Table of Content

26 November 2018, Volume 34 Issue 11

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Function Analysis of ZIP in Plant

LI Su-zhen, CHEN Ru-mei

2018, 34(11):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0464

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Zinc and iron are essential micronutrients for plant growth and development,and they play important roles in photosynthesis,respiration and other biochemical reactions. The normal growth and development of plant are ensured while zinc and iron in plant are in balance. ZIP plays important role in the uptake and transport,as well as the homeostatic regulation of Zn/Fe. At present there are research progress achieved in the ZIP gene family in plants. ZIP genes expression pattern,subcellular localization,yeast complementation assay,ZIP gene overexpression and knock down are summarized,and the function of ZIPin plant growth and development are explored. In sum,insight into the function of ZIP in uptake,transport and homeostasis of zinc/iron may contribute to the application of ZIP in agricultural production through genetic modification and conventional breeding.

Research Progress on Plant NAC Transcription Factors

WANG Chun-yu, ZHANG Qian

2018, 34(11):  8-14.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0443

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NAC（NAM,ATAF1,ATAF2,and CUC2）transcript family is the largest one in plant genome,and they are specific transcription factors in plants. More than 100 members are discovered and identified in the genome of varied land plants. NAC transcription factors function variously and play very important roles in multiple physiological processes such as plant growth and development,response to stress,regulating the resistance to disease,the regulation of secondary growth,the transduction of hormone signals,etc. Here,the progress on the basic structures and functions of NAC transcription factors are reviewed,to provide related research to plant NAC transcription factors for reference.

An Overview on Yellow Green Leaf Mutants in Rice

LI Su-zhen, YANG Wen-zhu, CHEN Ru-mei

2018, 34(11):  15-21.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0465

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Chlorophyll is the important pigment participating in photosynthesis in plant chloroplasts. The photosynthetic rate,the output and quality of crops are decided by synthesis of chlorophyll. Synthesis and catabolism of chlorophyll is a complicated process and many genes are involved in this process,in which any gene’s mutation would affect the synthesis and catabolism of chlorophyll and further lead to changing of the leaf colour or affecting the plant growth. The research of leaf colour mutant is an effective approach of verifying the gene function during the development of chloroplast. Therefore,it is of both important theoretical significance and application value to discover and identify the leaf colour mutant genes and to carry out the location,clone and function analysis of the chlorophyll-related genes. This paper reviews the research progresses on rice yellow green leaf mutants,which not only provides the promising materials for researching the chlorophyll biosynthesis pathway and the mechanism of photosynthesis,but also can be used as the marker traits in heterosis.

Research Progress on Maize Dwarf Genes

WANG Wen-xiu, WANG Lei

2018, 34(11):  22-26.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0444

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Corn has become the largest crop in China by planting area,and increasing the planting density is a key approach for improving maize yield. The plant type of corn has become one of the most critical researches on maize genetic breeding. Reasonable plant shape and lodging resistance is important trait affecting the corn yield,and is one of the important indexes for evaluating maize varieties. Molecular markers corresponding to dwarf genes can be developed through isolation and cloning of them,by which the dwarf or semi-dwarf corn plant may be cultivated;subsequently,the plant shape is improved,then the planting density can be increased,the photosynthetic efficiency of population is improved,and finally yield increases. Here we reviewed and summarized corn dwarf breeding,the genetic characteristics of corn dwarf genes,the phenotype of corn dwarf genes,and the effects of hormones on the corn dwarf,aiming at providing new clues for the application of dwarf gene and the expounding of molecular mechanisms.

Advances in Physiological,Biochemical and Response Mechanism of Sweet Sorghum Under Cadmium Stress

LI Jian-gang, LIU Rui-yuan, PENG Dan-ni, LI Wen-jian, DONG Xi-cun, MA Jian-zhong

2018, 34(11):  27-35.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0536

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The domestic waste,fertilizers,pesticides,and the development of manufacturing and mining have being caused heavy metal contamination in soils. This paper systematically summarizes the physiological and biochemical mechanisms of sweet sorghum under cadmium stress,tolerance and molecular mechanism. The physiological and biochemical mechanism mainly refers to the effects of cadmium stress on sweet sorghum seed germination and seedling growth,antioxidant enzyme activity,photosynthetic parameters,ultrastructure and microelement absorption. While the tolerance of sweet sorghum to cadmium includes the efflux mechanism and internal tolerance,the efflux mechanism is mainly the efflux of protein yellow stripe-like protein,the heavy metal ATPase 4,the plant cadmium resistance protein 1,and the pleiotropic drug-resistance protein 8 via heavy metals;the internal tolerance mainly has the chelation and the vacuolar compartmentalization of heavy metals. The mechanism of tolerance is mainly dependent on the regulation of glutathione pathway,the expressions of late embryogenesis abundant protein and natural resistance associated macrophage protein. In addition,the study on the cadmium uptake and accumulation and transport-related proteins in sweet sorghum mainly refers to the main process of cadmium entering plants,the accumulation order of calcium in plant,the transport of zinc-iron-ion regulated transporter protein,and natural resistance associated macrophage protein in the cell membrane. Finally,the advantages of applying sweet sorghum for bioremediation are explained and prospected,and the problems and future research directions are proposed,which may provide a basis for future breeding of sweet sorghum and the cultivation of new sweet sorghum varieties with high efficiency in remediating cadmium-contaminated soil .

Function of S-nitrosoglutathione Reductase Under Stresses in Plant

XIA Jin-chan

2018, 34(11):  36-41.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0514

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Nitric oxide（NO）as a signaling molecule is involved in diverse physiological processes such as germination,stomata closing,lateral root development and biotic and abiotic stress response. The predominant regulating way of NO action is S-nitrosylation,the reversible covalent attachment of NO to cysteine thiols. As a free radical,NO’s half-life is very short,which restricts their physiological function in cells;while the S-nitrosothiols（SNOs）from the interaction of NO with intracellular sulfhydryl-containing molecules are generally more stable in solution,and it participates in the transport,diffusion,and storage of NO,as well as the post-translational modifications of proteins. S-nitrosoglutathione（GSNO）from s-nitrosation with NO is the storage and transport form of NO,which can transfer its NO moiety to proteins and enable target protein to be in nitrosylation. As a type of conserved protein,S-nitrosoglutathione reductase（GSNOR）regulates the level of intracellular NO and nitroso mercaptan（SNOs）by reducing GSNO,thus which may protect the body from nitrosation stress and indirectly regulate the oxidative state cell. GSNO is a natural NO repository and GSNOR is the key gene that regulates the level of nitrosylation. This paper mainly summarizes the processes of plant growth and development,biological and abiotic stress involved in GSNOR. Exploring the mechanism of GSNOR in plant growth and stress response will help us to understand the physiological function of NO,aiming at providing a theoretical reference and thought for future GSNOR research.

Mechanism of Endophytic Bacteria Remediating Heavy Metal-Contaminated Soil

ZHANG Meng-meng, CAO Li-dong, WANG Ren-qing, SUN Rui-lian

2018, 34(11):  42-49.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0593

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Endophytic bacteria reside within plant hosts and have a close symbiotic association with host plants. Their excellent properties in heavy metal absorption,tolerance and detoxification provide a novel approach for the remediation of contaminated soil by heavy metals. This review concentrates on the fundamental mechanism of endophyte-assisted phytoremediation of heavy metal-contaminated soils,including the promoting the growth of host plants under heavy metals by producing plant growth regulating hormone and secreting ACC deaminases and chitinase and so on,reducing the damages of heavy metals to plants by altering the bioavailability /or toxicity of heavy metal,and improving the resistance of plants to heavy metal stress by forming joint remediating complex. Then recent progress in characterizing the diversity of endophytic bacteria from hyperaccumulator and its influencing factors are also reviewed. Furthermore,the main factors influencing the effect of endophytic bacteria in the phytoremediation are discussed,including the source and activity of endophytic bacteria and various biotic and abiotic factors of environmental stress. Special attention is paid to the future study in the field of endophyte-assisted phytoremediation technology,such as the mechanism of endophytic bacteria’s own survival and how to tolerate heavy metals,behavioral dynamics and metabolism of endophytic bacteria,and the ecological interaction between endophytic bacteria,plants and soil will be considered,aiming at promoting the large-scale application of endophytic bacteria in the phytoremediation of heavy metal-contaminated soil.

Research Progress on Zinc Finger Protein 6

XIE Wen-ya, ZHANG Chuan-hai, PAN Deng-ke, HE Xiao-yun

2018, 34(11):  50-55.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0449

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Zinc finger,BED-type containing 6（ZBED6）is a novel transcription factor that is closely related to the development of skeletal muscle and it regulates the expression of many genes including insulin-like growth factor 2（IGF2）. It is specific for placental mammals and highly conserved among species according to the available genome sequence data. The binding of ZBED6 gene with the intron 3 of key growth regulation factor IGF2 inhibits the expression of IGF2,which then regulate the cell proliferation and the growth of muscle. This article summarized the current research progress on the physiological function of ZBED6. It has been found that ZBED6 may repress the expression of IGF2 and promote the growth of skeletal muscle,heart,liver and kidney;it can reduce fat deposition,resulting in the changing of the metabolism of large molecules such as protein,lipid,etc.,and plays an important regulatory role in the development of diabetes and cancer. In addition,the article summarized the factors affecting the expression of ZBED6,including base mutations,DNA methylation,histone modifications and palindromic sequence. Finally,the article prospects the practical application potential of ZBED6,for instance,ZBED6,as a transcription factor,is closely related to the growth and development of muscle,knocking ZBED6 out may promote muscle growth,which provides new ideas for the genetic breeding of lean animals.

Circular RNAs：Research Progress and Its Significance in Birds and Livestock

XU Hai-dong, LENG Qi-ying, PATRICIA Adu-Asiamah, WANG Zhang, LI Ting, ZHANG Li

2018, 34(11):  56-69.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0403

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Circular RNAs（circRNAs）are a kind of newly discovered endogenous non-coding RNA molecule with covalently-formed closed loop structure. They are expressed abundantly in eukaryotic organisms,mainly from exons and/or introns,and have several traits,such as rich variety,high conservation,high specificity in cell and tissue,high stability,etc. The current studies show that the formation of circRNA is induced by lariat-driven circularization and intron pairing-driven circularization and regulated by varieties of cis and trans original. circRNA exerts their biological functions in numerous ways,such as combining with RNA binding proteins and then regulating the gene expression and protein translation,combining with miRNAs to play miRNA sponges function,and translating functional proteins or peptides by open reading frame in circRNAs and taking part in signal transcription among cells. The economic characters and disease control and prevention are the concerns of animal husbandry. The studies reveal that circRNAs play an important role in various physiological functions including livestock diseases control and prevention,breeding and reproducing,fat deposition,muscle and neural development,which have important research value in economic properties such as meat and dairy production and quality of livestock as well as disease control and prevention. This review summarized the characteristics,formation mechanism,main biological function of circRNA and research progresses of it in livestock and poultry species,aiming at providing certain theoretical basis and reference for the studies of livestock,providing new insights for the development of new molecular genetic markers,and providing novel techniques for enriching livestock and poultry breeding.

Research Progress on miR-155

LI Cong-cong, ZHAO Jin-yan, WU Jiao, XU Qiu-liang

2018, 34(11):  70-82.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0331

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microRNAs（miRNAs）represent a class of 19-25 nucleotides long,evolutionarily conserved,single-stranded,non-coding RNA molecules which are widely detected in eukaryotic organism. miRNAs play a post-transcriptional regulatory role by inhibiting the expression or translation of target genes. miR-155,an important member of the microRNAs family,has been increasingly studied due to its multifunctional characteristics. It is involved in many biological processes,such as the development and differentiation of hematopoietic cells,the development and differentiation of immune cells,inflammatory reaction,immune response,muscle development and adipose differentiation. miR-155 is highly expressed in many cancer tissues or cell lines,such as liver cancer,lymphoma,breast cancer,pancreatic cancer,lung cancer and so on,and closely related to tumorigenesis,tumor invasion and metastasis. With the continuous deepening of researches,miR-155 is likely to become a new tumor marker and a novel target for tumor gene therapy. miR-155 plays an irreplaceable role in various life processes and plays an indispensable role in the regulation of relevant signalling pathways. It is a typical important multifunctional miRNA. In this paper,we reviewed the research progresses on the major characteristics and relevant function of miR-155,aiming at discussing the important roles that miR-155 plays in the life activities of the organism and providing new ideas and new methods for the treatment of various diseases.

Research Progress on the Treatment of Wastewater from Poultry and Livestock Breeding Based on the Microalgae Cultivation

MA Hao-tian, LI Run-zhi, ZHANG Hong-jiang, HANG Wei, CUI Hong-li

2018, 34(11):  83-90.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0484

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At present,ecological environment pollution from large-scale poultry and livestock breeding has become increasingly serious due to the release of feces containing huge amount of nitrogen and phosphorus,heavy metals and organic pollutants;therefore,the duty of treating the wastewater is extremely urgent. Since the deficiency of traditional wastewater harnessing,the wastewater treatment based on microalgae biotechnology has become more attractive. Microalgae is a kind of unicellular organism that widely exist in water,has efficient ability of removing nitrogen and phosphorus and detergency;the major mechanism of which is as such,it adsorbs the nitrogen by anabolism,absorbs and precipitates phosphorus by phosphorylation,besides,the functional group of its cell membrane plays an important role on heavy metal enrichment. The adsorption rate of most microalgae for nitrogen,phosphorus and enrichment rate of heavy metals was 80% based on these physiological foundations. The researches about microalgae disposing the polluted compounds of breeding wastewater are focused on nitrogen,phosphorus and heavy metals,and the High Rate Algal Ponds（HARP）,active algae,immobilization technology,and the photobioreactor are usually choices for practical application. However,there are still a lot of challenges for this technology,such as the rare research on molecular mechanism and the deficiency of practical experience. Based on the mechanism of harnessing livestock wastewater by microalgae,this paper overviews the efficiency of several algae removing nitrogen,phosphorus and heavy metals,summarizes the status and existing problems in domestic and international researches of wastewater harnessing by microalgae,and prospects the future of microalgae-wastewater engineering.

A Review on Microlunatus phosphovorus in Enhanced Biological Phosphorus Removal System

LIU Cheng, JIANG Tian-yi, WANG Jing, CHEN Wen-bing, WANG Shan-shan, ZHONG Chuan-qing

2018, 34(11):  91-96.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0485

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Excessive phosphorus in water is the main reason for eutrophication. Enhanced biological phosphorus removal（EBPR）is one of the most effective approaches for removing phosphorus from wastewater,and phosphate accumulating organisms（PAOs）play important roles in EBPR. Firstly,the status and mechanism of typical PAOs in EBPR are summarized,i.e.,under anaerobic conditions,typical PAOs decompose Poly-P to synthesize PHA;and while under aerobic conditions,typical PAOs absorb a large amount of phosphorus to synthesize Poly-P by utilizing energy from the decomposition of PHA. Secondly,the role and mechanism ofMicrolunatus phosphovorus in EBPR are reviewed,M. phosphovorus as one of the PAOs occupies a large proportion in PAOs and has super effective capacity of removing phosphorus. Researches reveal that there is PHA in M. phosphovorus,but the synthetic system of PHA is different from that in typical PAOs. In addition,M. phosphovorus can directly use glucose as a carbon source,which is not available in other typical PAOs. Besides,its superior capacity of removing phosphorus is related to the effective capacity for the transport of phosphorus and the anabolic ability of Poly-P. Discussing and summarizing the function and mechanism of M. phosphovorus in EBPR system is of significance for further studying how to increase the efficiency of removing phosphorus by M. phosphovorus in wastewater treatment.

CRISPR/Cas9-mediated Targeted Knockout of Polyphenol Oxidase NtPPO1 Gene in Nicotiana tabacum

YAO Heng, YANG Da-hai, BAI Ge, XIE He

2018, 34(11):  97-102.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0560

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Polyphenol oxidases（PPOs）have many important roles in plants. In order to studying PPOs functions in Nicotiana tabacum,a PPO（Sequence ID：XM_016608009.1）was selected from the GenBank and named as NtPPO1. Then,the CRISPR/Cas9-mediated genome-editing tool was utilized to knockout the tobacco NtPPO1after NtPPO1 cDNA sequence amplified by PCR and determined by clone,and NtPPO1 relative transcript levels were analyzed by qPCR. As result,the full length of NtPPO1 cDNA was 1 746bp,and there were two kinds of alternative splicing in NtPPO1 transcripts. Additionally,1 bp or 2 bp deletion and 1 bp insertion at the target site in the NtPPO1 sequence of T₂ homozygous mutants occurred. Compared to the wild control plants,NtPPO1 relative expression in T₂ mutants by qPCR significantly reduced. Meanwhile,there were no obvious difference on the appearance between the T₂ mutants and wild ones of N. tabacum.

Construction of Genetic Linkage Maps for Armeniaca vulgaris×A. sibirica with Sequence-related Amplified Polymorphism and Simple Sequence Repeats

WANG Xiao-yi, KONG De-jing, AI Peng-fei

2018, 34(11):  103-110.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0594

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Sequence-related amplified polymorphism（SRAP）and simple sequence repeats（SSR）were used to construct draft linkage maps using F₁ population（98 individual plants）derived from a cross between Armeniaca vulgaris×A. sibirica “Longwangmao”and “Youyi’”. The molecular linkage maps of both “Longwangmao” and “Youyi” were constructed respectively using Join Map 4.0 software for the linkage analysis. Totally 132 SRAP and 17 SSR markers were obtained,The male map included 8 linkage groups with 53 SRAP and 9 SSR markers covering a total length of 694.8 centimorgans（cM）with an average of 86.58 cM;averagely there were 7.75 markers for each group and the average genetic distance between 2 markers was 11.21 cM. The female map included 8 linkage groups with 79 SRAP and 8 SSR markers and covered a total length of 924.8 cM with an average of 115.6 cM;there were average of 10.87 markers for each group and the average genetic distance between 2 markers was 10.63 cM.

Cloning,Expression and Bioinformatics Analyses of an ADC1 Gene in Hevea brasiliensis

Hou Lan-fei, Yang Hong, Deng Zhi, Dai Long-jun, Men Zhong-hua, Li De-jun

2018, 34(11):  111-119.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0498

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Arginine decarboxylase（ADC）is an important enzyme involved in polyamine biosynthesis in higher plants. In order to study the expression and bioinformatics of ADC gene from rubber tree,HbADC1 from Hevea brasiliensis was cloned with PCR and sequencing methods. The sequence length of HbADC1 was 2 594 bpwith a 2 175 bp open reading frame,encoding 724 amino acids. Bioinformatics results indicated that the deduced molecular weight and isoelectric point of HbADC1 were 77.7 kD and 5.14,respectively. Phylogenetic analyses showed that HbADC1 and ADCs from Manihot esculenta and Jatropha curcas was clustered into one. qRT-PCR analyses demonstrated that HbADC1 expressed without tissue specificity,with the highest in female flowers,followed by stem apexes,male flowers,leaves,barks,and latex. HbADC1 expression significantly varied in different developmental stages of rubber tree leaf,with the highest expression in light young stage and the lowest one in mature stage,suggesting that HbADC1 might be associated with leaf development. In addition,HbADC1 expression were regulated by wounding,low temperature,drought,high salt,hydrogen peroxide,and ethylene treatments,indicating that HbADC1 might be involved in stress and ethylene responses in rubber tree.

Genetic Cloning and Functional Verification of Hydroxycinnamoyltransferase in Ixeris sonchifolia Hance

LI Tian-zhen, ZHOU Wei, YIN Hua, ZHANG Tong-cun, LIU Tao

2018, 34(11):  120-126.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0340

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Phenolic acid compounds from Ixeris sonchifolia Hance,such as chlorogenic acid,etc.,are reported to have antioxidation and free radicals scavenging activities. Hydroxycinnamoyltransferase（HCT）is a key enzyme in the biosynthetic pathway of plant chlorogenic acid. A hydroxycinnamoyltransferase gene from I. sonchifolia Hance leaves was cloned using transcriptome sequencing,sequence analysis and RT-PCR strategies,and named as IsHCT（GenBank accession number：MH151086）. The open read frame of the gene was 1320 bp encoding a putative protein of 439 amino acids. Multiple sequence alignment and bioinformatics analysis results showed that IsHCT contained two conserved domains,and belonged to the HCT enzyme in the BAHD family. Phylogenetic tree analysis demonstrated that this gene had the highest homology with CiHCT from Cichorium intybus,and the similarity reached 95%.Finally,by overexpressing IsHCT and At4CL from Arabidopsis thaliana in Escherichia coli and feeding the substrates caffeic acid and quinic acid in the fermentation medium,and chlorogenic acid was detected in the fermentation liquid,indicating that the IsHCT catalyzed the chlorogenic acid synthesis from caffeyl coenzyme A and quinic acid,i.e.,it functioned as HCT

Sequencing and Analyzing Mitochondrial Genome of Cacopsylla coccinea

JIN Qiao, LIU Xia, GAN Zhi-Kai

2018, 34(11):  127-135.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0717

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In order to protect the medicinal plant resources such as Cacopsylla and to discover its biological properties，physiological and biochemical characteristics，we chosen Cacopsylla coccinea as the research object，conducted the determination of its mitochondrial（mt）genome，and analyzed its sequence characteristics. With the technology of PCR and gene cloning，the present study proved that the complete mitochondrial genome（MG）of C. coccineae was 14 832 bp of a full length. It encoded 37 genes，including 13 protein-encoding genes，22 transferred RNA genes，2 ribosomal RNA genes，and 1 non-coding region with high AT content. Using 3 insects of C. coccineae，Paratrioza sinica and Pachypsylla venusta as study material，we analyzed the molecular composition features of Psyllidae mitochondrial genome with comparative genomics and bioinformatics，and the results were as follows：（1）the sizes of MG from 3 insects were similar；（2）trnV gene in C. coccineae rearranged while compared with the other 2；（3）the interval in P. venusta was the longest，and the overlap area in P. sinica was the largest；（4）AT contents in 3 insects were similar，with about 70%；（5）Base mismatch in 3 insects existed，with the highest AT content of 3rd -site codon in H-strand and L-strand；and （6）the base substitution rate of nad 5，nad6 and cob genes in MG was < 1，indicating that they were under negative selection.

Cloning and Functional Identification of the 5' flanking Region of the aiiA Gene from Bacillus thuringiensis

CHEN Shao-wei, WU Cheng, SU Yue-hua, CAI Bin-bin, XIE Pan-pan, YANG Mei

2018, 34(11):  136-143.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0439

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This work is to determine the promoter region in order to study the expression regulation mechanism of N-acylhomoserine lactonase（AiiA）in Bacillus thuringiensis. The 5'-flanking region of aiiA（aP_-1930--1）was cloned by sub-cloning the flanking region while having gfp as a reporter gene,and the promoter’s activity was identified. Site-directed mutagenesis on the two continuous TATA boxes between base pairs -150 and -142 in the aiiA promoter sequence was carried out. The results showed that the flanking region sequence aP_-295--101 and aP_-295--1 functioned as promoters to allow the heterologous expression in Escherichia coli. The promoter sequence aP_-295--101 was more active than aP_-295--1,but the aP_-101--1 sequence failed to initiate GFP expression. Two TATA box mutations in the site-directed mutant pET28a-aP_-295--101-gfp vector significantly reduced GFP expression. It is suggested that -295--1bp is the promoter region ofaiiA,-429--296 and -100--1are the negative regulatory sequences of aiiA. Two TATA boxes in the -150--142 bp region play a key role in the promoter activity of the 5' flanking region of aiiA.

Cloning,Expression and Conversion Application of Glucose Isomerase from Alicyclobacillus sp.A4

DAI Chen-xia, CAO Hui-fang, LUO Hui-ying, YAO Bin, BAI Ying-guo, XU Bo

2018, 34(11):  144-151.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0408

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Glucose isomerase is a key enzyme for the commercial preparation of high fructose corn syrup by isomerizing D-glucose to D-fructose in vitro. The glucose isomerase（A4GI）gene from the thermophilic Alicyclobacillus sp. A4 strain was cloned and successfully expressed in Escherichia coli BL21. His-tag protein-purified magnetic beads were used to purify the crude enzyme solution,and the detailed enzymatic properties and conversion rate of the purified recombinant A4GI were determined. The results showed that the optimal temperature and pH of A4GI were 65℃ and pH 7.5,respectively. The enzyme was stable over a broad pH range of 6.0-11.0. After incubation at 37℃ for 1 h,the enzyme retained over 99% of its initial activity at pH 6.0-11.0. Under optimal reaction conditions,the K_m and V_max values of the recombinant enzyme for D-glucose were 99.8 mmol/L and 3.75 µmol/min/mg. In the experiment of glucose conversion at different concentrations,the conversion rate of A4GI tended to increase along with the increased concentration of D-glucose,and the conversion rate reached the peak of 52.7% at the substrate concentration of 3 mol/L. Thus,high-efficiency conversion under high concentration of glucose by A4GI may be achieved,resulting in the reduced cost of post-concentration,and there is great potential for it to be applied in industry.

Cloning and Expression Vector Construction of BLM Helicase Gene,and Its Expression Analysis

ZHAO Jia-fu, XU Hou-qiang, SONG Shu-xian, DUAN Zhi-qiang, CHEN Xiang

2018, 34(11):  152-159.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0461

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This work aims to clone the full-length CDS region of BLM gene and to study the expression of BLM in cells. The total RNA extracted from the PC3 cells of human prostatic carcinoma was transcribed into cDNA. Then the full-length CDS region of BLM obtained by subsection cloning was inserted into prokaryotic expression vector pET-32a and eukaryotic expression vector pEGFP-N3,and the recombinant expression vectors pET-32a-BLM and pEGFP-N3-BLM were constructed. The recombinant plasmids were transformed into competent cells BL21（DE3）or transfected into PC3 cells. Further the expressions and localizations of BLM helicase in the above two cells were studied by SDS-PAGE,Western blotting,and fluorescence localization analysis. The prokaryotic expression of BLM helicase showed that different IPTG concentration affected little BLM expression;low temperature was conducive to the increase of BLM expression level. It was further confirmed by Western blotting that the constructed recombinant prokaryotic and eukaryotic vectors of BLM helicase respectively expressed in BL21（DE3）and PC3 cells as 179 kD protein,which was consistent with the expected protein. The results of fluorescence localization indicated that human BLM helicase mainly was in the nucleus. In sum,the full-length CDS region of human BLM helicase is cloned successfully,and the constructed recombinant expression vector pET-32a-blm and pEGFP-N3-blm can be correctly expressed in corresponding host cells.

Combined Hepatotoxicity Assessment of Mycotoxins AFB1 and Zearalenone on Hepatocellular Carcinoma Cells HepG2 in vitro and Its Mechanisms

REN Ya-lin, LI Yun, WU Jing

2018, 34(11):  160-167.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0588

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Based on key apoptotic genes,early apoptosis signal indexes and apoptosis,this study used hepatocellular carcinoma Cells HepG2 cells to estimate combined hepatotoxic effects of aflatoxin B1（AFB1）and zearalenone（ZEN）and to explore its feasible mechanisms. The results showed that the combined effects of 1-10 μg/mL mycotoxins AFB1 and ZEN on the cells was synergetic,and the effect presented as weak antagonistic to toxic and further to strong synergetic with the increase of concentration. The early apoptosis indicator ROS rose with the exposure time prolonging;while GSH and MMP gradually was decreasing,and the change was the most significantly at 0 - 3 h. The ratio of Bax/Caspase-3 expression increased with the prolonging of the time;while the expressions of CYP1A2,CYP1A4 and CYP1A6 tended to be down,which was more obvious with the increase of the time. AFB1 and ZEN induced the combined hepatotoxicity via the interaction and regulation of gene Bax,Caspases-3,and CY450.

Transcriptome Sequencing and Analysis of Hepatopancreas from Carps Under Cold Stress

LIU Si-jia, TIAN Fei, ZHANG Cun-fang, QIAO Zhi-gang, ZHAO Kai

2018, 34(11):  168-178.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0503

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This work aims to detect the differentially-expressed genes（DEGs）of cold-tolerant and cold-sensitive carps and to understand the molecular response mechanisms for carps to adapt in cold environment. The transcriptome sequencing of hepatopancreas from cold-tolerant Songpu carp and a cold-sensitive Hebao carp were conducted and the enrichment analysis of DEG function and pathways were carried out. Totally,10 521 DEGs were detected via differentially-expressed analysis,and 5246 DEGs of them presented expression in cold-tolerant species. DEGS were enriched in 144 metabolic pathways including glycolysis/gluconeogenesis pathway. Five DEGs were selected to conduct qRT-PCR,and the correlation of the results between qRT-PCR and RNA-seq was 0.89.This study revealed that cold stress significantly changed the transcriptome of hepatopancreas in the experimental carp. The expressions of genes encoding the key enzymes of glycolysis/gluconeogenesis pathway were apparently higher than that in cold-sensitive ones,revealing that the functions of these genes facilitate the long-term cold-adaptation of the cold-tolerant carps.

Mechanism of Antimicrobial Peptide Scolopin 2-NH2 Isolated from Scolopendra subspinipes mutilans

LU Jia, DENG Qiu-ping, REN Wen-hua

2018, 34(11):  179-190.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0476

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This work aims to elucidate the secondary structure,antibacterial activity and physicochemical properties of antibacterial peptide Scolopin-2（S-2）and S-2-amidated Scolopin-2-NH2（S-2-N）,and to reveal their antibacterial mechanism at the cell and molecular level. The method of minimum inhibitory concentration was used to detect the antimicrobial activity of S-2/S-2-N against 4 strains,different treatments were applied to measure their physicochemical properties,and circular dichroism spectroscopy was to determine the secondary structure S-2/S-2-N. After Escherichia coli was treated with S-2-N and S-2 respectively,flow cytometry was used to detect the efficiency of peptides penetrating cell membrane and cell membrane integrity. Flow cytometry and RT-PCR technology were together to analyze the effects of peptides on cell cycle and the genes related to intracellular DNA replication and repair. In addition to the heat resistance,acid and alkaline resistance,the resistance to ionic strength and the ability of digestive enzymes,the S-2-N also had stronger antibacterial activity than the maternal peptide S-2. The results of flow cytometry showed that in a short period of time（30 min）both peptides caused cell membrane damaged,while the efficiency of S-2-N penetrating membrane was higher. The analysis of flow cytometry and RT-PCR demonstrated that the bacterial cell cycle was blocked by S-2-N and S-2,which lowered the expressions of gene dnaA,dnaB,dnaG and SSB,but promoted the expressions of RecA and RecN. In conclusion,the antibacterial peptide S-2-N amidated from S-2 has stronger antibacterial activity than maternal peptide,and both function by the same antibacterial mechanism,i.e.,destroying the bacterial cell membrane,combining bacterial DNA and RNA,affecting the secondary structure of DNA,blocking the cell cycle of bacteria,and affecting the expressions of genes associated with DNA replication and repair in cells.

Molecular Mechanism of Nucleoid-Associated Protein Lsr2 Repressing the Expression of Gene rv1057 in Mycobacterium tuberculosis

FU Jia-fang, ZHANG Pei-pei, ZONG Gong-li, WANG Xin-yuan, LIU Meng, CAO Guang-xiang

2018, 34(11):  191-197.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0566

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The objective of this work is to explore whether or not nucleoid-associated protein Lsr2 of Mycobacterium tuberculosis is a repressor protein in gene rv1057 expression. Electrophoretic mobility shift assay was used to analyze the in vivo specific binding capability of Lsr2 with the target gene promoter region.lacZ report gene and fluorescence quantitative PCR were adapted to detect the effects of different mutations of rv1057 promoter region on the transcription. Western blot was employed to analyze the protein expression level of target gene rv1057. The t test was used for statistical analysis. As results，Lsr2 was able to bind to rv1057 promoter. Both Lsr2 and the known repressor protein TrcR were able to bind to rv1057 promoter. The mutated rv1057 promoter lacking Lsr2 binding site was able to continuously activate the transcription of gene rv1057 during the growth cycle of M. tuberculosis. The relative expression of gene lsr2 was low in the early growth stage（24 h）of wild-type strain H37Rv，while the relative expression of gene lsr2 in H37Rv significantly increased both in the middle growth stage（48 h）and in the later growth stage（120 h），the difference was statistically significant（t=24.44，16.86，respectively；P<0.05）. The relative expression of the gene trcR of H37Rv and gene lsr2-deleted strain in the early growth stage（24 h）was significantly higher than in the middle growth period（48 h）and the later growth period（120 h），the difference was statistically significant（t=6.79，10.16，respectively；P<0.05）. In conclusion，Lsr2 is the repressor protein of gene rv1057 transcription. Both Lsr2 and TrcR may regulate the expression of rv1057. Lsr2 is mainly expressed in the middle and late growth period of the M. tuberculosis，and then represses the expression of rv1057；while TrcR represses the transcription of rv1057 in the early growth stage of the M. tuberculosis.

Optimization of Ultrasonic-assisted Extraction of Cordyceps militaris Proteins by Response Surface and Its Nutritional Assessment

HOU Ruo-lin, LIU Xin, XIANG Kai-kai, CHEN Lei, ZHENG Ming-feng, FU Jun-sheng

2018, 34(11):  198-204.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0516

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The method of ultrasonic-assisted extraction of proteins from Cordyceps militaris were optimized by response surface methodology,and the amino acid composition and nutritional value of the extracted C. militaris proteins were analyzed. The optimal conditions for the ultrasonic-assisted extraction of proteins were as follows：solid-liquid ratio of 1∶50,pH of 8.57,and ultrasonic at 150 W for 30 min,then the ultrasonic wave was turned off,the mixture was further stirred at 35℃ and extraction was conducted for 72 min. Under these conditions,the extraction rate of proteins reached 85.6%,which was 1.68 times of the non-ultrasound control group,and the relative error with the model prediction value was ±0.77%. Under the optimal conditions,the ratio of essential amino acids to total amino acids of C. militaris proteins was 44.4%,and the ratio of essential amino acids to non-essential amino acids was 79.84%,which was higher than the recommended values by the World Food and Agriculture Organization（FAO）and the World Health Organization（WHO）. It indicates that the extracted C. militaris proteins had high nutritional value,providing a reference for the efficient extraction of C. militaris proteins and the development of health food from C. militaris.

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDC_OV

KONG Wei-huan, Wang Jing-jing, WAN Peng-cheng, SHI Guo-qing

2018, 34(11):  205-209.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0365

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The aim of this study is to establish a double RT-PCR assay for simultaneously detecting porcine epidemic diarrhea virus（PEDV）and porcine delta corona virus（PDC_OV）. Two pairs of specific primers were designed according to the sequences of PEDV and PDC_OV genes published in GenBank. Firstly,the RT-PCR reaction technique was used to optimize the reaction conditions through the single amplification of the target genes of PEDV and PDC_OV viruses. Then,the establishment of double RT-PCR method was confirmed by specificity test,sensitivity test and clinical sample detection. Results showed as：The size of the amplified target gene PEDV-M was 750 bp,and 372 bp for PDC_OV-N fragment;PEDV and PDC_OV genes were detected simultaneously,but 3 viruses of PRV,CSFV and PPV were not detected. The minimum concentration limit of mixed nucleic acid concentration for optimizing and amplifying PEDV-PDC_OV was 100 pg/μL,thus it can be used to identify the clinical suspected virus infection. The results showed that a double RT-PCR detection method for PEDV-PDC_OV virus is established successfully,this method is highly sensitive and specific and a rapid and effective method for the clinical detection of PEDV-PDC_OV single virus infection or multi-virus mixed infection in pigs.

Screening and Identification of a Lovastatin-producing Strain of Aspergillus versicolor and Preliminary Optimization of Fermentation Conditions

XU Chu-xuan, WANG Jia-qi, JIANG Dong-hua

2018, 34(11):  210-215.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0453

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Total 86 Aspergillus strains were selected from different natural fermentation samples of food,soil,air and so on. Lovastatin yields of Aspergillus strains were measured by thin-layer chromatography（TLC）and high performance liquid chromatography（HPLC）methods. An Aspergillus strain（No. A-8）producing higher lovastatin was screened out,and the yield was of 58 g/L. Strain A-8 was identified as Aspergillus versicolor based on ITS sequence and morphology characteristics. Medium components and fermentation conditions of Aspergillus strain A-8 were optimized using single factor experiment. The results indicated that appropriate fermentation conditions were 28℃,initial pH 5.2,rotation speed 180 r/min,and inoculation size 10%. Optimal medium components were as follows：C source lactose,C content 100 g/L,N source peptone,N content 12 g/L,C/N 15：1.5. The lovastatin production of Aspergillus strain A-8 increased by 2.24 times and resulted in 130.04 μg/mL. The Aspergillus strain A-8 is one potential industrial strain.

Establishment of HPLC-Peptide Mapping Method for the Characterization of Anti-CD52 Monoclonal Antibody

QIAO Yu-ling, HUANG Zheng, QIN Hai-yan, SONG Lan-lan, CHEN Ji-jun, AN Chen, YE Xing, MAO Xiao-yan

2018, 34(11):  216-222.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0424

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This work is to establish a HPLC-peptide mapping method for specific identification of anti-human CD52 monoclonal antibody（CD52 mAb）. After denatured by guanidine hydrochloride and reduced by DTT,the free cysteine sulfhydryl of CD52 mAb was alkylated. Then digestion buffer was replaced by ultrafiltration,trypsase was digested,and the digestion reaction was terminated. HPLC conditions were as：An Agilent HPLC column（Eclipse XDB-C18 4.6×250 mm 5μm）adopted using water solution（containing 0.1% trifluoroacetic acid）as the mobile phase A and acetonitrile solution（containing 0.1% trifluoroacetic acid）as the mobile phase B;gradient elution of mobile phase A and B;detection wavelength 214 nm,30℃. The conditions for mass spectrometry were as：Data were obtained with positive ionization for 135 min;TOF MS+ scan range in 350-1 500 Da,Product Ion+ scan range in 100-1 500 Da,mass spectrometer resolution 40 000,and Exceeds in 150 Cps. The peptide fragments corresponding to heavy chain CDR1,CDR3 of CD52,and light chain CDR1 of CD52 were identified by mass spectrometry. The specific verification by HPLC-peptide mapping showed that the detection result was not affected by excipients and heterologous antibody. The results from precision verification demonstrated that the RSD% of the target peak area was in 1.7%-3.8% and the RSD% of relative retention time of the target peak was 0.1%-0.2%,which were less than 5% of acceptable standards. The results of durability indicated that the conditions of 3 μg trypsase,37℃ and 18 h were the optimal. In a sum,by HPLC-peptide mapping CD52 mAb may be qualitatively identified based on the CDR related peptides. The validation results of methodology reveal that the established method is appropriate to specific identification of anti-human CD52 mAb and can be used for quality control and batch release test.