Biotechnology Bulletin

Role of Acetylation in the Pathogenic Process of Plant Pathogens

WANG Li-chao, LI Huan, SHENG Ruo-cheng, LI Min, CHEN Feng-mao

2024, 40(5): 1-12. doi:10.13560/j.cnki.biotech.bull.1985.2023-1179

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Lysine acetylation, as a conservative post-translational modification, participates in a variety of biological processes. Numerous medical and plant pathological studies have revealed that acetylation plays an important regulatory role in the development of animal and plant diseases. Based on previous research progress, this article elaborates on the role of acetylation in plant pathogens from three perspectives involved in the pathogenesis of plant pathogens: Regulation of pathogen growth and pathogenicity, host plant resistance, and pathogen-host plant interaction. The aim is to understand the role of acetylation in the pathogenic process and to provide new ideas and theoretical support for the prevention and control of the pathogens.

Research Progress in the Effects of Microorganisms on the Growth and Development of Edible Mushrooms

CUI Man, SHAO Gai-ge, YANG Nuo-lin, FAN Qing-hao, ZHANG Jin-wei, TIAN Yu, ZHENG Su-yue, ZHANG Rui-ying

2024, 40(5): 13-22. doi:10.13560/j.cnki.biotech.bull.1985.2023-0933

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Edible mushrooms have become the fifth largest cultivation industry in China after grain, oil, fruit and vegetables. Edible mushrooms, as a type of macrofungi, are mainly cultivated using pure culture technology at present. However, edible mushrooms coexist with various microorganisms in nature, and have formed a complex interrelationship in the long-term evolution. According to their impact on edible fungi, the microorganisms can be divided into harmful and beneficial microorganisms. The common harmful microorganisms include competitive microorganisms and infectious pathogens. The beneficial microorganisms mainly include casing microorganisms, companionate microorganisms, bio-controlling microorganisms, etc. In crop production, a variety of microbial fertilizers and microbial agent with nutritional, growth promoting, and disease resistant functions have been developed according to the interrelationship between plants and rhizosphere microorganisms, and have effectively promoted the quality and yield of crops. However, those studies and development of edible mushrooms in hyphosphere microorganisms are still in the early stages. This article briefly summarizes the current research progress on the interrelationship between edible mushrooms and other microorganisms, aiming to promote relevant research and to provide new ideas for the domestication of new species of edible mushrooms in the next step, as well as high yield, stable yield, high quality, high resistance, wide suitability and green production.

Research Progress in the High-value Utilization of Lignocellulose Biomass by Steam Explosion

WU Wei, MA Qiu-gang, ZHU Xuan, WANG Jian

2024, 40(5): 23-37. doi:10.13560/j.cnki.biotech.bull.1985.2023-1154

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Lignocellulose biomass is a renewable resource with a large quantity, wide coverage, and low cost. It has gradually been developed and applied from biomass to biofuels, feedstuffs, and other value-added products. Such high-value transformation and comprehensive utilization have become important parts of “taking the path of green development and building a green production system”. However, the natural anti-degradation barrier and unique physicochemical properties of lignocellulose, as well as the rigid network of the three major components of cellulose, hemicellulose, and lignin, have been the long-term bottleneck of efficient conversion. The reasonable and effective pretreatment techniques are the key steps in the resource recovery process. This paper focuses on the analysis of basic composition and structural characteristics of lignocellulose biomass. On the basis of summarizing the advantages and disadvantages of traditional pretreatment methods such as physical, chemical, and biological methods, it emphasizes the development history, processing types, scope of application, working principle, reaction stages, technical characteristics, main parameters, and possible by-product effects of steam explosion. In addition, it involves research progress in the fiber modification, structural changes, dissolution characteristics, oligosaccharide preparation, active ingredient extraction, and ruminant feed utilization of biomass. It is also pointed out that the new trend in the post-treatment process after steam explosion assisted by microbial fermentation dominated by fungi and bacteria, as well as the exogenous addition of carbohydrase. Finally, the difficulties and challenges faced by steam explosion in future commercialization, industrialization, and large-scale production promotion are summarized. The corresponding breakthrough points and solution strategies are analyzed and proposed. The degradation effects of steam explosion technology on common by-product types of feed raw materials, as well as its reasonable application in the diet of monogastric animals, are discussed. It is aimed to provide new ideas and technical guidance for the potential development, value-added, and feed application of biomass resources using this means.

CRISPR-Cas9 Gene Editing Technology and Its Research Progress in Poultry

XIAO Yi-meng, YANG Wen, CHENG Yi-yi, LUO Gang

2024, 40(5): 38-47. doi:10.13560/j.cnki.biotech.bull.1985.2023-1135

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The CRISPR-Cas system, found in bacteria or archaea, serves as an adaptive immune system to counteract viral re-invasion by effectively cleaving nucleic acids. By leveraging the characteristics of Cas9 protein and its variants, researchers have devised a range of gene editing techniques that enable gene operations like gene knock-out or knock-in within cells, thereby facilitating genetic diversity in organisms. Additionally, these tools allow the manipulation of DNA or RNA through epigenetic modification to regulate gene expression, rendering them extensively employed in life science research. Poultry, as a significant agricultural species, plays a crucial role in supplying individuals with protein of superior quality. The utilization of CRISPR-Cas9 technology for gene editing and modification in avian genome is presently confined to the research phase, with remarkable advancements observed solely in chicken and quail. This paper presents an overview of CRISPR-Cas9 gene editing technology, encompassing its components, delivery methods, and optimization strategies, while also exploring its potential applications in poultry production and prevention and control of diseases. These insights lay the groundwork for the future development of CRISPR-Cas9 technology in the context of poultry system.

Functional Analysis of Exosomal MicroRNA in Antiviral Immunity

DOU Jin-ping, GAO Wei-song, WEI Shuang, GAO Xin-tao, LI Yi-nv

2024, 40(5): 48-57. doi:10.13560/j.cnki.biotech.bull.1985.2023-1151

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Exosomes are extracellular vesicles that contain various proteins and cellular regulatory factors, which participate in intercellular information transfer and regulate cell growth and physiological processes. Exosomes can indicate the level of gene expression of cells in immunological studies at the cellular level, as their functional variability is closely related to the type and state of the cells that secrete them. Viruses are small and simple in structure. They complete self-replication and escape host immunity by involving a complex immunoregulatory network. As a new focus of information exchange, the role of exosomes as “messengers” in this regulatory network deserves attention. MicroRNAs also play a crucial regulatory role in intercellular information exchange. After being selectively sorted into exosomes, as one of the exosome-loaded cargoes, exosomal microRNA can regulate biological functions more stably and participate in the regulation of viral immunity. In this paper, the composition, mechanism, and content of exosomes are briefly introduced. Taking the exosomal miRNA with a wide research range and more research content as a starting point, it is focused on its dual role in viral immunity. Understanding the regulatory role of exosomal microRNA in the same or different viruses would be of great significance to further analyze the mechanism of virus invasion into the host.

Research Progress in DNA Methylation Sequencing Technology

YUAN Ming-bo, YE Guo-hua, YANG Dan, SONG Dong-xue

2024, 40(5): 58-65. doi:10.13560/j.cnki.biotech.bull.1985.2023-1166

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DNA methylation is an important epigenetic modification that plays a key role in the growth and development of animals and plants, as well as in the occurrence of diseases and the regulation of gene expression. Clinically, DNA methylation tumor markers can serve as biological markers for the diagnosis, prognosis, and treatment of tumors. Accurate detection of DNA methylation is of great significance for elucidating the life mechanisms of biological growth, development, and disease occurrence. DNA methylation sequencing technology is a powerful tool for studying DNA methylation and is widely used for mapping DNA methylation in the genome. In recent years, in order to better detect DNA methylation site information, scientists have proposed a series of high-throughput DNA methylation sequencing technologies, which have improved sequencing detection sensitivity, reduced sequencing costs and experimental expenses, and greatly promoted the development of epigenomics. This article reviews a series of DNA methylation sequencing technologies, focusing on the technical principles and application scenarios of three sequencing technologies: WGBS, TAPS, and EM-seq. It also introduces sequencing methods for locating DNA methylation at the single-cell level, and finally prospects the future development direction.

Construction and Application of Potato Mitochondrial Targeted Expression Vector

ZHANG Zhen, LI Qing, XU Jing, CHEN Kai-yuan, ZHANG Chun-zhi, ZHU Guang-tao

2024, 40(5): 66-73. doi:10.13560/j.cnki.biotech.bull.1985.2023-1178

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【Objective】The genetic variations of mitochondria genome usually lead to cytoplasmic male sterility（CMS）in plants. Biotechnology is an important measure to produce variations resulting in CMS lines. In order to establish an efficient expression vector system for the research on potato mitochondrial genes, the optimal potato mitochondrial targeting expression vector was explored by comparing the expression level of eGFP, which was promoted by different combinations of promoters and mitochondrial targeting signal peptides（MTS）. 【Method】Three promoters AtUBI10, StUBI10, 2×35S and two mitochondrial transport signal peptides ATPγ and Rf1b were tested. The expression level of the target vector was verified by tobacco leaf transient expression system and PEG-mediated potato protoplast transformation system. 【Result】AtUBI10 :: ATPγ-eGFP among 6 expressing vectors had the best expression effect in tobacco and was accurately located in mitochondria. The second best ones were StUBI10 :: ATPγ-eGFP and 2×35S :: ATPγ-eGFP. The vector AtUBI10 :: ATPγ-eGFP also showed the best performance, followed by StUBI10 :: Rf1b-eGFP and AtUBI10 :: Rf1b-eGFP in the potato protoplast transformation system. The two vectors with 2×35S promoter were weakly expressed in protoplasts. 【Conclusion】The comparative studies showed the mitochondrial vector constructed by the promoter AtUBI10 and the mitochondrial transport signal peptide ATPγ is the best combination, and this vectors will be of great benefit to the relative study on mitochondrial genes.

Genetic Diversity Analysis and DNA Fingerprint Construction Based on SSR Markers for Xanthoceras sorbifolia

LI Si-qi, ZHANG Wen-chen, YANG Liu, FU Qing-xin, HONG Xin, ZHANG Hai-wang

2024, 40(5): 74-83. doi:10.13560/j.cnki.biotech.bull.1985.2023-1148

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【Objective】This work aims to quickly identify and utilize Xanthoceras sorbifolia germplasm resources, and explore the relationship of germplasm.【Method】Eleven primers with clear bands and good polymorphism were selected from 20 pairs of SSR primers. Fluorescence capillary electrophoresis technology was used for 29 X. sorbifolia varieties（lines）polymorphism detection. The neighbor-joining method was utilized for cluster analysis, while numerical and letter codes were assigned to create molecular identity cards. 【Result】A total of 66 alleles were detected, and each pair of primers detected 3-10 alleles with an average of 6. The Shannon information index（I）varied from 1.723 to 0.349，with an average of 1.16. The polymorphic information content（PIC）revealed by different primers ranged from 0.276 to 0.841，with an average of 0.66. The observed heterozygosity（Ho）ranged from 0.137 to 0.958, with an average of 0.739. The expected heterozygosity（He）ranged from 0.187 to 0.79, with an average of 0.648. Among the 11 loci, 7 loci had an average observed heterozygosity greater than the average expected heterozygosity. The genetic diversity coefficient varied from 0 to 1 with an average of 0.31. There were 15 individuals with a genetic similarity coefficient above 0.6, accounting for only 5.17% of the total data. At the genetic distance of 0.42, the 29 X. sorbifolia materials were divided into three categories by cluster analysis. Based on these results, the DNA fingerprint database composed of the digits 0 and 1 and the molecular identity card were established, which allowed unequivocal identification of all the varieties except a few unique ones. 【Conclusion】This indicates that the genetic diversity of the 29 germplasm resources of X. sorbifolia is relatively high, but there is also a certain degree of inbreeding. Molecular identity cards for X. sorbifolia using SSR markers is simple and feasible，which may provide technical support and scientific basis for genetic identification，rights protection，identity validation，traceability management，and new variety breeding of X. sorbifolia varieties.

Development and Application of Antibody Modified DNA Sequencing Enzymes

SONG Hui, CAO Wen-gang, XIAO Xiao-wen, DU Jun

2024, 40(5): 84-93. doi:10.13560/j.cnki.biotech.bull.1985.2023-1103

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【Objective】To develop a novel DNA sequencing enzyme that addresses the main challenges faced by first-generation sequencing technology when dealing with complex DNA structure templates, such as interruption of sequencing signals or rapid signal decay. 【Method】Taq DNA polymerase and single-strand binding protein SSB gene sequences were mined from the NCBI database, and a novel sequencing enzyme, Sso-Sequenase, was obtained using genetic fusion, site-directed mutagenesis, and gene design techniques. The purified sequencing enzyme was obtained through affinity chromatography and ion exchange chromatography. The performance of Sso-Sequenase was improved using antibody modification technology, and its hot start performance was characterized using STR technology. A variety of complex templates were selected, and the sequencing performance of the Sso-Sequenase sequencing enzyme kit was compared with the traditional BigDye sequencing kit using Sanger sequencing. 【Result】Sso-Sequenase was stably expressed in Escherichia coli, with a purity of over 95% and a yield of up to 10.5 mg/L. When the temperature was below 35℃, Sso-Sequenase demonstrated hot-start activity. In first-generation sequencing reactions with complex templates, such as those with repetitive sequences, high GC content, and hairpin structures, Sso-Sequenase successfully completed sequencing for all samples, with an average base quality value QV > 20. In contrast, the BigDye sequencing kit experienced significant signal decay or interruption when processing these complex samples. 【Conclusion】A DNA sequencing enzyme, Sso-Sequenase, has been developed to possess exceptional purity, remarkable yield, and hot-start activity. These advancements have significantly bolstered the sequencing success rate for intricate DNA templates, including those characterized by repetitive sequences, high GC content, and hairpin structures.

Design of Universal Tailed-sequence and Establishment of the Universal System Suitable for Multiple Species

SUN Bo, WANG Rui, HUO Yong-xue, GE Jian-rong, KUANG Meng, WANG Feng-ge

2024, 40(5): 94-102. doi:10.13560/j.cnki.biotech.bull.1985.2023-0967

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【Objective】Fluorescent capillary electrophoresis, for its high detection throughput and resolution, was in extensive applications in various scenarios, including individual identification, variety discrimination, and species identification. The tailed-sequence offers an efficient solution for the extensive application of the fluorescent capillary electrophoresis platforms. To address the limitation of existing tailed-sequences in meeting the demands of multi-species applications, this study developed an efficient Universal Tailed-Sequence（UTS）design tool based on encoding translation technology. Utilizing this tool, UTS were designed to construct a versatile PCR system and protocol suitable for multiple crops, thereby enhancing the throughput and flexibility of fluorescent electrophoresis. 【Method】An efficient UTS design tool was designed based on encoding translation technology and 3 755 commonly used Chinese characters were encoding translated with filtering criteria including GC content, hairpin, and the self-dimer annealing temperature. The BLAST tool was used to assess the homology of UTS generated by the design tool on various biological genomes. The UTS was experimentally evaluated on crops such as maize, tomatoes, peppers, and watermelons, constructing a species-universal experimental system and protocol. 【Result】The design tool, through encoding translation and screening, generated a total of 7 436 833 high-quality candidate UTS, accounting for 52.74% of all character combinations. Six selected UTS were demonstrated better specificity across 20 crop genomes by BLAST results compared to M13. With optimization of the general primer amplification procedure, the success rate of general primer amplification across multiple species reached or exceeded 95%, showing strong stability.【Conclusion】This study utilized encoding translation technology to develop UTS and paired them with a species-universal amplification system and protocol, which may provide a high-throughput, cost-effective universal detection method for fluorescent electrophoresis.

Establishment and Application of Dual RPA-LFD Detection Method for Capripox Virus and Orf Virus

WANG Ling-ling, MA Ai-hong, SONG Qian-jin, WANG Xiao-long, CAO Xiao-zhen, LIU Zhi-feng, CHEN Ya-fei, LI Ji-dong

2024, 40(5): 103-111. doi:10.13560/j.cnki.biotech.bull.1985.2023-1106

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【Objective】Based on recombinase polymerase amplification（RPA）detection technology combined with（lateral flow dipstick, LFD），it is aimed to establish dual RPA-LFD method for the rapid identification of capripox virus（CaPV）and orf virus（ORFV）. 【Method】The conserved fragments of CaPV P32 gene and ORFV 011 gene were selected for the amplification of target fragments and the construction of recombinant plasmids. Three pairs of primers for CaPV and ORFV RPA, and one probe for CAPV-HP and ORFV-HP were designed. Single base RPA primer screening test was performed. The reaction time and temperature of double RPA-LFD were optimized. The optimal primers and probe matching systems of dual RPA-LFD were explored. Dual RPA-LFD sensitivity, specificity and repeatability tests were performed. The method was used to detect 55 clinical samples. 【Result】The results of primer screening test showed that the primers had the strongest specificity and the highest amplification efficiency for CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1. The method had the best amplification efficiency at reaction temperature of 39℃ and reaction time of 11 min. The optimal primer ratio of CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1 was 0.8 μL∶1.6 μL, and the optimal probe ratio of CaPV-HP and ORFV-HP was 0.1 μL∶0.9 μL. The minimum detection limit of dual RPA-LFD sensitivity test was CaPV/ORFV 4.65/3.94×10⁰ copies/μL. The specific test results showed no cross-reaction with PPRV, IBRV, Sau and SPN. The positive rate of RPA-LFD was consistent with that of PCR. 【Conclusion】This study successfully established a dual RPA-LFD detection method for CaPV and ORFV, which can be used for the rapid differential diagnosis of CaPV and ORFV mixed infection in clinic.

Alternative Translation of Wheat Enolase-encoding Gene ENO2 and Its Prokaryotic Expression

ZHANG Na, LIU Meng-nan, QU Zhan-fan, CUI Yi-ping, NI Jia-yao, WANG Hua-zhong

2024, 40(5): 112-119. doi:10.13560/j.cnki.biotech.bull.1985.2023-1223

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【Objective】The objective of this study is to identify the genes（TaENO2s）encoding the enolase family member ENO2 in the important crop plant of wheat（Triticum aestivum L.）and to determine the alternative translation feature and protein enolase activity of TaENO2s.【Method】Wheat TaENO2s were identified by bioinformatics. One of the identified TaENO2s was selected as a representative for protoplast-based exogenous expression and characterization of alternative translation products. This selected TaENO2 was also subjected to prokaryotic expression for purification of recombinant protein. The enolase activity of the purified recombinant protein was determined with in vitro assays. 【Result】The hexaploid wheat genome had three homeologous TaENO2s which sequences encoded a conserved enolase catalytic center and had a putative alternative translation start site at the internal ATG codon encoding the residue M94（ATG^M94）of the enolase sequence. When exogenously expressed in protoplasts, the representative TaENO2 encoded two proteins, one full-length form of cytoplasmic and nuclear enolase protein and one N-terminal truncated form of nuclear transcriptional repressor TaMBP-1, while the variant of the same gene containing mutated ATG^M94only encoded the full-length form of enolase protein. Soluble recombinant GST-TaENO2 protein expressed in Escherichia coli was successfully purified and verified to have an in vitro enzymatic activity to catalyze the conversion from 2-phosphoglycerate（2-PGA）to phosphoenolpyruvate（PEP）. 【Conclusion】The wheat genome has three TaENO2 genes encoding the enolase protein ENO2. Under the condition of exogenous expression, TaENO2 encodes two protein products via mRNA alternative translation, one enolase protein translated from the first start codon and one TaMBP-1 protein translated from the internal start codon ATG^M94. The prokaryotically expressed recombinant protein GST-TaENO2 possesses in vitro enolase activity.

Genome-wide Association Study of Bolting Related Traits in Carrot

KONG Xiao-ping, CHEN Li-wen, LIU Si-si, YAN Xiang-ping

2024, 40(5): 120-130. doi:10.13560/j.cnki.biotech.bull.1985.2023-1120

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【Objective】Carrot plants are prone to early bolting under the influence of low temperature and long sunshine. Exploring SNP loci and candidate genes associated with carrot bolting traits would be beneficial for the breeding of new carrot varieties that are resistant to bolting. 【Method】This study used 240 carrot germplasm resources as materials to investigate the characteristics of carrot bolt（bolt time, bolt rate, bolt height, and bolt speed）in 2021 and 2022, respectively. Through DNA sequencing and SNP variation detection, high-quality SNP loci obtained from quality control were used for genome-wide association study（GWAS）of bolting related traits. 【Result】The bolting traits of 240 carrot germplasm resources demonstrated rich genetic diversity. After analyzing the 2-year data, it was found that there was significant variation in the phenotypes of each trait, with the highest coefficient of variation for bolting rate being 144.32% and 187.89%, and the lowest coefficient of variation for bolting time being 94.89% and 74.63%, respectively. The BLUE value reduced the error caused by the environment, with the lowest coefficient of variation being 22.53%. The correlation analysis results showed that the bolting rate in 2021 was significantly positively correlated with the bolting traits in 2022, and was extremely significantly correlated with the BLUE value. The BLUE value was highly significantly correlated with other traits except for no significant correlation with the bolt time in 2021. Based on the group structure analysis and cluster analysis, the associated groups were divided into four subgroups. Through a 2-year GWAS of the correlation among bolting traits, a total of 344 SNP marker loci were detected that were significantly correlated with bolting traits. Among them, 20 multi-effect loci were significantly correlated with 2 or more bolting traits, and the same loci can be considered as the same SNP. Based on the annotation information, 29 candidate genes related to bolting were screened, of which 14 genes had inhibitory effects on bolting and flowering, and 13 genes that promoted bolting and flowering were mainly related to the photoperiod pathway, vernalization pathway, and flowering integron. The mechanism of action of the other two genes was not yet clear and thus further verification is needed.【Conclusion】Multiple SNP loci associated with bolting traits were obtained by GWAS analysis, and related candidate genes were identified.

Cloning and Expression Analysis of Banana EARLY FLOWERING 3（ELF3）Genes

HAO Si-yi, ZHANG Jun-ke, WANG Bin, QU Peng-yan, LI Rui-de, CHENG Chun-zhen

2024, 40(5): 131-140. doi:10.13560/j.cnki.biotech.bull.1985.2023-1163

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【Objective】EARLY FLOWERING 3（ELF3）, an important component of the core circadian oscillator, has been identified to function greatly in regulating circadian and flowering, and stress responses of plants. Up to now, however, there has been no report on banana ELF3. Our present study is aimed to explore the functions of ELF in banana responses to stresses.【Method】Four ELF3 genes（MaELF3-1-MaELF3-4）identified from banana（Musa acuminata）genome were cloned using reverse transcription PCR and subjected to a series of bioinformatic analysis. Their expression patterns under treatments of high and low temperature, banana wilt disease pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4（FocTR4）, MeJA and ABA were analyzed using transcriptome data and quantitative real time PCR（RT-qPCR）. 【Result】The coding sequences of the four MaELF3s ranged from 2 058 bp to 2 301 bp, encoding proteins consisted of 685-766 amino acids. Except that MaELF3-4 had three exons, all the other three MaELF3s had four exons. Protein analysis revealed that all MaELF3s were nucleus-located unstable basic proteins; unlike the ELF3s from temperate plants, all ELF3s from banana and some other tropical plants did not contain the prion-like domain（PrD）. Phylogenetic analysis results showed that MaELF3-2 and MaELF3-4 shared the closest relationship with Arabidopsis ELF3（At2g25930）, while MaELF3-1 and MaELF3-3 was the closest to ELF3 from Ensete ventricosum and Musa balbisiana, respectively. The promoter regions of MaELF3s contained many light-, phytohormone（such as MeJA and ABA）- and stress（such as drought and low temperature）-responsive elements. Gene expression analysis revealed that, the expressions of all the four MaELF3s in the leaves were greatly influenced by JA and ABA treatments, and their expressions in the banana roots was significantly downregulated by FocTR4. Moreover, the expressions of all MaELF3s except MaELF3-4 were downregulated by both high and low temperature treatments. 【Conclusion】The results obtained in this study indicate that MaELF3s participate in the banana responses to different stresses.

Identification and Tissue Expression of Four Transcription Factors in Pholidota chinensis

LIU Bao-cai, ZHANG Wu-jun, ZHAO Yun-qing, HUANG Ying-zhen, CHEN Jing-ying

2024, 40(5): 141-152. doi:10.13560/j.cnki.biotech.bull.1985.2023-1096

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【Objective】Pholidota chinensis Lindl is a rare and endangered epiphytic orchid. The epiphytes have evolved many special morphological structures and physiological and biochemical characteristics compared to geophytes. The transcription factors play a role in regulating plant growth and development. The aim of this paper is to explore and discover transcription factors in P. chinensis that regulate its growth and development.【Method】This study used PacBio and Illumina sequencing techniques to obtain the full-length transcriptome data of P. chinensis and the gene expressions in the roots, rhizomes, pseudobulbs and leaves. After further bioinformatics analysis, the four transcription factor family members, physicochemical properties and tissue expression specificity of P. chinensis were revealed. The expression of C2H2 family and bHLH family was analyzed by RT-qPCR using sodium chloride（NaCl）and methyl jasmonate（MeJA）induced adversity stresses.【Result】The four identified transcription factors and their families members in P. chinensis were: 21 members in C2H2 family, 27 members in bHLH family, 30 members in WRKY family, and 19 members in bZIP family. These four families and their members were mainly hydrophilic unstable proteins, and their subcellular localization was mainly located in the nucleus. Some members of bHLH families were located in cytoplasm, chloroplast and other organelles. The gene family had typical conserved domain characteristics, and showed obvious tissue specificity in the roots, rhizomes, pseudobulbs and leaves of P. chinensis. Expressions of all genes in C2H2 families and bHLH families were affected by NaCl and MeJA, but family members differed in whether they were promoted or repressed in the expression and in the amount of their expression.【Conclusion】The number of four transcription factor family members in P. chinensis was identified, and the members of each family are mainly hydrophilic unstable proteins located in the nucleus with conserved structural domain features and tissue expression specificity.

Identification and Expression Pattern Analysis of Rosa roxburghii SOD Gene Family

WEN Jie, DU Yuan-xin, WU An-bo, YANG Guang-rong, LU Min, AN Hua-ming, NAN Hong

2024, 40(5): 153-166. doi:10.13560/j.cnki.biotech.bull.1985.2023-1138

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【Objective】Superoxide dismutase（SOD）is a natural scavenger of oxygen free radicals in plants, which plays a crucial role in protecting plants from environmental stress. The aim of this study is to investigate the role of SOD in response to drought stress in Rosa roxburghii and to provide a basis for further research on the function of the SOD gene family in Rosa roxburghii. 【Method】The SOD gene family of R. roxburghii was systematically identified and analyzed by bioinformatics method. The physicochemical properties, chromosome location, gene structure, subfamily evolution, cis-acting elements and WGCNA were comprehensively analyzed. Additionally, the expression pattern of the SOD gene family of R. roxburghii under drought stress was analyzed by RT-qPCR. 【Result】There was a total of nine members of SOD gene family in R. roxburghii, including four Cu/ZnSOD genes, three MnSOD genes, and two FeSOD genes. These genes were scattered across five chromosomes. The analysis of gene structure indicated that members within the same subfamily had similar motifs, but there were notable variations in the number and arrangement of introns/exons. Subfamily evolution analysis demonstrated that the MnSOD subfamily was the most primitive, with high conservation across all protein positions, followed by the FeSOD and Cu/ZnSOD subfamilies. The Cu/ZnSOD subfamily could be further divided into three subclasses due to significant sequence differences. Cis-element analysis revealed the involvement of the SOD gene family in various plant hormones, growth, and stress responses. Notably, the promoter region of RrCSD2 contained flavonoid biosynthetic elements（MBSI）. Transcriptome and WGCNA analysis further revealed that the genes of RrCSD2 module were significantly enriched in flavonoid and flavonol biosynthesis, as well as flavonoid biosynthesis and arachidonic acid metabolism, suggesting that RrCSD2 may be involved in flavonoid metabolism. RT-qPCR results showed that the expressions of RrMSD1 and RrMSD2 significantly decreased under drought stress, while the expressions of the other seven genes increased overall. Specifically, the expressions of RrCSD2 and RrCSD3 were significantly up-regulated. 【Conclusion】The SOD genes of R. roxburghii play a crucial role in drought stress response.

Identification and Expression Analysis of AcHSP20 Gene Family in Kiwifruit

HOU Ya-qiong, LANG Hong-shan, WEN Meng-meng, GU Yi-yun, ZHU Run-jie, TANG Xiao-li

2024, 40(5): 167-178. doi:10.13560/j.cnki.biotech.bull.1985.2023-1092

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【Objective】The identification and analyses of heat shock proteins（HSP20s/sHSPs）in kiwifruit would lay foundation for the functional studies of AcHSP20 genes and resistance breeding of kiwifruit. 【Method】Based on the genome-wide data of kiwifruit, the AcHSP20 gene family was identified, and the physicochemical properties, phyletic evolution, chromosome distribution, gene structure, subcellular localization as well as promoters were analyzed by bioinformatics. 【Result】The 34 members of AcHSP20 gene family were identified. They encoded proteins with 85-371 amino acids, 9.93-40.18 kD molecular weight（Mw）and 4.4-9.3 isoelectric points（pI）. Meanwhile, most AcHSP20 proteins were predicted to be located in cytoplasm and chloroplast. The 34 AcHSP20 genes were distributed on 17 chromosomes, and there were no or only one intron, mostly. Promoters of AcHSP20 genes had phytohormone and abiotic stress response elements. All the detected AcHSP20 genes expressed ubiquitously in the roots, stems and leaves of kiwifruit and the expressions changed significantly under abiotic stresses and phytohormone treatments. 【Conclusion】The AcHSP20 gene family plays important roles in high temperature, ABA, MeJA, NaCl stress responses in kiwifruit.

Genome-wide Identification and Expression Analysis of the SWEET Gene Family in Camellia oleifera

DU Bing-shuai, ZOU Xin-hui, WANG Zi-hao, ZHANG Xin-yuan, CAO Yi-bo, ZHANG Ling-yun

2024, 40(5): 179-190. doi:10.13560/j.cnki.biotech.bull.1985.2024-0021

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【Objective】To explore the involvement of SWEET（sugars will eventually be exported transporters）in sugar metabolism and response to abiotic stress in Camellia oleifera.【Method】Bioinformatics methods were used to analyze the gene structure, protein motif, chromosome localization, collinearity, cis-acting elements of promoter region and upstream regulators in CoSWEETs family, and RT-qPCR was used to analyze the expression pattern of CoSWEETs in different periods, tissues and stress responses.【Result】Total 14 CoSWEET genes were identified in Camellia oleifera for the first time, they were localized on 10 chromosomes, and there were differences in intron-exon numbers between different members. With 1-2 MtN3 conserved domain, the 14 CoSWEETs were divided into four subgroups by phylogenetic tree, and there were similar gene structures and motif in the same subgroup. There were differences in number of introns and exons among different members. Based on the analysis of promoter cis-acting elements and upstream transcription factor predictions, multiple cis-elements related to development, plant hormones and environmental stresses were found in the promoter region, and their expressions were regulated by transcription factors, like ERF, DOF, BBR-BPC and MYB. RT-qPCR results showed that CoSWEETs highly expressed in the fruit and root, and the expression in the seeds was related to the developmental stages. Moreover, several genes responding to low-temperature, drought or salt stress, such as CoSWEET1, CoSWEET2 and CoSWEET17 were mined based the expression profile of CoSWEETs members under abiotic stresses of low temperature, drought and high salt.【Conclusion】The expression of CoSWEETs is regulated by various hormones and transcription factors, and plays a crucial role in the development of seeds and the response to stress in Camellia oleifera.

Identification and Expression Analysis of the NRAMP Family in Seabuckthorn Under Lead Stress

LI Jia-xin, LI Hong-yan, LIU Li-e, ZHANG Tian, ZHOU Wu

2024, 40(5): 191-202. doi:10.13560/j.cnki.biotech.bull.1985.2023-1161

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【Objective】The NRAMP（natural resistance-associated macrophage protein）gene family plays a pivotal role in the transport of divalent metal ions at the cellular level. To identify the key NRAMP family genes in seabuckthorn (Hippophae rhamnoides) that confer resistance to heavy metal lead stress and to elucidate the associated molecular mechanisms.【Method】Using bioinformatics techniques, we identified seabuckthorn's NRAMP gene family members and comprehensively analyzed their physicochemical properties, phylogenetic relationships, gene structures, conserved motifs, cis-acting elements, expression profiles under varying Pb stress concentrations, intraspecific and interspecific variability, and protein interaction networks. 【Result】The 11 NRAMP gene family members were distributed across seven chromosomes and categorized into Groups 1-Group 3. Promoters of the NRAMP genes contained numerous hormonal, meristematic, and environmental stress response elements. Transcriptome analysis indicated differential expressions of NRAMP genes in seabuckthorn under varying lead ion concentrations, with eight genes significantly down-regulated at 1 000 mg/kg and seven up-regulated at 5 000 mg/kg lead levels. RT-qPCR validated the expression patterns of six chosen NRAMP genes in seabuckthorn. The seabuckthorn NRAMP genes showed greater interspecific co-linkage with monocotyledonous wheat than with the dicot Arabidopsis thaliana. Protein network analysis suggested that HrLNRAMP8 potentially regulates heavy metal uptake and translocation primarily via the ethylene pathway. 【Conclusion】A systematic identification of 11 HrLNRAMP family members from seabuckthorn was achieved at the whole-genome level. Members of the Group2 subfamily have the most complex gene structures and lower consistency. All seabuckthorn NRAMP members contain the unique transport domain motif1 of the NRAMP family. The study demonstrates preferential expression of different gene family members under lead stress conditions. This gene family regulates gene expression levels in response to heavy metal lead ion stress, thereby mitigating damage caused by heavy metal stress.

Analysis of Endogenous Hormone Regulation Mechanism for Carotenoid Synthesis in Sarcandra glabra

WU Di, YOU Xiao-feng, ZHENG Yi-zheng, LIN Nan, ZHANG Yan-yan, WEI Yi-cong

2024, 40(5): 203-214. doi:10.13560/j.cnki.biotech.bull.1985.2023-1047

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【Objective】This work aims to explore the possible regulatory mechanism of endogenous hormones on the differential accumulation of carotenoids in different organs of Sarcandra glabra. 【Method】In this study, metabolomics was used to analyze the differential distribution of endogenous hormones and carotenoid metabolites in different organs of S. glabra. Combined with transcriptomics technology, the differential enzyme genes related to carotenoids in S. glabra were excavated, and the transcription factors involved in the regulation of differential enzyme genes and their hormone-related cis-response elements were further predicted, so as to analyze the possible regulation of endogenous hormones on the differential accumulation of carotenoids. 【Result】The contents of seven endogenous hormones such as indole-3-carboxylic acid,[（-）-jasmonic acid], ethyl dihydrojasmonate, castasterone and brassinolide were high in the leaves. Consistent with the enrichment sites of 8 differential metabolites, such as canthaxanthin, echinenone, neoxanthin and nostoxanthin; and abscisic acid, gibberellin 4,5-methoxysalicylic acid, dihydrojasmonic acid and 5,6-dihydroxyindole was high in the roots, it was consistent with the accumulation site of 3 differential metabolites, abscisic aldehyde, 4,4'-aiapolycopenedial and antheraxanthin. The key transcription factor MYB106, SPL1, NAC015, ERF064, WRKY44 and BHLH116 responded to the stimulation of plant hormones such as AUX, SA, GA, ABA, and Me_ JA, and participate in the regulation of carotenoid synthesis pathway DXS, VDE, ABA2, AOG, ZEP, CYP97C1, DWARF27, CRTISO, LCYB expression of these 9 metabolic enzymes. Quantitative real-time polymerase chain reaction（RT-qPCR）results showed that the expression trends of 8 randomly selected differentially expressed genes were consistent with the transcriptome sequencing results. 【Conclusion】The 15 differential endogenous hormones and 11 differential metabolites related to carotenoids in the leaves and roots of Sarcandra glabra were screened, and 6 transcription factors were predicted to be involved in the regulation of 16 metabolic enzymes.

Response of Arabidopsis AtiPGAM2 Gene to Abiotic Stress

LI Jing-yan, ZHOU Jia-jing, YUAN Yuan, SU Xiao-yi, QIAO Wen-hui, XUE Yan-lei, LI Guo-jing, WANG Rui-gang

2024, 40(5): 215-224. doi:10.13560/j.cnki.biotech.bull.1985.2023-1139

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【Objective】The AtiPGAM2 gene is a member of the alkaline phosphatase superfamily in Arabidopsis and is involved in the glycolysis process. Studying the response mechanism of AtiPGAM2 gene under stress conditions lays the foundation for further studying the stress resistance function of AtiPGAM2.【Method】This study used PlantCARE online software to analyze the cis-acting elements of the AtiPGAM2 gene promoter. Real time fluorescence quantitative PCR was used to detect the response patterns of AtiPGAM2 under NaCl and ABA stress. The AtiPGAM2 gene was cloned and transferred it into Arabidopsis to obtain an overexpressing strain of the AtiPGAM2 gene. The Atipgam2 mutant was identified using the three primer method. Statistical analysis was conducted on the germination rate, green seedling rate, and phenotypes under various abiotic stresses, including mannitol, ABA, and MeJA, for overexpression, mutants, and wild-type under salt stress.【Result】There are multiple abotic stress and hormone responsive elements on its promoter, such as light, MeJA, low temperature, ABA, SA, GAs, etc. Fluorescence quantitative PCR analysis showed that AtiPGAM2 was induced to varying degrees under NaCl and ABA stress. AtiPGAM2 gene overexpression and mutant strains were obtained successfully. Under salt stress, the germination rate and green seedling rate of AtiPGAM2 overexpressing strains were higher than those of the wild type, while the mutant surface type was the opposite. The germination rate of the mutant under mannitol treatment was lower than that of the wild-type. The germination rates of mutant and overexpressing AtiPGAM2 strains treated with ABA within 48 h were lower than those of the wild-type. Under MeJA treatment, the number of overexpressed lateral roots was higher than that of the wild-type, while the mutant was the opposite.【Conclusion】AtiPGAM2 has the tolerance to salt, and this gene responds to mannitol, ABA, and MeJA treatments.

Comparison and Analysis of Endophytic Bacterial Communities in Different Perennial Rice Varieties

KONG De-ting, QI Xiao-han, LIU Xing-lei, LI Li-ping, HU Feng-yi, HUANG Li-yu, QIN Shi-wen

2024, 40(5): 225-236. doi:10.13560/j.cnki.biotech.bull.1985.2023-1088

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【Objective】To explore the structure and potential functions of endophytic bacterial microbiota in perennial rice, as well as the relationship between endophytic bacteria in perennial rice and host agronomic traits. 【Method】The diversity, composition and metabolic function of endophytic bacteria in different in perennial rice varieties（PR23, PR25, and PR107）were characterized via Illumina sequencing of the 16S rRNA gene. 【Result】These results showed that the beta diversity of endophytic bacterial communities significantly differed among the distinct genotypes of perennial rice. Due to the differentiation of tissue structures, perennial rice varieties shaped distinctive ecological niches for endophytes in different tissue compartments. Proteobacteria, Firmicutes and Bacteroidetes were the dominant endophytic bacterial phyla in the underground tissues of perennial rice, while Proteobacteria, Actinobacteria and Firmicutes were the dominant phyla in the aboveground tissues. These perennial rice varieties had different endophytic bacterial taxa with specific amplicon sequence variants（ASVs）, dominant genera and specific biomarkers. The endophytic bacterial communities of perennial rice were reassembled for various metabolic functions, including biosynthesis, degradation/utilization/assimilation, detoxification, generation of precursor metabolite and energy, glycan pathways, macromolecule modification and metabolic clusters. Significantly different MetaCyc pathways of endophytic bacteria in perennial rice varieties with distinct genotypes were involved in the biosynthesis and degradation of esters, alcohols and polysaccharides. 【Conclusion】These results indicated host genotypes and tissues differentiation affect the community structure, species composition and metabolic function of endophytic bacteria in perennial rice.

Study on the Microbial Community of Rhizosphere Soil in Ancient Tea Garden and Modern Organic Tea Garden in Jingmai Mountain

YU Li-jun, WANG Qiao-mei, PENG Wen-shu, YAN Liang, YANG Rui-juan

2024, 40(5): 237-247. doi:10.13560/j.cnki.biotech.bull.1985.2023-1104

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【Objective】It is to study the composition and diversity of rhizosphere soil microbial communities in tea gardens of different habitats, to identify the main driving factors affecting rhizosphere soil microbial communities, and to provide theoretical basis for the study of the relationship between rhizosphere soil microbial communities and pest control of tea trees, as well as for the function and utilization of soil microbials. 【Method】We used Illumina-MiSeq high-throughput sequencing technology to investigate the community composition and diversity of soil microorganisms in the rhizosphere soil communities of Jingmai Mountain Ancient Tea Garden（GS）and Jingmai Mountain Modern Organic Tea Garden（TS）, and used redundancy analysis to explore the relationship between soil nutrients and microorganisms.【Result】The cation exchange capacity, organic matter, humus, ammonia nitrogen, ammonium nitrogen, available phosphorus, and water content in the rhizosphere soil in TS were higher than those in the GS, showing significant differences; the dominant bacterial populations in both gardens were Actinobacteria, Acidobacteria, and Planctomycetes, and the dominant fungal populations are Ascomycota, Mortierellomycota, and Basidiomycota, except for the unclassified genera. The dominant bacterial genera in GS are very similar to those in TS, but the relative abundance of each genus varies greatly in two gardens. Redundancy analysis shows that cation exchange capacity, organic matter, humus, ammonia nitrogen, and ammonium nitrogen were the main environmental factors causing differences in the microbial community structure and diversity in the rhizosphere soil of two tea gardens. 【Conclusion】The nutrient and species richness of the rhizosphere soil of TS was significantly higher than that of Gs, and the composition of the dominant bacterial genera in the rhizosphere soil of two tea gardens was highly similar, but their abundance varied significantly, showing a high degree of correlation with soil nutrients.

Biocontrol Characteristics of Bacillus atrophaeus CNY01 and Its Salt-resistant and Growth-promoting Effect on Maize Seedling

SUN Ya-nan, WANG Chun-xue, WANG Xin, DU Bing-hai, LIU Kai, WANG Cheng-qiang

2024, 40(5): 248-260. doi:10.13560/j.cnki.biotech.bull.1985.2023-1174

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【Objective】A rhizobacterium with salt-resistant and growth-promoting effects was screened from the saline-alkali soil in Yellow River Delta to prepare an agent for alleviating high-salt stress of maize seedlings. 【Method】The salt-resistant and growth-promoting functions of the strain were identified on functional mediums, and the species of the strain was determined by 16S rDNA sequence and whole genome analysis. The strain was applied to maize rhizosphere to verify the salt-resistant and growth-promoting effects of the strain on maize seedlings.【Result】A strain Bacillus atrophaeus CNY01 was screened from the saline-alkali soil of Yellow River Delta. Strain CNY01 grew normally under the condition of 0-16% NaCl and pH 5-8. Strain CNY01 had the ability of degrading protein and dissolving potassium, and inhibited the growth of plant pathogens such as Ralstonia solanacearum and Fusarium moniliforme. The application of strain CNY01 promoted the growth of maize seedlings under high-salt condition and alleviated the stress of high salt on maize seedlings. When strain CNY01 was applied to maize seedlings in nutrient solution containing 100 mmol/L NaCl for 8 d, the physiological plant height, root length, and fresh weight of maize seedlings increased by 38.80%（P<0.01）, 23.73%, and 28.19%（P<0.01）, respectively. When strain CNY01 was applied to maize seedlings in pot experiments adding 100 mmol/L NaCl for 28 d, the physiological plant height, the aboveground fresh weight, and the underground fresh weight of maize seedlings increased by 10.84%, 41.87%（P<0.01）, and 23.29%, respectively. Genes that maintain cell osmotic pressure and synthesize stress proteins to alleviate high salt stress were also predicted in the genome sequence of this strain.【Conclusion】A strain of B. atrophaeus CNY01 is screened from saline-alkali soil of Yellow River Delta, which has the function of preventing disease and promoting growth, and has significant salt-resistant and promoting growth effect on maize plants. Meanwhile, strain CNY01 contains genes related to salt-resistant and growth-promoting. B. atrophaeus CNY01 is an important strain resource.

Degradation of Nematodes by Chitinase AO-492 from Arthrospora oligospora

ZHANG Jia-hua, ZHANG Hui-mei, MA Xi-xi, SUN Yan-sen, LI Ruo-bing, LI Ning-xing, CAI Xue-peng, QIAO Jun, MENG Qing-ling

2024, 40(5): 261-268. doi:10.13560/j.cnki.biotech.bull.1985.2024-0010

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【Objective】To explore the biological function of secreted protein AO-492 in the process of chitin induction from nematode-trapping fungus Arthrobotrys oligospora.【Method】The coding region of the main domain of chitinase AO-492 from A. oligospora was cloned and analyzed, and expressed in Pichia pastoris. The recombinant protein ReAO-Z492 was purified by nickel column affinity chromatography. The enzymatic activity of the recombinant protein under different temperature, pH and metal ion conditions was analyzed by NAG assay, and its biological function was analyzed by acting on Caenorhabditis elegans and eggs.【Result】The chitinase AO-492 had a signal peptide, no transmembrane domain, contained two chitin binding domains and a glycoside hydrolase 18 family domain, and the highly conserved substrate binding site of the glycoside hydrolase 18 family chitinase -SVGGWT- and the hydrolase active site -FDGGDLDWE-, as well as a typical TIM barrel molecular structure. Phylogenetic analysis showed that the protein had the closest genetic relationship with chitinase（EPS35099.1）. SDS-PAGE and Western Blot analysis showed that the molecular weight of ReAO-Z492 was about 59 kD, which specifically reacted with mouse polyclonal antibody against A. oligospora. The optimal temperature and pH of ReAO-Z492 were 40℃ and 7.0, respectively. Mg²⁺ promoted the enzyme activity, while Ag⁺, Cu²⁺, Fe³⁺ and Zn²⁺ inhibited it. ReAO-Z492 had strong degrading activity on the body wall and egg shell of C. elegans.【Conclusion】The chitinase AO-492 of A. oligospora has a strong degradation effect on C. elegans and its eggs.

Effects of Carbohydrate-binding Modules on the Enzymatic Properties of Xylanase

JIANG Wen-ping, RAN Qiu-ping, LIU Jia-shu, ZHANG Hui-min, ZHANG Di, JIANG Zheng-bing, LI Hua-nan

2024, 40(5): 269-279. doi:10.13560/j.cnki.biotech.bull.1985.2023-1032

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【Objective】This study is aimed to explore the binding ability of different sources of CBM to beech-xylanan, and to fuse exogenous CBM with high binding ability to the C-terminal and N-terminal of Streptomyces L10904 xylanase（XYN）, to explore the effects of exogenous CBM on the enzymatic properties of xylanase. 【Method】First, through the substrate adsorption method, the concentration of CBM in the solution before and after adsorption was detected by Coomassie Brilliant blue G250 method, and the substrate binding rate of CBM was calculated. CBM1 and CBM4 with better xylan binding ability were screened. In order to explore the effect of the fusion location of CBM with high substrate binding ability on the enzymatic properties of xylanase, CBM1 and CBM4 were fused with the C-terminal and N-terminal of XYN by flexible binding peptide, and four recombinant enzymes were obtained by expression in Escherichia coli BL21（DE3）. They were named CBM1-XYN, XYN-CBM1, CBM4-XYN, and XYN-CBM4.【Result】The binding rates of CBM1 and CBM4 to xylan were 89% and 95%, respectively. The specific activities of XYN, CBM1-XYN, XYN-CBM1, CBM4-XYN and XYN-CBM4 were 32 274.81, 49 342.21, 602.48, 230.42 and 2 362.24 U/mg, respectively, measured at 60℃ and pH 7.0. The specific activities of CBM1-XYN were 1.5 times higher than the specific activities of XYN. The analysis of enzymatic properties showed that CBM1 improved the temperature stability and pH stability of XYN, XYN and CBM1-XYN were incubated at 60℃ for 1 h, and the residual enzyme activity of CBM1-XYN and XYN was 81% and 28%, respectively. In the pH range of 3-11, CBM1-XYN maintained more than 90% enzyme activity after incubation at 4℃ for 12 h. 【Conclusion】Streptomyces derived xylanase was heterogeneically expressed in E. coli BL21（DE3）, and CBM1 and CBM4 with high substrate binding rates were screened. CBM was successfully fused to XYN by protein fusion technology, and CBM1-XYN with improved enzymatic properties was obtained, which improved the temperature stability, pH tolerance and specific enzyme activity of xylanase.

Prokaryotic Expression, Subcellular Localization and Expression Analysis of PcCHS Gene from Polygonatum cyrtonema Hua

PAN Ping-ping, XU Zhi-hao, ZHANG Yi-wen, LI Qing, WANG Zhong-hua

2024, 40(5): 280-289. doi:10.13560/j.cnki.biotech.bull.1985.2023-1100

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【Objective】This work aims to explore the role of chalcone synthase（chalcone synthase，PcCHS）gene in the synthesis of flavonoids in Polygonatum cyrtonema Hua, which may provide a reliable theoretical basis for the subsequent analysis of the function of PcCHS and the breeding of new varieties of P. cyrtonema Hua. 【Method】Using P. cyrtonema Hua as cDNA template, the coding sequence of PcCHS gene was cloned, and the gene was analyzed bioinformatically. The prokaryotic expression vector of PcCHS was constructed and the recombinant protein was purified to verify the expression activity of PcCHS in vitro. Transient overexpression system was used to investigate the changes of total flavonoids content after overexpression of this gene. Gateway technology was used to construct the subcellular localization vector 35S::PcCHS-GFP, and the subcellular localization of target protein was determined by the tobacco expression system. 【Result】The results showed that PcCHS was a hydrophilic protein with an open reading frame of 1 251 bp, a theoretical molecular weight of 44.63 kD and an isoelectric point of 5.89, and was closely related to AoCHS（Asparagus officinalis）. Prokaryotic expression experiment showed that pET28a-PcCHS was induced to express soluble recombinant protein by IPTG（isopropyl-β-d-thiogalactoside）. Western-blot showed that the size of pET28a-PcCHS was about 45 kD, which was consistent with the expected size. The purified protein had certain enzymatic activity and catalyzed the conversion of p-coumaryl-CoA and malonyl-CoA into naringin chalcone. In addition, in the transient overexpression of PcCHS, the expression level of PcCHS group was significantly higher than that of no-load K group, and the total flavonol content was also significantly higher than that of no-load K group, up to 1.83 times. Subcellular localization results showed that the gene plays a role in the cell membrane and nucleus. 【Conclusion】Prokaryotic expression of PcCHS gene has enzyme activity in vitro, and its subcellular location is in cell membrane and nucleus, and instantaneous overexpression significantly increase the total flavonoid content in the leaves of P. cyrtonema Hua.

Anaerobic Expression of Lactate Dehydrogenase to Improve the D-lactic Acid Optical Purity Procluced by Escherichia coli

WANG Zhou, YU Jie, WANG Jin-hua, WANG Yong-ze, ZHAO Xiao

2024, 40(5): 290-299. doi:10.13560/j.cnki.biotech.bull.1985.2023-0979

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【Objective】This work aims to solve the problem that the light purity of the final product D-lactic acid is reduced while using cheap raw materials（containing a small amount of L-lactic acid）such as agricultural rough processing or waste in the industrial fermentation production of D-lactic acid.【Method】The expressing plasmids of L-lactate dehydrogenase gene lldD, pUC19-PLD（PpflBp6）, pUC19-NLD（PnirB）and pUC19-PNLD（PpflBp6-PnirB）with different promoters, were constructed and transformed into Escherichia coli D-lactic acid engineering strain HBUT-D, and strain HBUT-D3, HBUT-D5 and HBUT-D7 was acquired respectively. Through LldD enzymatic activity test and TOPSIS multivariate evaluation on NBS fermentative experiments（supplemented with 1 g/L L-lactic acid）, one strain was screened by its capability for fast elimination of L-lactic and high fermentative efficiency of D-lactic acid, for following fermentative experiments with cheap raw materials. 【Result】HBUT-D7 was evaluated as the optimal strain based on the LldD specific enzyme activity of 64 U/g, L-lactate consumption rate of 34 mg/(L·h), and D-lactic acid productivity of 4.09 g/(L·h). When fermented with corn syrup, the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 10.41 mg/(L·h)and 34.75 mg/(L·h), respectively. The productivities of D-lactic acid were 4.24 g/(L·h)and 3.87 g/(L·h), respectively. The optical purities of D-lactic acid were 99.07% and 99.92%, respectively. When fermented with molasses, the L-lactic acid consumption rates of HBUT-D and HBUT-D7 were 6.87 mg/(L·h)and 17.18 mg/(L·h), respectively. The productivities of D-lactic acid were 1.93 g/(L·h)and 1.88 g/(L·h), respectively. The optical purities of D-lactic acid were 99.22% and 99.99%, respectively. 【Conclusion】The expressing plasmid of the anaerobic inducible promoter may increase the L-lactate dehydrogenase enzyme activity in strain HBUT-D7, thus it can eliminate the L-lactic acid to increase the optical purity of D-lactic acid while using cheap raw materials for fermentation.

Prediction of H5Nx Avian Influenza Virus Cross-reactivity CD4⁺ T-cell Epitopes

XIE Shuang, CHEN Yao, HUANG Jun-qiong

2024, 40(5): 300-309. doi:10.13560/j.cnki.biotech.bull.1985.2023-1006

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【Objective】To evaluate preexisting H5Nx cross-reactivity immune memory and to provide theoretical data for the prevention and treatment of human H5 avian influenza viruses and the development of broad-spectrum vaccines.【Method】Bioinformatics methods were used to screen the common conserved cross-reactivity CD4⁺ T cell epitopes of human H5Nx avian influenza viruses and seasonal influenza viruses, and further analysis of the nested CD8⁺ T cell epitopes and B cell epitopes of conserved epitopes and their coverage among the Chinese population.【Result】92.3%（513/555）of H5Nx CD4⁺ T cell epitopes were cross-reactivity in seasonal influenza A viruses, of which 172 epitopes nested CD8⁺ T cell epitopes and 5 epitopes nested B cell epitopes. These epitopes and the corresponding HLA-DRB1 alleles have a strong coverage rate of 88.65% in the world population.【Conclusion】Due to repeated exposure to seasonal influenza A viruses, there is a certain level of H5Nx cross-reactivity immune memory in the population that may provide partial protection from H5Nx infection.

Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage

WANG Xin-xin, GUAN Yu-zhu, LI Xiao-wei, HONG Wei, WU Dao-yan, KANG Ying-qian, LIU Yong-chang, CHEN Zheng-hong, CUI Gu-zhen

2024, 40(5): 310-320. doi:10.13560/j.cnki.biotech.bull.1985.2023-1114

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【Objective】Catalase（KatA）is an important virulence factor encoded by Helicobacter pylori, and plays an important role in bacterial resistance to host immune killing and vaccine development. The objective of this study is to identify the enzymatic properties of H. pylori catalase and to analyze its function in H. pylori oxidation tolerance.【Method】Firstly, katA gene was isolated from the genome of clinical drug-resistant strain Hp_G272, and KatA^G272protein was cloned and expressed in Escherichia coli. Then, the catalase activity of KatA^G272 was analyzed in vitro with CAT detection kit. Secondly, katA genetic engineering strains were constructed by homologous recombination, including knockout strains and complemented strains. Finally, the function of katA gene and its role in tolerance to oxidative damage of H. pylori were analyzed by comparing the differences in growth phenotype, decomposition and tolerance of hydrogen peroxide between wild and engineered strains.【Result】KatA^G272 was successfully cloned, expressed and purified from E. coli. Enzymatic analysis showed that KatA^G272 was a class of eosinophilic catalase. Gene knockout analysis showed that H. pylori lost its ability to decompose and tolerate hydrogen peroxide, while regained its ability to decompose and tolerate hydrogen peroxide after gene complement.【Conclusion】katA is the only functional gene that decomposes and tolerates hydrogen peroxide in H. pylori, and plays an important role in the oxidative tolerance of H. pylori.

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