Table of Content

26 February 2024, Volume 40 Issue 2

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Crosstalk Between Different Post-translational Modifications and Its Regulatory Mechanisms in Plant Growth and Development

CHEN Yan-mei

2024, 40(2):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0963

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Post-translational modifications（PTMs）have key regulatory roles in cell signal transduction networks. In addition to be a single PTM, many proteins undergo the regulation of multiple different types of PTMs，which functions in an orchestrated manner to adapt external or internal environmental cues. Such PTM crosstalk affects protein interactions, stabilities and subcellular localizations, and ultimately acts as a signaling hub to control diverse biological processes. Although PTM crosstalk has been extensively studied in humans and animals, the investigation in plants is just emerging. In this review, we discuss recent advances in crosstalk among three of the most common PTMs in plants, including phosphorylation, ubiquitination, and sumoylation，furthermore we highlight how this crosstalk may be connected to downstream signaling outputs.

Research Progress in the Dynamic QTL Analysis of Plant Growth and Development

FU Wei, WEI Su-yun, CHEN Ying-nan

2024, 40(2):  9-19.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0790

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Accurate and effective quantitative trait locus（QTL）mapping is the prerequisite for gene cloning and molecular breeding. Plant growth and development are affected by external environmental factors, and phenotypes at different developmental stages are formed due to the dynamic expressions of different major genes or QTLs. The conventional gene/QTL analysis based on the phenotypic data collected at stationary growth phase can only be used to estimate the cumulative effects of QTLs over a long period of time. It cannot reveal the actual effects and functional modes of the loci during the developmental process. Besides, the static QTL mapping method neglected the dynamic expression patterns of QTLs, leading to missing of information about dynamic variations of quantitative traits. Dynamic QTL analysis of the whole growth cycle provides an elite strategy for investigating the genetic mechanisms and dissecting major QTLs underlying plant growth and development. In this review, we briefly summarized the genetic models and analytical methods for dynamic QTL detection, as well as the research progress on mapping quantitative traits of plant development. We further proposed the current issues and future trends in the area of dynamic QTL analysis, which would provide references not only for dynamic QTL analysis during plant growth and development but also for molecular breeding based on marker-assisted selection.

Research and Application Progress in Chloroplast Genome of Tea Plant（Camellia sinensis）

YANG Yu-qing, TAN Juan, WANG Fang, PENG Shun-li, CHEN Jie, TAN Ming-yan, LYU Mei-yan, ZHOU Fu-yu, LIU Sheng-chuan

2024, 40(2):  20-30.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0451

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Most plant chloroplast genomes are double-stranded circular DNA with a conserved tetrad structure, non-recombination, haploid, uniparental inheritance, moderate evolutionary rate, and highly conserved sequence and structure, and may provide useful information for the evolution of plants. Due to abundant tea germplasm resources, their complex origin, evolution and classification remain largely elusive, resulting in hindering their efficient protection and innovative utilization. In recent, the rapid development of chloroplast genome sequencing technologies has been greatly promoted and being changed into the third-generation sequencing. About 33 sequenced chloroplast genomes from tea plants have been published on NCBI, and the gene types and gene sequence characteristics of chloroplast genomes in tea plants have been partially revealed, and used in the study of classification, identification, origin, evolution, and albino mechanism for tea plants. However, many chloroplast genomes of tea resources remain undeciphered. While reviewing the origin, inheritance, and basic characteristics of plant chloroplast genomes, the paper focused on the review of the sequencing technology, genomic features, gene types, and gene sequences of the published chloroplast genomes from tea plants. The paper briefly presented the application status of tea plant chloroplast genome and finally, and discussed the future application and development direction of tea plant chloroplast genomes.

Research Progress in the Reference Materials of Crop Pathogens in China

YANG Wen-li, ZHU Li-li, CHEN Jian, CHEN Yan-xin, YAO Juan, JIANG Da-gang

2024, 40(2):  31-37.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0607

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Crops are often infected by pathogenic bacteria during their growth and development stages, thus leading to huge economic losses. Timely and accurate detection for infected crop is helpful to provide reference for formulation of disease prevention and control methods. The reference materials of crop pathogens are important reference in the process of crop disease detection. They play a very important role in the qualitative and quantitative detection of crop disease. At present, there are few reports on the reference materials of crop pathogens, then it is urgent to support and develop them in an orderly manner. In this literature, we summarize the research progress of crop pathogen detection methods, detection standards and crop pathogen reference materials, aiming to provide theoretical direction for study on reference material of crop pathogens in China.

The Application Progress of Serum-free Suspension Culture Technology of MDCK Cells in Influenza Vaccine Study and Production

JIN Li-wu, ZHANG Zhen-yu, JIN Dong-wu, MA Hua, MA Yu-mei, QIAO Zi-lin, WANG Jia-min

2024, 40(2):  38-47.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0787

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Influenza is a common respiratory disease caused by influenza viruses. MDCK（Madin-Darby Canine Kidney）cells have the characteristics of easy culture and high production, which can support the replication and proliferation of influenza viruses, and are widely used in the research and production of influenza viruses. With the deepening of research on influenza virus, and in order to further optimize the ability of MDCK cells to be used for influenza virus research and industrial application, researchers began to develop the domestication technology of MDCK cells. Through long-term cultivation and selection, the domesticated MDCK cells strain can better adapt to the growth environment of influenza virus, and improve the production and infectivity of influenza virus. In conclusion, the domestication technology of MDCK cells plays an important role in influenza virus research and industrial application, which may improve the production and infectivity of influenza viruses and provide better tools for influenza vaccine production and anti influenza drug development. Concurrently we have to also pay attention to the safety of MDCK cells and take appropriate measures to ensure the safety and reliability of its application.

Establishment and Application of Ploidy Method for the Identification of Psidium guajava by Flow Cytometry

SHAO Xue-hua, LI Gui-lan, XIAO Wei-qiang, LAI Duo, ZHUANG Qing-li, QIN Jian

2024, 40(2):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0844

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【Objective】 The flow cytometry was used to identify the ploidy of Psidium guajava, which may lay the foundation for ploidy breeding and hybrid breeding of P. guajava. 【Method】 Using P. guajava as the material, the effects of sample parts, centrifugation treatment, and storage methods on ploidy detection results were compared. 【Result】 P. guajava petals had the highest number of collected nuclear DNA, with a CV value of 1.96% and the best peak shape. When preparing nuclear suspension, the best detection effect was achieved by directly staining and analyzing without centrifugation after filtration. The nuclear DNA content obtained from fresh P. guajava petals was significantly higher than that of refrigerated and other frozen treatment groups, with fewer background fragments and a clear main peak. The -80℃ freezing treatment caused the least damage to the samples, and the detection effect was second only to that of fresh P. guajava petals. The established flow cytometry method for ploidy analysis of P. guajava involved taking 0.50-1.00 cm² of pomegranate petals, mixing them with 1 mL of mGb lysis buffer, filtering, and adding 30 μL of PI staining for 1 min before analysis. 【Conclusion】 Using this established flow cytometry method, ploidy identification was performed on 33 P. guajava germplasm resources, resulting in the detection of 32 diploids and 1 hexaploid. This method is simple, efficient, and accurate, and may provide an effective approach for ploidy identification of P. guajava germplasm resources.

Establishment of A Bacterial Model of CRISPR/Cas9 Mediated adeG Gene Knockout in Escherichia coli

ZHU Tian-yi, KONG Gui-mei, JIAO Hong-mei, GUO Ting-ting, WU Ri-han, LIU Cui-cui, GAO Cheng-feng, LI Guo-cai

2024, 40(2):  55-64.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0841

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【Objective】 Acinetobacter baumannii is one of the important opportunistic pathogens that cause hospital infections, and the highly resistant A. baumannii is currently a thorny issue in prevention and treatment. Due to the fact that bacterial efflux pumps are one of the important causes of drug resistance, the distribution of the efflux pump gene adeLFGH in the resistance-nodulation-division（RND）of A.baumannii was detected by PCR to explore its relationship with drug resistance. Next, an adeG resistance gene model was established and preliminary targeted knockout studies using the CRISPR/Cas9 system was conducted. 【Method】 Broth microdilution method was used to detect drug resistance distribution of collected strains. PCR was to screen and analyze the distribution of adeLFGH efflux pump genes in 13 clinical isolates of multidrug-resistant A.baumannii. The key gene adeG of RND efflux pump was amplified and a model of adeG resistant bacteria was constructed. Specific sgRNA was designed, CRISPR/Cas9 system was used for targeted knockout, and drug sensitivity experiments were conducted to detect its knockout effect. 【Result】 13 clinical strains of A.baumannii were sensitive to polymyxin E, with a low resistance rate to levofloxacin, only 38.5%. The resistance rate to ceftazidime was 92.3%, the resistance rate to tobramycin was 84.6%, and the resistance rate to other common clinical drugs was 100%.The carrier rate of adeLFGH in 13 strains of multidrug-resistant A.baumannii was 100%. The drug sensitivity test results showed that the adeG model bacteria showed a transition from sensitive to resistant to piperacillin, ticarcillin/clavulanic acid, and piperacillin/tazobactam, while from sensitive to intermediary to chloramphenicol, meropenem, and minocycline. The targeted knockout efficiency of the CRISPR/Cas9 system mediated by specific sgRNA varies. The resistance of pCas9-sgRNA1（adeG）to piperacillin/tazobactam was reversed, and the resistance to tobramycin, tetracycline, doxycycline, and minocycline reduced to varying degrees. pCas9-sgRNA2（adeG）and pCas9-sgRNA3（adeG）restored the resistance of piperacillin/tazobactam from resistant to intermediary, but the reversal effect on other drugs was not significant. 【Conclusion】 The specific CRISPR/Cas9 system can specifically knock out drug-resistant genes and provide new ideas and methods for the treatment of drug-resistant bacteria.

Establishment of Quaking Knockout Mouse Embryonic Fibroblast Cell Line Using CRISPR/Cas9 Technology

GAO Deng-ke, MA Bai-rong, GUO Yi-ying, LIU Wei, LIU Tian, JIN Ya-ping, JIANG Zhou, CHEN Hua-tao

2024, 40(2):  65-72.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0879

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【Objective】 CRISPR/Cas9 technology was used to generate a mouse embryonic fibroblast cell line（NIH3T3）with a knockout of the Quaking gene and to investigate its impact on NIH3T3 cell proliferation. 【Method】 Initially, two sgRNAs targeting Quaking exons were designed using an online platform, and two CRISPR/Cas9 recombinant lentiviral plasmids targeting the first and second exons of the Quaking gene were constructed successfully. These constructs with pcDNA3.1-Quaking overexpression plasmids were co-transfected into HEK293T cells, and the knockout efficiency of Quaking protein was assessed through Western blot analysis. Subsequently, the recombinant lentiviral plasmid（LentiCRISPRv2-sgRNA1）with high knockout efficiency was co-transfected with auxiliary packaging plasmids into HEK293T cells for lentivirus packaging. After lentiviral transduction of NIH3T3 cells, positive monoclonal cell lines were selected using puromycin. Finally, we confirmed the knockout effect through Western blot and immunofluorescence staining, demonstrating the absence of Quaking protein in these cells. 【Result】 Sequencing confirmed the occurrence of a targeted gene segment deletion. CCK8 assays revealed that Quaking gene knockout significantly inhibited NIH3T3 cell proliferation. 【Conclusion】 This study represents the first successful utilization of CRISPR/Cas9 technology to establish a Quaking gene knockout cell line in mouse embryonic fibroblast cells（NIH3T3）, providing a valuable in vitro model for exploring the mechanistic role of the Quaking gene in the regulation of mouse physiological functions.

Construction of Pmepa1 Knockout TCMK1 Mouse Renal Tubular Epithelial Cell Line Using CRISPR/Cas9 Technology

ZHANG Hong-min, LONG Wen, LAO Xiao-qing, CHEN Wen-yan, SHANG Xue-mei, WANG Hong-lian, WANG Li, SU Hong-wei, SHEN Hong-ping, SHEN Hong-chun

2024, 40(2):  73-79.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0500

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【Objective】 CRISPR/Cas9 technology was used to construct Pmepa1 knockdown TCMK1 mouse renal tubular epithelial cell lines and to explore the effect of Pmepa1 knockdown on TGF-β-stimulated TCMK1 cell fibrosis, which provides a cell model for studying the role of Pmepa1 in fibrosis models. 【Method】 The pX333 vector was created and transfected into TCMK1 cells in accordance with the CRISPR/Cas9 design principle. Flow sorting of mCherry-positive cells, monoclonal cell expansion, and sequencing were used to identify the Pmepa1 knockout cells, and Western blot was used to confirm the knockout status of the Pmepa1 gene. Pmepa1 knockdown was confirmed using a Western blot. By using RT-PCR and Western blot, the effects of Pmepa1 knockdown on TGF-1-induced R-Smad activation and fibrosis were examined.【Result】 The sgRNA was designed in exon 2 of Pmepa1 48 h after transfection of TCMK1 cells with pX333-Pmepa1 plasmid, the expression of mCherry fluorescent protein was observed, and flow cytometric analysis of the transfection efficiency was about 17%. pX333-Pmepa1 plasmid transfection-positive cells were cultured by flow sorting and monoclonal amplification to obtain 19 cell clones. PCR amplification sequencing analysis of the sgRNA target sequence at the PMEAP1 gene locus revealed 2 double allele shift mutant cells, and Western blot verified the absence of Pmepa1 protein expression, indicating that the Pmepa1 knockdown TCMK1 cell line was successfully constructed, compared with the non-knockdown cells, the Pmepa1 knockdown cells were stimulated by TGF-β, p-Smad2 and p-Smad3 expression was significantly elevated in Pmepa1 knockout cells compared with non-knockout cells, and the expressions of the fibrogenic genes Fibronectin and Collagen I were promoted. 【Conclusion】 CRISPR/Cas9 technique was used to construct the renal tubular epithelial cell line of PMEPA1 knockout TCMK1 mice successfully, which established the cell model for the functional study of PMEPA1 protein and the important role of PMEPA1 in fibrosis.

Establishment and Application of Multiplex Droplet Digital PCR for SARS-CoV-2 Omicron Variant

ZHENG Qiao, LIN Hua, XU Hao, AN Wei, XUE Chang-hua, ZHANG Jing, HAN Guo-quan

2024, 40(2):  80-89.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0758

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【Objective】 This research is aimed to establish an accurate and efficient multiple droplet digital PCR（ddPCR）quantitative analysis detection method, and to develop a detection system that can simultaneously identify severe acute respiratory syndrome coronavirus（SARS-CoV-2）ORF1ab gene, N gene, E gene, and Omicron variant S gene, so as to improve the diagnostic efficiency of viral infectious diseases and the ability to monitor the risk of transmission. 【Method】 Based on it, conserved gene sequences were screened, specific primers and probes were designed, reaction systems and amplification procedures were optimized, and the specificity, sensitivity and stability of the method were evaluated. With clinical samples as experimental materials, the established ddPCR method was applied for testing and verification to determine the positive detection rate.【Result】 Each pair of primers effectively amplified the target fragment in the multiple ddPCR reaction system. The lower detection limits for SARS-CoV-2 ORF1ab gene, N gene, E gene, and Omicron variant S gene were 0.59, 0.68, 1.44 and 1.03 copies/µL, respectively. In the nucleic acid tests of 20 clinical samples, a total of 16 positive samples were detected, with a positive rate of 80%（16/20）. The coincidence rate was consistent with that of retesting by fluorescence quantitative PCR method. 【Conclusion】 The multiple ddPCR method established in this study is highly specific and sensitive, and can be used to accurately quantify trace amounts of SARS-CoV-2 in clinical samples.

Functional Analysis of Rice Heat Shock Transcription Factor HsfA2b Regulating the Resistance to Abiotic Stresses

ZOU Xiu-wei, YUE Jia-ni, LI Zhi-yu, DAI Liang-ying, LI Wei

2024, 40(2):  90-98.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0908

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【Objective】 Rice is often affected by various abiotic stresses during growth and development, which severely restricts rice yield. Heat shock transcription factors（HSFs）, as an important element of resistance to abiotic stresses in plant, can enhance plant resistance to abiotic stresses by regulating the expressions of a series of stress related genes. This study aims to investigate the function and mechanism of rice heat shock transcription factor OsHsfA2b regulating abiotic stresses, which may provide excellent gene resource and theoretical support for cultivating new rice varieties with stress resistance.【Method】 We constructed OsHsfA2b-overexpressed and RNAi rice transgenic plants, then their seedlings were treated with high temperature, low temperature, drought, and high salt, respectively, and their phenotypes were observed and survival rates were counted. The detection of reactive oxygen species（ROS）deposition by DAB staining, and expression levels of antioxidant pathway related genes OsSOD and OsCAT with RT-qPCR after abiotic stress treatment, aiming to analyze the regulatory effect of OsHsfA2b on the antioxidant pathway.【Result】 The rice HSF gene OsHsfA2b was induced significantly by abiotic stress conditions, such as high temperature, low temperature, drought, and high salt. Compared with the wild-type plants, OsHsfA2b-overexpressed transgenic plants significantly enhanced the resistance to abiotic stresses, as well as survival rate, and less damage. On the contrary, both resistance and survival rate of OsHsfA2b-RNAi plants to abiotic stresses decreased, and the plants were severely damaged. Moreover, the ROS deposition in OsHsfA2b-overexpressed plants decreased compared with NPB and RNAi plants under abiotic stress. Correspondingly, OsHsfA2b induced the expressions of antioxidant pathway related genes OsSOD and OsCAT, suggesting that OsHsfA2b suppressed ROS accumulation to reduce its damage caused by abiotic stress induction.【Conclusion】 The above results indicate that OsHsfA2b is induced by abiotic stresses and positively regulates rice resistance to abiotic stresses through antioxidant pathways, and it is an excellent gene resource for rice stress-resistance breeding.

Functional Study of Vitamin B₁ Synthesis-related Gene OsTHIC in Rice

ZHANG Chao, WANG Zi-rui, SUN Ya-li, MAO Xin-chen, TANG Jia-qi, YU Heng-xiu

2024, 40(2):  99-108.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0797

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【Objective】 Vitamin B₁ (VB₁) is a biologically essential micronutrient that acts as a cofactor for several enzymes and is involved in important cellular metabolic pathways. However, the synthesis pathway of vitamin B₁ in rice remains elusive. The aim of this study is to elucidate the biological functions of OsTHIC, a gene related to vitamin B₁ synthesis in rice.【Method】 We used a combination of mutagenesis techniques, pigment measurement, Mutmap+, gene editing, and non-targeted metabolomics analysis to clone the target gene and analyze its biological functions.【Result】 An albino lethal mutant, wll1 (white leaf and lethal 1), was obtained from EMS-induced mutant pool of japonica rice variety Nipponbare. The fourth leaf of wll1 had albino phenotype and the albino phenotype spread to other leaves, ultimately leading to seedling death. The photosynthetic chlorophyll and carotenoid contents of the wll1 mutant was significantly lower than that of the wild type (WT). The target gene OsTHIC involved in VB₁ synthesis was determined by MutMap+ and gene editing technology. OsTHIC gene was expressed in various tissues of rice, with the highest expression in the leaves and OsTHIC protein was localized in chloroplasts. The VB₁ content in the wll1 was significantly lower than that of the WT. Exogenous VB₁ restored the mutated phenotype of wll1. Exogenous VB₁ and OsTHIC mutation both affected the expressions of genes related to VB₁ synthesis. Non-targeted metabolome sequencing analysis showed that differential metabolites between wild type and wll1 were significantly enriched in amino acid biosynthesis, cofactor biosynthesis, ABC transporters, and aminoacyl-tRNA biosynthesis pathways.【Conclusion】 OsTHIC plays an important role in the growth and development of rice by regulating the content of VB₁ and participating in metabolic processes such as amino acid synthesis.

The Growth Promoting Effect of Bacillus amyloliquefaciens SQ-2 on Rice

LI Xue, LI Rong-ou, KONG Mei-yi, HUANG Lei

2024, 40(2):  109-119.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0770

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【Objective】 To study the growth-promoting characteristics of a strain of Bacillus amyloliquefaciens SQ-2 isolated and screened in the laboratory, and to determine the concentration range, mechanism of action, and changes in soil microbiota induced by this strain to promote rice growth.【Method】 The phosphorus solubility and nitrogenase activity of strain SQ-2 were detected using the molybdenum blue colorimetric method and ELISA nitrogenase kit. Inoculating10², 10⁴, 10⁶, 10⁸, and 3×10⁸ CFU/mL bacterial suspension into hydroponic and soil cultured rice, the dry and fresh weight, length, and thickness of the roots and stems of hydroponic and soil cultured rice were measured, respectively after 14 and 20 d of cultivation. Meanwhile, the activities of urease, protease, and sucrase in soil cultured rice were determined using the phenol sodium hypochlorite colorimetric method, the ninhydrin detection method, and the 3,5-dinitrosalicylic acid colorimetric method. Besides, the soil pH value was determined using the pH meter potential method and the content of nitrogen, phosphorus, and potassium available in soil and the activity of acid phosphatase were detected by utilizing the corresponding reagent kit. Analysis of rhizosphere bacterial community structure in soil cultured rice soil was inoculated with 3×10⁸ CFU/mL components.【Result】 The dissolution of tricalcium phosphate by strain SQ-2 was 229.63 mg/L, and the nitrogenase activity was 55.07 U/L. Compared with the control, the dry and fresh weight of hydroponic rice roots showed the most significant increase at an inoculum suspension concentration of 10⁴ CFU/mL. At a vaccination concentration of 3×10⁸ CFU/mL, the dry and fresh weight of soil cultivated rice rhizomes increased most significantly. As the concentration of the inoculum suspension increased, the activities of various enzymes and the concentration of available nitrogen, phosphorus, and potassium in the soil increased to varying degrees, the soil pH value while inoculating strain 3×10⁸ CFU/mL decreased from 7.83 to 7.26. The inoculant strain SQ-2 changed the colony composition in the rice rhizosphere soil and significantly increased Ace, Chao, Sobs and Shannon indices of soil α-diversity.【Conclusion】 B. amyloliquefaciens SQ-2 had different growth promotion effects on soil culture and hydroculture rice. In soil culture experiments, the growth-promoting effect is notably pronounced through the augmentation of soil enzyme activity, rapid increases in available nitrogen, phosphorus, and potassium levels, and alterations in the soil microbial community structure. Overall, these experimental findings offer valuable insights into new strain resources for the development of bacterial fertilizer.

Identification of SSR Loci and Development of Polymorphic Markers in Whole Genome of Oat

CHEN Kai-ling, WU Tao, XU Yi-qun, GAO Jia, ZHANG Mei-jun, LI Xin, JIA Ju-qing

2024, 40(2):  120-129.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0697

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【Objective】 Due to the limited number of SSR markers developed in oat, it is difficult to meet the needs of functional gene mapping and polymorphism marker development in oat.【Method】 Using the published reference genome, the SSR loci of the whole oat genome were retrieved by TBtools software, and their distribution characteristics were analyzed by Excel and SPSS software.【Result】 The SSR was abundant and varied in oat genome. A total of 828 138 SSR loci were identified in the whole genome, with an average density of 78.16 SSR/Mb and an average distance of 12.90 kb. Single, double and trinucleotide repeat types were dominant. The repetitions of the primitives were mainly 5 and 6, and the A/T, AG/CT and AAC/GTT repetitions were the dominant primitives. The SSR loci of oat genome showed moderate polymorphism, with length ranging from 11 to 1 587 bp. Through the systematic identification and analysis of the whole oat gene SSR locus, 52 pairs of polymorphic primers were developed and validated in the test material.【Conclusion】 The development of SSR primers in the reference genome of “Sanfensan” naked oat is effective and feasible, and can be used for further experimental research.

Genome-wide Identification of HD-Zip Gene Family in Gossypium hirsutum L. and Expression Analysis in Response to Abiotic Stress

WU Cui-cui, XIAO Shui-ping

2024, 40(2):  130-145.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0869

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【Objective】 HD-Zip（Homeodomain and Leucine zipper）family is one of the plant-specific transcription factor families, which plays an important role in plant growth and development and stress response. In this study, the members of GhHDZ gene family were identified from the whole genome of Gossypium hirsutum L. and the expression characteristics of related genes were analyzed to provide support for further research. 【Method】 The members of GhHDZ gene family were identified from TM-1 genome of G. hirsutum L. by bioinformatics, and their physicochemical properties, phylogenetic relationship, chromosome location, gene replication, gene structure and cis-acting elements in promoter region were analyzed. Transcriptome data and real-time quantitative polymerase chain reaction（RT-qPCR）were used to analyze the expression patterns of GhHDZ family genes under different abiotic stresses. The subcellular localization of the target protein was detected by tobacco transient transformation method, and the function of the target gene was verified by transgenic overexpression method. 【Result】 A total of 205 GhHDZ genes were identified in upland cotton genome, and the GhHDZ family was divided into 4 subgroups by phylogenetic analysis. Fragment replication was the main reason for the evolution of the GhHDZ gene family, and the gene family has undergone a strong purification selection. GhHDZ family members had potential diversity of biological functions. The analysis of abiotic stress transcriptome combined with RT-qPCR analysis of 10 GhHDZ genes showed that GhHDZ12, GhHDZ46 and GhHDZ119 positively responded to cold stress, GhHDZ15 and GhHDZ188 negatively responded to cold stress, GhHDZ15, GhHDZ46, GhHDZ50, GhHDZ76, GhHDZ116, GhHDZ146, GhHDZ176, GhHDZ188 positively responded to heat stress, and GhHDZ15, GhHDZ50, GhHDZ76, GhHDZ116, GhHDZ119, GhHDZ146, GhHDZ176, GhHDZ188 positively responded to salt stress. GhHDZ12, GhHDZ15, GhHDZ50, GhHDZ116, GhHDZ119, GhHDZ176, GhHDZ188 positively responded to drought stress, while GhHDZ76 negatively responded to drought stress, and it was found that the response times of these genes to various stresses were different. Further study on the function of GhHDZ146 showed that the gene was localized in the nucleus and heterologous overexpression in Arabidopsis, which significantly enhanced the tolerance to salt stress. 【Conclusion】 205 members of GhHDZ gene family are systematically identified from G. hirsutum L. within the whole genome. Different genes have different responses to various stresses, and there are differences in expression patterns. The positive response function of GhHDZ146 to salt stress is confirmed.

Cloning and Functional Analysis of MbbZIP43 Gene in ‘Hongmantang’ Red-flesh Apple

YANG Yan, HU Yang, LIU Ni-ru, YIN Lu, YANG Rui, WANG Peng-fei, MU Xiao-peng, ZHANG Shuai, CHENG Chun-zhen, ZHANG Jian-cheng

2024, 40(2):  146-159.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0676

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【Objective】 To investigate the regulatory role of basic leucine zipper（bZIP）transcription factors in anthocyanin metabolism in ‘Hongmantang’ apple.【Method】 We cloned a bZIP gene from ‘Hongmantang’ red-flesh apple, analyzed it and its sequence characteristics, studied the subcellular localization of its encoded protein using onion epidermis cell transient overexpression, and investigated its expression pattern in ‘Hongmantang’ red-flesh apple fruits at different ripening stages using quantitative real time PCR（RT-qPCR）. Furthermore, we explored its regulatory roles in the expressions of genes related to anthocyanin accumulation and biosynthesis based on the transient overexpression experiments in tobacco leaves, apple leaves and fruits.【Result】 This bZIP gene was intron-less and shared the highest similarity with MdbZIP43（XP_008393381.1）, thus was named as MbbZIP43. Its coding sequence was 522 bp, which encoded a 173 aa hydrophilic nucleus-localized protein with conserved bZIP_plant_GBF1 domain. RT-qPCR analysis revealed that MbbZIP43 showed a ‘rise-fall’ expression change pattern in ‘Hongmantang’ fruits during ripening, with the highest expression in the fruits at 11 weeks post flowering. Correlation analysis results showed that MbbZIP43 expression was positively correlated with the total flavonoids and anthocyanin contents in ‘Hongmantang’ fruits, indicating that it might play regulatory roles in flavonoid biosynthesis. Consistently, transient overexpression of MbbZIP43 improved significantly the anthocyanin accumulations by 17.42%, 25.66% and 48.99% in tobacco leaves, apple leaves and apple fruits, respectively. Moreover, RT-qPCR analysis results showed that, compared to the empty vector control, the expressions of anthocyanin biosynthesis related structural genes（CHI, F3'H, DFR and UFGT）in the apple leaves overexpressing MbbZIP43 were up-regulated by 2.27-, 1.84-, 2.39- and 2.89-fold, respectively. And their expressions in the peels of apple fruits overexpressing MbbZIP43 were up-regulated by 1.79-, 1.70-, 1.35- and 1.51-fold, respectively. These results indicated that the gene's transient overexpression may improve anthocyanin accumulation by activating the expressions of the anthocyanin biosynthesis related structural genes.【Conclusion】 MbbZIP43 can promote anthocyanins accumulation by activating the expression of anthocyanin biosynthesis related structural genes, including CHI, UFGT and so on.

Identification and Analysis of Grape（Vitis vinifera L.）CYP707A Gene Family and Functional Verification to Fruit Ripening

GONG Li-li, YU Hua, YANG Jie, CHEN Tian-chi, ZHAO Shuang-ying, WU Yue-yan

2024, 40(2):  160-171.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0856

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【Objective】 CYP707A is a subfamily member of cytochrome P450 family, encoding ABA8'-hydroxylase, which is a key enzyme in ABA catabolism and plays an important role in plant growth and development. Identification of grape（Vitis vinifera L.）CYP707A gene family may provide theoretical basis for further study of grape fruit ripening mechanism and biological breeding techniques.【Method】 Bioinformatics methods were used to analyze the gene structure, protein motif, chromosome localization, collinearity, phylogenetic relationship and cis-acting elements of promoter region of CYP707A family gene in the grape. RT-qPCR was used to analyze the spatiotemporal expression patterns of VvCYP707As gene in tissues, different grape varieties（early and medium maturity variety）and in response to the exogenous ABA treatment were analyzed by. Fruit transient transfection was applied to verify the function of Vitvi07g01751.【Result】 There were five VvCYP707As genes identified in grape genome, located on five chromosomes, among which Vitvi02g01269, Vitvi07g01751 and Vitvi18g00792 were fragment replicators. The CYP707A gene family had a high degree of conservation, sharing similarities in gene structure and protein motifs, and all subcellular localization were predominantly localized to the endoplasmic reticulum. The cis-acting elements of growth, development and hormone response were significantly enriched in the promoter regions of the CYP707A gene family, containing ABA hormone-corresponding elements. Furthermore, the RT-qPCR analysis demonstrated that VvCYP707A genes demonstrated both spatiotemporal- and tissue-specific expression patterns, with elevated expression levels during the early and expansion stages of fruit development as well as in stem and leaf tissues. Notably, Vitvi07g01751 gene was highly expressed in the fruits of the early maturity variety ‘Yong Zaohong’, and the expression pattern was consistent between these two varieties. The expression pattern of Vitvi07g01751first increased and then decreased with fruit development. Under exogenous ABA treatment, VvCYP707A showed two distinct expression patterns: it induced the high expression of 5 VvCYP707A genes in the grape flesh while significantly suppressing the expressions of these genes in the grape pericarp. Furthermore, upon transient expression of Vitvi07g01751 in the grape flesh, there was a slow accumulation of sugar content, accompanied by a significant upregulation of the ABA receptor VvPYL4 gene, suggesting that Vitvi07g01751 mediated ABA regulation of grape fruit ripens.【Cconclusion】Vitvi07g01751 plays an important role in fruit ripening of grape.

Identification of the Kiwifruit BBX Gene Family and Analysis of Their Transcriptional Characteristics

LU Yu-dan, LIU Xiao-chi, FENG Xin, CHEN Gui-xin, CHEN Yi-ting

2024, 40(2):  172-182.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0812

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【Objective】 BBX（B-Box）, as one of the subfamilies of zinc finger transcription factors, is widely involved in the plant growth and development process. To reveal the genetic characteristics and potential functions of the kiwifruit BBX family.【Method】 The kiwifruit BBX gene family was identified at the whole genome level. And the protein physicochemical properties, subcellular localizations, systemic developmental characteristics, conserved protein domains, and homologous characteristics were analyzed. Meanwhile, the expression patterns in different tissues and different storage treatments were analyzed.【Result】 A total of 48 AcBBX genes were unevenly distributed across 22 chromosomes. The amino acid length of AcBBXs was 126-515 aa, protein molecular weight was 14 172.09-57 905.84 Da, and four subcellular localizations were predicted. There were 0 to 4 introns in AcBBX family. AcBBX promoters contained various cis-acting elements involved in light responsiveness, plant hormone regulatory, biotic and abiotic stress regulatory, growth and development regulatory. Combined the evolutionary tree with conserved domains, they could be clustered into five groups, and gene tandem duplication and fragment replication were the main reasons for the expansion of family members. The expressions of AcBBXs varied in different organs of kiwifruit. Seven AcBBX members had higher expressions in the mature fruits compared to other parts, 3 members having higher expressions in the young fruits, 7 members having higher expressions in the leaves, and 3 members having higher expressions in the flowers. During the post-harvest storage of fruits, 3 members were upregulated by exogenous hormones and higher than other processing groups, while 6 members were upregulated after one week of storage at room temperature and higher than other processing groups. During 4℃ storage of fruits, 10 members were upregulated, while 5 members were downregulated.【Conclusion】 This study reveals the transcriptional characteristics of different members of the kiwifruit AcBBX family during its growth, development, and post-harvest storages, laying the foundation for subsequent functional verification of the AcBBX gene.

Identification and Function Analysis of AcMYB88 in Kiwifruit （Actinidia chinensis）

YUAN Xin-yu, ZHONG Cai-hong, ZHANG Long, ZHENG Hao, LI Ji-tao, ZHANG Qiong

2024, 40(2):  183-196.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0726

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【Objective】 MYB transcription factors play an important role in anthocyanin accumulation of kiwifruit. Identifying MYB transcription factors that regulate anthocyanin biosynthesis and verifying gene functions facilitate elucidating the molecular mechanism of anthocyanin accumulation.【Method】 It was reported that AcMYB110 was a key transcription factor promoting anthocyanin accumulation in kiwifruit. In PlantTFDB database, 12 MYB genes that showing high homology to AcMYB110 were identified from 97 kiwifruit MYB genes by phylogenetic tree analysis. Bioinformatics methods were employed to analyze their physical and chemical properties, hydrophilicity and hydrophobicity, protein phosphorylation sites, protein secondary structures, and phylogenetic relationships with other species.【Result】 The 12 MYB transcription factors encoded amino acids ranging from 200 to 423; their molecular weight ranged from 22.3 to 45.5 kD, and all of them were hydrophilic proteins located in the nucleus. The majority of potential phosphorylation sites in the 12 candidate MYB proteins were found to be at serine residues. Moreover, their secondary structures were primarily composed of random coils with some α-helices. Among the 12 MYB transcription factors, the expression pattern of AcMYB88 gene was very similar to that of AcMYB110 gene during kiwifruit development.【Conclusion】 Transient overexpression experiments in tobacco demonstrate that AcMYB88 acts as a positive regulator of anthocyanin accumulation, and AcMYB88 synergistically promotes anthocyanin accumulation with AcMYB110 and AcbHLH42. This study provides a theoretical foundation for understanding anthocyanin biosynthesis and breeding research in kiwifruit

Identification and Expression Analysis of the CBL-CIPK Gene Family in Sugarcane

XIN Qi, LI Ya-fan, YIN Zheng, ZHANG Xiao-dan, CHEN Ting, LIU Xiao-hua

2024, 40(2):  197-211.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0830

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【Objective】 Sugarcane is an important sugar crop, and temperature, salinity, water and other factors are the key environmental factors restricting its growth and development. Calcineurin B-Like protein（CBL）family represents a group of Ca²⁺ binding proteins and plays a key role in decoding calcium signals by specifically interacting with and regulating a family of protein kinases CIPKs（for CBL-interacting protein kinases）in abiotic stress signal transduction pathway. At present, the whole genome of sugarcane has been sequenced, but its CPL-CIPK gene family members have not been identified, and the interaction regulation mechanism is still unknown. Here, the members of CBL and CIPK in sugarcane were identified and the CPL-CIPK interaction relationship was revealed, which may provide gene resources and theoretical basis for studying the mechanism of CPL-CIPK interaction in sugarcane.【Method】 The expression levels of CBLs and CIPKs under low temperature, high temperature, NaHCO₃ and PEG were analyzed by RT-qPCR from sugarcane GT58. The interactions between SsCBLs and SsCIPKs were analyzed by yeast two-hybrid assay.【Result】 There were 19 CBL genes and 82 CIPK genes in the whole genome of sugarcane, which distributed in different evolutionary branches with gene replication. The physicochemical properties of gene family members were different, the structural domains and protein motifs were highly conserved, and the distribution of cis-acting elements was diverse. SsCBL7/SsCBL12/SsCIPK1/SsCIPK5 were specifically expressed under low temperature, high temperature, drought and high salt and other abiotic stress regulation. Meanwhile, SsCBL1 interacted with SsCIPK47 and SsCIPK81, and SsCBL8 interacted with SsCIPK47 and SsCIPK81.【Conclusion】 The CBL-CIPK signaling system may play an important role in response to abiotic stress during the growth and development of sugarcane.

Identification of HMA Gene Family and Cadmium Transport Function of SlHMA1 in Tomato

ZHAO Yao, WEN Lang, LUO Shao-dan, LI Zi-xing, LIU Chao-chao

2024, 40(2):  212-222.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0896

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【Objective】 The heavy metal ATPase（HMA）gene family is extensively involved in the absorption and transport of metal elements in plants. Therefore, this study is aimed to systematically identify the members of the tomato HMA gene family and their characteristics, as well as to investigate their functions in response to cadmium stress.【Method】 Tomato HMA gene family members were identified through bioinformatics, and their systematic evolution tree, protein physicochemical properties, gene structure, cis-regulatory elements, and gene expression patterns, etc., were analyzed. Additionally, yeast functional complementation experiments were conducted to validate the Cd²⁺ transport activity of SlHMA1.【Result】 Eight SlHMAs were identified in the tomato genome, categorized into 2 subgroups. Remarkable differences in gene structure were observed among SlHMAs genes and their homologs in Arabidopsis and rice. Notably, the promoter regions of SlHMAs members were found to harbor numerous cis-regulatory elements associated with stress responses, and RT-qPCR analysis also indicated that most SlHMAs genes showed tissue-specific responses to cadmium stress. Furthermore, yeast functional complementation experiments provided conclusive evidence for Cd²⁺ transport activity of SlHMA1, while evolutionary analysis indicated the widespread presence of HMA1 in the plant kingdom, with the motif DKTGT related to ATP hydrolysis activity within HMA1 also being highly conserved across the plant kingdom.【Conclusion】 The eight SlHMAs present typical characteristics of the HMA gene family while also having functional diversity. SlHMAs, along with their conserved amino acid motif DKTGT, are closely associated with metal ion transport and cadmium stress response, offering significant potential for application in the breeding of low cadmium-accumulating crop varieties.

cDNA Yeast Library Construction of Chinese Cabbage Seeds and Screening and Analysis of BrTTG1 Interacting Proteins

REN Yan-jing, ZHANG Lu-gang, ZHAO Meng-liang, LI Jiang, SHAO Deng-kui

2024, 40(2):  223-232.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0989

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【Objective】 Screening the interacting protein of WDR40 protein TRANSPARENT TESTA GLABRA 1（TTG1）by constructing the cDNA library of Chinese cabbage seeds, the molecular mechanism of TTG1 in the regulation of the formation of MBW ternary complex will be explored.【Method】 Seeds of brown-seeded Chinese cabbage ‘B147’ were used as materials for RNA extraction and cDNA library construction. The bait vector pGBKT7-TTG1 was constructed by gateway technology and then yeast two-hybrid screening were performed.【Result】 The library capacity was 1.2×10⁷ CFU and library titer was 5.0×10⁷ CFU/mL with average length of the insert greater than 1 000 bp. The bait vector had no self-activating activity in yeast. A total of 38 positive interacting proteins were obtained by hybridizing the bait vector pGBKT7-TTG1 with the cDNA library. Function prediction revealed one of the proteins annotated as a MYB73 transcription factor. Sequence analysis results showed that this gene contained R2R3-MYB type suppressor conserved motifs C1 and C2, speculated that this gene may be the R2R3-MYB suppressor involving in the seed coat color formation in Chinese cabbage, suggesting that there may be different MYB transcription factors in the regulation network affecting the formation of proanthocyanidins.【Conclusion】 In this study, a yeast two-hybrid cDNA library of Chinese cabbage seed-specific tissue is constructed and a total of 38 TTG1-positive interacting proteins are obtained. We found the R2R3-MYB type suppressor MYB73 that may affect the formation of proanthocyanidin color in cabbage seed coat for the first time. This study lays a good foundation for exploring the regulatory network of proanthocyanindins formation.

Genome-wide Identification of the BvBADH Gene Family in Sugar Beet（Beta vulgaris）and Their Expression Analysis Under High Salt Stress

LI Hao, WU Guo-qiang, WEI Ming, HAN Yue-xin

2024, 40(2):  233-244.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0874

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【Objective】 Betaine aldehyde dehydrogenase（BADH）is one of the key enzymes involved in glycine betaine synthesis and plays an important role in plant response to abiotic stress.【Method】 To explore the biological function and gene expression pattern of BvBADH gene family members in sugar beet（Beta vulgaris）, the physicochemical properties of protein, phylogenetic evolution, gene structure, chromosome localization, conserved motif, protein structure, and promoter cis-acting regulatory elements of BvBADHs were analyzed by bioinformatics method, and their expression patterns under salt stress were investigated by RT-qPCR.【Result】 A total of 9 BvBADH gene family members were identified. They contained 9-15 exons and 8-14 introns, the average amino acid number was 516, the average molecular weight was 55.84 kD, and the isoelectric point was 5.24-6.98. Phylogenetic analysis found that BADHs of higher plants were divided into three clusters, namely Clusters I, II, and III, among which Cluster III members can be further divided into two subgroups, including Cluster IIIa and IIIb. The members of BvBADH gene family were distributed in 3 clusters, respectively containing 3, 1, and 5 members. The promoter of the BvBADH gene mainly contained stress response elements, hormone response elements and growth and development response elements, the cis-acting regulatory element numbers of each member were 13-21. Further analysis of the expression pattern of the BvBADH genes in the sugar beet leaves showed that both 100 and 150 mmol/L NaCl significantly increased the expressions of all members of BvBADHs, but their expressions under 100 mmol/L NaCl were higher than those under 150 mmol/L NaCl.【Conclusion】 The BvBADH gene family plays an important role in sugar beet's response to salt stress, it provides excellent genetic resources for the genetic improvement of crop stress tolerance in northern China.

Cloning of the LkF3H2 Gene in Larix kaempferi and Its Function in Regulating Flavonoid Metabolism

LI Can, JIANG Xiang-ning, GAI Ying

2024, 40(2):  245-252.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1508

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【Objective】 The Larix kaempferi flavanone 3-hydroxylase 2（LkF3H2）gene in Larix kaempferi plays a crucial role in flavonoid biosynthesis. This study is aimed to investigate the function of the LkF3H2 in L. kaempferi and plant flavonoid biometabolism. 【Method】 The LkF3H2 gene of L. kaempferi was cloned based on preliminary transcriptome data, and bioinformatics analysis and tissue expression analysis were performed. The gene was then stably transformed into tobacco to examine the relationship between gene expression and flavonoid content using tissue expression analysis and flavonoid content determination. 【Result】 The cDNA sequence length of LkF3H2 gene is 1 074 bp, and its protein encodes 358 amino acids with a molecular formula of C₁₇₈₅H₂₈₀₇N₄₈₁O₅₄₁S₁₈ and a molecular weight of 40.24 kD, indicating that it is an unstable hydrophilic protein lacking a signal peptide. Moreover, phylogenetic analysis revealed that LkF3H2 is closely related to the F3H genes of Pinus taeda, Pinus radiata, Picea abies, and Picea glauca. In addition, the LkF3H2 gene had the highest expression in the leaves of one-month-old L. kaempferi seedlings and the stems of one-year L. kaempferi seedlings, as determined by tissue expression analysis. Moreover, it had the highest expression in the transgenic tobacco leaves; notably, transgenic tobacco leaves had the highest flavonoid content, followed by stems and roots, indicating a positive relationship between flavonoid content and LkF3H2 gene expression in transgenic tobacco. 【Conclusion】 The LkF3H2 gene of L. kaempfer belongs to the 2-oxoglutarate-dependent dioxygenase family and is pivotal in flavonoid biosynthesis.

Regulation of Nitrogen Application on Peanut Seed Germination and Spermosphere Bacterial Community Structure Under Salt Stress

XU Yang, ZHANG Rui-ying, DAI Liang-xiang, ZHANG Guan-chu, DING Hong, ZHANG Zhi-meng

2024, 40(2):  253-265.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0753

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【Objective】 Salt stress affects seed germination and peanut growth, and the internal regulatory mechanism of increasing seed germination rate and peanut yield by appropriate fertilization under salt stress was elucidated, and the relationship between the process and the bacterial structure of peanut spermosphere soil was analyzed, so as to provide theoretical and technical basis for improving peanut emergence and health rate, salt tolerance and stress resistance and peanut production capacity by regulating the microenvironment in spermosphere soil.【Method】 We set up three nitrogen levels of 0, 90, and 180 kg/hm² with a salt-tolerant peanut variety（Huayu 25, HY25）as experimental materials. Potted experiments and high-throughput sequencing techniques were used to study the effects of nitrogen fertilizer application on the spermosphere bacterial community structure, germination and yield of peanut under salt stress.【Result】 The application of nitrogen fertilizer increased the germination rate and peanut yield, and the optimal nitrogen application was 90 kg/hm². Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, Bacteroidetes, Chloroflexi, and Gemmatimonadetes were the dominant phyla in peanut spermosphere soil of different groups via 16S rRNA sequence. At the genus level, salt stress increased the relative abundance of beneficial bacteria Bacteroides, but induced a large number of harmful Streptococcus, whereas the relative abundance of the beneficial bacteria genera Bacillus, Sphingomonas, and Lysobacter decreased. Nitrogen application under salt stress may improve the spermosphere soil microenvironment and increase the relative abundance of beneficial soil bacteria Bacteroides, Bacillus, and Sphingomonas, which is helpful to soil restoration and fertility improvement, and can also enhance the stress resistance of peanuts.【Conclusion】 Appropriate nitrogen application under salt stress increases the relative abundances of beneficial bacteria in the spermosphere soil, thus which may improve the germination rate and salt tolerance, and finally increase the peanut yield under salt stress.

Bacterial Diversity and Structure in Rhizosphere Soil of Citrus Infested with Huanglongbing

LEI Mei-ling, RAO Wen-hua, HU Jin-feng, YUE Qi, WU Zu-jian, FAN Guo-cheng

2024, 40(2):  266-276.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0837

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【Objective】 This study aims to investigate the rhizosphere soil bacterial community of Citrus reticulata plants in the Nanping region of Fujian province, China. The objective is to analyze the impact of Huanglongbing（HLB）disease on the bacterial community and provide a scientific basis for HLB management strategies in the Nanping region.【Method】 We collected root-zone soil samples from both healthy and HLB-affected C. reticulata plants using the five-point sampling method. We used amplicon sequencing technology and bioinformatics methods to investigate the changes in diversity and composition of the bacterial community in the root-zone soil of HLB-affected C. reticulata plants. Additionally, we analyzed the correlation between soil physicochemical factors and the bacterial community.【Result】 The findings of the study demonstrate that the diversity of the bacterial community in the root-zone soil of C. reticulata plants affected by HLB was slightly higher when compared to the microbiota of healthy plants. Specifically, there was a significant increase in the relative abundance of the Proteobacteria, and a significant decrease in the relative abundance of the Actinobacteriota in the root-zone soil of HLB-affected C. reticulata plants. At the genus level, it was observed that the relative abundance of the Roseiarcus and Acidiphilium significantly increased in the bacterial community of HLB-affected C. reticulata plants. Furthermore, these genera presented a significant negative correlation with available potassium, available phosphorus, and organic matter, while showing a significantly positive correlation with pH values. Conversely, the relative abundance of the Conexibacter and Chujaibacter were significantly lower in HLB-affected plants than healthy ones. Additionally, these genera demonstrated a significantly positive correlation with available potassium, available phosphorus, and organic matter, but a significant negative correlation with pH values.【Conclusion】 The infection of the HLB pathogen altered the physicochemical properties of citrus soil and significantly reduced the abundance of beneficial bacteria in the rhizosphere soil. This, in turn, leads to considerable changes in both the diversity and composition of the rhizosphere bacterial community in C. reticulata plants. Concurrently, HLB-infected C. reticulata plants appear to counteract pathogen invasion by recruiting beneficial bacteria that not only fix nitrogen but also promote plant growth in the rhizosphere.

Identification of Three Strains of Bacillus from the Forest Soil of Wuliang Mountain and Mining of Their Bioactivities

WANG Nan, LIAO Yong-qin, SHI Zhu-feng, SHEN Yun-xin, YANG Tong-yu, FENG Lu-yao, YI Xiao-peng, TANG Jia-cai, CHEN Qi-bin, YANG Pei-wen

2024, 40(2):  277-288.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0918

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【Objective】 In order to provide efficient microbial resource for the development of green agricultural inputs, we identified potential high-activity microbial strains from the National Wilderness Preservation System of Mount Wuliangshan.【Method】 We used Fusarium oxysporum as a target to screen highly antagonistic strains, and observed the effect on hyphae growth and spore germination. We also tested the lipopeptide synthesis genes and the ability of the strain to produce enzymes, hydrolyze phosphorus, hydrolyze potassium, fix nitrogen and produce siderophore. In addition, the strains were identified according to their morphological, physiological and biochemical characteristics and 16S rRNA, gyrA and rpoB genes, and the effects of disease control and growth promotion were verified by pot experiment.【Result】 A total of 153 strains of culturable bacteria were isolated and screened. Among them, the inhibition rates of SH-53, N4471 and N9456 were 92.35%, 87.29% and 88.47%, respectively, it showed good antagonistic activity against many pathogens, and also inhibited the hypha growth and conidia germination of the pathogen. The three functional strains all had the abilities of dissolving zinc, producing amylase and protease, and the strains Sh-53 and N4471 also had the abilities of dissolving phosphorus, fixing nitrogen, secreting cellulase and producing siderophore. In addition, we found that srfA, fenA, ituA, ituC, ituD and bymC genes were found in the genomes of the three functional bacteria. Strain SH-53 was identified as Bacillus amyloliquefaciens, N4471 as B. cabrialesii, and N9456 as B. siamensis. The results of pot experiment showed that the control effects of the three functional bacteria were 84.66%, 54.96% and 59.74%, respectively. They promoted the agronomic characters of tomato seedlings, such as plant height, stem diameter and root length.【Conclusion】 As high-efficiency microbial resources, the three strains have broad-spectrum antibacterial activity, diverse biological activity, and have broad application prospects in the development of green agricultural inputs.

Characterization and Functional Analysis of Lytic Polysaccharide Monooxygenase TtLPMO9I from Thermothelomyces thermophilus

ZHENG Fei, YANG Jun-zhao, NIU Yu-feng, LI Rui-lin, ZHAO Guo-zhu

2024, 40(2):  289-299.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0877

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【Objective】 To explore a novel lytic polysaccharide monooxygenase（LPMO）enzyme and to elucidate its crucial role in cellulose degradation.【Method】 A new LPMO enzyme TtLPMO9I was cloned from Thermothelomyces thermophilus genome, and its sequence and structure were systematically analyzed to determine its evolutionary characteristics. The enzymatic properties of TtLPMO9I were characterized using the DNS method; the impact of different concentrations of ascorbic acid on the activity of TtLPMO9I was investigated by adding external electron donors to the reaction system. By detecting the production of reducing sugars, the synergistic effect between TtLPMO9I and cellulase was calculated when pretreated corn straw and Avicel were used as substrates. 【Result】 TtLPMO9I presented the highest activity at 60℃ and pH 5.0. After 12 h of incubation at 60℃, approximately 54% of its activity remained. TtLPMO9I still maintained initial enzyme activity at pH 6.0-8.0 for up to 12 h. Adding an external electron donor, ascorbic acid, to the reaction system, the activity of TtLPMO9I increased to 184%. In addition, TtLPMO9I has shown promising synergistic effects with cellulase in the degradation of pretreated corn straw and Avicel. Adding 50-200 μg of TtLPMO9I into the degradation system increased reducing sugar yields by 34%-142% and 6%-46% respectively. 【Conclusion】 TtLPMO9I not only has excellent thermal and pH stability, but also has outstanding performance in degrading lignocellulose, providing potential high-quality enzyme resources for industrial production.

Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis

LI Ya-nan, ZHANG Hao-jie, LIANG Meng-jing, LUO Tao, LI Wang-ning, ZHANG Chun-hui, JI Chun-li, LI Run-zhi, XUE Jin-ai, CUI Hong-li

2024, 40(2):  300-312.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0719

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【Objective】 Calcium-dependent protein kinases（CDPK or CPK）are widely involved in plant growth and development and environmental stress response. To explore their roles involved in Haematococcus pluvialis growth, development and astaxanthin accumulation, HpCDPK gene family was identified and analyzed. 【Method】 Bioinformatics tools were used to identify the HpCDPK gene family members, and to analyze the physical and chemical properties, phylogenetic evolution, conserved motifs and functional domains of the encoded proteins, as well as the gene expression profiles under nitrogen deficiency stress. 【Result】 The results showed that seven members of HpCDPK gene family were identified, and all HpCDPK proteins contained typical EF-hand motifs and protein kinase domains. Phylogenetic analysis showed that these HpCDPKs clustered with the CrCDPKs from Chlamydomonas reinhartii and separated from the AtCDPKs of Arabidopsis thaliana, suggesting that species-specific gene replication occurred in the process of evolution. Transcriptome data analysis showed that HpCDPK gene expression was induced by a variety of stresses, among which the transcription level of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency. In addition, the analysis of the correlation between HpCDPK and the expression of genes related to carotenoid synthesis showed that HpCDPK was closely related to the expressions of genes relevant to several carotenoid synthesis in H. pluvialis, especially HpCDPK2 was positively correlated with the expression of key genes of astaxanthin synthesis（BKT and BCH）. 【Conclusion】 Seven HpCDPK genes were identified, the expression of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency and HpCDPK2 may play an active role in the synthesis and accumulation of carotenoids and astaxanthin. The results provide a reference basis for further analysis of the function and molecular mechanism of stress response and carotenoids synthesis and accumulation mediated by HpCDPK in H. pluvialis.

Trait-regulated-genes Ontology Model Construction and Application by Integrating Cross-species Scientific Data

ZHANG Dan-dan, ZHAO Rui-xue, XIAN Guo-jian, XIONG He

2024, 40(2):  313-324.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0748

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【Objective】 With the proliferation of breeding data brought by new technologies and the new demand for knowledge services in computational breeding, in order to solve the problem of inefficient cross-species subject knowledge acquisition and difficult discovery of elite pleiotropic genes in crop breeding knowledge service.【Method】 We constructed trait-regulated-genes ontology model framework, and defined entity hierarchical structure and entity attributes in ontology model. Taking staple crops rice, corn, wheat and model plant Arabidopsis thalliana as data collection objects, a knowledge graph with trait-regulated-genes ontology model as model layer is constructed and experimented.【Result】 Finally, the trait-regulated-genes ontology model covering 13 entities, 16 data attributes and 14 object attributes was formed. This model is used as the knowledge graph of ontology layer to realize cross-species subject knowledge association retrieval, mining of elite pleiotropic genes and prediction of gene function across species.【Conclusion】 The method of trait-regulated-genes ontology model construction proposed in our study may achieve the correlation discovery of trait regulatory genes across species, improve the efficiency of cross-species subject knowledge acquisition, and support the gene discovery results of multi-dimensional data analysis. This study provides a feasible method path for the mining of pleiotropic genes and gene function prediction, and provides effective data support services for crop breeding scientific research.