Table of Content

25 July 2016, Volume 32 Issue 7

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Orignal Article

Studies on the Gene of Key Component GA20-oxidase for Gibberellin Biosynthesis in Plant

WU Jian-ming CHEN Rong-fa HUANG Xing QIU Li-hang LI Yang-rui

2016, 32(7):  1-12.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.001

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GA20-oxidase,as a key synthetizing and regulating enzyme in the biosynthesis of gibberellic acid,directly regulates the generation of biological GAs. It is also a multifunctional enzyme,and its most important feature is negative feedback regulation. GA20-oxidase plays an important regulating role in the development and physiological processes of plants. This article summarizes the recent studies on the molecular cloning and regulating expression analysis of GA20-oxidase genes and their effects on plant height,fiber formation,flowering,yield traits,etc.,and explains the interactions of GA20-oxidase gene with hormones,photoperiod,stress resistance and other factors,aiming at revealing the signal network system and its mechanism of GA20-oxidase.

Research Progress on Plantlet Development and Relevant Genes of Kalanchoë daigremontiana

JIANG Wen-ting ZENG Hui-ming

2016, 32(7):  13-20.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.002

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Kalanchoë daigremontiana reproduces asexually by forming plantlets on the leaves. During the developing process,the plantlets experience the various stages similar to that of zygotic embryo. The development of the plantlet share features of both organogenesis and embryogenesis. Research has shown that SHOOT MERISTEMLESS（STM）gene is required for the formation of plantlet. LEAFY COTYLEDON 1（LEC1）gene is expressed during the development of plantlet,however,it is not needed in the formation of the plantlet of K. daigremontiana. The functions of FUSCA3（FUS3）,SAHH and other relevant genes in the development of the plantlet of K. daigremontiana still needs further investigation. This paper summarized the development of K. daigremontiana plantlet and relevant genes,aiming at providing some references for related researches.

Research Progress on Epigenetic Regulation Mechanism in the Differentiation of Bone Marrow Mesenchymal Stem Cell

WANG Lian-qing ZHAI Qiao-li ZHAO Pei-qing LI Tao

2016, 32(7):  21-27.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.003

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Bone Marrow Mesenchymal Stem Cells（BMMSCs）,with the potential of self-renewal and multipotent differentiation,may differentiate to be several types of cells by induction,thus hold critical clinic values for the restoration of destroyed bones and cartilages. In order to investigate a medicine that may efficiently promote the directional differentiation of BMMSCs and thus using BMMSCs in clinic more safely and reasonably,it is necessary to illustrate the epigenetic regulation mechanism in the differentiation process of BMMSCs. Abnormal regulation of BMMSCs differentiation may lead to development of several diseases,thus appropriate control of BMMSCs differentiation mechanism has been the hotspot topic of research. Epigenetic regulation,such as DNA methylation,histone acetylation,histone methylation and noncoding RNA,play critical roles in the regulation of BMMSCs differentiation. This review summarizes the progress on major epigenetic regulation mechanisms involved in BMMSCs differentiation in recent years.

The Role of Focal Adhesion Kinase Signaling Pathway in the Bacterial Invasion of Non-phagocytic Cells

JIA Xiao-yang FU Yu WANG Xiao-nan WANG Zhi-gang HAO Hui-fang

2016, 32(7):  28-33.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.004

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The adhesion and invasion into eukaryotic cells are major steps in bacterial pathogenesis. In order to further study the entire process and molecular mechanisms of bacteria invading non-phagocytic cells,we reviewed the bacterial adhesion and invasion of non-phagocytic cells and the regulatory mechanisms of focal adhesion kinase（FAK）in this process. During the adhesion process,the interaction of surface adhesive molecules and special structures of bacteria with receptors of host cells activates the FAK to regulate the cytoskeletal rearrangement through FAK/Src-Cortactin-Arp2/3 and FAK/PI3K-Rac pathways,which promotes the bacterial invasion of non-phagocytic cells.

Technologies of Extracting Genome and Transcriptome from Mortierella alpina

LING Rui, SUN Li-jie, CHEN Xiang-song, YUAN Li-xia, WU Jin-yong, YAO Jian-ming

2016, 32(7):  34-39.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.005

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High-throughput sequencing of transcriptome and genome demands the high purity and content of sample,the purpose of this work is to determine the optimal extraction technology by studying the effects of Mortierella alpina in different culture conditions and using different hypha processing methods on the genome and transcriptome quality. The results showed that the mycelium from liquid fermentation medium after genome extraction presented a significant difference in agarose gel electrophoresis compared to the mycelium from PDA culture medium,the former gave degradation-free genome DNA but the latter was extracted genomic DNA of some degradation. During using domestic brand kit to study M. alpina tanscriptome RNA extraction,it was found that using liquid nitrogen fast-frozen method to break the hypha cell wall combined with applying β-Mercaptoethanol resulted in more likely to extract the high concentration and no-degradation transcription sample.

Transcriptome Analysis of Germinated Tartary Buckwheat Based on High-throughput Sequencing Technology

CHEN Chun-xu, LI Qi, GUO Yuan-xin, DU Chuan-lai, DING Zhi-gang

2016, 32(7):  40-47.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.006

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Illumina SolexaHiseq 2500 high-throughput sequencing technology was used to get the comprehensive transcriptome from germinated tartary buckwheat. As a result,42 953 962 sequence reads containing 5.37 Gb nucleotide sequence information were obtained. After de novo assembly by the software of Trinity,a total of 45 278 unigenes were generated,corresponding to a total of 39 Mb with an average length 862 bp. In addition,the data from the evaluation of the unigenes indicated fine sequencing quality and high reliability from the aspects of length distribution,GC content,and expression level. The comparison of sequence homology in database showed that 2 127 unigenes had various degrees of homology with other known biological genes. The unigenes in the transcriptome of germinated tartary buckwheat were correlated with cellular processes,cell and protein binding. According to KOG database,the unigenes were broadly divided into 24 categories. Referring to KEGG database,unigenes were located into 328 metabolic pathways,including ribosome,carbohydrate metabolism and so on. And 38 unigenes involved in the synthesis of GABA in oxidative phosphorylation metabolism were screened. Total 7 141 unigenes were found from 71 366 by SSR and the highest frequency was A/T,followed by AAG/CTT and AT/AT.

The Application of 454 High-throughput Sequencing Technology into Anlaysing the Diversity of Soil Fungi in the Field Planting Tamarix chinensis and Angiospermae

WANG Yan-yun GUO Du-fa

2016, 32(7):  48-53.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.007

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The study,which was related to the variety and diversity of soil fungi in the field planting Tamarix chinensis and Angiospermae,and the corresponding analysis of fungal diversity index and similarity were conducted by applying 454 high-throughput sequencing technology. It is aimed at providing the basic datas and theoretical gist for the futher study into the diversity of fungi in Yellow River Delta and the improvement of saline-alkali soil. And the result indicated four soil samples were divided into 5 phyla,17 classes,37 orders,48 families and 51 genus. The OTU number and diversity index showed that the diversity of soil fungal diversity was the richest in Tamarix chinensis surface（C1：0-20 cm）,which was accordingly 1.7 times,1.2 times and 2.4 times of OTU number,Shannon index and Chao1 index in Angiospermae substratum（Z2：20-40 cm）. In addition,the analysis of the distance heat map indicated the fungal communities of Z1 and Z2 had the most similarity,whose Unifrac metric value was 0.269 9 and the fungal communities of C1 and C2 have the least similarity,whose value was 0.690 2. In a word,the soil fungi in the field planting Tamarix chinensis was richer than one in the field planting Angiospermae.

Establishment of A Method for Determination of Protein Content in the Final Product Containing Recombinant Lysostaphin

XU Yan, ZHANG Yi-tao, YE Gui-zi, LU Hai-rong, HUANG Qing-shan

2016, 32(7):  54-58.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.008

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Recombinant lysostaphin（rLysn）,duo to its solid antibacterial activity to Staphylococcal aureus,is novel antibacterial medicine,and therapeutic agent containing this enzyme may efficiently control the infection of S. aureus. In order to control the quality of pharmaceutical preparations with rLysn,a method of measuring the protein content in the final product with rLysn was established and verified. Size exclusion high performance liquid chromatograph（SE-HPLC）was employed to measure the protein content in both physico-chemical control and the final product with rLysn under the conditions as below：a TSK gel 2000SWXL column（7.8 mm×30 cm,5 µm）,the mobile phase composed of phosphate buffer（20 mmol/L,pH7.0）and NaCl（0.3 mol/L）,the flow rate was 0.5 mL/min,the temperature was 25℃,and the detection wave length was 280 nm. As results,the rLysn（within 2-32 µg）was linearly correlated with the area of the elution peak（r² =1）. The weighted average recovery rate for physico-chemical controls in 3 rLysn concentrations of low,medium and high was 102.9%,while the weighted average recovery rate was 102.1%. Three supernatants were measured at different time,and RSD of protein content < 1%;while measurement was done for each supernatant in 1 h interval,RSD of the protein content was 0.98%;and 6 samples from one targeted solution were measured,RSD of the protein content was 1.71%. Above results reveal that SE-HPLC is simple and convenient,accurate,precise,stable and repeatable approach,thus may be used for the determination of protein content in the final product of rLysn.

Optimization of the Purification Method for Transmembrane Protein AgrC_TM6-7C

WANG Lu-lu, QUAN Chun-shan, XU Yong-bin, WANG Guan-tian, LIU Shuang, CHEN Ying

2016, 32(7):  59-65.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.009

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The core objective of this paper is to obtain the protein of AgrC_TM6-7C with high purity by optimizing the purification processes,and provide the materials for further research. We investigated the influence of imidazole concentration in buffer used in the metal ion affinity chromatography and buffer with different pH used in the ion exchange chromatography on the purification of AgrC_TM6-7C. Moreover,we discussed the effects of β-Mercaptoethanol on protein aggregation and kinase activity of AgrC_TM6-7C. The optimized purification method resulted in the obvious increase on the yield and purity of AgrC_TM6-7C,producing 15 mg with about 98% purity from one-liter culture medium.

Cloning of Bna-miR1140 Gene Promoter in Rape and Preliminary Identification of Its Expression Pattern

DONG Yun, WANG Yi, JIN Feng-wei, SUN Wan-cang, LIU Zi-gang, FANG Yan, XU Miao-yun, WANG Lei

2016, 32(7):  66-72.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.010

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In order to explore the expression pattern and regulation mechanism of Bna-miR1140,the upstream 1.5 kb fragment from Bna-miR1140 precursor was cloned in a cultivated variety Westar of rape by PCR approach on the basis of rapeseed miRNAs chip results in our laboratory,and their cis-acting elements were analyzed. Then plant expression vector of GUS reporter genes was constructed,the vector miR1140 pro∷GUS was transformed into variety Westar by Agrobacterium-mediated approach,and 5 positive transgenic lines were obtained. GUS chemically staining the varied tissues of T1 generation groups of positive rape strains,the results showed that the upstream 1.5 kb region of miR1140 precursor sequence presented the function of promoter,driving GUS expression in rapeseed,moreover,only specific expression in the petiole and leaf axil,indicating that miR1140Pro was a specific promoter.

Cloning and Expression Analysis of Actin Gene Fragment from Dioscorea opposite

GONG Ming-xia, ZHOU Yun-yi, WANG Ai-qin, LUO Hai-ling, HE Long-fei

2016, 32(7):  73-80.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.011

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The cloning and expression analysis of Actin gene fragment from Dioscorea opposite was studied in order to provide the foundation for studying its function and other genes expression and regulation in yam growth and development. A pair of degenerate primers were designed based on the conserved sequences of the published Actin genes of other plants in GenBank. The cDNA fragment of 1 Actin gene was isolated from yam tuber by RT-PCR and designated as DoActin. It’s length was 1 091 bp encoding a protein with 357 amino acids residues,and was deposited in GenBank（Accession number：KU669295）. Homologous alignment according to the nucleotide and protein databases showed that it shared over 83% nucleotide sequence similarity and over 97% amino acid sequence similarity withActins in other plants. The phylogenetic tree reconstructed on the base of amino acid sequences suggested that the DoActin had the nearest relationship with Phoenix dactylifera Actin-2 and Gossypium arboreum Actin-7. RT-PCR results revealed that the expression levels ofDoActin in different organs such as leaf,aboveground stem and underground tuber,as well as tubers and leaves in different developmental periods were relatively stable,indicating that DoActin can be used as an appropriate reference gene for D. opposite.

Molecular Cloning and Expression Analysis of Aquaporins SIP2-1 Gene from Banana

XU Yi, HOU Xiao-wan, XU Bi-yu, JIN Zhi-qiang, HU Wei, SONG Shun

2016, 32(7):  81-86.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.012

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In the present study,we isolated an aquaporin gene from banana,and designated as MaSIP2-1. MaSIP2-1’s complete ORF was 717 bp,and it encoded 239 amino acids. Alignment of amino acid sequences and phylogenetic analysis indicated that the protein encoded by MaSIP2-1 was in high similarity with AQP-encoding protein in the other known plants;and highly homologous with amino acid sequences in Musa acuminate,Elaeis guineensis,Jatropha curcas,and Camellia sinensis by 98%,74%,65%,and 63%,respectively. Organ-specific analysis showed that MaSIP2-1 constitutively expressed in roots,stems,leaves,flowers and fruits,and the highest in stems. Stress analysis demonstrated that MaSIP2-1 responded to the stress such as the drought,salt and waterlogging.

Expression Profile Analysis of Maize Resistance Under Osmotic Stress Induced by Secondary Metabolites of Endophytes

WANG Na, GONG Na, LIU Guo-li, MA Xiao-ying, YANG Zhen, YANG Tao

2016, 32(7):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.013

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Genechip technology was used to analyze the expression profile of differential genes in corn treated by metabolites of endophyte under 10% PEG osmotic stress. The results showed that there were 441 differentially expressed genes identified in this study including 147 up-regulated genes and 294 down-regulated genes,participating in 21 metabolic pathways. The genes with differential expressions were classified by GO-ranking methods through CapitalBio Molecule Annotation System V 3.0（MAS）. Among them 45% participated in various biological processes,18% were related to synthesis of cellular components,the coded products of 36% genes were related to performing a variety of molecular function,and 1% corresponded with other unknown sequences in GenBank. Thus the role of these genes still needs further study. Pathways analysis using the KEGG pathway database showed the differentially expressed genes broadly were involved in basic metabolism,energy metabolism,secondary metabolism and signal transduction pathways. The expression profile analysis indicate that the effects of secondary metabolites on corn under osmotic stress are synergistical processes by many genes in many pathways.

Analysis of Genetic Diversity in Chaenomeles Using Apple EST-SSRs

ZHANG Yan-yan, QI Hong, GUO Qing-mei, SUN Zhi-ying, ZHOU Feng-qin, LI Sheng-bo

2016, 32(7):  93-98.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.014

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EST-SSR markers of Malus pumila Mill. were selected to investigate the genetic diversity of germplasm resources in 33 cultivars of Chaenomeles,and hence to confirm the transferability of SSRs information from Malus to Chaenomeles. In this study,15 selected primers were employed for PCR amplification and 9 primers presented polymorphic bands;71 bands were generated by all EST-SSR,of which 59（83.10%）were polymorphic;the different cultivars listed here were successfully distinguished. The average of polymorphic information content（PIC）were 0.45. The average of Shannon index（I） and Nei’s genetic diversity index（H）were 0.71 and 0.45. UPGMA cluster based on the EST-SSR data,the materials were divided into 5 groups. Different samples of Chaenomeles were gathered together and could be distinguished by system tree. The results showed that EST-SSR markers developed from M. pumila showed high transferability to Chaenomeles,and they may be used in germplasm evaluation and genetic relationship for Chaenomeles.

Effect of Temperature on Growth and Chlorophyll Fluorescence of Ulva fasciata

HUANG Yan-hua, YANG Rui, SUN Qing-hai

2016, 32(7):  99-105.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.015

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In order to study the temperature adaptation mechanism of Ulva fasciata Delile,culturing the U. fasciata under the temperatures：5℃,10℃,15℃,20℃,25℃,30℃,33℃ and 35℃ for 7 d,and the growth change,chlorophyll content,and indexes of chlorophyll fluorescence of U. fasciata at different temperatures as well as the parameters of chlorophyll fluorescence after re-culturing at 25℃ were examined to understand the effects of different temperatures on the U. fasciata. Results showed that growth rate,chlorophyll content,the indexes of fluorescence（optimal photo-chemical quantum yield（F_v/F_m）,effective optimal photo-chemical quantum yield（F_v'/F_m'）,photosynthetic light use efficiency（α）,and photo-chemical quenching（qP））all reached the maximum at 25℃. All these physiological indexes of the seaweeds decreased gradually when the temperature were higher or lower than 25℃,and the lowest at 5℃ and 35℃;however,the non-photo-chemical quenching（NPQ）gradually rose with the increase or decrease of temperature,and till its maximum value at 10℃. The F_v/F_m and F_v'/F_m' values of the samples of 5℃ to 30℃ groups increased a bit after they were recovered at 25℃ for 2 d,and the highest increment occurred in 10℃ group. Meanwhile,the indexes did not change in the group of 33℃ and 35℃ after recovered cultivation. The results indicated that the most appropriate temperature for U. fasciata to grow and photo-synthesis was in a range of 15℃ to 30℃,5℃and 35℃ were the lowest and highest growth temperature respectively,and U. fasciata showed greater tolerance to low temperature than to high temperature.

Isolation and Identification of Streptomyces triostinicus C2 Antagonizing on a Variety of Plant Pathogenic Fungi

PENG Wei-fu, WU Zhi-ming, CHEN Wei, ZENG Yong-jun, LI Kun-tai

2016, 32(7):  106-111.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.016

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The research purposes of this work are to isolate and screen a strain against phytopathogenic fungi. The antagonistic strain was isolated and screened using plate confrontation culture with Rhizoctonia solani as indicator,and was further identified on the basis of morphological characteristics,physiological and biochemical characterization,and 16S rDNA sequence analysis. From 18 soil samples collected from Yunnan,Guangdong,Anhui,Hubei,and Jiangxi Provinces of China,the isolated actinomycete strain C2 presented a strong inhibitory effect on R. solani,and it was preliminarily identified as Streptomyces triostinicus,designated as Streptomyces strain C2 here. The bioassay results showed that Streptomyces strain C2 possessed a broad-spectrum inhibitory effect on a range of plant fungi such as R. solani,Pyricularia grisea,Colletotrichum gloeosporioides,Fusarium oxysporum f. sp. niveum,and Penicillium citrinum,and the inhibition percentage was 49.78%,56.62%,80.96%,18.59%,and 94.10%,respectively. Conclusively,Streptomyces strain C2 was an antagonistic strain with broad-spectrum in suppressing the growth of various plant fungal pathogens.

Identification of Antagonistic Strain Act0988,and Relationships Between Cultivation Conditions and Its Growth

ZHANG Jing MUO Li-hui LIU Zhi-wei KUANG Wei HUANG Si-mei DIAO Shu-ping JIANG Na GUAN Xi-guang

2016, 32(7):  112-118.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.017

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Strain Act0988 produces the metabolite with antifungal activity against many kinds of mould,such as Penicillium italicum et al. The strain identification and study of the relationship between cultivation condition and the strain growth were aimed at laying the foundation for the screening media and research of fermentation processes during the development of the metabolite in future. The strain was identified by its morphological and culture characteristics,physiological and biochemical characteristics and 16S rDNA sequencing. The relationships between carbon sources,nitrogen sources,temperature,pH,oxygen and strain growth were studied with shake flask cultivation. Strain Act0988 was identified as Streptomyces polychromogenes,the biomass of fermentative broth was 5.8 mg/mL if the optimal carbon source was soluble starch,the biomass was about 5.7 mg/mL while yeast extract and peptone were the optimal nitrogen sources,the biomass was 6.0 mg/mL when the optimal temperature was 28℃,the biomass was 6.2 mg/mL while the most suitable initial pH was 7.0,and the biomass was 6.3 mg/mL if the optimal volume of the medium was 60 mL per 500 mL shake flask. For being easy to be cultivated and fermentation broth owing high antifungal activity,the metabolites produced by strain Act0988 has the great potential of development and utilization.

Correlations Between Microbial Community and Physicochemical Indexes in Pit Mud of Different Ages

ZHONG Shu-xia DENG Jie WEI Chun-hui LUO Hui-bo WAN Shi-lü HUANG Zhi-guo

2016, 32(7):  119-125.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.018

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This experiment aims to study the diversity of major microbial community structure in pit mud of different ages and the variation pattern of pit mud’s physical and chemical index（pH,total nitrogen,humic acid,available phosphorus and total acid）,and to analyze the correlations among them. The results of Kjeldahl determination showed that：with the increase of pit age,pH of pit mud did not change basically,total acid tended to decrease slowly,total nitrogen rose firstly and then dropped,humic acid and available phosphorus increased slowly. The diversity index of bacteria and archaea by DGGE ranged as 1.62-2.58 and 1.79-2.33,respectively,and gradually increased with the increasing of pit age. The correlation coefficient between bacteria’s community structure and humic acid was 0.972（P<0.01）,-0.987（P<0.01）with total acid,and 0.878（P<0.05）with available phosphorus. The correlation coefficient of archaea’s community structure and humic acid was 0.986（P<0.01）,-0.954（P<0.05）with total acid,0.901（P<0.05）with available phosphorus,and no significant correlation with other indexes. Regarding bacteria,selected 11 dominant bands for sequencing,and most of them were found to be bacillus generally. Regarding archaea,selected 4 dominant bands for sequencing,and most of them were found to be methanogen. The physiochemical indexes and microbial community structure in pit mud showed a certain regular pattern with the change of the pit age,and some physiochemical indexes were strongly correlated with microbial community structure,these physiochemical indexes greatly concerned with the microbe’s domestication.

Conditions for Protoplast Preparation and Regeneration by Sphingomonas sp. ATCC 31555

ZHOU Ming-ming, LI Xiao-yan, REN Meng-nan, CHENG Ke-meng, HUANG Hai-dong

2016, 32(7):  126-130.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.019

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The effects of EDTA pretreatment,cell age and the concentration,temperature and work time of enzymolysis on protoplast formation and regeneration by Sphingomonas sp. ATCC 31555 were studied. The results showed that the formation rate and regeneration rate reached 97.3% and 33.9% respectively,when the cells cultivated 10 h,pretreated by 12 mmol/L EDTA,using 45 μg/mL of lysozyme,enzymolysis at 28℃ for 25 min. The result of this paper provides a reference for breeding fine microbial strains by protoplast fusion and genome shuffling.

Cloning and Expression Analysis of γ-actin Gene from Leucocortinarius bulbiger

ZHANG Hong ZHENG Rong

2016, 32(7):  131-137.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.020

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Designing the primers based on the conserved sequences of several fungal actin genes,the full-length cDNA sequence of γ-actin gene from Leucocortinarius bulbiger（Lb-act）was cloned using RT-PCR and RACE. The full-length of Lb-act cDNA sequence was 1 357 bp,consisting of a 1 137 bp open reading frame（ORF）,encoding a protein of 378 amino acids,a 5'-UTR with 92 bp and a 3'-UTR with 128 bp. The online analysis by Port Param software revealed that the putative amino acids had an isoelectric point of 5.12,a molecular weight of 95.022 kD,and 3 highly conserved regions of fungal γ-actin gene. Blast homology search indicated that the sequences of Lb-act amino acid had a high similarity with the sequences of Basidiomycetes actin,and it had the closest relationship with the amino acid sequence of Laccaria bicolor actin. In culture conditions of different carbon and phosphorus levels,the expression levels of Lb-act were almost the same,thus this research confirmed the reliability that actin gene could be used as intermolecular standard.

Gene Cloning of the Orange Carotenoid Protein from Cyanobacteria,and Its Ectopic Expression and Functional Evaluation in Escherichia coli

LIU Chang LUO Zhu ZHANG Meng-ru YANG Yu-mei LIU Xian GONG Ming ZOU Zhu-rong

2016, 32(7):  138-145.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.021

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The aim is to verify the orange carotenoid protein（OCP）in cyanobacteria can scavenge singlet oxygen（¹O₂）independent of carotenoids in vivo. The cyanobacterial OCP gene was amplified by PCR,and then subjected to prokaryotic inducible expression and further evaluation of anti-¹O₂ activity in Escherichia coli by bacterial dot-plating test and growth curve assay. Results showed that the gene（SyOCP）of OCP was successfully amplified from the metagenome of the cyanobacteria water,with a coding region of 954 bp. Bioinformatics analysis indicated that SyOCP was from the major cyanobacteria species—Synechocystis sp. PCC 6803. By IPTG induction,the SyOCP gene was highly expressed in E. coli with soluble protein. In addition,the ectopic expression of SyOCP in recombinant E. coli resulted in the notable enhancement of it on the tolerance to the common ¹O₂-induced photosensitizer dye,methylene blue. The intrinsic anti-¹O₂ role of cyanobacterial OCP（without binding carotenoids）was in vivo verified in E. coli.

Cloning and Expression Analysis of Lipid Transfer Protein LTP Gene in Carthamus tinctorius

HAN Yi-lai, CUI Qi, WU Yun-yun, GU Tian-yao, TENG Xiao-hua, HU Ren-ge, WU Guan-da, GUAN Li-li, LI Hai-yan, LI Xiao-kun

2016, 32(7):  146-151.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.022

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LTP（Lipid transfer protein）gene was cloned,its bioinformatics and expression were analyzed,providing the basis for the study the role of LTP gene in safflower resisting stresses. The sequence of LTP gene was cloned by RT-PCR technique,the protein characteristics were analyzed using bioinformatics,and the phylogenetic tree of LTP for safflower and other species was constructed. The expressions of LTP gene in the different tissues were analyzed using Real time-PCR. Sequence analysis showed that ORF of LTP was 294 bp,encoding a protein of 97 amino acids with a molecular weight of 7.46 kD,isoelectric point of 8.91. Safflower LTP protein contained a long signal peptide sequence of 29 amino acid residues,and the protein contained a serine phosphorylation site. Tertiary structure prediction indicated that the protein was a simple structure of 3 α-helix and 1 β-turn twining in random coils. Molecular evolution showed that the evolutionary relationship of LTP between Brassica of cruciferous and safflower was the most similar. Tissue-specific expression analysis of safflower LTP by fluorescence quantitative PCR showed that the gene expression levels in different tissues presented a significant difference,while high expression in seeds and flowers,and low expression in other tissues.

Cloning of Gene for a Glucose Oxidase from Penicillium notatum and Its Enzymatic Properties

GAO Qing-hua, HU Mei-rong, WU Fang-tong, TAO Yong, WANG Yun-peng, LUO Tong-yang, HU Chang-ying

2016, 32(7):  152-159.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.023

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The purpose of this work is to clone the gene of glucose oxidase（GOD）from Penicillium notatum,which was then in heterologous expression in Pichia pastoris,and the enzymatic properties of purified proteins were studied. GOD gene was cloned from genome DNA of P. notatum No. 8312 strain using PCR technique,the gene was ligated to the vector pMD-AOX and then expressed in strain X33 of P. pastoris,and finally the enzymatic properties of the purified proteins were analyzed. As results,the strain X33-GOD highly expressed the GOD with activity. Its activity of GOD in cultured supernatant reached 496 U/mL and the specific activity was 123.0 U/mg at 30℃ and pH6.5. The optimal temperature and pH of recombinant GOD was 40-45℃ and 6.0 respectively. The result of stability of the enzyme showed that the enzyme was stable when the temperature under 50℃ and the pH3.5-7.0,and it can be activated by 1 mmol/L Zn²⁺ and obvious inhibited by Ag⁺. The recombinant engineering P. pastoris strain with high-yield GOD has a higher fermentation activity and specific activity compared with GOD from P. notatum.

Construction of a Recombinant Protein Expression System in Neurospora crassa

GAO Ran-ran, LIU Qian, SUN Wen-liang, SUN Zhi-yong, LIU Hao, TIAN Chao-guang

2016, 32(7):  160-169.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.024

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To demonstrate the potential of the model filamentous fungus Neurospora crassa as a recombinant protein expression system,we used fungal hybrid technology to develop a sextuple gene disruptant LQ-1（Δ3βG∷Δ2cbh∷Δhis3）of it as the host strain of expressing the protein. The cellobiose was used to induce the expression of cellulase promoter in host strain,which removed the background band of glucosaccharase and was conducive to the expression and purification of target protein. We constructed 3 new efficiently-expressed vectors respectively consisting of its own strong inducible promoters（Pcbh-1,Pcbh-2 and Ptef-1）from N. crassa. Those vectors had fusion tag protein tev-6×his-gfp and efficiently and conveniently screened positive transformants,which was favourable for the purification of later target protein. Two endogenous cellulase（GH3-4 and CBH-1）were chosen as test proteins for recombinant expression. Then enzymatic activity measured by cellobiose-induced fermentation broth of recombinant strain,SDS-PAGE analysis,and Western blotting indicated that the recombinant proteins GH3-4-GFP and CBH-1-GFP were successfully expressed with secreted levels reaching as high as approximate 2.77 and 2.83 mg/L. The expression of recombinant protein with Pcbh-1 promoter was the highest,suggesting that promoting efficiency of Pcbh-1 in the cellobiose-induced system was the highest,and a protein expression system in cellobiose-induced N. crassa was preliminarily constructed.

Determination of Enzymatic Properties of a Laccase Lac1338,and Effects of Directed Mutants on the Degradations of Different Dyes

ZHANG Xue-ling CHEN Xiao-li LI He

2016, 32(7):  170-177.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.025

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In order to obtain the laccase with high expression and high thermal stability,a laccase gene lac1338 synthesized was cloned by codon optimization,ligated to the vector pET-32a（+）,then expressed in Escherichia coli BL21（DE3）,and the recombinant protein HIS-Lac1338 was obtained. Its enzymatic properties showed that while using ABTS as a substrate,the specific activity of HIS-Lac1338 was up to 22.8 U/mg,Km and Vmax of HIS-Lac1338 was 567 μmol/L and 2.8 mmol/（min·g protein）,respectively. The optimal temperature and pH of HIS-Lac1338 were 55℃ and 6.0 respectively. Its activity remained 50% at 55℃ for 2 h and in the pH range of 4-8. HIS-Lac1338 was strongly resistant to Cu²⁺,while its activity was promoted by Ca²⁺,Na⁺,K⁺ and inhibited by heavy metal ions such as Co²⁺,Fe²⁺,Hg²⁺,Ag⁺,etc. Comparing with HIS-Lac1338,mutant enzyme Lac16 of Lac1338 by sequential error-prone PCR improved the degradation rates of Acidviolet 7,Bromophenol blue,and Coomssie brilliant blue from 10.9%,20%,and 25% to 90.5%,67.8%,and 85%,respectively. Above results reveal that HIS-Lac1338 is stable to temperature and pH,the degradation rate of dye increases greatly with the mutant enzyme Lac16 via directed evolution of sequential error-prone PCR.

Effects of Inhibitors in Corncob Hemicellulose Hydrolysate on Butanol Fermentation

GU Chun-kai, WANG Gen-yu, LIU Hong-juan, WANG Ge-hua, WU Jian-rong, ZHANG Jian-an

2016, 32(7):  178-185.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.026

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Due to the toxicity of inhibitors in hemicellulose hydrolysate to microorganism,hemicellulose hydrolysate cannot be directly used as substrate for butanol fermentation. The effects of inhibitors to non-anaerobic butanol-producing symbiotic system TSH06 was studied,aiming at laying the foundation for the butanol fermentation using hydrolysate without detoxification. Hemicellulose hydrolysate of corncob diluted with P2 medium was used as substrate in butanol fermentation. The pH of culture media was adjusted to be neutral with NaOH and ammonia respectively. The inhibition effect of the hydrolysate on TSH06 was studied by qPCR with gene primes of key enzymes. Results showed that the existence of Na⁺ inhibited the growth of TSH06. Furthermore,TSH06 grew and fermented in 50% or lower degrees of diluted hemicellulose hydrolysate,tolerated and degraded the furfural and 5-HMF,and the ultimate butanol concentration reached 4.16-5.16 g/L,lower than that in P2 medium（butanol concentration of 8.83 g/L）. In diluted hemicellulose hydrolysate,acetic acid concentration remained above 3.18 to 4.16 g/L after 48 h,much higher than that in P2 medium（<2 g/L）. Compared with P2 medium,the transcription ratio of key genes in the organic acid production pathway rose significantly while TSH06 in the 50% hemicellulose hydrolysates and declined significantly in the organic acid reflux pathway and butanol production pathway. In conclusion,excessive acetic acid in the hemicellulose hydrolysate might damage cells and block the transition from acidogenesis phase to solventogenesis phase. As the accumulation of acid,bacterial cells tend to decline and death before the substrate is completely consumed,leading to the reduction of butanol production and the substrate is not fully utilized.

Optimization of Fermentation Conditions of Lipase-producing Pseudomonas stutzeri PS59 and Washing Performance of the Lipase

LIU Jin-hui, LI Xiao-lu, JIANG Yan, WANG Hai-kuan

2016, 32(7):  186-193.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.027

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It is aimed to optimize the fermentation conditions and detergent performance of lipase-producing Pseudomonas stutzeri PS59. The single-factor experiments and response surface analysis were adopted to select the best carbon and nitrogen sources. The impacts of enzyme dosage,time,temperature,pH,and adding dose of surface active agent on lipase detergent performance was also primarily evaluated for determining the optimal value of each factor. The results showed that the optimal carbon and nitrogen sources for producing lipase in laboratory culturing P. stutzeri for producing lipase were sucrose and soybean peptone. The optimal fermentation medium formula（g/L）were：soy peptone 22.39 g,sucrose10 g,K₂HPO₄·3H₂O 1 g,MgSO₄·7H₂O 0.5g,anhydrous CaCl₂ 0.05 g,olive oil 8.1 g,pH 8.1,temperature 30℃,and fermentation time 36 h,the average enzyme yield reached 36.12±1.32 U/mL,comparing with the original yield 15.65±4.81 U/mL,it increased by 1.3 times. To achieve the best washing effect,the optimal enzyme dosage was 100 U,the optimal washing time and temperature were 25 min and 25℃,respectively. Washing pH had little effect on the detergent performance. Under the optimal washing conditions,the washing performance while adding enzyme improved by 30%-40%.

Clone and Expression of WalR in Streptococcus pneumonia TCSs and Analyzation of Conservation and Antigenic Epitope

HAN Dao-bin, LUO Shi-lu, HUANG Jian, ZHU Jie-hua, CHEN Ze-hui, MIN Xun

2016, 32(7):  194-199.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.028

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This work aims to express WalR protein in Escherichia coli BL21 through constructing recombinant expression vector PET28a-WalR and to analyze the immunogenicity in the experimental animal C57 mice,as well as to test the conservation in differentStreptococcus pneumonia serotypes and B cell antigenic epitopes. Using the WalR gene of S. pneumonia D39 as the template,the recombinant expression vector PET28a-WalR was constructed and then transferred into Escherichia coli BL21 for induced expressing target protein. The conservation of WalR protein in varied S. pneumonia serotypes was analyzed by ClustalX 2.1 software,and the B cell antigenic epitopes was analyzed by DNASTAR Lasergene v7.1. As result,recombinant expression vector PET28a-WalR was constructed successfully and much target protein was obtained by inducement of IPTG,however mainly in inclusion body. The conservation of WalR protein was up to 99.4% among different S. pneumonia serotypes,and the antigenic epitopes of WalR protein were likely located in 9-16,35-42,95-111,113-135,150-161,and 200-218 of amino acid sequence. In conclusion,recombinant S. pneumonia WalR protein in the form of inclusion body was successfully acquired,and the conservation and antigenic epitope were analyzed.

Production of Monoclonal Antibodies Against Edwardsiella tarda,and Establishment of Double-antibody Sandwich ELISA Method

YANG Chuan QIN Yi-dan HU Qian LI Qiang

2016, 32(7):  200-205.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.029

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Using Edwardsiella tarda 2CDM001 as antigen and hybridoma technique,we prepared the monoclonal antibodies（mAbs）8D11,8H4,2F4,18C12,16E2,20D3,19G2,5F7,4E6,7C5 and 18H9 against E. tarda. Among these mAbs,8H4 and 2F4 had the poor specificity,presented varied cross reactions with control strains such as Vibrio damsela（ATCC33539）and Vibrio damsela（ATCC33539）. The other 9 mAbs had favorable specificity and no cross reactions with control strains except E. tarda used in the experiment. The result of detecting E. tarda isolates showed that only 20D3 and 8H4 reacted with all the E. tarda isolates,and the rest of mAbs did not reacted with all the E. tarda isolates. Due to the cross reaction of 8H4 with V. damsela（ATCC33539）,thus 20D3 was selected to establish a double-antibody sandwich ELISA for E. tarda with rabbit polyclonal antibody. The double-antibody sandwich ELISA possessed the strong specificity,and of which the limited detecting concentration reached 5 × 10⁷ CFU /mL. In conclusion,this study expanded the mAbs library for E. tarda,and also provided more reference data and alternative materials for further application of E. tarda mAbs.

Antitumor Activity of the Ethanol Extract of Pleurotus ferulae and the Extraction Procedure of Triterpenoid Components

WANG Wei-lan CHEN Kai-xu LIU Jun DUAN Wei-wei XU Ya-nan ZHANG Fu-chun

2016, 32(7):  206-216.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.030

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This work aims to explore the antitumor activity of alcohol soluble components from Pleurotus ferulae（PFECs）,and to optimize the extraction condition of triterpenoids from P. ferulae（PFTPs）for maximum extraction yield. MTT was used to detect the inhibitory activity of PFECs to 5 kinds of tumor cells,no significant inhibition to 2 kinds of normal cells was observed. Ultrasonic-assisted,hot-water leaching with ultrasonic-assisted,single-factor experiment,and response surface methodology were jointly employed to optimize the extraction process of PFTPS. The results showed that PFECs presented significant antitumor activity. The optimal process parameters for the PFTPs extraction were：the ratio of ethanol to material 1∶19（g/mL）,extraction time 22 min and extraction temperature 7℃,and the extracting yield of PFTPs was 4.42%.

Immunoregulative Action of Polysaccharides in Wild and Cultivated Artemisia rupestris in Xinjiang on Bone Marrow Dendritic Cells

YANG Yu, YANG Xiu-mei, ZHAO Gan, YU Yi, ZHANG Hui-zhen, ZHANG Ai-lian

2016, 32(7):  217-226.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.031

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This work is to study and compare the effects of crude polysaccharides in wild Artemisia rupestris（WARCP）and cultivated A. rupestris （CARCP）on the maturation and function of bone marrow dendritic cells（DCs）. The WARCP and CARCP were isolated by ultrasonic extraction,the protein was removed by Sevage,the polysaccharides content of them were measured by anthrone-sulfuric acid method,the maturation of DCs were detected by flow cytometry,and the expressions of IL-12 and TNF-α were examined by ELISA. As results,the polysaccharides contents of WARCP and CARCP were 26.18% and 22.14% respectively,while the polysaccharides contents of WARCP and CARCP after removing protein were 30.94% and 27.06% respectively. Both WARCP and CARCP significantly enhanced the expression level of CD40,CD86 and CD80 on the CD11c + DCs from mice’s bone marrow（P < 0.05）,significantly promoted the expression of IL-12 and TNF-α（P < 0.05）,significantly reduced the capabilities of DCs phagocytosis to FITC-Dextran,and their immunoregulative actions at the same dose of them was not significant（P > 0.05）,there was no significant differences on immunoregulative action of WARCP and CARCP between removing and not removing protein（P ﹥ 0.05）. In conclusion,there is little difference between WARCP and CARCP,and both enhance the maturation and function of mouse’s bone marrow DCs in little differencen.

The Construction of CYP2C8 and CYP3A4,and the Inhibition of Paclitaxel Metabolism by Small Kinase Inhibitor

LI Yuan HUANG Jie-qiong LI Jia-jun WANG Wei-peng ZHANG Hong-jian

2016, 32(7):  227-233.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.032

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The aims of this work are to establish the cell expression system of CYP2C8 and its 3 mutants,to study the effects of gene polymorphisms on its enzymatic activities,and to investigate the effects of small molecular kinase inhibitors on paclitaxel metabolism with the established CYP2C8 and CYP3A4 co-transfected cell lines. The gene fragments of CYP3A4,CYP2C8 and its three mutants（CYP2C8*2（805A>T）,CYP2C8*3（416G>A,1196A>G）,and CYP2C8*4（792C>G））were synthesized based on gene bank. Then those fragments were ligated to expression plasmid PEGFP-N1 for sequencing evaluation. Twenty-four hours after the wild and mutant plasmids were transfected into HepG2 cells,paclitaxel was added and incubation proceeded,and then the metabolites were quantitatively detected by well-constructed LC-MS/MS. Concurrently,the wild CYP2C8 and CYP3A4 plasmids in certain concentration ratios were transfected into HepG2 cells,to establish a co-expression system. Screening the plasmids with proper concentration ratio and transfecting them to the cells,adding small molecule kinase inhibitor while adding paclitaxel for incubation,the inhibition of paclitaxel metabolism by small molecule kinase inhibitors was investigated. The results showed that the metabolic enzyme of different CYP2C8 caused the significant difference of paclitaxel metabolic activity,of which the metabolic activity of CYP2C8*2,CYP2C8*3,CYP2C8*4 remained at about 80%（P < 0.05）,87%（P < 0.05）,and 65%（P < 0.01）,respectively,as compared to the wild type. Nilotinib completely inhibited the metabolism of paclitaxel and axitinib showed a 30% inhibition,while imatinib didn’t have any inhibitory effect. As conclusion,different genotypes of CYP2C8 differentially affect the overall paclitaxel metabolism,which might be the reason that results in the varied treatment effect in clinic. Small molecule kinase inhibitors while combined use with paclitaxel may inhibit the metabolism of paclitaxel to different extents.

Study on the Quinolone-resistant Mechanisms of Listeria monocytogenes

JIANG Xiao-bing, YU Tao, NIU Ya-bing, XU Ya-meng, SHI Lei, WANG Hai-lei

2016, 32(7):  234-241.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.033

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Using ciprofloxacin-resistant strains of Listeria monocytogenes（LM）isolated from food as research object,this study aims to understand the quinolone-resistant mechanisms of LM. The minimum inhibitory concentration（MIC）of ciprofloxacin by the strain was determined by the agar dilution method. The mutations in the quinolone resistance-determining region（QRDR）and the presence of plasmid-mediated quinolone resistance（PMQR）genes were investigated using PCR. Expression levels of lde gene under different conditions were detected by qRT-PCR. To address the role of efflux pump Lde in ciprofloxacin resistance,deletion mutant in lde was constructed using SOE-PCR. Results showed that the amino acid sequences of GyrA,GyrB,ParC,and ParE in 15 resistant strains showed 100% similarity with the sequences of sensitive strains,and no PMQR genes were detected in any of the resistant strains. In the presence of reserpine,an efflux pump inhibitor,the MICs of ciprofloxacin by strain L28 and L47 reduced to the 1/8 and 1/4 of original one. The expression of lde was observed in all sensitive and resistant strains by similar values. After treatment with ciprofloxacin,the resistant strains showed significant increases in lde,however,no obvious changes in expression of lde were observed in the sensitive strains. In the presence of reserpine,the expression of lde significantly inhibited in resistant strains. The mutant lacking the lde gene was susceptible to ciprofloxacin and its MIC of ciprofloxacin did not change in the presence of reserpine. Our data demonstrated that efflux pump Lde mediated quinolone resistance in L. monocytogenes.

Expression of AID and Dynamic Changes of Its DNA Methylation in Regulation Region During Bovine Early Embryonic Development

AO Xu-dong SA Ru-la WANG Jie WANG Hui-min YU Hai-quan

2016, 32(7):  242-249.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.034

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DNA methylation plays an important role in mammalian fertilization. The chromosome has undergone complex reconstruction and ultimately obtained totipotent during fertilization. Using activation-induced cytidine deaminase protein（AID,also known as AICDA with DNA active demethylation function,as research target,the expression changes of AID and its regulation mechanisms at different development stages of oocyte and in-vitro fertilization（IVF）embryo were detected for exploring the cell reprogramming mechanism. Real-time PCR,BSP（Bisulfite Sequencing PCR）,and immunofluorescence chemistry were used to analyze the effects of DNA methylation on the AID expression during bovine early embryonic development. The results showed that AID gene was regulated by DNA methylation in the bovine early embryonic development. Combining the expression of AID gene in the different tissues and DNA methylation status of its promoter region,the tissue-dependent and differentially methylated region（T-DMR）of AID gene was located at the transcriptional start site -88 bp--431 bp. During the maturing process of bovine oocyte,No. 2 and 3 CpG sites in the T-DMR region were obviously demethylated,whereas the other sites were not changed,and AID gene was correlated with the DNA methylation. At the different stages of early IVF embryo,though the expression of AID gene varied,only No. 2 and 3 CpG sites in the T-DMR region of AID were in the status of demethylation,while other sites were in methylation continuously,the changes of the expression probably was related to the embryonic genome activation. The above results revealed that low methylation of T-DMR in AID gene was correlated with its expression. Immunofluorescence detection discovered that AID proteins were uniformly distributed in the nucleus and cytoplasm from the oocyte maturation stage to morula stage. And a large number of AID protein concentrated in the inner cell mass at the blastocyst stage,which played the important role in the maintenance of pluripotency of the inner cell mass. In summary,accumulated AID during oocyte maturation presents an effect on fertilization,the DNA methylation in the promoter region is closely related to its expression,and the expression of AID gene,after activation of the embryonic genome,may be associated with embryo development.

The Effects of Preservation Methods and Temperature of Softening Specimens on DNA Extraction from Apis mellifera

ZHANG Hong-li JIA Xin-lei LIU Jian-xia ZHANG Yong-fang

2016, 32(7):  250-256.  doi:10.13560/j.cnki.biotech.bull.1985.2016.07.035

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This work aims to explore the effects of preservation methods and temperature while softening specimens before drying the specimen on DNA from different parts of bees. The phenol - chloroform method was used to extract the total DNA from bee specimens preserved in different methods and from those dried bee specimens softened at different temperatures. The extracted DNA was analyzed and identified using agarose gel electrophoresis and the PCR amplification. The electrophoresis images showed that fine-quality DNA was extracted from all specimens,including specimens collected freshly,specimens soaked with alcohol, and specimens dried under natural conditions,and the quality of genomic DNA from the head and the leg of these specimens was better than that from other parts. The total extracted DNA results from dried bees softened under different bath temperatures and preserved half year showed that 65℃ was the optimal softening temperature,and the damages to bee’s DNA was the least,and the ideal part for extracting DNA was the bee’s leg. The results of orthogonal experiment also revealed that the leg and softened at 55℃ was the optimal combination for extracting DNA from the dried specimen. Furthermore,the results of PCR amplification confirmed that these DNA extracted in our experiment were used to successfully amplify mitochondrial 16S rRNA and CO I fragments. In summary,DNA extraction was explored from the three aspects：preservation methods,extracted parts and softening operations,which could provide better alternative proposals for collecting DNA from bees.