Table of Content

25 April 2017, Volume 33 Issue 4

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Orignal Article

Research Progresses on Plant Sucrose Transporters and Physiological Functions

TU Wen-rui, CAI Yu-meng, YAN Jing, LU Jiang ,ZHANG Ya-li

2017, 33(4):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.001

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Sucrose is the primary product of photosynthetic CO₂ fixation which is used for the distribution of assimilated carbon within higher plants. The transmembrane transport of sucrose requires the participation of sucrose transporters. Currently,studies have shown that sucrose transporter are widely present in higher plants and played main role in seed,flower,leaf cells,phloem,root and other organs. By the way of subcellular localization,sucrose transporters have been found mostly to be located in the cell membrane and vacuole membrane. In this paper,we review the progresses on different aspects of SUTs,such as the protein structure,classification,physiological functions,functional verification and subcellular localization;then we discussed the research significance and remained problems in studying sucrose transporter for better understanding the action mechanism of sucrose transporter proteins in plants.

Research Progress on the Oxidative Modification of Plant Proteins Mediated by Reactive Oxygen Species

ZHOU Wen-fei, BAI Juan ,GONG Chun-mei

2017, 33(4):  8-18.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.002

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Post-translational modification of protein is an important process for precursor protein to its carrying-out function. Some redox-sensitive proteins can be modified reversibly or irreversibly by reactive oxygen species in stress conditions. At present,research on oxidative modification of plants is still in the initial stage. With growing demand on plant developing the ability to defense the harsh environment,more attentions have been focused on the reversible oxidative modification of proteins caused by reactive oxygen species,and some corresponding progresses have been finished in its mechanism and research methods. This review describes the way and sites of oxidative modification of plant proteins and regulation mechanism of various oxidoreductase,aiming at illuminating the significance of reversible oxidative modification in the oxidative stress resulted from defensing the stress to plant. The review also summarizes the effective methods for studying the reversible oxidative modification of proteins. Currently,via mutations of specific sites and proteomics it is feasible to determine whether or not there are reversible oxidative modifications and in which extent the oxidative modification is. Moreover,in future,it is aimed to dissect the reduction and regeneration mechanism by comprehensive experiments to control the proteins involved in reactions.

Research Progress on 4-Coumarate：Coenzyme A Ligase（4CL）in Plants

TIAN Xiao-ming, YAN Li-hong, XIANG Guang-feng, JIANG Li-yuan

2017, 33(4):  19-26.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.003

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4CL（4-coumarate：coenzyme A ligase,EC6.2.1.12）is a key enzyme in the lignin biosynthesis pathway,and it catalyzes a hydroxycinnamic acids and its derivatives to generate the corresponding thioester. Concurrently,4 CL is also the third step in the metabolic pathway of phenylpropane,ligating the precursor of lignin and varied branch pathways,playing the critical regulating role in the lignin synthesis. It has become a research hotspot that uses the genetic engineering way to alter lignin biosynthesis,to reduce lignin content,and to decrease the pollution in pulp and papermaking process. Combining the new reports about 4CL in recent years,we summarized the research progresses on phylogenetics,enzymatic activities,crystal structure and catalytic mechanism and regulation of 4CL in lignin biosynthesis. The future directions of researches on 4CL were also suggested, This paper provides reference information for 4CL in the future study.

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

HU Li-ping, ZHANG Feng, XU Hui, LIU Guang-min, WANG Ya-qin, HE Hong-ju

2017, 33(4):  27-37.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.004

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SWEETs（sugars will eventually be exported transporters）are a novel class of recently identified sugar transporters. SWEETs appear to function as a bidirectional uniporter/facilitator,facilitating diffusion of sugars across cell membranes along a concentration gradient. SWEETs play central roles in a number of biochemical processes,such as phloem loading for long-distance sugar transport,nectar secretion,seed filling,pollen nutrition,plant-pathogen interaction and stress regulation,so they had attracted much attention in recent years. SWEETs are widely found in plants. However,only a few species,such as Arabidopsis and rice,of the SWEETs have been functionally identified. This article focuses on the advances of the SWEET gene family,including details about their discovering,characteristics of protein structure,physiological functions and regulation in plants. It will help to elucidate the molecular bases of their function in plants more in-depth and comprehensive in the future.

Plant Transposon and Gene Expression Regulation

HE Hu-yi, TAN Guan-ning, TANG Zhou-ping, YANG Xin, LI Li-shu, HE Xin-min

2017, 33(4):  38-43.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.005

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Plant transposon is a mobile DNA repeat sequence of plant genome,playing an important role in plant genome evolution,gene expression regulation,system development,and evaluation of genetic diversity. This paper reviews the classification,origin,and transposition mechanism of plant transposons,and epigenetic control between transposon and host genome,and elaborates the regulatory pattern of different transposons on gene expressions,and the future research is prospected,aiming at providing reference for fully understanding the function of plant transposons.

Effects of Virus-Plant Interaction on Biological Characteristics of Insects as Vectors

GUAN Gui-jing, ZHAO Heng-yan, WANG Hong-su, LIU Jin-xiang

2017, 33(4):  44-50.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.006

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Most plant viruses are transmitted by vectors,among which insect is the most important category. The tripartite interaction of plant-virus-vector is complex and specific. So far,virus has become a powerful booster for co-evolution of plant-virus-vector due to its rapid reproduction and fast variation,which then is conducive for its effective transmission. The effects of virus on vector insects can be direct and indirect. The indirect effect refers to such process,i.e.,virus infection to host plants results in the changes on the photosynthesis,secondary metabolism,nutrition components,and signal pathways of plants,which thus affects insects’ biological properties,such as their behavior of host selection,growth and development,fertility and so on,and finally influences virus’ transmission efficiency. The biological impacts of virus-infected host plants on piercing-sucking insects are reviewed here,aiming at providing evidences for the prevention and control of plant virus diseases.

Research Progress on Blue-photoreceptors and Its Functions in Eukaryotic Microalgae

CUI Hong-li, CHEN Jun, HOU Yi-long, WU Hai-ge, QIN Song

2017, 33(4):  51-62.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.007

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Eukaryotic microalgae,as a kind of photosynthetic eukaryotes,originate from endosymbiotic events. Their broad varieties,wide distribution,and complexity of evolutionary history make it become an ideal experimental material for research in algal stress physiology. Light is one of the important signal factors in the environment,it is not only the energy source for eukaryotic microalgae,but also provides information,and regulates the growth and development. For adapting the information of light intensity,quality and cycles,eukaryotic microalgae form a set system of fine light signal receiving and transducing during the long-time evolution. Photoreceptor plays the critical linkage role of receiving and transforming in the light signal pathway. Based on the various wavelengths of light,there are four types of photoreceptors,i.e.,red/far-red,blue,green and ultraviolet. Blue-photoreceptors can perceive 320-400 nm light and play key roles in the regulation of multiple plant physiological processes. Referenced by the study advances on the structures and functions of blue-photoreceptors in higher plant Arabidopsis thaliana,we summarized the study advances on the photoreceptor of eukaryotic microalgae,mainly focusing on their gene cloning,protein structure,molecular evolution,photochemical characteristics,physiological functions,and light signal transduction. Moreover,we suggested the future issues and focuses on the studies of blue-photoreceptors in eukaryotic microalgae,which provide reference for the research of blue photoreceptors from eukaryotic microalgae.

Application and Research Progress of Inter-simple Sequence Repeat（ISSR）Marker in Medicinal Plants

REN Meng-yun, CHEN Yan-jun, ZHANG Dun, DU Le-shan, LIU Fang, GUAN Xiao, ZHANG Yin-dong

2017, 33(4):  63-69.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.008

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Inter-simple sequence repeat（ISSR）has been known as a novel molecular maker technology based on simple sequence repeat（SSR）. At present,the research and development of medicinal plants are facing the problems such as unclear drug substance,uncontrollable quality and exhausted resources. DNA molecular marker technology,especially ISSR,has provided a new solution for the above problems. Comparing the merits and demerits of several commonly used molecular markers,finally,we mainly introduced the principles and characteristics of DNA molecular marker ISSR applied in current research of the medicinal plants,and summarized the new advances on ISSR molecular technology and its application prospective in the genetic diversity and structure of medicinal plants,identification of germplasm resources,quality identification of traditional Chinese medicine,and genetic map,aiming at providing better reference for the development and utilization of medicinal plants.

Current Issues and Progress in the Application of CRISPR/Cas9 Technique

YUAN Wei-xi, YU Yun-mei, HU Chun-cai, ZHAO Zu-guo

2017, 33(4):  70-77.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.009

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CRISPR/Cas9[clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9]is a RNA-guided genome-editing technique,which can be applied in the deletion,insertion or replacement of specific DNA sequence thus to verify the function of genes or remedy pathogenic genes. Owing to its simplicity,affordability,multiple-site editing and scalability,the technique of CRISPR/Cas9 is undergoing rapid development and perfection and has been one of the first three prevailing genome-editing technology following that of Zinc finger nucleases and transcription activator-like effector nucleases. In this review,we focus on the current issues of CRISPR/Cas9,including low efficiency of precise-editing,sequence-recognizing limitation from PAM,off-target,unavoidable mosaicism,together with corresponding tactics for improvement. Our aim is to provide the objective understanding on the current disadvantage of CRISPR/Cas9 technique to researchers and the systematic review for reasonable application and improvement of this technology.

The Latest Progress on the Methods for in Vitro Screening Aptamers

LI Ya-nan, ZHAO Jie, ZHANG Ao-zhe, TAN Yan, HUA Qian, ZHANG Zi-jian

2017, 33(4):  78-82.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.010

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Aptamers are single-stranded oligonucleotides that are screened by systematic evolution of ligands by exponential enrichment（SELEX）in vitro,which can selectively bind to different target with high affinity and high specificity,including protein,small molecules,organic compound,metal ions,drugs,etc. This technology has attracted more attentions for its advantages and thus it has been applied widely in many aspects such as biological sensor,gene chip,new drug development,nano technology,etc. However,the traditional SELEX method is cumbersome,which usually takes several months to screen out the target of nucleic acid in high specificity. With the rapid development of SELEX,many novel screening methods have emerged in recent years. The screening cycle and the screening efficiency are improved with these new methods,and the application of aptamers is expanded. This review introduces several new screening methods in recent three years,including multiple GO-SELEX,SWCNTs-assisted cell-SELEX,on-chip Cell-SELEX,Sequence-constructive SELEX and High-Fidelity（HI FI）SELEX,It is helpful for us to know more about the latest progress on the methods for in vitro screening aptamers and to promote the application of aptamers in various fields..

Research Progress on the Prediction of Protein Stability Based on Amino Acid Sequence and Simulated Structure

YI Hua-Wei, TANG Xiao-Feng

2017, 33(4):  83-89.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.011

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Stability is one of the important properties of protein. Protein’s stability is not only demanded in industry,but also the prerequisite for its deriving new functions in protein evolution. Improving the stability of protein,especially protein of unknown structure,is a very challenging task,i.e.,traditional protein modification methods such as directed evolution are time-consuming and laborious,while rational design is difficult to replicate by other researchers. At present,there are many reported predictors for protein stability. However,the majority of these tools require the pre-measurement of three-dimensional structure of protein,which restricts the stability prediction of protein in unknown structure. Recently,a number of tools to predict the effects of mutations on the stability of unknown proteins have been developed using amino acid sequences and simulated structures of proteins,here we introduce the research progress on them,aiming at providing guidance for protein engineering.

iTRAQ-based Quantitative Proteomic Analyses of Differentially Expressed Proteins in Nicotine-induced SH-SY5Y Cells

ZHU Bei-bei, LI Xiang-yu, CHEN Huan, WANG Hong-juan, HOU Hong-wei, HU Qing-yuan

2017, 33(4):  90-97.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.012

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SH-SY5Y,induced by nicotine,was used to seek the effects of nicotine exposure on the alteration of proteins expression profile. The differentially expressed proteins in nicotine-induced neuronal cells were identified using the iTRAQ and nano-LC-Q-TOF MS proteomics,and their bioinformatics was also analyzed. The results showed that the levels of 132 proteins were differentially altered in response to nicotine（| ratio | ≥ 1.5,P<0.05）,including proteins involved in gene expression,MAPK cascade,GTP signal transduction and neurotrophin TRK receptor signaling. Together,our data suggested that the nicotine might elicit dependence by altering the neuronal balance of proteasome subunits,with the MAPK cascade and ubiquitin-proteasome pathway playing a pivotal role.

Genome-wide Association Study of Chlorophyll Content in Maize Leaves

TENG Shou-zhen, WANG Hai, LIANG Hai-sheng, XIN Hong-jia, LI Sheng-yan, LANG Zhi-hong

2017, 33(4):  98-107.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.013

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The chlorophyll content of plant leaves is closely related to photosynthetic efficiency and yield potential,thus it is an important physiological indicator of all crop species. However,currently most cloned genes controlling chlorophyll content are from Arabidopsis and rice,and the genetic basis of natural variation in chlorophyll content in maize is still unclear. In this research it was discovered that the chlorophyll content of the first leaves at the seedling stage was highly correlated with that of ear leaves at silking stage,and the former had a higher heritability than the latter. This study further analyzed the chlorophyll contents of first leaves from 287 maize inbred lines. Genome-wide association study using 558 269 SNP markers revealed 9 SNPs significantly associated with the measured trait,leading to the discovery of 16 candidate genes. Two genes potentially controlling chlorophyll content in maize were identified by sequence analysis and functional annotation of these candidate genes：a homolog of the Arabidopsis Tic22 and a homolog of rice SAG12 relating to aging.

Cloning and Expression Analysis of New KAH Genes from Stevia rebaudian

ZHU Jing-wen GUO, Shu-qiao, SHU Hong-mei, GONG Yuan-yong, JIANG Lu, NI Wan-chao

2017, 33(4):  104-107. 

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This work is to further reveal the molecular mechanism of the biosynthesis of steviol glycosides（SGs）and analyze the reasons caused the different accumulation of SGs in stevia leaves. Different stevia materials were utilized to clone the coding genes of kaurenoic acid hydroxylase（KAH）at the key point of a branch pathway，and the biological function of the obtained nucleic acid sequences were analyzed and predicted. Finally six highly homologous nucleotide sequences were cloned. The key base mutations of nucleotide sequences caused significant differences in the location and size of the open reading frame（ORF），and totally 7 different KAH genes were identified. Both KAH1 and KAH2 were found in SR1，SR3，Xinfeng 3，Puxing 1 and Shoutian 3，KAH3 was cloned in Jiangtian 1 and Shoutian 3，KAH4 were found in Jiangtian 3，and KAH5，KAH6 and KAH7 all existed in SR2. Furthermore，SrKAHs showed the highest expression in stevia varieties and tissues with high accumulation of SGs. P450 conserved domains were in the all of them，and further evolution analysis and amino acid homology results showed that they belonged to the CYP716 family. And the predicted results of subcellular location showed KAH2 and KAH4 localized in cytoplasm while the other five were localized in the chloroplast. Differences of gene numbers，amino acid length and location in subcellular structures of KAH genes in Stevia maybe cause the different expressions and functions of SrKAHs，finally resulting the different accumulation of SGs.

Comparative Analysis of Identification Methods of CP4-EPSPS Transgenic Cotton Plants

GUO Wen-fang, WANG Nan, LI Gang-qiang, XU Fang-fang, YANG Cai-feng, LIU De-hu

2017, 33(4):  114-118.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0250

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CP4-EPSPS isolated from Agrobacterium strain CP4 is a glyphosate resistant gene and one of the most commonly used marker genes in transgenic plant research. However,we found that investigated results of the transgenic plants were not always completely accurate only using simple glyphosate resistance assay in the identification of CP4-EPSPS transgenic plants,which resulted in false-positive plants or missing positive transgenic plants. In this study,by use CP4-EPSPS transgenic cottons that we had gained,the four screening methods of CP4-EPSPS transgenic plants were compared,which included PCR,ELISA,resistant test strips and glyphosate resistance assay. The results show that the accuracy of ELISA and test strip are significantly higher than PCR and 0.3% glyphosate,but the test strip test is more convenient than ELISA basing on the comprehensive analysis,accuracy,timeliness,ease of use and price in identification of transgenic plants. Therefore we think combination glyphosate resistance assay with resistant strip test is the best choice. Our finding provides significant guidance for people engaging in the identification of CP4-EPSPS transgenic plants both in the laboratory and field.

Cloning of a CAC Gene in Dove Tree（Davidia involucrata Baill.）and Evaluating its Potential as a Novel Reference Gene

REN Rui, DAI Peng-hui, CAO Fu-xiang, DONG Xu-jie, LI Meng

2017, 33(4):  119-129.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.015

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In this research,a gene named as DiCAC（GenBank accession number KX268525）,encoding the clathrin adaptor complexes（CAC）,has been cloned from dove tree（Davidia involucrata Baill.）. The sequence of DiCAC includes a ORF（open reading frame）of 1317 bp,which encodes a protein with 438 amino acids. Homologous alignment and cluster analysis show that DiCAC shared over 94% amino acid sequence identity with CAC genes in other 15 plants,and it has the highest amino acid homology with grape（Vitis vinifera）CACgene. To evaluate the feasibility of using DiCAC as a reference gene in dove tree,combined with five housekeeping genes of dove tree（ACT7,EF1a,GAPDH,β-TUB and 18S rRNA）,we analyzed the expression stability of DiCAC in different tissues and in bracts of different developmental stages. Furthermore,expression analysis of DiCAC detected by semi-quantitative PCR and qPCR showed that the expression levels of DiCAC are stable in both different tissues and bracts of different developmental stages. Remarkably,the expression of DiCAC showed more stability than the expression of common housekeeping genes. Therefore,it could be used as a reference gene of related gene expression research in dove tree. In this study,we have cloned the CAC gene from dove tree firstly and have confirmed the feasibility of its utilization as a reference gene. Our finding provides a novel candidate reference gene resources for the further research gene expression analysis in dove tree.

Identification and Biological Efficacy of Antagonistic Strain Lh-1 Suppress Watermelon Fusarium Wilt

ZHAO Jia, HUANG Jing, CHEN Zhe, NIE Yuan-jun, LIANG Hong

2017, 33(4):  130-136.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.016

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Aims：Identify the strain Lh-1 and evaluate its capability to suppress watermelon Fusarium wilt. Methods：Test the morphological,physiological and 16S rRNA gene sequence characteristics of strain Lh-1. Detect its antagonistic spectrum. A pot experiment was designed to evaluate the effects of applying strain Lh-1 combined with organic fertilizer（T2）,compared with an organic fertilizer（T1）and a chemical fertilizer（CK）. Results：the strain Lh-1 was identified as Bacillus amyloliquefaciens. It can suppress a variety of plant pathogens with a broad-spectrum antimicrobial activity,especially for F. oxysporum f. sp. niveum. Pot experiment showed that the disease incidence is only 17.2%,the control efficacy reached 78.5%,the SOD,POD and CAT activity increased significantly and the MDA content reduced obviously. The number of bacteria and actinomycetes increased but the number of fungi,especially Fusarium oxysporum decreased obviously in the rhizosphere microflora after Lh-1 treated. Conclusion：the application of theBacillus amyloliquefaciens Lh-1 can suppressed watermelon Fusarium wilt.

The Establishment of Northern Blot Hybridization Detection System for Tobacco Mosaic Virus Liaoning Isolate

LI Yan-li, AN Meng-nan, WANG Guan-zhong, WU Yuan-hua

2017, 33(4):  137-142.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.017

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In order to establish a more sensitive and specific molecular hybridization detection system of Tobacco mosaic virus Liaoning isolate（TMV-LN）,total viral RNA was extracted from purified TMV-LN viral particles and amplified by reverse-transcript PCR. The PCR products were ligated into pUC119 vector to construct pUCTMV-PP that express RNA detection probe using Digoxigenin（DIG）Northern Starter Kit. DNA probe was also constructed using DIG DNA hybridization kit. Dot-blot hybridization and Northern blot hybridization analysis were performed to study the specificity and sensitivity of the RNA probe and DNA probe constructed above. Both of the probes showed high specificity and sensitivity in TMV-LN detection through Dot-blot hybridization and Northern blot hybridization detection system. Comparison of two hybridization systems,Dot-blot hybridization system is suitable for virus qualitative detection,whereas,Northern blot hybridization has its advantage in relative quantification of viral genome RNA.

Preliminary Study on the Function of Polyphenol Oxidase in Cassava Resistance to Mite

LIANG Xiao, LU Fu-ping, LU Hui, WU Chun-ling, CAO Xian-hong, CHEN Qing

2017, 33(4):  143-148.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.018

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So far,the function of polyphenol oxidase（PPO）in cassava resistance to mite has not been understood clearly. In order to validate this function,we used quantitative real-time PCR（qPCR）and enzymatic assay to determine the changes of expression and activities of PPO in either cassava or Tetranychus cinnabarinus after the mite-resistant and mite-susceptible cassava cultivars were damaged by T. cinnabarinus. The results showed that,in one hand,compared to those in the same leaves before damaged,the transcriptions of MePPO and total activities of PPO only increased to 0.99-,1.02-fold and 1.05-,1.03-fold while the mite-susceptible cassava cultivar BRA900 was damaged by T. cinnabarinus for 1 d and 8 d,respectively. However,the corresponding values in 1 d- and 8 d-damaged leaves of mite-resistant cassava C1115 increased to 1.78-,1.74-fold and 1.74-,1.72-fold,respectively,thus significantly higher than those of mite-susceptible cassava. In the other hand,compared with those before feeding,the expressions of TcPPOand activities of PPO in T. cinnabarinus were 0.98-,0.97-fold and 0.98-,1.10-fold,respectively after feeding on BRA900 for 1 d and 8 d;while the corresponding values in T. cinnabarinus feeding on C1115 decreased to 0.52-,0.64-fold and 0.54-,0.57-fold,respectively. This study preliminarily validated the function of PPO in cassava resistance to T. cinnabarinus.

The Application Potential of Ectomycorrhizal Fungus Pisolithus tinctorius Assisting Plant in Phytoremediation of Cu-contaminated Soils

WEN Zhu-gui, WANG Jie, TANG Yang-ze, SHI Liang, HONG Li-zhou, SHEN Zhen-guo, CHEN Ya-hua

2017, 33(4):  149-156.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.019

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The purposes of this paper are to study copper tolerance of Pisolithus tinctorius（Pt）and the effects of Pt on host pine（Pinus thunbergii）seedlings growth,nutrients and heavy metal（Cu）absorption,and to probe its potential for mitigating Cu-contaminated soil. Pt was treated by Cu at concentrations of 0（control）,5,50,100,250,and 500 µmol/L in pure culture with the pot experiment. The growth,nutrients,and Cu concentrations of different parts of seedlings（inoculated with or without Pt）were analyzed. Results showed that Pt strain presented greater Cu tolerance,and its 50% tolerance index（TI）was about 200 µmol/L. Inoculation with Pt significantly improved the growth of host plant（F = 44.57,P = 0.003）and the contents of nutrients such as P and Ca in roots and stems compared with non-inoculated ones,thus mitigating the toxic damage of Cu to host pine. The extraction efficiency of heavy metals in the soil could be improved by increasing biomass of host plants,though Cu concentration in the shoots of inoculated seedlings was decreased. Besides,inoculation with Pt reduced the toxicity of Cu to host plants via reducing the soil exchangeable Cu content. Conclusively,Cu-tolerant fungus Pt may improve the growth and nutrient absorption of host seedlings,as well as reduce the toxicity of Cu to host,improve the extraction efficiency of P. thunbergii to Cu. It shows application potential in phytoremediation and vegetation restoration in Cu-contaminated soil.

An Evaluation on Heat Tolerance of a Desert Plant Karelinia caspia Seedlings

WANG Yan-qin, SHI Xin-jian, LI Zhi-jun

2017, 33(4):  157-163.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.020

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In order to explore and utilize the feature of heat tolerance of a desert plant Karelinia caspia,its heat tolerance during K. caspia seedling was evaluated. The 5 physiological indexes,relative conductivity,MDA content,activity variation of SOD,POD and CAT,as well as trypan blue staining in the K. caspia seedling leaves were measured at varied timeslots under different temperature treatments（40,45 and 50℃）by traditional methods. The reliability of using these indexes in the evaluation of the heat tolerance of K. caspia was comprehensively analyzed by subordinate function method. Results showed that：（1）Under 40℃,the relative conductivity and MDA content tended to increase during early stage of treatment and then reduce in K. caspia seedling with the stress time increasing. The activities of SOD,POD and CAT rose with treatment time longing. There was no coloration on the leaves of K. caspia by trypan blue staining.（2）Under 45℃,each index remained stable at 2-8 h after treatment. The MDA content and relative conductivity rose sharply at 12 h after treatment,but the enzymatic activities declined. With the extension treatment time,the relative coloration area of K. caspia leaves by trypan blue staining enlarged gradually.（3）Under 50℃,the MDA content and relative conductivity rose gradually to the significant level. The activities of SOD,POD,and CAT reached peak at 1 h after treatment,but rapidly decreased as treating time extending. With the extension of treatment time,the relative coloration area of K. caspia leaves by trypan blue staining enlarged gradually,and the color became heavily. The results of subordinate function method showed that these 5 physiological indexes could be used as the indicators of evaluating the feature of heat tolerance in K. caspia. In addition,theK. caspia was not susceptible to 40℃,and the tolerant time was 8 h under 45℃ and 1 h under 50℃,indicating that K. caspia is a highly tolerant plant to high temperature.

Effects of Nano Carbon on the Growth and Differentiation of Several Plants in Vitro Culture

FENG Lu ,WANG Yu-guo, WEN Yin-yuan, ZHAO Juan, LIU Yuan, REN Jian-hong, HE Mei-lin

2017, 33(4):  164-168.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.021

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This work is to study the effects of different concentrations of nano carbon on growth and differentiation of several plants’ explants. The test tube seedling of edible lily,Anthurium andraeanum,Cymbidium hybridum,and Jinchang jujube as experimental material was cultured on MS medium with different concentrations of nano carbon. Results showed that,MS + 6-BA 0.8 mg/L + NAA 0.2 mg/L + nano carbon 0.10 g/L was conducive to the multiplication of lily bulb with the multiplication coefficient 2.11;when the concentration of nano carbon was 0.50 g/L,the growth and rooting of lily in vitro was significantly better. The optimal medium for induction and differentiation of Anthurium ‘s axillary buds was MS+ 6-BA 1.5 mg/L + IBA 0.2 mg/L + nano carbon 0.04 to 0.06 g/L,the induction rate was over 90% and the differentiation rate was 97%. The addition of nano carbon 1.0 to 2.0 g/L obviously inhibited the browning of C. hybridum protocorm subculture. The optimal medium for the multiplication cultivation of Jinchang jujube was MS + 6-BA 1.5 mg/L + IBA 0.5 mg/L + nano carbon 0.05 g/L,the multiplication coefficient reached 9.76;bud growth was robust when the concentration of nano carbon was 0.15 g/L.

Identification and Analysis of Secretory Proteins in Bacillus thuringiensis

WANG Zhi-wen, CHEN Hai-bo, SONG Fu-ping, GUO Shu-yuan

2017, 33(4):  169-176.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.022

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The study on Bacillus thuringiensis（Bt）secretory protein types and their contents,especially the extracellular proteases,is of great significance for fully understanding the insecticidal mechanism and expanding the application of this bacterium. In our research,the secretory protein samples in the transition phase of Bt_HD73 were detected by mass spectrometry,then the types,contents,functions and signal peptides of secretory proteins were analyzed. The results showed that Bt_HD73 secreted 54 kinds of extracellular proteins in transition phase,and most of them were enzymes,accounting for 66.30% of all secretory proteins. Moreover,most of the enzymes were proteases,and the followed was virulence protein,accounting for 14.53% of all secretory proteins. Among the 54 secretory proteins,27 proteins were found to contain the classic Sec-type signal peptides,and the lengths were between 24 and 40 amino acid residues. Therefore,Bt_HD73 has a strong secretory ability,and it can secrete large amounts of enzymes especially proteases in the transition phase. Concurrently,the signal peptide library of this strain was established in this study,including 27 signal peptides with potential for guiding the secretion of heterologous proteins.

Recombinant Expression and Characterization of Trehalase Tre F from Escherichia coli str. K-12 substr. MG1655

YU Lin-gang, SU Ling-qia, WU Jing

2017, 33(4):  177-184.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.023

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Trehalase is a trehalose hydrolase,which can distinctively catalyze trehalose into two molecules of glucose. In order to have a recombinant expression and application of the trehalase gene Tre F from Escherichia coli str. K-12 substr. MG1655 in E. coliBL21（DE3）,the Tre F was amplified by PCR,and a recombinant strain E. coli BL21（DE3）/pET-24a（+）-Tre F was constructed. The highest activity reached 107 U/mL when the strain was cultured in shake flask for 24 h at induction temperature 25℃and IPTG induction concentration 0.4 mmol/L. Further study of the enzymatic properties indicated that the optimal pH and temperature was 7.0 and 50℃,respectively. The crude enzyme was used in the hydrolysis of trehalose. The reaction conditions for the conversion was as the following：initial trehalose concentration 300 g/L,initial pH 7.0,reaction temperature 30℃,enzyme concentration 84 U/g. When the reaction was performed under this specified condition,the glucose yield reached the maximum of 98.4 % at 36 h. This paper is the first report on the recombinant expression of trehalase gene Tre F of E. coli str. K-12 substr. MG1655 in E. coli BL21（DE3）.

Cloning,Expression,and Insecticidal Activities of Gene cry2Ab34 from Bacillus thuringiensis

ZHANG Yue, LI Hai-tao, LIU Rong-mei, GAO Ji-guo

2017, 33(4):  185-190.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.024

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In order to identify and detect insecticidal activities from Bacillus thuringiensis strain,the genomic DNA was extracted from the strain Bt BJH500 with high insecticidal activity,from which the insecticidal gene cry2Ab34（GenBank accession number：KX357382）was cloned by PCR. The gene was designated as cry2Ab34 by International Gene Nomenclature Committee of Bt. Gene cry2Ab34 was expressed and the weight of expressed product was approximate 70 kD. The gene was expressed in Escherichia coli BL21 for the determination of biological activity,results showed that this gene presented certain insecticidal activity to Plutella xyllostella and Helicoverpa armigera of Lepidoptera pests,the corrected mortality of 10 µg/mL was 31.25%,of 100 µg/mL was 62.5 %,of 10 µg/mL was 29.4 %,and of 100 µg/mL was 52.9 %.

Screening and Identification of Rhamnolipid-producing Strains and Characterization of the Product

WANG Jiang, JU Jiu, CAO Hai-long, GUO Wei-hua, YIN Heng

2017, 33(4):  191-197.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.025

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Rhamnolipid is the most common biosurfactant that has been deepest studied and the most widely applied. Total 25 strains were isolated from soil of the near oil field and biogas plant. The strains that produced rhamnolipid were screened and characterized by sulfuric acid-phenol reaction,emulsification experiment,oil-spreading experiment,and thin layer chromatography experiment. The strains were identified by 16S rDNA sequence analysis. The sulfuric acid-phenol reaction showed that 2 strains might produce rhamnolipid;the fermentation supernatants of 5 strains showed emulsified effect and the emulsifying index of the strain was up to 58.97%. The oil-spreading diameter of the fermentation supernatant,diluted 10 times,of one strain reached 3.53 cm. The thin layer chromatography experiment verified that the strain “Qihong” did produce rhamnolipid. The rhamnolipid in the strain “Qihong” was detected by all four experiments,confirming that strain “Qihong” produced rhamnolipid. The 16S rDNA sequence analysis revealed that the strain “Qihong” belonged to Shewanella,and was the most similar to Shewanella putrefaciens LMG 26268（T）.

Screening and Identification of a Marine Alginate-degrading Bacterium and the Utilization Capacity of Polysaccharide

XU Chao, XIONG Ya-ru, LU Ming-qian, LIAO Wei, ZHANG Yun-kai, HUANG Shu-shi

2017, 33(4):  198-204.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.026

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The purpose of this work is to obtain a strain with an ability to degrade alginate. With sodium alginate as a sole carbon source,a strain of marine bacterium with the solid ability to degrade alginate was screened and purified from the cankered gulfweed,coded as X511. According to the morphological observation,physicochemical indexes and the techniques of molecular biology,the strain was identified as Vibrio,designated as Vibrio sp. X511. The exponential phase of strain X511 was about 5-16 h,and the suitable salinity for its growth was around 2%-6%（W/V）. It was capable to grow in the culture medium with a sole carbon source like glucose,mannitol,or starch,et al. Also,4%-6%（W/V）of the salinity,the kelp powder,and the laminarin extended its stationary phase. The activity of intracellular crude alginate lyase reached 12.68 ± 0.13 U/mL when the X511 fermented in the screening medium for 24 h. The intracellular crude alginate lyase was extracted,and its ability to digest alginate sodium,poly mannurono acid,and poly guluronic acid was measured as such order：alginate sodium > poly mannurono acid > poly guluronic acid. The results of the Thin Layer Chromatography（TLC）showed that the enzymatic degradation product of alginate sodium was trisaccharide. The above results indicate that the X511 is a marine strain with strong salt tolerance,short growth cycle,and it simultaneously utilizes the sodium alginate,poly mannurono acid,and poly-guluronic acid as its growing carbon resource.

Screening of Photoelectron-response Microbes as well as Their Growth and Metabolism

XIANG Sha, LIU Ming-xue, ZHANG Ge-ge, LUO Lang, WEI Hong-fu, DONG Fa-qin

2017, 33(4):  205-213.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.027

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Screening microbial strains that have promising response to semiconductor photoelectron brings broad application prospects in the study of interaction between minerals and microorganisms. Using the uranium tailing area soil，the microbial strains with photoelectron-response were screened in the media of containing 1/2，1/4，1/8，1/10，1/50，1/100，and 1/1 000 semiconductor material TiN under illumination condition. Then the strain was identified through its morphological，physiological and biochemical characteristics，and the molecular phylogeny. Ultraviolet-visible absorption spectra and three-dimensional fluorescence spectra were used to analyze the growth of the strain and the changes of protein，humic acid，and other metabolites. The strain Y-5 screened from soil was identified to be Alcaligenes faecalis，which had strong response to photoelectron of TiN. The optoelectronic effect was not obvious due to rich nutrient in the 1/10 substrate concentration；but in 1/50 and 1/100 low substrate concentration，the growth rate was accelerated by photoelectron due to poor nutrient，and the protein and humic acid increased correspondingly. A photoelectron-response microbial strain A. faecalis was screened from an uranium tailing area soil，and the photoelectron promoted its growth and metabolism. This method demonstrated certain feasibility in screening the photoelectron-response.

Optimization of Culture Conditions for the Enhancement of Surfactin Production from Bacillus substilis B006

WANG Jun-qiang, WANG Lian-guo, GUO Rong-jun, MA Gui-zhen, LI Shi-dong

2017, 33(4):  214-221.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.028

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Bio-surfactin produced by Bacillus spp. is one kind of cyclic lipopetide with important antibiotic and biofilm-promoting func-tions,and the amount and component constitution affect its functions. In order to improve its production from Bacillus strain B006,the liquid culture medium and fermentation conditions were optimized by single-factor experiment and orthogonal experiment,and the effects of carbon source,nitrogen source,inorganic salts,and other nutrients,as well as inoculation,incubation time,liquid volume and initial pH value on surfactin production of Bacillus B006 in shake flask were investigated. The surfactin amount in fermentation broth was determined by the oil dispersing activity in the optimal tests,and its amount as well as component constitution were finally analyzed and determined by HPLC-MS method. Results showed that,the optimized culture mediums of surfactin production were as follows：beef extract 10 g/L,corn flour 15 g/L,NH₄N0₃ 3 g/L,and NaCl 3 g/L. The optimized fermentation conditions for surfactin production in shaking flask were as follows：initial pH7.0,the inoculation ratio 10%,volume in shake flask 60 mL/500 mL,and culture time 64 h. Under the optimal culture medium and fermentation conditions,the surfactin amount reached 314.73 mg /L,with an improvement of 74.88%,and the amount of surfactin components C16 and C17 increased obviously,raised by 1.64 and 8.34 times,in comparison with no optimization. In a conclusion,under optimized medium components and culture conditions,the surfactin production from Bacillus strain B006 is improved and its component constitution change. All the above results lay a foundation for surfactin production in large scale and further research in metabolic and regulation of surfactin produced by strain B006.

Molecular Cloning of Gene for Enolase in Highly Virulent Strains from Streptococcus suis serotype 2 and Its Protein Biological Function

SUN Wen, ZHENG Feng

2017, 33(4):  222-230.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.029

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To investigate the role of enolase in the pathogenesis of highly virulent strains from Streptococcus suis serotype 2,its molecular cloning,protein location analysis,enzymatic activity assay,and immune related function were studied. Based on the whole genome sequencing of S. suis 2 05ZYH33,bioinformatics of gene eno encoding enolase was analyzed. The recombinant expression plasmid pET30a∷eno was transformed into Escherichia coli BL21 competent cells,and the expression was induced. Recombinant protein enolase was obtained by the purification with His-Beads and FPLC. Then the enzyme activity of enolase was detected,and its basal metabolic function was identified. Further,FCM was used to analyze the localization of enolase in S. suis 2. Finally,the effect of enolase on the activity of peripheral blood monouclear cells（PBMCs）was detected by MTT test. Homology analysis showed a highly homologous of eno with that in many others. SignalP and TMHMM analysis revealed that enolase had neither signal peptide sequence nor transmembrane domain structure. Molecular cloning and sequencing illustrated that the eno fragment length was 1 308 bp. The 75 kD protein was acquired after the recombinant plasmid was induced to express and purification. Enzyme activity assay showed that the purified recombinant enolase had the ability to convert 2-PEG into PEP. FCM analysis demonstrated that enolase also existed on the surface of bacteria. The results of MTT test showed that enolase resulted in the decrease of PBMCs activity. In conclusion,enolase is not only involved in the activities of glucose metabolism in S. suis 2,but also exists on its surface,probably involving in the infection process by destroying the mononuclear cells.

Inhibition of Brominated Furanone to Quorum Sensing Regulating Behaviors of Vibrio anguillarum

PAN Yu-rong, ZHANG Cai-li, ZHU Su-qin, ZENG Ming-yong

2017, 33(4):  231-237.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.030

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The present study aims to investigate the inhibition effect of a quorum sensing（QS）inhibitor,brominated furanone（BMF）,on the QS-related genes and pathogenic factor of Vibrio anguillarum. The effects of BMF on the expressions of QS-related genes（vanI/R and luxS）were determined by RT-PCR at sub-minimal inhibitory concentration（MIC）. The role of BMF in the formation of biofilm and flagellum was observed by flagellum staining,and the role of BMF in the extracellular enzyme were detected by plate diffusion method. The results indicated that BMF with sub-MIC significantly suppressed the expression of vanI/R and luxS,reduced the capacity of V. anguillarum secreting protease and gelatinase,and inhibited the growth of flagellum and formation of biofilm,i.e.,the alleviated its moving ability.

Optimization of Biotransformation Technology for 10-DAB Production from Baccatin III Using Response Surface Methodology

ZHU Feng-zhi, CHENG Cheng, LIU Xiang-sheng, ZHANG Kun WANG, Li-shuang ,WANG Min, LUO Jian-mei

2017, 33(4):  238-246.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.031

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10-Deacetyl baccatin III（10-DAB）is the synthetic precursor of docetaxel-a taxanes drug with anti-tumor activity. The biotransformation technology conditions for 10-DAB production from baccatin III by Pantoea agglomerans P2-A-8 was optimized by response surface methodology（RSM）. Using Box-Behnken test and variance analysis,the transformation conditions for baccatin III to 10-DAB were optimized from the followings：cell culture time,cell concentration OD ₆₀₀,substrate final concentration,transformation temperature,and transformation time. The optimized biotransformation conditions were as follows：after the second stage cultivation for 12 h,cells were transferred and the initial OD₆₀₀ of cells was adjusted to 3.0 and the final concentration of baccatin III solution（dissolved in methanol）was 0.007 mg/mL The biotransformation was performed at 32℃ on a rotary shaker（200 r/min）for 4 d. Under the above optimized conditions,the yield of 10-DAB reached 24.5%,which increased by 446.8% compared with that before optimization. This study proved that applying RSM for modeling of 10-DAB production from baccatin III by Pantoea agglomerans P2-A-8 was reasonable and feasible. This result has important application value for docetaxel biosynthesis.

Characterization of Recombinant Toxin rCCK_96-104PE38 Targeting Colon Cancer

ZHANG Song, LIU Xi-lin, LI Meng, CHANG Jiang, GAO Shi-qi, HU Pan, LIU Zeng-shan

2017, 33(4):  247-252.  doi:10.13560/j.cnki.biotech.bull.1985.2017.04.032

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This work aims to conduct the prokaryotic expression and purification of a novel recombinant toxin rCCK_96-104PE38,and to verify the targeted killing effect on colon cancer cells. A reversed cholecystokinin（rCCK_96-104）was conjunct to pseudomonas exotoxin（PE38）by gene amplified technology to construct a prokaryotic expression vector. Ni-NTA affinity chromatograph was used for purification. The anti-tumor ability of rCCK_96-104PE38 was verified by in vitro cytotoxicity of colon cancer cells and vivo experiment with nude mice model. Results showed that the amplified gene of rCCK_96-104PE38 was in correct sequence as designed,i.e.,the active expression was successfully achieved using prokaryotic expression system of pET-28a. The purified recombinant protein induced the apoptosis of colon cancer cells in vitro,and inhibited the tumor growth in nude mice model. Conclusively,it was successful to produce the recombinant toxin rCCK_96-104PE38 in high purity and with no tags,the vitro and vivo experiments confirmed that it had inhibitory ability on colon cancer.