Biotechnology Bulletin

Systematic Control of Antibiotic Resistance in Bacteria: From Unknown Mechanisms to Novel Strategies

SUN Jian, LIU Ya-hong

2022, 38(9): 1-3.

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Antimicrobial Resistance：Biochemical Mechanisms and Countermeasures

LIU Cheng-cheng, HU Xiao-fang, FENG You-jun

2022, 38(9): 4-16. doi:10.13560/j.cnki.biotech.bull.1985.2022-0759

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The improper use of antibiotics promotes the emergence and global dissemination of antimicrobial resistance（AMR）. AMR is listed as the 3^rd cause leading to human death,seriously threatening public health and food security. Therefore,it is urgently demanded to enhance the studies on AMR mechanisms,and pursue novel prevention and control strategies against AMR. This review summaries major AMR biochemical mechanisms,including drug uptake limitation,drug target modification,drug inactivation,and active drug efflux. Then the review outlines its prevention and control approaches such as antimicrobial peptide therapy,immunotherapy,phage therapy,antimicrobial photodynamic therapy,probiotic therapy,antimicrobial nanoparticle technology,antisense oligonucleotide and gene editing technology,host-directed therapy,etc. This review aims to popularize the science of bacterial drug resistance and provide a reference for relevant studies on drug resistance.

Advances in the Discovery of Novel Antibiotic-resistant Genes Based on Functional Metagenomics

LU Zhao-xiang, WANG Xi-ran, LIAN Xin-lei, LIAO Xiao-ping, LIU Ya-hong, SUN Jian

2022, 38(9): 17-27. doi:10.13560/j.cnki.biotech.bull.1985.2022-0537

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The widespread use of various antibiotics in the treatment of bacterial infections and the widespread clinical use of antibiotics drives the continuous evolution of various antibiotic resistance genes（ARGs）, leading to the increasing problem of drug resistance. The global spread of antibiotic-resistant genes and bacteria severely threatens the public health and results in tremendous economic losses in recent decades. Of note, the excessive usage of antibiotics not only led to the occurrence of antibiotics-resistant bacteria, but also constitutively elevated the selection pressure of environmental microbiome, indirectly accelerated the development and evolution of antibiotics-resistant genes, making environmental microbiome as the storage reservoir of antibiotics-resistant genes. Therefore, the extensive monitoring of antibiotics-resistant bacteria and the early exploration of new antibiotics-resistant genes are of great clinical significance and research value. However, traditional methodologies for ARGs identification and profiling largely depend on the molecular characterization of cultivable bacteria, hardly providing a comprehensive landscape of the resistome in a given ecological niche. To cope with the increasing necessity to uncover the ARGs in uncultivable bacteria, the functional metagenomics emerged as a promising toolkit that combines the phenotypic profiling with next high-throughput sequencing, it does not depend on the culture of specific bacteria with target genes, and thus it is deemed as a powerful alternative to reveal all novel functional genes. In the present review, we systematically summarized the latest advances in the field of functional metagenomics and dated their recent applications in unveiling the unknown ARGs from environments. This review offers a solid theoretical foundation for in-depth understanding the resistant mechanism of antibiotics.

Research Progress in Colistin（COL）Resistance in Bacterial and Its Reversal Mechanism

HU Gong-zheng, CUI Xiao-die, ZHAI Ya-jun, HE Dan-dan

2022, 38(9): 28-34. doi:10.13560/j.cnki.biotech.bull.1985.2022-0384

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Colistin（COL）is presently considered the “last resort” of defense against human infections caused by multidrug-resistant Gram-negative bacteria. With the increasing use of COL,bacterial COL resistance is also increasing worldwide. It is extremely difficult to develop new antibacterial drugs with new targets,and combination drugs to reverse drug resistance are cost-effective strategies to combat MDR bacteria. Antibiotic adjuvants target non-essential functions of bacteria and enhance antibiotic activity,providing a cost-effective way to reverse bacterial resistance. Based on the summary of the action mechanism of COL and resistant mechanism in bacteria,this paper focuses on the reversal role of adjuvants on COL resistance in Gram-negative and its molecular mechanism are mainly discussed. More than 60 adjuvants that can reverse COL resistance have been reported to date. It has been proved that the disruption of the bacterial cell envelope,efflux pump inhibition are the expression decrease of mcr-1 gene or activity inhibition of MCR-1 protein the important mechanism of COL resistance reversal. The research status and existing problems on the reversal role of adjuvants on COL resistance and its molecular mechanism of adjuvants is analyzed,and its development trend is prospected. Screening of the efficient reversal adjuvants,dose-effect relationship of the reversal roles and in-depth mechanistic studies will be important development direction in the future.

Research Progress in Plasmid Conjugation and Its Inhibitors

LIU Yi-yun, DENG Li-min, YUE Hui-ying, YUE Chao, LIU Jian-hua

2022, 38(9): 35-46. doi:10.13560/j.cnki.biotech.bull.1985.2022-0688

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With the rapid development of antimicrobial resistance（AMR）and the increasing emergence of new antimicrobial-resistant bacteria, there are fewer and fewer antibiotics available for therapeutic options. Controlling the dissemination of resistance genes, without directly killing pathogens, is becoming a novel strategy to combat AMR. Plasmid acts as one of the most important vectors of resistance genes. It can be disseminated globally to bacterias of diverse sources（e.g., animal, human being, foods, and environment）through conjugation, and plays an important role in the transmission of AMR. Thus, inhibiting the conjugative transfer of plasmids is considered as a promising approach to control AMR and has been attracting more and more attentions of the researchers. This review briefly introduced the mechanism of AMR transfer, expounded the process and mechanism of plasmid conjugation, and summarized the progress of conjugation inhibitors,aiming to provide reference for the development of new strategies to control AMR.

Overview of CLSI,EUCAST,and Susceptibility Breakpoints in China

RUAN Zi-han, HUANG An-xiong, WANG Xiu-juan, HUANG Ling-li, HAO Hai-hong

2022, 38(9): 47-58. doi:10.13560/j.cnki.biotech.bull.1985.2022-0695

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The significance of establishing susceptibility breakpoints is to interpret the results of drug susceptibility tests,and to select appropriate drugs and optimal dosing regimens in treating animals,which is an important means to control drug resistance. At present,institutions to establish susceptibility breakpoints have been established in many developed countries,among which the most influential and authoritative institutions are the Clinical and Laboratory Standards Institute（CLSI）and the European Committee on Antimicrobial Susceptibility Testing（EUCAST）. The research on drug resistance monitoring in China started relatively late,and mainly drew on the susceptibility breakpoints established by the CLSI. In 2017,with the support of the European Society for Clinical Microbiology and Infectious Diseases and EUCAST,the Chinese Committee on Antimicrobial Susceptibility Testing was established in China. Leading by the China Institute of Veterinary Drug Control（IVDC）in recent years,researchers from various research institutions have also made breakthroughs in susceptibility breakpoints and drug resistance monitoring,laying a solid foundation for controlling the development of antimicrobial resistance in China. This article first introduced the definition and significance of susceptibility breakpoints to provide a basic understanding for microbiological researchers. Then,it reviewed the basic situation of the CLSI and the EUCAST,and compared the differences in the establishment of susceptibility breakpoints between the two. Finally,this article analyzed and summarized the results of China's veterinary antimicrobial resistance monitoring and susceptibility breakpoints. In general,China is in a state of steady growth in the control of veterinary antimicrobial resistance,and many established standards for the determination of resistance are of great significance for controlling the development of resistance. However,there are still many veterinary antibiotics lacking corresponding susceptibility breakpoints,which requires more researchers to find more rapid,simple and accurate methods in the future,and fill and update the susceptibility breakpoints of veterinary antibiotics.

Research Progress in Heteroresistance of Escherichia coli

ZHAO Yan-kun, LIU Hui-min, MENG Lu, WANG Cheng, WANG Jia-qi, ZHENG Nan

2022, 38(9): 59-71. doi:10.13560/j.cnki.biotech.bull.1985.2022-0854

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Escherichia coli is a commonly pathogen in humans and animals,which has developed resistance to a variety of antibiotics. Due to the increasing selection pressure of antibiotics,the E. coli strains are more adaptable and pathogenic,the spread of antimicrobial resistance is faster,and the colonization rate is higher,that poses great challenges to epidemiology and clinical treatment. In recent years,researches related to heteroresistance（HR）of E. coli have been reported. HR refers to the fact that the homologous subpopulations in bacteria show different susceptibility to a certain antibiotic,which is the intermediate stage of sensitive bacteria evolving into drug-resistant bacteria. Different from full resistance,it is difficult for HR to be detected clinically in time,which often leads to the failure of clinical therapy. Therefore,in order to effectively prevent and control E. coli infection and the evolution of antimicrobial resistance,it is particularly important to deeply analyze the epidemiological characteristics and possible mechanisms of E. coli's HR. This paper sorts the studies on HR,and reviews the phenotypic characteristics,detection methods and possible mechanisms of HR. It is aimed to strengthen the understanding and research on the HR of E.coli,and to provide a comprehensive understanding of E. coli antimicrobial resistance process and mechanism,optimizing antibiotics use strategies,and slowing down the emergence of resistant strains.

Research Progress of Main Virulence Factors in Salmonella

LIU Li-hui, CHU Jin-hua, SUI Yu-xin, CHEN Yang, CHENG Gu-yue

2022, 38(9): 72-83. doi:10.13560/j.cnki.biotech.bull.1985.2022-0687

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Salmonella is one of the most common foodborne pathogens and is one of the leading causes of bacterial gastroenteritis worldwide. Its serotypes are numerous,more than 2 600 ones have been reported,and different serotypes of Salmonella have different pathogenicity in humans and animals. Its pathogenicity is closely related to virulence factors. In this paper,the main virulence factors,secretion systems and/or virulence genes encoded by Salmonella and their functions are systematically introduced,the relationship between virulence genes and virulence is further analyzed,and the feasibility of whole genome sequencing（WGS）in predicting virulence levels is also discussed. It is conducive to exploring the mechanism of interaction between various virulence factors and the host in Salmonella,to understanding salmonellosis at the genetic level and facilitating the control of Salmonella at the source,thus providing new ideas for the prevention and treatment of salmonellosis.

Research Progress on Contamination Control of Fluoroquinolone Antibiotics and Drug Resistance Genes

LI Liu, MU Ying-chun, LIU Lu, ZHANG Hong-yu, XU Jin-hua, YANG Zhen, QIAO Lu, SONG Jin-long

2022, 38(9): 84-95. doi:10.13560/j.cnki.biotech.bull.1985.2022-0497

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Fluoroquinolone antibiotics belong to quinolone antibiotics,is a kind of common antibiotics for human and livestock. In recent years,it has been widely used in human and animal husbandry,aquaculture and other aquaculture fields. However,its massive use has caused continuous residue and accumulation in the environment,posing a great threat to the natural environment and human health. Existing studies show that microbial degradation is one of the effective methods to remove the residual contamination of fluoroquinolone antibiotics. This paper summarized and introduced the practical application of fluoroquinolone antibiotics in microbial degradation of single strain and mixed bacteria,microbial degradation enzymes,degradation pathways and fluoroquinolone antibiotics in recent years,and analyzed the existing issues in the study of fluoroquinolone antibiotics microbial degradation. In addition,the paper also discussed the research focus of fluoroquinolone antibiotics microbial degradation in the future,aiming to provide reference for the follow-up research.

Research Progress in Non-antibiotic Active Substances Against Helicobacter pylori

LIU Xiao-li, TONG Zhen-yi, ZHAO Liang, YIN Li, LIU Chen-guang

2022, 38(9): 96-105. doi:10.13560/j.cnki.biotech.bull.1985.2022-0089

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Helicobacter pylori infection is closely related to multiple gastric diseases,it is global epidemics threatening human health. In past few decades,the main treatment of H. pylori infection were combination of a variety of antibiotics. However,due to long-term abuse of antibiotics and nature defects of threatment,H. pylori obtained resistance to clinical drugs,leading to the clinical cure rate declined steadly. In response to this issue,non-antibiotics antibacterial active substances gained more attention from researchers in recent years because it induce less drug resistance. This review summarizes the updated status and progress of non-antibiotic substances（includes natural product from plant,glycolipids,polyunsaturated fatty acids,antimicrobial peptide,urease metabolism inhibitor and probiotics）against H. pylori infection,while focusing on the antibacterial mechanism（such as lysing membrane,anti-biofilm,inhibiting H. pylori urease,competitively inhibiting adhesion and secreting bacteriocin）and effect of host body（such as anti-inflammation,inhibiting cell apoptosis,promoting the repair of mucosa and uclers,and regulating digestive canal flora）. At the same time,the differences of their clinical application approaches of different active substances. Finally,the current key challenges are also included,aiming to provides theoretic foundation and reference for the development of such substances as clinical drugs.

Identification and Drug Resistance of Escherichia coli Simultaneously Producing Carbapenemase NDM-1 and NDM-5

LI Hai-li, LANG Li-min, ZHANG Qing-xian, YOU Yi, ZHU Wen-hao, WANG Zhi-fang, ZHANG Li-xian, WANG Ke-ling

2022, 38(9): 106-115. doi:10.13560/j.cnki.biotech.bull.1985.2022-0073

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In order to understand the prevalence of carbapenemase-carrying NDM-1 and its subtypes in pig farms,87 strains of Escherichia coli were isolated and identified from piglet diarrhea cases. Carbapenemase-resistant genes NDM-1 and NDM-5 were amplified and sequenced. An E. coli strain carrying both NDM-1 and NDM-5 was identified from 87 strains of E. coli and named HN2106. In order to further understand the whole genome of HN2106 and the plasmids and drug-resistant genes carried by HN2106,16S rRNA strain identification of NH2106 was conducted,bla_NDM drug-resistant gene was confirmed,and whole genome sequencing was performed. BLAST and MEGA software were used for bioinformatics analysis and phylogenetic tree analysis. The results showed that the strain contained 7 plasmids（plasmid A,plasmid B,plasmid C,plasmid D,plasmid E,plasmid F,and plasmid G）,in 3 types,were as Col,IncFII,and IncQ1. The strain simultaneously carried gene NDM-1 and NDM-5,they belonged to a β -lactamase of group B,in which bla_NDM-1 was located at plasmid pNDM5-HN2106（plasmid C）,bla_NDM-5 was located at plasmid pNDM5-HN2106（plasmid B）. They mediated bla_NDM-1 and bla_NDM-5 resistance genes,respectively,and were important vectors for the transmission of drug-resistant bacteria. The prediction of whole genome resistance gene demonstrated that HN2106 also carried 21 types of antibiotic resistance genes. The resistances of HN2106 strain to 85 antibiotics were tested,and the results showed that HN2106 was resistant to imipenem,penicillin G,et al.,as well as it was sensitive to sulfamethoxazole,cefmetazole,et al. The above results reveal that NDM-1 drug-resistant plasmid is an important vector of NDM-1 and its subtype resistance genes,which may bring great hidden dangers and threats to healthy breeding and public health safety in the breeding industry. In order to provide theoretical reference for the prevention and treatment of clinical drug-resistant bacterial infection,it is necessary to strengthen the rational application of antibacterial drugs and the prevention and control measures of infection,and pay more attentions to the monitoring of bacterial resistances.

Comparison in Antibiotic Resistance Genes Carried by Bacteriophages and Bacteria in Farmland Soil Amended with Different Fertilizers

HU Xue-ying, ZHANG Yue, GUO Ya-jie, QIU Tian-lei, GAO Min, SUN Xing-bin, WANG Xu-ming

2022, 38(9): 116-126. doi:10.13560/j.cnki.biotech.bull.1985.2021-1544

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This study is aimed to elucidate the effect of the long-term application of organic and chemical fertilizers on the diversity and abundance of antibiotic resistance genes（ARGs）carried by bacteriophages,and to compare them with ARGs carried by bacteria in farmland soil. Soil antibiotic resistance was divided into bacterial and bacteriophage fractions,and droplet digital PCR（ddPCR）was employed to analyze the abundance of 25 ARG subtypes and class I integrase gene（intl1）in DNA of soil bacteria and bacteriophages. The total abundance and detection rate of ARGs and intl1 abundance carried by bacteriophages were evidently lower than those in soil bacterial fraction. Twenty ARG subtypes were detected in bacteriophage DNA. Detection rates of target ARGs in bacteriophages were 68%,72%,and 76% in unfertilized,chemically fertilized,and organically fertilized soils,respectively. The total abundance of ARGs in bacteriophages in the soil amended with organic fertilizer was the significantly higher than that in the soil amended with chemical fertilizer and the unfertilized soil（P<0.05）,and the gene abundances of multi-drug,macrolide-lincosamide-streptogramin group B（MLSB）,and β-lactam resistance dominated bacteriophages. Apart from β-lactam resistance gene blaTEM_,the abundance of other ARG subtypes in bacteriophages was significantly lower than that in soil bacteria（P<0.05）. The significantly positive correlations of ARG abundance were observed between soil bacteria and bacteriophages under different fertilization treatments（P<0.05）. The results of redundancy analysis showed that fertilization may affect the occurrence characteristics of ARGs in bacteria and bacteriophages by changing soil pH,heavy metal and nutrient factor levels. The findings of this study indicate that bacteriophages are another important reservoir of ARGs in farmland soil besides bacteria. The application of organic fertilizer simultaneously increased the diversity and abundance of ARGs carried by soil bacteria and bacteriophages.

Biological Characteristics and Genome Analysis of a Novel Multidrug-resistant Shigella flexneri Phage

WEN Chang, LIU Chen, LU Shi-yun, XU Zhong-bing, AI Chao-fan, LIAO Han-peng, ZHOU Shun-gui

2022, 38(9): 127-135. doi:10.13560/j.cnki.biotech.bull.1985.2021-1549

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Antibiotic resistance of bacteria caused by over use and abuse of antibiotics has been more and more seriously,and phage therapy as an precision treatment to treat multi-drug-resistant pathogens has being received more attentions. Double-layer agar plate method was applied to isolate a phage P300,a multi-drug resistant Shigella flexneri,from activated sludge and identified as Myoviridae. The measurement of biological characteristics demonstrated that the optimal multiplicity of infection（MOI）of phage P003 was 10,and the latent period was 10 min. Phage P003 remained active under the conditions of 4-70℃ and pH 3.0-10.0. The whole genome sequencing of phage P003 revealed that the length was 68 721 bp,contained 99 ORFs and 46.14% GC content,and no known drug resistance genes and virulence genes were discovered. Whole genome multi-sequence linear comparison and phylogenetic tree analysis showed that P003 was highly similar to Escherichia coli phage,but it cannot infect E. coli,indicating that P003 was a novel phage. In sum,a novel multidrug resistant phage S. flexneri P003 is isolated from active sludge,which may provide a new strain resource for the treatment of multidrug-resistant pathogen S. flexneri infection by phage therapy in the future.

Genome Construction of Achromobacter 77 and Its Characteristics on Chemotaxis and Antibiotic Resistance

LI Ji-hong, JING Yu-ling, MA Gui-zhen, GUO Rong-jun, LI Shi-dong

2022, 38(9): 136-146. doi:10.13560/j.cnki.biotech.bull.1985.2022-0112

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Achromobacter sp. 77,the dominant hyphosphere bacterium of Fusarium oxysporum f. sp. cucumerinum（Foc）,actively migrates along with Foc hyphae growth. To clarify the colonization mechanism of strain 77 in Foc hyphosphere,the whole genome of strain 77 was sequenced by using Illumina Hiseq+PacBio sequencing method,and its characteristics on chemotaxis and antibiotic resistance were examined. The genome of strain 77 was 5 868 070 bp with one chromosome and GC content of 65.89%. Total of 2 696,4 862 and 4 212 genes were annotated against KEGG,COG and GO database,respectively. Gene annotation demonstrated that strain 77 contained multiple important chemotactic genes such as mcp,che and mot in its genome. Annotation of its genome against CARD database showed that it harbored antibiotic resistance efflux pump genes such as drr,emr and gene clusters resistant to multiple antibiotics. Antibiotic sensitivity tests showed that strain 77 was resistant to chloramphenicol,kanamycin and ampicillin at the concentration < 300 µg/mL,and resistant to tetracycline at the concentration < 50 µg/mL,indicating that strain 77 was a multi-drug resistant strain. The chemotactic test indicated that strain 77 showed obviously chemotactic response to 1 mmol/L p-hydroxyphenylacetic acid,salicylic acid,α-ketoglutaric acid,fumaric acid,succinic acid and malic acid. Moreover,the growth of strain 77 was promoted by the above compounds at the concentration of 0.5 mmol/L and by 0.5-1.0 mmol/L of fusaric acid. These results indicated that strain 77 showed chemotaxis toward root or fungal exudates and utilization them,which might be the reason that the abundance of Achromobacter increased in Foc hyphosphere after continuous cultivation of cucumber in Foc infested soil.

Effect of ε-Polylysine on the Cell Structure and Biofilm Formation of Cronobacter sakazakii

SHI Cheng-long, WANG Xi-wu, LI An-qi, QIAN Sen-he, WANG Zhou, ZHAO Shi-guang, LIU Yan, XUE Zheng-lian

2022, 38(9): 147-157. doi:10.13560/j.cnki.biotech.bull.1985.2022-0396

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ε-polylysine（ε-PL）,as a cationic natural peptide with strong antibacterial activity,high safety and good thermal stability,has been applied as a food preservative. Having Cronobacter sakazakii as the test strain,the bacteriostatic mechanism of ε-PL was revealed,which may provide theoretical support for the study of bacterial drug resistance and food preservative measures. The effects of ε-PL on the physiological characteristics of C. sakazakii,such as the minimum inhibitory concentration（MIC）,membrane wall permeability,cell surface hydrophobicity and motility,were studied. The morphological changes of C. sakazakii cells treated with ε-PL were observed by transmission electron microscope,and the scavenging and inhibitory effects on the biofilm（BF）of C. sakazakii were investigated,and the permeability of the cell in the biofilm was also observed by fluorescence staining. ε-PL had positively concentration-dependent antibacterial activity against C. sakazakii,the MIC of ε-PL to C.sakazakii ATCC51329 was 256 μg/mL. Physiological studies on bacteria have found that ε-PL enhanced the permeability of cell membrane wall,and transuded cell contents such as nucleic acid and alkaline phosphatase,thus presented the bactericidal effect on C.sakazakii. Meanwhile,ε-PL reduced the surface hydrophobicity and motility of C. sakazakii,and then affected the formation of the biofilm. ε-PL demonstrated a significant effect on the inhibition and eradication of biofilm,and the removal efficiency of biofilm can be greatly improved when combined with physical oscillation. ε-PL destroyed the cell structure of C. sakazakii and the bacteriostatic effect was achieved. By reducing the cell surface hydrophobicity and motility of C. sakazakii,ε -PL also inhibited the formation of biofilm,and removed the mature biofilm.

Identification and Gene Functional Analysis of Salinity-hypersensitive Mutant ss2 in Rice

CHEN Guang, LI Jia, DU Rui-ying, WANG Xu

2022, 38(9): 158-166. doi:10.13560/j.cnki.biotech.bull.1985.2021-1444

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A library of ethane methyl sulfonate-induced Japonica rice mutants was screened under salinity conditions to explore putative salt stress-responsive genes,and a particularly salinity-sensitive mutant was identified. When challenged with 100 mmol/L NaCl,the mutant wilted heavily,became chlorotic and took up substantially more Na⁺ into shoots than its wild type（WT）. The mutant plants' leaves suffered from a significantly greater decrease in both photosynthetic rate and leaf chlorophyll content than the WT plants' leaves after exposure to salinity,while the sucrose contents of the leaves increased and that in the roots decreased to 83% of WT ones. The expressions of several genes involved in sugar transport in the source leaves were down-regulated, indicating the impairment of sugar partitioning to the roots of the tissue organs. The lower sucrose contents in the sink tissues of the mutant could not meet the carbohydrate demands of plant growth,thereby the salinity tolerance reduced. The candidate gene was identified by map-based cloning and was denoted here as SS2（salinity sensitive 2）. Functional complementation using the WT restored the salt sensitivity of the mutants. There was no significant difference between the performance of the complementary transgenic lines and the WT plants grown under salinity stress. The conclusion is that SS2 plays an important role in seedling growth and the response of rice to salinity stress by influencing sugar transport.

Genome-wide Identification of CONSTANS-Like Family Genes and Expression Analysis in Wanlut

YUAN Xing, GUO Cai-hua, LIU Jin-ming, KANG Chao, QUAN Shao-wen, NIU Jian-xin

2022, 38(9): 167-179. doi:10.13560/j.cnki.biotech.bull.1985.2021-1459

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The genome-wide analysis of the COL family in walnut（Juglans regia）was used to identify the COL（CONSTANS-Like）family members in walnut,and then to reveal molecular evolution and function of the COL family in walnut. Having ‘Xinxin 2'（Juglans regia cv. Xinxin No. 2）as research materials,the genome-wide identification and comprehensive analysis of the COL gene family in walnut were conducted,and their expressions in the female flower bud in different tissues and stages were analyzed. Totally,55 members of COL genes were identified from the whole genome of walnut,and they were unevenly distributed over 16 chromosomes and 1 Scaffold,designated as JrCOL1-JrCOL55,amino acids were 117-789,the molecular weight was between 12.99 to 86.08 kD,and the theoretical isoelectric point ranged from 4.00 to 8.94. The JrCOL proteins were classified into 3 subgroups by evolutionary tree analysis,and the conserved motifs and gene structure of each subgroup were similar. Cis-acting element analysis in the promoter region showed that JrCOLs contained a large number of functional domains related to light signal,plant hormone and stress. Spatial expression pattern analysis showed that JrCOL gene expression was tissue specific and the expression of JrCO1,JrCO7a-c,JrCO8,JrCOL23,JrCO31a-c,JrCO33,JrCOL35 and JrCOL50a-f was the highest in the leaves. And the RT-PCR analysis revealed that JrCOL1,JrCOL35,JrCOL50a-f and JrCOL52 showed consistent expression trend in female flower buds of ‘Xinxin 2',and JrCOL50a-f played an important role in the differential process of female flower buds of ‘Xinxin 2'. And 55 JrCOL members in 3 subgroups were identified in the walnut,and were unevenly distributed in 16 chromosomes and 1 Scaffold,with conserved motifs distributions and diverse expression patterns. It is suggested that JrCOL family gene plays key role in the development of walnut leaves.

Assembly of Pepino Genome Based on PacBio's Third-generation Sequencing Technology

SI Cheng, ZHONG Qi-wen, YANG Shi-peng

2022, 38(9): 180-190. doi:10.13560/j.cnki.biotech.bull.1985.2022-0344

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Pepino（Solanum muricatum）has a variety of biological activities such as antioxidation,antitumor activity,antidiabetic activity. To enrich genomic information and evolutionary development of Solanaceae crops,we obtained the whole genome sequencing information of pepino,which lays the foundation for pepino-related molecular studies. Illumina HiSeq sequencing platform was used to construct a small fragment library for pepino characterization and evaluation while using plant tissues of pepino as experimental material. Then third-generation sequencing technology PacBio's sequencing technology and Hi-C technology were used to construct a whole genome database of the pepino. Different bioinformatics methods were to study assembling the obtained pepino genomes,function annotating and evolutionary analysis. The results showed that a total of 54.11 Gb of Illumina HiSeq data were acquired. First,55.08 Gb of PacBio data were obtained with an average reads length of 14 179 bp. The obtained chromosome conformation capture（Hi-C）was 143 Gb and total length contig sequemce of the assembled genome was 1.16 Gb,with a scaffold N50 of 22.63 Mb,chromosomes with a total of 1.12 Gb length of sequence that can be mapped to 12 chromosomes,accounting for 97.16% of the total genome sequence,respectively. Among the sequences,the length of sequences for which the order and orientation could be determined was 1.08 Gb,96.11% of the total length genes were localized chromosomal sequences. Based on the estimated genome size（1.25 Gb）,the 64.22% repeat sequences were predicted,including 41 571 genes and 99.06% of which could be annotated to NR,GO,KEGG and other databases. Noncoding RNAs included 4 360 tRNAs,5 677 rRNAs,154 miRNAs,and a total of 449 pseudogenes were identified. The evolutionary time between pepino and potato was at approximately 12.82 MYA.

Cloning and Activity Analysis of SikCDPK1 Promoter from Saussurea involucrata

SHI Guang-zhen, WANG Zhao-ye, SUN Qi, ZHU Xin-xia

2022, 38(9): 191-197. doi:10.13560/j.cnki.biotech.bull.1985.2022-0570

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The objective is to clone the SikCDPK1 promoter of Saussurea involucrata and analyze its activity, so as to lay the foundation for further understanding the transcriptional regulation mechanism of the SikCDPK1 gene in S. involucrata. TAIL-PCR was used to clone the promoter of SikCDPK1 from S. involucrata, and PlantCARE to analyze the promoter cis-acting elements. The recombinant expression vector P0∷GUS, P1∷GUS, P2∷GUS and P3∷GUS driven by full-length promoter or 5'-deletion promoter were constructed and transferred into Agrobacterium tumefaciens for transient transformation. The activities of different length promoters were analyzed by GUS staining, and the activity of GUS under low temperature and drought was determined respectively. As results, a 1 042 bp promoter sequence of SikCDPK1 was obtained. pSikCDPK1 had the core elements of eukaryotic promoter TATA-box and CAAT-box, and also contained several cis-acting elements related to stress, hormone and light response. All transformed tobacco leaves showed blue color after GUS staining, and the activity was in P0>P1>P2>P3. The GUS activity changed after low temperature and drought. In sum, the promoter of SikCDPK1 is successfully cloned and has the activity of driving downstream reporter gene expression.

Cloning of Rd3GT1 in Rhododendron delavayi and Its Effect on Flower Color Formation of Petunia hybrida

SUN Wei, ZHANG Yan, WANG Yu-han, XU Hui, XU Xiao-rong, JU Zhi-gang

2022, 38(9): 198-206. doi:10.13560/j.cnki.biotech.bull.1985.2022-0510

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Flavonoid 3-O-glycosyltransferase（3GT）is an enzyme at the end of anthocyanin biosynthetic pathway, responsible for transferring glycosylated donors to the 3-OH site of anthocyanidin, and plays an important role in increasing the stability and water-solubility of anthocyanin. To study the function of 3GT in Rhododendron delavayi during anthocyanin biosynthesis would provide the basis for the research on R. delavayi flower color formation and its regulation. The full length CDS of Rd3GT1 was cloned by RT-PCR and analyzed by bioinformatics, then the pBI121-Rd3GT1 plant expression vector was constructed by DNA recombination technology. Subsequently, petunia hybrida was transformed by Agrobacterium-mediated method,and the regenerated plants were identified by transgene detection, phenotype identification, gene expression and anthocyanin detection analysis. The results showed that the full length CDS of Rd3GT1 was 1 395 bp encoding 464 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that Rd3GT1 belonged to 3GT subfamily. Compared with the wild type, the flower color of Rd3GT1 transgenic petunias changed from white to pink, anthocyanin and flavonol accumulations as well as expressions of several flavonoid-related biosynthetic genes significantly increased. In conclusion, Rd3GT1 plays a vital role in anthocyanin synthesis and can be used to improve flower color in other plants.

Gene Cloning of an Aldehyde Dehydrogenase from Bursaphelenchus xylophilus and Biochemical Characterization

LI Wen-shuo, WANG Lin-song, DU Gui-cai, GUO Qun-qun, ZHANG Ting-ting, YANG Hong LI Rong-gui

2022, 38(9): 207-214. doi:10.13560/j.cnki.biotech.bull.1985.2021-1450

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Pine wood nematode（PWN）,Bursaphelenchus xylophilus,is a causal pathogen of pine wilt disease（PWD）that is devastating to dozens of pine species. An gene aldh of aldehyde dehydrogenase from PWNs was successfully cloned through RT-PCR,its length was 1 353 bp and encoded a protein containing 451 amino acids residues. Prior studies demonstrated a nematicide fomepizole resulted in the up-regulation of aldh expression. Sequences alignment showed that the conserved catalytic position was Leu residue,which was different to Cys residue in most other aldehyde dehydrogenases,and its adjacent residue at C-terminal was His rather than the common nonpolar amino acid residue such as Leu. The aldh-encoding aldehyde dehydrogenase was over expressed in E. coli BL21（DE3）transformed with pET-15b-aldh via IPTG induction,and the recombinant aldehyde dehydrogenase was purified by Ni-NTA affinity chromatography. The Km of the aldehyde dehydrogenase with formaldehye as substrate was 27.87 mmol/L,the optimal pH value and temperature was 7.5 and 25℃,respectively. The enzyme activities were enhanced by Fe³⁺and Ni²⁺,while inhibited by Ca²⁺,Mn²⁺,Na⁺ and K⁺. The recombinant aldehyde dehydrogenase showed different catalytic activities towards 5 aldehydes,and vanillin was found to be its optimal substrate in the 5 tested aldehydes. This study lays a foundation for investigating the roles of aldehyde dehydrogenase in PWD and offers a clue to explore aldehyde dehydrogenases utilized in industry.

Optimization of Producing Cellulase by Aspergillus fumigatus A-16 and Its Enzymatic Properties

ZHANG Kai-ping, LIU Yan-li, TU Mian-liang, LI Ji-wei, WU Wen-biao

2022, 38(9): 215-225. doi:10.13560/j.cnki.biotech.bull.1985.2021-1507

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The aim of this work is to screen efficient straw degradable strains and study their process of cellulase and characteristics. A cellulase-producing strain was isolated from soil under decayed wood using the Congo red staining test,and then was identified through morphological characteristics and 18S rDNA sequence comparisons. The condition of enzyme production was determined by single factor experiment and response surface experiments,and the stability of cellulase was investigated. As results,the isolated strain was named Aspergillus fumigatus（Aspergillus fumigatus A-16）. The response surface test showed that the optimal process parameters of producing cellulase were as follows：straw powder 7 g/100 mL,pH 6.0,fermentation temperature 65℃,and fermentation time 5 d. Under this optimal fermentation conditions,the carboxymethyl cellulase（CACase）and filter paper activities（FPA）was 2 954.76 U/mL and 1 086.37 U/mL,and the total enzyme activity was 26.4% higher than that before optimization. The suitable reaction temperature was 70℃ and pH 6.0 of CMCase and FPA. The relative enzyme activity of the enzyme retained at over 80% after being heated for 90 min at 80℃,indicating that it had a good thermal stability. Meanwhile,the relative enzyme activity of the enzyme was stable in the pH 5.0-7.0 and could be remained > 70% after 1 d. This study may provide basic support for exploiting clean energy and food-grade glucose resource by using cellulose-rich biomass as a raw material.

Effects of Pseudomonas sp. PR3,a Pyrene-degrading Bacterium with Plant Growth-promoting Properties,on Rice Growth Under Pyrene Stress

GAO Xiao-rong, DING Yao, LV Jun

2022, 38(9): 226-236. doi:10.13560/j.cnki.biotech.bull.1985.2022-0539

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The accumulation of polycyclic aromatic hydrocarbons（PAHs）in farmland not only seriously hinder crop growth,but also increase food security risks. Screening the microorganisms with both the functions of promoting plant growth and degrading pollutants is an effective means to solving the above problems. Strain PR3 was isolated from the root surface of plants growing near the oil field,which had the ability of degrading pyrene,and also showed the properties of promoting plant growth like phosphorus dissolution,indole acetic acid and siderophore production. The results of 16S rDNA sequence analysis showed that the strain was classified as Pseudomonas sp. After growing in PMM medium containing 20 mg/L pyrene for 14 d,the degradation rate of pyrene by strain PR3 reached 94%. The degradation rates of naphthalene（50 mg/L）,phenanthrene（50 mg/L）and benzo（a）pyrene（10 mg/L）at 14 d were 92%,84% and 47%,respectively. Furthermore,the maximum dissolved phosphorus amount of strain PR3 was 756.25 mg/L in 7 d,the synthesis amount of IAA in 2 d was up to 14.46 mg/L,and the synthetic siderophore activity unit in 4 d reached 58.53%. Greenhouse pot experiments under different pyrene contamination concentration demonstrated that the inoculation of strain PR3 significantly improved the growth of rice and increased the degradation of pyrene near rhizosphere,and the removal rate reached 72.02%-86.22%. Meanwhile,the pyrene contents in the roots and shoots decreased by 21.81%-53.01% and 49.81%-57.17%,respectively. These results indicated that strain PR3 could contribute to the ecological remediation of pyrene-contaminated soil and reduce the risk of pyrene exposure to crops.

Colonization on the Peanuts of Two Plant-growth Promoting Rhizobacteria Strains and Effects on the Bacterial Community Structure of Rhizosphere

LI Ying, LONG Chang-mei, JIANG Biao, HAN Li-zhen

2022, 38(9): 237-247. doi:10.13560/j.cnki.biotech.bull.1985.2021-1577

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This work aims to investigate the growth-promoting mechanism of two plant growth-promoting rhizobacteria（PGPR）strains,Tsukamurella tyrosinosolvens P9 and Burkholderia pyrrocinia P10 on peanut. The two strains were labeled with GFP and rifampicin,respectively,and colonization dynamics of 2 strains in peanut tissues were studied,combined with scanning electron microscopy. Bacterial diversity of rhizosphere soil inoculated with 2 strains was analyzed by 16S rRNA whole-length sequencing. It showed that rifampicin-labeled strains P9 and P10 had good genetic stability,and their growth and growth-promoting characteristics were consistent with original strains,respectively. Two GFP-labeled strains colonized in the root tip and meristematic region of peanut. Rifampicin-labeled strains were stabilized in the roots and stems of peanut plants,and the colonization number remained 10⁴ CFU/g after 30 d of inoculation. In addition,the bacterial communities in inoculation groups of P9 and P10 and mixed strains were more similar than those in the un-inoculated control group. In three inoculation groups,the relative abundance of Flavihumibacter and unidentified_Rhizobiaceae increased significantly,and that of Bacillus and Streptomyces increased in different degrees compared with CK,while the abundance of Lysobacter,Achromobacter and Pseudoxanthomonas decreased. The two PGPR strains could not only directly colonize in peanut tissues,but also indirectly affect soil bacterial community structure to promote the growth of peanut. The inoculation of mixed strains had better growth promotion effect on peanut. The results clearly elucidate the growth-promoting mechanism of the two PGPR strains,and provide scientific basis for the further application of the strains.

Effects of Fermented Corn Xiaoqu Distiller's Grains Feed on the Intestinal Microbiota of Growing-Finishing Pigs

WANG Song, JIAN Xiao-ping, PAN Wan-shu, ZHANG Yong-guang, WANG Tao, YOU Ling

2022, 38(9): 248-257. doi:10.13560/j.cnki.biotech.bull.1985.2022-0273

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The aim of this study is to reveal the effects of fermented corn Xiaoqu distiller's grains feed on the intestinal microbiota of growing-finishing pigs. The fermented feed group and the control group fed a basal diet were compared on bodyweight gain and intestinal bacterial diversity and functions of growing-finishing pigs. After 47 d of feeding,no significant effects were found on bodyweight gain in growing-finishing pigs,and on meat moisture,intramuscular fat,and pH in pork（P>0.05）. The abundance of Burkholderia increased from 2.6% to 50.0% in small intestine bacteria community,as well as the abundance of Ralstonia,Herbaspirillum,Lactobacillus and Streptococcus significantly increased,while the abundance of unspecified blautia,bifidobacterial,Psychrobacter,Gemmiger,Clostridium and Macrococcus significantly decreased. KEGG analysis showed that the effect of fermented feed on the intestinal bacteria function was mainly in the relative enhancement of metabolism and the relative restriction of bacterial growth,suggesting it promote the transformation of substances in small intestine. This study provides a scientific basis for the application of fermented corn Xiaoqu distiller's grains feed in the breeding of growing-finishing pigs.

Effects of Proanthocyanidin on the Proliferation of Sheep Follicular Granulosa Cells in vitro

YANG Xiao-feng, QIN Xiao-wei, GUO Ze-yuan, LV Li-hua

2022, 38(9): 258-263. doi:10.13560/j.cnki.biotech.bull.1985.2021-1177

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The aim of this study is to investigate the effects of proanthocyanidin（PC）on the proliferation of sheep follicular granulosa cells in vitro. Sheep follicular granulosa cells（GCs）were cultured in complete medium with different concentrations of PC（20,30,40,50,60,70 and 80 μg/mL）for 24,48 and 72 h,respectively. The proliferation rate of GCs was determined by MTT method. And the mRNA abundances of cell cycle related genes p21,p27 and cell apoptosis-related gene caspase-8 were detected by RT-qPCR. The results showed that the proliferation rate of GCs was the highest when the concentration of PC was 50 μg/mL when cultured for 48 h in vitro. And the relative mRNA abundances of three genes（p21,p27 and caspase-8）significantly decreased,respectively（P<0.01）. In sum,PC could promote the proliferation of sheep follicular GCs in vitro,and which was verified at relevant mRNA level.

Establishment and Application of ERA Real-time Fluorescence Method for Rapid Detection of Mycoplasma pneumoniae

HU Hai-yang, YING Wan-qin, HE Jun, LV Zhi-xian, XIE Xiao-ping, DENG Zhong-liang

2022, 38(9): 264-270. doi:10.13560/j.cnki.biotech.bull.1985.2021-1529

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This work is to establish a real-time fluorescence detection method of Mycoplasma pneumoniae by the enzymatic recombinase amplification（ERA）technology. Specific primers and probe were designed for M. pneumoniae P1 gene,reaction conditions were optimized,the sensitivity and specificity were evaluated,and the method to clinical samples was validated. The ERA real-time fluorescence method had the amplification ability at 25-40℃,and the amplification effect was the optimal at 35℃,and the amplification was completed within 20 min. The detection limit of M. pneumoniae was 10³ copies/μL. There was no cross reaction with other 6 respiratory pathogens,showing good specificity. The diagnostic sensitivity,specificity,positive predictive value and negative predictive value of ERA real-time fluorescence method for 34 clinical samples were 96.15%,100%,100% and 88.89%,respectively,when the results of quantitative PCR detection were used as the standard. The study established a rapid,sensitive and specific method for early and rapid diagnosis of M. pneumoniae nucleic acid,which meets the needs of on-site rapid detection in primary health institutions.

Establishment of a Triple Droplet Digital PCR Quantitative Detection Method for Typical Pathogenic Bacteria in Livestock and Poultry Manure

CHENG Shen-wei, ZHANG Ke-qiang, LIANG Jun-feng, LIU Fu-yuan, GAO Xing-liang, DU Lian-zhu

2022, 38(9): 271-280. doi:10.13560/j.cnki.biotech.bull.1985.2022-0944

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In order to accurately and rapidly detect typical pathogenic microorganisms in livestock and poultry manure and reduce the risk of transmission of zoonotic diseases,three common pathogenic bacteria,Staphylococcus aureus,Escherichia coli（O157：H7）and Salmonella enteritidis,were used as the research object. By screening its specific primers and probes as well as optimizing the reaction system,a fast and stable multiplex droplet digital PCR（ddPCR）reaction system was established. The specificity of the system was verified by detecting different strains,the detection limit of pathogenic bacteria in livestock and poultry waste was determined,and a rapid detection method of multiple droplet digital PCR was developed. The results showed that each pair of primers and probes amplified the target strains without cross-reaction in the ddPCR system. The absolute quantitative lower limit of detecting S. enteritidis was 0.68 copies/µL,and that for S. aureus was 0.79 copies/µL,and that for E. coli was 1.02 copies/µL. The high-efficiency and high-precision detection of three typical pathogenic bacteria in livestock and poultry manure may be achieved by the method established in this study.

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2022, 38(9): 281-281.

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