Table of Content

26 October 2019, Volume 35 Issue 10

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Synthetic Biology Research of Plant Nitrogen-fixation Organelle

LUO Li

2019, 35(10):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0585

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With the rise of synthetic biology,the long-established field of biological nitrogen fixation has ushered in new development opportunities. After the introduction of the principles and techniques of synthetic biology into nitrogen-fixing biology,a new interdisciplinary subject of nitrogen-fixing synthetic biology was born. Symbiotic nitrogen fixation is the most efficient of the three forms of biological nitrogen fixation. In the symbiotic nitrogen-fixing system,the nitrogen-fixing bacteria stably exist in the cytoplasm of the host plant as the organelles,and the relatively stable nitrogen fixation reaction is carried out by using the favorable conditions of micro-oxygen,and sufficient material and energy. However,the limitation of symbiotic nitrogen fixation is host specificity,that is,symbiotic nitrogen-fixing bacteria are difficult to complete infection and nitrogen fixation on most economic crops. Therefore,an important challenge for nitrogen-fixing synthetic biology is how to break through the host specificity of nitrogen-fixing bacteria,and achieve symbiotic or independent nitrogen fixation on main economic crops. In order to solve this difficult problem,domestic and abroad researchers have made fine research progress through hard exploration. This review will give a brief overview of some major advances and issues in the biology of nitrogen-fixing synthesis.

Advances in Mechanisms and Regulation of Iron Uptake and Metabolism in Rhizobia

JIAO Jian, LIU Ke-han, TIAN Chang-fu

2019, 35(10):  7-17.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0767

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Rhizobia are a collection of facultative symbiotic bacteria that possess the ability to establish symbiotic associations with legume hosts and carry out nitrogen fixation. Hence,they play important roles in the global nitrogen cycle and green sustainable agriculture. In order to adapt to the changing living environment such as soil,rhizosphere and infected host cells,it is vital for rhizobia to develop powerful strategies to sense and acquire nutrients. Iron is not only a limiting nutrient element for rhizobial growth and reproduction in the soil,but also directly involved in the synthesis of functional proteins closely related to symbiotic nitrogen fixation,such as nitrogenase,leghemoglobin and electronic respiratory chain components. Therefore,how rhizobia obtain iron under free-living and symbiotic conditions is highly concerned by scientists in the field of biological nitrogen fixation. Here we review recent researches on the uptake pathways of iron in different types such as Fe²⁺,Fe³⁺-siderophore complex,and heme and summarize regulation mechanisms of iron homeostasis mediated by RirA,Irr and other regulators in rhizobia,aiming to provide reference and insight for subsequent intensive researches.

Research Progress of Horizontal Gene Transfer in Rhizobia Evolution

CHEN Xue-lian, JIANG Gao-fei, ZHONG Zeng-tao

2019, 35(10):  18-24.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0742

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Legume-Rhizobium symbiosis is the most important biological nitrogen fixation system favoring the crop production worldwide. This symbiotic interaction is highly specific. Each rhizobium strain only forms a symbiotic association with a specific group of legumes,and vice versa. Recently,horizontal gene transfer（HGT）of symbiotic genes and within-host adaption has been well documented as major drivers of rhizobia evolution. This review summarizes the mechanisms and roles of HGT in rhizobia evolution and the extension of their host range,discusses the issues of strains with symbiotic traits building symbiotic systems and amenable solutions,which provides clues for the better application of symbiotic nitrogen fixation in agricultural production.

Research Progress on the Immune Regulation of Symbiotic Nitrogen Fixation Between Legumes and Rhizobia

DONG Ru, CAO Yang-rong

2019, 35(10):  25-33.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0716

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In the long-term evolution,rhizobia and legume have formed a specific mutualistic relationship：symbiotic nitrogen fixation. Rhizobia and legume interact in a very similar way as that pathogens stimulate plant pathogen response. However,rhizobial invasion and colonization do not activate the excessive defense response in their host plants,and plants also have evolved special symbiotic signal transduction and nodule development pathways to “invite” rhizobia. In addition,plant innate immunity regulates the host specificity of rhizobia to legumes. Recent studies have shown that plant defense response plays a key role in the regulation of rhizobia recognition,invasion,colonization and bacteroid development. We review the recent progress of symbiotic interaction between rhizobia and legume from the perspective of plant immune response,and discuss the regulatory mechanism of immune response induced by rhizobial MAMP and effectors through comparing interaction between pathogen and plant.

Mechanism of Nitrate Regulating Symbiotic Nitrogen Fixation Between Legumes and Rhizobium

LUO Zhen-peng, XIE Fang

2019, 35(10):  34-39.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0780

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Nitrogen is one of major macronutrients to support plants growth and development. Nitrate-nitrogen not only can be directly absorbed and utilized by plants,also affects the development and growth of plants via acting as a molecular signal to regulate the expressions of genes related to nitrogen-responding,absorbing and metabolizing. Legumes plants can obtain the demanded nitrogen for plant growth by establishing a symbiotic interaction with rhizobia;however,nodule formation and nitrogen fixation are energy-consuming processes for plants. When there is high concentration of nitrogen in soil,nitrogen as a signal molecule will affect the function of symbiotic nitrogen fixation gene and thus inhibit the process of symbiotic nitrogen fixation. Current researches reveal that nitrate inhibits symbiotic nitrogen fixation through a local and systemic regulation pathway. Autoregulation of nodulation（AON）and NIN-like proteins（NLPs）play a critical role in symbiotic nitrogen fixation. Combined with the latest researches,this review focuses on discussing the roles of NLPs transcription factors and AON pathways in symbiotic nitrogen fixation.

Research Progresses on the Mechanism of High Nitrogen Inhibiting Nodulation and Nitrogen Fixation in Legumes

KE Dan-xia, XU Qin-zhen, YANG Na, BAI Meng-yan, GUAN Yue-feng

2019, 35(10):  40-45.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0657

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Legumes form nodules and fix nitrogen by symbiosis with rhizobium. The nodulation and nitrogen fixation in legumes is of great significance in agriculture,such as reducing chemical fertilizer application,increasing farmers' benefit and improving soil environment. However,high nitrogen inhibits the nodulation and nitrogen fixation in legumes,forming a “nitrogen repression” effect. This paper discusses the molecular mechanism of high nitrogen inhibiting nodulation in legumes,including the recent progress in inhibiting the number of nodulations and development via pathways such as autoregulation of nodulation,NLP transcription factor and plant hormone signal,and explores te hypothesis and controversy on high nitrogen inhibiting the nodular nitrogenase activity,including nitrite toxicity and carbon starvation,aiming at providing theoretical basis for legumes to cope with nitrogen repression.

Biological Nitrogen Fixation in Association with Sugarcane：Retrospect and Prospect

LIN Li, LI Yang-rui, AN Qian-li

2019, 35(10):  46-56.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0901

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In Brazil certain sugarcane varieties are able to obtain large contributions of nitrogen via biological nitrogen fixation. The research on rhizosphere and endophytic diazotrophs associated with sugarcane led the research on associative nitrogen fixation in non-legume plants. The endophytic diazotroph Gluconacetobacter diazotrophicus shows some distinctive characteristics,and the diazotroph consortium containing G. diazotrophicus,Herbaspirillum seropedicae,Herbaspirillum rubrisubalbicans,Nitrospirillum amazonense and Paraburkholderia tropica is able to fix nitrogen in association with sugarcane and promote sugarcane growth. Recent studies reveal that some diazotrophs belonging to Bradyrhizobium,Rhizobium and other rhizobial genera are in the core nitrogen-fixing microbiota associated with sugarcane and actively express the gene encoding dinitrogenase reductase in sugarcane without nodulation. This review introduces some featured studies on these diazotrophs associated with sugarcane and discusses the strategies to optimize the associative nitrogen fixation with sugarcane.

Rapid Validation of Target Rice miRNAs Genes in Transient Expression System

HU Ji-xiang, CAO Ya-qian, ZHU Xiu-mei, YU Chao, TIAN Fang, YANG Feng-huan, CHEN Hua-min, HE Chen-yang

2019, 35(10):  57-63.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0175

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The aim of this study is to develop an accurate,rapid and convenient assay for experimental validation of target rice miRNA genes in transient expression systems and to lay a foundation for study the biological function of rice miRNAs. Many studies have confirmed that osa-miR169o may cleave the mRNA of OsNF-YA4（LOC_Os03g48970.1）. Here,the precursor gene miR169o（MIR169o）was co-expressed respectively with LUC empty vector,LUC-48970,and LUC-48970m3 fusion vector in transient expression systems in rice protoplast and tobacco. The LUC activity of each reaction was measured by the CCD imager and Luminometer. Combined with the miR169o levels detected by stem-loop qRT-PCR at the given time points,an optimized investigative system to validate targets of miRNAs was developed. In the rice protoplast system,LUC activity and miR169o expression level were increased gradually after transformation;the LUC activity attached the peak at 24 h. Meanwhile,the level of miR169o increased about 10-fold. Thus,the optimal detection time in rice protoplast was between 24 h and 36 h after transformation. In Agrobacterium-mediated transient expression in tobacco,LUC activity peaked at 72 h and the level of miR169o increased to 20 folds at 48h after co-infiltration. The optimal detection time was between 48 h and 72 h after co-infiltration in tobacco. In sum,this system may provide an easy,fast,and almost in vivo method to validate the candidate target gene of rice miRNAs.

Alleviation Effect of Exogenous Jasmonic Acid on Detrimental Effect of Rhododendron chrysanthum Pall Under UV-B Radiation

LIANG Dong-yu, WANG Yu-jia, SU Ao-chun, XU Hong-wei, ZHOU Xiao-fu

2019, 35(10):  64-70.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0541

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The objective of this work is to explore the feasible role of jasmonic acid in UV-B radiation and the mechanism of its mitigation. Jasmonic acid pretreated Rhododendron chrysanthum for 4 d,then PAR and UV-B radiation was conducted for 48 h,and chlorophyll fluorescence parameters,superoxide dismutase（SOD）,catalase（CAT）,malondialdehyde（MDA）,NADPH oxidase and anthocyanin were measured.UV-B radiation induced a significant reduction of Fm,Fv/Fm,Fv'/Fm',Fv/Fo,qP,anthocyanin content,and CAT activity,and increased the decreased amplitude of qP and rETR rapid photoresponse curves,while significantly increased the activity of SOD and NADPH oxidase. JA pretreatment obviously mitigated the detrimental effect of UV-B on R. chrysanthum by increasing Fm,Fv/Fm,Fv'/Fm',Fv/Fo,qP,anthocyanin content,and SOD and NADPH oxidase activities,and maintained MDA content and CAT activity at the control level,and the qP,NPQ and rETR fast light response curves presented the optimal state. In conclusion,these results confirm that the hypothesis of the external application of jasmonic acid can make R. chrysanthum resistant to stress,partially offset the negative effect of UV-B radiation on R. chrysanthum.

Physiological Mechanism of Exogenous Ethylene and Sulfur in Alleviating Cadmium Stress in Portulaca oleracea

WANG Zhu-cheng, LIU Hui, LI Rong-hua, CHEN Xin, LI Xin, LU Zhi-yuan

2019, 35(10):  71-79.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0303

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To uncover the physiological mechanism of exogenous ethylene and sulfur（S）in response to cadmium（Cd）stress,the effects of ethephon（ethylene donor）on the oxidative stress,S assimilation,glucose content,ethylene biosynthesis,and photosynthesis in leaf of P. oleracea were detected. The results revealed that exogenous ethylene and S-treatment decreased the Cd content in the leave and root of P. oleracea under Cd stress,also decreased H₂O₂accumulation and malondialdehyde（MDA）content via enhancing the activities of ATP sulfase（ATP-S）and glutathione reductase（GR）as well as increasing the content of reduced glutathion（GSH）in P. oleracea leaves. Moreover,the application of S or ethylene resulted in the glucose content reducing and Rubisco enzyme activity and photosynthesis increasing via increasing the activity of 1-aminocyclopropane carboxylic acid enzyme（ACS）and ethylene content in leaves. Simultaneous addition of exogenous ethylene and S enhanced above promoting and inhibitory effects,but the application of ethylene action inhibitor norbornadiene（NBD）resulted in the reverse effects of above indexes. The results suggest that exogenous S and ethylene alleviates the Cd- induced oxidative stress and glucose-mediated photosynthesis inhibition in P. oleracea through decreasing Cd absorption,enhancing internal ethylene signaling pathways,promoting GSH production,and decreasing glucose content.

Comparative Analysis of Antioxidant Activities of Camellia oleifera Oil Extracts from Different Areas and Their Nutritional Assessments

YE Zhou-chen, WU You-gen, YU Jing, ZHANG Jun-feng, YANG Dong-mei, HU Xin-wen

2019, 35(10):  80-88.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0433

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In order to investigate the quality differences of Camellia oleifera oils between from Hainan Island and other areas,gas chromatography mass spectrometry（GC-MS）and high performance liquid chromatography（HPLC）were used to determine their fatty acids and active ingredients,and their antioxidant activities were evaluated by applying 4 analytical methods. The results indicated that the content of oleic acid（81.353%）in Hainan（stir-fried）oil was significantly higher than that in Jiangxi（steamed）oil（70.849%）,and linoleic acid content（8.080%）was the highest in Hainan（steamed）oil,while it was not detected in Hainan（stir-fried）oil. In addition,some secondary metabolites such as squalene（2.300%-15.237%）,vitamin E（0.400%-2.900%）,stigmasterol（0%-2.507%）and 6 kinds of polyphenols were detected in the samples,and the level of quercetin in Hainan（stir-fried）oil extract was 59.025 µg/g,which was about 20 times higher than those in Hainan（steamed）oil and Jiangxi（steamed）oil,thus the difference was significant. Furthermore,the oil extracts of C. oleifera exhibited clear antioxidant activities in a dose-dependent manner,among them,the free radical scavenging capacity of Hainan（stir-fried）oil exceeded those of the other samples,concluding that this cultivar had potential utilization values as well as medical and economic benefits.

Preparation of Selenized Derivatives from Tobacco Leaf Polysaccharides and Its Antioxidant Activity in Vitro

ZHANG Hao, WEI Yue-wei, JI Xiao-ming, PAN Ting-ting, WANG Hui

2019, 35(10):  89-94.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0323

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Using flue-cured tobacco leaves as raw material,the crude polysaccharide was extracted by ultrasound-assisted and refined polysaccharides were purified and selenization of them was done for derivatives. The derivatives were characterized by infrared spectroscopy and their degrees of selenization substitution were measured. On the basis of this,the antioxidative activity of tobacco leaf polysaccharide and selenized tobacco leaf polysaccharide were studied in vitro. The results showed that tobacco leaf polysaccharide TPP1 and TPP2 were isolated when the polysaccharide was eluted with 0.30 mol/L and 0.40 mol/L NaCl solution,respectively. The selenization substitution degree of the two polysaccharides was 0.20 and 0.24,respectively. The antioxidant activity of selenized tobacco leaf polysaccharides was significantly greater than that of tobacco leaf polysaccharides in vitro antioxidant experiments,and the greater the degree of substitution was,the more obviously the ·DPPH and ·OH radical scavenging rate increased.

Correlation Between Genes Expression Levels of CaXMT1,CaDXMT1,CCS1 in Caffeine Biosynthesis Pathways and Caffeine Content in Coffee arabica

LI Fen, LI Li-jiang, XU Dan-ni, CHEN Chun-lin, WANG Wei, LI Mei

2019, 35(10):  95-101.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0294

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This work aims at exploring the correlation between expression levels of 3 key enzyme genes CaXMT1,CaDXMT1,CCS1 and caffeine synthesis in the biosynthetic pathways of caffeine in coffee（Coffee arabica）plants,as well as studying the synthetic mechanism of caffeine,thus providing theoretical evidences for the breeding of coffee trees. HPLC was used to measure the caffeine contents in the different tissues of coffee（C. arabica）plants at different developmental stages,as well as PCR（qRT-PCR）to measure the mRNA levels of CaXMT1,CaDXMT1 and CCS1,and the correlations between genes expression levels and caffeine content were conducted. The results suggested that caffeine content in different tissues presented the order as tender leaves > immature fruits > mature fruits > mature leaves. The caffeine content in immature tissues was significantly higher than that in mature ones（P<0.01）. Gene expression levels of CaXMT1 in the immature tissues was markedly less than that in the mature tissues（P<0.01）,whereas,there was no significant difference among the relative expressions CaDXMT1 and CCS1 in different development stages at the same tissue. Gene expression levels of CaXMT1 and caffeine content in immature fruits demonstrated a significantly negative correlation（R²=0.93,P<0.01）,while,there was no significant correlation between the relative expressions of CaDXMT1,CCS1 and caffeine content in different tissues at different development stages. The biosynthetic pathway of caffeine in coffee（C. arabica）plants is a very complex process,the expression levels of key enzyme genes and their correlations with the caffeine content vary,which might result from their enzymatic properties.

Difference Expression Analysis in Different Tissues of Agrotis ipsilon Reveals the Possible Mechanism of Wing Development

ZHENG Wei-gang, JIN Ming-hui, SWAPAN Chakrabarty, XIAO Bin, XIAO Yu-tao, YUAN Hai-bin

2019, 35(10):  102-110.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0336

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Black cutworm（Agrotis ipsilon）is a typical migratory agricultural pest,its wing development plays an important role in the insect migration process,and the identification and functional analysis of its specific control genes need to be further studied. The transcriptome from black cutworm whole larvae,adult head,chest,and abdomen transcriptome were sequenced and assembled. The assembled transcriptome size was 243.4 Mb and N50 contig size was 668 bp. A total of 429 288 transcripts,37 744 independent genes and 373 KEGG database annotation pathways were assembled. Wnt signaling pathways are the key ones for insect wing development. In Wnt signaling pathways in the black cutworm transcriptome,53 elements for example CaN,Frizzled and Siah-1 were annotated. CaN,Frizzled and Siah-1 in the thoracic tissues were up-regulated compared to the head and larval tissues;while CaN,Notum and Siah-1 in the thoracic tissues were up-regulated compared to the abdominal tissues. In sum,these genes regulation may be related to the muscle development and wings morphogenesis of the black cutworm.

Identification and Biocontrol Activity Analysis of Bacillus sp. BS-6 Based on Genome-wide Data

QI Jia-ming, SUN Shan-shan, ZHANG Dong-xu, XU Zhi-wen, XU Yan-ping

2019, 35(10):  111-118.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0394

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Based on the genome-wide data,the taxonomic status of Bacillus sp. BS-6 isolated from soil was clarified. The antagonistic effect of Bacillus sp. BS-6 on multiple plant pathogens was validated to uncover its potential biocontrol functions. Phylogenetic trees were constructed by 16S rRNA and gyrA gene sequences analysis to identify Bacillus sp. BS-6. Moreover,whole-genome sequencing analysis was also employed to determine the taxonomic status of strain BS-6. Plate isolate-antagonistic experiments were applied to explore the antagonistic effect of strain BS-6 on Fusarium oxysporum,Rhizoctonia solani and Botryospuaeria berengeriana. antiSMASH was used to analyze and predict the antibiotic genes of strain BS-6 among secondary metabolite biosynthesis gene clusters and to explore its biocontrol potential. According to the results of phylogenetic trees,average nucleotide identity and digital DNA-DNA hybridization,BS-6 was characterized as Bacillus subtilis. The BS-6,with 7 important or unique secondary metabolite gene clusters of Bacillus and 4 unknown function secondary metabolite gene clusters in genome,exhibited a fine ability of inhibiting the growth of fungi. Bacillus subtilis BS-6 has strong antagonistic ability against plant pathogens and promising application prospects in agriculture.

Transcriptome Analysis of Clavariadelphus pistillaris Fruiting Bodies at Different Development Stages

TANG Xian, DING Xiang, DONG Ming-ming, ZHU Miao, SONG Zhi-qiang, HOU Yi-ling

2019, 35(10):  119-129.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0233

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This work aims to study the differential expressed genes（DEGs）in Clavariadelphus pistillaris fruiting bodies between the period of juvenile mushroom（CP-1）and mature mushroom（CP-2）and to explore the key genes and metabolic pathways during the growth and development of C. pistillaris. RNA-Seq sequencing technology was used to sequence the C. pistillaris fruiting bodies,and their bioinformatics was analyzed. The sequencing results demonstrated that a total of 8.78 G（CP-1）and 8.28 G（CP-2）clean reads were obtained. Functional annotations of gene KOG and GO enrichment analysis showed that the metabolic processes,cellular and cellular parts,and oxidoreductase activities were the key gene categories in the development of C. pistillaris fruiting bodies. The analysis of gene expression level revealed that the main high-expression genes in CP-1 and CP-2 were polyphenol oxidase,ribosome,and glycoside hydrolase family,etc,which played an important role in protein and cell wall synthesis. The results of DEGs showed that oxidative phosphorylation and citrate cycle（TCA cycle）were the key metabolic pathways for the growth and development of CP-1,and the environmental oxygen content had an important effect on the growth and development of C. pistillari;meanwhile,the molecular mechanism of regulating the concentration of C1 compound and the response to various environmental stresses at CP-2 changed. KEGG pathway enrichment showed that MAPK was a key signaling pathway involved in regulating the growth and development of C. pistillaris fruiting bodies,and it played an important role in regulating the polar growth of mycelia,cell cycle and the morphological transformation of fungal cells.

Pan-Genome Analysis and Secondary Metabolic Pathway Mining of Bacillus circulans

YAO Cai-miao, ZHAO Wen-ya, WANG Bu-qing, ZHENG Li-yan, ZHANG Li-ping, LIU Hong-wei

2019, 35(10):  130-136.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0266

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This study aimed to deeply understand the genomes of Bacillus circulans and to mine these secondary metabolic pathways. The genomes of 9 B. circulans were downloaded from NCBI database and analyzed by phylogenetic analysis software，pan-genome analysis software and secondary metabolite mining software. The genome size of 9 strains was between 5.01-9.63 Mb and was divided into two branches in the evolutionary tree. Through the analysis of pan-genome and core genome，it was found that the pan-genome contained 9 572 cluster genes，the core genome was composed of 3 622 cluster genes，and a total of 4 593 specific cluster genes were identified. Among them，strain NCTC2610 had the most specific cluster genes（3 030）and strain NBRC 13626 had the least specific cluster genes（39）. After the analysis of secondary metabolite synthesis gene clusters，6 types and 32 secondary metabolic gene clusters were found in 9 B. circulansgenomes，and the most repeated metabolic pathways were lanthipeptide，lassopeptide and terpene compounds synthesis pathways. In sum，through this study the pan-genome and core genome of 9 B. circulans were clarified，and their secondary metabolic pathways were predicted. These results will help us to fully understand B. circulans，and will provide us some clues to better use those strains.

Research on Quorum Sensing Inhibitory Activity and Culture Condition of a Marine Streptomyces parvulus

ZHOU Heng, JIANG Yun, XU Ye-xiang, QIAN Sheng-hui, MIAO Li

2019, 35(10):  137-143.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0341

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We used filter paper method to investigate the quorum sensing inhibitory activity and culture condition of a marine Actinomycetes Streptomyces parvulus（HY026）that was isolated from Lianyungang surface water in our previous study. It was found that,the metabolites of S. parvulus（HY026）significantly inhibited the violacein production in the quorum sensing reporter bacterium Chromobacterium violaceum 12472,showing the strong inhibition to quorum sensing activity. The carbon and nitrogen sources of HY026 medium were optimized by single factor experiment. It was revealed that the quality and quorum sensing inhibitory activity of the HY026 crude extract were the highest when corn meal was used as carbon source,and the quality and activity of the crude extract were the highest when soybean meal was used as nitrogen source. According to the results of the single factor experiment,corn meal and soybean meal were selected as the best carbon and nitrogen sources,and 4 factors,including salinity and potassium hydrogen phosphate,were used to carry out L16（4⁵）orthogonal experiment. The quality and quorum sensing activity of the crude extracts from each treatment were analyzed. The order of importance of the four factors was as potassium hydrogen phosphate > salinity > corn flour > soybean flour. The optimal medium recipe was potassium hydrogen phosphate 1.0 g/L,salinity 34.0 g/L,corn flour 20.0 g/L and soybean flour 5.0 g/L. The quality and quorum sensing inhibitory activity of the crude extracts from S. parvulus HY026 fermentation broth increased by 398% and 19%,respectively,when cultured in this optimal recipe.

Cloning and Expression Analysis of Interferon Regulatory Factor 3（IRF3）Gene in Hybrid Grouper

LU Xiao-ying, HUANG Bao-song, MA Qian, CHEN Gang, WANG Zhong-liang, HUANG Jian-sheng, ERIC AMENYOGBE, XIE Rui-tao, DENG Wen-xin

2019, 35(10):  144-151.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0324

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Interferon regulatory factor（IRF）family has antiviral and immunoregulatory functions. Interferon regulatory factor 3（IRF-3）is a member of the IRF family and is also an immune-related factor. To understand the immune response of gene IRF3 in the hybrid grouper（Epinephelus fuscoguttatus,♀）×（E. polyphekadion,♂）to exogenous virus stimulation,the IRF3 gene of the hybrid grouper was cloned by RACE（rapid-amplification of cDNA ends）. The full-length cDNA of this gene is 2 529 bp,including 5' non-coding region（5'-UTR）325 bp,3' non-coding region（3'-UTR）916 bp,open reading frame（ORF）1 377 bp,and encoding 458 amino acids. The amino acid sequence comprises of 3 domains,i.e,one N-terminal DNA binding region（DBD）（1-108 aa）,one C-terminal interferon related region（IAD）（255-435 aa）,and a tryptophan rich region（SRD）（440-450 aa）. Phylogenetic analysis showed that the hybrid grouper's IRF3 and the Japanese sea perch's（Lateolabrax maculatus）IRF3 clustered into one,thus the genetic relationship was close. Real-time quantitative PCR was used to detect the expression of gene IRF3 in 9 tissues of liver,stomach,gill,intestine,spleen of hybrid grouper,etc. as well as the temporal expression of peripheral blood lymphocytes（PBL）. The results showed that gene IRF3 expressed in 9 tissues,and the expression in the liver,stomach and intestine was significantly higher than that in other tissues,and the expression in head and kidney was the lowest. The expression of gene IRF3 in PBL increased gradually after stimulation for 1 h at PolyI：C,and reached the maximum at 4 h（about 9.3 times of the control group）,and gradually decreased after 8 h.

Research on SNP Associated with Multiparous Trait in Double-lamb of Tan Sheep by SNaPshot Assay

TIAN De-hong, LIU Si-jia, DING Ning, LI Xue, ZHAO Kai

2019, 35(10):  152-161.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0167

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The SNPs（Single Nucleotide Polymorphism）detection and correlation study of multiparous traits were carried out on the double-lamb group of Tan sheep,aiming at promoting the application of SNPs in the genetic breeding of double-lamb in China. On the basis of SNPs screening at the previous studies,13 SNPs sites of 3 genes were determined as candidate sites by the flight mass spectrometry of candidate genes. Population differences were analyzed and multiparous candidate genes were screened by detecting polymorphism of the double-lamb group of Tan sheep. The association analyses showed that 5 of 13 loci were associated with multiparous performance,3 genetic models were confirmed,whereas haplotype constructed by BMPR1B gene was not associated with multiparous performance. The study is aimed at providing reference for molecule-assisted breeding techniques,such as molecule-marker species selection and breed selection for double-lamp group of Tan sheep in the protection and also a sustainable utilization of rare germplasm genetic resources of indigenous domestic animal,therefore accelerating the breeding progress of high quality multiparous mutton sheep.

Correlation Analysis Between LHX3 Gene Polymorphism and Growth Traits in Three Sheep Breeds

WANG Yan-xin, LIAO Yuan-yuan, A Yimuguli, QI Ao-qiong, LI Hai-jian, XU Hong-wei, YANG Ju-tian, CAI Yong

2019, 35(10):  162-168.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0384

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This work aims to provide a theoretical basis for high quality sheep breeding by finding molecular markers related to the sheep growth traits. Agarose gel electrophoresis and first-generation sequencing technology were used to detect the three genotypes of LHX3 genes in Tan sheep（TS）,Small-Tailed Han sheep（STHS）and Lanzhou Fat-Tailed sheep（LFTS）,which include insertion（AA）,insertion/deletion（AB）and deletion（BB）of 29 bp in LHX3 gene. Genetic polymorphism of LHX3 in three different sheep breeds was analyzed and the correlation between LHX3 different genotypes and the growth traits of three different breeds were analyzed by general linear model. The polymorphic information content（PIC）of LHX3 in the different breeds was as 0.25<PIC≤0.5,indicating a high genetic variation of this point. The LHX3 gene affected one or more growth traits in the three different sheep breeds. In Tan Sheep,the body length（BL）,body weight（BW）,chest depth（ChD）and cannon circumference（CaC）were significantly higher（P<0.05）in LHX3 gene deletion genotype than insertion genotype and insertion/deletion genotype. In Small-Tailed Han sheep,the chest circumference（ChC）,ChD and CaC were significantly higher（P<0.01）in LHX3 gene deletion genotype than the other two genotypes. In Lanzhou Fat-Tailed sheep,the ChC,BL,BW,hip width（HW）and CaC were significantly higher（P<0.01）in LHX3 gene deletion genotype than the other two genotypes. The 29 bp-deletion genotype in LHX3 may be selected as a DNA marker site in sheep molecular breeding.

Effect of CO₂ Partial Pressure on Subculture of Anchorage-dependent Cells MRC-5

ZHANG Yi-li, LIU Zhao-ping, LU Min-yi, CAI Ming-yong, WU Gen-peng

2019, 35(10):  169-173.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0390

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This work aims to study the effect of pH stability and CO₂ employing on the proliferation,subculture and virus production level of MRC-5 for increasing its cells subculture capacity. Preparing cell culture medium with serial pH value by adding NaHCO₃ and monitoring the dynamic variation of pH combined with CO₂,the cell morphology and proliferation during passages in different condition were evaluated. Furthermore,the cell sensitivity to virus using varicella-zoster virus（VZV）was observed. When cell culture medium with the NaHCO₃ as buffer,the pH of culture medium in the sealed group cultured without CO₂ partial pressure fluctuated in wide range,correspondingly the cells grew aberrantly and subculture to passage 35-37 was in a significantly weakened trend. The VZV production on passage 31 cells of sealed groups was 4.64 Lg PFU/mL. On the contrary,cells under CO₂ partial pressure condition presented a superior state because pH varied gently,and this group of MRC-5 cells could subculture to passage 41 with steady cell viability upon 80%,and the VZV production on the passage 31 was 4.83 Lg PFU/mL. In conclusions,employing NaHCO₃ buffer paired with proper CO₂partial pressure is conducive to the optimization and improvement of MRC-5 cells morphologies.

Eukaryotic Expression Vector Construction of Human RhoB Gene and Its Expression Analysis

WANG Mei-lin, NIU Jing-ling, JIN Ruo-qi, XU Ying-ying, CAI Jing-jing, CHEN Qun-li

2019, 35(10):  174-179.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0213

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This work aims to construct a eukaryotic expression vector carrying Flag-taged human RhoB gene and to validate its expression in cells. First,total RNA from human embryonic kidney cell HEK-293T was extracted and its cDNA was obtained by reverse transcription. Then,primers were designed and ORF sequence of RhoB gene was amplified by PCR. The amplified sequence was then inserted into a modified eukaryotic expression vector pCMV5 containing Flag tag at the loci between endonuclease SalⅠand Xba Ⅰ,thus the eukaryotic expression vector Flag-RhoB was obtained,and its accuracy was sequenced and validated by enzymatic digestion. The constructed plasmid Flag-RhoB was transfected into breast cancer MCF7 cells,cervical cancer HeLa cells or colorectal cancer HCT116 cells. Western Blotting was employed to determine RhoB protein expression. Flag-RhoB was transfected into Mv.1.Lu cells,immunofluorescence were employed to determine its sub-cellular localization. As results,the ORF region of RhoB gene was successfully amplified,then linked into vector pCMV5 and the recombinant plasmid Flag-RhoB was acquired. A 590 bp fragment was obtained by double digestion,and sequencing results showed that the cDNA sequence of RhoB gene was consistent with that in GenBank. Results from Western Blotting demonstrated this Flag-RhoB plasmid expressed well in the MCF7 cells,HeLa cells or HCT116 cells. Immunofluorescence revealed that over-expressed RhoB protein was mainly located in the plasma membrane,and little in cytosol. In sum,human RhoB eukaryotic expression vector Flag-RhoB is successfully constructed and expresses well in cells.

Effects of Plant Virus Infection on Biological Factors and Their Interactions in the Ecosystem

HU Ya-ping, ZHOU Xu, CHEN Shui-fei, GE Xiao-min, DING Hui

2019, 35(10):  180-188.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0290

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Plant virus can harm or gain host plants,and plant virus infection can change the ecological adaptability,growth and development characteristics and behavior characteristics of arthropods and their natural enemies. There are interactions between plant viruses and vector-arthropods,non-vector arthropods,vector natural enemies,other pathogenic microorganisms. Risk assessment is an important means to prevent the invasion of alien species. Clarifying the various biological factors and their interactive relations that affect the host ecosystem is the research basis of ecological risk assessment caused by plant virus infection. Here we make an overview on the interactive relationships among the plant viruses and the host plant,vector-arthropods,non-vector arthropods,vector natural enemies,other pathogenic microorganisms etc. We also discuss the future research directions of plant virus ecology on the perspectives of invasion ecology and plant virus ecology,aiming at providing an important study basis for the ecological risk assessment after plant virus invades an ecosystem as well as the control and prevention of plant virus disease.

Research Progress in PGPR Improving Plant's Resistance to Salt and Alkali

JIANG Huan-huan, WANG Tong, CHEN Na, YU Shan-lin, CHI Xiao-yuan, WANG Mian, QI Pei-shi

2019, 35(10):  189-197.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0860

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Soil salinization has become one of major factors limiting plant growth and reducing crop yield,thus seriously restricting the development of agriculture. Enhancing plant's resistance to salt and alkali may lay the foundation for improving the sustainable and efficient development of agriculture in China. Here the research status of plant growth-promoting rhizobacteria（PGPR）and the diversity of halotolerant PGPR are briefly reviewed. Furthermore,mechanisms of PGPR inducing plants to build the resistance and tolerance to salt and alkali are discussed,i.e.,changing plant physiology and substance metabolism via producing physiologically active substances such as phytohormone,1-aminocyclopropane-1-carboxylic acid（ACC）deaminase,antioxidant defense substances,osmotic adjustment substances,exopolysaccharides（EPS）,and volatile organic compounds（VOCs）. In addition,some PGPR can also regulate the expressions of genes and proteins related to enhancing plant resistance to salt and alkali. The prospect of research on interaction between halotolerant PGPR and plants is carried out,aiming at promoting the large-scale application of PGPR to alleviate salt stress damage and increase yield of plants in saline-alkali soil.

Production and Process Development of Anticancer Drug Epothilone

TIAN Lu, XUE Ren-zheng, ZHANG Zhi-min, XUE Chun-xian, SHEN Lan-fang, GONG Guo-li

2019, 35(10):  198-204.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0126

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Epothilone is a class of macrolide-centered compounds with anticancer activity,and it has a strong inhibitory effect on breast cancer and rectal cancer. Its mechanism of action is very similar to the paclitaxel's,that is,mitosis can be prevented at G2-M phase by inducing tubulin polymer to form superstable state,which can inhibit the proliferation of cancer cells and even induce their death. However,its antitumor activity is 10-1000 times higher than that of paclitaxel. As a secondary metabolite of bacteria,epothilone can be produced by microbial fermentation at a large scale,which makes it not restricted by sources like paclitaxel. In addition,because of its relatively simple molecular structure,there is a bright potential for epothilone to have chemical structural modification. Most importantly,epothilone has also shown high activity against some cancer cells that have developed drug resistance. Therefore,its huge medicinal value and market potential can make it a new type of anti-cancer drug after paclitaxel,thus is has an important application value. This paper reviews the research progress of chemical synthesis,biosynthesis,isolation and extraction of epothilone and how to improve the yield of epothilone,so that new researchers in this field have a general understanding of the recent research situation of epothilone,and it may play a guiding role in their later research ideas.

Research Progress on Biotransformation Modification of Anthocyanins

ZHAO Xiang-jie, YANG Wen-jun, YANG Rong-ling, WU Ting-ting, WANG Zhao-yu, XU Ning-ning, HE Jia-mei

2019, 35(10):  205-211.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0262

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Anthocyanins,as a natural water-soluble food coloring,possess a variety of important physiological functions and biological activities as well as high medicinal and health care value,and thus demonstrate great commercial potential. Anthocyanins are formed by the condensation reaction of anthocyanidin and saccharides in glycosidic bonds. The mother nucleus of the anthocyanin is 2-phenylbenzopyryalium cationic aglycone,and there are multiple active hydroxyl groups in the structure,which have poor stability and are sensitive to pH value,light,temperature and other factors,and easily leading to the loss of color or activity. In addition,anthocyanin is strongly hydrophilic and poor soluble in hydrophobic environment,thus it is not easy to pass through the phospholipid bilayer biofilm,resulting in a low bioavailability. Therefore,anthocyanin ester derivatives with improved physical and chemical properties and enhanced stability and good bioavailability may be obtained via acylation modification of anthocyanins on the basis of maintaining the original basic structure,which is an important approach to solve the problem in applying anthocyanins and also a hot and difficult topic in the current research. At present,biological acylation modification of anthocyanins mainly includes plant cell transformation,enzymatic transformation and whole cell microbial transformation. In this paper,the stability,bioactivity and acylation modification with biotransformation of anthocyanins are reviewed,which may provide useful reference for further research on the structural stability and physiological activity of anthocyanins and their broad application in pharmaceutical,food and cosmetic industries.

Optimization of Liquid Fermentation Conditions for Soybean Milk with Nattokinase

NI Nan, XING Zhi-hong, XU Bin, ZHANG Li-jing, WANG Yan, JIANG Jun-ping, WANG Feng-zhong, LI Shu-ying

2019, 35(10):  212-219.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0071

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Cardio-cerebrovascular diseases have seriously threatened human health,and morbidity and mortality from them are the highest among all kinds of diseases. Nattokinase is widely concerned as an ideal naturally functional food for prevention and treatment of thrombosis,thus the research on the scale production of nattokinase is of great significance to promote the development of thrombolytic functional food. Based on the previous optimization study of fermented soybean milk technology and slightly adjusting the factor sequence and factor level,the fermentation conditions were optimized by single factor and orthogonal experiments,while using whole soybean milk as fermentation medium,nattokinase as function evaluation index,and the slag rate and finial pH as auxiliary indexes. Result showed that the optimal fermentation conditions were determined as follows：the fermentation time was 30 h,the liquid volume in flask was 5%,the initial pH was 5.5,and the amount of bacteria was 1.5%. The nattokinase activity was 660 184.14 IU/mL under this condition,which was 8.31 times of that before optimization（79 434.68 IU/mL）and 1.54 times of previous optimized study（429 869.34 IU/mL）. The enzyme production significantly increased by adjusting initial pH and the order of inoculation even with lower amount of inoculation,compared to the optimized process of whole bean milk with inoculation 1.75% . This study confirmed that the cost can be eliminated while the nattokinase production can be improved,which is significant for the large-scale production of nattokinase.

Establishment of the Real-time and Label-free Screening System for Tumor Cell Apoptosis

LI Yan-wei, SONG Xing-hui, WANG Jia-jia, LIU Li, HUANG Ying-ying, GUO Chun

2019, 35(10):  220-226.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0399

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A real-time and label-free tumor cell apoptosis screening system via combining real-time xCELLigence-RTCA analysis system and flow cytometry technology. Cisplatin was used to induce the apoptosis of non-small lung cancer cells A549,and xCELLigence-RTCA system through 96 h continuous monitoring was applied to acquire the dynamic growth curve of A549 cells. The optimal testing time of apoptosis and 50% inhibitory concentration was deduced from the growth curve. Combined with precise quantitative flow cytometry technology,apoptotic rate of A549 cells treated with cisplatin was analyzed. These results confirmed the feasibility and reliability of the real-time and free technology for apoptosis screening of tumor cells.