Table of Content

26 January 2018, Volume 34 Issue 1

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The Applications of Metagenomics in the Detection of Environmental Microbes Involving in Nitrogen Cycle

WANG Zhu-jun, WANG Shang, LIU Yang-ying, FENG Kai, DENG Ye

2018, 34(1):  1-14.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0024

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The global nitrogen cycle is one of the most important biogeochemical cycles on earth,which is predominantly driven by a large number of microorganisms in various ecosystems. The application of metagenomics to reveal the total amount and diversity of functional microbial communities involved in nitrogen cycle,is one of the hotspots of environmental microbiology in recent years. In this paper,we briefly summarized the recent discovery on the functional microorganisms involved in nitrogen cycle. In addition,we focused on the selection of functional genes that could indicate certain nitrogen biological processes（including nitrogen fixation,nitrification,denitrification,anammox,assimilatory/dissimilatory nitrogen reduction,ammonification and assimilation）,and also highlighted the applications of these functional genes to detect the distribution of functional microbial communities in natural environments. Finally,we pointed out the importance of molecular detection techniques and data analysis platforms for future functional microbiological studies.

The Research Progress of the Endoplasmic Reticulum（ER）Stress Response in Plant

CHEN Qian, XIE Qi

2018, 34(1):  15-25.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0996

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Endoplasmic reticulum（ER）is a crucial site for the synthesis of membrane proteins and secretory proteins,as well as the preliminary modification of new peptides. Only the peptides that finished their preliminary modification like disulfide bonds formation,hydroxylation and glycosylation,they can enter the Golgi apparatus for further modification and eventually reach their correct protein conformation. However,the folding and modification process is complex and error-prone. If the cells face some endogenous or exogenous stresses,the probability of errors is greatly increased. The misfolded proteins were recognized and retained in ER by ER quality control system. The over-accumulated of the misfolded proteins in ER caused a series of response,which called ER stress response. They are mainly formed by UPR,ERAD,ERQC autophagy and cell death. These processes have been systematically studied in the field of yeasts and animals. This review mainly summarized the conserved mechanism in higher organism and the advances in the plant research. We hope to provide a reference for researchers who are concerned with the process of plant growth,development and plant stress response.

Research Advances on the F-box Gene Family in Plants

XU Ke-heng, ZHANG Yun-tong, ZHANG Ying, WANG Bin, WANG Fa-wei, LI Hai-yan

2018, 34(1):  26-32.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0636

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The F-box gene family is one of the largest gene families in plants. According to its protein C-terminal domain,F-box genes are divided into different subfamilies,for its number is large. Proteins encoded by F-box gene can regulate a variety of life activities,such as delaying plant senescence,regulating plant flowering and response to biological stress,drought and salt stress. In recent years,with the improvement of whole genome sequencing,more and more F-box genes of different species were identified. Most of the F-box proteins encoded by the F-box gene can form SCF complexes by binding with proteins Skp1,the skeletal proteins Cullin 1 and Rbx1,which then participates in the ubiquitin-protease pathway（UPP）. A small number of F-box proteins play a role in non-SCF complexes. UPP is one of the key regulatory mechanisms,and through which the most of the intracellular proteins are degraded. This review mainly summarizes the protein structures,function pathways and biological functions of F-box gene,and explores the life activities involved by F-box gene,aiming at laying the foundation for intensively investigating F-box.

Research Advances on the Structure,Function and Regulation of Defensins Gene Family in Plants

GUAN Li-li, CUI Qi, LI Sheng-zhao, DONG Cheng, LI Hai-long, PENG Jian-hao, LI Xiao-kun

2018, 34(1):  33-39.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0457

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As the most ancient antimicrobial peptides（AMP）,plant defensins are small cysteine-rich proteins of 45-54 amino acids and a cysteine-stabilized αβ motif that are closely related to insect and mammalian defensins. Most plant defensins are synthesized as precursors with a signal peptide sequence and are secreted to extracellular space. Plant defensins play an important role in the regulation of host immune by not only inhibiting a range of bacteria and fungi from plants and human and also killing some tumor cells and protozoon. Plant defensins are widely existing in flowers,stems,leaves,fruits,roots and seeds of plants,and have the functions of anti-bacteria,anti-tumor,inhibiting enzyme activity,making ion blockers and increasing tolerance,thus can be used as novel fungicides or antibiotics-type medicine. With the appearance of drug-resistant strains resulted from using chemical antibiotics,the study of plant defensins becomes extremely important,especially because of their potent antifungal activity,they have huge potential for engineering disease resistance in crops. This paper reviewed progresses on the discovery,structural characteristics,classifications,exogenous expression,functions and mechanism of defensins in plants. It will help to elucidate the molecular bases of their function in plants more in-depth and comprehensive in future.

Research Advances on WRKY Transcription Factors

ZHANG Fan, YIN Jun-long, GUO Ying-qi, YUE Yan-ling

2018, 34(1):  40-48.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0703

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WRKYs is one of the largest transcription factor families（TFs）in higher plants. It has a special structure of WRKY domain,which allows WRKY transcription factors to have different transcriptional regulatory functions. WRKY TFs can regulate their stress responses not only by regulating the plant hormone signal transduction pathways,also by activating or inhibiting the expressions of downstream genes while binding to the W-box（TGACC（A/T））in the target gene promoter. In addition,WRKY protein can regulate plant defense responses to various stresses not only by interacting with other TFs,also by self-regulation while identifying and combining the W-box in its own target gene. Therefore,WRKY TFs play important roles in the responses of plants to biological and abiotic stresses. However,the current research on the regulatory roles of WRKY TFs in higher plants is rare and simple in recent years. In order to provide theoretical reference and ideas for future research on WRKY TFs,here we mainly summarized the structural features and classification of WRKY TFs,the roles in plant biological and abiotic stresses,and the regulations to various stresses via hormone signal transduction pathway,MAPK signal cascade and self-regulation.

Application and Prospect of Synthetic Biology in Improving Intestinal Health

DU Ruo-xi, GUO Ming-zhang, XIE Zi-xin, HE Xiao-yun, HUANG Kun-lun, XU Wen-tao

2018, 34(1):  49-59.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0648

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Synthetic biology is a novel research field,and it is referred to design and construct new artificial biological pathways,organisms,devices or redesign natural biological systems. Synthetic biology could be used to rebuild the symbiotic bacterial strains in our gut,which may effectively improve the intestinal health status of the host by the targeted regulation of the intestinal flora or intestinal cell state. Due to the strong flexibility,a wide range of controllable targets,target specificity and few side effects of this method,it has been gradually applied in the treatment of intestinal diseases. We summarized the advances on synthetic biology in the following aspects,first,intestinal pathogens killing and intestinal microbiota balance maintaining;second,nutrient metabolism assisting and metabolic diseases curing;third,intestinal diseases diagnosing,tumor tissue locating and intestinal immune system regulating. Further we analyzed the advantages and issues while synthetic biology used in improving intestinal health,and based on which,we proposed the technological route and management system of “synthetic biology-based novel probiotics system to achieve personalized medical treatment of intestinal health”.

Research Progress on Mitochondrial Epigenetics

LIU Ying, GAO Li, FENG Jun-rong

2018, 34(1):  60-66.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0649

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As the “power house” in the cell,mitochondrion is the main organelle to carry out the reaction of oxidative phosphorylation and to generate most of the ATP. Traditionally,it is suggested that mitochondria was lack of any epigenetic mechanism,but the discovery of DNA methyltransferase,5-methylcytosine,and 5-hydroxymethylcytosine in mammalian mitochondria in 2011 contradicted with this statement. In mitochondria,the DNA methyltransferase and the patterns of both DNA methylation and DNA hydroxymethylation are quite different compared with the nuclear genome DNA,and the changes of environmental factors would cause the impacts on the mitochondrial DNA methylation . In addition,epigenetic factors of mitochondrial DNA also include mitochondrial long noncoding RNA,mitochondrial miRNA and mitochondrial DNA binding protein. With the continuous improvement of research techniques,the use of mitochondrial DNA methylation as biomarker will become more extensive and its association with nuclear epigenetics will be further revealed.

The Biological Effects Induced by Heavy Ion Radiation and Its Application in Life Science

JIA Rong, SU Feng-tao, HU Bu-rong

2018, 34(1):  67-78.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0735

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The heavy ions are the ionized particles that are heavier than element He of second one in the element periodic table. Because of the strong local ionization on the pathway of penetration,the high-energy heavy ions induce the biological effects of more serious radiation damage compared with the traditional photon radiation,such as X-rays or γ-. The researches onmechanism show that radiation by heavy ions results in multiple-type and multiple-number damages at one or two helixes of DNA,i.e.,the clustered DNA damage. The complex damages impede the binding of DNA repair enzyme with DNA segment and the consequent repairing of cells,resulting in the subsequent death of cells or improper repair（i.e.,mutation）. The characteristic of heavy ion radiation,easily inducing mutant,is popular in mutation breeding of plant and microorganism. In addition,due to the high cell killing capability and the advantage of inverted dose profile,the exact targeted therapy can be achieved using heavy ions to treat tumor,because it delivers high doses of radiation to the deep regions of tumor cells to efficiently kill them but causes relatively little damages to the surround tissues at low dose;thus,heavy ion beam is deemed a most promising technology of tumor radiotherapy. Here we review and deeply analyze the clustered DNA damage induced by heavy ions and its repairing mechanism,as well as the research advances on the 2 key repair pathways. Besides,we summarize the application of heavy ions in mutation breeding and cancer therapy. It is expected that this review may provide a convenience for researchers in radiation biology of heavy ion to understand clustered DNA damage and repair mechanisms,and culture the research direction for better estimating the risks of heavy ions to human health and setting up better protection strategy as well as the application in life science.

Research Progress on Cell Reprogramming Induced by Small Molecule Compounds

GU Shan, ZHAO Gao-ping, LI Xi-he

2018, 34(1):  79-83.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0613

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Cell reprogramming refers to the transformation of gene expression within a cell from one type to another,and that is defined as the converting process of somatic cells to pluripotent or totipotent stem cell by differentiation or to another type of somatic cells by transdifferentiation under certain conditions. Cell reprogramming provides infinite cell resources for clinical patient-specific cell therapy. It can be achieved by somatic cell nuclear transfer,transfecting specific transcription factor,and induction with small molecule compounds. However,the somatic nuclear transfer technique is thought to have ethical issues because of using oocytes;and the integration of transcription factor into the host genome leads to gene mutations,which greatly limits their clinical application. Small molecule compounds are easy to be synthesized,have fine cell permeability and flexible biological effects. The use of small molecule compounds to induce cell reprogramming avoids the ethical issues from nuclear transfer the potential harm from gene manipulation. At present,the small molecule compounds are used to induce safer iPSCs（induced pluripotent stem cells）,ciCMs（chemically induced functional cardiomyocyte cells）,and ciNSLCs（chemical-induced neural stem cell-like cells）from somatic cells. Here we summarize different kinds of cell identity conversions induced by small molecule compounds,referring to induced pluripotent stem cells,induced extended pluripotent stem cells and induced cell transdifferentiation,and finally prospect the future development of induction by small molecule compounds,the purpose is to provide reference for future research in this field.

Characterization of Polyclonal Antibody Against Pseudomonas syringae pv. actinidiae Effector Hopz5 by Enzyme Linked Immunoabsorbent Assay

HU Yue, CHEN Hang, YANG Xun-zhe, LI Qing, YANG Hui

2018, 34(1):  84-89.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0668

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In order to establish a serological detection method for Pseudomonas syringae pv. actinidiae,we used highly pathogenic PSA3 group specific effector Hopz5 to prepare the polyclonal antibody（PAb-Hopz5）. The characteristics of PAb-Hopz5 was comprehensively analyzed by detecting sample type,specificity,sensitivity and field samples via enzyme linked immunoabsorbent assay . The results showed that PAb-Hopz5 could be used for detecting not only the kiwifruit tissues,but also the PSA of artificial culture;and not only the protein of disease tissue but also the soak solution of tissues could be used for the detection. Besides,the samples of PSA different infection periods could be detected by PAb-Hopz5. No cross-reaction were observed with other testing bacteria,including P. syringae pv. tomato,P. fluorescens and P. putida,etc,indicating that the specificity of PAb-Hopz5 was solid. The minimum detectable concentration of bacterial suspension by ELISA was 3×10⁵CFU/mL,which was significantly lower than that by PCR,but for the detection of disease samples in the field it was better than that by PCR. Overall,the results showed that PAb-Hopz5 could be used as the test reagent for the PSA3 group detection of P. syringae pv. actinidiae,thus it provided a new detection tool for P. syringae pv. actinidiae.

Genetic Diversity and Population Structure Analysis of Japonica Rice Varieties from Yunnan Province

GUAN Jun-jiao, YANG Xiao-hong, ZHANG Jian-hua, WANG Jiang-min, ZHANG Peng, Li Yan-gang

2018, 34(1):  90-96.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0788

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The objective of this study is to analyze the genetic diversity,genetic structure and genetic relationships among japonica rice varieties from different breeding organizations of Yunnan province,and to provide genetic basis for breeding japonica rice varieties. The genetic diversity,genetic structure and cluster analysis of 163 japonica rice varieties from 7 breeding organizations（Geographic regions）of Yunnan province were conducted using 30 pairs of polymorphic SSR markers. The total number of alleles detected from all tested 163 accessions was 207,and the alleles per locus ranged from 2 to 17 with the mean value of 6.6. The index of gene diversity varied from 0.024 4 to 0.823 9 with an average of 0.479 8. The polymorphism information content（PIC）changed from 0.024 1 to 0.802 5 with the average of 0.440 7. The dendrogram of japonica rice varieties was constructed based on Nei’s genetic distance,showed that all rice varieties were subdivided into 4 clusters. The genetic structure analysis was performed based on model,and the varieties of japonica rice were classified into 2 subgroups. The varieties from the Dali Academy of Agricultural Sciences and the Baoshan Academy of Agricultural Sciences were all in the first subgroup,and the varieties of Yunnan Academy of Agricultural Science and Agricultural Scientific Promotion Institute of Chuxiong Yi Autonomous Prefecture were mainly concentrated in the second subgroup. The varieties from Lijiang Academy of Agricultural Science and Qujing Academy Agricultural Sciences were evenly in the two subgroups. It was concluded that the rice varieties in one breeding organization had closer genetic relationship with each other and the genetic diversity in Yunnan province was not rich at present. The genetic relationship among japonica rice varieties was partly correlated with the geographical distribution. The rich japonica rice resources have not been fully exploited yet in Yunnan province.

Gene Cloning,Structure Variation,and Expression Analysis of MeTPP1 in Cassava

DING Ze-hong, TIE Wei-wei, FU Li-li, HU Wei

2018, 34(1):  97-103.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0714

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This work is to reveal the roles of MeTPP1 gene in abiotic stresses such as drought and cold in cassava. Homology-based cloning method was used to clone MeTPP1 gene from cassava leaves,MEGA software to construct its neighbor-joining phylogenetic tree,DnaSP software to analyze its structural variations,and quantitative RT-PCR（qRT-PCR）to explore its expression characteristics under different abiotic treatments. The results showed that gene,MeTPP1 had a 1 131 bp open reading frame encoding 376 amino acids,and contained a conserved domain of TPP gene family. Phylogenetic analysis revealed that MeTPP1 had close genetic relationship to its homologues from Populus trichocarpa and Salix purpurea,and the sequence similarity was 77.8% and 74.5%,respectively. Genetic structural variation showed that a total of nine mis-sense mutations,which might be related to the expression of MeTPP1,were identified between cassava wild and cultivated species. qRT-PCR analysis demonstrated that the expression of MeTPP1 significantly changed in response to drought,cold,and ABA treatments. Together,these results indicate that MeTPP1 is involved in ABA-mediated drought and cold stresses of cassava at the transcriptional level,and can be served as a candidate to further study its functions in resistance of abiotic stress in cassava.

Comparison on Microstructure and Chlorophyll Fluorescence Parameter in Cassava

AN Fei-fei, CHEN Ting, YANG Long, CHEN Song-bi

2018, 34(1):  104-109.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0673

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In this study,wild cassava（Manihot glaziovii）,wild relative W14（M. esculenta ssp. flabellifolia）and SC205（M. esculenta cv. SC No.205）were used as materials. Leaf anatomical structures and chlorophyll fluorescence parameter were analyzed in order to show the difference in its structure and photosynthetic activity. The microstructures of cassava functional leaves were observed by paraffin sections,95% ethanol extraction method to extract chlorophyll directly,using PAM-2500 to measure chlorophyll fluorescence kinetics parameters of light systemII（PS II）. Result showed that wild cassava and SC205 leaves were with "unique" green bundle sheath cell structure,but no typical “kranz structure” of C4 plant. The overall shape of green bundle sheath cells in W14 was slightly like the “kranz structure”,but was not typical as C4 plant,while mesophyll cells were adjacent to the external of the green bundle sheath and they were arranged at slightly loose. Content of chlorophyll A and total chlorophyll in W14 leaves were significantly lower than wild cassava and SC205. Chlorophyll fluorescence parameter ΦPS II in them can be sequenced as SC205 > wild cassava> W14;however,Fv/Fm in W14 was higher than wild cassava and SC205,speculated that W14 might have a greater potential of photosynthetic capacity. Wild cassava and SC205 belong to C3 plant,W14 was a kind of cassava germplasm between C3 and C4. Their photosynthetic efficiencies in ΦPS II were in descending order of SC205,wild cassava,and W14.

Genome-wide Identification of MYB Gene Family and Cloning & Expression Analysis of JcMYB308 Gene from Jatropha curcas

XIN Hu, YAN Yue-hui, DING Xue-mei, LIU Chao, GAO Yong, DAI Dong-qin, TANG Li-zhou, WANG Hai-bo

2018, 34(1):  110-118.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0621

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MYB transcription factor family involves in the processes of the plant growth and development and the response to environmental stresses. Based on the database of Jatropha curcas genome,MYB308 gene family was identified,then JcMYB308 of J. curcas was cloned,and the functional domains,phylogenetic relationship,gene structure,and chilling expression characteristics were analyzed. The results demonstrated that the whole genome was identified to have 213 members of gene family,and they were clustered as 6 subfamilies. The cloned JcMYB308 gene fragment length was 713 bp,and the gene structure was highly conserved,with two exons. In addition,the phylogenetic tree showed that its amino acid sequence shared 62.7% identity with Ricinus communis that belonged to the same family of Euphorbiaceae. qRT-PCR expression analysis revealed that JcMYB308 expressed in different organs,abundantly in root,but scarcely in leaves,and at the highest level in stem and root at 24 h after cold-induction.

Cloning and Characteristics Analysis of Gene ItSnRK2.1 from Isatis tinctoria

ZHANG Chun-lan, MAN Li-li, XIANG Dian-jun, Winner, LIU Peng

2018, 34(1):  119-128.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0692

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As crucial protein kinase in ABA pathway,SnRK2 regulates the expressions of stress responsive genes by regulating the activity of downstream related proteins,and which then significantly improves the resistance of plants to abiotic stresses such as high salinity and drought. The degenerate primers were designed according to the conserved sequences of UTR domains from SnRK2 cDNA sequences. And a full-length cDNA sequence of SnRK2,named as ItSnRK2.1,was cloned from Isatis tinctoria by RT-PCR method. The length of full-length cDNA（GenBank accession number：MF423122）was 1 305 bp containing a complete open reading frame of 1 071 bp and encoding 356 amino acids. The theoretical isoelectric point and putative molecular weight of the ItSnRK2.1 protein were 5.64 and 30.60 kD,respectively. The ItSnRK2.1 was a hydrophilic protein with 42 phosphorylation sites,contained two significant transmembrane domains and no signal peptide,and played a physiological role in the cytoplasm. The secondary structure of the ItSnRK2.1 protein contained 151 α-helixes,109 random coils,66 extended strands,and 30 β-turns. The ItSnRK2.1 protein contained an ATP binding site and a serine/threonine enzyme activity domain,and was at the same evolutionary branch with AtSnRK2.1,AtSnRK2.5 and StSnRK2.6. RT-PCR results showed that ItSnRK2.1 gene expressed mostly in roots,following in leaves,and marginally in stems of I. tinctoria seedlings. In conclusion,ItSnRK2.1 gene actively involved in the responses to high salt and drought stresses,but not sensitive to ABA stress,suggesting that the function of this gene was possibly associated with plant stress resistance,and independent of ABA regulatory pathway.

Cloning and Expression Analysis of Invertase Inhibitor Gene Families from Dendrobium officinale

MIAO Xiao-rong, NIU Jun-qi, MO Zhao-zhan, WANG Ai-qin, HE Long-fei

2018, 34(1):  129-136.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0746

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Invertase inhibitor（InvInh）controls the activity of invertase and plays a significant role in the sucrose metabolic processes. This study aims to clone member of InvInh gene in a rare and endangered medicinal plant Dendrobium officinale,and to quantitatively detect the expression in different organs and to conduct bioinformatics analysis. As results,three InvInh genes were cloned,named as DoInvInh1,DoInvInh2,and DoInvInh3,and they encoded 176,177,and 177 amino acids,respectively. Bioinformatics analysis of 3 DoInvInh proteins showed that there were 1 signal peptide,2 transmembrane structures,and the conservative domain structure of PMEI protein. Though the homology of InvInh protein amino acid sequence was very low among different species,there were 4 conservative Cys and SP sites in all of them. The results of quantitative real-time PCR demonstrated that the expressions of all 3 DoInvInh genes were detected in the roots,stems,leaves,and flowers. The DoInvInh2 and DoInvInh3 genes expressed the lowest in roots,while the highest in flowers. The DoInvInh1 and DoInvInh3 presented a similar gene expression pattern in stems at different growth years of D. officinale,i.e.,the highest expression in the stems of one year,the medium in the stems of two years,and the lowest in stems of three years. The DoInvInh2 gene was at the highest expression in the stems of two years. The expressions of DoInvInh1 and DoInvInh3 genes were significantly in negative correlation with CIN activities,speculated that two genes probably played regulatory roles in CIN activities.

Cloning and Expression Analysis of the Promoters of OfLCYB and OfLCYE in Osmanthus fragrans

SHEN Zi-you, ZHANG Chao, DONG Bin, FU Jian-xin, HU Shao-qing, ZHAO Hong-bo

2018, 34(1):  137-143.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0706

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Lycopene cyclization is an important branching point in the synthesis of carotenoids. There are two kinds of lycopene cyclase LCYB and LCYE in plants. Previous studies have found that the differential expressions of OfLCYB1,OfLCYE1 and other genes determine the contents of downstream products of carotenoid metabolism in different varieties of Osmanthus fragrans. In this study,1028 bp and 904 bp of the OfLCYB and OfLCYE promoters were cloned by genome walking with the genomic DNA of cultivar ‘Yanhong Gui’ as the template. The bioinformatics analysis discovered two promoter sequences that contained TATA-box and CAAT-box,also the TCA-element of salicylic acid,and some photoresponsive elements such as Sp1,ACE,Box4,and G-box. The OfLCYB promoter also includes abscisic acid response element ABRE,gibberellin response element P-box. OfLCYE promoter includes gibberellin response element GARE-motif and thermal response element HSE. The cloned promoter fragment was recombined with pBI121 vector,and the plant expression vector was constructed and transferred into Agrobacterium tumefaciens. The tobacco leaf was used for transient expression. The results proved that OfLCYB and OfLCYE promoter fragments all had the function of driving downstream reporter gene expression.

Cloning and Expression Analysis of CsDXS1 Gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Synthase in Camellia sinensis

GUO Ya-fei, WANG Jun-ya, GUO Fei, NI De-jiang

2018, 34(1):  144-152.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0652

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1-deoxy-D-xylulose-5-phosphate synthase（DXS）is the first key enzyme of the MEP terpenoids synthetic pathway,thus it plays an important role in the biosynthesis of plant terpenoid. In this study,the complete ORF of CsDXS1 gene was cloned from Camellia sinensis var. fudingdabaicha based on the previous tea plant transcriptome database,and it’s length was 2 154 bp encoding 717 amino acids. The bioinformatics analysis indicated that CsDXS1 gene belonged to the class I of DXS gene family. The deduced CsDXS1 protein shared high similarity（85%-90%）with other homologous protein,and it was an instable and hydrophilic protein with no signal peptide and transmembrane domain. There were 15 high-probability phosphorylation sites. Furthermore,CsDXS1 had two typical conserved domains of transketolase superfamily. The expression profiles of CsDXS1 gene in different tissues of tea plant or under hormonal treatments were detected using quantitative real-time PCR analysis. Results showed that CsDXS1 exhibited the highest expression in the third leaf,and higher expressions in the young leaves than in the steam and old leaves. The tissue expression profiles showed that the successive order as the third leaf > the fourth leaf > the first leaf > the second leaf > young stem > old stem >old leaf. The CsDXS1 gene expression were induced at varied degrees in response to six hormonal treatments,also the occurrence times of expression peak differed. The expressions of CsDXS1 gene was in the peak（4 h）under IAA and ABA treatments,which was 1.5 times of the control. CsDXS1 gene was at the lowest expression（24 h）under MeJA treatment,which was only 1.1 times of the control.

Cloning and Expression Analysis of FGF21 Gene in Ctenopharyngodon idellus

HUANG Long, WU Ben-li, HE Ji-xiang, SONG Guang-tong, CHEN Jing, WANG Xiang, WU Song

2018, 34(1):  153-159.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0729

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In this study,the Reverse Transcription PCR（RT-PCR）was used to clone full-length sequence of FGF21（Fibroblast growth factor）gene from grass carp liver,and the quantitative real-time PCR（qPCR）was used to detect the expression levels of FGF21 gene in different juvenile tissues. Sequence analysis revealed that the full-length of cDNA of the FGF21 gene（GenBank accession number：MF_094727）was 615 bp,containing 51 bp 5'-untranslated region,564 bp open reading frame（ORF）,and encoding 187 amino acids. Alignment analysis showed that the amino acid sequence of FGF21 gene in grass carp were 78.86%,77.66%,73.94%,53.29%,52.41%,43.71%,35.96%,31.87%,and 30.39% identical to that from zebrafish（Danio rerio）,horned golden-line barbel（Sinocyclocheilus rhinocerous）,common carp（Cyprinus carpio）,rainbow trout（Oncorhynchus mykiss）,Atlantic salmon（Salmo salar）,Nile tilapia（Oreochromis niloticus）,Japanese medaka（Oryzias latipes）,human（Homo sapiens）,and house mouse（Mus musculus）,respectively. qPCR analysis indicated that the FGF21 gene expressed in all examined tissues including liver,foregut,midgut,hindgut,heart,muscle,kidney,and brain,and the highest in muscle,foregut,and midgut. These results indicate that the FGF21 gene may play an important role in muscle and intestine of grass carp.

Molecular Cloning and Immune Function Analysis of TLR3 Gene in Anguilla japonica

YU Li-li, LIN Peng, GUO Song-lin, WANG Yi-lei, ZHANG Zi-ping, WANG Ting-ting, FENG Jian-jun

2018, 34(1):  160-171.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0656

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Toll-like receptor 3（TLR3）is one of the most significant members in TLRs family which is renowned as pattern recognition receptor（PRR）to recognize double-stranded RNA（dsRNA）. To identify the structure and function of TLR3 gene in Japanese eel（Anguilla japonica）,a full-length cDNA sequence of AjTLR3 was cloned by RT-PCR and RACE. The expressions of AjTLR3 in various tissues of Japanese eel as well as in the Japanese eel liver cells were analyzed and examined via quantitative real-time polymerase chain reaction（qRT-PCR）. The full-length cDNA sequence of AjTLR3 was composed of 3 383 bp with an open reading frame（ORF）of 2 766 bp encoding a polypeptide of 921 amino acid residues. Analysis of the deduced amino acid sequence indicated that AjTLR3 protein had three main structural domains,extracellular domain which contained 16 leucine-rich repeats（LRRs）motifs,a transmembrane region and a Toll/interleukin-1 receptor（TIR）domain where had a highly conserved amino acid residue Tyr⁷⁷⁸. qRT-PCR analysis revealed a broad expression for AjTLR3 in a wide range of tissues,with the predominant expression in liver. The AjTLR3 expressions in blood,intestine,liver,spleen,skin,heart,and muscle were significantly induced after injection with the viral mimic poly I∶C,while only in the liver and intestine significantly improved with the stimulation LPS. In vitro,the AjTLR3 transcripts of Japanese eel liver cells significantly up-regulated with poly I∶C treatment at 12 h and 24 h,whereas there were significantly enhanced after 24 h by PGN and CpG-DNA stimulation. The expression level of AjTLR3 gene significantly increased and reached the peak at 24 h and 12 h after the bacterial concentration was 10⁷CFU/mL and 10⁸ CFU/mL,respectively. These results collectively suggest that AjTLR3 play an important role in Japanese eel’s immune responses to viral and bacterial infection.

Studies on Acute Toxicity and Oxidative Stress of Benzisothiazolin to Danio rerio Embryos

LÜ Peng, XU Jia-qing, WANG Sen, YAN Yan-chun

2018, 34(1):  172-182.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0665

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Benzisothiazoline（BIT）,as one of the most efficient preservatives,is widely used in industry. It is broadly distributed in environmental water condition,and has recently been regarded as a potential threat to aquatic organisms,especially to the fish. Zebrafish was used as a test organism,and the developmental toxicity and oxidative damage of BIT to zebrafish embryos were investigated by 96 h acute exposure test,oxidative stress test and related gene expression analysis. The results showed that BIT severely inhibited the hatching rates of 51 hours post-fertilization embryos under BIT exposure,and resulted in their malformation. The 96 h half lethal concentration and 96 h half teratogenic concentration were 3.65 mg/L and 1.31 mg/L,respectively. The activities of stress-related oxidative enzymes（SOD,CAT,and GST）were affected at varied level,and the expressions of related genes（Mn-sod,cat,gstp-2,nqo-1,cox-1,and ucp-2）were interfered,indicating that the organism was severely in oxidative damage. Meanwhile,the expression patterns of apoptosis-related genes（bcl-2 and bax）also changed toward the direction of apoptosis promotion,indicating that oxidative damage led to apoptosis. In summary,BIT has high developmental toxicity to zebrafish embryos at early stage and induces oxidative damages to zebrafish embryos,and leads to the death of embryo at the end. This study provides a scientific basis for environmental risk management of BIT.

Gene Cloning,Sequence Analysis,and Three Dimensional Structure Prediction of Cathepsin B in Spodoptera frugiperda and Its Molecular Docking Simulation

CHENG Xing-an, YE Jing-min, JIANG Xu-hong, LIU Zhan-mei, Hu Mei-ying

2018, 34(1):  183-194.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0818

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Cathepsin B plays an important role in development and metabolism of insect.In present study,Cathepsin B（SCB）gene was cloned from Spodoptera frugiperda（J.E.Smmith）（GenBank accession number：HQ110064）.Bioinformatics analysis showed that the open reading frame（ORF）of SCB was 1 026 bp,encoding 341 amino acids,and the predicted N-terminus hydrophobic region contained 20 amino acid residues displaying the characteristic features of a signal peptide. The predicted molecular weight（MW）and isoelectric point（pI）of SCB was 35.6 kD and 6.59 respectively while excluding signal peptide. The similarity of Cathepsin B amino acid between S. frugiperda and other 15 species ranged from 51.2%-96.2%,while at the highest of 96.2% with Spodoptera exigua（Hiibner）.The three dimension structure of SCB was predicted by homology modeling method,SCB was found to fold into a tight global structure with the dynamic optimization and contain 6 disulfide bonds responsible for the stability of the structure,and hydrophilic amino acids were mainly coated on the surface of the protein. Moreover,molecular docking simulation showed that the SCB binding pocket was relatively shallow,wide and irregular. ARG19,VAL23,and ASN24 were the key amino acid residues at active sites of SCB,and the inhibitor CA-074me（CA）interacted strongly with these three key amino acid residues by mainly electrostatic interaction energy. CA formed 2 hydrogen bonds with VAL23 and ASN24 respectively. This study lays a foundation for the further research on structure and function of SCB by using biological experimental methods,and for the development of cathepsin inhibitor pesticides.

Structure and Characteristics Analysis of Chitin Synthase 1 from Sogota furcifera

CHEN Jing, ZHANG Dao-wei, QIAN Zheng-min

2018, 34(1):  195-201.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0569

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Chitin synthase 1（CHS1）is a crucial enzyme in chitin synthesis and plays a key role in the chitin formation in insect tissues. In order to explore the gene structure and function of Sogota furcifera chitin synthase 1（SfCHS1）,transcriptome sequencing combined with PCR amplification technology were employed to study the structure characteristic of SfCHS1 gene,then quantitative PCR technology to study the spatio-temporal expression characteristics of of SfCHS1 gene,and the method of micro injection of RNAi to study the RNAi efficiency of SfCHS1 gene. The SfCHS1 cDNA contained an open reading frame of 4 719 bp and encoded 1 572 amino acids with the putative molecular weight of 180.6 kD. Homology analysis indicated that SfCHS1 included 6 N-glycosylation sites and 16 transmembrane helixes. In addition,2 transcripts（CHS1a and CHS1b）resulting from exclusively alternative splicing were identified in S. furcifera by PCR amplification and sequencing.Phylogenetic analysis suggested that SfCHS1 shared 97% identity with the known CHS1（Laodelphgax striatellus and Nilaparvata lugens）. The analysis of temporal expression characteristics showed that SfCHS1 was most highly expressed in the first day of every instar. SfCHS1 was mainly expressed in epidermis and then in trachea,a little in gut. Efficient silencing of the SfCHS1 gene through micro injection of RNAi led to rapid and significant reduction levels of SfCHS1 mRNA and resulted in S. furcifera deaths. This study suggests that SfCHS1cDNA has two variants（SfCHS1a and SfCHS1b）,and SfCHS1 is highly expressed in chitin biosynthesis stages and tissues. RNAi injection-based significantly reduces the expression level of SfCHS1 gene and causes their high-rate deaths.

Identification and Analysis of Biocontrol Proteins from a Strain of Bacillus amyloliquefaciens

GUO Ji-ping, MA Guang, WANG Zhi-jie, QI Shan-hou, WANG Bao-mei, SU Chang-qing

2018, 34(1):  202-207.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0630

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It is aimed at the identification of protein composition with biocontrol function in the secretory protein while a strain of being antagonistic grape downy mildew,Bacillus amyloliquefaciens,was cultured in NA liquid medium,which was carried out by protein profile. The proteomics of the identified proteins was analyzed by gene ontology and KEGG pathway for screening the proteins related to biocontrol. The results showed that 53 proteins were identified from the secreted proteins. Their relative molecular weights were mostly（79.24%）in 0-100. The proteins involved in the processes of carbohydrate metabolism,energy metabolism,lipid metabolism,amino acid metabolism,bio-defense,etc. They also were the cellular components in structural composition. Six proteins involved in the interaction of plant and pathogen were discovered,and among them,two belonged to the aminopeptidase family,and four were involved in the biodegradation of chitin.

Screening and Study of a New Promoter with Effective Expression of Alkaline Protease

CHEN Kun, YUAN Fei-yan, CHAI Hao-nan, LIU Huan, WANG Xing-ji, ZHANG Hui-tu, LU Fu-ping

2018, 34(1):  208-214.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0748

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The alkaline protease has extensive applications but in short supply,for which the efficient expression system of alkaline protease is constructed. A new type of promoter pChi was screened from the Bacillus licheniformis by SDS-PAGE and mass spectrometric detection. With the other two strong constitutive promoters pShuttle-09 and pyxiE as the control,pChi’s expression activities in the alkaline proteases from B. clausii and B. licheniformis was investigated. Further,six types of recombinant expression vectors and bacteria were constructed. The results showed that the functional expression of pChi was 1.25 times of pShuttle-09 and 2 times of pyxiE,which offers the foundation for the gene heterologous expression in mediated B. subtilis expression system.

Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated

WEI Rui-yang, BAI Jie, XU Bin, XI Yan-yan, WANG Lin-yi, WANG Gai-li, FU Chen, WEI Feng-xian, LI Shao-yu

2018, 34(1):  215-222.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0731

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The attempt of this work is to investigate the anti-inflammatory activity of Musca domestica lectin subunit alpha（Lsa）toward Staphylococcus aureus. The well-constructed pLEX-Lsa and pLEX were transfected into RAW264.7 cells with recombinant lentiviruses,then the stably-expressed RAW-pLEX-Lsa and RAW-pLEX were obtained after screening,and RAW-pLEX was as a negative control. S. aureus was used to stimulate RAW-pLEX-Lsa cells and RAW-pLEX cells,then both un-stimulated cells and stimulated cells for 3 h,6 h,12 h,and 24 h were collected,and the cell culture supernatants of 6 h un-stimulated and stimulated cells were also collected. Real-time quantitative reverse transcription PCR（RT-PCR）was performed to analyze the mRNA expression of TNF-a,IL-1β,NFκB-1 and NFκB-2,and the protein level of TNF-a and IL-1β in the supernatant were detected by ELISA. The results of RT-PCR showed that the stimulation of RAW-pLEX-Lsa by S. aureussignificantly down-regulated the mRNA expression of TNF-a transcript variant 1（TNF-a-tv-1）at 6 h（P<0.05）and TNF-a transcript variant 2（TNF-a-tv-2）at 6 h and 12 h（P < 0.05）,compared with the RAW-pLEX cells. The stable transfection of Lsa in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1β-T at 3 h,6 h and 12 h（P < 0.05）and the mRNA expression of IL-1β at 3 h and 6 h（P < 0.05）,compared with the RAW-pLEX cells,while the mRNA expression of NFκB-1 and NFκB-2 in RAW-pLEX-Lsa had insignificant decreasing. The results of ELISA demonstrated that the protein level of TNF-a in the supernatants of RAW-pLEX-Lsa cells at 6 h after stimulation by S. aureus significantly down-regulated（P < 0.05）. In conclusion,Lsa possesses potent anti-inflammatory activity,and stable transfection of Lsa in RAW264.7 cells inhibits the expression and production of TNF-α and IL-1β. However,the inhibition of TNF-α and IL-1β is not resulted from blocking the activation of NF-κB signal pathway.

The Fermentation Characteristics and Application in Chili Fermentation of Strain CICC 6287

XIE Jiu-yan, ZHAI Lei, SONG Zhen, YANG Yu-xin, CHENG Chi, YAO Su

2018, 34(1):  223-229.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0527

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This work aims to study the fermentation characteristics of strain CICC 6287 isolated from Xinjiang dairy products,and to evaluate the advantages of strain CICC 6287 as fermenting strain by chili fermentation experiments. Taxonomic status was identified by polyphasic taxonomy technique. The fermentation traits were characterized by detecting the acid- and salt-resistance,the abilities of acid production and nitrite degradation,the activities of amino acids decarboxylases,and antimicrobial properties. Based on the properties mentioned above,the strain was applied in Xinjiang chili fermentation to evaluate the function of strain CICC 6287. The results revealed that strain CICC 6287 was identified as Lactobacillus kefiranofaciens. The strain degraded nitrite,tolerated 8% NaCl and pH 3,inhibited the growth of Escherichia coli,Staphylococcus aureus,Listeria monocytogenes,and Salmonella,and was negative for amino acids decarboxylases. The nitrite content of fermented chili inoculated with strain CICC 6287 was 1.06 mg/kg,bioamine content was 5.62 mg/kg,and total acid content was 2.54%. The results indicate that strain CICC 6287 has promising fermentation characteristics,and is applicable for chili fermentation to improve its security.

Effects of Continuous Cropping on Bacterial Flora Structure in Soybean Rhizosphere Soil

YIN Ji-zhong, LI Liang, JIE Wei-guang, CAI Bai-yan

2018, 34(1):  230-238.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0728

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The flora structure of soil bacteria is an important part of soil ecological environment quality. In order to find out the changes of bacterial flora structure in the rhizosphere soil after continuous cropping of soybean,soybean rhizosphere soil samples of zero- and 2-year of continuous cropping were selected,and their bacterial flora structures were studied using Illumina high-throughput sequencing technique. The results showed that the bacterial abundances and diversity indexes in the rhizosphere soil of zero-year continuous cropping were higher than those of the rhizosphere soil of 2-year continuous cropping. The abundances of these beneficial bacteria Cytophagaceae,Sphingomonas,Bradyrhizobium,and Streptomyces varied insignificantly. Soybean continuous cropping reduced the bacterial diversity of the soil and changed the bacterial flora structure. However,during the short-term continuous cropping process,the variation pattern of rhizosphere microbes in different crops differed,thus it was difficult to infer a common phenomenon resulted from continuous cropping. The results are aimed to provide a theoretical basis for alleviating the short-term soybean continuous cropping obstacle and increasing the yield of soybean.

Effects of CO₂ Concentrations on the Physiological and Biochemical Parameters of Haematococcus pluvialis

CHEN Jia-ni, LIN Li-chun, XU Nian-jun, ZHANG Lin, SUN Xue

2018, 34(1):  239-246.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0669

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In order to explore the response of microalga Haematococcus pluvialis to different CO₂ concentrations,the effects of two kinds of CO₂ concentrations on the algal growth,pigment content,chlorophyll fluorescence parameter and activities of two carbon metabolism-related enzymes,were investigated by the physiological and biochemical methods. Results showed that at the green stage,compared to the air CO₂,the growth rate of H. pluvialis increased 3.08 times（10 th）under quadruple air CO₂ condition;the chlorophyll and carotenoid content,and the extracellular carbonic anhydrase activity all decreased;non-photochemical quenching（NPQ）,most of quantum yield of photo-chemical electron transport_（Ф_PSII）,and maximal photo-chemical efficiency of PSII（F_v/F_m）almost significantly increased,whereas the photo-chemical quenching（qP）and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase（Rubisco）differed insignificantly.At the red stage,the algae cultured with quadruple air CO₂ grew slower,however,their astaxanthin content increased by 20.23%（8 d）of that under air CO₂ condition. The study indicated that appropriately high CO₂ concentration promoted its growth rate and astaxanthin accumulation in H.pluvialis.

Construction of SGK3 Gene Lentiviral RNA Interference Vector and Effects on Cell proliferation and Apoptosis of Breast Cancer Cell Line MB-474

GUO Hong-yan, GAO Han, WU Qi, SUN Xiao-jie, LIU Xiu-cai, ZHAO Li-qun

2018, 34(1):  247-252.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0557

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This study is aimed to construct the lentiviral RNA interference（RNAi）vector of SGK3 gene,and to observe its effect on the proliferation and apoptosis of human breast cancer cell line MB-474. The lentiviral vector of shRNA sequence targeting SGK3 was constructed and transfected into human breast cancer MB-474 cells to silence the SGK3 gene. The expression of SGK3 mRNA and protein in transfected MB-474 cells was detected by real-time PCR and Western bloting,and the effect of SGK3 gene silencing on the proliferation,cell cycle and apoptosis of MB-474 cells was detected by MTT assay and flow cytometry. Gene sequencing showed that 4 RNAi lentiviral vectors targeting the SGK3 gene were successfully constructed. The lentiviral vectors were packed successfully by 293T cells and the virus titer was（3-8）×10⁸ TU/mL. After the recombinant lentiviral vector was transfected into MB-474 cells,real-time PCR and Western bloting demonstrated that the expression of SGK3 in interference group was lower than that of control group. Among the 4 lentiviral vectors,the one with best interference effect is the PGC-LV3-SGK3-1 sequence. SGK3 silencing inhibited MB-474 cell proliferation and promoted apoptosis,but had no significant effect on cell cycle progression. In short,RNAi lentivirus vector targeting SGK3 gene was successfully constructed,and SGK3 gene silencing affected cell proliferation and apoptosis of breast cancer.