Table of Content

26 August 2024, Volume 40 Issue 8

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Research Progress in Light Signal and Circadian Rhythm Regulating the Perception of Plants to Cold Stress

LI Wen-cui, PENG Yu-jia, LIU Yong-bo

2024, 40(8):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0234

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Temperature and light are important environmental factors in regulating plant growth and development, and plants perceive temperature changes and respond to cold stress by altering gene expression patterns and other responses. The responses are affected by light signal and circadian rhythms. However, the molecular regulatory networks involved in plant response to cold stress are not well understood. This review focuses the roles of light signal and circadian rhythms in the perception of plants to cold stress. Light signal is involved in cold stress mainly through photopigment-induced CBF gene pathways to activate the expressions of cold genes. It includes two main pathways: one is that photosensitive pigments directly regulate cold tolerance by modulating CBF and COR gene expression; the other is the activation of COR（COLD REGULATED）genes by HY5, a positive regulator factor. Circadian rhythms are involved in cold stress mainly through their components, CCA1/LHY and RVE4/RVE8, regulating the expression of DREB1 downstream gene. Clarifying the roles of light signal and circadian rhythms in cold perception and conduction pathways not only contributes to a better understanding of the mechanisms of cold resistance in plants, but also helps in the trade-offs between plant growth and stress responses. It provides a theoretical basis for enhancing the response sensitivities of plants to diurnal and nocturnal temperature variations.

Research Progress of Crop Resistance to ACCase-inhibitor-like Herbicides

WANG Yao, WANG Rong-huan, FENG Ling-yang, ZHANG Lu, ZHAO Qi, WANG Jia-le, ZHAO Jiu-ran

2024, 40(8):  13-23.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0251

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Weeds are one of the important adverse factors that seriously affect the yield and quality of crops, and weed control is an important aspect of field management. The ACCase is rate-limiting enzyme for fatty acids synthesis from acetyl-CoA, and ACCase-inhibitor herbicides inhibit ACCase activity, resulting in the obstruction of fatty acid synthesis and killing weeds. With the extensive application of herbicides in agricultural production, the issue of herbicide-resistant weeds has become increasingly serious, and the impact on crop yield and quality is particularly obvious. The development of elite herbicide-resistant germplasms and breeding new varieties with resistance to herbicide are effective strategies to control weeds in the field, especially for compound planting of mono-/dicotyledonous crops. The effective mutation sites were discovered in many crops and weeds through natural mutation, chemical mutagenesis, transgenic and gene editing techniques, and used in breeding applications. Here, we reviewed the properties and action mechanisms of ACCase in plants, the classification of ACCase-inhibitor herbicides, the resistant mechanism of ACCase, and their effective variations in crops. Finally, we proposed novel highly-effective breeding programs and strategies to improve high resistance to ACCase-inhibitor herbicides in crops.

Mechanism and Industrial Application of Bacillus Tolerance to Stress Conditions

HAN Zhong-rao, HUO Yi-xin, GUO Shu-yuan

2024, 40(8):  24-38.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0183

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Bacillus, as a highly potential chassis strain, can grow in various industrial and agricultural waste and extreme conditions. They can also produce a variety of industrial products such as food, feed, probiotics, plant growth promoters, enzymes, and bioactive compounds. However, despite their advantages in efficient production and low cost, Bacillus still face several bottlenecks in fermentation production, limiting the full exploitation of their industrial potential. One of the key issues is that the growth and production efficiency during fermentation are easily restricted by various stress conditions, leading to incomplete and inefficient fermentation production. Therefore, exploring the influencing factors and modification strategies of Bacillus stress response, and exploring the relationship between its various metabolic activities and growth traits, can enhance the tolerance of Bacillus to stress and improve their quality and quantity in industrial applications.This article first analyzes the various stress response mechanisms of Bacillus, aiming to further enhance its stress tolerance and to construct a high-efficiency production strain with excellent resistance, and then summarizes the irrational design strategies for screening resistant strains. It also elaborates on the construction methods of stress-resistant gene circuits and high-tolerance microbial chassis. These efforts provide strategies and insights to advance research on stress tolerance mechanisms in Bacillus and expand its industrial applications.

Integrating Histological Image Information to Enhance Cell Clustering Resolution in Spatial Transcriptome

WANG Rui, QI Ji

2024, 40(8):  39-46.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0334

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【Objective】Increasing the spatial resolution of transcriptome gene expression can improve the accuracy of cell lineage and type changes in genetic development and disease studies, providing more detailed molecular phenotypic information.【Method】Using image segmentation to simulate the spatial distribution of cells in a spatial transcriptomics grid, the high-resolution spatial gene expression using linear interpolation was reconstructed. Graph clustering methods were used to reveal the spatial preferences of cell distribution within tissues.【Result】Application of SpaGMM on the 10X Visium dataset of the mouse posterior brain helped to accurately identify various spatial domains of the mouse brain. Compared with several commonly used methods for spatial transcriptome clustering, the results showed that the clustering results by SpaGMM were more consistent with the annotated histological regions, which were supported by the spatial expression of many marker genes. SpaGMM also recognized layers corresponding to Purkinje cells and Bergmann glial cells from mouse cerebellum, revealing complementary gene expression patterns in different cell layers.【Conclusion】SpaGMM may reveal the fine structure of tissue domains by improving the spatial resolution of spots.

Studies on the Regulation of MBD1-induced Expression by Dox in the Tet-On System

LU Xi, YUAN Yue, LI Dan, ZHANG Peng

2024, 40(8):  47-52.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0015

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【Objective】This work aims to construct the Tet-On inducible expression plasmid of methyl-binding protein 1（MBD1）and 293T cells, to explore the effect of doxycycline（Dox）on the expression of MBD1, and to reflect the regulatory mechanism of MBD1 by analyzing the CpG methylation level of the plasmid promoter in the cells.【Method】The MBD1 expression sequence of 293T was amplified using MBD1 primers and ligated into the polyclonal site of Tet-On inducible expression plasmid. The successfully constructed plasmid was transfected into MBD1 KO 293T by electro-transfection, and the obtained MBD1-restored 293T was verified using fluorescence microscope and Western blot（MBD1 RE）. MBD1 expression was induced using different concentrations of Dox, and the expression of MBD1 in monoclonal cells was detected by Western blot. MBD1 expression was induced using the same concentration of Dox, and the cells were collected at 12, 24, 48, 96, 144 and 192 h. The expression of MBD1 in the cells was detected by Western blot; and MBD1 expression in the cells was detected by bisulfite sequencing. The expression of MBD1 in the cells was detected by Western blot, and the CpG methylation of inducible plasmid promoter was detected by bisulfite sequencing after Dox induction.【Result】Tet-On inducible MBD1 plasmid was successfully constructed; MBD1 protein was re-expressed in the transfected cells after Dox induction compared with uninduced cells. The expression of MBD1 in the cells increased gradually with the increase of Dox concentration; the expression of MBD1 in the cells increased gradually from 12 to 96 h after induction with the same concentration of Dox, but the expression decreased after 144 h. The expression of CpG methylation of plasmid promoter was detected by bisulfite sequencing method; the CpG methylation of plasmid promoter was detected by bisulfite sequencing method. CpG methylation of the plasmid promoter showed that the methylation level of the plasmid promoter gradually increased at 144 and 192 h compared with that at 96 h. The methylation level of the plasmid promoter decreased and the expression of MBD1 increased after the addition of the methyltransferase inhibitor, decitabine, at 144 h. The expression of MBD1 in the cells also decreased by the addition of the methyltransferase inhibitor, decitabine, at 144 h after the induction.【Conclusion】Tet-On induced the re-expression of MBD1 protein in the cells and the expression was positively correlated with the Dox concentration, but the elevated level of intracellular plasmid CpG methylation after prolonged induction affected the expression of MBD1 in the cells.

Genome-wide Association Analysis of γ-aminobutyric Acid in Rice Grains

SUN Zhi-yong, DU Huai-dong, LIU Yang, MA Jia-xin, YU Xue-ran, MA Wei, YAO Xin-jie, WANG Min, LI Pei-fu

2024, 40(8):  53-62.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0058

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【Objective】Identification of single nucleotide polymorphisms（SNPs）and candidate genes significantly associated with the content of γ-aminobutyric acid（GABA）in rice grains may provide a theoretical basis for further exploring the molecular mechanism of GABA enrichment in rice grains, and lay the foundation for cultivating high-GABA content varieties.【Method】This study performed genome-wide association studies（GWAS）for GABA content with re-sequencing in 139 early japonica rice core germplasm resources in Northwest China. The candidate genes were screened based on haplotype analysis and gene annotation, and expression pattern of candidate genes were analyzed by expression profile database and fluorescence quantitative PCR.【Result】GABA content in grains showed abundant variation with a coefficient of variation of 31.25%. Genome-wide association analysis showed a total of 6 significant and representative SNPs on chromosomes 6, 8, 9, 11 and 12 were detected, and OsBADH2 and OsGABA-T2, which have been cloned in relation to GABA content. In addition, six candidate genes were predicted for GABA content in the rice grains, among which LOC_Os09g10720 was identified as highlight candidate gene, and the feasibility of its involvement in the decomposition of GABA in rice grains was further verified.【Conclusion】A total of 6 SNPs significantly associated with GABA content in rice grain are detected. And 6 candidate genes that might be associated with GABA content are screened out, they are LOC_Os09g09750, LOC_Os09g09760, LOC_Os09g10210, LOC_Os09g09820, LOC_Os09g10280 and LOC_ Os09g10720. Among them, LOC_Os09g10720 was used as a focus candidate gene.

Genome-wide Identification and Stress Response Analysis of Soybean GS Gene Family

WU Shuai, XIN Yan-ni, MAI Chun-hai, MU Xiao-ya, WANG Min, YUE Ai-qin, ZHAO Jin-zhong, WU Shen-jie, DU Wei-jun, WANG Li-xiang

2024, 40(8):  63-73.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0205

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【Objective】Glutamine synthetase（GS）is a key enzyme in the GS-GOGAT cycle of nitrogen metabolism. This work is aimed to investigate the family members of GS in soybean and the response to external stress.【Method】Based on bioinformatics methods, we comprehensively identified GS genes in soybean（Glycine max）, clarified the location and structure of soybean GmGSs genes, the physicochemical properties of proteins, and the tissue expression patterns, and also studied the responses to abiotic stresses.【Result】The total of eight GS genes were identified from the soybean, located on eight chromosomes, corresponding to coding amino acid sequences with lengths ranging from 356 to 432 aa. Among the ten conserved motifs, all eight GmGS contained nine conserved motifs, and GmGS7 and GmGS8 had one more motif 10 conserved motif than other members. Analysis of promoter's cis-acting elements showed that the promoters of GmGSs contained abundant light-responsive elements, hormone-responsive elements, and adversity stress-responsive elements. Transcriptome data analysis showed that GmGSs genes were expressed in all tissues, with GmGS3 and GmGS4 having higher expression in all tissues. Fluorescence quantitative qPCR results revealed that GmGS4, GmGS5, and GmGS7 of soybean GS family responded most significantly to the late stage of low concentration ammonium treatment after different concentrations of ammonium chloride. And after high salt stress treatment, the expression of GmGS5 decreased in the root, stem and leaf tissues; while the expression of GmGS7 increased in root and stem tissues.【Conclusion】There are eight GS members in soybean, of which GmGS7 is involved in response to both ammonium chloride treatment and salt stress.

Genome-wide Identification of Non-specific Phospholipase C Gene Family in Hordeum vulgare L. and Stress Expression Analysis at Seedling Stage

CUI Yuan-yuan, WANG Zhao-yi, BAI Shuang-yu, REN Yu-zhao, DOU Fei-fei, LIU Cai-xia, LIU Feng-lou, WANG Zhang-jun, LI Qing-feng

2024, 40(8):  74-82.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0201

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【Objective】Non-specific phospholipase C（NPC）is a type of phospholipase C, which plays an important role in plant growth and development and response to abiotic stresses. Identifying the NPC gene family members from the whole barley genome, and analyzing the expression characteristics of the related genes would provide a theoretical basis for the study of barley NPC gene function and genetic improvement.【Method】Based on the barley genome-wide data included in the EnsemblPlants database, bioinformatics methods were used to identify NPC gene family in barley and analyze their physicochemical properties, subcellular localization, phylogenetic relationship, gene structure, protein structural domains, and promoter cis-acting elements. Transcriptome data and RT- qPCR were used to analyse the expression characteristics of HvNPCs in different tissues as well as at seedling stage under low temperature, drought and salt stress.【Result】Five NPC members were identified in the barley genome, and the encoded proteins were structurally similar to NPC genes from other plants, all of which had a phosphoesterase structural domain ; the sequence lengths of the HvNPC proteins ranged from 477 to 553 aa, the isoelectric points ranged from 5.22 to 8.83, and the molecular weights ranged from 53.12 to 61.33 kD. Subcellular localization showed that the HvNPC gene was localized in chloroplasts, membrane-intrinsic proteins, and cytoplasm. The HvNPC promoter region contained a large number of cis-acting elements associated with adversity stresses, and real-time PCR analysis confirmed that the HvNPC gene was induced to express by drought, high salt and low temperature. Among them, HvNPC2, HvNPC3 and HvNPC5 were induced to be expressed by low-temperature stress and drought stress; and HvNPC3 and HvNPC5 were induced to be expressed by salt stress.【Conclusion】A total of five HvNPC genes were identified in the barley genome, and members of the HvNPC genes were organ-specofc expression and different genes responded differently to abiotic stresses.

Screening and Functional Analysis of Gene CqSTK Associated with Gametophyte Development of Quinoa

LIN Tong, YUAN Cheng, DONG Chen-wen-hua, ZENG Meng-qiong, YANG Yan, MAO Zi-chao, LIN Chun

2024, 40(8):  83-94.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0156

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【Objective】SEEDSTICK（STK）, a member of the MADS-box transcription factor family, plays a key role in controlling gametophyte development and seed size and morphology. This study is aimed to investigate the role of STK in quinoa gametophyte development.【Method】Transcriptomic data from two quinoa varieties with different photoperiod characteristics under different photoperiod treatments were used to screen differentially expressed genes regulated by photoperiod and related to gametophyte development. The identified gene was cloned and subjected to bioinformatics analysis, expression pattern analysis, subcellular localization analysis, and functional verification through heterologous expression in Arabidopsis. 【Result】A differentially expressed gene, AUR62022366-RA, was identified. The expression pattern of this gene was consistent with phenotypic differences in plants. In short-day materials, high expression of the gene during the grain-filling stage resulted in fertile plants, while low expression led to infertile plants. Bioinformatics analysis revealed that the full-length coding sequence（CDS）of CqSTK was 672 bp, encoding 223 amino acids. Phylogenetic analysis of STK homologous genes showed that CqSTK clustered with STK genes from spinach and sugar beet, indicating a close evolutionary relationship. Furthermore, CqSTK shared a similar tertiary structure with Arabidopsis STK. Haplotype analysis indicated that the exonic sequences of CqSTK were completely conserved across ten quinoa varieties, while single nucleotide polymorphisms（SNPs）were present in the intronic regions. This gene was localized in the nucleus and cell membrane of tobacco leaf epidermal cells. Fluorescence quantitative PCR showed that CqSTK was highly expressed during flower organ formation and grain development, especially during grain development when its expression reached its peak. Overexpression and complementation experiments in Arabidopsis demonstrated that overexpressing and complementation plants had significantly delayed flowering time compared to mutants and wild-type plants, and their pod length and seed number per pod were significantly higher than those of stk mutants and complementation plants.【Conclusion】The overexpression of CqSTK delays flowering and positively regulates seed length and fruit set in Arabidopsis thaliana.

Effect of Immune Inducer ZNC on the Prevention and Control of Wheat Scab and the Yield of Wheat

ZHANG Xiao-ying, MAO Mi, WANG Hong-feng, DING Xin-hua, ZHU Shu-wei, SHI Lei

2024, 40(8):  95-105.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0192

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【Objective】To seek an effective and safe medicament to control wheat scab, and to reduce the incidence rate of wheat scab and the effect of medicament itself on wheat yield and quality.【Method】We selected three types of fungicides（carbendazim）and plant immune inducers（ZNC, aminooligosaccharides）to investigate the inhibitory effects of different agents on the pathogen of wheat scab, Fusarium graminearum, using the plate inhibition method. Then we verified the effectiveness of different pesticides through pot experiments. Also we explored the effects of different pesticides on wheat growth through pot experiments, as well as the field control effects of different pesticides on wheat scab under artificial inoculation with F. graminearum.【Result】Carbendazim has a significant inhibitory effect on the growth of F. graminearum, and its field control effect is also significantly higher than that of ZNC and aminooligosaccharides, with a control effect of 77.90%. ZNC and aminooligosaccharides did not have a significant inhibitory effect on F. graminearum in the plate inhibition test but showed antibacterial effects in both potted and field control experiments, and significantly reduced the field disease index, which was 47.59% and 49.93% lower than CK, respectively. Carbendazim inhibited the growth and yield of wheat. Compared with CK, the plant height, stem diameter, root length, number of roots, fresh weight, and dry weight of wheat decreased by 8.69%, 2.90%, 21.28%, 12.50%, 12.83%, and 15.15%, respectively, and the yield of wheat decreased by 13.70%. ZNC promotes the growth of wheat and increases wheat yield. Compared to CK, wheat yield increases by 7.96%, wheat grain protein content increases by 14.45%, and wheat grain vomiting toxin（DON）content decreases by 6.00%.【Conclusion】ZNC may promote wheat growth, increase wheat yield, and improve wheat grain quality while preventing and controlling diseases. Plant immune inducer ZNC provides a new option for preventing and controlling wheat scab.

Transcriptome Analysis of the Anthocyanin Biosynthesis in the Fruit Development Processes of Lycium ruthenicum Murr.

NIE Zhu-xin, GUO Jin, QIAO Zi-yang, LI Wei-wei, ZHANG Xue-yan, LIU Chun-yang, WANG Jing

2024, 40(8):  106-117.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0247

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【Objective】To explore the molecular mechanism involved in anthocyanin biosynthesis during the fruit development of Lycium ruthenicum Murr. is important for deeply understanding the regulatory network of anthocyanin biosynthesis.【Method】Transcriptome sequencing was performed on the samples at the five stages of fruit development: green fruit, early color-changing, late color-changing, ripeness, and complete ripeness. The candidate genes related to anthocyanin biosynthesis were mined.【Result】With the development of L. ruthenicum Murr. fruit, the anthocyanin content gradually increased. The anthocyanin content in the fruits at the ripeness and complete ripeness stages were significantly higher than that at the green fruit, early color-changing, and late color-changing stages. A total of 13 540 differentially expressed genes（DEGs）were identified from the five stages of fruit development. Using the green fruit stage as a control, the number of DEGs gradually increased with the development of the fruit. GO analysis showed that DEGs were co-enriched GO terms including phenylpropanoid metabolic process, polysaccharide catabolic process, phenylpropanoid biosynthetic process, flavonoid biosynthetic process, cell wall macromolecule metabolic process, anchored component of membrane, and nutrient reservoir activity. KEGG analysis indicated the significant enrichment of DEGs in flavonoid biosynthesis, phenylpropanoid biosynthesis, plant hormone signal transduction, stilbenoid, diarylheptanoid and gingerol biosynthesis, as well as cutin, suberine, and wax biosynthesis. The correlation between DEGs expression and anthocyanin content was performed and 36 candidate genes involved in anthocyanin biosynthesis during the L. ruthenicum fruit development were screened, including ten structural genes of anthocyanin biosynthesis pathway, four genes of transcription factors, nine, eight, and five genes of ABA, GA, and JA signal transduction pathway. Ten DEGs were selected for RT-qPCR analysis, and the results were consistent with RNA-seq data.【Conclusion】DEGs related to structural genes and transcription factors involved in anthocyanin biosynthesis, as well as genes involved in the ABA, GA, and JA signal transduction pathways, affect fruit coloration during the development of L. ruthenicum Murr. fruit.

Analysis of DoWRKY40 Gene Expression Characteristics and Screening of Interacting Proteins in Yam

XING Li-nan, ZHANG Yan-fang, GE Ming-ran, ZHAO Ling-min, CHEN Yan, HUO Xiu-wen

2024, 40(8):  118-128.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0177

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【Objective】WRKY, as a plant idiosyncratic transcription factor, is involved in plant growth and development. DoWRKY40 gene in yam（Dioscorea opposita Thunb.）was cloned and its expression pattern was analyzed. The protein interacting with DoWRKY40 was screened by yeast two-hybrid technique to explore the regulatory mechanism of WRKY40 in the process of yam expansion.【Method】DoWRKY40 gene was cloned from ‘Dahechangyu’ yam, and bioinformatics analysis, expression pattern analysis and subcellular localization were performed. Yeast libraries of yam tuber at different developmental stages were constructed and DoWRKY40 interacting proteins were screened.【Result】The length of open reading frame of DoWRKY40 was 927 bp; DoWRKY40 belonged to group II of the WRKY family and contained a typical WRKY transcription factor conserved domain, which was localized in the nucleus. It was closely related to Dioscorea cayenensis subsp. The expression of DoWRKY40 gene was the highest at 120 d after the growth and development of yam tuber, and it was expressed in the leaves, stems and tubers with tissue specificity. The library capacity of yam cDNA library was 1.484×10⁸; pGBKT7-WRKY40 decoy vector had no self-activation activity. Four kinds of proteins interacting with DoWRKY40 were screened from yam library by yeast two-hybridization, which were involved in plant growth and development, cell cycle regulation and cell expansion, signal transduction and environmental stress response, respectively.【Conclusion】The DoWRKY40 gene is cloned, which responds to the growth and development process of yam. The four interacting proteins are screened, suggesting that it plays a regulatory role in the expansion and signal transduction of yam cells.

Genome-wide Identification and Expression Analysis of the SRO Gene Family in Brassica juncea L.

YANG Wei, ZHAO Li-fen, TANG Bing, ZHOU Lin-bi, YANG Juan, MO Chuan-yuan, ZHANG Bao-hui, LI Fei, RUAN Song-lin, DENG Ying

2024, 40(8):  129-141.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0179

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【Objective】SRO gene family is a class of plant-specific transcription factors, which plays an important role in plant growth and development and stress response. The study of the SRO gene family in Brassica juncea L. provides a theoretical basis for analyzing the function of SRO genes in B. juncea L. and its genetic improvement.【Method】Bioinformatics was used to identify members of the SRO gene family in the genomes of two types of oilseed mustard and vegetable mustard, the software and database such as TBtools, MEGA, Cytoscape, NCBI, STRING, EggNOG were used for the analysis of physicochemical properties, sequence characterization, evolutionary relationships, regulatory networks, etc., as well as RT-qPCR to analyze the expression patterns under salt stress.【Result】There were differences in the number of members of the SRO gene family between the two types of B. juncea L., and they belonged to two major classes. Among them, class A SRO proteins contained WWE, PARP and RST structural domains, whereas class B proteins were lack of WWE domains. The promoter region of the SRO genes contained a variety of cis-acting elements for abiotic stress response and hormone response. The microRNA-BjuSRO-target genes constructed a complex regulatory network, which was involved in apoptosis, root morphogenesis, immune response, cellular response to heterologous stimuli, and response to reactive oxygen species, and other biological processes. The RT-qPCR results showed that BjuVA1a, BjuVA1e, BjuVA2a, BjuVA3a, BjuVB1b, and BjuVA2a were significantly upregulated under salt stress.【Conclusion】The SRO gene family in the mustard has functional diversity, and BjuVA1a, BjuVA1e, BjuVA2a, BjuVA3a, BjuVB1b, and BjuVA2a are closely related to the response to salt stress, which can be used as candidate genes for breeding new salt-tolerant varieties of B. juncea L.

Identification of the bHLH Gene Family and Selection of Genes Related to Color Formation in Camellia reticulata

ZHOU Lin, HUANG Shun-man, SU Wen-kun, YAO Xiang, QU Yan

2024, 40(8):  142-151.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0257

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【Objective】The preliminary screening of bHLH genes related to C. reticulata flower color by bioinformatics method provides support for further study of C. reticulata flower color.【Method】Based on the full-length transcriptome data of C. reticulata, the members of the bHLH gene family were identified by bioinformatics method, and their physical and chemical properties, secondary structure, phylogenetic relationships and domains were analyzed. The second-generation transcriptome and metabolome data were used to mine the bHLH genes related to the flower color of C. reticulata. Real-time quantitative polymerase chain reaction（RT-qPCR）was used to verify the expression of bHLH gene during flowering of C. reticulata among different varieties.【Result】Total 71 CrbHLHs genes were identified in the full-length transcriptome of C. reticulata. Their molecular weight ranged from 22 994.58 to 102 996.96 Da, and their equipotential ranged from 4.98 to 9.51. All of them were hydrophilic proteins. The cluster analysis with Arabidopsis thaliana showed that the bHLH gene family members of C. reticulata could be distributed into 14 subfamilies. Multiple omics analysis showed that CrbHLH8, CrbHLH61 and CrbHLH63 were positively correlated with cyanidin, while CrbHLH59 was negatively correlated with cyanidin. CrbHLH13 and CrbHLH71 were positively correlated with proanthocyanidins, and CrbHLH8 and CrbHLH61 were members of the IIIf subfamily.【Conclusion】The 71 CrbHLHs family members were identified from the full-length transcriptome of C. reticulata, of which 6 CrbHLHs were related to C. reticulata flower color.

Genome-wide Identification and Expression Pattern Profiling of the Trehalose-6-phosphate Synthase（TPS）Gene Family in Tea Plant（Camellia sinensis）

LIU Dan-dan, WANG Lei-gang, SUN Ming-hui, JIAO Xiao-yu, WU Qiong, WANG Wen-jie

2024, 40(8):  152-163.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0252

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【Objective】Trehalose-6-phosphate synthase（TPS）is a key enzyme in the trehalose biosynthetic pathway in plants. The TPS gene family of the tea plant[Camellia sinensis（L.）O. Kuntze]was identified, analyzed, and its expression pattern under adverse stress was studied. This study aims to provide a foundation for the functional validation of TPS genes.【Method】Utilizing biological information methods, we identified members of CsTPS gene family and analyzed the physical and chemical properties of their encoded protein, conserved motifs and domains, chromosomal location, phylogenetic relationships, promoter cis-acting elements, and other biological information. Subsequently, we employed transcriptome data in conjunction with RT-qPCR technology to investigate the expression patterns of CsTPS family members under drought, salt, and cold stress.【Result】The 16 TPS gene family members were identified in C. sinensis genome, which were classified into two subfamilies: Class I and Class II based on phylogenetic study. Members of the same subfamily presented similar motif composition and gene structure. The covariance and evolutionary studies revealed that CsTPS genes were duplicated within the tea plant genome and experienced purifying selection during the evolutionary process. RT-qPCR results revealed that the six candidate genes, CsTPS3, CsTPS5, CsTPS6, CsTPS9, CsTPS11, and CsTPS14, were all induced by PEG, NaCl, and low-temperature stress, and their expression patterns were similar with transcriptome information.【Conclusion】Sixteen TPS family members were identified systematically across the tea plant's genome. The CsTPS3, CsTPS5, CsTPS6, CsTPS9, CsTPS11, and CsTPS14 genes all are sensitive to drought, salt, and low-temperature stresses, though their sensitivity levels differed.

Cloning of SgTPS7 in Sindora glabra and Its Function in Terpene Synthesis and Abiotic Stress

YU Niu, LIU Fan, YANG Jin-chang

2024, 40(8):  164-173.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0237

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【Objective】Terpenes play important roles in plant physiology and defense. The terpene synthase gene（TPS）is a key enzyme in the terpene biosynthetic pathway. The stems of the tropical oil-secreting tree species Sindora glabra contain a large amount of terpenoid resin oil. This study explores the role of S. glabra SgTPS in terpene synthesis and abiotic stress response. These will provide important genetic resources for the production of terpenes through synthetic biology, and help expand the understanding of plant terpene biosynthesis pathways and regulatory mechanisms.【Method】The SgTPS7 gene was cloned from the stem tissue of S. glabra using genome and transcriptome data, and bioinformatics analysis was performed on it. The SgTPS7 protein was expressed and purified and its catalytic function was identified using GC-MS, and RT-qPCR technology was used to analyze the expression pattern of SgTPS7.【Result】The full length sequence of the coding region of SgTPS7 of S. glabra was 1 650 bp. Homology comparison analysis found that SgTPS7 had the highest similarity with CoTPS2 of Copaifera officinalis, at 92.3%. It was distantly related to plants of other families and genera, and it belonged to the unique TPS genes of Caesalpinioideae. In vitro enzyme activity experiments showed that SgTPS7 mainly catalyzed farnesyl pyrophosphate to produce germacrene D, and catalyzed geranyl pyrophosphate to produce linalool. The expression of SgTPS7 gene was significantly different under different stress treatment conditions. After treatment with high-temperature stress, the expression of SgTPS7 in the leaves and stems reached the peak at 24 h, while the expression in the roots showed a downward trend. Long-term drought treatment significantly affected the expression of SgTPS7, and the expression of SgTPS7 in the leaves and stems reached the highest after 5 days of treatment, while the expression of SgTPS7 in the roots reached the highest after 10 d.【Conclusion】SgTPS7 is a multifunctional terpene synthase gene, which plays an important role in responding to abiotic stresses such as high temperature and drought.

Cloning and Functional Analysis of bHLH Gene Related to Anthocyanin Synthesis in Paeonia qiui

LI Yu-qing, WU Nan, LUO Jian-rang

2024, 40(8):  174-185.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0149

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【Objective】Using Paeonia qiui as the experimental material, the regulatory mechanism of PqbHLH1 gene on anthocyanin synthesis during leaf development was studied.【Method】Based on the transcriptome data obtained in the early stage, primers were designed and the bHLH1 gene was cloned from the leaves of Paeonia qiui using PCR technology. Bioinformatics methods were used to analyze the physicochemical properties, hydrophobicity, theoretical isoelectric points, protein secondary structure, and evolutionary relationships of the PqbHLH1 gene. Fluorescence quantitative PCR technology was used to detect its expressions in different tissues and stages. Agrobacterium mediated method was employed to overexpress it in Arabidopsis thaliana and detect the effect of overexpressing PqbHLH1 on the anthocyanins of peony leaves.【Result】The total length of the coding region of PqbHLH1 gene was 1 920 bp, encoding 639 amino acids. The PqbHLH1 protein was hydrophilic and does not contain signal peptides or transmembrane structures, and was localized in the nucleus. Phylogenetic analysis showed that the PqbHLH1 protein in peony ovale had the closest genetic relationship with VvMYCA1 in grapes. Fluorescence quantitative PCR analysis showed that the expression of PqbHLH1 gene in peony leaves had a trend of first decreasing and then increasing, and the expression was significant in the S6 stage of leaves. The expressions of anthocyanin synthesis structural genes PqCHS, PqF3'H, PqDFR, and PqANS showed a trend of first increasing and then decreasing with leaf development. In A. thaliana strains overexpressing the PqbHLH1 gene, the expressions of structural genes related to anthocyanin synthesis was significantly higher than that of wild-type Arabidopsis, and the expression trend of anthocyanin content was consistent with that of structural genes AtCHI, AtF3'H, and AtUFGT, while the expression trend of flavonol content was consistent with that of structural gene AtFLS.【Conclusion】The PqbHLH1 gene positively regulates the synthesis of anthocyanins and flavonols in peony leaves by regulating the expressions of genes related to anthocyanin synthesis.

Genome-wide Identification and Expression Features Analysis of the bZIP Family in Rhododendron henanense subsp. lingbaoense

LI Yong-hui, BAO Xing-xing, DUAN Yi-ke, ZHAO Yun-xia, YU Xiang-li, CHEN Yao, ZHANG Yan-zhao

2024, 40(8):  186-198.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0822

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【Objective】To identify the members of the basic leucine zipper（bZIP）family of Rhododendron henanense subsp. lingbaoense genome, and to explore their expression patterns under abiotic stress.【Method】We identified the RhlbZIPs（bZIP TFs in R. henanense subsp. lingbaoense）using bioinformatic methods, and investigated their physical and chemical properties, gene structures, conserved motifs, evolutionary relationships, cis-acting elements, and expression patterns. In addition, real-time quantitative fluorescent PCR（RT-qPCR）was used to analyze the expression of RhlbZIP gene in abiotic stress.【Result】A total of 57 RhlbZIP genes were identified and divided into 11 subfamilies, and the same subfamily members having similar gene structure and conserved motifs. RhlbZIP genes were unevenly distributed in 13 chromosomes, and collinearity analysis revealed 45 replication events（3 pairs of tandem replications and 42 pairs of fragment replication）. Cis-acting elements in the promoter region of RhlbZIP genes included four categories responding to transcription, development, abiotic or biological stress and hormone. GO annotation analysis showed that RhlbZIP gene family was associated with transcriptional regulation, metabolism, and biological processes. Transcriptome data showed that most bZIP family genes（82.46%）were expressed in 5 tissues of root, stem, leaf, flower, and hypocotyl, but there were significant differences in expressions. RT-qPCR analyses demonstrated that RhlbZIP4 was significantly up-regulated under low temperature treatment. RhlbZIP24 was significantly upregulated under drought and ABA treatment. RhlbZIP16 was significantly upregulated under high salt and MeJA treatment. 【Conclusion】A total of 57 RhlbZIP genes have been identified in the R. henanense subsp. lingbaoense genome, and all selected genes are expressed under different treatments, and the major genes showing increase in expression in response to low temperature, drought, high salt, ABA and MeJA stress are RhlbZIP4, RhlbZIP24, RhlbZIP16, RhlbZIP24 and RhlbZIP16, respectively.

Identification and Expression Analysis of the YABBY Gene Family in Sunflower

WANG Qian, ZHOU Jia-yan, WANG Qian, DENG Yu-ping, ZHANG Min-hui, CHEN Jing, YANG Jun, ZOU Jian

2024, 40(8):  199-211.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0139

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【Objective】The identification of HaYABBY gene related to flower development in sunflower（Helianthus annuus L.）is conductive to exploring its biological function and regulatory mechanism in the floral development of sunflower, and to provide important molecular clues for sunflower variety breeding.【Method】Based on sunflower genome database and our transcriptome data, bioinformatics analysis was conducted to analyze the physicochemical properties, chromosome localization, phylogenetic relationship, gene structure, conserved motif, protein structure, promoter cis-action regulatory element, and tissue expression pattern, etc. RT-qPCR was used to analyze the differences of HaYABBY gene expression between WT sunflower and cb1（crazy broccoli 1）mutant with the defection of meristem determinacy and floral organ differentiation at st2-st8.【Result】Twelve members of YABBY family were identified, belonging to 4 subfamilies, and located in 8 chromosomes； meanwhile, the members in the same subfamily shared similar gene structure and conserved motifs. Results of tissue expression pattern showed that 8 members presented high expressions during flower development in sunflower, including HaYABBY1a, HaYABBY1c, HaYABBY3, HaYABBY4b, HaYABBY5a, HaYABBY5b, HaCRABS CLAWa, and HaCRABS CLAWb, among which HaYABBY3, HaYABBYY5a, and HaYABBYY5d expressed with up-regulation at the late stages of flower development in cb1 mutant; while the expression of HaYABBYY4b and HaCRABS CLAWa was strongly inhibited in cb1 mutants, corresponding to their expressions at early floral developmental in WT plants. The analysis of cis-regulatory elements showed that all the HaYABBY members contained multiple promoter cis-acting elements, some acting elements only existing in specific genes. Among them, endosperm expression elements were presented in HaCRABS CLAWa and HaYABBY4a. Auxin responders were in HaCRABS CLAWa, HaCRABS CLAWb, HaYABBY4a, HaYABBY4b and HaYABBY5c. Meristem expression elements existed in HaYABBY1a, HaYABBY1c, HaYABBY4b, HaYABBY5a, and HaYABBY5d. Gibberellin response elements were included in HaCRABS CLAWa, HaYABBY5b, HaYABBY5c, and HaYABBY5d. Seed-specific regulatory elements presented in HaYABBY5a, HaYABBY5c, and HaYABBY5d.【Conclusion】Five HaYABBY genes of HaYABBY3, HaYABBY4b, HaYABBY5a, HaYABBY5d and HaCRABS CLAW play important roles in the process of floral meristem transformation and floral organ formation in sunflower, and they might depend on the regulation of auxin, gibberellin and meristem expression related regulators during meristem transformation and floral organogenesis, and need the involvement of the auxin response factor ARFs, gibberellin response factor MYBs and flower organ development regulator MADS-boxes.

Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium

XU Xue-fei, YANG Pan-pan, ZHANG Wen-liang, BIAN Guang-ya, XU Lei-feng, LIU Hui-chao, MING Jun

2024, 40(8):  212-220.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0229

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【Objective】Lily mottle virus（LMoV）is one of the major viruses that jeopardize the important food and medicinal lily Lilium lancifolium Thunb, causing billions RMB yuan losses to the lily cultivation industry every year. We clarified the distribution of LMoV in the apical meristematic tissues of L. lancifolium by in situ hybridization to provide support for the cultivation of lily detoxification.【Method】Cultivated L. lancifolium planted in Qingzhen city, Guizhou province, was used as the test material. We identified the virus species carried by the test material by RT-PCR and LMoV isolates from different places were downloaded from the NCBI website to construct an evolutionary tree using the material of L. lancifolium. RNA flourescent probes were designed and prepared based on the LMoV genome sequence, and in situ hybridization was carried out in the stem tip and root tip regions to observe the distribution of LMoV.【Result】RT-PCR showed that the L. lancifolium material was infested with LMoV, and the construction of evolutionary tree revealed that the isolates in this experiment were most closely related to the LMoV isolates from Jilin and Liaolin, and in situ hybridization showed that the LMoV hybridization signals were mainly distributed in the tissues 0.15-0.2 mm below the shoot apical meristem, and there were weak viral hybridization signals in the base of the primary leaf adjacent to the first leaf primordium. The fluorescence signals of the virus were strong in the apices of mature leaves close to the primary leaves, and the size of the avirulent zone in the center of the LMoV-infected L. lancifolium meristem was (0.4-0.6) mm×(0.15-0.2) mm in longitudinal section. There was no distribution of hybridization signals in the 0.25 mm×0.2 mm meristem of the root tip. The signals were the strongest in the cortex and weaker in the root crown and root elongation zone.【Conclusion】No viral signals were found in the apical meristem and the closest apical leaf primordium[（0.4-0.6）mm×0.2 mm in size]. These regions can be used as a starting material for stem tip detoxification. The outer root crown of the apical meristem had virus signals and was not suitable for use as an explant in the culture of L. lancifolium detoxification.

Cloning and Functional Analysis of NtPRR37 Gene in Nicotiana tabacum L.

LI Yi-jun, YANG Xiao-bei, XIA Lin, LUO Zhao-peng, XU Xin, YANG Jun, NING Qian-ji, WU Ming-zhu

2024, 40(8):  221-231.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0112

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【Objective】Pseudo response regulators（PRRs）are important genes that regulate the flowering pathway in higher plants. The aim of this study is to clone the NtPRR37 gene from Nicotiana tabacum L. and analyze its response to different photoperiods and its effect on flowering, providing a target gene for tobacco flowering regulation.【Method】We cloned NtPRR37 gene from N. tabacum L. by homologous cloning method. Then we performed bioinformatics analysis, tissue-specific expression analysis, and expression pattern analysis of NtPRR37 gene under different light duration treatments. Concurrently, we used Virus Induced Gene Silence（VIGS）to reduce the expression of NtPRR37 and observed the phenotype and detected the expression changes of flowering-related genes.【Result】The fulllength CDS of NtPRR37 was 2 472 bp, encoded a peptide of 823 amino acids with a relative molecular weight of 90.16 kD. It contained REC and CCT domains, which were typical conserved domains of the PRRs gene family. Through homology evolution analysis, it was found that tobacco NtPRR37 belonged to the same branch as PRR37 from Nicotiana tomentosiformis, Nicotiana sylvestris, and Nicotiana benthamiana in evolution. The quantitative reverse transcription-PCR（RT-qPCR）analysis showed that, the expressions of NtPRR37 varied in various tissues of N. tabacum L. at full-bloom stage, with the highest expression in pistils and the lowest expression in lateral roots. Under different photoperiod treatments, the expression of NtPRR37 increased with the prolongation of light exposure. In different light duration treatments, the expression of NtPRR37 increased with the extension of light time, with the lowest expression under darkness, and demonstratted circadian rhythmicity. In NtPRR37-silenced plants, the expression of NtPRR37 was significantly downregulated, and the flowering period was advanced, which may be related to the significant upregulation of flowering-related genes（NtFT4, NtAP1, NtCO, NtSOC1）. 【Conclusion】The expression of NtPRR37 is regulated by the photoperiod, and NtPRR37 acts as a flowering repressor during the flowering process of tobacco.

Optimal Conditions for L-ascorbic Acid Combined with Ultrasonic Treatment of Fresh-cut Taro

CHEN Lin, CHEN Li-li, CHEN Lin-lin, ZHANG Min, CHEN Bing-zhi, JIANG Yu-ji

2024, 40(8):  232-243.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0088

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【Objective】The objective of this study is to determine the optimal conditions for the combined treatment of fresh-cut ‘June Red Taro’ with L-ascorbic acid（AA）and ultrasound（US）, and to investigate its preservation effects.【Method】Yongding ‘June Red Taro’ was used as the test material. Initially, 11 types of various preservative solutions，including tap water, pure water, deionized water, 9% sucrose solution, 1% sodium carbonate solution, 1% sodium chloride solution, 1% AA solution, and slightly acidic electrolyzed water（20, 40, 60, and 80 mg/L）, were used to identify the most effective one for preserving fresh-cut ‘June Red Taro’. Subsequently, the preservative effects of AA and ethanol mixture on fresh-cut ‘June Red Taro’ was studied. The experimental results indicated that AA showed the best preservative effect. Based on this finding, a further exploration was conducted to investigate the specific impacts of AA and US combined treatment（AS）on the storage quality of fresh-cut ‘June Red Taro’. Metrics such as browning degree, hardness, pH value, relative conductivity, and sensory evaluation scores were measured to determine whether the AS treatment further enhanced the preservative effect.【Result】The experimental results demonstrated that the sensory quality of fresh-cut ‘June Red Taro’ could be effectively maintained when the AA concentration was 2%, soaking time was 15 minutes, US treatment time was 10 minutes, US frequency was 53 kHz, and US power was 400 W. The AS treatment effectively inhibited the increase in browning degree, maintained acidity stability, slowed down the decrease in hardness, preserved the integrity of cell membranes, and maintained a high sensory score. Therefore, these parameters were determined as the optimal conditions for AS treatment.【Conclusion】AA is selected as the best color-protecting solution from different soaking solutions, and the optimal conditions for the AS treatment of fresh-cut taro are determined. The AS treatment effectively improved the preservative effect of fresh-cut ‘June Red Taro’.

Evaluation of Drought Resistance of Three Chlorella Strains

HAN Kai, ZHOU Yong-shun, ZHANG Kai-yue, WANG Lu, GAO Jian-feng, CHEN Fu-long

2024, 40(8):  244-254.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0101

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【Objective】In order to study the physiological response of desert Chlorella to drought stress, we isolated and cultured Chlorella with strong drought resistance, laying a foundation for the development and production of desert microalgae resources.【Method】Two strains of Chlorella vulgaris isolated from Taklimakan Desert and Gurbantunggut Desert in Xinjiang and one strain of C. vulgaris purchased from Chinese Academy of Sciences Freshwater Algae Seed Bank were used as materials, and Bold's Basal Medium（BBM）as the culture medium. The growth rate and physiological and biochemical indicators of three strains of C. vulgaris under drought stress were measured by polyethylene glycol（PEG6000）to simulate different degrees of drought stress, and the drought resistance capacity of three strains of C. vulgaris was evaluated by principal component analysis, membership function, comprehensive drought resistance measurement value and other methods.【Result】 Under drought stress, the cell density（OD₆₈₀）and biomass（dry weight）decreased, chlorophyll a and chlorophyll b content decreased, while the malondialdehyde（MDA）content and superoxide dismutase（SOD）activity increased. However, soluble sugar, protein, proline（Pro）content, and catalase（CAT）activity varied with the degree and time of stress. The principal component analysis table showed that the sugars, Pro, SOD, CAT, and chlorophyll b played important roles in the drought resistance process of C. vulgaris. According to the analysis of drought resistance coefficient, the soluble sugars, proteins, Pro, SOD, and CAT of desert Chlorella had stronger effects than ordinary ones during severe drought.【Conclusion】The comprehensive evaluation shows that the drought resistance of the three strains of Chlorella is in the following order: Desert Chlorella TLD 6B > Desert Chlorella GTD 8A1 > Common Chlorella FACHB-2338, and desert Chlorella has advantages in growth rate, sugar content, and protein content compared to common Chlorella.

Screening and Identification of Excellent Strains of Endophytic Bacteria Promoting Rice Iron Absorption from Wild Rice

LI Qing-mao, PENG Cong-gui, QI Xiao-han, LIU Xing-lei, LI Zhen-yuan, LI Qin-yan, HUANG Li-yu

2024, 40(8):  255-263.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0178

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【Objective】To identify siderophilic endophytic bacteria that efficiently promote iron absorption in rice, and to investigate the siderophore-producing capacity and iron absorption promoting effect of different genera of endophytic bacteria from four wild rice.【Method】This study analyzed the siderophore contents of 198 endophytic bacteria isolated from four types of wild rice including Oryza longistaminata, O. rufipogon, O. officinalis, and O. minuta. The 82 strains with highly efficient siderophore-producing ability were screened from 128 strains with siderophore-producing ability. Analysis of the 16S rRNA gene sequences revealed that these 82 strains belonged to 18 different genera or species. Based on their siderophore production abilities, 18 candidate strains were selected to assess their abilities of promoting iron absorption in rice.【Result】The results demonstrated that different genera had varying effects on promoting rice iron absorption, with Bacillus（class）, Pseudomonas, and Saccharomycetes showing significant effects. Five high-efficient siderophore producing strains were selected from 18 strains and further confirmed that endophytic bacteria partially restored the iron deficiency phenotype of a rice iron-absorption-deficient mutant（osbhlh156）.【Conclusion】This study elucidates that siderophore produced by plant-interacting microorganisms can be utilized by rice, thereby enriching the microbial resources associated with iron absorption in rice. Additionally, it provides valuable bacterial resources and a theoretical basis for promoting iron absorption in rice through rice-microbial interactions.

Effects of Compound Microbial Agent on the Growth, Quality and Rhizosphere Environment of Grape

CHE Jian-mei, LAI Gong-ti, LI Si-yu, GUO Ao-lin, CHEN Bing-xing, CHEN Xing, LIU Bo, LAI Cheng-chun

2024, 40(8):  264-274.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0230

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【Objective】Grape is one of the five varieties of fruit trees widely cultivated in China. Long-term excessive application of chemical fertilizer will destroy the microbial ecology, thus affecting the yield and quality of grapes. Compound microbial agent of different strains may be a more active and effective measure to improve the quality of grapes.【Method】The compound microbial agent（Brevibacillus brevis FJAT-0809-GLX, Bacillus velezensis FJAT-55034 and B. brevis FJAT-10623）were used to irrigate the roots, and the effects of the compound microbial agent on grape growth, fruit quality, rhizosphere soil enzymatic activity and diversity of culturable Bacillus were analyzed.【Result】The compound microbial agent promoted the growth of grape leaves, increased the contents of chlorophyll a and carotenoid, and the activities of POD and PPO. The chlorophyll b content, SOD and PPO activities of young grape fruits in the treatment group was 100%, 1.77% and 66.67% higher than that in the control group, respectively. The application of compound microbial agent promoted early ripening, fruit color transformation and quality of grapes. The single fruit weight was 29.06% higher than that of control group, and the soluble solid and soluble protein increased by 7.31% and 94.74%, respectively, compared with the control group. The PPO enzyme activity of ripe grape fruit in the treatment group was significantly higher than that in control group. In addition, the compound microbial agent significantly improved the activities of amylase, catalase, cellulase, sucrase, and the number and species of Bacillus in grape rhizosphere soil. Correlation analysis showed that Bacillus zanthoxyli and Bacillus pseudomycoides promoted grape leaf growth and fruit quality.【Conclusion】The compound microbial agent may improve soil, promote grape growth and increase fruit quality.

Effects of Simulated Mutational Biosynthetic Regulation on the Secondary Metabolites of Aspergillus terreus C23-3

MA Xiao-xiang, MA Ze-yuan, LIU Ya-yue, ZHOU Long-jian, HE Yi-fan, ZHANG Yi

2024, 40(8):  275-287.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0110

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【Objective】Butyrolactone I is a small molecular natural product of Aspergillus terreus with versatile bioactivities, thus the exploration of its structural diversity extension is significant. A marine-derived A. terreus strain C23-3 featuring producing butyrolactone I was used as the base strain to study the effects of butyrolactone I precursor analogs and precursor synthase inhibitors on its secondary metabolism.【Method】The strain was statically fermented under chemically regulative conditions established by seawater potato liquid medium supplied with three precursor small molecules and three 4-hydroxyphenylpyruvate synthase inhibitors. The high-performance liquid chromatography（HPLC）, HPLC -ion trap mass spectrometry（MS）, and MS based molecular networking together with database mining were used to further analyze the yields and diversity of secondary metabolites.【Result】The most of the precursor analogs and synthase inhibitors suppressed the synthesis of butyrolactone I to different extents. Moreover, the precursor analog 3,4-dihydroxyphenylpyruvate at a high dose, the combination of the two 4-hydroxyphenylpyruvate synthase inhibitors and the combination of these three demonstrated remarkable inhibitory effects. In addition, the global synthesis for sorts of secondary metabolites including butenoids, territrems, lovastatins, etc., was downregulated when butyrolactone I was inhibited for production. However, the yield of a cyclopeptide was upregulated by more than ten folds under this situation, which was supposed to be a stress response to the synthetic suppression of butyrolactone I as a quorum sensing molecule and inducer of global transcriptive factor lae A. The detailed mechanism deserves further investigation.【Conclusion】The present study manifests that 4-hydroxyphenylpyruvate is the precursor of butyrolactone I and reveals that 3,4-dihydroxyphenylpyruvate and 4-hydroxyphenylpyruvate synthase inhibitors can be used as small molecule tools to suppress the production of butyrolactone I, which may be applied in further study on simulated mutational biosynthesis to generate diverse butyrolactone derivatives and induce the production of cyclopeptide.

Enrichment of 1, 2-dichloroethane Degrading Bacterial Consortium, and Isolation and Identification of Ancylobacter sp. BL0 of a Key Degrading Bacterial Strain

ZHANG Zhi-mei, ZHANG Yan-meng, XIE Dong-ming, YANG Xiu-yun, WANG Lang, ZUO Zi-han, WU Zhi-guo

2024, 40(8):  288-298.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0108

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【Objective】The pollution of 1, 2-dichloroethane（1,2-DCA）will seriously harm human health and environmental ecological safety. In order to obtain efficient 1,2-DCA biodegrading microorganism resources, the characteristics of bacterial flora were studied, and then the rules and methods for separating and screening efficient 1,2-DCA degrading strains were explored.【Method】The bacteria utilizing 1,2-DCA as the sole carbon source and energy were enriched and cultured from soil contaminated by 1,2-DCA. The growth of bacteria and degradation of 1,2-DCA in different batches were investigated by ultraviolet spectrophotometry and gas chromatography respectively. The species diversity and relative abundance of different enriched solution were analyzed by high-throughput sequencing. The strain was identified by 16S rDNA gene sequence analysis. The degraded products were determined by gas chromatography-mass spectrometry, and the degradation pathway was analyzed.【Result】Bacterial consortium BG1 for degradation was gained, and the experimental results showed that the degrading rate of 12.5 mg/L 1,2-DCA by enriched bacterial consortium in batch 6-11 and 15 increased at the beginning and then decreased within 24 h, and the microbial biomass（OD₆₀₀）increased from 0.03 to 0.095, but the relative abundance of Ancylobacter sp. in the flora decreased with continuous enrichment process. A strain named BL0 was screend from the batch 9 of enrichment solution and identified as Ancylobacter sp., which degraded 120 mg/L 1,2-DCA within 6 h. It was inferred that the metabolic pathway of degradation of 1,2-DCA by BL0 was hydrolysis of 1,2-DCA to 2-chloroethanol at first, and 2-chloroethanol was oxidized to chloroacetic acid, which was then completely degraded and utilized.【Conclusion】Highly efficient 1,2-DCA degrading bacterial consortium BG1 and strain Ancylobacter sp. BL0 are obtained, and it is found that a long period of sample enrichment is required for screening and separation of 1,2-DCA degrading strains during the process of sample enrichment and low microbial growth is observed, which is not suitable for over-enrichment. Strain Ancylobacter sp. BL0 is competitive with the other strains in the enrichment of bacterial consortium.

Optimization of Fermentation Culture Conditions for Macrolactins Yield of Bacillus siamensis Using Response Surface Methodology

ZHANG Meng-fei, YU Lian, LI Fei, LI Zhe, SU Xin-ying, LAN Cai-bi, ZHU Hu, QIN Ying

2024, 40(8):  299-308.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0040

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【Objective】Macrolactins（MLNs）are a class of 24-membered macrolides formed by cyclization of polyketones, which have a variety of biological activities such as antibacterial, antifungal, antiviral, and anti-tumor. However, due to the low yield of macrolides in traditional fermentation, its development and application are limited. Therefore, this article aims to increase the yield of MLNs synthesized by Bacillus siamensis K53, in order to provide scientific basis for the mass production of MLNs.【Method】Based on previous research, single factor experiments was used to study the effects of carbon, nitrogen and amino acids on the MLNs yield of strain K53 and to determine the key nutritional factors. Response surface analysis was used to optimize the key nutritional factors for obtaining the optimal parameters in fermenting MLNs.【Result】In the single factor experiment, glucose was the optimal carbon source in the fermentation medium of strain K53. The yeast extract and (NH₄)₂SO₄ were the optimal organic and inorganic nitrogen sources, respectively. The addition of histidine and valine increased the yield of MLNs. In response surface optimization, the optimal fermentation medium for strain K53 was 22.0 g/L glucose, 3.0 g/L (NH₄)₂SO₄, 1.0 g/L yeast extract, 6.1 g/L valine, and 5.9 g/L histidine. The valine and histidine were precursors for the synthesis of MLNs. The MLNs yield in this medium reached 5.37×10² μg/mL, which was very close to the maximum value（5.46×10² μg/mL）predicted by theory, and 4.10 times higher than that in the non-optimized medium（1.31×10² μg/mL）.【Conclusion】The optimized medium formula by response surface methodology is conducive to the MLNs yield of the strain K53，and the results provide theoretical bases for the consequent development and application of the strain K53.

Preparation of Low-phenylalanine Casein by Novel Phenylalanine Ammonia-lyases Derived from Rhodotorula

ZHANG A-na, HAN Xue, GU Tian-yi, XIN Feng-jiao, WANG Yu-lu

2024, 40(8):  309-319.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0092

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【Objective】To explore the phenylalanine ammonia-lyase（EC 4.3.1.24; PAL）with high activity and stability, and to lay a foundation for the subsequent preparation of special dietary foods with no（low）phenylalanine.【Method】The genes RmPAL and RdPAL were cloned from Rhodotorula mucilaginosa and Rhodotorula diobovata, respectively, and the sequence and structural features of the two enzymes were analyzed by bioinformatics. The two enzymes were heterologously expressed and purified in Escherichia coli, and the optimal reaction conditions and substrate specificity were determined. In addition, the ability of RmPAL and RdPAL to convert L-Phe in casein acid hydrolysate（CAH）was determined by high-performance liquid chromatography（HPLC）and phenylalanine assay kit.【Result】The fungal-derived RmPAL and RdPAL contained three domains: the MIO domain, the core domain, and the shielding domain, with the catalytic amino acid Tyr and the substrate-specific amino acid His in the active pocket. Both RmPAL and RdPAL existed as tetramers in solution. The optimal pH and temperature of the two enzymes were 8.9 and 50℃, respectively, and they demonstrated broad pH and temperature stability, which was superior to commercial PALs derived from Rhodotorula. Furthermore, both enzymes catalyzed L-Phe and L-Tyr, with a higher catalytic efficiency towards L-Phe, approximately five times that of L-Tyr. The conversion rates of L-Phe removal from CAH were 88% and 93% for RmPAL and RdPAL, respectively.【Conclusion】RmPAL and RdPAL have strong stability, L-Phe hydrolysis preference, and ability to remove L-Phe from food-derived protein.

Analysis of LncRNA Differential Expression in Mammary Tissue of Cows with Staphylococcus aureus Mastitis

ZHOU Ran, WANG Xing-ping, LI Yan-xia, LUORENG Zhuo-ma

2024, 40(8):  320-328.  doi:10.13560/j.cnki.biotech.bull.1985.2024-0159

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【Objective】Long non-coding RNA（lncRNA）is involved in inflammatory responses, however, their specific molecular regulatory mechanism in dairy mastitis remains unclear. In this study, differentially expressed lncRNA（DElncRNA）and its function in mammary tissues of cows with Staphylococcus aureus（S. aureus）-type mastitis were analysed to support subsequent in-depth studies.【Method】Transcriptome sequencing（RNA-Seq）technology and bioinformatics methods were used to carry out lncRNA sequencing, DElncRNA analysis, and GO and KEGG functional enrichment analysis of mammary tissues from healthy cows and cows with S. aureus-induced mastitis.【Result】A total of 1012 lncRNA are detected in the mammary tissues of both groups. Compared to the healthy dairy cows, total 75 lncRNA are up-regulated and 114 down-regulated in cows with S. aureus-induced mastitis. Further analysis reveals that DElncRNA TCONS_00042123, TCONS_00068055, TCONS_00108420 and TCONS_00076361 may be involved in the MAPK, NLR, Jak-STAT and TLR signaling pathways related to inflammation, regulating the occurrence and development of S.aureus-type mastitis in the dairy cows.【Conclusion】189 DElncRNA were obtained from mammary tissues of cows with S. aureus-induced inflammation, which may regulate the process of onset and progression of S. aureus-type cow mastitis through potential target genes.

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2024, 40(8):  329. 

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2024, 40(8):  330. 

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2024, 40(8):  331. 

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