Table of Content

11 July 2017, Volume 33 Issue 7

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CONTENTS

Auxin and Cytokinin Modulate Root Microbe Interactions

HOU Peng-fei,JIA Zhen-hua ,SONG Shui-shan,

2017, 33(7):  1-6.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0111

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Plants and microorganisms form a complex and subtle symbiotic system in the process of co-evolution. Plant hormones such as auxin and cytokinin not only regulate the plant development，but also play an important role in plant-microbial interactions. This review summarizes recent advances in our understanding of how auxin and cytokinin regulate the interaction between plant roots and soil microbes，including fungi，beneficial/pathogenic bacteria，aiming at revealing the similarities and specificities of auxin’s and cytokinin’s regulatory functions，thereby providing theoretical and practical guidance for crop production and resistance to microbial disease.

Research Advances of Transcriptomics in Horticulture Plants Pigments Metabolism

LI Xia, WANG Shun-li

2017, 33(7):  7-14.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0018

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Pigments mechanism is one of the most important research fields in horticultural plants. Related researches mainly include anthocyanin and carotenoid biosynthesis，which decide the quality and ornamental character of horticultural plants. Using transcriptomic technology，the transcriptional regulation mechanism of horticulture plants can be investigated at the transcriptome level. This paper summarized the recent reports about the isolation of code genes，branch mechanism，new genes isolation，regulatory mechanism，and environment- pigments interaction of fruit trees，vegetables and ornamental plants. The current problems in application were analyzed. The development prospect of transcriptomics in horticultural plants pigments metabolism was also prospected. We hope that it will provide useful information for the regulation mechanism study，important gene isolation and quality orientation breeding of horticulture plants by using transcriptomic technology.

Recent Advances on the Regulation of Phytochrome in Plant Defense Resistance

JIANG Min, LI Wei, DONG Zheng ,LI Li-hua ,DAI Liang-ying

2017, 33(7):  15-21.  doi:10.13560/J.cnki.biotech.bull.1985.2017-0099

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Phytochrome is a red and far-red light receptor，which not only plays an important role in plant morphogenesis，but also is involved in the regulation of plant defense resistance signaling pathway. Here，we present the reaction and mechanism of phytochrome and its interacted transcription factors regulating plant resistance to biotic stress，such as pathogens and pest，by inducing plant hormone signaling pathways. And we also review the mechanisms of phytochrome regulating abiotic stress，such as the competition of neighboring plants，drought，low temperature and high temperature. In addition，we discuss and prospect the challenges and future directions in the research of phytochrome.

Research Advances on Sugarcane Streak Mosaic Virus

FENG Xiao-yan, WANG Wen-zhi, SHEN Lin-bo ,FENG Cui-lian ,ZHANG Shu-zhen

2017, 33(7):  22-28.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0084

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Sugarcane streak mosaic virus（SCSMV）is one of the main pathogens causing sugarcane mosaic disease. It generally occurs in the major sugarcane growing areas of the world and seriously threatens the development of sugarcane industry. In this paper，we review the research advances of biological characteristics，occurrence and harm，identification and detection，genome structure and function，control strategy of SCSMV in order to provide some references for the further study of SCSMV and the diseases caused by it.

Study Progress on Circular RNA

ZHANG Ying-yue ,MA Yue-hui ,ZHAO Qian-jun

2017, 33(7):  29-34.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0009

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Circular RNA（circRNA）is a novel class of endogenous non-coding RNAs（ncRNA）with closed loop structure and is formed by alternative splicing from a precursor RNA（pre-mRNA）. Prior studies have discovered that circRNA widely and very stably exists in all eukaryotes. At present，the research of circRNA has become a new hotspot in the field of RNA research. It is found that circRNA accounts for a large proportion in transcripts，and some of the expressions are even higher than other transcripts. CircRNA plays a regulating role in gene expression，and an essential role in the process of biological development，such as miRNA sponges，endogenous RNAs and biomarkers，as well as critical role in the diagnosis and treatment of diseases. Some studies have revealed that circRNAs play key roles in the development of some diseases，including atherosclerosis，nerve system disorders，diabetes and cancers. In this paper，we review the research progress of circular RNA，including the characteristics of circRNA，the formation process，the expression，the relationship between circRNA and disease，as well as the function of circRNA. Moreover，we discuss the significance and existing problems in studying circRNA.

Research Advances on Whole Genome Sequencing of Chicken

LI Dong-hua, WANG Xin-lei ,LI Zhuan-jian ,SUN Gui-rong ,KANG Xiang-tao, YAN Feng-bin

2017, 33(7):  35-39.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0194

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Chicken possesses unique biological traits. Whole genome sequencing is the sequencing of all genes in a biological genome，mainly including de novo sequencing and whole genome re-sequencing. With the rapid development of sequencing technology in recent years，there have been great progresses in chicken genome research，which plays a critical role in explaining the biological characteristics of chicken and shortening the period of molecular breeding. This paper mainly describes the de novo sequencing completion of whole genome in different chicken cultivars，as well as the wide application of whole genome re-sequencing technology in analyzing genetic diversity，revealing evolutionary mechanisms and genetic mechanisms of quality traits，and further discusses the existing issues and potential prospects in the chicken whole genome sequencing. The aim is to provide a resource for genetic improvement and breeding programs.

Research Progress on Identification and Evaluation Methods，and Mechanism of Drought Resistance in Plants

LI Rui-xue,SUN Ren-jie,WANG Tai-chu, CHEN Dan-dan,LI Rong-fang,LI Long,ZHAO Wei-guo,

2017, 33(7):  40-48.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0023

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Drought affects the growth and development of plants at every phase and various physiological metabolic processes in plants，and ecological environment. It is one of the most disastrous limiting factors for growth and survival of plants. With the development of molecular biology，studying the regulation mechanisms of stress resistances at molecular scale has been become the key task in agricultural research. This paper describes the advances in drought resistance of plants in recent years based on two aspects：the identification and evaluation system of drought resistance in plant，and the mechanism of drought resistance，which aims at providing reference and strategies for plant drought resistance molecular mechanism research and molecular genetic breeding in plant drought resistance improvement.

Research Progress on the Isothermal Nucleic Acid Amplification Techniques in Rapid Detection of Microorganisms

WANG Da-zhou ,GUO Tian-xiao ,ZHENG Shi, SHANG Ying, XU Wen-tao

2017, 33(7):  49-61.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0106

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In recent 20 years，isothermal amplification technology has been rapidly developed. Relying on the advantage of the isothermal reaction，it is gradually replacing the traditional in vitro PCR（polymerase chain reaction）amplification，and plays an important role in different fields such as clinical medicine，laboratory medicine，molecular biology，genomics，food safety，and especially in the detection of pathogenic bacteria，viruses and other microorganisms. This article reviews and compares more than 10 kinds of high-profile isothermal amplification technologies，mainly focusing on the reaction principles，advantages，disadvantages as well as the development and application，and finally prospects the trends of isothermal amplification techniques，in order to play a positive role in the development of the related research fields.

The Research Progress of the Functions and Mechanism of Extracellular Proteins in Bioleaching

YU Run-lan ,LIU A-juan ,LIU Ya-nan, LIU Xue-duan, QIU Guan-zhou, ZENG Wei-min

2017, 33(7):  62-68.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1197

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Mineral-microbe-solute multi phase interface is the key space for bioleaching of sulfide minerals. Microbial proteins at the interface as the key components of extracellular polymeric substances（EPS）play a key role in the biofilm formation，structure maintainance and sulfide mineral dissolution. This article reviews the developments，existing methods and their applicability for studying the extracellular proteins in EPS of metallurgical microorganisms during bioleaching，and prospects a vista of future research on the extracellular protein produced by bioleaching microorganisms. These will offer important theoretical basis for studying the structure and functional mechanisms of extracellular proteins in the bioleaching process.

Prokaryotic Expression of Wheat Autophagy-related Factor ATG5 and Preparation of Its Antiserum in Rabbits

ZHANG Jia-zi ,LI Kai-xin ,YU Bao-jia ,YUE Jie-yu, WANG Hua-zhong

2017, 33(7):  69-74.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0020

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Autophagy is closely implicated in plant growth and development，as well as responses to environmental stimuli. ATG5 is one of the core factors involved in autophagosome assembly. Two wheat ATG5-encoding genes，TaATG5a and TaATG5b were cloned previously and are under deep functional study in our lab. In order to meet the need of specific antibodies in immunological methods used in further functional gene and protein study，a recombinant protein TaATG5a was acquired by vector construction，prokaryotic expression，and purification via the Ni-column affinity chromatography method，then the antiserum was prepared in rabbit immunization. The titer and specificity of the prepared antiserum were tested by the ELISA and Western blot assays. The results showed that the expression of TaATG5a cloned in vector pET30a was efficiently induced in Escherichia coli by IPTG，and gradually increased with the extension of IPTG induction time over 0 - 8 h. The apparent molecular weight of recombinant protein TaATG5a was close to its theoretical value. The purified recombinant protein was in high-purity that satisfied the requirements for antibody preparation. The prepared antiserum had a high titer of 1∶25 600 and specifically recognized TaATG5a in E. coli or wheat total protein. In addition，the ATG12-ATG5 in conjugated form，i.e.，the main form of wheat ATG5 in leaves，was also recognized by the prepared antiserum through Western blot assay.

The Sequence and Expression Analysis of MdAFS Gene in‘White Winter Pearmain’Apple

WANG Ru-ru ,DENG Shuai,DING Rui-rui ,ZHAO Rong-rong ,ZHANG Yuan-hu

2017, 33(7):  75-82.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0082

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This work is to study the full-length gene structural characteristics and encoded protein properties of α-farnesene synthase（MdAFS）from ‘White Winter Pearmain’ apple，and analyze the induced expression patterns of MdAFS gene. Bioinformatics software was used for analyzing the MdAFS gene and its encoded protein，secondary structure，and tertiary structure，then MEGA6.0 for constructing the phylogenetic trees with AFS from different species，and qRT-PCR for studying the expression pattern of MdAFS gene. The full-length of MdAFS gene（GenBank accession number：AY563622）was 1731 bp，contained the complete ORF，encoding 576 amino acids. The prediction analysis of secondary structure and tertiary structure showed that the protein was α-helix as the main parts of the mixed type proteins. The sequence similarity of α-farnesene synthase amino acid in different species was low，but the N-terminal RRx8W and C-terminal the H-α1loop of α-farnesene synthase was highly conservative. Abiotic stress and hormones induced MdAFS expression up-regulated，and more remarkably up-regulated expressions were observed in MV，ABA and MeJA treatments. In conclusion，α-farnesene synthase in ‘White Winter Pearmain’ apple has low similarity in amino acid sequence，but similar protein 3D structure. α-farnesene synthase presents monoterpene synthases characteristics in the protein structure and function. It is revealed that the abiotic stress and hormonal signal induce the up-regulation expression of key enzyme MdAFS gene in the α-farnesene metabolic pathway in ‘White Winter Pearmain’ apple.

Expression Analysis of Fusarium Wilt Resistance-Related Genes at the Flower Bud Differentiation Stage of Banana

WANG Gui-hua, ZHANG Xin ,QI Yan-xiang ,WANG Yu-guang ,PENG Jun ,XIE Yi-xian

2017, 33(7):  83-88.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0071

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A large number of differentially expressed genes were obtained from transcriptome analysis of ‘Brazilian’ and ‘Formosana’ bananas infected by Fusarium oxysporum f. sp. cubense race 4（Foc4）at the flower bud differentiation stage. The expression variations of 10 disease-resistance related genes from diseased ‘Brazilian’ and ‘Formosana’ banana roots were detected by real-time quantitative PCR（qPCR）. The results showed that these 10 genes expressed in both ‘Brazilian’ and ‘Formosana’ at flower bud differentiation，but the expression levels of these genes varied. Compared with the control of ‘Brazilian’ banana，the expression levels of the nine genes（laccase-4（B），periplasmic beta-glucosidase precursor，glutathione-S-transferase Cla47，class III peroxidase 136 precursor，peroxidase N，similar to T-phylloplanin，snakin-1，putative chitinase，and ellulose synthase catalytic subunit 12）up-regulated except gene laccase-4（A）. Among them，the expression of peroxidase N significantly up-regulated，and the relative expression was 1 682 times as much as in ‘Brazilian’. These results suggested that 10 differentially expressed genes may be involved in defense responses against Foc4 at the flower bud differentiation stage of ‘Formosana’，which lay a foundation for the further study on molecular mechanisms of ‘Formosana’ defense against fusarium wilt.

Cloning and Expression Analysis of Jc5β-StR Gene in Jatropha curcas

HUANG Yao-yao, WEN Jin-fen ,DENG Ming-hua, GONG Ming ,CHEN Hao-wei

2017, 33(7):  89-95.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1114

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The CDS sequence of Jc5β-StR gene was cloned with Jatropha curcas cDNA as template，and analyzed by bioinformatics analysis. The opening reading frame（ORF）of Jc5β-StR was 1 173 bp encoded 390 amino acids. The predicted molecular weight was 44.23 kD，and theoretical isoelectric point（pI）was 5.22. Blast searching and phylogenetic analysis showed that Jc5β-StR had highest identity（87%）and closest relationships with the 5β-StR of Ricinus communis. Jc5β-StR was a short-chain reductase，containing a progesterone 5β reductase（5β-POR）in a family of short chain reductase（SDRs），and also 6 conserved domains of SDRs. Jc5β-StR expressed in different tissues of root，stem，leave，flower，seed capsule，and seed，the 1highest in seed；moreover，the expression gradually rose with the development of seed，and declined after peak in the later stage of seed development（50 d）. The abiotic stress such as NaCl，PEG，4℃，and mechanical damage resulted in the up-regulation of Jc5β-StR expression in varied level，indicating that Jc5β-StR involved in the response to abiotic stress.

Effect of Salicylic Acid on Chlorophyll Fluorescence Parameters in Wheat Under Cadmium Stress

WANG Rui-bo

2017, 33(7):  96-99.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1188

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The effect of salicylic acid on chlorophyll fluorescence parameters in wheat（Xinmai 18）under cadmium stress was analyzed by using chlorophyll fluorescence technique. The results showed that under cadmium stress，cadmium content in wheat root decreased significantly. Moreover，root length，chlorophyll content，chlorophyll fluorescence parameters Fv/Fm，φPSⅡ，qP and qN through salicylic acid（SA）pretreatment were significantly less than CK. It was concluded that salicylic acid inhibited the cadmium into the root of wheat，resulting in higher photosynthetic efficiency. The above results revealed that salicylic acid pretreatment significantly increased the wheat’s tolerance to cadmium.

Effects of Nitrogen Fertilizer on the Expression of nifH Gene of Endophytic Azotobacter in Sugarcane Leaves

LI Jia-hui, YUAN Dan, LU Jian-ming, YANG Li-tao, LI Yang-rui ,XING Yong-xiu

2017, 33(7):  100-106.  doi:10.13560/j.cnki.biotech.bull.1985.20170002

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In order to investigate the effects of nitrogen fertilizer on the expression of nifH gene of endophytic diazotrophs in sugarcane leaves，we carried out a field experiment with and without nitrogen fertilization，and used RT-PCR and Western blot technology to analyze the differences of nifH expression and the relative expression quantity of nitrogenase in 3 sugarcane varieties，GXB9，ROC22 and GT11. The results showed that the expressions of nifH gene in endogenous azotobacter were detected at gene and protein levels in the leaves of sugarcane growing in field both with nitrogen fertilization and without nitrogen fertilization. Nitrogen fertilization treatment inhibited or decreased the expression of nifH gene in nitrogen-fixing bacteria，but it was beneficial to the expression of nifH at the protein level. The relative expressions of the nifH gene in the leaves of three sugarcane cultivars were all higher under nitrogen application than the control without nitrogen application；especially，the difference was significant for cultivar ROC22.

Research on Rhizosphere Soil Microbial Diversity of Two Typical Kinds of Disease in Yam

KANG Jie,ZHANG Shu-yan, HAN Tao,SUN Zhi-mei, LUO Tong-yang

2017, 33(7):  107-113.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0067

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For understanding the microbial community structure of soil rhizosphere with two typical diseases and clarifying the correlation between soil diseases and microbial diversity，high-throughput sequencing was applied to compare and analyze the microbial diversities of the rhizosphere with 2 typical diseases. As results，the population diversity and enrichment of bacteria in soil sample were the scab disease > CK > root-rot disease. While in fungi，the changes were more obvious，fungal diversity and enrichment in the soil with 2 diseases were higher than the control group. The species annotation showed that comparing with the control group，the bacterial species of different treatments were almost the same，but just the proportion of each were not identical. There were some specific fungal species which may be related to disease occurrence，and variation of proportion was significant，indicating that the changes of soil microbial structure affect the happening of the disease in a certain extent.

Screening，Identification，and Inhibitory Effect of Antagonistic Actinomycetes Against Macrophoma kuwatsukai Causing Winter Jujube Ring Grain Disease

FAN Yan-hui ,WANG Jun

2017, 33(7):  114-119.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0119

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The objective is to select actinomycete which has strong antagonistic ability against winter jujube ring grain disease. Total 198 actinomycetes were isolated by culture-dependent method from the shell sediment in the Seashell Islands of Yellow River Delta，and the strain BK with the strongest antagonistic ability was further studied. Strain BK was identified based on its morphology，physio-biochemical characters and 16S rRNA sequence analysis，and its ability against winter jujube ring grain disease was also studied，providing a theoretical basis in biological control of winter jujube ring grain disease. The results showed that BK significantly inhibited the mycelium growth of Macrophoma kuwatsukai，with inhibitory rate of 72.3%. The control effect of the culture filtrate against winter jujube ring grain disease was 73.7%，which had no significant difference from that by the 50% carbendazim. Strain BK was identified as Streptomyces purpureus. To sum up，strain BK is an ideal antagonistic one，and presents a potential application for the biological control of winter jujube ring grain disease.

Research on Growth Characteristics and Algicidal Effects of Bacillus subtilis

CHENG Xin ,LI Kun-tai, HUANG Lin

2017, 33(7):  120-125.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0013

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Algicidal microorganisms rapidly kill cyanobacteria，and subsequently the purpose of controlling algal bloom in water bodies is achieved. To investigate the inhibition of Bacillus subtilis against Microcystis aeruginosa and Chlorell，the Bacillus subtilis cultures were used to study its inhibition and mode of action against these two kinds of algae. The MDA，GSH，SOD and CAT activity in M. aeruginosa when cultured with B. subtilis were measured. The results showed that B. subtilis had a solid adaptability to environment and inhibited the growth of the algae by secreting extracellular substances. The chlorophyll a and protein contents of M. aeruginosa in treated groups were significantly lower than that in the control group，while the MDA and GSH contents as well as the SOD and CAT activity increased obviously when M. aeruginosa was cultured with B. Subtilis.

An Efficient Transformation of Salinity-resistant Peppermint Stem Mediated with Agrobacterium tumefaciens

ZHAO Zhong-juan, WEI Yan-li ,LI Ji-shun, WANG Yi-lian ,YANG He-tong

2017, 33(7):  126-132.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1199

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Transgenic technology is an important means to improve the essential yield and quality of peppermint（Mentha piperita L.）oil. An Agrobacterium tumefaciens-mediated transformation using bud peppermint stem was established，in order to solve the problems in adventitious bud induction of salinity-resistant peppermint leave，such as easy browning and difficult differentiation. The orthogonal test was carried out to screen the optimal concentration of A. tumefaciens，transformation time and co-culture time. The results showed that the effect of the three factors on transformation displayed as the transformation time（B）> bacterial concentration（A）> co-culture time（C）. The bacterial concentration OD600 = 0.8，the transformation time 15 min and the co-culture time 3 d proved to be the optimal transformation conditions. The optimal composition of transformants was screened to be 1/5MS + 0.5 g/L MES（2-（N-morpholino）ethanesulfonic acid）+ 1% glucose + 2% sucrose and PH 5.4. The transformation was conducted using different plant expression vectors，and the positive plants were detected by genomic PCR and GUS staining. And the results revealed that this transformation system was suitable for the transformation of many plant expression vectors，and the regenerated plants were easily obtained. This provides a new method and system for gene transformation of salinity-resistant peppermint.

Effect of Ethanol-induction on the Metabolism of Acetaldehyde in Lactobacillus rhamnosus HCCB 20591

ZHONG Jian-hong, YIN Yu, CHEN Dai-jie

2017, 33(7):  133-137.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0051

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Acetaldehyde is a flavor substance in alcoholic beverages. Excessive acetaldehyde not only affects the flavor of alcoholic beverages，but also causes health damages to human body. This study is to screen edible probiotic strains with the ability to metabolize acetaldehyde，and explore the relationship between acetaldehyde metabolism and ethanol-induction. Strain HCCB 20591 of Lactobacillus rhamnosus with strong acetaldehyde metabolism ability was obtained via a resting cell system，and its metabolic ability was induced by ethanol. When the ethanol concentration was 3% and the induction time was 12 h，the acetaldehyde conversion ratio by HCCB 20591 was 41.13%. GC-MS analysis of the acetaldehyde metabolites showed that in addition to acetic acid，ethanol was also generated by HCCB 20591. RT-PCR method was used to analyze the transcription level of the related genes. It was found that acetaldehyde metabolism was the combined catalytic result of alcohol dehydrogenase，aldehyde dehydrogenase and aldehyde oxidoreductase.

Preparation and Detection of Polyclonal Antibody of GAPDH Protein from Bombyx mori

CHEN Ying, WANG Yuna-zhuo, YANG Juan-juan, GUO Xing-guo, QIAO Hui-li

2017, 33(7):  138-144.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0104

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This work is to prepare and detect the polyclonal antibody of GAPDH protein in Bombyx mori. GAPDH gene was cloned from silkworm by PCR，then prokaryotic expression vector was constructed and transformed into Escherichia coli competent cell. The recombinant protein was induced and purified. The purified protein was used as antigen to prepare the polyclonal antibody by immunizing New Zealand purebred rabbit. The titer and specificity of the polyclonal antibody was detected by enzyme-linked immune sorbent assay（ELISA）and Western blot. As results，GAPDH/pET-28a vector was constructed successfully，and recombinant GAPDH fusion protein in high purity was obtained. SDS-PAGE and anti-His monoclonal antibody detection showed that the molecular weight of the purified protein was consistent with that predicted. GAPDH polyclonal antiserum was obtained by immunized New Zealand purebred rabbit for four times using the purified protein as an antigen. The titer of GAPDH polyclonal antibody was 1：8 000 by ELISA，and the antiserum was able to bind specifically with the total protein of B mori. Conclusively，the polyclonal antibody of GAPDH is successfully prepared，which lays a solid foundation for further studying the physiological function of different proteins in B. mori.

Purification and Activity Analysis of Tag-free Protein Kinase BIN2

YUAN Min, QI Yu-rong ,WANG Rui-ju

2017, 33(7):  145-149.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1167

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BIN2 is a kind of Ser/Thr protein kinase，and plays important roles in BR signal transduction pathway. In order to obtain the active and tag-free BIN2 protein，the BIN2 gene was cloned into the prokaryotic expression vector pPAL8 to obtain BIN2-pPAL8 expression vector. The correct plasmid BIN2-pPAL8 was transformed into E.coli expression strain BL21（DE3）. After exploring the proper induction conditions，the 51 kD eXact-BIN2 protein was successfully induced and purified using eXact purification system. The high-quality and tag-free BIN2 protein was obtained through cleaving the eXact tag. The purified BIN2 protein is highly active，which could phosphorylate MBP-BZR1 in vitro.

Expression and Purification of orf Virus Protein ORFV035 and Preparation of Polyclonal Antibody

CHEN Hui-qin ,WANG Xiao-ping ,LUO Shu-hong ,WANG Li-jie ,CHEN Qian-qian, HAO Wen-bo

2017, 33(7):  150-154.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1182

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This work aims to express and purify recombinant orf virus encoded protein 035（ORFV035）in Escherichia coli and prepare a polyclonal antibody（pAb）against ORFV035 for immunoassays，as well as lay foundation for the further researches on the replication，assembly，morphogenesis，and mature process . The gene of ORFV035 was amplified by PCR from orf viral genome，and sub-cloned into the expression vector pET-30a（+），and recombinant plasmid pET-30a-035 was constructed by enzymatic digestion and ligation of BamH I and Hind III. Then the plasmid pET-30a-035 after double enzyme digestion and sequencing was transformed into Escherichia coli BL21，the transformants were induced by IPTG，and SDS-PAGE was used to identify the expression of the protein. The His-tagged fusion protein ORFV035 was ultrasonic-treated and then purified through Ni-chelating affinity chromatography. The purified ORFV035 was used to immune into BALB/c mouse for producing anti-ORFV035 pAb. The results showed that the recombinant plasmids pET30a-035 was successfully constructed，and the protein ORFV035 in E. coli BL21 was in inclusion body. The ORFV035-his fusion protein was obtained after washing，dissolving，and purifying the inclusion body，and the pAb anti-ORFV035 was prepared. The Western blot analysis showed that this pAb recognized natural ORFV035.

Effects of Salinity on the Growth and Biochemical Properties of a Freshwater Algae Scenedesmus sp.

LI Jia-ying,LI Tao, TAN Li,WU Jia-yi ,XIANG Wen-zhou, LIU De-hai

2017, 33(7):  155-161.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1159

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The aims of this work are to analyze and compare the effects of different salt concentration（0-40‰）on the growth and biochemical characteristics of a freshwater algae Scenedesmus sp. incubated in small tubular bioreactors，and evaluate the industrialization potential of culturing this algae in seawater as well explore the adaptive mechanism at high salinity，which provides theoretical references for further study on seawater domestication. The growth of the microalgae，the content of protein，lipid and polysaccharide，and the composition of pigments and fatty acids were analyzed correspondingly by microscopic observation，Soxhlet extraction，Kjeldahl method，phenol sulfuric acid determination，gas chromatography technology，and HPLC. The results showed that cells grew bigger and naturally precipitated at high salinity，meaning that it can be harvested at low cost. The study also found that growth of Scenedesmus sp. was gradually inhibited with the increase of salinity，and completely inhibited at the salinity of 40‰；however，it still grew well at the salinity of 30‰ with biomass of 2.84 g/L，and higher protein content（95.40% higher than 0‰ group）with similar protein productivity as that by freshwater. Combined with the great cost reduction of producing protein at high salinity，culturing Scenedesmus sp. in seawater for protein resource is of high potential. Moreover，with the increase of salinity，Scenedesmus sp. tended to accumulate more β-carotene，astaxanthin，and even soluble sugar，which might be the key physiological basis of adapting salt-stress.

Recombinant Expression and Fermentation Optimization of Sulfolobus acidocaldarius ATCC 33909 Maltooligosyltrehalose Synthase in Bacillus subtilis

HAN Chang ,SU Ling-qia, WU Jing

2017, 33(7):  162-168.  doi:10.13560/J.cnki.biotech.bull.1985.2017-0086

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To obtain the recombinant expression of gene treY for maltooligosyltrehalose synthase（MTSase）from Sulfolobus acidocaldarius ATCC 33909 in Bacillus subtilis，the target gene was PCR-amplified using plasmid pET-24a（+）-treY as template，and ligated with the expression vector pHY300PLK，then transformed into the expression host Bacillus subtilis CCTCC M 2016536. The activity of MTSasereached 17.5 U/mL after cultivated in TB culture for 48 h. By single factor test（nitrogen source，nitrogen proportion，nitrogen concentration，carbon source，glucose concentration，initial pH，and induction temperature）and orthogonal test（nitrogen concentration，glucose concentration，initial pH，and induction temperature），the optimal fermentation condition was determined as：48.0 g/L of nitrogen source（industrial peptone∶cottonseed powder=3∶1），10.0 g/L of glucose，initial pH of medium=7.0，and the optimal temperature was 30℃. Under optimal conditions，the production of MTSase reached 41.5 U/mL，which was 2.4 times of that before optimization.

Molecular Cloning and Induced Expression Analysis of CD8 Gene from Humphead Snapper

HUANG Yu-cong, LIANG Xiu-quan ,CAI Shuang-hu,LU Yi-shan,JIAN Ji-chang, WU Zao-he

2017, 33(7):  169-178.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0095

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This work aims to characterize CD8 gene of humphead snapper，Lutjanus sanguineus. The full length cDNA sequences of CD8α and CD8β genes were cloned by homologous cloning and rapid amplification of cDNA ends（RACE）from humphead snapper，and their tissue distribution and expression pattern after induction with immunostimulants were also analyzed by real time quantitative PCR（qRT-PCR）. The CD8α’s cDNA consisted of 1 576 bp encoding 225 amino acids，and the CD8β’s cDNA consisted of 1 486 bp encoding 210 amino acids. The predicted CD8α and CD8β proteins were similar in structure，both contained a signal peptide，immunoglobulin superfamily variable domain，hinge region，transmembrane domain and cytoplasmic tail. The qRT-PCR analysis showed that the highest level of CD8α and CD8β mRNA expression was found in thymus，followed by kidney，gill，intesine，skin，spleen，and head kidney. Furthermore，the mRNA levels of CD8α and CD8β in head kidney leucocytes was significantly up-regulated after 8 h treated with the stimulants lipopolysaccharide（LPS），concanavalin A（ConA），and polyinosinic-polycytidylic acid（PolyI∶C），respectively（P < 0.01）. And their expression levels in gill，anterior kidney，spleen，and intesine also increased at 24 h after stimulated with formalin-inactivated Vibrio harveyi.

Expression Analysis of lc3b Gene in Gonadal Development of Triploid Female Rainbow Trout

HUANG Tian-qing, WANG Xing-ran ,WANG Yan-na, SUN Hui-zhi, HAN Ying

2017, 33(7):  179-184.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1166

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This work aims to show a new light on the relationship between autophagy and gonadal regression of triploid female rainbow trout. Autophagy gene lc3b of rainbow trout was cloned by RT-PCR，and its expressions in gonads of triploid female rainbow trout were analyzed at different developmental period in mRNA and protein level. Gonadal ultrastructures were observed under transmission electron microscopy. Results showed clear evidence that lc3b gene was highly expressed in gonads of triploid female rainbow trout during the period of 200dpf to 270 dpf，in which autophagosome structures were identified. In this stage，the conversion of LC3B-I to LC3B-II was greater. These results demonstrated autophagy played pivotal roles in gonadal regression of triploid female rainbow trout.

Expression and Purification of Outer Membrane Protein 34 of Acinetobacter baumannii and Analysis of Its Bioactivity

AN Zhi-yuan ,SU Jian-rong

2017, 33(7):  185-194.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0102

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The present study is to clone and purify the outer membrane protein 34（Omp34）from Acinetobacter baumannii ATCC 19606，and to analyze its biological structure and effects on the apoptosis of HEK293 cells. First，the gene of outer membrane protein 34 of A. baumannii ATCC 19606 was amplified by PCR，it was cloned into the pET28a（+）vector to generate the pET28a（+）-Omp34 expression vector，and the vector was transformed into Escherichia coli BL21 for expression. Then，the recombinant protein was expressed by IPTG induction and purified by Ni-affinity chromatography. L-arginine and GSH/GSSG were added in the dialysis buffer to facilitate the Omp34 protein correct folding. The biological structure of Omp34 was predicted by bioinformatics. The secondary structure of the Omp34 was detected by circular dichroism（CD）.The apoptosis of HEK293 cells induced by Omp34 were measured by flow cytometry and Western blot. As results，the pET28a（+）-Omp34 recombinant expression vector was successfully constructed，the Omp34 was highly expressed in prokaryotic expression system，and the soluble protein was obtained by refolding. Bioinformatics predicted that Omp34 was beta barrel-shape. CD confirmed that the secondary structure of purified Omp34 was beta sheets，of which the α-helix，β-sheets，and β-turn，and random coil of the Omp34 were 12%，39%，22%，and 27%，respectively. The re-natured Omp34 was able to induce the apoptosis of HEK293 cells. In summary，the Omp34 of A. baumannii can be expressed by molecular cloning technology. Moreover，its secondary structure was analyzed and it is demonstrated that Omp34 can lead to apoptosis of HEK293 cells. This work provides a basis for further study of the biological functions and antibiotic resistance mechanism of A. baumannii Omp34.

Expression of Channel Catfish C-type Lysozyme in Pichia pastoris and Its Bacteriostatic Activity

FENG Ya-dong, TAO Yan, LI Wen, CUI Xu, WANG Qiang-hou

2017, 33(7):  195-202.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0094

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C-type lysozyme is one of various lysozymes in different tissues of all organisms，and is a key immune protein in cells and possesses stable structure and excellent bacteriostatic activity. Thus，it is considered to be promising food preservative and feed additives. In order to develop fish C-type lysozyme，the present study established a preparation method of channel catfish（Ictalurus punctatus）C-type lysozyme，based on Pichia pastoris expression system. A cDNA fragment（cflyC）added with the Xho I and Xba I restriction sites at 5 ‘and3' ends respectively，encoding the C-type lysozyme of channel catfish，was obtained by PCR. This cDNA encoded a peptide consisting of 127 amino acids. The cflyC fragment was ligated to pPICZαA vector，and a recombinant expression vector pPICZαA-cflyC was constructed，and transformed into competent Pichia pastoris X-33. The yeast transformants containing multi-copy gene insertions were screened using zeocin in different concentrations and PCR identification. The target protein was induced for 144 h with 0.5% methanol at pH 6.0，29℃，and 250 r/min，and the expression product was purified by immobilized metal affinity chromatography（IMAC）. Tricine-SDS-PAGE analysis showed that molecular mass of the purified recombinant protein was about 15.1 kD. MALDI-TOF-TOF analysis demonstrated that it was the expected recombinant cflyC. Folin-reagent method indicated that the expression yield of recombinant cflyC was 2.75 mg/L. Agar well diffusion and activity assays proved that the recombinant cflyC presented antibacterial activity against Bacillus subtilis. The present study firstly realized the recombinant DNA expression of the channel catfish C-type lysozyme in P. pastoris，which provides key basis for its large-scale preparation.

Comparing and Analyzing the Disease Resistances of Four Disease-resistant Selected Lines of Macrobrachium rosenbergii

LI Yu-feng, DAI Xi-lin,YUAN Xin-cheng,ZHOU Xun,HU Yan-jie,DING Fu-jiang

2017, 33(7):  203-209.  doi:10.13560/J.cnki.biotech.bull.1985.2017-0096

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Expressions of 4 disease-resistant genes（heat shock protein70（HSP70），Se-GPx，CAT，and SOD）from 4 selected lines（A，B，C，and D）of Macrobrachium rosenbergii were analyzed with real-time PCR. Infected with Vibrio alginolyticus，the disease-resistant abilities of the 4 selected lines were compared based on the survival rates of 4 selected lines and the expression differences ofHSP70，Se-GPx，CAT，and SOD among the 4 selected lines. The results showed that relative expression of HSP70 mRNA of A and B was obviously higher than that of C and D in the hepatopancreas（P<0.05）. The expressions of Se-GPx，CAT，and SOD changed significantly within 96 h，and were different among the 4 selected lines at different time；however，the general trend was that expressions in A and B were higher than those in C and D. The rate of A，B，C，and D was 65.0%，65.8%，51.7%，and 53.3% after infecting with V.alginolyticus for a week，it is inferred that disease-resistant ability of A and B is higher than that of C and D.

Transcriptome Sequencing Analysis of Hepatocellular Carcinoma HepG2 Cells Induced by Antitumor Peptide 9R-P201

LIU Wen-rong ,DING Ruo-fan, ZHANG Yi-ming ,LI Yu-peng, LI Ling ,GUO Zhi-yun

2017, 33(7):  210-215.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0053

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This wok aims to elucidate the regulation of 9R-P201 on hepatoma cells（HepG2）at transcriptome level by investigating the gene fusion，SNP（Single Nucleotide Polymorphism）mutation，alternative splicing and other events after 9R-P201 treating HepG2，and analyzing the biological processes and signaling pathways involved by differentially expressed genes. The expression differences of the genes before and after the 9R-P201 treating HepG2 cell line were detected by transcriptome sequencing. Meanwhile，gene fusion，SNPs，and alternative splicing were identified by tophat-fusion，SAMTOOLS software，and rMATS respectively. Functional enrichment analysis of differentially expressed genes were performed by GO（Gene Ontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）. As results，276 alternative splicing events，5 557 SNP sites，and 45 gene fusion events were detected in the transcriptome sequencing. In addition，403 differentially expressed genes including 269 up-regulated and 134 down-regulated were detected. Gene GO and KEGG enrichment analysis showed that differentially expressed genes were significantly involved in the biological processes of cell growth，locomotion，as well as a number of cancer-related signaling pathways. In conclusion，the study revealed that the differentially expressed genes resulted from 9R-P201 treating HepG2 were significantly correlated with the biological processes and signaling pathways related to cancer and hepatocellular carcinoma HepG2 cell line，and a large number of alternative splicing events，SNP mutations，gene fusion events after 9R-P201 inducement occurred，suggesting this peptide may be used as a potential drug for subsequent hepatocellular carcinoma interventional therapy.

Bioinformatics Analysis and Functional Verification of p53 Regulating miRNA-3661 in Hepatoma Cell HepG2

LI Yu-peng ,ZHANG Yi-ming,HU Hai-bi ,KANG Cheng-yu, LI Mu-zhou ,GUO Zhi-yun

2017, 33(7):  216-223.  doi:10.13560/j.cnki.biotech.bull.1985.20170005

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The aims of this work are to have the bioinformatics analysis of hsa-miR-3661 regulated by p53，which was found in doxorubicin（Dox）inducing the DNA damages of hepatoma cell HepG2 in previous experiment，and to verify its function by molecular biological experiment，providing theoretical basis for the regulating mechanism of miR-3661 in hepatoma cells. After acquiring the structure and sequence information of miR-3661，we predicted the target genes and used DAVID to do the functional enrichment analysis of miRNA target gene. We then analyzed the binding sites of p53 and miR-3661，and built the regulatory network by the interaction among genes. Finally，multiplication experiment verified the functions of miR-3661 in restraining tumor. The results showed that miR-3661 sequence was conserved，and the promoter region existed in the binding site of p53，suggesting that there was direct regulation between p53 and hsa-miR-3661. We predicted that there were 1009 target genes，and 369 genes of them significantly enriched in the biological procedure related to tumor，such as cell cycle regulation，proliferation，apoptosis and so on（P < 0.05），mainly involved in cancer pathway，MAPK signaling pathway，ErbB signaling pathway（P < 0.05）. Using the interactions of 2830 groups of genes，we constructed the regulatory network among p53，hsa-miR-3661 and target genes，and analyzed key target gene participating in several tumorous biological process from the perspective of system biology. It was confirmed in the experiment that the overexpression of miR-3661 significantly inhibited the proliferation of hepatoma cell HepG2 （P-value= 0.00146）. In conclusion，miR-3661 is directly regulated by p53 and its target genes significantly enrich in various biological processes and signal pathways related to tumor. Moreover，the overexpression of miR-3661 significantly inhibits the proliferation of hepatoma cell.

Effects of PDSW Combined with Hyperthermia on COX-2 and Bcl-2 Expressions in Gastric Cancer Cells and Tissues

TANG Hui-rong, CUI Jin ,ZHANG Jia-hua, DAI You-guo ,LI Wei-ming ,LIAO Chen

2017, 33(7):  224-230.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0691

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This work aims to explore the effect of PDSW combined with hyperthermia on COX-2 and Bcl-2 expressions in gastric cancer cells SGC-7901 and tissues，and to clarify its anti-cancer effects on gastric cancer cells SGC-7901 in vitro and in vivo. The SGC-7901 cells were cultured in vitro and then were randomly divided into experimental，control and blank groups；the experimental group was added with PDSW，control group was given with normal saline，blank group was in no other treatment，and each group were cultured under 37℃，40℃ and 43℃ respectively. Concurrently，the SGC-7901 cells were injected into the nude mice subcutaneous to establish the transplantation tumor model. The injected mice were randomly divided into 5 groups，and then given the treatment with H2O+RT，H2O +40℃，H2O +43℃，PDSW+40℃，and PDSW+43℃ respectively when the tumor cells grew to 1 cm diameter. qRT-PCR and Western blot were used to detect the mRNA of gene COX-2 and Bcl-2 and the protein expression of COX-2 and Bcl-2 in the cells and tissues. As results，in SGC-7901 cells and nude mice subcutaneous transplantation tumor tissues，the expressions of gene COX-2 and Bcl-2 mRNA and their protein expressions decreased under the treatment of hyperthermia. Compared with the treatment of normal saline group，the expressions of gene COX-2 and Bcl-2 and their protein expression decreased remarkably under the treatment of PDSW combined with hyperthermia. Conclusively，PDSW combined with hyperthermia may inhibit the expressions of gene COX-2 and Bcl-2 and their protein expressions in gastric cancer SGC-7901 cells and tissues，so as to effectively inhibit the proliferation of gastric cancer cells.