Table of Content

26 August 2023, Volume 39 Issue 8

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Dense Planting for High Yield - The Key Goal of Maize Breeding in China

YAN Jian-bing, ZHAO Jiu-ran

2023, 39(8):  1-3. 

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High-yield Contests in Maize Facilitate the Vitalization of China’s Seed Industry

LENG Yan, MA Xiao-wei, CHEN Guang, REN He, LI Xiang

2023, 39(8):  4-10.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0776

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High-yield contests have been widely conducted globally, covering major food crops such as maize, wheat, soybeans, and rice. Successful experiences from international cases demonstrate that organizing yield contests may effectively promote the integration of superior seeds and agronomic practices, enhance crop breeding capabilities, and improve crop yields. Given the new challenges to food security in our country, it is necessary to explore how to draw on successful experiences of other nations to enhance our own potential for increasing crop production. The National Center of Technology Innovation for Maize adopts a high-yield technology dissemination chain centered around “quality seeds and good practice”, develops a biotechnological breeding innovation chain of high-yield genes and high-quality seeds varieties, and establishes a growth chain that spans from small-scale trials to large-scale applications. The goal is to establish a nationally replicable and scalable high-yield maize model, contributing to the vitalization and development of the seed industry.

Molecular Design of Ideal Plant Architecture for High-density Tolerance of Maize Plant

WANG Bao-bao, WANG Hai-yang

2023, 39(8):  11-30.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0660

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Maize（Zea mays L.）is the most productive grain crop in the world. Sufficient and stable production of maize is of great importance for ensuring worldwide food security. Long-term studies and maize production practice have shown that increasing density tolerance and planting density of maize varieties is crucial for increasing its yield, and breeding an ideal plant architecture is an important way to improve maize density tolerance. Previous reports indicate that more erected leaf angle, lower ear height, fewer tassel branches, and earlier flowering were important components of ideal plant architecture of maize for adapting to high-density planting. Hereby we intend to summarize the research progress in their relationship of four morphological traits with high-density tolerance, their morphological development, and genetic regulation. We also propose several directions for further research on improving density tolerant plant architecture in maize, aiming to provide some useful references for future breeding high-density tolerant maize.

Epigenetic Regulation of Yield-related Traits in Maize and Epibreeding

ZHANG Dao-lei, GAN Yu-jun, LE Liang, PU Li

2023, 39(8):  31-42.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0378

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Crop phenotypic diversity is influenced by many factors, and epigenetic variation can control crop traits and stress response through epigenetic modification to regulate gene expressions, and then affect crop yield. The main agronomic traits affecting maize yield include plant height, leaf angle and root. In addition, germplasm resources, biotic stress and abiotic stress are also key factors affecting maize yield. The main epigenetic regulation modes in crops include histone modification, DNA modification, RNA modification, non-coding RNA and chromatin remodeling. In this review, the epigenetic mechanism of the main agronomic traits in maize is summarized from the perspective of epigenetic regulation, and the new strategy of epigenetics-mediated crop breeding（epibreeding）to improve maize yield is proposed in combination with CRISPR/Cas-based epigenome editing technology.

Research in Maize Dwarf Genes and Dwarf Breeding

WANG Tian-yi, WANG Rong-huan, WANG Xia-qing, ZHANG Ru-yang, XU Rui-bin, JIAO Yan-yan, SUN Xuan, WANG Ji-dong, SONG Wei, ZHAO Jiu-ran

2023, 39(8):  43-51.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0504

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Plant height is an important agronomic trait which affects maize architecture and planting density. Breeding plants with dwarf/semi-dwarf architectures could tolerate high-density planting and contribute to yield increase. However, most dwarf mutants have severely reduced yield and are difficult to be applied in breeding. Therefore, it is particularly important to explore the regulation mechanism of maize plant height and to find out the excellent allele variation in plant height genes. It can improve the plant-type of maize, increase the light use efficiency and enhance the tolerance of population to water and fertilizer. Here we reviewed the quantitative trait locus of plant height excavated so far. We also explained that the cloned genes related to plant height are mainly regulated by plant hormones, microtubule-associated proteins and flower factors. Then we summarized the application and limitations of brachytic2 gene in maize dwarf breeding research, and looked forward to the important value of dwarf favorable alleles and their molecular markers, as well as modern biotechnology in the creation of dwarf germplasm resources. These efforts will promote the research on the genetic mechanism of maize plant height, and provide reference for the molecular breeding of dwarf maize.

Current Status and Prospects of Maize Super High Yield Research in China

LIU Yue-e, XU Tian-jun, CAI Wan-tao, LYU Tian-fang, ZHANG Yong, XUE Hong-he, WANG Rong-huan, ZHAO Jiu-ran

2023, 39(8):  52-61.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0555

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Maize, as the largest grain crop with the largest planting area and highest total yield in China, is crucial for food and feed security in China. In the increasingly tense situation of arable land resources, the space for expanding the area of crop in China is limited in the future, and the increase in crop production mainly relies on the increase in yield per unit area. In 2023, the No. 1 Central Document of the Central Government clearly proposed that we should pay full attention to grain production, carry out the construction of 1 t grain yield per 667 m², and implement the project of increasing maize yield per unit area. The super high yield of maize is the embodiment of the highest level of excellent cultivars, production conditions, and cultivation management techniques. This article comprehensively elaborates on the policy support, current status of high yield records, distribution and meteorological characteristics of super high yield fields, and technical key points for creating super high yield. On this basis, suggestions are put forward for the large-scale high-yield creation of maize in China, including increasing scientific research investment, strengthening the breeding of new cultivars, strengthening the integration of cultivation techniques for demonstration and promotion, and strengthening the construction of farmland infrastructure.

Gene Editing Reshaping Maize Plant Type for Increasing Hybrid Yield

SHI Jia-xin, LIU Kai, ZHU Jin-jie, QI Xian-tao, XIE Chuan-xiao, LIU Chang-lin

2023, 39(8):  62-69.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0533

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Compact plant architecture in maize achieves increased yield through dense planting. The key trait for improving the plant architecture is leaf angle. By creating hybrid varieties with compact plant architecture, the increased yield can be achieved. Using gene editing technology and the DTM（Desire Targeted Mutation）strategy, the gene ZmLG1 underlying leaf angle was mutated in the female parent（Named as CX1）of hybrid Zhongdan88. An improved inbred line（Named as CX1-lg1）with a compact plant architecture was developed. Then, by crossing the female parent CX1-lg1 with the male parent CX2, a hybrid with a compact plant architecture was obtained, named as Zhongdan88M, which contained ZmLG1Zmlg1 alleles. The seed production efficiency of CX1-lg1 and the yield of the improved hybrid variety Zhongdan88M under dense planting were evaluated. The results showed that: 1）Improved plant architecture of the female parent improved the corresponding hybrid's plant architecture. 2）The improved female parent CX1-lg1 had higher seed production efficiency than that of CX1 under the condition of 5 000 plants/667 m². 3）The improved hybrid Zhongdan88M had a more compact plant architecture than that of Zhongdan88 under different environments and densities, and achieved the increased yield at a high density. These results indicate that gene editing technology is powerful in the precise regulation of phenotype via targeting specific genes. Moreover, modifying one parent's genotype through the DTM strategy lead to improvements in desired phenotype in the corresponding hybrids.

Effects of Planting Density on the Stem Quality and Root Phenotypic Characters of Summer Sowing Maize

ZHANG Yong, XU Tian-jun, LYU Tian-fang, XING Jin-feng, LIU Hong-wei, CAI Wan-tao, LIU Yue-e, ZHAO Jiu-ran, WANG Rong-huan

2023, 39(8):  70-79.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0493

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The experiment was carried out to provide useful reference and guidance for the selection and breeding of maize varieties with high-resistance to density and lodging. The 24 widely planted maize varieties were selected to compare the differences of stem qualities and root traits under two plant densities of 60 000（D1）and 90 000（D2）plants/hm². The results showed that: 1）The lodging resistances of the tested 24 maize varieties differed largely, no lodging occurred during the growth period for 18 varieties such as Jingnongke728 in which used Jing2416 as the male parent. The lodging rate of Zhengdan958 reached 89.4% at physiological maturity under D2 density, but no lodging before physiological maturity. The root lodging rate of Xianyu335 reached 68.56% at the V12 stage, and no lodging occurred during the last stage. 2）Plant heights, ear heights and ear height to plant height ratio of the tested 24 maize varieties increased with the increasing of plant density. Plant height, ear height and ear coefficient of 18 varieties such as Jingnongke728 in which used Jing2416 as the male parent averaged 272.13 cm, 106.44 cm and 39.11%, and significantly lower than those of Zhengdan958 and Xianyu335. 3）The rind penetration strength, snapping resistance, root length and dry weight of the tested 24 maize varieties averaged 40.61 N/mm², 205.06 N, 217.35 m/plant and 14.29 g/plant respectively, and decreased with the increasing of plant density; but the yield showed an increasing trend, with an average of 11 724.48 kg/hm². The rind penetration strength, snapping resistance, root length and dry weight of 18 varieties such as Jingnongke728 which used Jing2416 as the male parent had obvious advantages than those of Zhengdan958 and Xianyu335, and the varieties such as Jingnongke728 were the high-yield and density-tolerance varieties.

Molecular Mechanisms of Rice Grain Size Regulation Related to Plant Hormone Signaling Pathways

YAO Sha-sha, WANG Jing-jing, WANG Jun-jie, LIANG Wei-hong

2023, 39(8):  80-90.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0273

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Rice is a major food crop for humans, and how to effectively improve its yield and quality is a major scientific concern. Rice grain size is one of the main factors affecting yield, and research on the regulation of rice grain development is an important guide for using molecular design breeding to improve yield and quality. Grain size is determined by a combination of the length, width and thickness of the grain; it is a quantitative trait regulated by multiple genes, and is one of the crucial determinants of rice yield and quality. In recent years, a large number of quantitative trait loci（QTLs）related to grain size have been identified through the study of mutants with seed development defects in rice, and some related genes have been cloned and identified, and the complex signalling pathways regulating grain size in rice are gradually being elucidated. Several studies have found that functional genes regulating rice seed development are involved in the synthesis, catabolism and transport of plant hormones as well as the signal transduction pathways of plant hormones. This review outlines the basic process of rice endosperm development, summarizes the overall the overall understanding of the dynamic changes of plant hormones during endosperm development, focuses on the current research status of QTLs related to phytohormone signalling pathways associated with rice grain size, summarizes and analyses the relationship between the pathways related to cytokinin, brassinosteroid, growth hormone, gibberellin, ethylene and jasmonic acid and grain size regulation, and further sorts out the regulation network of rice grain shape-related phytohormone signaling. It is aimed to provide a reference for identifying and analyzing the molecular mechanisms of phytohormone regulation of rice grain size, and to provide new ideas for rice molecular design breeding.

Research Progress in the Biosynthesis and Regulation of β-ionone in Plants

YE Yun-fang, TIAN Qing-yin, SHI Ting-ting, WANG Liang, YUE Yuan-zheng, YANG Xiu-lian, WANG Liang-gui

2023, 39(8):  91-105.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0208

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β-ionone（β-ionone, ΒI）is an important natural aroma volatile compound widely distributed in plants, which is an apocarotenoid produced by carotenoid cleavage dioxygenases cleaving β-carotene at the 9, 10 and the 9', 10' positions. As a representative of cyclized isoprene, β-ionone has various biological activities such as antibacterial, antiviral and antitumor. Therefore, studies on the biosynthesis and metabolic regulation of β-ionone in plants has gradually become a hot topic. This paper summarizes the progress of β-ionone bioactivity, biosynthesis and regulation in plants, focusing on the β-ionone biosynthesis pathway and its related enzymes, followed by the control of β-ionone biosynthesis by carotenoid cleavage dioxygenase genes at the molecular level, as well as transcription factors such as WRKY, NAC, ERF and MYB regulate the expressions of related structural genes and thus affect β-ionone biosynthesis. And the paper describes the effects of environmental factors such as temperature, light, water and soil salinity on β-ionone biosynthesis in plants. It also introduces the effects of genes involved in the biosynthetic pathway of β-ionone on the color and aroma of plant leaves, flowers and fruits, as well as the improvement of β-ionone biosynthesis in plants using gene overexpression, gene silencing and other genetic engineering techniques, and makes reasonable prospects for the problems to be solved in the research on β-ionone in plants, aiming to provide theoretical assistance for the deeper research on the biosynthesis and regulatory mechanism of β-ionone in plants, and provide a theoretical foundation for improving plant floral aroma substances and obtaining a high yield of natural β-ionone.

Research Progress in Plant Leaf Color Mutation Induced by Radiation

ZHAN Yan, ZHOU Li-bin, JIN Wen-jie, DU Yan, YU Li-xia, QU Ying, MA Yong-gui, LIU Rui-yuan

2023, 39(8):  106-113.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1561

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Leaf color mutants are ideal materials for studying a range of physiological metabolic process in higher plants, such as the structure of photosynthesis system, chlorophyll metabolism, chloroplast development, photosynthesis and hormone physiology, as well as important materials for genetic and plant breeding research. Radiation is the main method of leaf color mutagenesis, producing leaf color mutants with theoretical research and practical application. In this paper, we briefly introduced the type of leaf color mutants induced by different radiation sources, and a brief sketch about the physiological and molecular mechanism of leaf color mutations. Finally, we discussed the issues in the research of the leaf color mutant induced by radiation and prospected the further development. It is expected to provide references for improving the efficiency of plant radiation mutation breeding and promoting the application of leaf color mutants.

Research Progress in Mechanisms of Microbial-enhanced Phytoremediation for Lead-contaminated Soil

JIANG Run-hai, JIANG Ran-ran, ZHU Cheng-qiang, HOU Xiu-li

2023, 39(8):  114-125.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0125

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Lead（Pb）is one of the heavy metals that cause soil pollution and pose a serious threat to biological safety. Microorganisms play an important role in alleviating the toxicity of lead to plants. In the process of interacting with the rhizosphere, soil microorganisms improve the microecological environment of the rhizosphere and promote the repair of plants to lead and reduce the toxicity of lead by various means. This article focuses on the biochemical interaction between soil microorganisms and heavy metal lead, reviews the mechanism of microorganisms'role in repairing lead pollution in plants, and concludes as follows: 1）There are a large number of binding sites and negatively charged functional groups that have strong adsorption of lead on the cell walls of microorganisms. 2）Cations exchange ions with heavy metals in the process of interaction with heavy metals. 3）Microorganisms secret extracellular substances in metabolism, which form stable lead complexes with heavy metals. 4）The forms of heavy metals in soil are changed or transformed by oxidation-reduction reactions, or the lead are oxidized and reduced to insoluble precipitation, thus reducing the toxicity of heavy metals in soil. 5）The growth hormone secreted by microorganisms can promote the growth and development of plants, improve the resistance of plants, and its metabolic products can change the characteristics of heavy metals in the rhizosphere, which promotes the absorption of lead by plants or fix lead in the rhizosphere of plants to reduce the toxicity of lead to plants. A comprehensive discussion of the main mechanisms of lead adsorption by microorganisms was conducted, and the relationship between microorganisms and plants in the repair of lead pollution in soil was summarized, providing a basis for further understanding the interaction mechanism among plants, microorganisms and heavy metals in the process of soil heavy metal remediation.

Research Progress in Gut Microbiota and Metabolites Regulating Host Intestinal Immunity

SHA Shan-shan, DONG Shi-rong, YANG Yu-ju

2023, 39(8):  126-136.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1530

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The gut microbiota is a stable and complex microecosystem. After a long period of evolution, these microorganisms have established a stable symbiotic relationship with the host. The activity of the microorganism directly affects the health of the host. They not only play an important role in digestion and metabolism of host nutrients and body development, but also are closely related to host immunity and disease. The interaction mechanism between the gut microbiota and the body's immune system is very complex, and it has not yet been fully elucidated by various environmental factors. Microbial metabolites and microbial-body co-metabolites play an important role in regulating immune function, which gradually attract researchers’ attention. Therefore, on the basis of introducing the intestine and its role in host defense, this article reviews how intestinal microbes and metabolites in the intestine promote the development of the host's immune system and regulate the host's immune response. The purpose is to provide a reference for further research on the interaction between gut microbiota and metabolites and the body's immune system, and concurrently to provide a theoretical basis for nutritional measures to improve the intestinal health of livestock and poultry.

Modification of Pichia pastoris for Erythritol Production by Metabolic Engineering

ZHAO Si-jia, WANG Xiao-lu, SUN Ji-lu, TIAN Jian, ZHANG Jie

2023, 39(8):  137-147.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0138

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The aim of this study is to construct an erythritol-producing strain using Pichia pastoris as the chassis cell. An erythritol-producing P. pastoris strain was developed by regulating the expression of phosphofructokinase gene（pfk）in glycolysis pathway, knocking out the genes associated with the production of by-products arabitol and ribitol, and overexpressing 4-phosphate erythrose phosphorylase, erythrose reductase and sugar alcohol phosphatase genes derived from different organisms. Next, we investigated the effects of overexpressions of erythrose reductase and two key enzymes transketolase（TKL）and ribulose-phosphate epimerase（RPE）involved in pentose phosphate pathway on the erythritol production. The results showed that strain C8 harboring pyp1 gene derived from Saccharomyces cerevisiae and yidA gene derived from Escherichia coli had the ability to produce erythritol. The erythritol production of the C8 strain in shake-flask fermentation was 30 mg/L. Furthermore, the overexpression of tkl and rpe genes enhanced the erythritol production of C10 strain by about 40-fold. The erythritol production of C10 reached 1.2 and 10.6 g/L in the shake-flask and high-cell-density fermentations, respectively. Further the overexpression of erythrose reductase did not cause the erythritol production increased, while caused the production of by-products increased. In this study, the erythritol synthetic pathway was successfully constructed in P. pastoris for the first time, which laid a foundation for engineering P. pastoris for efficient production of erythritol and other high-value compounds.

Advances in CRISPR/Cas9 System Modifying Saccharomycescerevisiae

CHEN Xiao-ling, LIAO Dong-qing, HUANG Shang-fei, CHEN Ying, LU Zhi-long, CHEN Dong

2023, 39(8):  148-158.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1534

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Saccharomyces cerevisiae is one of widely used industrial strains, it is extremely important to increase its performance via modifying S. cerevisiae; however, there are cumbersome steps and long-cycle by traditional modifying method. CRISPR/Cas9 system is an adaptive defense system found in bacteria and archaea. At present, the CRISPR/Cas9 system has become a powerful tool for genome editing, which can simultaneously modify multiple genes of S. cerevisiae. This review provided a brief overview of the function and construction of CRISPR/Cas9 system in S. cerevisiae gene editing, introduced the design rules and representative design tools of sgRNA targeting sequences, as well as summarized the multiple-gene editing strategy. Moreover, the review discussed the challenges in application of CRISPR/Cas9 system, including off target and low editing efficiency, and their control measures, which may provide the references for better applying CRISPR/Cas9 system modifying S. cerevisiae.

Establishing a Stable Cell Line with Site-specific Integration of ERK Kinase Phase-separated Fluorescent Probe Using CRISPR/Cas9 Technology

YANG Yu-mei, ZHANG Kun-xiao

2023, 39(8):  159-164.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0173

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This work is aimed to construct a KYSE-150 cell line containing an inducible expressed ERK-SPARK kinase fluorescent sensor into the AAVS1 locus. The homologous recombination donor plasmid of the inducible ERK-SPARK expression cassette with 750 bp homology arms at both ends was constructed according to the DNA sequence design of the AAVS1 locus and the published base sequences of the ERK-SPAEK fluorescent probe. Secondly, three sgRNAs targeting the AAVS1 locus were designed using an online design tool, and cloned into the pX330 backbone plasmid. Three CRISPR/Cas9 targeting vectors capable of site-specific cutting of the AAVS1 locus were obtained. The homologous recombination donor plasmids and the expressed CRISPR/Cas9 targeting vectors were co-transfected into KYSE-150 cells, after Dox-induced expression for 24 h, puromycin resistance screening and flow cytometry were performed, and cell subpopulations resistant to puromycin and expressing GFP were obtained. Then PCR was used to identify the genotype, and Western blot was used to detect protein expression. Genomic DNA was extracted from the obtained cell population and the genotype was identified by PCR. The results showed that the size of the obtained fragment was consistent with the size of the inducible ERK-SPARK expression cassette. The results of protein expression detection showed that the cell population expressed the ERK-SPARK probe fusion protein. The results of fluorescent probe imaging experiments demonstrated that the cell line had a very small amount of leakage expression under the condition of no Dox-induced expression. After Dox-induced expression, the GFP fluorescent protein was evenly distributed in the cytoplasm without obvious aggregation into spots. After adding EGF to activate ERK kinase, the green fluorescence of GFP that was uniformly distributed in the cytoplasm gathered into high-brightness green droplets. A stable cell line that expressed the SPARK fluorescent probe after induction for detecting ERK kinase activity were successfully obtained, which may solve the problem that the unstable efficiency of the transiently expressed ERK-SPARK probe affected the accuracy of the experimental results, and may lay the preliminary research foundation for subsequent establishment of screening platform of a SPARK-based high-throughput protein kinase inhibitors.

Establishment of YFV17D Non-infectious Reporter Replicon and Pseudovirus Packaging System

HE Yu-hang, HU Tao, WU Zhen, HE Yu, CHENG An-chun, CHEN Shun

2023, 39(8):  165-172.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1402

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The YFV17D noninfectious replicon and pseudovirus packaging system was constructed to further elucidate the molecular mechanism of replication and assembly of yellow fever virus. In this study, the single-copy YFV17D reporter replicon based on the SP6 promoter was transformed into a low-copy YFV17D reporter replicon driven by the CMV promoter using reverse genetics. At the same time, the packaging plasmid expressing YFV17D structural protein and reporter replicon plasmid were co-transfected into BHK-21 cells to establish a YFV17D trans-pseudovirus packaging system. NanoLuc luciferase activity（Nluc）, oxGFP and mCherry fluorescent protein expression, and indirect immunofluorescence assay were used to monitor viral double-stranded RNA to determine the replication and packaging effect of replicon in BHK-21 cells. The results showed that Nluc activity, oxGFP and mCherry fluorescent protein expression increased steadily in wild-type replicon compared with replication-deficient replicon, while dsRNA also increased. After co-transfection of the replicon plasmids and packaging plasmids into BHK-21 cells, the supernatant was collected to re-infect BHK-21 cells. The success of pseudovirus packaging was confirmed by detecting Nluc activity and oxGFP and mCherry protein expression. In conclusion, the YFV17D replicon carrying different reporter genes was successfully constructed in this study to monitor the replication of viral genomic RNA. The structural proteins provided by the packaging plasmid can successfully package the non-infectious YFV17D subgene replicons into a YFV17D pseudovirus system with single round infection ability.

Identification of Rice Plant Height-associated QTL Using BSA-seq and RNA-seq

WU Yuan-ming, LIN Jia-yi, LIU Yu-xi, LI Dan-ting, ZHANG Zong-qiong, ZHENG Xiao-ming, PANG Hong-bo

2023, 39(8):  173-184.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0386

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The stability of rice yield is significantly influenced by plant height, making it as a crucial factor. The identification of plant height-associated QTLs and mining of candidate genes are conducive to comprehending the molecular regulatory mechanisms that determines plant height, which may lay a foundation for breeding ideal rice varieties. This study employed 285 CSSL populations derived from the parental strains of Oryza sativa var. Youzhan 8 and wild rice from Guangxi as the experimental cohort. The study utilized SNP and InDel molecular markers, in conjunction with NGS and BSA-seq techniques, to conduct an association analysis aimed at identifying genomic regions that are potentially associated with plant height. As results, the molecular markers Δ（SNP-index）were discovered to have an association with candidate genomic regions of 3.205 and 1.311 Mb in Chr.7 and Chr.10, respectively. The genomic regions linked with Δ（InDel-index）markers were of sizes 2.848 and 1.292 Mb, and were entirely encompassed within the intervals associated with Δ（SNP-index）. Through the integration of functional annotation in GO, KEGG, Uniprot, and eggNOG databases, as well as high-quality polymorphic site screening and existing transcriptome data pertaining to plant height, five candidate genes located in Chr.7 were ultimately linked to this trait, including the previously characterized OsTCP21 function and mechanism. The qRT-PCR findings were consistent with prior research, revealing differential expression of OsTCP21 in tall and dwarf plants. Specifically, LOC_Os07g05050 and LOC_Os07g02850 demonstrated elevated expressions in tall plants, whereas LOC_Os07g04220 and LOC_Os07g02770 had higher expressions in dwarf plants. The regulation of rice plant height is significantly influenced by five candidate genes, with OsTCP21 being identified as a pivotal regulatory gene.

Salt Tolerance at Seedling Stage and Analysis of Selenium and Folic Acid Content in Seeds in Two Sets of Wheat-Dasypyrum villosum Chromosom Additional Lines

HAN Zhi-yang, JIA Zi-miao, LIANG Qiu-ju, WANG Ke, TANG Hua-li, YE Xing-guo, ZHANG Shuang-xi

2023, 39(8):  185-193.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0003

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Dasypyrum villosum as a related species of wheat has many excellent genes, and it is an ideal material to improve the salt tolerance and nutritional quality of wheat. In this study, wheat-D. villosum #2 and wheat-D. villosum #3 disomic addition lines were applied for the identification of salt tolerance and determination of selenium and folic acid contents in seeds. It was found that the germination rates of the two sets of additional lines under salt stress were 16.67%-43.33% and 26.67%-70.00%, respectively, which were higher than the control Chinese Spring（CS）（6.65%）. The germination rates of the addition lines DA3V#3, DA7V#3, DA2V#3 and DA5V#2 under salt stress were 70.00%, 56.67%, 53.33% and 43.33%, respectively. The additional lines grew normally under salt stress, and the plant height and root length of the additional lines under salt stress were higher than those of the control CS, among which the plant height of DA4V#2 and DA2V#3 reached 15.2 cm and 16.1 cm, respectively, and the root length of DA2V#3 was 3.4 cm. The highest grain selenium content was tested in the addition line DA2V#2（8.47 μg/g）and DA2V#3（7.60 μg/g）containing chromosome 2V in the 14 addition lines. The folic acid content in the seeds of wheat-D. villosum additional lines #2 and #3 was 9.00-26.10 μg/100 g, among which most of the addition lines folic acid content were higher than the control（10.98 μg/100 g）. The difference between DA4V#2 or DA6V#2 and the CS was extremely significant in folic acid content, and the folic acid content in DA 6V#2 was 2.4-fold higher than that in CS. The results inferred that there might be salt tolerance related genes on chromosomes 2V#3, 3V#3, and 5V#2, grain selenium enrichment related genes on chromosome 2V, and grain folic acid biosynthesis related genes on chromosome 6V#2.

Cloning, Subcellular Localization and Expression Analysis of the Transcription Factor Gene FtbHLH3 in Fagopyrum tataricum

LYU Qiu-yu, SUN Pei-yuan, RAN Bin, WANG Jia-rui, CHEN Qing-fu, LI Hong-you

2023, 39(8):  194-203.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0034

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bHLH（basic helix-loop-helix）transcription factors play crucial roles in the biosynthesis of flavonoids in plants. In order to explore the function and molecular regulation mechanism of bHLH transcription factor in flavonoid biosynthesis in tartary buckwheat, RT-PCR was used to clone the FtbHLH3, one bHLH transcription factor. Then the bioinformatics subcellular localization, transcriptional activation activity, gene expression, gene co-expression, and the correlation between gene expression level and total flavonoid content of it were conducted. As results, the CDS length of FtbHLH3 was total 783 bp and it encoded total 260 amino acids. Conserved domain and phylogenetic analysis showed that FtbHLH3 was a member of non-IIIf subfamily of bHLH transcription factor family. Subcellular localization and transcriptional activation assay suggested that FtbHLH3 was located in the nucleus and had no transcriptional activation activity. Gene co-expression analysis revealed that FtbHLH3 was co-expressed with the Arabidopsis TT8 homologous gene FtTT8 and 12 structural genes of flavonoid biosynthesis in the tartary buckwheat. The correlation analysis between gene expression level and total flavonoids content in different tissues indicated that there was a highly positive correlation between the expressions of FtbHLH3 and the contents of total flavonoids. All these results suggest that FtbHLH3 is a non-IIIf subfamily bHLH transcription factor that was located in the nucleus and has no transcriptional activation activity. It may regulate the flavonoids biosynthesis in tartary buckwheat through interacting with other transcription factors.

Cloning and Expression Analyses of C-glycosyltransferase Gene FtUGT143 in Fagopyrum tataricum

WANG Jia-rui, SUN Pei-yuan, KE Jin, RAN Bin, LI Hong-you

2023, 39(8):  204-212.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0098

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Tartary buckwheat[Fagopyrum tataricum（L.）Gaertn]is rich in flavonoid C-glycosides, and it has many health effects, such as anti-oxidation, anti-cancer and anti-inflammation. In this study, in order to explore the flavonoid C-glycosyltransferase gene in tartary buckwheat, a glycosyltransferase gene named FtUGT143 was cloned from tartary buckwheat by RT-PCR（reverse transcription-polymerase chain reaction）, and its bioinformatics, molecular docking, gene expression, correlation between gene expression and metabolites content were also analyzed. The results showed that the full-length CDS sequence of FtUGT143 was 678 bp, encoding a protein of 226 amino acids. The codon preference analysis indicated that FtUGT143 had the codon usage preference of dicotyledons. Multiple sequence alignment and phylogenetic tree analysis suggested that FtUGT143 belonged to the carbon glycosyltransferase subfamily of plant glycosyltransferase gene family. The molecular docking analysis implied that FtUGT143 interacted with apigenin and luteolin, which were the substrates of flavonoid C-glycosides（vitexin, isovitexin, orientin and isoorientin）. RT-qPCR results showed that FtUGT143 gene was expressed in all tissues of tartary buckwheat, but it was significantly expressed in the roots, stems and leaves at seedling stage, and its expression in different tissues had a good correlation with the accumulation of four flavonoid glycosides. The research results showed that FtUGT143 is a C-glycosyltransferase which may participate in the biosynthesis of flavonoid C-glycosides, revealing the important role of FtUGT143 in the synthesis mechanism of flavonoid C-glycosides in tartary buckwheat.

Cloning of IbHQT1 Promoter and Identification of Upstream Regulatory Factors in Sweet Potato

XU Jing, ZHU Hong-lin, LIN Yan-hui, TANG Li-qiong, TANG Qing-jie, WANG Xiao-ning

2023, 39(8):  213-219.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0128

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Hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase（HQT）is the last rate-limiting enzyme in the chlorogenic acid biosynthesis. IbHQT1 is a key gene involved in chlorogenic acid biosynthesis in sweet potato. To further reveal the role and the transcriptional regulation mechanism of IbHQT1, the promoter sequence of IbHQT1 was cloned, a yeast one-hybrid cDNA library of sweet potato leaf was constructed and the upstream regulatory factors interacting with the IbHQT1 promoter were screened by yeast one-hybrid technology. The results showed that 1 500 bp IbHQT1 promoter contained a variety of hormone and defense-related cis-elements, as well as transcription factor-binding elements. The capacity of the constructed library was 1.15×10⁷ CFU, and the average length of inserted fragments was about 1 200 bp. Two transcription factors, IbMYB11 and IbTGA2.2, were screened and identified to interact with the IbHQT1 promoter. Furthermore, IbMYB11, IbTGA2.2 and IbHQT1 demonstrated similar expression patterns in different tissues and at developmental stages of sweet potato（QS80-12-11）, and significantly correlated with the accumulation of chlorogenic acids.

Characteristics Analysis of Seed Microrhizome Gene Expression of Polygonatum cyrtonema

LIU Bao-cai, CHEN Jing-ying, ZHANG Wu-jun, HUANG Ying-zhen, ZHAO Yun-qing, LIU Jian-chao, WEI Zhi-cheng

2023, 39(8):  220-233.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0115

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The germination process of Polygonatum cyrtonema seeds have a special germination phenomenon of micro rhizomes formation. The purpose of explaining the changes of key genes in the germination process is to lay a foundation for related research on seed physiology, morphological structure development and so on. In this paper, based on high-throughput sequencing technology, the transcriptome sequencing and bioinformatics analysis were performed on the seeds of P. cyrtonema in different germination states. The results showed that 17 907 differentially expressed genes were identified in microrhizome formation, compared to radicle breakthrough seed coat, these genes were in significantly different expressions in terms of metabolic process, catalytic activity, protein phosphorylation, etc. Pathway significance enrichment indicated that the differential genes were mainly enriched in plant hormone signal transduction, starch and sucrose metabolism, flavonoid biosynthesis, and other pathways. Genes enriched were abundantly expressed in plant hormone signal transduction pathways. Especially, the expression genes were up-regulated in brassinosteroid biosynthesis pathway. There were 26 833 significant differences Unigenes before and after the microrhizomes turned green, mainly enriched in metabolic process, peptide biosynthesis process, catalytic activity and other pathways. The differential genes were mainly enriched in, starch and sucrose metabolism, photosynthesis and other pathways by KEGG pathway enrichment analysis. The germination process of P. cyrtonema seeds is a complex regulatory network of a series of genes, and genes related with in brassinosteroid biosynthesis pathway may have an important role in the formation of microhizome. The results also provides a reference for in-depth research on the micro-rhizome formation physiology, micro-rhizome rapid expansion in production, and microhizom exploitation.

Cloning, Expression and Function of Iron Regulated Transporter VvIRT1 in Wine Grape（Vitis vinifera L.）

SONG Zhi-zhong, XU Wei-hua, XIAO Hui-lin, TANG Mei-ling, CHEN Jing-hui, GUAN Xue-qiang, LIU Wan-hao

2023, 39(8):  234-240.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1552

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The genes VvIRT of iron regulated transporters（IRT）family from wine grape was cloned, its expression pattern and potential biological function were analyzed, which may provide theoretical foundation for Fe absorption and efficient utilization mechanisms of fruit trees. Homology cloning technology was used to clone the VvIRT1 gene from wine grape, real-time quantitative PCR was applied to analyze tissue-specific expression characteristics and their responses to different Fe supplies, and yeast heterologous expression system was to analyze the functional verification. The iron regulated transporter VvIRT1 from diploid wine grape ‘Marselan’ was isolated and determined. Phylogenetic tree analysis showed that VvIRT1 was closely clustered with citrus CsIRT1（Rutaceae）and upland cotton GhIRT1（Malvaceae）that the genetic distance among them was relatively close. VvIRT1 was specifically expressed in the roots of both 5-year-old mature trees and tissue-cultured seedlings. Fe depletion significantly induced the expressions of VvIRT1 in the whole tested seedlings, while high Fe toxicity significantly reduced the expressions of VvIRT1 in all tested seedlings in the roots of tissue-cultured seedlings. In addition, VvIRT1 recovered the normal growth of yeast mutant DEY1453 and had ability of transporting external Fe²⁺.

Cloning, Subcellular Localization and Expression Analysis of CnbHLH79 Transcription Factor from Camellia nitidissima

LI Bo, LIU He-xia, CHEN Yu-ling, ZHOU Xing-wen, ZHU Yu-lin

2023, 39(8):  241-250.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1562

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In order to explore the regulation mechanism of CnbHLH79 in the flower color formation of Camellia nitidissima, the coding sequence of CnbHLH79 was cloned and analyzed by bioinformatics, and the subcellular localization of CnbHLH79 protein was performed. Subsequently the expression pattern of CnbHLH79 was analyzed by fluorescence quantitative PCR（RT-qPCR）in different tissues and petals of different flowering stages, and the correlation between the expression of CnbHLH79 and value of chromaticity b* and the content of flavonoid compounds was analyzed. It was found that the coding sequence of CnbHLH79, with bHLH_AtBPE_like conserved domain, was 855 bp encoding 284 amino acids. CnbHLH79 had the closest relationship with bHLH79 protein of Camellia sinensis. Subcellular localization analysis showed that CnbHLH79 mainly had functions in the nucleus. CnbHLH79 highly expressed in the roots and flowers of C. nitidissima and gradually decreased at flowering stages by RT-qPCR. In addition, correlation coefficient between the expression of CnbHLH79 and the value of chromaticity b* and content of Qu7G was -0.92 and -0.70 respectively, which had negative relationship. This study lays a foundation for elucidating the mechanism of CnbHLH79 transcription factor regulating the synthesis of flavonoids in C. nitidissima and the mechanism of regulating the yellow color of petals.

Growth Differences Among Grafted Seedlings with Two Rootstocks of Catalpa bungei and Comparative Analysis of Transcriptome

FU Yu, JIA Rui-rui, HE He, WANG Liang-gui, YANG Xiu-lian

2023, 39(8):  251-261.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1557

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Grafting is the main way of reproducing and expanding Catalpa bunge of superior cultivar. In order to investigate the growth differences of two ‘Suqiu No. 1’ superior cultivars, taking Catalpa ovata and Catalpa fargesii as rootstocks, the survival rate, new shoot length, new shoot thickness, scion diameter/stock diameter, leaf area of the two cultivars were studied for screening the better rootstock. Transcriptome sequencing was performed on the leaves and terminal buds of the two grafted seedlings by high-throughput sequencing platform. The differentially expressed genes were confirmed by quantitative real-time PCR. The results showed that the survival rate and the growth of the grafted seedlings with C. ovata as the rootstock were significantly higher than those with C. fargesii as the rootstock. Total 559 differentially expressed genes were screened by transcriptome sequencing, including total 192 up-regulated genes and total 367 down-regulated genes. GO enrichment analysis showed that total 559 differentially expressed genes were significantly enriched in metabolic process, cellular process, organelles, binding and catalytic activities; KEGG enrichment analysis showed that total 213 differentially expressed genes were significantly enriched to total 66 metabolic pathways in leaves, and total 137 differentially expressed genes were significantly enriched to total 45 metabolic pathways in terminal buds. The RT-qPCR trends of four differentially expressed genes（Unigene0040270, Unigene0006320, Unigene0036149 and Unigene0007805）were consistent with the transcriptome data. The results of this study showed that there were significant differences in the growth of the two grafted seedlings, among which the growth parameters of the grafted seedlings with C. fargesii as the rootstock were better. At the same time, the differences were preliminarily analyzed from the perspective of transcriptome, providing a certain reference value for the study of C. bungei grafting molecules.

Screening and Identification of Antagonistic Bacillus spp. Against Cucumber Fusarium wilt and Its Biocontrol Effect

CHU Rui, LI Zhao-xuan, ZHANG Xue-qing, YANG Dong-ya, CAO Hang-hang, ZHANG Xue-yan

2023, 39(8):  262-271.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1574

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Cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum seriously affects the sustainable development of the cucumber industry. In order to obtain high-efficiency antagonistic Bacillus spp. against cucumber Fusarium wilt and clarify its growth-promoting effect, hte biocontrol strain with a strong antagonistic effect against F. oxysporum were screened from Bacillus with a certain resistance to Fusarium solan, and its morphological, physiological and biochemical, genetic characteristics and plant growth-promoting characteristics were evaluated. The results showed that strain N1、N3 and N6 presented significant inhibitory effects on F. oxysporum, with inhibitory rates of 77.71%, 75.94% and 74.76%, respectively. Three strains were all identified as Bacillus velezensis, all the three strains showed releasing potassium ability, and produced protease, chitinase, pectinase and siderophores. In the seedlings disease-prevention and growth-promotion test, the disease index of inoculated strain N1, N3 and N6 significantly reduced, the control efficiency was 41.93%, 20.97%, and 66.13% respectively on the 18th day after inoculation. Inoculation with N1, N3 and N6 strains effectively promoted the growths of cucumber seedlings compared with single inoculation of F. oxysporum f. sp. treatment, including plant height, stem thickness, leaf area, chlorophyll content and aboveground and subsurface fresh weight, the growth promoting effect of N6 treatment was the most significant. It increased by 21.56%, 56.13%, 47.95%, 76.18%, 222.89% and 70% respectively. Compared with the control, the growth characteristics of treated plants significantly decreased except for the fresh weight of underground parts. Under inoculation F. oxysporum f. sp., the addition of N6 significantly increased the stem thickness and aboveground fresh weight. In conclusion, N6 can be used as potential biocontrol resources to control cucumber Fusarium wilt caused by F. oxysporum f. sp. cucumerinum and promote the growth of seedlings for sustainable and efficient cucumber production.

Isolation, Identification and Growth-promoting Characteristics of Endohyphal Bacterium 7-2H from Endophytic Fungi of Spiranthes sinensis

FANG Lan, LI Yan-yan, JIANG Jian-wei, CHENG Sheng, SUN Zheng-xiang, ZHOU Yi

2023, 39(8):  272-282.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0038

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Endohyphal bacteria are a special class of bacteria that play an essential role in host fungi and ecosystems. To explore the endohyphal bacteria resources with growth-promoting functions in endophytic fungi of Spiranthes sinensis and explore their biological functions，the endohyphal bacteria were isolated by mycelial tissue grinding and identified by colony morphology, physiological and biochemical properties and molecular characteristics, after detecting the presence of endohyphal bacteria in the mycelium of Epicoccum sorghium by fluorescence in situ hybridization（FISH）. Then the endogeneity of the bacteria was verified by specific primers, and the growth-promoting characteristics were determined by Salkowski colorimetry, CAS colorimetry and Mo-Sb colorimetry. The growth-promoting ability of endohyphal bacteria was preliminarily verified by rice seed growth-promoting experiments, and the whole genome sequencing and growth-promoting functional gene analysis of endohyphal bacteria were performed. The presence of bacteria within the mycelium of the fungus E. sorghinum was observed after hybridization with the fluorescein-labeled single-stranded nucleic acid probe, which might confirm the mycelium contains bacteria. An endohyphal Rhizobium oryzihabitans was isolated from the mycelium and named as 7-2H, which had the ability of producing indole-3-acetic（IAA）and siderophore as well as solublizing phosphate, and significantly increased the shoot length, root length, fresh weight and dry weight of rice seedlings. The average nucleotide identity（ANI）value between the genome of strain 7-2H and the standard strain Rhizobium oryzihabitans M15 was 96.98%. In the genome of strain 7-2H, genes related to IAA production, siderophore and phosphate solubilization were revealed. An endohyphal Rhizobium oryzihabitans is isolated, which has fine plant growth-promoting property and has a potential to be developed to be microbial fertilizer.

Isolation and Identification of a Cellulose-degrading Strain of Olivibacter jilunii and Analysis of Its Degradability

RAO Zi-huan, XIE Zhi-xiong

2023, 39(8):  283-290.  doi:10.13560/j.cnki.biotech.bull.1985

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In order to obtain the new cellulose-degrading bacteria at normal temperature, a bacterial strain named as 18B with cellulose-degrading ability was isolated and purified from garden compost in this study. Congo red staining experiment and filter paper degradation experiment were conducted to verify its cellulose-degradation ability. 16S rRNA sequence alignment and whole genome alignment confirmed that it belonged to the genus Olivibacter, and was the closest to Olivibacter jilunii 14-2A^T. Further comparative physiological and biochemical characterization revealed differences with O. jilunii 14-2A^T in terms of growth temperature, oxidase and glycolysis. The strain 18B grew in the range of 12-48℃, and the optimal growth temperature was 30-37℃. O. jilunii 14-2A^T grew at 4-42℃. Strain 18B was positive for oxidase and negative for glycolysis, while O. jilunii 14-2A^T had no oxidase activity and was positive for glycolysis. Combined with the results of the genome-wide covariance alignment, it was known that strain 18B had a certain evolutionary relationship with O. jilunii 14-2A^T. The cellulose-degradation ability was found that the cellulase activity of 18B was up to 82.14^+0.99_-9.90 U/L at 37℃, 200 r/min until the 5th d. Meanwhile, the enzyme activity could be maintained without degradation under the incubation conditions with sodium carboxymethyl cellulose as the only carbon source and only inorganic nitrogen source added. O. jilunii 14-2A^T had no cellulose degrading ability and could not survive in a culture environment where sodium carboxymethylcellulose was the only organic carbon source. The results of whole genome sequencing showed that there were cellulase genes in the genome of strain 18B. The cellulase genes were heterologous expressed in Escherichia coli, and the degradation ability of the expressed products was tested. The results showed that the cellulase genes were active. The above results finally identified strain 18B as a new physiological strain of O. jilunii with cellulase activity.

Whole Genome Sequencing and Comparative Genome Analysis of a Fish-derived Pathogenic Aeromonas Hydrophila Strain XDMG

GUO Shao-hua, MAO Hui-li, LIU Zheng-quan, FU Mei-yuan, ZHAO Ping-yuan, MA Wen-bo, LI Xu-dong, GUAN Jian-yi

2023, 39(8):  291-306.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1507

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The objectives of this study are to explore the mechanism of pathogenic Aeromonas hydrophila XDMG strain infecting fish, and to compare its genome structure, function and evolutionary relationship with the reference strain. The whole genome of A. hydrophila XDMG strain was sequenced, and the original data obtained by sequencing were assembled, spliced and annotated using Newbler, SeqMan, Glimmer and other software. Based on the constructing phylogenetic tree analysis from housekeeping genes and determining evolutionary relationships, the genomes of A. hydrophila were compared with those of four A. hydrophila strains in different regions. The complete genome sequence of A. hydrophila XDMG strain was 4.99 Mb in length and the GC content was 60.84%. A total of 4 935 coding genes were predicted, 4 137 genes with clear function were found, 99 tRNAs and 3 rRNAs were found; 4 061 genes had ortholog family classification, 3 228 genes were related to gene ontology, and 2 663 genes related to metabolic pathways; 101 tandem repeats and 14 gene islands were predicted in the genome of A. hydrophila XDMG strain, with genes related to three QS systems: AI 1, AI 2, and AI 3. Through genome comparative analysis, A. hydrophila XDMG was closely related to domestic A. hydrophila J-1 strain and US-derived A. hydrophila AL0971 strain; it was more conserved with US-derived A. hydrophila AL0971 in the comparison of virulence genes, outer membrane proteins, and O-antigen gene clusters. Comprehensive genome annotation and comparative analysis of the genome of A. hydrophila XDMG strain provide basic data for the study of functional genomics and genetic engineering vaccines of A. hydrophila.