIE Yong-xin, PAN Guang-tang
    2022, 38(7):  160-170.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1285
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    AOX(alternative oxidase,AOX)is a terminal oxidase in the respiratory pathway of plants. Through the identification and analysis of AOX genes in 8 species of Zea mays,Oryza sativa,Arabidopsis thaliana,Brachypodium distachyon,Solanum lycopersicum,Solanum tuberosum,Sorghum bicolor and Glycine max,we aim to further explore the function and regulatory mechanism of AOX gene. A total of 35 genes encoding AOX protein in these 8 species were identified,and these 35 genes were divided into 5 subfamilies by cluster analysis. The phylogenetic analysis and comparison of the chromosome position exon-intron structure and the conserved motif composition of AOX further supported the subfamily division model,and revealed that the AOX gene maintained a strong conserved nature during the evolution process. The prediction of cis-acting elements,miRNA target sites and the GO annotation analysis of AOX protein revealed that AOX gene may play an important role in plant growth and development and stress response. After stress treatment at the seedling stage of maize,the qRT-PCR analysis of 4 AOX genes in maize showed that the 4 AOX genes presented different degrees of inducible expression,suggesting that they were closely related in response to abiotic stress. These results lay a foundation for further understanding the biological function of AOX genes and their regulatory mechanism in response to abiotic stress.

    Genetic Analysis and Gene Mapping of Sorghum Double-grain Mutant Dgs
    ZHOU Shi-chen, YI Zhi-ben, WANG Xin-yi, YANG Xiao-ying, SUN Li-na, LUAN Wei-jiang, LIANG Shan-shan
    2022, 38(7):  171-177.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1294
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    Grain number per panicle is very important for crop yield. In order to identify and clone the gene controlling grain number per panicle,a double-grain mutant gene of sorghum[Sorghum bicolor(L.)Moench]was located,for laying a foundation for gene cloning and functional analysis and providing evidences for the regulation and analysis of sorghum yield traits. The flower organ phenotypes of wild-type sorghum plant and double-grain mutant were observed by stereoscopy. A F2 population was constructed from the cross between S. bicolor Jin 5 and the double-grain mutant to analyze the inheritance pattern of the mutant and gene mapping. BSA-seq sequencing and map cloning were used for linkage analysis and gene mapping. The results showed that,compared with wild type,the mutant mainly showed an increase in the number of pistil and stamen,ultimately two kernels in one pair of glumes. Genetic analysis indicated that the double-grain trait was controlled by a pair of dominant genes. Gene mapping showed that the target gene was located between markers Indel2930 and SSR7060 on the chromosome 6 of S. bicolor with physical distance of 404 kb. Dgs is a novel sorghum double-grain mutant gene.

    Analysis of Expression Characteristics of CmABCG8 Gene in Cucumis melo L.
    HONG Tian-shu, HAI Ying, ENHE Ba-ya-er, GAO Feng
    2022, 38(7):  178-185.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1117
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    ABC transporter is a kind of proteins with extensive functions,which can actively mediate the transport of various molecules,but its study in melon(Cucumis melo L.)is still unclear. In this paper,the characteristics of ABC transporter subfamily G member CmABCG8 were analyzed by bioinformatics method,and the expression characteristics of CmABCG8 in different fruits and tissues at different periods and their responses to stress were detected by fluorescence quantitative PCR. The phylogenetic tree results showed that CmABCG8 had the closest relationship with cucumber(Cucumis sativus L.)CsABCG23. CmABCG8 was expressed in all tissues of melon,and was highly expressed in the gynoecium and leaves. CmABCG8 had 10 interacting proteins. The results of RT-qPCR showed that the expression of CmABCG8 was the highest at 10 d. CmABCG8 expression was the highest in the leaves among different tissues of melon. After treatment with cytokinin,brassinosteroid and hydrogen peroxide,the expression of CmABCG8 presented an upward trend with the increase of the treatment concentration. After treatment with Fe2+,Cu2+ and Mn2+,the expression of CmABCG8 showed a downward trend. Therefore,it was analyzed that CmABCG8 had a certain influence on the early development of melon fruit and had different degrees of response to plant hormones and metal ions. Plant hormones could effectively induce the expression of CmABCG8.

    Cloning and Functional Analysis of Gynostemma pentaphyllum GpMIR156a and GpMIR166b
    YU Qiu-lin, MA Jing-yi, ZHAO Pan, SUN Peng-fang, HE Yu-mei, LIU Shi-biao, GUO Hui-hong
    2022, 38(7):  186-193.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1072
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    This study aims to deeply understand the role of two important miRNAs involved in the Gynostemma pentaphyllum aerial stem-to-rhizome transition,GpmiR156a and GpmiR166b,in regulating plant growth and development. GpMIR156a and GpMIR166b genes were obtained by homologous cloning and analyzed by bioinformatics. The effects of GpMIR156a and GpMIR166boverexpression on plant phenotype were investigated by constructing their overexpression vectors and then transforming Arabidopsis thaliana. The overexpression of GpMIR156a increased the number of leaves and branches of A. thaliana seedlings,and accelerated seed germination and root elongation,indicating that GpmiR156a promoted vegetative organ development and seed germination,but inhibited juvenile-to-adult transition. However,the overexpression of GpMIR166b led to the premature and dwarf plants,i.e.,increase in the number of trichomes on the inflorescence stems,as well as delayed seed germination but promoted root elongation. These findings indicated that GpmiR166b affected the normal development of vegetative organs and delayed seed germination to a certain extent,whereas promoted juvenile-to-adult transition. The results of this study broaden the understanding of miR156a and miR166b in regulating plant growth and development.

    Genetic Transformation of Plant Cells Mediated by Mesoporous Silica Nanoparticles
    LU Xin-hua, SUN De-quan, ZHANG Xiu-mei
    2022, 38(7):  194-204.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1040
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    With the rapid development of nanobiotechnology,nanoparticles have been applied as vectors for gene transformation in plant cells. In this study,mesoporous silica nanoparticles(MSNs)with different particle sizes were synthesized and then amine-functionalized. The transformation efficiencies of amine-functionalized MSNs(Am-MSNs)for delivering plasmid DNA encoding green fluorescent protein(smGFP)were investigated in the protoplasts of Arabidopsis thaliana. As results,the Am-MSN-20 exhibited monodispersed spheroids with average particle size of 20 nm,while Am-MSN-50 showed a flower-like structure and had an average particle size of 50 nm. Both amine-functionalized MSNs were positively charged. Am-MSN-50 had higher binding capacity of pDNA than that of Am-MSN-20. The Am-MSNs/pDNA complex showed good stability,which suggested that both Am-MSNs protected bounded pDNA efficiently against degradation by cellular nucleases. The MSNs also exhibited no cytotoxic effects on the protoplasts of A. thaliana. The Am-MSN-50 enabled much higher transformation efficiency of the pDNA encoding smGFP compared to that of Am-MSN-20. The results suggest that Am-MSNs can be employed as promising carriers for safe and effective gene delivery.

    Characteristics of Soil Bacterial Community Structure in the Different Developmental Stages of Desert Grassland Caragana korshinskii Kom. Nebkhas
    WANG Zi-yin, LIU Bing-ru, LI Zi-hao, ZHAO Xiao-yu
    2022, 38(7):  205-214.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0248
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    To understand the soil bacterial community structure and diversity characteristics and the influencing factors at different developmental stages of Caragana korshinskii Kom. nebkhas in arid and semi-arid desert grasslands,C. korshinskii Kom. nebkhas in Ningxia desert grasslands at three different developmental stages of growing,mature,and recession were selected. At the same time,high-throughput sequencing technology was applied to analyze their soil bacterial community structure and diversity characteristics,and to investigate the influencing factors of developmental stages and soil physicochemical factors evolution on soil bacterial community structure and function. Finally,PICRUSt method was to predict the functions of soil bacteria at different developmental stages of C. korshinskii Kom. nebkhas. The results showed that the obtained 15 383 operational taxonomic units(OTUs)belonged to 50 phyla,146 classes,364 orders,590 families,and 1 111 genera,with the largest number of bacteria at the growing stage and the dominant phyla being 9 of Actinobacteriota,Proteobacteria,Acidobacteriota,Chloroflexi,Crenarchaeota,and nine others. Proteobacteria,Acidobacteriota,Chloroflexi,and others,accounting for > 90% of the total number of microorganisms,and the dominant phyla were similar to most studies. There were significant differences in soil AP and EC in different development stages of C. korshinskii Kom. nebkhas,showing mature> growing > recession. Other soil nutrients were not significantly different,but the values were also expressed as mature> growing > recession. There was no significant difference in the diversity and abundance of bacterial communities,but the numerical value was the highest in the mature period. AP and SOC were the main factors influencing the diversity of soil bacterial communities in C. korshinskiiKom. nebkhas. Other environmental factors such as EC and pH also significantly affected the richness of multiple bacterial communities. Based on the prediction of 16S rRNA sequences of soil bacterial genomes in different developmental stages of C. korshinskii Kom. nebkhas,it was found that metabolism was the main function of soil bacteria. The above results can enrich the theoretical knowledge of soil microbial diversity in semi-arid desert grasslands and provide a reference for the restoration of C. korshinskii Kom. vegetation.

    Effects of Soil Moisture on the Microbial Community Under Continuous Cropping of Panax notoginseng
    ZHAO Lin-yan, GUAN Hui-lin, WANG Ke-shu, LU Yan-lei, XIANG Ping, WEI Fu-gang, YANG Shao-zhou, XU Wu-mei
    2022, 38(7):  215-223.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1287
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    Using 8-year replanting P. notoginseng soil as study object,we investigated the effects of SMC(10%,20%,30% and 40%)on the soil physicochemical properties and microbial community by using the high-throughput DNA sequencing and in-door soil-cultivated experiment. The results showed that the relative abundance of Fusarium was the highest when the SMC was maintained at 10% and 20%. However,when SMC was 30%,the relative abundance of Fusarium decreased and that of Trichoderma increased significantly(P<0.05),which was benefit to controlling the soil-borne diseases of P. notoginseng. When SMC was raised to 40%,the relative abundance of Fusarium increased while Trichoderma decreased(P<0.05). Comparing to bacterial community,the fungal community varied more obviously with the increase of SMC. Moreover,when SMC was maintained at 40%,the content of NH4+-N significantly increased,while NO3--N content decreased(P<0.05). Our study provided scientific basis for reducing the obstacle of replanting P. notoginseng from the perspective of field moisture management.

    Effects of Trichoderma harzianum ST02 on the Growth of Peppermint and Physicochemical Properties of Root Zone Soil Under Salt Stress
    ZHAO Zhong-juan, YANG Kai, HU Jin-dong, WEI Yan-li, LI Ling, XU Wei-sheng, LI Ji-shun
    2022, 38(7):  224-235.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1251
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    The salt-resistant Trichoderma harzianum strain ST02 and salt-resistant peppermint were used in this study to analyze the effects of Trichoderma on plant salt resistance and saline soil improvement. The physiological and growth traits of peppermint,the soil physicochemical property and soil enzyme activity of root zone soil under salt stress were analyzed. And the main soil factors related to the physiological and growth of peppermint were analyzed through the correlation analysis and principal component analysis(PCA). The results showed ST02 relieved the inhibition of NaCl on peppermint growth and photosynthesis and promoted its growth and photosynthesis intensity under salt stress. When treated with NaCl,ST02 reduced the accumulation of maldehyde aldehyde(MDA),soluble protein(SP)and proline(Pro)in peppermint cells,enhanced superoxide dismutase(SOD),peroxide dismutase(POD),and catalase(CAT)activities,and enhanced the salt resistance of peppermint through antioxidant response. The physiological/growth comprehensive index(PGCI)of peppermint treated with NaCl was -2.391,ST02 promoted the physiological and growth of peppermint,as index was -0.025. ST02 influenced the soil physicochemical properties for root zone soil of peppermint under NaCl stress,reduced salinity by 17.36%,and significantly enhanced the content of alkali-hydrolyzed nitrogen(AN). The soil enzyme activity for the root zone soil of peppermint was also affected by ST02 strain,and the activity of soil cellulase(SCL)and catalase(SCAT)increased remarkably. The soil physicochemical property and soil enzyme activity affected the physiological and growth traits under NaCl stress through the correlation analysis. And the main soil factor for physiological and growth of peppermint was:EC(electrical conductance)> SCL >SSC(soil saccharase)/SNEP(soil neutrality phosphatase)>AP(available phosphorus)>pH.

    Physiological and Biochemical Response Characteristics of Bacillus sp. ZJS3 Under As3+ Stress
    YUAN Cun-xia, LI Yan-nan, ZHANG Xiao-chong, YANG Rui, LIU Jian-li, LI Jing-yu
    2022, 38(7):  236-246.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1234
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    We aim to investigate the response mechanism of strain Bacillus sp. ZJS3 to As3+ stress under optimal growth conditions. The tolerance range was determined by the growth of the strain Bacillus sp. ZJS3 in solid medium. The activities of antioxidant enzymes,the content of malondialdehyde(MDA)and the contents of protein and polysaccharide in extracellular polymeric substances(EPS)were determined under the influence of different arsenic concentrations. The mechanism of tolerance of Bacillus sp. ZJS3 was studied by photographing biological scanning electron microscope(SEM),measuring the adsorption rate and redox activity,detecting tolerance genes. The tolerance range of Bacillus sp. ZJS3 to As3+ was 100-300 mg/L. According to the single factor experiments,the suitable culture conditions were pH 7.0,rotation speed 200 r/min,and temperature 30℃. Through silver nitrate staining and arsenic molybdenum blue method,it was detected that Bacillus sp. ZJS3 oxidized As3+ to As5+. Under different arsenic concentration stress,the contents of peroxidase(POD),catalase(CAT),superoxide dismutase(SOD)and MDA increased significantly,and the polysaccharide content in EPS increased continuously,while the protein increased first and then decreased. It was observed by SEM that the cell morphology gradually lengthened and granular substances appeared on the surface after arsenic pollution. Within 24 h,the maximum adsorption rate of arsenic by the strain was 25.00% and the adsorption capacity was 3.23 mg/g. In conclusion,Bacillus sp. ZJS3 can oxidize high toxic As3+ to low toxic As5+,and has adsorption effect on arsenic. Its antioxidant enzymes and extracellular polymers play an important role in the process of arsenic stress.

    Isolation of Halophilic Heterotrophic Nitrification-aerobic Denitrification Bacterium Bacillus megatherium N07 and Its Denitrification Characteristics
    GAO Yu-xuan, JIN Jing-chen, XU Li-shan, GAO Ya-Juan, ZHANG Wen-tian, LI Chen-chen, ZHANG Guo-wei, JIN Yong-sheng
    2022, 38(7):  247-257.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1111
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    Biological nitrogen-removing technology based on heterotrophic nitrification and aerobic denitrification bacteria may effectively remove nitrogen in sewage. There are many shortcomings in the treatment of high salt wastewater,such as inhibiting microbial growth and low nitrogen removal efficiency. In order to solve the problem of difficult nitrogen removal from high salt wastewater,a strain of heterotrophic nitrification and aerobic denitrification with high salt concentration of 8%(W/V)was selected from chemical wastewater of agricultural pharmaceutical factory and named N07. The denitrification rate of the strain was measured under different salinity and dissolved oxygen conditions,and the effects of single environmental factors on the denitrification performance of the strain was investigated. The results showed that strain N07 was identified as Bacillus megatherium. It tolerated 8% salinity and reproduced vigorously. The removal rates of ammonia nitrogen and nitrite nitrogen were up to 73.61% and 83.56%,belonging to moderate halophilic bacteria. The bacterium showed high denitrification ability under aerobic conditions,with nitrate and nitrite removal rates of 11.87 mg/(L· h)and 8.26 mg/(L· h)at 12 h. The best nitrification capacity was obtained when carbon source was sucrose,C/N was 25,pH was 7,rotation speed was 160 r/min and temperature was 28℃. B. megatherium N07 can degrade nitrogen in high salinity environment,which provides reference for the practical application of heterotrophic nitrification and aerobic denitrification bacteria in the treatment of high salinity nitrogen wastewater such as coastal aquaculture industry.

    Screening and Degradation Characteristics of a CP-7 Strain of Dephosphorization Bacteria
    WANG Ya-jun, SI Yun-mei
    2022, 38(7):  258-268.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1271
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    In order to achieve the biodegradation and effective treatment of phosphorus in domestic sewage,a strain of dephosphorization bacteria was isolated from a domestic sewage treatment plant in Lanzhou to carry out enhanced biological phosphorus removal research. 1)Combined with physiological and biochemical characterization,16S rDNA sequencing analysis and phylogenetic tree construction,the results showed that the strain had a high homology with the Klebsiella oxytoca strain. 2)The bacterium was inoculated into the double screening medium at the inoculation rate of 5%(V/V)and cultured in a constant temperature oscillation incubator at 30℃ and 150 r/min. The concentration of TP was measured every 12 h. The preliminary study on the degradation characteristics of the bacterium showed that the degradation rate of TP was 58.52% within 72 h. 3)The optimal conditions of carbon source,nitrogen source and C/N were investigated. The optimal carbon source was glucose,nitrogen source was ammonium sulfate,and the optimal C/N was 5∶1. 4)By investigating the adaptability of inoculation amount,pH,temperature,initial NaCl concentration and initial phosphate concentration,it was concluded that the degradation rate of TP was the best when the inoculation amount was 8%(V/V),pH was 9,temperature was 30℃,initial NaCl concentration was 2.5 g/L and initial phosphate concentration was 70 mg/L. The degradation rates of TP were 75.94%,90.19%,65.48%,57.02% and 52.26% respectively. At the same time,when the inoculation amount was 2%-14%(V/V),pH was 4.0-9.0,temperature was 15-40℃,initial NaCl concentration was 0-15 g/L,initial phosphate concentration was 45-90 mg/L,the strain demonstrated varied degrees of degradation ability to TP. Correlation analysis showed that the TP degradation rate of the strain was significantly correlated with inoculation amount,pH,initial NaCl concentration and initial phosphate concentration. 5)The interaction between the strain and other strains was investigated,and the strain was mixed with a Pseudomonas aeruginosa strain. It was found that the two strains synergistically enhanced phosphorus removal. When the ratio of the two strains was 4∶1,the TP degradation rate reached 99.43%. This experiment lays a foundation for the practical application of dephosphorization bacteria in the next stage,and provides more bacterial species selection for biological enhanced phosphorus removal technology.

    Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA
    WANG Guang-li, FAN Chan, WANG Hui, LU Hui-fang, XIA Ling-yin, HUANG Jian, MIN Xun
    2022, 38(7):  269-277.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1299
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    It is aimed to express and purify hemolysin HlyA protein of Vibrio cholerae in prokaryotic cells,and to prepare and identify the polyclonal antibody of mouse anti-HlyA. The hlyAgene was amplified from the genome Vibrio cholerae via PCR and cloned into the expression plasmid pET28a,pET32a and pCold TF to construct the recombinant plasmid pET28a-hlyA,pET32a-hlyA and pCold TF-hlyA, and they were transformed into E. coli BL21(DE3). The conditions used to induce expression were optimized and the expressive forms were identified. The soluble form of hlyA protein was purified by Ni-NTA column. The purified HlyA protein was used as antigen for immunization of BALB/c mice to prepare polyclonal antibody. The antibody titer was determined to test the immunogenicity by indirect ELISA. The antibody specific recognition of HlyA protein in Vibrio cholerae was analyzed by Western blot and verified by mass spectrometry. The hemolytic activity of the purified hlyA protein and the neutralizing activity of the antibody were analyzed. The HlyA protein was expressed as inclusion bodies in the pET28a-hlyA and pET32a-hlyA vectors,and as soluble form in the pCold TF-hlyA vector. HlyA was purified by affinity chromatography on an Ni-NTA agarose column to get higher purity. The recombinant HlyA protein could not lyse rabbit red blood cells,but immunized mice can obtain polyclonal antibody with higher titer. The results of Western blot and mass spectrometry showed that the HlyA polyclonal antibody can specifically recognize the HlyA protein in Vibrio cholerae,and the antibody can effectively inhibit the hemolytic activity of Vibrio cholerae. The soluble form of HlyA protein and the high titer anti-HlyA polyclonal antibody were successfully obtained,which laid the foundation for the follow-up study of the role of HlyA protein in the pathogenic process of V. cholerae.

    Effects of Glucose and Sodium Butyrate on the rHSA Yield in CHO-rHSA Engineering Cell Line
    NIU Yu-hui, LI Xiang-rong, WU Bei, LI Hong-shan, LI Dian-yu, CHEN Lei, WEI Suo-cheng, FENG Ruo-fei
    2022, 38(7):  278-286.  doi:10.13560/j.cnki.biotech.bull.1985.2021-1238
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    At present,suspension CHO cells are widely used as the most potential engineered cell lines for exogenous proteins. The culture conditions of promoting its high density growth and high expression of exogenous protein have also become the research hotspot. Glucose and sodium butyrate play an important role in promoting the growth of CHO cells and the expression of exogenous proteins. Therefore,study the effect of glucose and sodium butyrate on the expression of recombinant serum albumin in CHO-rHSA engineered cells is of great value. Firstly,the effect of glucose and sodium butyrate on the expression of recombinant serum albumin in CHO-rHSA engineering cells was determined by single factor test,and the optimal addition conditions were selected. Then the combination culture conditions of glucose and sodium butyrate which effectively increased the expression of rHSA were optimized by the two-factor combination experiment. The yield of rHSA increased 121.93% by adding 7 g/L glucose every 48 h from the 3rd day of cell culture,and 110.01% by adding 1.0 mmoL/L sodium butyrate on the 2nd day of cell culture. The combination of sodium butyrate and glucose prolonged the culture time from 7 d to 14 d and increased the expression of rHSA to 85.642 mg/L,which was 212.49% higher than that of untreated cells,134.85% higher than glucose alone,and 88.35% higher than sodium butyrate alone. The culture condition of CHO engineering cell line was determined to add 1.0 mmol/L sodium butyrate and 3.0→7.0 g/L glucose when cells cultured for 48 h. Sodium butyrate combined with glucose can promote foreign protein expression more efficiently,and glucose can weaken the inhibition of sodium butyrate on the growth of CHO cells,which provides a new theoretical basis for enhancing the expression of foreign protein in CHO engineering cells.

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    2022, 38(7):  290-290. 
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    2022, 38(7):  291-291. 
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    2022, 38(7):  292-292. 
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