Table of Content

26 October 2020, Volume 36 Issue 10

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Physiological Responses of Soybean to Phenanthrene and Its Tolerance Mechanism

YANG Dan, WANG Gang, WANG Wu-teng, FAN Ya-jun, XIAO Wei-wei, ZHANG Si-qi, LI Qian, JI Jing

2020, 36(10):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1167

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In order to investigate the tolerance mechanism and physiological response of plants under phenanthrene stress,foliar application experiment to soybean（Glycine max. L）was conducted,and the effects of different concentrations of phenanthrene to the growth of soybean seedlings,including phenotypic difference,biomass,oxidative damage and antioxidant enzyme activities,were explored. The results showed that the biomass of leaves increased under low concentration of phenanthrene stress（≤50 μmol/L）. Also the content of chlorophyll and carotenoid increased. The content malondialdehyde（MDA）,the activity of proline,superoxide dismutase（SOD）,and peroxidase（POD）were significantly higher than those in control group. Under the concentration of phenanthrene（75-100 μmol/L）,the content of MDA increased along with the increasing of phenanthrene concentration,while the biomass of the soybean seedlings,content of chlorophyll,the content of permeable stress substance,the activity of SOD were inhibited,and the carotenoid content and POD activity in the leaves were high. The above results reveal that plants eliminate the effects of applying phenanthrene in leaves on the biomass of the plants,content of chlorophyll,and oxidative stress by increasing POD and SOD activities as well as the contents of carotenoid and permeable stress substance. In addition,POD and carotenoid maintain high levels under high concentration of phenanthrene,suggesting that they play a more stable role in the corresponding phenanthrene stress.

Role of Alternative Respiratory Pathway in Brassinosteroids Inducing Heat Stress Response in Nicotiana benthamiana

ZHANG Rui-ping, YANG Feng, CHEN Le-zhang, DENG Xing-guang, ZHANG Da-wei

2020, 36(10):  8-14.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0591

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Brassinosteroids（BRs）,a class of steroid hormones,play important roles in regulation of plant growth and development,as well as the response to stress. To date,the crosstalk between BR and mitochondria and signaling mechanism of BR in regulating mitochondria under stress remain largely unknown. In this study,the role of mitochondrial alternative respiratory pathway in BR inducing heat stress tolerance in Nicotiana benthamiana was investigated. External application of BR increased the resistance of N. benthamiana to heat stress,accompanied by the induction of alternative respiration and up-regulation of NbAOX1 expression. Chemical inhibition of alternative respiration or inhibition of NbAOX1 through virus-induced gene silencing assay compromised BR-induced heat resistance in N. benthamiana. In conclusion,our results indicate that the alternate respiratory pathway induced by BR can avoid the superfluous reactive oxygen species accumulation and protect the photosystems in plants under heat stress condition.

Verticillium dahliae Elicitor PevD1 Activates MAPKs in Nicotiana benthamiana

JIA Feng-lian, LI Ze, LIANG Ying-bo, LI Guang-yue, YANG Xiu-fen

2020, 36(10):  15-24.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0230

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Plant resistance enhancement via activating plant immune system is one of the important approaches in green management system of plant diseases and pests. Mitogen activated protein kinase（MAPK）cascade is a vital signal transduction pathway in plant innate immune system. In order to investigate the molecular mechanism of Verticillium dahliae elicitor PevD1 inducing broad-spectrum disease resistance,transcriptome sequencing（RNA-seq）data from Nicotiana benthamiana response to PevD1 was obtained and a large number of differential gene expression（DGEs）were significantly enriched in the MAPK pathway. In this paper,DGEs enriched in the MAPK pathway were functionally classified and further analyzed. The involved functions by these DGEs were broad,including protein kinases（LRR- RLK）associated with plant recognition,ERF and WRKY transcription factor family for the regulation of gene expression,chitinase gene involved in disease resistance,calmodulin participated in calcium signaling,breathing outbreak oxidase（Rboh）regulating reactive oxygen species（ROS）and catalase（CAT）clearing ROS,etc. Expression patterns of 10 genes from the DGEs enriched in MAPK pathway were verified by qPCR and it was consistent with the RNA-Seq result. The antibody hybridization technique of specific phosphorylation sites was used to confirm that PevD1activated MAPK in N. benthamiana,i.e.,salicylic acid-induced protein kinase SIPK and wound-induced WIPK were activated by PevD1.

Screening of Actinomycetes Against Phytophthora Root Rot of Soybean and Its Growth Promotion and Disease Control

ZHANG Hong-yan, GAO qing, ZHANG Lin-yuan, LIN Guo-li, LI Ru-lian

2020, 36(10):  25-31.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0092

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To screen the strain with good antagonistic effect against the phytophthora root rot of soybean,actinomycete XS1-5 with significant antagonism was isolated from soybean rhizosphere soil by agar block method and mycelia growth assay while taking the Phytophthora sojae of phytophthora root rot pathogen. Strain XS1-5 was identified as Streptomyces albosporeus based on morphological,physiochemical characters as well as molecular methods. The pot experiment was carried out with soybean seeds coated with different spore concentrations of the XS1-5. The results showed the application of the XS1-5 significantly promoted soybean growth,and increased chlorophyll content,root vitality and the protective enzymes in the leaf and root of soybean. When spore concentration was 1.0×10 ⁸ CFU/g,the plant height,root length,fresh weight and dry weight increased by 19.92%、28.26%、87.40% and 34.51%;meanwhile,chlorophyll content（7.53 mg/L）by 50.6% and root vitality（685.41 μg/g·h）by 2.4 folds increased,compared with the control,respectively,as well as the CAT,SOD and POD activity of leaf increased significantly by 158.86%,459.71% and 135.45%. The above results indicate that the antagonistic strain XS1-5 screened in this study has a certain disease-control and growth-promoting effect on soybean.

Identification of SA Synthetic Pathways Responding to Rhizoctonia solani Infection in Zoysia japonica

XU Qiang, ZUO Hui, CHAO Yue-hui, ZENG Hui-ming

2020, 36(10):  32-39.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0103

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To reveal the main salicylic acid（SA）biosynthetic pathway in Rhizoctonia solani-infected Zoysia japonica,the key genes was recognized with aligned homologous sequences,the responsive pathway was identified according to transcriptional profile during pathogenesis,SA concentration was measured,and phenylalanine ammonia lyase（PAL）activity was verified. As typical alleles of PAL,genes ZjPAL1-ZjPAL5 were up-regulated continuously in the diseased root shown by their corresponding Unigenes,meaning an active response to infection at transcription level. About genes encoding isochorismate synthase,only ZjICS1 transcripts were observed,whose abundance was always less than one-tenth of that of ZjPAL1-ZjPAL5 and tended downwards with infection increasing. The PAL activity in the root reduced after mycelial invasion into the endodermis of the root. The more the activity was weakening,the faster the decrease of synthesized SA concentration was. However,the SA concentration in the non-infected leaves remained high by enhanced PAL activity. Phenylalanine pathway regulated by PAL gene plays a major role in SA synthesis when Z. japonica is infected by R. solani. Responsive intensity increases with the infection deepening,and the reduction of PAL activity will result in SA concentration decreasing.

Comparative Analysis on miRNA Transcriptome of Skeletal Muscle Between Wuzhishan Pig and Landrace

SUN Rui-ping, WANG Feng, CHAO Zhe, LIU Hai-long, XING Man-ping, LIU Quan-wei, ZHENG Xin-li, HUANG Li-li, WEI Li-min

2020, 36(10):  40-48.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0137

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In order to investigate the differences of microRNAs（miRNAs）in growth and development of skeletal muscle between Wuzhishan and Landrace pigs and to explore the effects of differential miRNAs on post-transcriptional regulation of skeletal muscle development,we used the longissimus dorsi muscle of 8-month-old Wuzhishan pig and of Landrace pig to screen their differential miRNAs related to the development of skeletal muscle via Solexa sequencing combined with bioinformatics analysis. A total of 311 known porcine miRNAs were identified from 2 libraries,300 and 293 known miRNAs were identified in Wuzhishan and Landrace pigs respectively,and new identified miRNAs were 96 and 79 respectively. Fifteen of 17 screened differential miRNAs significantly down-regulated in the Landrace pig,while 2 miRNAs significantly up-regulated. A total of 574 target genes were predicted by the 17 differential miRNAs that enriched to 30 significantly different biological pathways（P<0.05）,of which 5 miRNAs enriched to the insulin signaling pathway（ssc-miR-362,ssc-miR-455-3p,ssc-miR-497,ssc-miR-499-5p,and ssc-miR-874）. This study reveals the differences in the growth and development of skeletal muscle between 8-month-old Wuzhishan pig and Landrace pig at the level of miRNA and provides basic data for further research on the molecular mechanism of skeletal muscle growth.

Effects of Gut Microbiota on Bile Acid Profile and Bile Acid Metabolism in Piglets

LI Meng-ying, ZHOU Hua, DING Yu-chun, LIU Zuo-hua, SUN Jing, LI Zhou-quan

2020, 36(10):  49-61.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0269

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The aim of this study is to investigate the effects of gut microbiota on bile acid profile and bile acid metabolism in liver,jejunal contents and feces of pig models. Eleven piglets from aseptic caesarean section were randomly divided into germ-free（GF）group（5 piglets）and fecal microbiota transplantation（FMT）group（6 piglets）. GF group remained germ-free from beginning to end,and the FMT group was implanted with healthy porcine fecal bacteria at the age of 7 d. Liver,jejunal contents and fresh feces were collected for bile acid quantification at 42 d,as well as liver and jejunal tissues were collected for the quantification of genes related to bile acid metabolism. The results showed that：compared with the GF group,the total bile acid level in the piglet feces of the FMT group was significantly higher than that of the GF group（P<0.01）. The proportion of secondary bile acids in the jejunal contents and feces of the piglets in the FMT group to total bile acids was significantly higher than that in the GF group（P<0.05）,and the same trend was found in the liver（P<0.05）. Compared with the GF group,cholesterol 7-alpha hydroxy-lase gene（CYP7A1）,bile acid amino acid converting enzyme gene（BAT）,and farinol X receptor gene（FXR）,as well as genes related to bile acid synthesis and transport in the FMT group were down-regulated,while the expression of the intestinal transporter MRP2 gene was up-regulated. Thus,the transfer and colonization of gut microbiota can not only increase the bile acid excretion of piglets,increase the proportion of secondary bile acid,change the bile acid profile of piglets,but also affect the hepatointestinal circulation of bile acid.

Development and Optimization of Serum-Free Medium for High-density Culture of Suspended BHK-21 Cells and High-yield of FMD Virus

CHEN Min, LIU Xu-ping, ZHAO Liang

2020, 36(10):  62-71.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0343

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Based on the high-density suspension culture of BHK-21 cells,this study is to optimize the serum-free medium in order to improve proliferation of the BHK-21 cells and amplification of foot-and-mouth virus（FMDV）. Firstly,the key nutrients in the serum-free medium were determined based on the metabolic characteristics and dynamics analysis of the BHK-21 cells. Then,the composition of the medium was optimized. Finally,the growth and FMDV-producing ability of the BHK-21 cells between before and after the optimization were compared and verified in bio-reactor. The maximum viable cell density reached 1.78×10⁷ cells/mL during batch culturing of the BHK-21 cells in optimized serum-free medium（Opt-SFM）,and the situation of accumulating metabolic by-products was improved. Compared with non-optimized medium containing 1%（V/V）serum, TCID₅₀ of the produced FMDV in the Opt-SFM showed a similar level at 7.25 lgTCID₅₀/0.1 mL,while the yield of 146s particals increased by 50.7% to 15.6 g/mL. In conclusion,based on the requirements of cell growth and metabolic characteristics,optimizing nutrients and reducing metabolic by-products accumulation may effectively eliminate “cell density effect” and achieve ultra-high cell density and high yield of foot-and-mouth virus.

Stability of Suspended MDCK Cells for Avian Influenza Virus Production

BAI Chun-li, YE Qian, JI A-mei, LIU Xu-ping, ZHANG Xu, LIU Zhi-liang, ZHU Ming-long, ZHAO Liang, TAN Wen-song

2020, 36(10):  72-79.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0434

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The aim of this study is to determine the stability of growing and virus producing for suspension MDCK cells during long-term passaging culture. Suspended MDCK cells were subcultured for more than 200 d. Taking suspended MDCK cells at different passaging time points,their growth patterns and the difference in virus production of H9N2 avian influenza virus（H9N2-AIV）were investigated. The effects of different process parameters and conditions such as medium dilution ratios and MOI on the production of H9N2-AIV from the suspended long-term passaging MDCK cells was explored. The reasons for the difference in H9N2-AIV production capacity between the lowest passaged cells（P1）and the highest passaged cells（P111）were preliminarily investigated from two aspects,i.e. the intracellular ROS level and cell cycle. The results showed that the MDCK cells at the different passages presented similar morphology,and insignificant difference was observed concerning cell growth between all subcultures and batch cultures,which indicated that the growth of the MDCK cells were stable during long-term passaging. The HA titer increased with elevated cell passaging time,the HA titer of P111 was about 0.5 Log₂HAU/100 μL higher than that of P1. Meanwhile,P111 cells produced higher virus than P1 cells under different tested processes,indicating that the H9N2-AIV production capacity of the suspended MDCK cells had slightly increased with the increase of cell passaging time. It was also found that P111 cells had lower ROS level and higher proportion of G0/G1-phase cells,which may be the main reasons for the increased yield of H9N2-AIV from MDCK cells.

Genomic Characterization of a Totiviridae from the Ustilaginoidea virens Strain GZ-14-07

ZHANG Ting-ting, TENG Li, CAI Xiao-yao, LONG Hui, LI Sheng-yu, LIU Hong-mei

2020, 36(10):  80-87.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0479

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Ustilaginoidea virens is a pathogen that causes fungi-like disease Ustilaginoidea oryzae. A mycovirus isolated from strain GZ-14-07 of U. virens was designated as Ustilaginoidea virens RNA virus 6（UvRV6）and sequenced. Mycovirus UvRV6 was extracted from the strain GZ-14-07 using CF-11 cellulose powder method and its full-length genomic sequence was obtained by random-primer amplification method. Sequence analysis,alignment and phylogenetic tree of the UvRV6 were performed using DNAMAN,BlastP and MEGA software. The complete genome sequence of the UvRV6 was determined to be 5 043 bp in length,with a GC content of 48.8%,and contained two open reading frames（ORF1 and ORF2）. ORF1 had a length of 2 280 bp and encoded a 760-amino-acid protein（79.6 kD）,and ORF2 was 2 385 bp long and encoded a 830-amino-acid protein（91.5 kD）. ORF1- and ORF2-encoded protein had a high percentage of sequence similarity to the capsid protein and the RNA-dependent RNA polymerases（RdRps）of genus Victorivirus in the family Totiviridae,respectively. Moreover,there was an AUGA between ORF1 and ORF2,and a sequence forming H-type pseudo-knot existed in the upstream of the AUGA. These 2 sequence elements allowed ORF1 and ORF2 to be merged as a bigger ORF via a mechanism of re-initiating translation after translation ended. A phylogenetic analysis based on the alignment of RdRps of UvRV6 and other totiviruses showed the UvRV6 was clustered with the members of Victorivirus in the family Totiviridae. Therefore,UvRV6 is confirmed as a new member of Victorivirus in the family Totiviridae.

Secondary Metabolic Pathway Mining and Pan-genome Analysis of Bacillus coagulans

QIAN Wen-jiang, WANG Bu-qing, LI Wei-xi, YANG Xue-miao, LIU Hong-wei, ZHANG Li-ping

2020, 36(10):  88-98.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0097

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This study was to further understand the genome of Bacillus coagulans and to explore its secondary metabolic pathways. The genomes of 33 B. coagulans were downloaded from the NCBI database and analyzed by pan-genomic analysis software and secondary metabolite mining software. The genome size of 33 strains of B. coagulans was between 2.059 47-3.694 84 Mbp and the GC content ranged from 46.2% to 47.5%. Pan-genome analysis of 11 strains of B. coagulans with complete genome level revealed that the pan-genome contained 5 899 cluster genes,the core genome consisted of 2 152 cluster genes,and 2 255 strain-specific genes were identified. Based on the analysis of secondary metabolite synthesis gene clusters,79 secondary metabolite gene clusters of 8 groups were found in the 33 B. coagulans genomes. The major synthetic gene clusters were bacteriocins,lactones and sugars. In brief,the pan-genome and core genome sizes of B. coagulans and their secondary metabolites through can be predicted in this study,which is conducive to understanding B. coagulans and providing relevant information further better utilization of this strain.

Identification and Analysis of a Strain of Enterobacter hormaechei Capable of Degrading Polyethylene

GAO Chao, HAO Kong-li, ZHAO Yu-ting, MAO Ying-xiang, CHI Ming-yan, ZHANG Jie

2020, 36(10):  99-104.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0246

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With the development of modern industry and agriculture,polyethylene plastic products are widely used due to their excellent properties. Because of their large molecular weight,strong surface hydrophobicity,low surface energy,and lack of functional groups that can be used by microbial enzyme systems,their degradation in natural environments is very slow,thus causing serious environmental problems. In this study,a strain LB-1 capable of degrading polyethylene was isolated from the intestinal tract of larvae of Indian loquat（Plodia interpunctella（Hübener））. It was identified Enterobacter hormaecheipartial sequence” by combining morphological,physiological and biochemical characteristics and molecular biological measurements. After the strain was cultured in a medium with a polyethylene film as the sole carbon source for 60 d,the analysis were conducted by a contact angle tester,a scanning electron microscope（SEM）,a Fourier transform infrared spectrum（FTIR）,and a gas chromatography-mass spectrometer（GC-MS）. And results showed that the surface of polyethylene became less hydrophobic,the surface morphology changed,the surface was oxidized to have -C= O-functional groups,the weight of polyethylene film was loss,and the soluble products were generated,all these proved that LB-1 degraded polyethylene. This study provides a theoretical basis for the degradation of polyethylene by E. hormaecheipartial sequence” and a green way for polyethylene pollution.

Screening of an Effective Sulfur-oxidizing Strain and Its Main Bio-oxidation Metabolic Pathway of S^2-

ZHANG Lin-yi, SONG Chen, XU Yao-yao, WANG Jia-ning, WANG Jin, YUE Zheng-bo, LIU Xiao-ling

2020, 36(10):  105-115.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0282

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Inorganic sulfur（S^2-）was used as the target pollutant,and an indigenous microorganism capable of efficient S^2--oxidizing was isolated from Dongsha river,a black-odor water body in Beijing. Through 16S rRNA sequencing experiment,this strain was identified as Citrobacter and named as Citrobacter sp.sp1. The optimal S ^2--oxidizing conditions of this strain sp1 were as follows：25℃,initial pH of 7.0,and initial glucose concentration of 1.00 g/L along with initial cell concentration of 1.00 g/L. Under the above optimal conditions,the highest S^2--oxidizing ratio of artificial sulfur-containing wastewater by this strain sp1 reached 97.1%. Five chemical forms of sulfur including S⁰、S₂O₃^2-、SO₃^2-、S₄O₆^2- and SO₄^2- were produced in the whole bio-reaction process,and the concentration of SO₄^2- increased slowly with the decreasing of S^2-. Results of high-throughput sequencing showed that the strain sp1. transformed the unsteady-state S^2-into steady-state SO₄^2-via two main S^2--oxidizing metabolic pathways of Paracoccus sulfur oxidation（PSO）and S4 intermediate（S4I）. In PSO pathway,S^2- partially was oxidized to be S⁰ and then the generated S⁰ continuously was oxidized to be SO₃²,while other part of S^2- were directly bio-converted to SO₃²,and intermediate SO₃^2- were further oxidized to SO₄^2- by both directly and indirectly oxidation pathways;in addition,S⁰ reacted with SO₃^2- spontaneously to form S₂O₃^2-,while S₂O₃^2- released SO₃^2- and S⁰ by the disproportionate reaction. In S4I pathway,S₂O₃^2- partially was oxidized to S₄O₆^2-,and S₄O₆^2- subsequently was then oxidized to SO₄^2-.

Construction of Tetracycline-degrading Bacterial Co-culture System and Community Analysis of Wastewater Remediation

WU Xue-ling, ZHOU Xiang-yu, WU Xiao-yan, LUO Kui, GU Yi-chao, ZHOU Han, LIAO Wan-qing, ZENG Wei-min

2020, 36(10):  116-126.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0232

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Tetracycline-containing wastewater widely exists in environment,and it has attracted much attention due to issues such as resistance gene transmission,human drug resistance,etc. After comparing the ability of bacteria degrading tetracycline,the co-culture system mainly with strain Raoultella sp. XY-1 and strain Pandoraea sp. XY-2 was clearly constructed. Then this system was applied in actual wastewater for bioremediation,and the removal efficiency of pollutants and bacterial community changes were recorded. The results showed that degradation percentage by this co-culture system for 10 d was > 80% in glucose medium with 80 mg/L tetracycline. The co-culture system presents a favorable effect on the removal of pollutants from actual tetracycline-containing wastewater,and the results by high-through sequencing community indicated that the strain Raoultella sp. XY-1 became the dominant bacterium.

Isolation,Identification and Characterization of Salt-alkali-tolerant and Phosphorus-dissolving Bacterium Y2R2

YANG Hai-xia, LIU Xi-min, PAN Yi-chen, ZHAO Xiang-lin, HUANG Hai-dong

2020, 36(10):  127-134.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0236

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The objective of this study is to screen phosphorus-dissolving bacteria with fine environment adaptability to coastal salt-alkali land. Strain Y2R2 with the ability of salt-alkali-tolerant and phosphorus-dissolving was isolated from salt-alkali soil samples collected from Tianjin Binhai New Area. Strain Y2R2 was identified as a novel species of Halomonas according to poly-phase taxonomic characteristics. Strain Y2R2 grew at the temperature 10℃ to 45℃,and tolerated the condition of pH 14 and 15% NaCl. Strain Y2R2 showed ability of dissolving phosphorus under pH 7-13 and NaCl content 0-12%. In inorganic phosphorus medium,under the fermentation conditions in which the initial pH was 8,NaCl content was 9% and fermentation time was 48 h,the phosphate-dissolving quantity by strain Y2R2 reached its highest value,247.6 mg/L. Strain Y2R2 screened in this study is a novel species of indigenous microorganisms that adapted to the soil and climate environment of coastal salt-alkali land,and has high salt-alkali tolerance and phosphate-dissolving capacities. The strain is expected to be used as salt-alkali phosphorus-dissolving bacterial agent.

Screening and Identification of High-yield Feruloyl Esterase Strains and Optimizing of the Enzyme Activity Assay Conditions

WANG Xiang-feng, WANG Qiao, YUAN Hui-jun, WANG Li

2020, 36(10):  135-141.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0304

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Using transparent zone and solid fermentation,two strains with high feruloyl esterase（FAE）yield were selected from 106 macrofungi that were collected from field and deposited in the lab an. They were identified as Cerrena unicolor and Pycnoporus sanguineus by ITS sequence analysis and morphological identification. Then the FAE activity of two screened strains were determined using rice bran as induction medium and the crude enzyme with the highest activity was selected separately to optimize the enzyme activity determination conditions. The results demonstrated that the activity of FAE by C. unicolor and P. sanguineus reached the peak at 4 d and 6 d respectively. The optimal determination conditions for FAE activity in the crude enzyme solution of C. unicolor was 40℃,25 min reaction time,30 μL crude enzyme and 400 μL the methyl ferulate. The optimal determination conditions for FAE activity in the crude enzyme solution of P. sanguineus was 30℃,35 min reaction time,20 μL crude enzyme solution and 400 μL methyl ferulate.

σ Factor SigW/ Anti-σ Factor Involving in the Toxicity of Pseudomonas donghuensis HYS to Caenorhabditis elegans

WU Ting-ting, GUI Zhe, QIN Ying-qiu, XIE Zhi-xiong

2020, 36(10):  142-149.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0106

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Pseudomonas donghuensis HYS is toxic to Caenorhabditis elegans but its mechanism is not fully understood. This study aims to investigate the roles of σ factor SigW and anti-σ factor RsiW in the toxicity of P. donghuensis HYS to C. elegans. Through the bioinformatics analysis of the mutation site sequence in the toxicity-reduced mutant of P. dongshanensis HYS,the target gene knockout mutant was constructed. Then with the help of research methods such as yeast two-hybrid,fluorescent quantitative PCR and P. dongshanensis -C. elegans interaction,etc.,the protein functions of related gene were verified. The results revealed that：（1）the gene rsiW was related to the toxicity of P. donghuensis HYS to C. elegans. （2）RsiW interacted with neighboring SigW（σ²⁴）on the genome at the protein level,forming a pair of σ factor and anti-σ factor. （3）The interaction between RsiW and SigW was involved in the toxicity of P. donghuensis HYS to C. elegans and there was a dose effect,i.e.,there was reducing effect when SigW expression was relatively strong. This study discovers that a pair of σ factor and anti-σ factor are involved in the toxicity of P. donghuensis HYS to C. elegans and provides new clues and ideas for further exploring the mechanism of virulence factors of Pseudomonas.

Effects of Biotin on L-Glutamate Efflux Mediated by MscCG in Fermentation Process

NIE Zhi-hua, ZHU Lei-lei

2020, 36(10):  150-155.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0003

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The L-glutamate produced by the fermentation of Corynebacterium glutamicum is mainly released by the L-glutamate exporters’ opening the channel when the cell membrane is stretched. The biotin in medium can affect the components of cell membrane and thus affect the membrane tension,and the concentration of biotin can indirectly affect the capacity of L-glutamate exporter and the accumulation of extracellular L-glutamate in the fermentation broth. In this study,we investigated the effect of L-glutamate exporter MscCG on the L-glutamate production from the fermentation of C. glutamicum,and online monitored the changes of biomass,dissolved oxygen and pH in the fermentation process using the M2P BioLector micro-bioreactor. The results showed that biotin had a great influence on the efflux of L-glutamate by MscCG. 0.5 μg/L biotin was the suboptimal under the condition of this experiment,at which the amount of L-glutamate exported by MscCG reached the highest. When the concentration was lower than that,the growth rate of the bacteria was slow and the efflux of L-glutamate reduced. When the concentration was > 0.5 μg/L,the growth rate of bacteria was fast but the concentration of L-glutamate was low,and the lack of dissolved oxygen led to the accumulation of lactate. Therefore,the concentration of biotin,the dissolved oxygen and pH control should be adjusted in a combined way for maximum productivity of L-glutamate.

Studies on the Antioxidant Activities of Six Red Yeasts

WANG Yue, OUYANG Dan, TANG Wei, LIU Shi-bo, GU Yan, HE Zeng-guo

2020, 36(10):  156-164.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0406

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Armed with strong environmental adaptability and broad resources,red yeasts has shown decent antioxidant application effects in aquatic farming practice,thus it is of great necessity to investigate their antioxidant potentials. In order to investigate the antioxidant activities of red yeast,6 red yeasts（LW1-LW6）were isolated recently from marine and terrestrial niches,their contents of total carotenoids and the tolerance to H₂O₂ were measured,and the antioxidant activities were comprehensively evaluated by measuring their reducing powers,the scavenging free radicals abilities,and the total antioxidant capacities,as well as the superoxide dismutase activities. The results indicated that the carotenoid contents of 6 red yeasts varied in 117.78-162.91 μg/g,and they had decent tolerances to H₂O₂ at 9 mmol/L. The order of reducing powers followed the pattern of fermentation supernatant > pigment extract > intact cell,whereas the DPPH· scavenging powers went with pattern as intact cell > fermentation supernatant > pigment extract. Although the scavenging ·OH potentials of the intact cell and the pigment extract were at the same level,but both were significantly higher than that for the fermentation supernatant. In addition,decent T-AOC and SOD activities presented in the fermentation supernatant and intact cell of the two isolates LW3 and LW5,with former demonstrated slightly better antioxidant capacity. In this study,the antioxidant activities of the 6 red yeast strains are systematically studied by different means,revealing the application potential of the studied 6 red yeast strains.

Functional Analysis and Validation of Mg²⁺ Binding Sites of Intron-encoded Protein

CUI Gu-zhen, CHEN Xiang-hao, HONG Wei, ZHANG Zheng-rong, QI Ting-na, CHEN Zheng-hong

2020, 36(10):  165-172.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0012

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This work aims to screen and construct the mutants of having intron-encoded protein Mg²⁺ binding site,and to verify its effect on the “Retrohoming” efficiency of group II intron. Bioinformatics technology was used to screen key sites,site-directed mutation technology to construct mutants,and Targetron and blue-and-white spot counting methods to verify its “Retrohoming” efficiency. Results showed that 2 sites,D308 and D309,were identified as the core catalytic sites for group II intron-encoded protein Mg ²⁺ binding,and three mutants of these sites were successfully constructed,including two single-site mutants（D308A and D309A）and a double-site mutant（D308A/D309A）. The experimental results in Escherichia coli showed that all three mutants completely inactivated the “Retrohoming” function of group II intron. In conclusion,this work confirms that the Mg ²⁺ binding sites of group II intron-encoded protein is the core catalytic site of it playing function.

Advance in Relationship Between Heat Shock Protein 90s and Thermo-Tolerance in Plants

TANG Jia-le, XU Hai, YUAN Ping, HE Ke-jia, WANG Ren-cai, BU Fan-wen

2020, 36(10):  173-179.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0264

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Plant heat shock protein 90s（Hsp90s）is a kind of heat shock protein gene family that is wide existing and highly conserved. Hsp90s plays an important role in assisting protein folding,intracellular transport,maintenance and degradation,and promoting cell signaling under heat stress. In recent years there has been a great progress in the production,classification and location of Hsp90s of plant under heat stress,as well as in the regulation of its gene expression and biological functions. This paper systematically discussed the structure of plant Hsp90s as well as the relationship between Hsp90s and thermo-tolerance in plants,aiming at providing references for the improvement of crop heat resistance by genetic engineering.

Advances on the Structural Characteristics and Function of Dof Gene in Plant

LIU Jun, JIN Yu, WU Yao-song, LIU Yan, WANG Wen-bin, REN Shan-shan, DIAO Song-feng, CHEN Yu-long

2020, 36(10):  180-190.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0112

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DNA binding with one zinc finger（Dof）is a kind of specific transcription factors only in plant,which are encoded by multiple genes. The N-terminus contains a highly-conserved C2-C2 single zinc finger domain consisting of 52 amino acid residues,which could bind and interact with DNA and proteins. The C-terminus is a specific transcriptional regulatory domain,its variable amino acid sequence results in the functional diversity of Dof genes. With the development of genomics and bioinformatics,Dof proteins of multiple species have been reported in recent years. The Dof genes are mainly involved in biological processes,such as plant growth and development,carbon and nitrogen metabolism,abiotic stress,and flowering regulation. Hereby we review the structural characteristics of Dof transcription factors,the number of reported Dof genes and their biological functions. We also discuss the current issues and the future prospects in studying moso bamboo flowering,aiming to provide a reference for the further study of Dof transcription factors.

Advances of Bryophytes in Response to Heavy Metal Stress

CHEN Xuan, LIU Xiang-long, TANG Ting

2020, 36(10):  191-199.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0385

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With more and more serious heavy metal pollution from intensifying human activities,the phytoremediation technology for heavy metal contaminated soil has become a research hotspot due to the advantages of low cost,environmental friendliness and high remediation efficiency. However,the research in this field mainly focuses on angiosperms. Bryophytes possess special physiological structures and development processes,including the leaf composed of single cell layer,the underdeveloped stratum corneum but relative large body surface area,and strong electrostatic adsorption capacity,which enable them to adapt well to environmental heavy metal stress. This paper reviewed the impact of heavy metal stress on bryophytes as well as their stress responses,and proposed that mechanisms contributing to heavy metal tolerance might be conserved between bryophytes and other terrestrial higher plants,aiming at providing the ideal materials for the ecological restoration and biological monitoring of heavy metal polluted area.

Research Progress on the Occurrence and Control of Fusarium Crown and Root Rot of Tomato

DU Jian-feng, WU Wei, ZHANG Xiao-ying, LI Yang, DING Xin-hua

2020, 36(10):  200-206.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0260

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Fusarium crown and root rot of tomato is one of the most serious soil-borne diseases in greenhouses during winter and spring in recent years. It is caused by Fusarium oxysporum f. sp. radicis-lycopersici（FORL）,which caused huge losses to tomato production in China. FORL and pathogen causing Fusarium crown and root rot of tomato were different specializations of the same species of Fusarium oxysporum;however,its host was broader and it was more harmful than the tomato Fusarium oxysporum that only infects tomato. This article reviews the research progress on the occurrences of Fusarium crown and root rot of tomato,pathogen morphology and detection methods,pathogenesis and its prevention and control,and prospects on the research of Fusarium crown and root rot of tomato,aiming to provide useful reference for the control on Fusarium crown and root rot of tomato in China.

Research Progress on Methionine Regulating the Oxidative Stress of Livestock and Poultry

LIU Rong, CUI Kai, BAI Fu-heng, DIAO Qi-yu

2020, 36(10):  207-214.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0271

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Methionine is an essential amino acid for livestock and poultry,and has extensive and important biological functions in the body. Recent studies have shown that methionine restriction（MetR）in the diet can lead to many physiological reactions,such as prolonging life span,preventing metabolic diseases and participating in the regulation of inflammatory diseases. As an important indicator for evaluating the health of the body,oxidative stress has received more and more attention. Many studies have confirmed the positive effects of MetR on oxidative stress. MetR can participate in the regulation of oxidative stress by reducing the production of mitochondrial reactive oxygen,regulating key signaling pathways and changing expressions of miRNAs,which has a wide range of effects on the health of livestock and poultry. This paper summarizes the effect of MetR on oxidative stress in livestock and poultry,and expounds the discovered molecular mechanism of MetR and oxidative stress.

Classic Post-translational Modification in Ribosomally Synthesized and Post-translationally Modified Peptides Biosynthesis

WANG Jin-xiu, ZHANG Qi, DING Wei, CHEN Tuo

2020, 36(10):  215-225.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0136

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Ribosomally synthesized and post-translationally modified peptides（RiPPs）are a large class of natural products synthesized by ribosome via post-translational modification which feature a wide range of structural and biological diversity. We reviewed the various classes of RiPPs and their post-translationally modification mechanisms,discussed the significance of post-translational modifications,and prospected the development of RiPPs using genome mining under bioinformatics.

Research Advance in Polyethylene Terephthalate Hydrolytic Enzymes

SHI Li-xia, GAO Song-feng, ZHU Lei-lei

2020, 36(10):  226-236.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0336

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Polyethylene terephthalate（PET）is one of the most widely used plastics and accumulation of the discarded PET has become a global environmental issue. PET hydrolytic enzymes mainly hydrolyze PET into soluble building blocks：terephthalic acid（TPA）and ethylene glycol（EG）at moderate conditions. Enzymatic degradation of the recalcitrant plastic PET may provide a more environmentally friendly approach to solve the problem of “white pollution.” Here,we reviewed the 3D structures,catalytic mechanisms and PET degradation activity of four PET hydrolases,including cutinases,PETase,lipases and MHETase,mainly focusing on the protein engineering effort of cutinases and PETase. We also discussed the key scientific challenges on the development of high efficient enzymatic degradation of PET,aiming to provide theoretical basis for the engineering of more efficient PET hydrolytic enzymes.

Applications of Single-cell Sequencing Technology in Microbial Ecology

WANG Dan-rui, SHEN Wen-li, WEI Zi-yan, WANG Shang, DENG Ye

2020, 36(10):  237-246.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0335

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Single-cell sequencing technology,allowing nucleic acid molecules sequenced at the level of single cell,has become a hot spot in molecular biology,and from it there are remarkable achievements in medicine,biochemistry,life science,thus it become an important part of single-cell ecology. The combination of single-cell sequencing and amplicon or metagenomic techniques can more accurately identify microbial species,explore population heterogeneity,intensively study the function of specific species,and obtain the complete genome of rare species. Here,we briefly review the generation and development process of single-cell sequencing,focus on introducing the new technologies of cell isolation and genome amplification,and illustrate the application of single-cell sequencing in microbial ecology.

Perfusion Process Development of MDCK Suspension Cells for Influenza Virus Production

WU Yi-xiao, Thomas Bissinger, Yvonne Genzel, LIU Xu-ping, Udo Reichl, TAN Wen-Song

2020, 36(10):  247-255.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1228

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This work aims to increase the production efficiency of influenza vaccine,i.e.,overcome the current drawback of low production,and to meet the growing market demand for influenza vaccines and be full preparedness to pandemic outbreaks. Process development was carried out using MDCK suspension cells by virus adaptation,extended batch cultivation to evaluate cell growth and virus production,semi-perfusion to verify the feasibility of MDCK high cell density cultivation,and ATF perfusion process verification in the bioreactor. Faster infection with adapted seed virus contributed to high virus titer of（3.57±0.17）log₁₀（HAU/100 μL）in extended batch cultivation. The high cell concentration over 40×10⁶ cells/mL and high virus titer over 4 log₁₀（HAU/100 μL）in the semi-perfusion model proved the feasibility of high cell density cultivation using MDCK suspension cells. MOI and trypsin concentration presented significant impacts on the cell growth and virus propagation during the infection phase. Similar cell growth profile and higher virus titer of 4.37log₁₀（HAU/100 μL）in the ATF-based perfusion process were obtained in the bioreactor;and comparable cell-specific virus yield（CSVY）to the batch cultivation was maintained by implementing the temperature reduction strategy during infection phase,which overcame the “cell density effect”. In conclusion,the platform of producing influenza virus based on the MDCK suspension cell ATF perfusion process is established,and it enhanced the influenza virus productivity by increasing the cell density and CSVY,which provides a new option for the cell-based industrialization of influenza vaccine.

Research on a Preparation Process of Deoxynivalenol

DUAN Jia-qi, LI Bei-bei, REN Jie, QIU De-wen, LI Guang-yue

2020, 36(10):  256-262.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0325

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Deoxynivalenol（DON）is a mycotoxin produced by Fusarium. It is cytotoxic and can cause embryo deformity,which seriously threatens human and animal health. To provide enough high-purity DON for scientific research in toxicology and detoxification,we developed a simple,economical and effective process for the separation and purification of DON. 1. Extraction：84% acetonitrile aqueous solution was used to extract DON from the rice culture of Fusarium graminearum PH-1 to obtain DON crude extract. 2. Cleaning：Petroleum ether and saturated saline aqueous solution were used to remove the impurity from the DON crude extract. 3. Refining：The product obtained in step 2 was washed by ethyl acetate：petroleum ether (1∶1 of V/V). Then more impurity was removed by liquid-liquid distribution based on the polarity difference between ethyl acetate and saturated saline aqueous solution. 4. Purification：Helped by preparative high performance liquid chromatography DON in high purity was obtained. With this method 61.36 mg highly purified DON was obtained from one kilogram PH-1 culture. Its main feature is that the purity of the refined DON has reached 80%,which could be directly applied in conventional scientific research. If further purification needed via preparative liquid chromatography,the column loss can be remarkably reduced,therefore the economic cost decreases.