Table of Content

26 June 2025, Volume 41 Issue 6

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Current Progress and Applications of CRISPR/Cas12a Gene Editing Technology in Plants

HUO Guan-zhong, ZHANG Xin-ru, TIAN Shi-jun, LI Jun

2025, 41(6):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1160

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‍The CRISPR/Cas system provides bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. The development and optimization of CRISPR/Cas system have provided a variety of gene editing tools for life science research, such as CRISPR/Cas9 genome editing technology, CRISPR/Cas12a genome editing technology, base editing, and prime editing. These tools are capable of precisely editing target genomes to generate various types of desired mutants, and show great application potential in functional genomics, construction of disease models, gene therapy and crop breeding. CRISPR/Cas12a genome editing technology is developed based on Class II type V CRISPR. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA. Besides, it presents distinct characteristics, such as the ability to utilize TTTV PAM, easy to engineer, and multiplex genome editing. Due to these advantages, CRISPR/Cas12a has been successfully applied in diverse species including animals and plants since the first report. Therefore, CRISPR/Cas12a will have a high prospect of providing important technical support for gene therapy and crop breeding. In this review, we provide a detailed introduction to the CRISPR/Cas12a genome editing technology, as well as the precise genome editing technologies developed based on CRISPR/Cas12a. We also focus on the strategies for optimizing CRISPR/Cas12a through improving editing efficiency, expanding the targeting scope, and enhancing specificity. Finally, we summarize the current applications of CRISPR/Cas12a in the creation of new plant germplasm. This review is aimed to facilitate the further application of CRISPR/Cas12a in crop improvement.

Research Progress in RNA Binding Proteins in Plant Disease Resistance

LYU Yue, ZHANG Jie-wei, WANG Bo

2025, 41(6):  12-26.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0056

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During their growth and development, plants are continuously exposed to complex environmental stresses that severely constrain their growth, agronomic traits, and productivity. To combat biotic stresses such as pathogen infection, plants have evolved multilayered sophisticated regulatory networks. In recent years, post-transcriptional regulation has emerged as a novel research hotspot in plant immunity, demonstrating unique advantages in the resistance to disease through dynamic regulation of messenger RNA (mRNA) metabolism. RNA-binding proteins (RBPs), functioning as core executors in plant resistance-to-resistane networks, act as "molecular switches" in plant-pathogen interactions by recognizing specific RNA motifs to regulate critical processes including pre-mRNA alternative splicing, mRNA stability, alternative polyadenylation (APA), translation efficiency, and RNA modifications. This review systematically elaborates RBP-mediated post-transcriptional regulatory mechanisms and their functions during plant-pathogen interactions. For instance, at the pathogen-recogned stage, RBPs regulate mRNA stability of immune receptors to enable rapid activation of defense signals. During disease resistance responses, RBPs mediate alternative splicing of resistance genes to generate transcript variants with distinct subcellular localization or functional activities. Recent studies also reveal novel pathways in plant immunity where RNA epigenetic modifications (e.g., m⁶A) regulate RBP recruitment efficiency. This article provides in-depth analysis of the multilayered defense systems constructed through RBPs and their molecular regulatory mechanisms, while proposing future research directions including deciphering RBP-mediated disease resistance mechanisms, modifying RBP regulatory elements through multi-omics integration, and developing novel disease-resistant breeding strategies. Comprehensive understanding of RNA regulatory codes in plant immunity will offer theoretical foundations for creating broad-spectrum resistant germplasm and provide crucial references for developing innovative green control strategies.

Research Progress in the Function and Their Action Mechanism of Plant PPR Protein

WANG Yi-fan, ZHU Hong-liang

2025, 41(6):  27-37.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1185

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RNA editing is a post-transcriptional level process of processing and modification that makes mature RNA different from its DNA template strand by base insertion, deletion, or replacement, which is a universal correction mechanism in plants to restore conserved amino acids that have been altered by DNA mutations. PPR proteins (pentatricopeptide repeat protein) are an important class of RNA editing factors that are widely distributed in plants and constitute one of the largest families of higher plants. At present, many studies have shown that it plays an important role in plant growth and development. Its main function is to affect the expression of organelle genes by participating in the processing of RNA precursors, such as achieving the transformation of RNA C-U, participating in intron splicing, affecting mRNA stability and translation, etc., thus affecting photosynthesis, respiration, plant development and environmental response. In this paper, the classification, localization, function and mechanism of PPR protein reported in recent years are reviewed, the application prospect of PPR protein is also prospected, aiming to provide theoretical basis for the subsequent research and analysis of the function of PPR protein and its application.

Research Progress in the Mechanisms and Functions of Gene Loss in Genome Evolution

ZHOU Yi, LIU Yong-bo

2025, 41(6):  38-48.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1239

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Gene loss is widespread among organisms and is one of crucial mechanisms in the evolution of genomes, contributing significantly to environmental adaptation and speciation. However, how gene loss happens and its implications remain unclear. This review synthesizes research progress in understanding the molecular mechanisms, functional consequences, and bias patterns of gene loss. Gene loss primarily arises from intrinsic mechanisms such as DNA replication errors, transposon activities, and chromosome structural variations, with additional influences from genetic drift, natural selection and other evolutionary forces. While gene loss can enhance the survival and reproductive capacities of organisms by optimizing resource utilization, streamlining metabolic pathways, and improving environmental adaptability, it likely inhibits adaptive flexibility to environmental changes due to the loss of critical functional genes, thereby increasing survival risks. Gene loss demonstrates non-random preferences, which is influenced by gene function, expression level, dosage sensitivity, genomic location, and protein network topology. Gene loss interacts with gene duplication, horizontal gene transfer (HGT) and other mechanisms to maintain a dynamic balance between genome reduction and expansion. In the future, it is essential to investigate the trade-offs and risks associated with gene loss, to clarify the mechanisms of gene loss in regulating adaptive strategies and its impacts on environmental adaptation, particularly in speciation and adaptive evolution, and ultimately to advance its applications in genetic and bioengineering breeding.

Advance on the Changes of Rhizosphere Microbial Communities in the Growth Stages of the Four Major Staple Crops

CHEN Cai-ding, SONG Yun-jie, TIAN Meng-qing

2025, 41(6):  49-60.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1140

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Rhizosphere microbes are selectively recruited and aggregated from the surrounding soil microbiota, forming a unique community influenced by both biotic and abiotic factors. These microorganisms, closely associated with plant roots, collectively form the rhizosphere microecosystem, which plays a crucial role in plant growth and development. In recent years, breakthroughs in high-throughput sequencing and metagenomic technologies have shifted research focus from model plants to four major staple crops, rice, wheat, maize, and potato, gradually unveiling the dynamic evolutionary patterns of rhizosphere microbiomes throughout crop life cycles. Studies demonstrate that the structure and functionality of rhizosphere microbiota show significant temporal heterogeneity across developmental stages. During plant growth, the alpha diversity of rhizosphere microbiota typically follows a parabolic trend (“low-high-low”), peaking during the vegetative growth phase. This stage-specific succession is closely linked to plant nutrient demands, compositional shifts in root exudates, soil environmental fluctuations, immune responses, and microbial interactions. This review synthesizes recent advances in rhizobacterial community dynamics across growth stages of four staple crops and their underlying mechanisms, providing insights for rhizosphere beneficial microbe research and microbial inoculant applications during crop cultivation. Future efforts should integrate microbiome engineering with agronomic practices to develop growth-stage-specific microbial amendments, thereby offering theoretical foundations and technical support for precise regulation of plant-microbe interactions.

Research Progress in Single-base Mutation Detection Methods and Applications

LIU Hua, SONG Jie, ZENG Hai-juan, WANG Jin-bin, QIAN Yun-fang

2025, 41(6):  61-70.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0933

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Single nucleotide mutation refers to a type of genetic mutation in which a single nucleotide in the genome sequence changes, which has been proven to be an important cause of biological organisms' hereditary traits, susceptibility to diseases, and resistance to drugs. It has significant research significance in many fields, including genetics, disease diagnosis, and biological evolution. With the continuous development of nucleic acid detection technology, single nucleotide mutation detection technology provides critical assistance in assisting plant and animal breeding, detecting disease or microbe-related mutation sites, and guiding the use of therapeutic drugs. This review summarizes several common single nucleotide mutation detection methods, briefly introduces the principles, advantages, and limitations of each method, and lists the application situations of the technology in hereditary traits, disease diagnosis, virus detection, food adulteration, plant and animal breeding, and microbial resistance detection. It focuses on describing the rapid detection strategy of single nucleotide mutation based on the CRISPR/Cas system, and elaborates on the application of the system in different fields based on the precise identification of the target type, and analyzes it in combination with nucleic acid amplification technology. It also discusses the application and development trend of single nucleotide mutation detection technology in the future.

Cutting-edge Omics Technology Innovations Empower Livestock and Poultry Biological Breeding

LI Chen-ying, KONG Da-shuai, LI Ruo-nan, ZHANG Yu-bo, YAN ping, LI Kui, KONG Si-yuan

2025, 41(6):  71-86.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0733

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The growth of the global population has led to an increase in the demand for livestock and poultry products, while climate change and environmental pressures pose challenges to the survival and production of livestock and poultry. The independently selected core livestock and poultry breeds in China is rather lack, and thus "biological breeding" has become a "national strategy" for the New Quality Productivity of Agriculture. Therefore, it is necessary to breed new livestock and poultry breeds with excellent traits and strong adaptability. The continuous development of omics and molecular biology technology has promoted the development and improvement of genomics, transcriptomics, epigenetics, three-dimensional genomics, metagenomics, and single-cell and other multi-omics technologies, and successfully combined and applied to genome-wide association analysis, marker-assisted selection, genome-wide selection and other livestock and poultry bio-breeding methods. With the innovation and development of cutting-edge genomics technologies, the potential molecular genetic mechanisms of livestock and poultry biomorphogenesis have also been realized to be resolved. In order to make better use of Genomic & Multi-omics Breeding techniques and concepts to help livestock and poultry biological breeding, this review summarizes the contents and characteristics of the main livestock and poultry biological trait formation and molecular breeding-related genomics technologies. It firstly describes the principles and applications of classical genomics technologies, which are mainly related to genomes, transcriptomes, proteomes, etc. Meanwhile, it also pointed out the limitations of these technologies, and introduced a series of new omics technologies, including three-dimensional (3D) genomics, gut microbiomics and single-cell omics. It also summarizes the application of the Genomic & Multi-omics Breeding concept in the analysis of economically important traits and molecular biological breeding of livestock and poultry and outlines the application scenarios and challenges of these genomics technologies. Finally, the review discusses the comprehensive application of multi-omics technology, and looks forward to the future development trend of genomics technology. It aims to provide new references for the research of important traits of livestock and poultry and the development of breeding field, and to promote the development of livestock and poultry breeding towards a more precise, efficient, and economical direction.

Genome-wide Identification and Expression Analysis of the Rice BXL Gene Family

WU Hao, DONG Wei-feng, HE Zi-tian, LI Yan-xiao, XIE Hui, SUN Ming-zhe, SHEN Yang, SUN Xiao-li

2025, 41(6):  87-98.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1215

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Objective BXL (β-D-xylosidase) belongs to the third family of glycoside hydrolases, which catalyzes the degradation of xylan in the cell wall, and plays a key role in regulating plant growth and development as well as abiotic stress response. Evolutionary and expression analysis of the OsBXL gene family will facilitate further exploration of their functions in rice. Method Bioinformatic technologies were used for analyzing the phylogenetic relationships, replication events, gene structure, conserved motifs, and tissue expression profiles of OsBXLs, and fluorescence quantitative PCR was used to investigate the expression pattern of OsBXLs under abiotic stress and hormone treatments. Result There are 10 members in rice BXL gene family, which can be divided into three subfamilies, with Group III specific to monocots. The gene structures and structural domains of OsBXLs within each group showed a high similarity. Gene location analysis indicated that OsBXL family members were unevenly distributed on 4 chromosomes, with OsBXL5/6/7/8 on chromosome 4 in a gene cluster. Transcriptome data revealed that OsBXL1/2/3/4/8 showed high expressions in a majority of detected tissues/organs, while OsBXL5/6/7/10 were expressed at low levels. Notably, the expressions of OsBXL7/10 were significantly high at the certain stage during the maturation of young inflorescence and seed. Moreover, three haplotypes of OsBXL7 were identified in the natural population, and significant differences in grain length, width, and thousand-grain weight were observed among different haplotypes. Fluorescence quantitative PCR revealed different expression patterns of OsBXLs under drought, saline-alkali, abscisic acid and methyl jasmonate treatments. Conclusion The OsBXL gene family is highly conserved, OsBXL7 is possibly involved in regulating rice grain size, while OsBXL1/3 simultaneously respond to salt-alkali stress and methyl jasmonate treatment. Together, these results provide a key guide for future functional analyses of OsBXL genes in rice.

Functioal Analysis on GmKTI1-like Gene of Soybean Resistance to Bean Pyralid (Lamprosema indicata)

TAN Yu-rong, CHEN Dong-liang, YANG Shou-zhen, LAI Zhen-guang, TANG Xiang-min, SUN Zu-dong, ZENG Wei-ying

2025, 41(6):  99-108.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1136

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Objective Kunitz-type trypsin inhibitors play an important role in enhancing plant resistance to pests. Prior research conducted by our group has demonstrated a significant association between the Kunitz-type trypsin inhibitor GmKTI1-like and resistance to the soybean leaf borer. This study is aimed to investigate the function of the GmKTI1-like gene and investigate its role to provide key resistance genes and new breeding materials for the cultivation of insect-resistant soybean varieties. Method GmKTI1-like was cloned from soybean leaves. Then bioinformatics methods were used to analyze their physicochemical properties, protein structure, physical positioning of chromosomes and subcellular localization, and RT-qPCR was used to analyze the expression pattern of GmKTI1-like in different soybean tissues. Trangenic plants were created using the Agrobacterium-mediated. Following extensive molecular biology research and phenotypic characterization over serveral years, a new transgenic soybean germplasm was identified. This germplasm, which carries the target genes and exhibits genetic stability, was evaluated for its significant application potential. The evaluation included assessments of its insecticial proper ties and measurements of trypsin inhibitor enzyme activity. Result GmKTI1-like was located on chromosome 1, and its encoded GmKTI1-like contained one KTI structural domain and one transmembrane structural domain, and the results of subcellular localization showed that GmKTI1-like was a membrane protein. Fluorescence quantitative PCR showed that GmKTI1-like had the highest expression in soybean leaves. Compared with the wild type (WT), the transgenic soybean plants showed significantly enhanced resistance to insect and high levels of trypsin inhibitor under Lamprosema indicata borer stress. Conclusion The overexpression of GmKTI1-like transgenic soybean lines significantly increase theresistance to insect, indicating its pivotal role in the soybean defense mechaism against the L. indicata borer.

Identification and Functional Analysis of Gravity Response Regulatory Genes in Potato

LUO Ji-lin, LI Jin-ye, JIA Yu-xin

2025, 41(6):  109-118.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0003

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Objective This study aims to provide a reference for elucidating the molecular mechanisms underlying gravity response in potato (Solanum tuberosum L.) and to offer potential genetic resources for the genetic improvement and breeding of potato. Method Paraffin sectioning was employed to examine the histological structures of potato stolons and aerial stems. Gravity-responsive regulatory genes in potato were identified through homology alignment, with their expressions validated by RT-qPCR. Expression patterns were further analyzed using transcriptome sequencing. Utilizing the tetraploid potato variety Desiree as a background, we constructed transgenic lines that overexpressed the gravity-responsive key gene StLAZY1-1, followed by observation and statistical analysis of their phenotypes. Result Significant morphological differences in tissue structure were observed between the stolon and aerial stem, particularly regarding the arrangement of vascular tissues and the deposition of starch granules. Gravity-responsive genes, including PRAF/RLD FAMILY MEMBER (RLD), SCARECROW (SCR), SHOOT-ROOT (SHR), BRX-LIKE4 (BRXL4), and PIN-FORMED3 (PIN3), demonstrated conserved domains and distribution patterns across various species, whereas LAZY family genes displayed diverse domain distributions. The expressions of gravity-responsive genes were found to be tissue-specific and regulated by light signals. Functional verification of the StLAZY1-1 gene in transgenic potato lines demonstrated significant alterations in plant height and stolon number when compared to wild-type plants. Conclusion The key gravity-responsive regulatory gene StLAZY1-1 in potato influences plant height and the number of stolons.

Analysis of the Potato SUMO E3 Ligase Gene Family and Cloning and Expression Pattern of StSIZ1

XU Hui-zhen, SHANTWANA Ghimire, RAJU Kharel, YUE Yun, SI Huai-jun, TANG Xun

2025, 41(6):  119-129.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1252

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Objective This study is aimed to clone, characterize, and analyze the subcellular localization and tissue-specific expression of the StSIZ1 in potato, providing theoretical foundation for elucidating its functional role. Method Homology search was conducted using the Arabidopsis thaliana SUMO E3 ligase gene sequence to obtain members of the potato SUMO E3 ligase gene family, and have bioinformatics analysis of them. The StSIZ1 was cloned from the potato variety Atlantic using reverse transcription PCR (RT-PCR). The expression patterns of StSIZ1 in various potato tissues and its responses to abiotic stresses and hormone were analyzed using quantitative real-time PCR (RT-qPCR). A pCEGFP-StSIZ1 subcellular localization vector was constructed, and the localization of StSIZ1 was confirmed via Agrobacterium-mediated transient expression in tobacco. Result The coding sequence (CDS) of the StSIZ1 is 2 634 base pairs (bp) in length and is located on chromosome 11. RT-qPCR results revealed that StSIZ1 has the lowest relative expression in stems and the highest in tubers. The gene presents significant responsiveness to various abiotic stress treatments, including osmotic, salt, and high-temperature stress. Subcellular localization analysis using fluorescence detection indicated that StSIZ1 primarily functions within the nucleus. Conclusion The StSIZ1 is responsive to a wide range of abiotic stresses and functions in the nucleus, highlighting its potential role in tolerance to stress.

Genetic Diversity and Principal Component Analysis of 125 Potato Germplasm Resources

DUAN Yong-hong, YANG Xin, YU Guan-qun, XIA Jun-jun, SONG Lu-shuai, BAI Xiao-dong, PENG Suo-tang

2025, 41(6):  130-143.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1285

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Objective To clarify the genetic relationship between potato germplasm resources, and to provide a research basis for the creation of new germplasm. Method In this paper, 125 potato varieties (lines) were used as research materials. Morphological markers and SSR molecular markers were used to analyze the differences among germplasm resources, and principal component analysis was used to evaluate the genetic diversity. Result The coefficient of variation of the eight phenotypic traits ranged from 19.40% to 63.90%, and the diversity index ranged from 0.990 7 to 1.001 0. According to phenotypic traits, the tested materials could be divided into four categories, most of which were clustered according to phenotypic index characteristics. Twenty-seven pairs of polymorphic SSR primers were screened, and 910 alleles and 832 polymorphic sites were amplified, with a polymorphism site ratio of 90.93%. The average Nei’s genetic diversity (H) and Shannon’s index (I) were 0.228 6 and 0.362 1, respectively. According to SSR test results, the test materials were clustered into five categories. Most of the varieties were clustered according to their geographical origin, and the new strains were clustered according to their parents. PCA analysis of SSR marker clusters showed that the coordinated distribution results of each group were basically consistent with the clustering results, and the two analysis results supported each other. Conclusion There are differences in the classification of groups between phenotypic traits and SSR marker clustering results, but some potato materials show consistent in the two clustering methods, and the materials from the same geographical origin are clustered into one group. Phenotypic traits are affected by environmental conditions and human factors, and cannot reflect all genomic information, but molecular markers directly detect molecular differences in the genome. The combination of the two methods should be used in the analysis and evaluation of genetic diversity of potato germplasm resources, which may provide reference for potato germplasm innovation and genetic improvement.

Cloning of GhSWEET9 in Upland Cotton and Functional Analysis of Resistance to Verticillium Wilt

ZHANG Yong, SONG Sheng-long, LI Yong-tai, ZHANG Xin-yu, LI Yan-jun

2025, 41(6):  144-154.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0002

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Objective To investigate the correlation between GhSWEET9 gene and cotton plant’s resistance to Verticillium wilt, and provide theoretical basis for exploring the molecular mechanism of cotton plant’s resistance and breeding a cultivars of cotton plant combating Verticillium wilt. Method Bioinformatics software was utilized to analyze the sequence characteristics, phylogenetic relationships, and subcellular localization of the GhSWEET9 gene. Yeast heterologous complementary system was used to clarify the sugar transport function of GhSWEET9 protein. Virus induced gene silencing (VIGS) technology was applied to study the function of this gene in combating Verticillium wilt. Result The GhSWEET9 protein has 7 transmembrane domains, and phylogenetic tree analysis shows that it belongs to the SWEETs Clade II subfamily. The subcellular localization results show that GhSWEET9 is located on the cytoplasmic membrane. Yeast heterologous complementation experiments show that GhSWEET9 protein can transport galactose, mannose, glucose, and fructose. Using VIGS technology to silence GhSWEET9, glucose content measurement reveals that the glucose content in the root system of silenced plants (pTRV2:GhSWEET9) was significantly higher than that of the empty vector control (pTRV2:00). Using the Verticillium dahliae Vd991 strain to infect control plants and silenced plants, the glucose contents in the roots of the silenced plants were still significantly higher than that in the roots of control plants after 2 and 6 d of infection. After 14 d of infection, compared with the control plants, the leaves of silenced plants show more severe yellowing and wilting, more obvious browning of vascular bundles, and a higher disease index. Conclusion The silencing of GhSWEET9 gene is beneficial for the accumulation of glucose in cotton roots, which may promote the infection of cotton roots by V. dahliae, thereby reducing the resistance of cotton to Verticillium wilt.It is speculated that this gene may play an important role in cotton's resistance to Verticillium wilt.

Cloning of CsGR-RBP3 and Its Functional Roles in Cold Tolerance of Harvested Cucumber

WANG Bin, LI Jian-rong, ZHAN Zhao-xia, YUAN Xiao

2025, 41(6):  155-166.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1144

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Objective Cucumber (Cucumis sativus L.) is a cold-sensitive vegetable prone to chilling injury during postharvest storage, which limits the application of cold storage technology in postharvest cucumbers. Cold treatments induce the expression of CsGR-RBP3 (glycine-rich RNA-binding protein 3) in harvested cucumber fruit. Cloning CsGR-RBP3 and studying its functional roles in chilling tolerance of postharvest cucumber can provide candidate gene for the cultivation of new cold-resistant cucumber varieties. Method The coding sequence of CsGR-RBP3 was cloned using cDNA from cucumber peels as templates. Virus-induced gene silencing (VIGS) technology was used to knock-down CsGR-RBP3 expression in postharvest cucumbers to study the effects of the reduced expression on chilling injury in cold-stored cucumbers. RNA-Seq was used to identify potential pathways modulated by CsGR-RBP3 regulating chilling tolerance of cucumber fruit. Result Cucumber CsGR-RBP3 encoded a protein spanning 168 amino acid residues with a conserved RRM (RNA-recognition motif) domain, which might be a mitochondria-related protein and had low sequence similarity with Arabidopsis AtGR-RBPs. Reducing cold-inducible CsGR-RBP3 expression aggravated chilling injury of postharvest cucumbers during cold storage, downregulated the expression of cold-inducible genes, and reduced mitochondrial antioxidant enzyme activity and gene expression. Furthermore, differentially expressed genes (DEGs) potentially regulated by CsGR-RBP3 were significantly enriched in the phenylalanine metabolism, phenylpropanoid biosynthesis and plant-pathogen interaction pathways. Moreover, the expressions of genes in these three pathways were overall downregulated when cold-inducible expression of CsGR-RBP3 was restricted. These results indicated that the CsGR-RBP3 expression was positively correlated with cold resistance in cold-stored cucumbers, and it might maintain mitochondrial redox balance by regulating activities of mitochondrial-related antioxidant enzymes and integrating multiple defense pathways in cucumber fruit. Conclusion CsGR-RBP3 genehad important roles in cold tolerance of harvested cucumber fruit, which may might regulate cold tolerance through multiple defense pathways.

Coupling Effect of AM Fungi on the Root Morphology and Leaf Structure of Xanthoceras sorbifolium Bunge under Drought Stress

ZONG Jian-wei, DENG Hai-fang, CAI Yuan-yuan, CHANG Ya-wen, ZHU Ya-qi, YANG Yu-hua

2025, 41(6):  167-178.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0759

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Objective The study intends to investigate the effect of arbuscular mycorrhizal (AM) fungi on the root morphology, leaf physiology, and anatomical structure of Xanthoceras sorbifolium Bunge under drought stress, and to clarify the irrigation threshold. Method Funneliformes mosseae was used as the test strain, and one-year-old X. sorbifolium Bunge seedlings were selected for pot experiments. Four types of drought stress (well water supply (WW), light drought (LD), moderate drought (MD), and serious drought (SD)) were set up, with a total of eight treatments. All data were evaluated by conducting correlation and principal component analysis. Result AM fungi had a positive infection effect on X. sorbifolium Bunge, which enhanced root vitality, root volume, and the longest lateral root length, increased the accumulation of aboveground biomass higher than that of underground biomass. As the drought intensity intensified, the integrity of leaf anatomical structure was damaged after LD stress, but the damage to AM fungi-inoculated seedlings was less. Notably, mycorrhizal colonization markedly improved the accumulation of aboveground biomass than that of underground aboveground biomass. Moreover, AM fungi symbiosis led to a highly significant increase in the thickness of lower epidermis and palisade tissue (P<0.001), and the AM group were still high in comparison with the NM group under SD stress. In addition,the mycorrhization process led to a strong increase of the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in comparison with NM plants under the same stress. Unlike, MDA content, and membrane damage reduced by AM symbiosis. Correlation analysis showed that the biomass of X. sorbifolium Bunge had a significant impact on various indicators of the root system. Furthermore, there was a significant positive correlation (P<0.05) between leaf thickness, upper epidermal thickness, and root vitality. On the other hand, the thickness of leaf sponge tissue was significantly positively correlated with root vitality and volume quantity (P<0.001). Principal component analysis showed that MD treatment was the critical point for the drought resistance of X. sorbifolium Bunge itself, and X. sorbifolium Bunge bacterial root seedlings had the best adaptability in LD drought environment. Conclusion AM fungi can contribute to better growth performance exposure to drought stress by enhancing root vitality, root volume, as well as improving the thickness of leaf palisade group, and leaf thickness. In general, X. sorbifolium Bunge can adapt to drought through activating antioxidant system of leaves, root traits, and synergistic strategies between root traits and leaves.

Identification of TCP Gene Family in Medicago ruthenica and Their Expression Pattern Analysis under Drought Stress

WANG Miao-miao, ZHAO Xiang-long, WANG Zhao-ming, LIU Zhi-peng, YAN Long-feng

2025, 41(6):  179-190.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1205

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Objective TCP (Teosinte-branched 1/Cincinnata/Proliferating) is a class of transcription factors that regulate plant growth and development and response to stress. Dissecting the sequence structure and physicochemical properties of the TCP gene family members (MruTCP) in Medicago ruthenica will help to understand the role of MruTCP genes under drought stress and provide genetic resources for molecular breeding of forage. Method MruTCP genes were identified based on the whole genome level and analyzed for bioinformatics and expression patterns under drought stress. Result A total of 20 TCP genes were identified in M. ruthenica, and distributed on seven chromosomes. The results of phylogenetic analysis showed that the MruTCP proteins were categorized into three subfamilies: PCF, CIN and CYC/TB1, with differences in the motif types and order of the proteins, but all of them contained a common conserved motif: Motif 1, and the structure of the family members was relatively simple. The results of the covariance analysis indicated that there were two fragment duplication events within the alfalfa TCP family and the closest affinity with soybean. The results of promoter cis-acting element analysis indicated that the MruTCP family members were more complex in function and played roles in signaling, response to abiotic stress, light signaling response, and hormone regulation. Transcriptomic data and qRT-PCR results under drought stress indicated that the expression patterns of MruTCP gene family members differed at different time points and under different concentrations of drought treatments. Among them, MruTCP05 and MruTCP09 contained abundant hormone-responsive elements and stress-responsive elements and were strongly induced by drought stress. Conclusion Combined with the bioinformatics analysis and expression pattern analysis of the TCP gene family, this study hypothesized that MruTCP05 and MruTCP09 are candidate genes for regulating drought tolerance in M. ruthenica.

Identification of HD-Zip Gene Family in Camellia oleifera and Analysis of Its Expression under Abiotic Stress

HUANG Dan, PENG Bing-yang, ZHANG Pan-pan, JIAO Yue, LYU Jia-bin

2025, 41(6):  191-207.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1146

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Objective To identify the HD-Zip gene family members in Camellia oleifera, analyze their structure, characteristics and functions, as well as examine their expression patterns under abiotic stress,including salt, drought, low temperature and high temperature. Method Based on the whole genome data of hexaploid common C. oleifera, members of CoHDZs gene family were screened and identified by bioinformatics method. The analysis included an examination of gene structure, conserved motifs, cis-acting elements, chromosomal locations, and gene colinearity. Additionally, expression patterns of CoHDZ genes were analyzed during various developmental stages of C. oleifera, including leaves, buds, petals, pistils, seeds, calyxes, stamens, and fruits. Furthermore, real-time fluorescence quantitative PCR was utilized to investigate the transient expression characteristics of CoHDZs under different stress conditions. Result The CoHDZ gene family comprises 46 members (CoHDZ1-CoHDZ46) that are categorized into four distinct subfamilies: HD-Zip Ⅰ, HD-Zip Ⅱ, HD-Zip Ⅲ, and HD-Zip Ⅳ. Members of the CoHDZ genes within the same subfamily have similar structural characteristics. Analysis of cis-acting elements indicated that members of the C. oleifera HD-Zip gene family are significantly involved in light response, hormone response, and abiotic stress response. Chromosomal localization and collinearity analysis revealed that the 46 CoHDZ genes are unevenly distributed across 26 chromosomes and 2 chromosome scaffolds. Whole genome duplication (WGD) events or segmental duplications are likely the primary drivers of the evolution of CoHDZ genes. Transcriptome analysis demonstrated that CoHDZ genes have pronounced tissue-specific expression. Real-time fluorescence quantitative PCR experiments confirmed that members of the C. oleifera HD-Zip gene family are highly expressed under conditions of salt, drought, low temperature, and high temperature stress. Furthermore, the expressions of different CoHDZ genes vary under the same stress conditions, and the expression of the same gene also differs across various stressors. Notably, CoHDZ1, CoHDZ3, CoHDZ14, CoHDZ18, CoHDZ20, CoHDZ22, CoHDZ42, CoHDZ44, and CoHDZ45 are identified as key genes in the C. oleifera HD-Zip family responding to abiotic stress. Conclusion CoHDZs genes are widely involved in the growth and development, hormone signaling and abiotic stress response of C. oleifera, and play an important regulatory role.

Functional Study of CjRAV1 from Camellia japonica in Regulating Flowering Delay

GUO Tao, AI Li-jiao, ZOU Shi-hui, ZHOU Ling, LI Xue-mei

2025, 41(6):  208-217.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0019

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Objective The study aims to investigate the functional roles and molecular mechanisms of the Camellia japonica related to ABI3 and VP1 (CjRAV1) gene in flowering time regulation, providing a theoretical foundation for molecular breeding of everblooming C. japonica. Method A comprehensive approach combining bioinformatics analysis, gene expression profiling, transgenic technology, and DAP-seq was employed to systematically examine the function and regulatory mechanisms of CjRAV1. Bioinformatics analysis was conducted to identify the gene structure, conserved domains, and phylogenetic relationships of CjRAV1. The expression patterns of CjRAV1 under exogenous hormone induction, in different tissues, and during various developmental stages of flower buds were analyzed using RT-qPCR technology. Transgenic Arabidopsis plants of overexpressing CjRAV1 were generated to observe phenotypic changes, particularly alterations in flowering time. Finally, DAP-seq technology was utilized to identify potential DNA binding sites and downstream regulatory genes of CjRAV1, elucidating its molecular regulatory network. Result Bioinformatics analysis showed that the CDS of the CjRAV1 was 1 101 bp, encoding 366 amino acids and possessing conserved AP2 and B3 domains. Systematic evolutionary analysis revealed that the CjRAV1 protein in C. japonica was most closely related to the CsRAV protein in Camellia sinensis, suggesting potential functional similarities between the two proteins. Subcellular localization analysis confirmed that the CjRAV1 transcription factor was localized in the nucleus, suggesting its potential direct involvement in transcriptional regulation. The expression pattern analysis showed that CjRAV1 had the highest expression in the leaves; during the maturation process of flower buds, the expression of CjRAV1 showed a gradually decreasing trend overall. The overexpression of CjRAV1 in transgenic Arabidopsis thaliana demonstrated the delayed flowering phenotype. A potential downstream regulatory gene CjERF of CjRAV1 was obtained by DAP-seq screening. Conclusion Overexpression of CjRAV1 leads to delayed flowering phenotype in transgenic Arabidopsis plants, and this function might be performed in collaboration with the potential regulatory gene CjERF.

Cloning of MYB Transcription Factor Gene CeMYB154 and Analysis of Salt Tolerance Function in Cyperus esculentus

CHENG Shan, WANG Hui-wei, CHEN Chen, ZHU Ya-jing, LI Chun-xin, BIE Hai, WANG Shu-feng, CHEN Xian-gong, ZHANG Xiang-ge

2025, 41(6):  218-228.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1182

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Objective The MYB transcription factor gene CeMYB154 is involved in the salt stress response process in oil chestnut(Cyperus esculentus). Cloning and characterization of CeMYB154 gene in oil chestnut and analysis of its salt tolerance function may provide molecular basis and genetic resources for salt tolerant breeding of oil chestnut. Method Based on the reference genome information of oil chestnut, the full-length CDS sequence of CeMYB154 was cloned. Bioinformatics software was used to analyze its amino acid sequence characteristics and cis acting elements on its promoter, and molecular biology techniques were used to perform subcellular localization and transcriptional activation verification. CeMYB154 overexpression vector was constructed, Arabidopsis thaliana was transformed using Agrobacterium mediated method, and the salt-tolerant phenotype and physiological indicators of the overexpression strain were analyzed. Result The CeMYB154 CDS sequence with a length of 780 bp was successfully cloned, which encodes two typical MYB domains in the protein sequence and belongs to the R2R3 MYB transcription factor. Protein sequence alignment analysis showed that CeMYB154 protein is highly conserved across multiple species. The promoter region contains multiple hormone (such as ABA) response and MYB response related cis-acting elements. In addition, subcellular localization and transcriptional activation analysis showed that CeMYB154 is a nuclear transcription factor with transcriptional activation activity. Transgenic A. thaliana overexpressing CeMYB154 was successfully created and a homozygous T3 transgenic positive strain was obtained. Under salt stress, transgenic plant seedlings showed better growth status (leaf size, color, and root length) compared to wild-type A. thaliana. Moreover, the levels of MDA and H₂O₂ in the transgenic plants significantly reduced, while the activities of CAT, POD, and SOD antioxidant enzymes significantly enhanced, indicating that overexpression of the CeMYB154 gene improved salt tolerance in A. thaliana. Conclusion Preliminary evidence has shown that the CeMYB154 gene in C. esculentus has the function of positively regulating salt stress response. The overexpression of the CeMYB154 gene may enhance antioxidant enzyme activity and improve salt tolerance in transgenic plants.

Comparative Analysis of Glehnia littoralis from Different Geographic Regions Based on the Characteristics of Chloroplast Genome

AN Chang, XU Wen-bo, LU Lin, LI Deng-lin, YAO Yi-xin, LIN Yan-xiang, YANG Cheng-zi, QIN Yuan, ZHENG Ping

2025, 41(6):  229-242.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0005

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Objective This study aims to investigate the characteristics of chloroplast genomes and their phylogenetic relationships across different populations of Glehnia littoralis, with a focus on revealing genetic variation and population differentiation within the species from the perspective of organelle genomes. The findings provide a genetic basis for the breeding of new varieties and the conservation of germplasm resources. Method The chloroplast genome of G. littoralis from Fujian was assembled and annotated. Subsequently, comparative analysis was conducted on the chloroplast genomes of six samples from different geographic regions, examining genomic structure, distribution of repetitive sequences, codon usage preference, nucleotide polymorphism, genomic boundary features, and phylogenetic relationships. The study explores the genetic diversity and adaptive evolution traits among populations from different regions. Result All six chloroplast genomes had a typical double-stranded circular quadripartite structure, with genome lengths ranging from 147 445 bp to 147 552 bp and GC content ranging from 37.51% to 37.52%, demonstrating high structural conservation. A total of 129 genes were annotated, including 85 protein-coding genes, 8 ribosomal RNA genes, and 36 transfer RNA genes. The number of simple sequence repeats (SSRs) ranged from 77 to 79, consisting of six types, predominantly A/T repeats. Codon usage analysis indicated that G. littoralis from Fujian and Taiwan (China) preferentially utilized the CGA codon for arginine, whereas those from Shenzhen and Zhejiang favored CGT. Genomic boundary genes showed a degree of conservation, with differences primarily occurring at the JLB and JLA boundaries. Nucleotide polymorphism analysis identified 12 polymorphic genes and 12 intergenic polymorphic regions. Phylogenetic analysis revealed that the Korean population of G. littoralis was evolutionarily distinct from all other samples, while the Taiwan population diverged earliest, showing unique genetic characteristics. The Fujian population followed in divergence, while the Shenzhen and Zhejiang samples formed a minor clade. Conclusion The chloroplast genome structure of Glehnia littoralis from different regions presents high conservation; however, variations are observed in codon usage, genome boundary regions, and nucleotide polymorphisms. Phylogenetic analysis reveals genetic differentiation among populations, reflecting their adaptive evolutionary characteristics.

Positive Regulation of Anthocyanin Biosynthesis by PfMYB80 Transcription Factor in Perilla frutescens

LI Rui, HU Ting, CHEN Shu-wei, WANG Yao, WANG Ji-ping

2025, 41(6):  243-255.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1211

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Objective The R2R3-MYB transcription factor is mainly involved in regulating the biosynthesis pathways of secondary metabolites such as flavonoids and anthocyanins. To verify its function in the anthocyanin biosynthesis of perilla (Perilla frutescens (L.) Britt.) would lay the foundation for elucidating the role of R2R3-MYB transcription factors in regulating plant anthocyanin synthesis. Method This study employed bioinformatics analysis to identify the R2R3-MYB transcription factors across the entire genome of perilla, and to predict their physicochemical properties, phylogenetic evolution, chromosome localization, and cis-acting elements of the promoter. R2R3-MYB members potentially involved in regulating the biosynthesis ofperilla anthocyanin were screened through correlation analysis, and the highly expressed PfMYB80 gene coding sequence in leaves was cloned to explore the regulatory effect of PfMYB80 on anthocyanin synthesis in perilla and its response mechanism to red and blue light stress. Result A total of 186 R2R3-MYB members were identified. Phylogenetic analysis revealed that PfMYB80 and PfMYB146 from perilla are most closely related to the genes in subgroup S6 of Arabidopsis thaliana, which are known to regulate anthocyanin synthesis in plants, suggesting their potential involvement in the regulation of anthocyanin synthesis. Analysis of promoter cis-acting elements indicated that the promoter regions of Perilla R2R3-MYB genes contain light stress-responsive elements. RT-qPCR results showed that the expression of the PfMYB80 gene gradually increases during different developmental stages of Perilla leaves, consistent with the trend of anthocyanin synthesis, suggesting its possible role in anthocyanin biosynthesis. This protein is localized in the nucleus. Expression profile analysis of PfMYB80 and anthocyanin synthesis-related structural genes revealed that the expression of the LDOX gene in Perilla aligns with that of PfMYB80 and matches the trend of anthocyanin accumulation, indicating that the PfMYB80 transcription factor might regulate anthocyanin synthesis by directly controlling the transcriptional expression of the LDOX gene. Functional analysis through transgenic tobacco demonstrated that PfMYB80 responds to blue light induction and positively regulates anthocyanin synthesis. Conclusion The PfMYB80 transcription factor in Perillapositively regulate the biosynthesis of anthocyanins and respond to light response. Overexpression of PfMYB80 in tobacco under blue light treatment increases anthocyanin accumulation and enhances SOD enzyme activity and reduces POD enzyme activity and MDA content.

Identification and Expression Analysis of bHLH Transcription Gene Family in Lonicera macranthoides

QU Mei-ling, ZHOU Si-min, ZHANG Jing-yu, HE Jia-wei, ZHU Jia-yuan, LIU Xiao-rong, TONG Qiao-zhen, ZHOU Ri-bao, LIU Xiang-dan

2025, 41(6):  256-268.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1105

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Objective To study the structure and function of bHLH transcription factor family members in Lonicera macranthoides, and to analyze their expression characteristics, so as to lay a foundation for further study on the function of bHLH gene family in flower development of L. macranthoides. Method Based on the flower transcriptome data of two varieties of L. macranthoides, the members of bHLH gene family were screened, and bioinformatics analysis was carried out by online tools to obtain the genes with significant expression differences between the two varieties of L. macranthoides. The expression of bHLH was verified by RT-qPCR. Result A total of 55 bHLH transcription factor family members were identified. Phylogenetic tree analysis showed that the bHLH protein of L. macranthoides was highly conserved with Arabidopsis thaliana, and the functions of some bHLH proteins were predicted based on the phylogenetic tree. Expression analysis showed that there were significant differences in bHLH genes between the two varieties of L.macranthoides. LmbHLH4/21/35 was highly expressed in the ‘Changgui’, and LmbHLH25 was highly expressed in the flowering stage 5-7 of ‘Xianglei’. The results of RT-qPCR showed that the relative expression was basically consistent with the transcriptome sequencing data, which confirmed the reliability of the transcriptome sequencing results. Conclusion LmbHLH4/21/15/25/35 may be a key transcription factor involved in the regulation of flower growth and development of L. macranthoides, which provides a reference for analyzing the molecular mechanism of flower difference phenotype of two varieties of Lonicera macranthoides.

Populus × canescens AHL17 Negatively Regulates Tolerance to Cadmium in Arabidopsis thaliana

FENG Bing, YAN Cai-xia, LIU Yi, DONG Kai-yue, ZHAO Nan, ZHAO Rui, CHEN Shao-liang

2025, 41(6):  269-283.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0074

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Objective We delved into the molecular mechanisms by which AHL17 (AT - hook motif nuclear - localized proteins, AHLs) in Populus×canescens regulates the plant’s response to cadmium stress. The findings of this research offered a reference for the breeding of stress-resistant varieties and the application of ecological restoration. Method Wild-type (WT), vector control (VC), and Arabidopsis thaliana overexpressing PcAHL17 (PcAHL17-OE1, PcAHL17-OE, and PcAHL17-OE3) were used as experimental materials, the mechanism of PcAHL17 regulating plant response to cadmium stress was studied. Result After cadmium treatment, the expressions of PcAHL17 in the roots, stems, and leaves of Populus×canescens changed. The germination rate and root length of the overexpressed lines showed a significant decrease compared to those of WT and VC. The activity and transcription level of antioxidant enzymes in the overexpressing strains both increased, but still were lower than those in WT and VC. The degree of membrane damage in the overexpressing strains was significantly higher than that in WT and VC, and the accumulation of Cd²⁺and H₂O₂ in roots, as well as the influx of Cd²⁺, were significantly higher than those in WT and VC. Overexpressed strains had lower chlorophyll content, chlorophyll fluorescence parameters, and photosynthetic rate than WT and VC. Conclusion The overexpression of PcAHL17 gene may negatively regulate tolerance of transgenic Arabidopsis thaliana to cadmium.

Effects of PtoMYB61 on Lignin Biosynthesis and Salt Tolerance in Populus tomentosa

LI Xiao-huan, CHEN Xiang-yu, TAO Qi-yu, ZHU Ling, TANG Ming, YAO Yin-an, WANG Li-jun

2025, 41(6):  284-296.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0006

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Objective The effects of PtoMYB61 on secondary cell wall formation and stress defense of Populus tomentosa were studied, so as to lay a foundation for studying wood development and stress resistance breeding. Method RT-PCR was used to clone the PtoMYB61 gene from P. tomentosa. Phylogenetic tree analysis and homology comparison was to predict its biological function. RT-qPCR was applied to analyze the tissue expression specificity of PtoMYB61 and its response to biotic and abiotic stresses. Leaf disc method was to transform P. tomentosa and the lines of overexpressed and knocked-out PtoMYB61 were obtained transformed. Tissue section, toluidine blue staining, lignin content determination, and expressions of key enzyme genes in secondary cell wall biosynthesis pathway were used to analyze the effect of PtoMYB61 on poplar secondary development. Transgenic lines were treated with 150 mmol/L NaCl, and the effects of PtoMYB61 on the tolerance of P. tomentosa to salt were analyzed by phenotypic observation and physiological index determination. Result PtoMYB61 gene encodes a R2R3-MYB transcription factor consisting of 309 amino acids, which is highly expressed in axillary buds, leaves and stems, and is induced by salt, ABA and fungal diseases. Overexpressed and knocked-out lines have no significant difference in growth phenotype compared with wild type, but the number of xylem cells in the overexpressed plants of PtoMYB61 increase, the lignin content significantly increases, and the expressions of key enzymes in secondary cell wall biosynthesis are up-regulated, while knocking PtoMYB61 out leads to the decrease of xylem cell layers, the decrease of lignin content and the down-regulation of key enzymes in secondary cell wall biosynthesis. Salt treatment shows that compared with wild type, PtoMYB61-knockout plants are more sensitive to salt stress, while the overexpressed plants have certain tolerance. Conclusion PtoMYB61 responds to the induction of salt stress and influences the tolerance of poplar to salt stress by regulating the development of secondary cell wall.

Cloning of UpNOA1 Gene from Ulmus pumila L. and Screening of Its Interacting Proteins

LIU Xiao-tian, HE Yu-qi, LIU Chang, XUE Hua

2025, 41(6):  297-306.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1143

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Objective Nitric oxide-related protein NOA1 is associated to the levels of NO in plants, which may be involved in various physiological activities of plants. The UpNOA1 gene was cloned, its tissue-specific expression in elm (Ulmus pumila L.) was analyzed, and the proteins interacting with UpNOA1 was screened, which may provide the basis for studying the function of UpNOA1 in elm. Method Ulmus pumila L. was used as the experimental material, and the NO-related UpNOA1 gene was cloned. The domain architecture and phylogenetic relationships were analyzed using bioinformatics approaches. Tissue-specific expression of UpNOA1 was assessed via RT-qPCR. A vector was constructed for prokaryotic expression analysis, and the resulting UpNOA1 protein was purified. Finally, the UpNOA1 interaction proteins were screened and analyzed by His-PULL DOWN. Result The UpNOA1 gene possesses a 1 698 bp open reading frame, encoding 565 amino acids and containing a conserved GTP/Mg²⁺ binding sites. Subcellular localization prediction using Plant-mPLoc indicated that the UpNOA1 protein is targeted to chloroplasts. Phylogenetic analysis revealed that the UpNOA1 protein shows the highest sequence homology to UaNOA1 protein from American elm (Ulmus americana) and clusters with it in the phylogenetic tree. RT-qPCR analysis revealed that the expression of UpNOA1 gene is the highest in the leaves, followed by the seeds. A total of 131 proteins were identified as UpNOA1-interacting proteins via His-PULL DOWN assay. KEGG and GO enrichment analyses indicated a predominance of ribosomal proteins is associated with genetic information processing among the interacting proteins. Following annotation and comparison, a list of 25 candidate proteins potentially involved in stress resistance and GTP binding was compiled. Conclusion UpNOA1 gene is cloned. The highest expression is found in leaves, followed by elm seeds. A total of 131 UpNOA1 interacting proteins are identified, of which 25 are associated with stress resistance and GTP binding.

Effects of Intercropping and Nitrogen Application on the Diversity and Functions of Soil Bacteria around Sorghum Rhizosphere

ZHAO Qiang, CHEN Si-yu, PENG Fang-li, WANG Can, GAO Jie, ZHOU Ling-bo, ZHANG Guo-bing, JIANG Yu-wen, SHAO Ming-bo

2025, 41(6):  307-316.  doi:10.13560/j.cnki.biotech.bull.1985.2025-0001

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Objective To explore the effects of different planting modes and nitrogen application treatments on the bacterial community of sorghum roots. Method Two planting modes (SW: sole sorghum planting; WS: sorghum intercropping with soybean) and three nitrogen application levels (N0: no nitrogen; N1: conventional nitrogen; N2: high nitrogen) were set up, 16S rRNA sequences in sorghum rhizosphere soil under different treatments were determined, and bacterial diversity and function were analyzed. Result The abundance of bacterial community increased with increasing N application in sole cropping while decreased with increasing N application in intercropping. No significant difference was observed between the treatments in Shannon index and Simpson index. The planting modes and nitrogen application levels significantly affected the composition of rhizosphere bacterial communities. There were six dominant phyla in sorghum rhizosphere soil, i.e., Proteobacteria, Acidobacteriota, Actinobacteria, Gemmatimonadota, Bacteroidota, and Chloroflexi. Bacterial function prediction using FAPROTAX centered mainly on chemoheterotrophic, autotrophic chemoheterotrophic, prephagous or ectoparasitic, nitrate reduction, and animal parasites or symbionts. Correlation network analysis showed that there were more interactions of bacterial community in intercropping and more complex networks than sole cropping under the conditions of N0 and N1. Conclusion Intercropping and different nitrogen application levels affected the composition of bacterial community in sorghum rhizosphere soil. In a certain range of nitrogen application level, intercropping may improve the bacterial diversity in sorghum rhizosphere soil and increase the relative abundance of Actinobacteria and Bradyrhizobium.

Screening of Nitrogen-fixing Microorganisms in Rhizosphere Soil of Desert Plants and Their Stress-resistant and Growth-promoting Characteristics

ZHANG Jun-jie, LIU Shuang, HU Ming-zhu, SHI Xue-rui, DAI Jin-xia

2025, 41(6):  317-326.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1242

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Objective Studies on the diversity, resistance and growth-promotion characteristics of nitrogen-fixing microorganisms in the rhizosphere soil of desert shrubs may provide a basis for exploring desert microbial resources. Method Nitrogen-fixing microorganisms were isolated and screened from the rhizosphere soil of four desert scrubs using isolation and culture methods and PCR amplification of the nitrogen-fixing enzyme gene nifH. Nitrogen-fixing strains were identified through observation of colony morphology, determination of physiological and biochemical characteristics, and analysis of the 16S rRNA gene sequences. The tolerance to salt alkali and the resistances of the strains to drought were tested by NaCl, NaOH and PEG6000 stresses. The growth-promoting characteristics of strains such as phosphorus-solubilizing ability, IAA production, siderophore production, and ACC deaminase were detected by colorimetric method. The growth-promoting effects of the strains were verified through the pot inoculation experiment of alfalfa. Result A total of 61 strains of nitrogen-fixing microorganisms were screened from the rhizosphere soil of four shrubs. Most of the strains were gram-negative bacteria, and the H₂O₂ enzyme reaction were positive. The results of 16S rRNA sequence analysis showed that the strains belonged to 38 genera, among which Pseudomonas, Bacillus, Azotobacter and Ensifer had higher isolation frequency, and the diversity of nitrogen-fixing microorganisms in the rhizosphere of Caragana korshinskii was the most abundant. All strains grew normally under the condition of pH10, and most strains tolerated 5% NaCl and 15% PEG6000, showing strong stress resistance. The growth-promoting characteristics of the strains varied, total 17 strains had the ability of dissolving phosphorus, and these strains' maximum amount of phosphorus to be dissolved was 135.84 mg/L; total 15 strains produced siderophores. Eight strains had ACC deaminase activity, the highest enzyme activity was 10.63 U/mg; nineteen strains had the ability to secrete IAA, and these strains' IAA production was between 3.41-56.93 mg/L. There were 6 strains with more than 3 kinds of growth-promoting potential. Among them, the SDQ-1 and MC-20 strains had good comprehensive performance. The alfalfa pot inoculation experiment showed that the inoculation treatment significantly promoted the growth of alfalfa seedlings, and the growth-promoting effect of compound microbial agent group F was the most significant. Compared with the control CK, the seedling height, fresh weight, root length and root area significantly increased by 55.88%, 147.53%, 292.65% and 306.38%, respectively. Conclusion The nitrogen-fixing microbial diversity in the rhizosphere soil of the desert shrub in Ningxia is extremely abundant, and most of the strains have good stress resistance and multiple growth-promoting potentials, which have the value of further development and utilization.

Discovery and Verification of a Functional Gene Influencing the Growth and Development of Pleurotus ostreatus

PEI Jing-qi, ZHAO Meng-ran, HUANG Chen-yang, WU Xiang-li

2025, 41(6):  327-334.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1257

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Objective This study is aimed to investigate the role of the functionally unknown gene g13394 in the growth and development of Pleurotus ostreatus, specifically in the transition from mycelium to primordium and fruiting body formation. Method The g13394 gene was cloned from the P. ostreatus strain CCMSSC00389 using Polymerase Chain Reaction (PCR), and its biological characteristics were analyzed through bioinformatic tools. A homologous recombination approach was used to construct both overexpression and RNA interference (RNAi) vectors, which were then introduced into P. ostreatus via Agrobacterium tumefaciens-mediated transformation. Gene expressions in the transformed strains were validated by quantitative real-time PCR (qRT-PCR) to select the g13394-overexpressed and RNAi strains. The phenotypic differences between the overexpression, RNAi, and wild-type strains were assessed at the mycelial growth stage and during fruiting body development. Result Bioinformatic analysis revealed that g13394 encoded a nuclear-localized protein with unknown functional domains and the subcellular localization was predicted in the nucleus. Two strains were successfully constructed and obtained for g13394 overexpression and RNAi, respectively. Overexpressed strains showed significantly faster mycelial growth and accelerated fruiting body development compared to the wild-type. In contrast, RNAi strains showed slower mycelial growth, delayed primordium formation, and extended fruiting body development time. These results suggest that g13394 positively regulates both mycelial growth and the timing of fruiting body formation. Conclusion The gene g13394 promotes mycelial growth and accelerates the formation of fruiting bodies.

Effect of Soil-casing Cultivation of Pleurotus ostreatus on Antibiotic Resistance Genes in Soil

MI Chun-xia, Shu XU, WANG Shou-xian, LIU Yu, SONG Qing-gang, SONG Shuang

2025, 41(6):  335-343.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1126

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Objective To investigate the feasibility of reducing the abundance of antibiotic resistance genes (ARGs) in soil using edible fungi soil-casing cultivation mode. Method The abundance of ARGs and mobile genetic elements (MGEs) in soil under soil-casing cultivation mode of Pleurotus ostreatus was investigated by high-throughput fluorescence quantitative PCR. The differences in soil bacterial communities before and after the formation of fruiting bodies were analyzed by using 16S rRNA sequencing. Co-occurrence patterns among ARGs, MGEs and bacterial communities were analyzed by using network analysis. Result After P. ostreatus cultivation, the total relative abundance of ARGs decreased by 34.62% (P<0.01), and the total absolute abundance decreased by 48.56% (P<0.01), with significant reductions observed in aminoglycoside, sulfonamide, β-lactamase, macrolide-lincosamide-streptogramin B, and tetracycline ARGs. The total relative abundance of MGEs decreased by 20.63%, and the total absolute abundance decreased by 32.99% (P<0.01). Bacterial community structure changed significantly (P<0.01), explaining 31.50% of the variation in ARGs, while the combined effect of bacterial community structure and MGEs explained 8.01% of the variation. Conclusion The proliferation and development of P. ostreatus mycelium in the soil changed the structure of microbial community and achieved the effect of reducing the abundance of ARGs in the soil. The method of using edible fungi soil-casing cultivation is easy to operate and requires no high temperature or fermentation treatment, which not only reduces the abundance of ARGs in the soil, but also enables the harvesting of edible mushroom, providing a new perspective for bioremediation of agricultural soils contaminated with ARGs.

Effects of Transcription Factor CREA Knockout on the Morphology and the Secretion of β-glucosidase in Aspergillus niger

CHENG Hui-juan, WANG Xin, SHI Xiao-tao, MA Dong-xu, GONG Da-chun, HU Jun-peng, XIE Zhi-wen

2025, 41(6):  344-354.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1125

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Objective Transcription factor CREA in Aspergillus niger was knocked out to explore its regulatory effects on hyphal development and BGL expression, which may provide effective regulatory targets for the design of high-yield BGL cell factories. Method The A. niger An-1 was used as the research strain, and the operation plasmid and donor fragment for the knockout creA gene were constructed. After protoplast transformation, resistance screening, and mycelial PCR verification, the knockout strain ΔcreA was obtained using the CRISPR-Cas9 genome editing technology. The effect of the transcription factor CREA on the fungus morphology and fermentation performance was examined by plate culture and shaking flask fermentation, respectively. The impact of carbon catabolite repression on BGL biosynthesis was explored under different concentrations of glucose. Result The creA gene in the A. niger An-1 was precisely knocked out, and the deletion mutant strain ΔcreA was obtained. Compared with the wild-type strain, the colony morphology of the creA strain showed a wheel-like wrinkle on potato dextrose agar and stronger β‍-glucosidase solid secretion ability on the esculin color plate. With cellobiose and p-nitrophenyl-‍α‍-D-glucopyranoside as substrates, the BGL activity of the ΔcreA strain was 1.5 times and 1.8 times higher than that of the wild-type strain, respectively. Fluorescence quantitative PCR showed that the knockout of creA increased the expression of the bglA gene by 8.4 times. During the fermentation process with different concentrations of glucose added, the enzyme activity of the ΔcreA strain was higher than that of the wild-type, showing the derepression of carbon catabolite repression. Conclusion The transcription factor CREA regulates the hyphal growth and BGL expression of A. niger An-1. Its knockout alleviates the carbon catabolite repression and enhances the ability of A. niger-An to produce BGL. This study provides theoretical support for regulatory modification of A. niger to optimize the production performance of BGL.

Effect of Carbon Source in Methanogenic Communities Regulation and Methane Production Capacity

AN Miao-miao, LIN Xiang-hao, LIANG Rui-na, ZHAO Guo-zhu

2025, 41(6):  355-366.  doi:10.13560/j.cnki.biotech.bull.1985.2024-1180

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Objective Methanogens play a pivotal role in methanogenesis during anaerobic digestion. To obtain an efficient methanogenic microbial community, the regulatory effects of different carbon sources on the methane production potential, methanogen abundance, and microbial community structure were investigated. Method Three distinct carbon source culture media (Group A: CH₃COONa and CH₃OH;Group B: CH₃COONa; Group C: CH₃COONa and HCOONa) were designed to enrich methanogenic communities by inoculating with anaerobic digestion liquid from kitchen waste (FW) and activated sludge suspension (SS). The performance and diversity composition of methanogenic microbial community were analyzed using gas chromatography, real-time quantitative PCR (qPCR), fluorescence microscopy, and amplicon sequencing. Result The microbial communities enriched by carbon sources A, B, and C demonstrated a descending order of methane yield when inoculated with the same inoculum. Group 1A-2 achieved the highest cumulative methane yield over 49 d, with a methane yield of 35.9 mL per gram of available carbon (C) in a unit volume (L) of culture medium. The copy number of mcrA gene and fluorescence microscopy observations indicated a successive decrease in methanogen abundance in communities enriched by carbon sources A, B, and C. Specifically, carbon source A enriched methylotrophic methanogens, while carbon source B significantly boosted the relative abundance of aceticlastic Methanothrix compared to the other groups. Carbon source C notably enhanced the relative abundance of hydrogenotrophic methanogens. Conclusion The type of carbon source significantly influence the diversity, richness, and community structure of methanogens and bacteria in the enriched microbial communities. The microbial communities enriched with CH₃COONa and CH₃OH (Group A) showed the highest methanogen abundance, mainly including Methanothrix, Methanoculleus, Methanomethylovorans, Candidatus Methanoplasmaand Methanosarcina, and demonstrated the highest methanogenic capacity. Carbon source A promotes methane (CH₄) production by enriching syntrophic bacteria, balancing the metabolic pathway distribution of methanogens, and facilitating direct interspecies electron transfer (DIET).

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2025, 41(6):  367. 

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2025, 41(6):  368. 

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2025, 41(6):  369. 

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