Biotechnology Bulletin

Research Progress of Metabolomics in Plant Stress Biology

ZHANG Feng, CHEN Wei

2021, 37(8): 1-11. doi:10.13560/j.cnki.biotech.bull.1985.2021-0861

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In recent years,with continuous and complex environmental changes,biotic and abiotic stresses frequently burst out in nature,and many stresses seriously affect plant normal growth and development,especially crop yield. The metabolic remodeling under stresses are the consequences of interactions between genotypes and surrounding environments,are the direct reflections of plant physiological phenotypes and biochemical activities,and largely reflects the plant response and defense to stresses. The rise of metabolomics provides a reliable way to study the metabolic remodeling in different tissues and under stresses in plants. Meanwhile,the integrations of metabolome with genome,transcriptome,proteome and phenome,especially metabolome-genome association analysis by the integration of genome and metabolome play an important role in revealing the genetic basis of plant response and adaptation to stresses,in improving crop yield and in developing stress-tolerant crop varieties. In this paper,the metabolome research method,the diversity of the metabolic remodeling and the genetic basis of plant metabolome under stresses are reviewed,and the application prospects and limitations of plant metabolomics in plant stress biology are also prospected.

Engineering Non-conventional Yeast Cell Factory for the Biosynthesis of Natural Products

YE Min, GAO Jiao-qi, ZHOU Yong-jin

2021, 37(8): 12-24. doi:10.13560/j.cnki.biotech.bull.1985.2021-0815

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Natural products have been widely applied in medical,cosmetic and food industries. With the increasing of people’s demand,the green sustainable producing process is urgently needed. Recently,microbial cell factories for synthesizing natural products have been rapidly developing,and the current common-used cell factory hosts include Saccharomyces cerevisiae and Escherichia coli. Non-conventional yeasts have becoming very potential cell factory hosts due to their specific advantages,such as high-cell-density aerobic fermentation,tolerance to a wide range of temperature and pH,and wide substrate spectrum（long-chain hydrocarbons,fatty acids and methanol）. In this review,we summarized recent progresses on engineering non-conventional yeasts for the synthesis of natural products mainly terpenoids and flavonoids,and introduced common engineering methods and strategies. On this basis,we systematically discussed their strengths and weaknesses of novel non-conventional yeasts cell factories,and finally listed the challenges and opportunities while using non-conventional yeasts cell factories for the synthesis of natural products.

Research Strategies of Natural Products Biosynthesis Pathways and Key Enzymes in Medicinal Plants

ZHOU Zheng, LI Qing, CHEN Wan-sheng, ZHANG Lei

2021, 37(8): 25-34. doi:10.13560/j.cnki.biotech.bull.1985.2021-0949

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Natural products derived from medicinal plants are important sources of drug research and development. The traditional researches about natural products from medicinal plants mainly focus on their extraction and purification,chemical structure,biosynthesis and biological function. With the rapid development of genomics,bioinformatics,molecular biology,molecular genetics,synthetic biology and other disciplines and extensive cross integration among them,there are emerging opportunities for the natural products of medicinal plants. This paper systematically summarizes the research strategies of natural products biosynthesis and key enzymes in medicinal plants,included six aspects：speculation of natural products biosynthesis pathway,discovery and prediction of key enzymes in natural product biosynthesis,expression characteristics of enzymes,function of enzymes in vivo,catalytic characteristics of enzymes,analysis and optimization of enzyme structure and synthetic biology studies. Moreover,the trend of research strategies of natural products biosynthesis pathway and key enzymes in medicinal plants are prospected.

Effects of Endophytes on Biosynthesis of Secondary Metabolites and Stress Tolerance in Plants

LIANG Zhen-ting, TANG Ting

2021, 37(8): 35-45. doi:10.13560/j.cnki.biotech.bull.1985.2021-0735

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Plant secondary metabolites have broad pharmacological activities and various biological functions. Biosynthesis of secondary metabolites has attracted much attention due to its low natural yield in plants and increasing demand of medicine. The plant and endophytes are long-tern mutual beneficial and symbiotic,and affects many metabolic processes and physiological activities of plants,which provides important source to solve the shortage of secondary metabolites. Here this paper reviews the biosynthesis of secondary metabolites,such as terpenoids,flavonoids and alkaloids,promoted by endophytes,as well as the regulation of secondary metabolites by endophytes to enhance plant resistance to biotic and abiotic stresses. The paper also discusses the production path and application prospect of endophytic associated plant secondary metabolites in modern biological field,aiming at providing ideal microbial resources for the biosynthesis of medicinal secondary metabolites and the improvement of crop stress tolerance.

Research Progress on Biosynthesis and Metabolic Regulation of Ganoderic Acids

YUAN Kai, HE Wei, YANG Yun-li, ZHU Wei-yu, PENG Chao, AN Tai, LI Li, ZHOU Wei-qiang

2021, 37(8): 46-54. doi:10.13560/j.cnki.biotech.bull.1985.2021-0734

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Ganoderic acid（GA）is the secondary metabolites from medicinal Ganoderma lucidum and main active component of it,which has been studied deeply in anti-cancer and other medical fields. However,due to the low yield of GAs,long growth cycle of spores and fruiting bodies,and high growth environment requirements,and the submerged liquid fermentation of G. Lucidum was developed. Although some breakthroughs have been made,the problem of low GAs yield is still existed. The analysis and regulation of GAs biosynthesis pathway is of significance for solving low yield in the fermentation of producing GAs. Therefore,this paper reviews the research progress of GAs biosynthesis and metabolic regulation,proposes the direction of future research,and provides a reference for further improving GAs yield through genetic engineering and metabolic pathway regulation.

Research Progress on Patchoulol Molecular Regulation and Synthetic Biology in Pogostemon cablin

ZHANG Chan, YAO Guang-long, ZHANG Jun-feng, YU Jing, YANG Dong-mei, CHEN Ping, WU You-gen

2021, 37(8): 55-64. doi:10.13560/j.cnki.biotech.bull.1985.2021-0126

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Patchoulol is one of the main active components in Pogostemon cablin,and it is an important index for evaluating P. cablin in Chinese Pharmacopoeia. It can be obtained by natural extraction or synthetic biological engineering. The little amount of patchoulol in P. cablin is the bottleneck of its large-scale application. This paper reviews the research progress on patchoulol synthesis pathway,molecular regulation mechanism and synthetic biology engineering,providing reference for patchoulol development and application.

Genome-wide Identification and Bioinformatics Analysis of R2R3-MYB Transcription Factors in Artemisia annua

LI Qi, WANG Yi-chao, LIU Chang, TAN He-xin

2021, 37(8): 65-74. doi:10.13560/j.cnki.biotech.bull.1985.2021-0173

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The transcription factors in MYB family are widely involved in a variety of plant biological processes,among which R2R3-MYB is the most abundant type and has the most extensive functions. Previous studies have shown that some R2R3-MYB transcription factors can regulate the development of glandular secretory trichomes and the biosynthesis of artemisinin in Artemisia annua,however,the relevant studies are still little. Based on the genome data of A. annua,132 sequences of R2R3-MYB transcription factors were identified by local BLAST and HMMsearch,and their conserved domains were analyzed. Meanwhile,a phylogenetic tree of R2R3-MYB transcription factors between A. annua and Arabidopsis thaliana was constructed,and the functions of R2R3-MYB transcription factors were predicted with BLAST2GO. In addition,the expression patterns of R2R3-MYB genes in different tissues were analyzed based on transcriptome databases. Synthesizing the above bioinformatics analysis data,the genes of R2R3-MYB transcription factor that may participate in artemisinin biosynthesis are obtained,which provides the evidences for screening the genes of R2R3-MYB transcription factors involved in key biological processes.

Optimization of Hairy Roots Culture System of Broccoli Aiming at the Yield of Secondary Metabolites

ZHANG Xiu-min, MA Shao-ying, YANG Jie, BAO Jin-yu, ZHANG Xiao-ling, TIAN Peng, LU Ya-qi, LI Sheng

2021, 37(8): 75-84. doi:10.13560/j.cnki.biotech.bull.1985.2021-0143

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This study focuses on the relationship between the release of glucoraphanin（GRA）and sulforaphane（SF）in broccoli hairy root culture system under different single factors. Through 10 mmol/L methyl jasmonate（MeJA）treatment,the effects of different inoculation amount,medium volume,pH,culture temperature and rotating speed on the release of GRA and SF in hairy roots were investigated. On the basis of single factor experiments,the culture temperature,hairy root inoculation amount and pH were subjected to response surface experiments. The results showed that total GRA and SF yields in the broccoli hairy roots were 2.08 mg under culture temperature of 25.44℃,hairy root inoculation amount of 0.15 g and pH of 5.56,which was close to the theoretical total yield of 2.44 mg predicted by regression model. The results demonstrated that the amount of GRA and SF released into the medium varied under different culture conditions.

Cloning and Functional Identification of Trichothecene Mycotoxin Biosynthesis Gene Promoter from Paramyrothecium roridum

CEN You-fei, ZHU Mu-zi, YE Wei, LI Sai-ni, ZHONG Guo-hua, ZHANG Wei-min

2021, 37(8): 85-94. doi:10.13560/j.cnki.biotech.bull.1985.2020-1386

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The promoters of trichothecene mycotoxin biosynthetic genes tri5 and tri12 from endophytic fungus Paramyrothecium roridum were cloned by genome walking,and the core regions of the promoters were identified using prokaryotic,eukaryotic and luciferase expression systems,respectively. The results showed that 12-f1,the promoter fragment of tri12,was of the highest transcriptional efficiency for the expression of ampicillin resistance genes and hygromycin resistance genes;meanwhile,5-f0,the promoter fragment of tri5,demonstrated the highest transcriptional efficiency for luciferase gene expression. Analysis of the functional components of the promoters revealed that tri5 contained TATA box and CAAT box,while tri12 was a TATA-less promoter,but contained CAAT box and GC box. This study lays a foundation for the screening of strong promoters and efficient biosynthesis of trichothecenes.

LC-MS/MS Based Molecular Network Analysis of the Effects of Chemical Regulation on the Secondary Metabolites and Biological Activities of a Fungal Strain Aspergillus terreus C23-3

MA Xiao-xiang, LIU Ya-yue, NIE Ying-ying, LI Yan-mei, WANG Yuan, XUE Xin-yi, HONG Peng-zhi, ZHANG Yi

2021, 37(8): 95-110. doi:10.13560/j.cnki.biotech.bull.1985.2020-1398

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A marine-derived Aspergillus terreus strain C23-3 featuring of producing butyrolactone I was used as the starting strain to screen out chemically regulative cultural conditions under which abundant secondary metabolites can be produced. The strain was cultured on 24-well plates under chemically regulative conditions established by two types of basic media supplied with 9 small molecule compounds respectively. Based on the changes of mycelial morphology and the thin layer chromatography bioautography,the liquid chromatography-tandem mass spectrometry and the MS based molecular networking were used to further analyze the yield and diversity of secondary metabolites. Four chemical inducing conditions,e.g.,brown rice medium supplied with（E,E）-farnesol（1 µmol/L）,farnesyl pyrophosphate ammonium salt（5 μmol/L）,menadione sodium bisulfite（10 µmol/L）,or 3-amino-l-tyrosine（1 mmol/L）induced remarkable changes of secondary metabolites,especially with prominent diversity and yield of butyrolactones,territrems and lovastatin derivatives. The secondary metabolites of strain C23-3 have changed to different extents after chemical regulation,which provides a basis for the discovery of active compounds.

Cloning and Functional Verification of GhD6PKL2 from Gossypium hirsutum

LIU Yuan-yuan, YANG Dong-jie, ZUO Dong-yun, CHENG Hai-liang, ZHANG You-ping, LV Li-min, WANG Qiao-lian, SONG Guo-li

2021, 37(8): 111-120. doi:10.13560/j.cnki.biotech.bull.1985.2020-1591

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This work aims to investigate the functionality of gene of serine/threonine-protein kinase（STPK）during the development of cotton fiber,to provide an insight of development and differentiation mechanism of cotton fiber cells. The gene GhD6PKL2 from Gossypium hirsutum genetic standard line TM- 1 was cloned,and its sequence and structure were analyzed by bioinformatics,expressions and phenotypic observation in the overexpression of Arabidopsis GhD6PKL2,with a molecular mass of 49.74 kD,and a pI of 6.17,contained typical STPK conserved sequence sites and a number of serine and threonine phosphorylation sites. The analysis of expression pattern indicated that GhD6PKL2 significantly expressed at 20 d of flowering,which was consistent with fiber elongation stage. Both prediction by software and results of fluorescence protein localization in tobacco leaves showed that the protein encoded by GhD6PKL2 was localized on the plasma membrane. Bait self-activation assay confirmed that GhD6PKL2 was of no self-activation and no toxicity. The overexpression of GhD6PKL2 in Arabidopsis plants resulted in the increase of the number of trichome and lateral roots,while the length of main root shorting. These results imply that GhD6PKL2 may play a certain role in regulating the Arabidopsis’s trichome and lateral roots,and thus can be a potential candidate gene in cotton fiber elongation stage.

Cloning,Expression and Preliminary Bioinformatics Analysis of the Alkaline Tolerant Gene GhZAT12 in Gossypium hirsutum

FAN Ya-peng, RUI Cun, ZHANG Yue-xin, CHEN Xiu-gui, LU Xu-ke, WANG Shuai, ZHANG Hong, XU Nan, WANG Jing, CHEN Chao, YE Wu-wei

2021, 37(8): 121-130. doi:10.13560/j.cnki.biotech.bull.1985.2020-1516

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Some members of zinc finger protein（ZFP）transcription factor family play important roles in regulating plant growth,development,and response to abiotic stress. In this study,we aim to determine the role of GhZAT12,a C2H2-type transcription factor,in responding to adversity and stress. Bioinformatics analysis of the protein showed that the gene had the full length of 474 bp in coding region,encoding 157 amino acids with a predicted molecular mass of 17.074 kD. GhZAT12 protein did not contain a transmembrane domain. It was a non-secreted hydrophilic protein with 7 phosphorylation sites and no signal peptide. GhZAT12 contained two C2H2-type zinc finger domains,with a conserved L-box at the N-terminus,and a EAR-Motif at the C-terminus. Phylogenetic tree analysis demonstrated that GhZAT12 was closely related to the zinc finger protein in cocoa（XP_017982131.1）. The CDS sequence of GhZAT12 gene was cloned from cotton,and a subcellular localization vector was constructed. The GhZAT12-GFP fusion protein was observed for subcellular localization using the tobacco transient expression system. The results indicated that the GhZAT12 protein was localized in the nucleus. qRT-PCR analysis results showed that the relative expression of GhZAT12 in the root of Gossypium hirsutum was the highest,followed by the leaves and the lowest in the stem. At different times after alkaline stress,the expression of GhZAT12 gene generally increased first and then decreased,suggesting that GhZAT12 gene may play an important role in alkaline stress.

Sequencing and Analysis of Full-length Transcriptome from Medicago truncatula

SHANG Xiao-yao, ZHOU Ling-fang, YIN Qian-qian, CHAO Yue-hui

2021, 37(8): 131-140. doi:10.13560/j.cnki.biotech.bull.1985.2021-0191

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For deep analysis and exploring of the complete structure of mRNA from Medicago truncatula,a model plant in Leguminosae,the single-molecule long-read sequencing technology（SMRT）was used to sequence and analyze the full-length transcriptome of M. truncatula. A total of 7 728 183 subreads and 509 014 full-length non-chimeric reads（FLNC）were obtained. By comparing the reference genomes of R108 and A17,94.36% and 93.01% isoforms were identified as mapped reads,respectively. A total of 8 406 alternative splicing events was identified and the majority was intron retention（RI）. The 23 926 genes were detected,of 12 049 genes had 295 545 transcripts,in which at least there was one poly（A）site. In addition,3 223 transcription factors,6 595 long non-coding RNAs（lncRNA）and 479 fusion transcripts were identified. The results showed the feasibility of deep sequencing M. truncatula transcriptome by SMRT,which provides data supplement for better utilizing the genome resource of M. truncatula.

Identification and Tissue Specific Expression Analysis of AKR Gene Family in Grape

MA Ya-nan, LU Xu, WEI Yun-chun, LI Kang, WEI Ruo-nan, LI Sheng, MA Shao-ying

2021, 37(8): 141-151. doi:10.13560/j.cnki.biotech.bull.1985.2021-0068

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Aldo-Keto Reductase（AKR）is a kind of NADP-dependent oxidoreductase. Through the identification and analysis of grape AKR genes,the aim is to reveal the molecular biological function and genetic regulation mechanism of AKR genes. Based on the latest assembly reference genome data of grape in 2020,bioinformatics was used to analyze the physicochemical properties,gene structure,Motif analysis,cis-acting elements,subcellular localization,secondary structure prediction,and tissue-specific expression analysis of grape AKR genes. Finally,fluorescence quantitative analysis was used to analyze the change of grape AKR gene expressions under salt stress. Thirteen members of AKR gene family were identified at the genome-wide level and divided into 2 subfamilies. Genetic structure analysis demonstrated that the number and location of exons among different members varied. Motif analysis showed that the conserved domains of AKR protein were highly similar. The cis-acting element analysis showed that the upstream promoter elements of this gene not only had core elements and enhancement elements,but also had various hormone and stress response elements,organ specific elements and light regulatory elements. The prediction of secondary structure and subcellular localization indicated that the gene family was mainly expressed in the nucleus,and was dominated by α-helix and irregular coil. Analysis via gene chip expression profile showed that the expression levels of AKR family members were significantly different and had tissue specificity. Fluorescence quantitative results showed that the expression levels of VvAKR02,VvAKR05,VvAKR07,VvAKR11,VvAKR12 and VvAKR13 were up-regulated under salt stress. In conclusion,13 AKR family genes were identified from grape and their expression levels under salt stress were analyzed,which may provide a theoretical and experimental basis for mining salt-tolerant genes in AKR family of grape.

Antioxidant Activity,Antibacterial Activity and Volatile Components of Extracts from the Branches and Leaves of Torreya fargesii Franch.

WANG Xiao-he, GU Xi-rong, QI Shun-ju, LI Jie, CUI Yao, LI De-xia, YANG Li-hui

2021, 37(8): 152-161. doi:10.13560/j.cnki.biotech.bull.1985.2021-0130

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Our aims are to explore the antioxidant capacity,bacteriostatic activity,and volatile components of the extracts from the branches and leaves of T. grandis,and to provide scientific basis for the development and utilization of the edible or medicinal value of T. fargesii Franch. The exactions from the branches and leaves of T. fargesii Franch. were conducted by 90% ethanol,antioxidant capacity was determined using free radical scavenging rate,antibacterial activity was analyzed by Oxford Cup Method,and volatile essential components and their relative contents were detected by head sead spacesolid phasemicro extractions and gas chromatography mass spectrometry. First,the extracts from the branches and leaves of T. fargesii Franch. have different degrees of scavenging effects on DPPH,ABTS⁺,and hydroxyl radicals,among them,the branch extracts of T. fargesii Franch. in Kaizhou district had the highest scavenging rates of DPPH and ABTS⁺ free radicals,39.9% and 95.3% respectively;the leaf extracts of T. fargesii Franch. in Kaizhou district had the highest scavenging rates of hydroxy radical,reaching 69.8%. Second,the extracts from the branches and leaves of T. fargesii Franch. significantly inhibited the growth and reproduction of Escherichia coli,Bacillus subtilis and Staphylococcus aureus. Among them,the branch extracts of T. fargesii Franch. in Kaizhou district had the strongest inhibitory activity against B. subtilis,with the diameter of inhibition zone reaching 26.9 mm. Third,> 98% of the volatile components from the branches and leaves of T. fargesii Franch. Were terpenes,mainly monoterpenes and sesquiterpenes. In conclusion,the extracts of branches and leaves of T. fargesii Franch. have strong antioxidant and antibacterial activities,the main components of volatile components are monoterpene and sesquiterpene.

Analysis of Differential Metabolites and Bacterial Community Structure in Soils of a Pineapple Orchard in Different Continuous-cropping Years

LIU Chuan-he, HE Han, HE Xiu-gu, LIU Kai, SHAO Xue-hua, LAI Duo, KUANG Shi-zi, XIAO Wei-qiang

2021, 37(8): 162-175. doi:10.13560/j.cnki.biotech.bull.1985.2021-0273

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In order to investigate the effects of continuous-cropping pineapple on the soil metabolites and bacteria community structure,taking the soil of uncultivated land（T0）on the edge of the orchard as the control,untargeted metabolomics and bacterial 16S high-throughput sequencing were combined to compare and analyze the metabolites of the soils continuous-cropping pineapple for 5（T5）and 15（T15）years respectively as well as differences in bacterial community. The results of metabolites analysis indicated that there were totally 120 and 80 differential metabolites in the soil of T5 and T15 when compared with T0. And 11 significantly different metabolites（VIP>1,P<0.05）were detected between T0-T5 group and T0-T15 group in total,including 4 organic acids,3 carbohydrates,2 amino acid derivatives,2 alcohols. KEGG metabolic pathways analysis suggested that the differential metabolites were mostly enriched in 11 key metabolic pathways,e.g.,biosynthesis of unsaturated fatty acids. The bacterial 16S high-throughput sequencing profiling indicated that the bacteria diversity and abundance of the soil in T5 increased when compared with T0. The bacteria diversity increased while the abundance decreased in T15 soil when compared with T0. And there were significant differences in bacterial community structure between T5 and T15. In the levels of phyla and genus,the relative abundances of soil bacteria in T5 and T15 were significantly higher than that in T0. The proportion of two dominant bacteria communities in the soil of the pineapple orchard,Proteobacteria and Acidobacteriota,increased in T5 but decreased in T15 when compared with T0. CCA analysis showed that ccontinuous-cropping years were in significantly negative correlation with pH. This work would contribute to provide a reference for related research on the impacts of ccontinuous-cropping pineapple on the soil environment and contribute to provide the guidance for improving soil management level of pineapple orchard.

Classification and Identification of Bacillus LM Based on Whole Genome and Study on Its Antibacterial Principle

CAI Guo-lei, LU Xiao-kai, LOU Shui-zhu, YANG Hai-ying, DU Gang

2021, 37(8): 176-185. doi:10.13560/j.cnki.biotech.bull.1985.2020-1303

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Bacillus LM,which inhibits Aspergillus niger,was analyzed with genomics and UPLC-Q-TOF-MS. Combined with morphological characteristics,16S rRNA sequence and gyrA gene cluster analysis,the strain was primarily identified as Bacillus amyloliquefaciens. The analysis of whole genome showed that the genome size of the strain was 4 000 885 bp and GC content was 46.24%. The analysis of antiSMASH demonstrated that there were 11 secondary metabolite gene clusters and most of them encoded antibacterial substances. UPLC-Q-TOF-MS analysis revealed that the molecular weights of 10 chemical compounds were consistent with those by 3 predicted gene clusters via antiSMASH,which were lipopeptide antibiotics-like surfactin,iturin and fengycin. It could be speculated that the B. amyloliquefaciens activity of suppressing A. niger was associated with those 3 substances.

Isolation,Purification and Characterization of Laccase LacT-1 from Cerrena unicolor

TIAN Jia-hui, FENG Jia-li, LU Jun-hua, MAO Lin-jing, HU Zhu-ran, WANG Ying, CHU Jie

2021, 37(8): 186-194. doi:10.13560/j.cnki.biotech.bull.1985.2020-1408

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Laccase may react with many types of substrates and has a broad application prospect,but the laccase properties from different strains differ. In this study,the laccase in the fermentation supernatant of a newly isolated fungus Cerrena unicolor was separated and purified sequentially by（NH₄）₂SO₄ precipitation,hydrophobic chromatography（phenyl sepharose）,ion exchange chromatography（DEAE sepharose）and gel chromatography（TSK gel G2000SWxl）,and named as LacT-1. The molecular weight of purified LacT-1 was approximately 65 kD with specific activity of 41.31 U/mg. The recovery rate was 6.57% after 21.86 times of purification. The optimal temperature of LacT-1 was 60℃ and the optimal pH was 4.8. The LacT-1 activity was relatively stable with the temperature at 0-30℃ or pH 5.4-8. TCA promoted laccase activity. The Michaelis constant K_m of LacT-1 with ABTS as substrate was 0.075 mmol/L,while the corresponding V_max was 250 000 mmol/（L·min）. The degradation rates of methyl orange,acid red 1,reactive black 5,Coomassie brilliant blue R250,malachite green,crystal violet and bromophenol blue ranged in 71.64% to 95.17%. The above results indicate that the LacT-1 has practical application potential because of excellent pH stability,strong binding force with ABTS and it can effectively degrade some common dyes.

Molecular Cloning and Functional Verification of Histone Deacetylase Gene cobB in Vibrio alginolyticus

FAN Chen-long, DING Yu

2021, 37(8): 195-202. doi:10.13560/j.cnki.biotech.bull.1985.2020-1361

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This work aims to study the function of the histone deacetylase cobB gene in Vibrio alginolyticus. The clone of cobB gene,its bioinformatics analysis,and induction,expression,purification and function of CobB protein were carried out. The relative molecular mass was 27.09750 kD,theoretical isoelectric point was 5.15,the chemical formula of the protein was C₁₁₈₆H₁₈₆₅N₃₃₉O₃₆₉S₁₀,and hydrophilic average coefficient grand aural of hydropathity（GRAVY）：-0.409,showing that it was a hydrophilic protein. CobB presented high homology with that of Vibrio diabolicus,Vibrio harveyi,Vibrio cholerae,and Vibrio parahaemolyticus,and was relatively conserved among Gram-negative bacteria. The fusion protein was about 53 kD,and the molecular weight of the CobB protein after tag removed was about 27 kD. The optimal expression condition of the CobB protein was 37℃ and inducing time 4 h with 0.4 mmol/L IPTG. Functional analysis showed that the CobB had deacetylation effect on acetylated protein.

Cloning,Expression and Bioinformatics Analysis of the CDS Region of Chicken CEBPA Gene

DU Zhen-wei, ZHU Shuai-peng, MA Xiang-fei, LI Dong-hua, SUN Gui-rong

2021, 37(8): 203-212. doi:10.13560/j.cnki.biotech.bull.1985.2020-1410

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This work is to investigate the amino acid sequence characteristics of CCAAT/Enhancer Binding Protein Alpha（CEBPA）and the mRNA expression profile of its coding gene in different tissues of Gushi chicken. The CDS sequence of CEBPA with a total length of 975 bp was cloned by PCR,and it encoded 325 amino acids. The bioinformatics analyses showed that there was no transmembrane structure and signal peptide in CEBPA,and the protein structure was mainly composed of irregular crimp and α-spiral,and most of them were distributed in the nucleus. The construction of protein interaction network indicated that CEBPA might interact with TRBs family and Runx transcription factors family. Amino acid sequence comparison found that chicken and quail were closely related and had 13 different promoters and a 1 640 bp CpG island on its -2 000 bp sequence of the 5' regulatory region. qRT-PCR results showed that the expression of CEBPA gene in the breast muscle and leg muscle was significantly lower than that in other tissues of 12-week Guishi chickens（P<0.05）. The expression of CEBPA in the breast muscle of 22-week chicken was significantly lower than that in 6-week and 55-week ones（P < 0.05）. The expression of CEBPA in the breast muscle of Ross chicken was always significantly higher than that in the breast muscle of Gushi chicken（P<0.05）. The above results provide a theoretical basis of further exploring the biological function of chicken CEBPA gene in the lipid deposition process of chicken breast muscle.

Comprehensive Transcriptome Analysis of Intestinal Epithelial Cells of Cyprinus carpio Exposed to Lipopolysaccharide

CHEN Jian-jun, ZHAO Yi-di, CAO Xiang-lin

2021, 37(8): 213-220. doi:10.13560/j.cnki.biotech.bull.1985.2021-0055

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This work aims to explore the effect of lipopolysaccharide（LPS）on the genes and functions of intestinal epithelial cells in Cyprinus carpio. The intestinal epithelial cells of C. carpio were taken as the research object,normal treatment was as the control group,and lipopolysaccharide treatment was as the experimental group. Transcriptome sequencing was performed on the two groups of the intestinal epithelial cells of C. carpio treated for 24 h. A total 44.77G of high-quality data from transcriptome sequencing results were obtained,of which,nearly 81.83% data could be aligned to the C. carpio genome. Analyzed using the DESeq software and compared with the control group,590 differentially expressed genes（DEG）were induced by LPS treatment. Among them,303 were up-regulated and 287 were down-regulated. Gene Ontology（GO）functional enrichment analysis showed that DEG accounted for the highest proportion in cellular processes,single organism processes,metabolic processes,cells,cell part,organelle,binding and catalytic activity functional subclasses. The Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis revealed that DEG was significantly enriched in cell cycle,autophagy and apoptosis pathways. The 10 randomly selected differential genes were verified by quantitative real-time PCR,and the results showed that the transcriptome data were reliable. In this study,transcriptome sequencing technology was used to comprehensively and rapidly obtain all transcriptome information during LPS exposure to C. carpio intestinal epithelial cells,and to systematically prove that LPS blocked the cell cycle of C. carpio intestinal epithelial cells,induced autophagy and apoptosis,thus providing an important theoretical basis for the molecular mechanism of LPS regulation in C. carpio intestinal epithelial cells.

Preparation of @ZIF-8 Immobilized Enzyme by Using Cysteine as Auxiliary Reagent and Its Characterization

SUN Bao-ting, QIU Meng-xia, WANG Zi-chen, WANG Zi-yuan, CUI Jian-dong, JIA Shi-ru

2021, 37(8): 221-232. doi:10.13560/j.cnki.biotech.bull.1985.2020-1326

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Immobilization of enzyme in ZIF-8 metal-organic framework by co-precipitation embedding strategy may significantly improve the catalytic stability of the enzyme. However,immobilization efficiency and enzyme activity recovery of the prepared enzymes@ZIF-8 composites are very low. For solving this problem,phenylalanine ammonialyase（PAL）was encapsulated into the ZIF-8 using cysteine（Cys）as an auxiliary reagent by “co-precipitation” approach to prepare PAL@ZIF-8 immobilized enzymes. The preparation conditions of PAL@ZIF-8 immobilized enzymes were optimized by single factor experiment and response surface experiment. Moreover,catalytic properties of PAL@ZIF-8 immobilized enzymes including pH tolerance,temperature tolerance and reusability were investigated. The results of the response surface experiment showed that 2-methylimidazole concentration,zinc acetate concentration,and Cys concentration significantly affected the activity recovery of the enzyme,and there was an interaction between zinc acetate concentration and Cys concentration on activity recovery. The maximum activity recovery of the PAL@ZIF-8 immobilized enzymes was（56.15±0.94）% under 200 mmol/L 2-methylimidazole,60 mmol/L zinc acetate,4 mg Cys,and 2 mg polyvinylpyrrolidone（PVP）. Compared with the PAL@ZIF-8 without Cys,the activity recovery of the PAL@ZIF-8 immobilized enzymes increased about 40%. Furthermore,the PAL@ZIF-8 immobilized enzymes presented better temperature and pH tolerance than free PAL,and retained 60% of the initial activity after 7 cycles,showing good reusability. Above results indicate that this method of embedding enzyme co-precipitation in ZIF-8 using Cys as auxiliary reagent is an efficient strategy for enzyme immobilization.

Synergetic Enhancing the Soluble Expression of Thermotoga maritima α-Glucan Phosphorylase by Chemical Chaperones and Induction Condition Optimization

DUAN Xu-guo, ZHANG Yu-hua, HUANG Ting-ting, DING Qian, LUAN Shu-yue, ZHU Qiu-yu

2021, 37(8): 233-242. doi:10.13560/j.cnki.biotech.bull.1985.2020-1473

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In order to efficiently prepare α-glucan phosphorylase from Thermotoga maritima,a recombinant strain of producing α-glucan phosphorylase was constructed and the condition of fermentation was optimized. The preliminary study uncovered that the activity of the enzyme was only 5.2 U/mL when the recombinant strain was cultured under induction at 37℃ in shake flasks for 24 h. SDS-PAGE electrophoresis also revealed that the recombinant enzyme mostly existed as insoluble inclusion bodies,while the soluble protein in the supernatant was very low. In order to reduce the formation of inclusion body and to increase the soluble expression of recombinant α-glucan phosphorylase,both of the induction conditions and chaperones were optimized. Firstly,the induction conditions,including inducer types,inducer dosage,temperature,and induction time were optimized in shake flasks. Under optimized induction condition,the maximal enzyme activity reached 27.0 U/ml,which was 5.2 times of that before optimized. In addition,it also showed that the best chemical chaperone was inositol and the optimal dosage and additive time of inositol was 20 mmol/L and 0 h,the maximal enzyme activity was 62.0 U/mL,which was 11.9 times of that before optimized. However,the molecular chaperone plasmid（pG-KJE8,pGro7,pKJE7,pG-Tf2 and pTf16）did not enhance the soluble expression of recombinant α-glucan phosphorylase.

Research Progresses on the Drought Resistance of Medicago at Molecular Level

LI Qian, JIANG Wen-bo, WANG Yu-xiang, ZHANG Bo, PANG Yong-zhen

2021, 37(8): 243-252. doi:10.13560/j.cnki.biotech.bull.1985.2021-0057

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Drought stress is one of the most serious environmental constraints for global agricultural production,and it causes severe losses in pasture production. In this paper,drought induction related genes and the main progresses on the breeding of 4 important Medicago plants with drought resistance were reviewed,and the prospects and issues in this field were also discussed. It is suggested that biological technology can be utilized to explore drought-resistance related gene resources,then to preliminarily verify the function of target genes in the model legume plant Medicago truncatula. Further the mechanism of drought resistance response network in M. truncatula was clarified,and then the drought-resistance related genes from Medicago falcata was cloned. Finally,cultivating new alfalfa varieties with high drought-resistance was proposed by using molecular breeding approaches. Cultivating new alfalfa varieties with drought-tolerant is an effective way to improve Medicago growth and yield under drought stress. It is of great significance to obtain drought-tolerant alfalfa germplasm through biotechnology approaches for genetic improvement,and to provide new cultivars during the implementation of the Grain to Feed Policy of China,and ultimately to promote the healthy and sustainable development of grass and animal husbandry.

Advance on Epigenetic Modification During Plant Callus Induction

WANG Jian-yong, ZOU Yong-mei, GE Yan-bin, WANG Kai, XI Meng-li

2021, 37(8): 253-262. doi:10.13560/j.cnki.biotech.bull.1985.2020-1302

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Plant tissue culture is a complex development process,and it can be divided into three main stages：callus induction,adventitious shoot regeneration and adventitious root induction. Effective callus induction is of great significance for biological science research. Plant callus induction refers to differentiated cells reversed into pluripotent stem cells,and is a result of the interaction of genes in differentiated cells under the stimulation of complex external factors,which is not only regulated by many external signals,but also affected by internal diverse epigenetic modifications. Changes in epigenetics information in an organism leads to more profound impact than changes in genomic DNA sequences,and can affect every aspect of plant tissue culture. Early studies show that epigenetic modification plays an important role in the induction of plant callus. Therefore,this paper reviews the effects of some epigenetic modifications such as DNA methylation,histone modification,small RNA,transposon and chromatin remodeling on the induction of plant callus,analyzes the regulatory mechanism,summarizes the major issues of epigenetics modification in this field,and prospects for future research directions,which is not only conducive to deepening the understanding of callus induction process,also to callus induction work of recalcitrant plants and even to promoting the development of transgenic plants.

Research Progress on Active Compounds Against Drug-resistant Microorganism from Plant Endophytes

WANG Nan, SU Yu, LIU Wen-jie, FENG Ming, MAO Yu, ZHANG Xin-guo

2021, 37(8): 263-274. doi:10.13560/j.cnki.biotech.bull.1985.2020-1471

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With the increase of bacterial resistance caused by irrational use of antibiotics year by year,bacterial resistance has become a serious problem all over the world. In order to solve this increasingly serious problem,searching new antimicrobial drugs is undoubtedly an important measure of solving this problem,in addition to the reasonable and standardized application of antibiotics. Plant endophytes are a new type of microbial resource that urgently needs to be developed,and its rich diversity of microbial classification and secondary metabolites provide the possibility of finding new active compounds against drug-resistant microorganism,showing great potential for research and development. In this paper,the recent research progress on active compounds against drug-resistant microorganism in endophytes is reviewed,aiming to provide a reference for the research of new drugs against drug-resistant microorganism.

Research Progress on Bacterial Periplasmic Chaperone LolA

HE Xiao-li, GUO Lei-zhou, HAN Jia-hui, TANG Yin, YUAN Yuan, DAI Qi-lin, PING Shu-zhen, JIANG Shi-jie

2021, 37(8): 275-283. doi:10.13560/j.cnki.biotech.bull.1985.2020-1247

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Bacterial lipoprotein is a lipid-modified membrane protein,which participates in many important physiological processes such as cell membrane synthesis. The formation of lipoproteins depends on the Lol transport system. The protein is first synthesized in the form of precursors in the cytoplasm,and then processed into mature lipoproteins on the cell membrane and anchored to the perimembranous sides of bacteria. Lol system is composed of 5 proteins LolA-E,among which the lipoprotein is transported by the chaperone LolA in the periplasmic space. LolA transports the lipoprotein from LolCDE to LolB in the way of “mouth to mouth”,thereby completing the localization of lipoprotein. This review focuses on the structure,involved transport system and biological functions of periplasmic molecular chaperone LolA,aiming to provide more drug targets for the treatment of infectious diseases by understanding the molecular mechanism of lipoprotein transport.

Construction of a Strain with Fluorescence Labeling of Cytoskeleton in Colletotrichum gloeosporioides

LIU Na, LIU Shi-ke, WANG Qian-nan

2021, 37(8): 284-293. doi:10.13560/j.cnki.biotech.bull.1985.2020-1467

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Colletotrichum gloeosporioides spp. is fungal pathogen that devastates crop plants worldwide. The structure,reorganization and polarization of cytoskeleton play essential roles in growth,morphogenesis and pathogenicity of filamentous fungi. However,the organization and dynamics of cytoskeleton during hyphae growth and infection of C. gloeosporioides is known little. To construct fluorescent labeled strains in the microfilament and microtubule skeleton of C. gloeosporioides,Lifeact-EGFP,MBD-EGFP and CgTUB1-EGFP labeled recombinant strains were successfully constructed by random recombination and homologous recombination. Laser confocal microscopy showed that Lifeact-EGFP clearly labeled the microfilament skeleton of C. gloeosporioides,and CgTUB1-EGFP clearly labeled the microtubule skeleton;whereas MBD-EGFP was not applicable for microtubule labeling. Further phenotypic analysis suggested that the fluorescence labeling of cytoskeleton by Lifeact-EGFP and CgTUB1-EGFP did not impact the normal physiological function. These 2 labeled strains can be used for further experiment,and lays a foundation for further investigating the dynamics of cytoskeleton and the functions of cytoskeleton-binding proteins.

Research Advances in Isolation and Cultivation of Rumen Bacteria

YAN Hui, HU Wen-yue, SONG Xiao-qing, JI Shou-kun, LIU Xuan, HUANG Zhuo-han, LIU Yue-qin, ZHANG Ying-jie

2021, 37(8): 294-306. doi:10.13560/j.cnki.biotech.bull.1985.2020-1314

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The fermentation of rumen microorganisms enables ruminants to effectively utilize nutrients such as crude fibers. As a natural fermentation tank of feedstuffs,the rumen supplies important resources for the development of microecological agents,including cellulose,xylan,fat and toxin-degrading bacteria,non-protein nitrogen utilization bacteria and methanotrophic bacteria,etc. However,most of the microorganism in the rumen cannot be cultured,and non-standard artificial culture methods and processes further restrict the development and utilization of rumen bacterial resources. Therefore,based on the existing research progress in the isolation of rumen bacteria at home and abroad,this article summarizes the separation standard steps,the current research status and improvement opinions. It aims to provide a more comprehensive technical reference and an overview of the current situation for the separation of rumen functional bacteria.

Comparative Study on Methods of Analyzing Proteome in Blood Samples

WANG Zhi-bo, WANG Dao-ping, MIAO Lan, LI Ying, PAN Ying-hong, LIU Jian-xun

2021, 37(8): 307-318. doi:10.13560/j.cnki.biotech.bull.1985.2021-0183

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The objective is to compare and optimize the technologies of blood sample preparation and mass spectrometry analysis,for further studying and mining proteomic information in blood samples. A Q-Exactive Plus mass-spectrometer was used to compare the proteome composition of R. norvegicus blood samples prepared from plasma,serum and serum with high-abundance protein removed. The efficiency of trypsin digestion of serum proteins was also investigated by using conventional digestion,45℃ digestion,heat assisted digestion,repeated heat assisted digestion,urea assisted digestion and variable temperature digestion. Then the qualitative and quantitative characteristics of three mass spectrometry analysis methods,Data-Dependent Acquisition（DDA）,Data Independent Acquisition（DIA）and Parallel Reaction Monitoring（PRM）were compared. Finally,the blood sample of R. norvegicus was analyzed based on optimized proteomic technologies. After removing high-abundance protein from serum samples,the number of identified proteins was higher and the quantitative repeatability was better. The number of identified proteins and peptides,and the rate of matched mass spectrum were relatively high when serum samples digested by heat assisted digestion and variable temperature digestion. The efficiency of these two digestion methods and the repeatability of their qualitative and quantitative data were acceptable. Compared the three mass spectrometry methods,DDA was more convenient,DIA was highly reproducible and PRM was more accurate. The 490,490 and 504 proteins were identified in 3 repeated experiments respectively when high-abundance protein was removed from serum samples and digested proteins with heat assisted-variable temperature digestion and collected data by DDA method. While a total of 590 proteins were detected,and repetition rate of identified protein was 69.8%. In conclusion,the optimized method has advantages of simple operation,high protein identification rate and good repeatability,and is suitable for proteomic analysis of blood samples.

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2021, 37(8): 319-319.

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