Table of Content

26 March 2024, Volume 40 Issue 3

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Research Progress in Genomic Selection Breeding Technology for Crops

WANG Xin, XU Yi-yi, XU Yang, XU Chen-wu

2024, 40(3):  1-13.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1079

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Genome selection（GS）breeding builds a genetic model based on the association between genotypes of molecular markers on the whole genome and phenotypes of the training population, and then estimates the breeding values or predicts the phenotypes of the candidate population with known genotypes, so as to achieve efficient and accurate selection of the population for breeding. Compared with the commonly used molecular marker-assisted selection breeding, GS breeding does not require marker significance testing, and is particularly suitable for quantitative traits controlled by minor polygenes. It can shorten breeding cycle and reduce breeding cost, and has become a cutting-edge technology in the field of animal and plant breeding. However, for quantitative traits such as crop yield that are greatly affected by environment, it is still bottleneck issue to improve the accuracy of genomic prediction. This article first analyzes the main factors that affect the efficacy of GS in crop breeding, and then elaborates on the research progress of GS technology in crop breeding from the aspects of models with non-additive effects, population construction schemes, multi-trait and multi-environment prediction, multi-omic prediction and the current status of breeding chip technology. Then the article points out the issues and development prospects of the research, and provides the strategies and ideas for further research on crop GS breeding technology.

Research Advance and Applications in Maize Wild Relatives Genomes

HU Yi-wa, CHEN Lu

2024, 40(3):  14-24.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1131

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Global change of climate has made great challenges to the crop productions and economical stabilities. The creation of adaptive species under the extreme environmental change is one of the primary goals in modern crop breeding process. As one of the most important crops in the world, maize have lost substantial genetic diversities during domestication, especially the loci that respond to the biotic and abiotic stress. Finding and utilizing the genetic diversity lost during maize domestication in wild species and landrace will provide new light for the creation of new adaptive varieties. In this review, we firstly introduce the discoveries and classification of teosinte（wild relatives of maize）, and then summarize the traits and genomic difference between teosinte and maize, the genetic mechanism of divergence of maize and teosinte basing on genetics, population genetics, molecular biology, revealing the importance of teosinte in the development of maize genetics from multiple perspectives. Finally, we prospect the key role of teosinte in maize future breeding process under climate changes.

Regulation of PIN-FORMED-mediated Polar Auxin Transport in Plant Gravitropism

WANG Xian, PENG Ya-kun, CHEN Meng, KONG Meng-juan, TAN Shu-tang

2024, 40(3):  25-40.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0882

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Plant tropisms are the directional growth response of plants to environmental stimuli such as light or gravity. In gravitropism, plant organs sense directional environmental cues to control growth orientation to promote their survival. The phytohormone auxin and its polar transport serve as a major coordinative signal in plant gravitropism. The PIN family auxin exporters, through their dynamic polar subcellular localizations at the plasma membrane（PM）, redirect polar auxin transport in response to environmental stimuli, thus the auxin gradients across tissues or organs underlie differential cell elongation and bending, which regulates the morphological formation and growth process of plants. In this review, we focus on recent advances in the mechanisms underlying the perception and signal transduction of gravity that take place within the sensing cells in the early process of gravitropism, the regulatory mechanisms of PIN-mediated auxin polar transport, PIN polar localization, and PM protein abundance.

Phytomelatonin: An Emerging Signal Molecule Responding to Abiotic Stress

ZHOU Hong-dan, LUO Xiao-ping, TU Mi-xue, LI Zhong-guang

2024, 40(3):  41-51.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0880

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Compared with other five traditional hormones, the identification of melatonin（MT）was done in only two decades. MT is an emerging plant hormone, which is a pleiotropic signaling molecule with multiple physiological functions in organisms. In plants, MT is called phytomelatonin, which is not only involved in the regulation of seed germination, root system architecture, stomatal movement, biological rhythm, flowering, and senescence, but also in the response of plants to biotic and abiotic stresses by activating the activities of antioxidant system, scavenging ROS, followed by reducing osmotic stress, oxidative stress, protein denaturation, and cell damage caused by biotic and abiotic stress, thus plants respond to biotic and abiotic stresses. Based on the latest research progress of MT metabolism in plant and its response to abiotic stress, the anabolism and catabolism of MT in plants are summarized. The plant resistance to high temperature, low temperature, drought, salt, heavy metals, ultraviolet radiation and waterlogging is improved ultimately under stress by MT directly removing ROS and/or triggering the signal transduction pathway, up-regulating resistance related gene expression, and then activating the vitality of osmoregulation system and antioxidant system, promoting the synthesis of stress protein and secondary metabolic substances, stable photosynthesis and carbon metabolism, and thus reducing the accumulation of ROS and cell oxidative damage. This paper lays the theoretical foundation for understanding the metabolism, physiological function, and cellular signal transduction of MT in plants, and point out the future research directions.

Mechanisms of Root Exudates-mediated Plant Resistance to Soil-borne Diseases

FANG Tian-yi, YUE Yan-ling

2024, 40(3):  52-61.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0924

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Root exudates have gained increasing attention as underground defense substances. In recent years, studies have gradually revealed the expression characteristics of root exudates in plants under pathogenic stress, as well as their roles in plant disease resistance. Some resistance regulatory mechanisms mediated by root exudates have also been simulated. Given the growing losses caused by soil-borne diseases, understanding how root exudates resist various pathogenic bacteria has become a research focus. This article comprehensively reviews the plant resistance mechanism mediated by root exudates under soil pathogen stress. It examines the expression characteristics of root exudates under pathogen stress, the role of root exudates in the process of plant disease resistance, and the functions of secretions and plant resistance mediated by resistant roots. The article also discussed the impact of root secretions on plant resistance under current plant disease conditions, aiming to provide future insights into the study of resistance mechanisms of soil-borne diseases.

Giant Cells Induced by Root-knot Nematodes and Its Formation Mechanisms

LIU Rui, ZHAO Jian-long, XIE Bing-yan, LI Hui-xia, MAO Zhen-chuan

2024, 40(3):  62-74.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0938

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Root-knot nematodes（Meloidogyne spp.）are obligate endoparasitic nematodes with features of wide distribution, various hosts, and serious harm. It causes huge economic losses to crops annually, and is one of the most harmful nematodes on crops. When infecting plant root systems, root-knot nematodes induce the re-differentiation of vascular cells at the root tips into multinucleate giant cells, which serve as feeding sites and sole source of nutrients for the nematodes. These cells become the sole source of nutrients for the growth and development of the nematodes. Forming and maintaining ning giant cells are crucial for the growth and reproduction of the nematode. Based on forming of the giant cells and their regulatory mechanisms induced by root-knot nematodes, this paper focuses on the structure of giant cells, cell cycle changes during giant cell formation, nutrient transport, hormone regulation, and the recognition, defense and expression patterns of host plants, and discuses the functions of root-knot nematode effector proteins in the process of giant cell formation. Through the review of the multi-level research progress on the formation and regulation mechanism of giant cells and the molecular mechanism of nematode-plant interaction, it sheds light on the pathogenic mechanisms of root-knot nematodes, aiming to inspire innovative strategies for future research and control of these nematodes.

Research Progress in the Biocontrol Secondary Metabolites of Bacillus velezensis

XU Pei-dong, YI Jian-feng, CHEN Di, PAN Lei, XIE Bing-yan, ZHAO Wen-jun

2024, 40(3):  75-88.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0867

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Bacillus velezensis is an important representative of biocontrol strains in the genus Bacillus. It is widely used as the core strain of biological pesticide in the biocontrol of plant diseases. B. velezensis has endophytic properties in plants, and its biocontrol mechanism mainly includes three aspects. Firstly, it can produce the secondary metabolites against pathogens. Secondly, it can improve host plant microbial community to promote host nutrition and growth. The last, it can stimulate the plant to produce a defense response and develop systemic resistance. Among them, the production of secondary metabolites is the most important mechanism of biocontrol. B. velezensis contains multiple biosynthesis gene clusters encoding secondary metabolites, including gene clusters encoding polyketide synthase（PKS）and non-ribosomal peptide synthase（NRPS）, as well as gene clusters for ribosomal pathway synthesis of secondary metabolites. B. velezensis can produce plantazolicin, amylocyclicin, and mersacidin through the ribosomal pathway, and produce lipopeptides, polyketides, bacilysin, and bacillibactin through non-ribosomal pathways. These bioactive secondary metabolites have become repositories of natural new drugs and candidate antibiotics, which is of great significance for analyzing the mechanism of biocontrol action. This paper reviews the naming and alteration, the types of secondary metabolites, synthesis and regulation genes, and target pathogens of B. velezensis. It aims to provide a scientific basis for new theories and applications of strain improvement and plant disease biological control.

Research Progress in the Molecular Mechanisms of Microalgae Responding to Salt Stress

SHEN Tian-hong, QI Xiao-bo, ZHAO Rui-feng, MA Xin-rong

2024, 40(3):  89-99.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0684

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Microalgae, as one of the ancestors of photosynthetic microorganisms on earth, are widely exploited for its short growth cycle, fast growth rate and potential to produce high value-added products. However, their scale-up cultivations have been substantially hindered by those adverse effects resulting from various abiotic stresses including high salinity during industrial production processes. Therefore, it is extremely essential to understand the molecular mechanism of salt stress responses in microalgae, which may contribute to the build-up of genetically engineered microalgae or even higher plants. In this mini review, we summarized various proteins involved in the response of eukaryotic microalgae and prokaryotic cyanobacteria to salt stress and their specific mechanisms including transporters maintaining ion homeostasis, accumulating osmotic regulators, antioxidant defense mechanisms, signal proteins and lipid remodeling. Meanwhile, we outlined the recent research progress of halotolerant microalgae including Dunaliella salina, Aphanothece halophytica, Picochlorum sp. and Chlamydomonas W80, and their salt-tolerant genes that are mostly under active investigations. Finally, we briefly discussed the value and application prospect of typical important salt-responsive genes in the breeding of algal species.

Research Progress in Increasing Yield of Outer Membrane Vesicles from Gram Negative Bacteria

LIU Can, YAN Xiao-yang, ZENG Yan, OU Xiang-long, LIAO Yong-hong

2024, 40(3):  100-108.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0540

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Outer membrane vesicles（OMVs）are vesicle-like structures released by gram-negative bacteria, and OMVs play an important role in gram-negative bacteria growth, infection and communication with the environment. Because OMVs are inactive, non-replicable and possess high immunogenicity, OMVs are widely applied for research and development of novel vaccines. However, the natural OMVs yield is too low to meet scale-up production. To increase OMVs yield is the key technical issue that should be resolved for scale-up production. We mainly review on three aspects for increasing OMVs yield, modification on bacterial genes, optimization on bacterial growth conditions and OMVs production and purification, aiming to provide informative reference for industrial research and development of OMVs vaccine.

Research Progress in cGAS-STING Signaling Pathway in ASFV Antagonizing Host

ZHAO Hong-yuan, LIU Qiang, CHENG Wen-yu

2024, 40(3):  109-117.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0681

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African swine fever virus（ASFV）possesses multiple strategies for escaping host immune responses, making it difficult for the virus to be cleared by the host. The cGAS-STING pathway, which mediates host innate immune response, is critical for fighting ASFV infection. Nevertheless, numerous viral proteins targeting different molecules involving in the cGAS-STING pathway inhibit type I interferon production. The deletion of partial of these ASFV genes using genome editing technology attenuated the virulence of the virus, which provides targets for the development of gene deletion vaccine. In this review, we summarized current knowledge on the viral proteins of ASFV modulating host cGAS-STING signaling pathway and elucidate the mechanism of these proteins escaping cGAS-STING signaling pathway, which may deepen the understanding of ASFV immune evasion strategy and provide theoretical basis for the research of ASFV pathogenic mechanism and vaccine development.

Strategies for Increasing Heterologous Protein Expression in Pichia pastoris

RUZHA Yelizhati, YANG Yu

2024, 40(3):  118-134.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0857

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The production of heterologous proteins via recombinant expression system is a focus of genetic engineering research. Because of its ease of genetic manipulation, high degree of heterologous protein secretion, and post-translational modification, Pichia pastoris, a methylotrophic yeast, has become an essential strain for protein production in industrial applications and is frequently employed in the manufacturing of enzymes. Although some heterologous proteins can be expressed in P. pastoris, the amount of expression is modest and has to be increased. Therefore, it is critical to investigate the principle and method of boosting the expression of heterologous proteins in P. Pastoris in order to lower industrial production costs and improve economic benefits. In this review, the optimizing strategies for improving heterologous protein expression in P. Pastoris in recent years are summarized from six perspectives: Gene level, transcription level, translation level, protein folding and secretion levels, cell resistance level, and fermentation technology, with the goal of demonstrating comprehensively how to improve the expression of heterologous protein in P. Pastoris expression system.

Analyzing the Growth and Caproic Acid Metabolism Mechanism of Rummeliibacillus suwonensis 3B-1 by Tandem Mass Tag-based Quantitative Proteomics

CHEN Xiao-song, LIU Chao-jie, ZHENG Jia, QIAO Zong-wei, LUO Hui-bo, ZOU Wei

2024, 40(3):  135-145.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0862

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【Objective】 This work aims to elucidate the growth and caproic acid metabolism mechanism of Rummeliibacillus suwonensis from protein level, and to further improve its metabolic capacity. It provides a technical basis for the genetic engineering of R. suwonensis. 【Method】 The differential expression proteins（DEPs）of strain R. suwonensis 3B-1 were extracted by tandem mass tags（TMT）proteomics in aerobic and anaerobic conditions. Its subcellular localization analysis, Gene Ontology（GO）functional analysis, KEGG signaling pathway annotation analysis, and protein-protein interaction were analyzed.【Result】 A total of 810 DEPs were identified, including 423 up-regulated proteins and 387 down-regulated proteins. A total of 725 DEPs were subcellular mapped to 6 items, mainly involving cytoplasmic proteins, cytoplasmic membrane proteins and cellwall proteins. GO enrichment of function analysis indicated that, biological processes such as peptide biosynthesis, translation, and peptide metabolism, molecular functions such as structural constituent and structural molecular activity of ribosomes, and cellular components such as ribosomes and ribonucleoprotein complexes have undergone significant changes. A total of 810 DEPs were annotated into 113 KEGG signaling pathways in KEGG database, which mainly were involved cofactor biosynthesis, two-component system, pentose phosphate pathway, glycolysis/gluconeogenesis and oxidative phosphorylation. The phenylalanine-tRNA ligase β subunit and the riboflavin biosynthesis protein RibD had the highest correlation in the protein interaction network. 【Conclusion】 Under anaerobic conditions, the expression of pyruvate dehydrogenase and pyruvate kinase in the glycolytic pathway was down-regulated. Additionally, proteins related to amino acid metabolism and biotin protein ligase cofactors were also downregulated. This indicates that the bacterium is better suited for growth in an aerobic conditions. Regarding the synthesis of caproic acid, the expression of acyl-CoA thioesterase was significantly up-regulated. In addition, the glycolysis/gluconeogenesis pathway, the tricarboxylic acid cycle and the pentose phosphate pathway provided sufficient precursors and reduction equivalents for caproic acid synthesis, which jointly promoted caproic acid synthesis.

Association Analysis and Exploration of Elite Alleles of Plant Architecture Traits in Gossypium hirsutum L.

WANG Juan, WANG Xin, TIAN Qin, MA Xiao-mei, ZHOU Xiao-feng, LI Bao-cheng, DONG Cheng-guang

2024, 40(3):  146-154.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0746

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【Objective】 Plant architecture is a key factor affecting the mechanization and yield of cotton. The association analysis of plant architecture traits in upland cotton can provide elite marker sources and excellent germplasm materials for molecular breeding of cotton plant architecture. 【Method】 In this study, taking 403 upland cotton germplasms as materials, an association analysis of four plant architecture traits in six environments was implemented using 201 pairs of polymorphic SSR markers covering the whole genome, and the elite alleles were identified. 【Result】 The average coefficients of variation of the plant height, height of first fruit branch node, the first fruit branch node and fruit branch number were 13.70%, 21.51%, 14.18% and 11.51%, respectively, and the range of broad-sense heritability was 46.24%-74.15%. A total of 394 allelic variation loci were generated from 201 markers. A total of 38 loci were repeatedly detected in best linear unbiased prediction（BLUP）（P < 0.01）and more than two environments at a significant level（P< 0.05）. Of 38 loci, 16 were associated with plant height, six were associated with height of first fruit branch node, 11 were associated with the first fruit branch node, and five were associated with fruit branch number. In addition, five loci were associated with multiple traits simultaneously. Thirty one materials containing elite alleles of target traits were identified, and six of them carried multiple elite alleles concurrently. 【Conclusion】 Thirty-eight marker loci associated with four plant architecture traits were identified in 403 natural populations, and 31 typical materials containing excellent alleles were identified.

Genome-wide Identification of the FAX family in Gossypium hirsutum and Functional Analysis of GhFAX1

YANG Wei-cheng, SUN Yan, YANG Qian, WANG Zhuang-lin, MA Ju-hua, XUE Jin-ai, LI Run-zhi

2024, 40(3):  155-169.  doi:10.13560/j.cnki.biotech.bull.1985.2023-1075

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【Objective】 Fatty acid export（FAX）can mediate the transport of fatty acids from plant cells to the outer plastid, and play an important role in plant growth and development and response to abiotic stress. This work is to study the FAX family gene and function analysis of upland cotton（Gossypium hirsutum）, and to provide new ideas for clarifying the molecular mechanism of oil accumulation and oil metabolism engineering of upland cotton. 【Method】 In order to reveal the potential function of the upland cotton FAX gene family, the GhFAX gene family was identified at the whole genome level, and the GhFAX1 protein was to have subcellular localization, and the function of GhFAX1 was verified by the genetic transformation of yeast and tobacco.【Result】 A total of 12 GhFAXs genes were identified in the genome of upland cotton, and all members of the family had similar gene structure and protein physicochemical properties. Sequence alignment analysis showed that only a few amino acids of GhFAX protein were highly conserved in evolution, suggesting their importance to function. Phylogenetic tree analysis showed that GhFAX gene was closely related to Asian cotton（Gossypium arboreum）and Raymond's cotton（G. raimondii）. Transcriptome data suggested that GhFAXs may be involved in the regulation of upland cotton response to stress. Real-time fluorescence quantitative PCR showed that GhFAX4 was highly expressed in flowers, suggesting that GhFAX4 was involved in pollen development. The expression of GhFAX9 was high in the stems and GhFAX1 was high in all tissues of upland cotton. GhFAX1 was selected for subcellular localization analysis, and it was confirmed that GhFAX1 was localized in plastid. The GhFAX1 vector was constructed to overexpress GhFAX1 in Saccharobacteria cerevisiae. The results showed that the total fat of GhFAX1 overexpressed yeast was increased by 3.53%. Substrate bias experiments showed that GhFAX1 was selective to C16:0. GhFAX1 overexpressing tobacco was cultivated through genetic transformation of tobacco. The results showed that the chlorophyll of overexpressed tobacco increased by 13.24%, the fluorescence parameters NPQ increased by 14.17%, F_m increased by 34.94%, F₀ decreased by 35.28%. The total fatty acids of the leaves and seeds increased by 5.2% and 6.52% respectively. The 1 000-grain weight of seeds increased by 19.1%. At the same time, the protein content decreased significantly.【Conclusion】 GhFAXs can increase the oil content of yeast and tobacco, participated in the transfer of palmitic acid from plastid, and causing the carbon source from protein synthesis pathway to oil synthesis pathway.

Genome-wide Identification of Potato WOX Gene Family and Its Expression Analysis in in vitro Regeneration and Abiotic Stress

ZHANG Yu, SHI Lei, GONG Lei, NIE Feng-jie, YANG Jiang-wei, LIU Xuan, YANG Wen-jing, ZHANG Guo-hui, XIE Rui-xia, ZHANG Li

2024, 40(3):  170-180.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0947

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【Objective】 The WUSCHEL-related homeobox（WOX）gene family is plant-specific, which plays important roles in plant growth and development, regulation of stem cell differentiation, and response to adverse stress. Identification of potato WOX gene family may provide a theoretical basis and gene resources for potato genetic improvement.【Method】 Based on the WOX protein sequences of Arabidopsis thaliana, tomato, tobacco and rice, all members of potato WOX gene family were identified by HMMER 3.0 and BLASTP, MCScanX was used for collinear analysis, and the phylogenetic tree was constructed using neighbor-joining method. Physical and chemical properties, gene structure, protein motif, and transcription factor binding sites of potato WOX members were analyzed by ExPASy, GSDS and other software. Expression patterns of StWOXs were analyzed based on potato transcriptome data from PGSC database. RT-qPCR assay was carried out to analyze relative expression of StWOX5 in four potato varieties（lines）with different in vitro regeneration abilities.【Result】 11 members of potato WOX gene family were identified, distributed on five chromosomes, and classified into three clades, namely WUS, intermediate and ancient, with differences in gene structure, motif composition, transcription factor binding sites. The results showed that members of potato WOX gene family were involved in in vitro regeneration and abiotic stress responses. StWOX4/5/11/13 were specifically expressed in callus, StWUS and StWOX1/3c/4/5/13 were up-regulated under mannitol treatment, StWOX3a/11 were up-regulated under salt treatment, and StWOX4/5/13 were up-regulated under heat stress. The relative expression of StWOX5 was positively correlated with the differentiation rate.【Conclusion】 Different potato WOX genes had potential functional diversity. StWOX5 may promote potato regeneration in vitro and participate in abiotic stress responses.

Cloning and Expression Analysis of StDof5 Gene in Potato

MEI Xian-jun, SONG Hui-yang, LI Jing-hao, MEI Chao, SONG Qian-na, FENG Rui-yun, CHEN Xi-ming

2024, 40(3):  181-192.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0960

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【Objective】 The DNA binding with one finger（Dof）gene family is a plant-specific transcription factor family that plays important roles in plant growth, development and stress response. Studying the function and mechanism of Dof transcription factors responding to abiotic stress, especially salt stress in potato, provides some theoretical and technical basis for obtaining new varieties resistant to salt stress.【Method】 StDof5 was cloned, and it was analyzed via bioinformatics. StDof5 overexpression vector was constructed by homologous recombination, potato was transformed to obtain positive plants lines, and their expressions were analyzed.【Result】 CDS of StDof5 is 498 bp, it encodes 165 amino acids, and the molecular formula is C₈₀₀H₁₂₃₆N₂₄₂O₂₄₆S₉. The number of StDof5 atoms is 2 533, the relative molecular weight is 18 468.63 kD, the isoelectric point is 8.47, the aliphatic index is 51.45, and the instability coefficient is 45.16, the protein is predicted to be an unstable protein. The predicted number of StDof5 transmembrane helices is 0, thus it is a non-transmembrane protein. The average value of hydrophilicity of StDof5 protein is -0.876, predicting that it is hydrophilic. The predicted secondary structure of StDof5 protein showed it has 20% α-helices, 0 β-turn, 9.7% extended strands and 66.06% random coils. Salt stress phenotype experiments showed that the plant heights and fresh weights of overexpressing plants（OPs）were significantly higher than those of wild type（DES）plants, but there was no significant difference in root length, root number and leaf number between them. 【Conclusion】 Overexpressing plants（OPs）can resist salt stress and demonstrate desensitization phenotype to salt stress. StDof5 has significant responses to salt stress and tissue specificity.

Cloning and Preliminary Functional Analysis of CaPI Gene in Capsicum annuum L.

WU Xing-xing, HONG Hai-bo, GAN Zhi-cheng, LI Rui-ning, HUANG Xian-zhong

2024, 40(3):  193-201.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0957

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【Objective】 The PISTILLATA（PI）gene belongs to the typical type II MADS-box gene family and is a class B gene in the ABC（D）E model, and plays an important role in plant development. However, functions of the PI homologous genes in pepper（Capsicum annuum L.）have not been reported. This study explored the function of pepper PI gene, laying a foundation for in-depth research on the functional mechanism of plant PI homologs.【Method】 We cloned a PI homologous gene CaPI from the cDNA of floral organ tissues of pepper using RT-PCR, and analyzed its physicochemical properties, subcellular localization, protein structure, and phylogenetic relationship through bioinformatics methods. The expression pattern of CaPI in different tissues of pepper was performed using real-time quantitative PCR（RT-qPCR）method. A plant overexpression vector of 35S:CaPI was constructed and transformed into Arabidopsis using floral-dipping method.【Result】 The CaPI gene contains an open reading frame of 648 bp and encodes a peptide of 215 amino acids with a relative molecular weight of 25.13 kD. Multiple amino acid sequence alignment indicated that the N-terminus of CaPI contains a conserved motif of ‘MGRGKIEIKRIEN’. Phylogenetic tree analysis with homologous proteins of other species showed that the CaPI gene was closely related to the PI homologous genes of potato, tomato, and petunia. The RT-qPCR analysis showed that CaPI was predominantly expressed in flowers, with the highest expression level in the sepals, followed by petals, with low expression in stamens and almost no expression in pistils. The overexpression of CaPI in Arabidopsis revealed that, compared to wild type（Col-0）plant, the 35S:CaPI transgenic plants exhibited phenotypes such as increased number of rosette leaves and branches, but did not affect the development of floral organs.【Conclusion】 CaPI in the pepper functions in promoting plant branching development.

Genome-wide Analysis of Codon Usage Bias in Saccharum Species and Its Phylogenetically Related Species Erianthus fulvus

TIAN Chun-yan, LI Xu-juan, LI Chun-jia, MAO Jun, LIU Xin-long

2024, 40(3):  202-214.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0982

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【Objective】 To clarify the codon usage characteristics and improve expression efficiency of exogenous genes in sugarcane. 【Method】 Codon usage bias were analyzed by Python and CodonW1.4.2 tools based on their genomes data of three species including Saccharum officinarum (LA-purple), S. sponaneum (NP-X and AP85-441), and Erianthus fulvus (Yunnan2009-3). Neutrality-plot, ENC-plot, and Parity Rule 2（PR2）-plot analysis were also used to explore the possible factors that affect the formation of codon usage bias. Simultaneously, correlation analysis between gene expressions and codon usage bias parameters was carryd out based on transcriptome data. Finally, a comparison analysis of codon usage patterns were performed between sugarcane and the other seven main model organisms, namely, Zea mays, Sorghum bicolor, Oryza sativa, Arabidopsis thaliana, Nicotiana tabacum, Escherichia coli, and Saccharomyces cerevisiae based on the average RSCU value of three species. 【Result】 S. officinarum, S. sponaneum, and E. fulvus were rich in GC base pairs, the average GC content was 56.3%, and GC3 was the highest, followed by GC1 and GC2, indicating that sugarcane preferred to use synonymous codons ending with a C/G base. The average ENC value was 48.45, suggesting that sugarcane had a lower codon usage bias. According to the results of neutral-plot, ENC-plot, and PR2-plot analysis, the codon usage bias of four genomes was affected by natural selection, mutation pressure, and so on, and the natural selection dominated in them. Correlation analysis showed that codon preference parameters were significantly correlated with the actual transcription expressions of genes, but the correlation was not strong. Based on the RSCU and ∆RSCU values, thirteen identical optimal codons were confirmed, and all of them were ended with C/G base pairs. The codon usage characteristics were not different at genome-wide and chromosome level. In addition, the codon usage bias of sugarcane was similar to that of Z. mays, S. bicolor, and O. sativa, while it had a significant difference with A. thaliana, N. tabacum, E. coli, and S. cerevisiae. 【Conclusion】 The codon usage bias among S. officinarum, S.sponaneum and E. fulvus was highly similar, and its formation was affected by natural selection and mutation pressure factors. Meanwhile, Z. mays, O. sativa and S. bicolor could be used for elite gene function research on sugarcane as a exogenous expression system on account of their codon usage bias patterns.

Regulatory Genes Mining Related to Transcriptome Sequencing and Phenolic Metabolism Pathway of Canarium album Fruit with Different Fresh Food Quality

XIE Qian, JIANG Lai, HE Jin, LIU Ling-ling, DING Ming-yue, CHEN Qing-xi

2024, 40(3):  215-228.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0747

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【Objective】 The category and content of phenolic compounds in fruits are important quality traits closely related to the nutrition and flavor of Chinese olives（Canarium album）. This work is to explore the molecular regulation mechanism of phenolic biosynthesis in Chinese olives.【Method】 The fruits of Chinese olives（low-phenol/high-phenol）with significant differences in total phenol content were taken as test materials, and the transcriptome analysis was carried out 80-160 d after flowering, and the differential genes of shikimic acid-hydrolyzed tannin/phenylpropane-flavonoid biosynthesis pathway were characterized, and the differentially expressed genes were analyzed by WGCNA, from which the transcription factors related to phenolic metabolism pathway were mined.【Result】 The 296 314 Unigenes were obtained, of which 73% were annotated to the database. A total of 1 628 differentially expressed genes（DEGs）were identified in the mature fruits of four varieties（lines）of Chinese olive. KEGG analysis showed that DEGs was significantly enriched in the “flavonoid biosynthesis” pathway of phenolic metabolism pathway. Furthermore, DEGs of shikimic acid-hydrolyzed tannins/phenylpropane-flavonoids biosynthetic pathway in fruit ripening process was characterized. Combining R² and P values of each module and character in WGCNA analysis, four key modules were screened out. According to the regulation relationship of genes in modules, MCC topological analysis was used to mine key transcription factors in modules with degree value ≥1. A total of 137 transcription factors Unigenes were found to be co-expressed with 30 phenolic synthetic structural genes Unigenes. The functional annotations of the transcription factors Unigenes were from 35 gene families, the most of which were zinc finger proteins（C2C2, C3H, C2H2 and PHD）, followed by B3, HB, MYB and NAC gene families.【Conclusion】 This study initially analyzed the differences in phenolic metabolic pathways in Chinese olive fresh food quality from a transcriptomic perspective, and concurrently excavated the differential genes regulating phenolic metabolism, which may provide an important basis for further investigation of the molecular mechanisms underlying the differences in Chinese olive fresh food quality.

Identification and Expression Analysis of 14-3-3 Gene Family in Dendrobium officinale

JIANG Lin-qi, ZHAO Jia-ying, ZHENG Fei-xiong, YAO Xin-yi, LI Xiao-xian, YU Zhen-ming

2024, 40(3):  229-241.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0839

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【Objective】 14-3-3 protein, also known as general regulatory factor（GRF）, encodes through multi-gene family, and plays a central role in encouraging plant growth and development and stress-related adversity responses. The identification of GRF gene family in Dendrobium officinale may provide theoretical basis for the study of D. officinale GRF gene function and genetic improvement.【Method】 Bioinformatics was used to identify the members of 14-3-3 gene family, and analyze their physicochemical properties, chromosomal localization, phylogenetic tree, gene structure, and cis-acting elements of each DoGRF member. Simultaneously, their expression profiles in different tissues and under the treatment of low-temperature or salt stress were detected using the real-time quantitative PCR.【Result】 Results demonstrated that seventeen DoGRF gene family members were presented in D. officinale, which harbored two subfamilies（ε group and non-ε group）. They were unevenly located on 7 chromosomes, and harbored seven tandem repeat gene pairs. The gene structure, conserved motifs, and secondary structure of DoGRF members within the same subfamily were similar. The cis-acting elements at the promoter regions of DoGRF family genes contained abundant hormone and stress response elements. DoGRF genes family were expressed in various tissues（flower buds, sepals, labellum（lip）, pollinia, gynostemium（column）, stems, leaves, roots, green root tips, and white part of roots）, and most of them were highly expressed in floral organs, followed by stems and roots. Besides, they were differentially expressed under treatment of low-temperature or salt-stress, suggesting that they might be involved in regulating the stresses of low-temperature and salinity. Especially, DoGRF2 might play a crucial role in the process of stress responses.【Conclusion】 17 members of DoGRF family were systematically identified from D. officinale at the genome level. Different genes possessed different responses to abiotic stresses, and exhibited tissue-specific expression profiles. DoGRF2 showed positively response to low-temperature and salt stress.

Impact of Different Water Management Cultivation Methods on the Rhizosphere Bacteria Community of Shanlan Upland Rice

LIU Jia-ning, LI Meng, YANG Xin-sen, WU Wei, PEI Xin-wu, YUAN Qian-hua

2024, 40(3):  242-250.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0884

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【Objective】 The cultivation way of Shanlan upland rice is single, and the traditional slash and burn cultivation method damages the environment. Cultivation in water can be a new way for Shanlan upland rice. This work is to explore the changes in rhizosphere microorganisms of Shanlan upland rice under water cultivation environment in large fields. 【Method】 The cultivation treatment of Shanlan upland rice was set up with normal irrigation and drying management, and 16S rRNA gene amplification sequencing was performed on the bacterial community in the rhizosphere soil of Shanlan upland rice. The sequencing data was combined with soil physicochemical properties for comprehensive analysis. 【Result】 The changes in cultivation methods have significantly affected the composition of rhizosphere bacterial communities. In particular, the relative abundance of Nitrosprota and Proteobacteria in the normal irrigation is lower than that in the drying management, and the relative abundance of Firmicutes is higher than that in drying management. The top 10 bacterial communities with relative abundance are correlated with soil environmental factors. The correlation network analysis shows that more bacterial communities interact in normal irrigation, and there is a more complex bacterial network under normal irrigation. 【Conclusion】 Significant changes in bacterial community composition occure in the rhizosphere of Shanlan upland rice under different water management cultivation methods. The bacteria groups of Nitrospirota and Proteobacteria may improve the stress resistance of Shanlan upland rice by promoting nitrogen uptake at the root of rice. In normal irrigation, more bacterial community interactions enhance the stability of the ecosystem.

Mn²⁺ Promotes the Biofilm Formation of Bacillus altitudinis LZP02

SHAN Xin, HUANG Dong-hui, XU Wei-hui, WANG Zhi-gang, CHEN Wen-jing, HU Yun-long

2024, 40(3):  251-260.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0888

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【Objective】 Bacillus altitudinis LZP02 is a plant growth-promoting rhizobacteria（PGPR）in the rice rhizosphere, which can colonize rice rhizosphere to form biofilm, and Mn²⁺ can promote the biofilm formation of LZP02 strain, but the regulation mechanism is not clear. The purpose of this study is to explore the mechanism of Mn²⁺ promoting the biofilm formation of LZP02 strain. 【Method】 Crystal violet staining and anthrone-sulfuric acid method were used to perform quantitative analysis of biofilm and exopolysaccharide yield, respectively. Scanning electron microscope（SEM）was used to observe LZP02 colonization in rice rhizosphere. And differently expression genes（DEGs）were analyzed by transcriptome sequencing technology.【Result】 The addition of 4 mmol/L Mn²⁺ and 8 mmol/L Mn²⁺ significantly enhanced the film-forming ability of LZP02 strain and the production of exopolysaccharide, scanning electron microscopy showed that 4 mmol/L Mn²⁺ and 8 mmol/L Mn²⁺ improved the colonization ability of LZP02 strain in rice rhizosphere. The number of DEGs increased significantly with the increase of Mn²⁺ concentration, which was mainly enriched in spore formation, toxin metabolism pathway and two-component system. The expression of skf operon gene was up-regulated in 1 mmol/L Mn²⁺ and 4 mmol/L Mn²⁺ treatments. Scanning electron microscope observation showed that there was damage in LZP02 cells in 1 mmol/L Mn²⁺ and 4 mmol/L Mn²⁺ treatments. The genes of KinE and Spo0A in the two-component system of 4 mmol/L Mn²⁺treatment group were significantly up-regulated. 【Conclusion】 Mn²⁺ can improve the biofilm-forming ability of strain LZP02 by “cannibalism” and KinE gene activates Spo0A~P in two-component system.

Fungal Electrochemical Remediation of Herbicide-contaminated Soil: Preliminary Study on Degradation Kinetics

HAO Da-cheng, ZHENG Yu-wei, WANG Fan, HAN Lei, ZHANG Ze

2024, 40(3):  261-272.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0895

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【Objective】 Microbial fuel cell（MFC）, which produces electricity while removing pollutants, is a promising means of ecological restoration. We aim to construct a fungal-augmented MFC device, compare the effects, advantages and disadvantages of electrokinetic（EK）, fungal and MFC remediation against herbicides in the polluted soil, and explored the application potential of MFC in the remediation of organic pollutants. 【Method】 A novel MFC with fungal bioaugmentation was designed. The contaminated soil by two herbicides was remediated by three methods of EK, fungi and MFC remediations, and the screened and domesticated Talaromyces dalianensis and Myrothecium verrucaria strains were implemented in the latter two approaches. The effect of fungal augmentation on the removal of herbicides was studied. Soil pH, electrical conductivity, herbicide removal, and MFC electrical performance were quantified, and degradation products of two herbicides were identified by gas chromatography-mass spectrometry. 【Result】 In EK remediation, when simulated electrolyte, carbon fiber and 10 V were applied for 7 d, the removal rate of florpyrauxifen-benzyl（F）and haloxyfop-P（H）was 71% and 38%, respectively. Compared with H, F was more easily degraded, and fungal and MFC treatments completely removed it. Compared with T. dalianensis, M. verrucaria strain had better degradation capacity to both herbicides. MFC based on two fungi had the removal rate of 62.5% and 24.1% respectively toward H. The degradation product of F and H was halauxifen and 4-methoxybenzyl acetate, respectively, and the degradation pathways and kinetics were speculated. The degradation of F by three methods and the degradation of H by EK followed the first-order kinetic reaction, while the degradation of H by fungi and MFC followed the second-order reaction. 【Conclusion】 Compared with EK and fungal remediations, MFC remediation had a better effect, which can remediate the soil without the need for additional power supply, making it a cost-effective self-sustaining remediation strategy.

Screening and Growth-promoting Characteristics of Multifunctional Exopolysaccharides-producing Bacteria

CHANG Hai-xia, LI Ming-yuan, MAIRIYANGU·Yasheng , ZHOU Qian, WANG Ji-lian

2024, 40(3):  273-285.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0942

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【Objective】 To explore the effect of exopolysaccharides-producing strains in promoting plant growth and improving soil properties in saline soils, which may lay a foundation for the development of microbial bacterial agents applicable to saline soil remediation.【Method】 Strains with high-yield exopolysaccharide were isolated from the rhizosphere soil of saline-alkali grassland in Kashi, Xinjiang, and species identification was performed based on 16S rRNA gene sequence comparison and phylogenetic analysis. Moreover, the single factor and response surface method were used to optimize the medium components of the excellent strains to increase the yield of exopolysaccharides, and its effects on plant seedlings cultivated in saline-alkali soil and soil aggregate formation were verified by inoculation test.【Result】 A total of 19 exopolysaccharides-producing strains were screened, which were divided into 7 genera, of which Pseudomonas were the dominant. The exopolysaccharides yield was 236-1 544 mg/L, especially Pantoea sp. MQ A0. In addition, MQ A0 was capable of nitrogen fixation, indole-3-acetic acid（IAA）secretion, siderophores production and phosphorus solubilization. The optimal fermentation medium of MQ A0 was 12.5 mL/L glycerol, 9.0 g/L peptone, 5.5 g/L yeast exact and 5.1 g/L CaCO₃. Compared with the initial fermentation process, the exopolysaccharides yield reached 2 436 mg/L, which increased by 57.8%. Pot experiments showed that the inoculation of Zea mays with the fermentation broth of MQ A0 had a significant promoting effect on seedling in saline-alkali soil, including the development of plant height, fresh weight, stem diameter, root number and total root length, which increased by 41.2%, 203.0%, 42.7%, 30.4%, 99.7%, respectively. And the formation of soil aggregate was significantly promoted by the EPS（P<0.05）.【Conclusion】 The high-yield exopolysaccharide strain MQ A0 had various growth-promoting properties, and showed positive effects on plant growth and soil improvement in saline-alkali soil.

Influence and Mechanism of Bacteroides fragilis Type VI Secretory System on the Intestinal Barrier

LEI Qi-yi, XU Yang, LI Peng-fei

2024, 40(3):  286-295.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0840

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【Objective】 To investigate the effect and mechanism of Bacteroides fragilis type VI protein secretion system（T6SS）on the intestinal barrier. 【Method】 Suicide vector was used to construct B. fragilis T6SS mutant strain. The dextran sulfate sodium（DSS）salt-induced colitis mouse model was constructed, and supplemented mice with PBS, B. fragilis WT, and B. fragilis ΔT6SS, respectively. The disease characters and intestinal barrier integrity were compared among the three groups. Quantitative PCR and immunohistochemistry were used to detect the expressions of tight junction proteins. Untargeted metabolomics was used to compare gut differential metabolites in each group of mice.【Result】 The deletion of T6SS did not affect bio-viability of B. fragilis. B. fragilis WT showed the improvements in body weight loss and colon length compared to PBS controls, demonstrating the protection to DSS-induced colitis; while this protection was lost with the absence of T6SS. The fluorescein isothiocyanate dextran concentration in the mouse serum and histological examination in intestinal pathological sections indicated that B. fragilis T6SS improved the integrity of the intestinal barrier in mice. T6SS mutation affected intestinal tight junction protein expression, confirmed by quantitative PCR and immunohistochemistry. Untargeted metabolomics analysis revealed 96 up-regulated differentially expressed metabolites in the B. fragilis WT group, compared to the B. fragilis ΔT6SS group, with multiple metabolites enriched in cholinergic synaptic metabolism and glycerophospholipid metabolism-related pathways. 【Conclusion】 B. fragilis T6SS alters intestinal metabolome and increases the expression of tight junction protein in intestinal cells, thus improving the permeability of intestinal barrier, being involved in the protection of B. fragilis to the intestinal barrier.

Biological Characteristics and Application of Enteroinvasive Escherichia coli Phage DK-13

WANG Meng-ya, LIU Jia-qi, JIANG Hai-lin, LI Jing-hua, ZHAO Chun-yan, HUANG Hong-lan

2024, 40(3):  296-304.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0902

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【Objective】 Selection and isolation of phage that can lyse Enteroinvasive Escherichia coli (EIEC), analyzing its biological characterization, and investigating its sterilizing effect on contaminated pork. 【Method】 The double-layer agar method was used to isolate phages, and then the optimal multiplicity of infection，one-step growth curve and other biological characteristics of the isolate were determined. The genetic genomes were sequenced and analyzed, and the lysis efficiency was evaluated.【Result】 A bacteriophage that specifically lysed EIEC was isolated from hospital sewage and named DK-13. It has a typical tadpole-like morphology, consisting of a head and a contractile helical symmetric tail. It belongs to the family Myoviridae. The biological characterization showed that the optimal multiplicity of infection was 0.01, the incubation period was about 10 min, the lysis period was about 70 min, and the phage DK-13 was able to survive at 50℃ and pH 5.0-10.0. The whole genome sequencing showed that the phage genome was about 172 275 bp in length, with a GC content of 40.18%, and was predicted to have a total of 293 open reading frames（ORFs）, and no resistance genes or virulence genes were found. the sterilization effect in the contaminated pork was good, and the number of host bacteria significantly reduced. 【Conclusion】 A new virile phage DK-13 was isolated and identified, which has the advantages of short incubation period and high cleavage efficiency, and has great potential for application in food safety.

Isolation and Identification of Rumen Skatole-degrading Bacteria and Analysis on Their Degradation Characteristics

WANG Lu, LIU Meng-yu, ZHANG Fu-yuan, JI Shou-kun, WANG Yun, ZHANG Ying-jie, DUAN Chun-hui, LIU Yue-qin, YAN Hui

2024, 40(3):  305-311.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0934

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【Objective】 The aim of this study is to isolate skatole-degrading bacteria from the rumen of sheep and estimate its skatole-degrading ability and growth performance, to develop suitable direct-fed microbials for ruminants to reduce odor.【Method】 The MSM medium containing skatole was used for enrichment and isolation from the rumen fluid of sheep. The bacterial colony morphology was used for primary classification. Amplification, sequencing and phylogenetic analysis of 16S rRNA gene were carried out for further identification. The growth curve was drawn, and degradation curve of skatole was determined using HPLC.【Result】 A total of 25 skatole-degrading strains were obtained, and 11 representative strains（MSML1-11）of them were selected based on colony morphology for further study. MSML2 and MSML6 belonged to Bacillus subtilis, MSML4, MSML5, MSML7 and MSML10 belonged to Priestia aryabhattai, MSML3 and MSML11 belonged to Burkholderia contaminans. MSML1, MSML8 and MSML9 belonged to Pseudomonas chengduensis. Among these strains, MSML5 grew at the fastest rate. Its stable phase appeared after 12 h, and the bacterial concentration in the stable phase was the highest. MSML3 and MSML8 grew slowly in the first 16 h and reached a stable phase after about 32 h. In terms of skatole degradation, MSML2 showed the highest skatole degradation efficiency of 23.03% within 48 h, followed by MSML7, MSML8 and MSML10, with a degradation rate higher than 20%.【Conclusion】 This research successfully isolated skatole-degrading bacteria from the rumen of sheep, including four different species. Bacillus subtilis, Priestia aryabhattai and Pseudomonas chengduensis with skatole-degrading ability are reported for the first time, which provides valuable resources for the development of microbial agents directly fed to ruminants.

Effect of γ-aminobutyric Acid on Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells

SHAN Xin-yu, LI Tai-chun, YANG Ruo-chen, DUAN Xiang-ru, KANG Jia, ZHANG Ying-jie, LIU Yue-qin

2024, 40(3):  312-321.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0784

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【Objective】 This study was conducted to investigate the effects of γ-aminobutyric acid（GABA）on the apoptosis and the secretion of steroid hormones in ovine ovarian granulosa cells（GCs）. 【Method】 The GCs were treated with 0, 10^-8, 10^-7, 10^-6 and 10^-5 mol/L GABA for 24 h, respectively. Then, the cell proliferation and apoptosis were detected by CCK-8 and flow cytometry. And the appropriate culture concentration of GABA was screened for subsequent experiments. ELISA was used to detect the secretion levels of estrogen（E₂）and progesterone（P₄）in the culture supernatant after GCs were treated with the appropriate concentration. The qRT-PCR and Western blot were to detect the mRNA and protein expressions of GCs proliferation-promoting genes（PCNA, CCNB1, and CCND1）, anti-apoptosis genes（Bcl-2）, apoptosis-promoting genes（Bax, Caspase-3）and steroid hormone synthesis-related genes（StAR, 3β-HSD, CYP11A1, CYP19A1）.【Result】 The 10^-6 mol/L GABA treatment for 24 h significantly promoted GCs proliferation（P < 0.05）and significantly inhibited its apoptosis（P < 0.05）. The mRNA and protein expressions of PCNA, CCNB1, CCND1 and Bcl-2 in the experimental group were higher than those in the control group（P < 0.05）. Compared with the control group, the mRNA and protein expressions of Bax and Caspase-3 in the experimental group decreased significantly（P < 0.05）, as well as Bax/Bcl-2 decreased significantly（P < 0.05）. Treatment of ovine ovarian GCs with 10^-6 mol/L GABA for 24 h significantly promoted the synthesis of P₄（P < 0.05）and significantly inhibited the synthesis of E₂（P < 0.05）. Compared with the control group, the mRNA and protein expressions of StAR, 3β-HSD and CYP11A1 in the experimental group increased significantly（P < 0.05）and the mRNA and protein expressions of CYP19A1 decreased significantly（P < 0.05）. 【Conclusion】 The results demonstrate that GABA treatment could promote GCs proliferation as well as inhibit GCs apoptosis by regulating the expression of CCNB1, CCND1, PCNA, Caspase-3, Bax and Bcl-2, and promote the synthesis of P₄ as well as inhibit the synthesis of E₂ by regulating the expressions of StAR, 3β-HSD, CYP11A1 and CYP19A1.

Study on the Properties and Functions of LchAD Protein, a Key Module of Lichenysin Synthase

YANG Wei-jie, YANG Zhou-lin, ZHU Hao-dong, WEI Yu, LIU Jun, LIU Xun

2024, 40(3):  322-332.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0996

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【Objective】 Lichenysin, as a kind of lipopeptide substance, can promote the formation of Baijiu flavor. To study the properties and functions of thioesterase module LchAD protein in lichenysin synthase, Bacillus strains were isolated from Daqu using traditional separation and purification methods.【Method】 The fermentation products were detected by primary and secondary mass spectrometry, and the species relationship of strains was analyzed by morphological, physiological and biochemical experiments and phylogenetic tree construction. LchAD gene was cloned using genomic DNA of a selected strain as template, and LchAD protein was heterologously expressed in Escherichia coli and purified by nickel column. Bioinformatics analysis was performed with online software to explore the properties and functions of LchAD protein.【Result】 The strain YC7 isolated from Daqu was Bacillus licheniformis, and its fermentation products contained the homologue of lichenysin A（D or G）, indicating that strain YC7 was a lichenysin producing bacterium. The study on the soluble expression of LchAD protein showed that LchAD protein was induced to form soluble protein at the temperature of 20℃ and the final concentration of IPTG was 100 μg/mL, with a molecular weight of 27.62 kD. The results of nickel column purification showed that the relatively pure LchAD protein was obtained when the imidazole concentration was 250 mmol/L. The results of bioinformatics analysis showed that LchAD protein was a stable hydrophilic protein without signal peptide, with a theoretical isoelectric point（pI）of 7.23 and a conserved GrsT domain. The secondary structure of LchAD protein consisted of 43.5% α-helix, 13.82% β-folding and 5.69% β-turn, and its tertiary structure had a high similarity with the thioesterase module of surfactant synthetase. 【Conclusion】 The isolated B. licheniformis strain YC7 can produce lichenysin, and the protein encoded by the LchAD gene of this strain is a hydrophilic protein of 27.62 kD. Soluble LchAD protein can be obtained by heterologous expression, which may be functionally similar to the thioesterase module of surfactant synthetase.