Biotechnology Bulletin

Research Advance on Chlorophyll Degradation in Plants

DING Yue, WU Gang, GUO Chang-kui

2016, 32(11): 1-9. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.001

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Chlorophyll degradation is closely related to crop production. Delayed chlorophyll degradation in crops increases photosynthetic capacity at late stages,leading to the increase of crop production. With the development of structural biology,genome sequencing and bioinformatics in recent years,the regulatory mechanism of plant chlorophyll degradation pathway has witnessed some great progresses;especially PAO（Pheide a oxygenase）pathway,the primary chlorophyll degradation pathway,has been largely elucidated. In this review,we summarized the advances on 3 aspects of chlorophyll degradation pathways,the regulatory mechanism and stay-green mutants,and we also discussed some future research directions,aim to supply idea for the crop breeding and efficient use of light in crop.

Advances on Cysteine-rich Receptor-like Kinases in Plants

ZHENG Chao, LI Deng-gao, BAI Wei

2016, 32(11): 10-17. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.002

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Receptor-like kinases（RLKs）are protein kinases existing in plants,and play an essential role in many plant signal transduction pathways. As receptors localized on plasma membrane,RLKs perceive environmental stimuli and are involved in intercellular signal transductions through phosphorylation. Cysteine-rich receptor-like kinases（CRKs）,also called domain of unknown function 26（DUF26）receptor-like kinases,are a large subgroup of RLKs. Recently,CRKs are found to be involved in plant disease resistant defenses. In this paper,we reviewed the structural characters of CRKs and summarized biological functions in abiotic and biotic stresses and their roles in plant growth and development,moreover,we prospect the future research,aim to follow-up stuclies.

The Application of the Functional Molecular Marker in Wheat Breeding

LIU Guo-sheng, ZHANG Da-le

2016, 32(11): 18-29. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.003

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Based on the polymorphic sequence of a particular area in a functional gene closely related to phenotype, functional marker is a novel dominant molecular marker developed by correlation analysis, spectrum analysis, RNA interference and QTL mapping method, etc. Using these functional markers, whether or not the target allele from different genetic backgrounds exist can be directly and rapidly determined. In this work, the concept and characteristics of functional markers are briefly introduced, mainly exploring the application and prospect of functional markers as an assistant means in wheat breeding, in order to provide reference for the development of related molecular markers

Research Progress on Population Genomics of Marine Fishes

LIU Ying, GAO Li, FENG Jun-rong

2016, 32(11): 30-37. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.004

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Over the recent years,advances in high-throughput sequencing and big data analysis have facilitated genome-wide research in many species. Using the methods of genome sequencing,single nucleotide polymorphism（SNPs）analysis and population transcriptomics,the evolutionary progress and regulatory mechanisms of adaptive genetic variation were understood in more details,which poses great significance for theoretical research and productive practice. Furthermore,research on population genomics will deepen our understanding of important events in microevolution such as population divergence,hybridization and speciation. Therefore,the related research would be conducive for us to better understand the evolution of marine organisms,and provide theoretical evidences for development of aquaculture and management of marine fishery resources.

Research Progress on Polysaccharide-degrading Enzymes from Marine Microorganism

WEN Xia, ZHOU Shao-lu, YANG Xiu-jiang, SUN Ting-li, XIE Xiao-bao

2016, 32(11): 38-46. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.005

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Polysaccharide-degrading enzymes are a class of hydrolase that can catalyze the cleavage of glycosidic bond in polysaccharide molecules,consequently reduce the degree of polymerization,and eventually produce oligosaccharides. There are huge number of microbes on the Earth,thus the variety of polysaccharide-degrading enzymes is broad. Particularly polysaccharide-degrading enzymes from marine microorganism,due to their specific catalytic activities,presented an important industry application prospect. With the rapid development of marine biotechnology,the development and utilization of polysaccharide-degrading enzymes from marine microorganism have gradually attracted researchers' attentions. In this paper,the main types and the status of current research on polysaccharide-degrading enzymes from marine microorganism are reviewed,and the trends of application and development are summarized,aiming at providing references for the research and development of polysaccharide-degrading enzymes from marine microorganism.

Advances on Regulation of Plant Disease Resistance by N-acyl-homoserine Lactones of a Bacterial Signal Molecule

JIN Xiao-yang, HOU Peng-fei, ZHANG Xiao-han, ZHAO Qian, SONG Shui-shan

2016, 32(11): 47-51. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.006

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N-acyl-homoserine lactones（AHLs）play an important role in the biological communication,provide chemical bases for the intraspecific and interspecific interactions of gram-negative bacteria,and regulate varied bacterial physiological processes. Besides bacteria,plant may perceive and response to AHLs. This article summarizes researches of AHLs regulating plant resistances in recent years,which is conducive to explore mechanisms how AHLs influence host plants,and will provide theoretical basis for its application in agriculture.

Research Progress on p53-involved Metabolic Regulation

CHEN Ke, DING Yan-ping, WANG Jian-lin, SHAO Bao-ping

2016, 32(11): 52-58. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.007

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As a tumor suppressor,p53 not only is involved in the stress regulation of genetic toxicity,but also plays an important role in the regulation of metabolic balance. When the cells are in different physiological stresses,the activated p53 will affect various metabolic pathways by participating in the regulations of glucose metabolism,fatty acid metabolism,and ROS level. Consequently,the metabolic stress was generated by firstly inducing the occurrence of cell cycle arrest,repair,senescence or apoptosis and finally regulating the organisms and cells. This review summarized the current advances of p53 pathxxray, and discussed the role that p53 played in cancer and metabolic syndrome, which might benefit the study of p53 involved in metabolic regulation.

Immobilization of Lipase and Its Application in Food Industry

WEI Xue, SUN Li-chao, LI Shu-ying, WANG Feng-zhong, XIN Feng-jiao

2016, 32(11): 59-64. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.008

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Lipase（EC 3.1.1.3）is a kind of enzyme that can hydrolyze long chain fatty acid esters into fatty acid and monoglyceride,diglyceride or glycerol,and it is mainly from animals,plants and microorganisms. Considering the great application potential of lipase in the food industry,optimization of lipase performance is of significance for achieving continuous and low-cost production. Immobilized enzyme technology significantly enhanced the lipase performance by increasing the enzymatic activity,stability and the recovery rate. Therefore,immobilized lipase is widely used in the food industry,including the enzymatic synthesis of glycolipids,oil modification,and the synthesis of aromatic ester compounds and ascorbyl esters. This article introduces the lipase and the immobilized enzyme technology,and mainly reviews the application of immobilized lipase in the food industry in last seven years,finally prospects how to improve the performance of lipase.

Research Progress on Gene Isolation and Clone Technology of Tropical Fruit Tree

AN Na, WU You-gen, CHEN Ping

2016, 32(11): 65-71. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.009

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Firstly, we summarized the isolations of the genes from several common tropical fruits' genes（banana, papaya, mango, pineapple, litchi, etc.）by frequently used technologies of rapid-amplification of cDNA ends（RACE）and reverse transcription-polymerase chain reaction（RT-PCR）, also the analyses and expressions of cloned target genes and fragments. Finally, we discussed the advantages and disadvantages of gene isolating and cloning technologies, and prospected the new gene isolating and cloning technologies, aiming at laying the foundation for studying tropical fruit tree and agriculture at biological level of genetic molecule.

Application Progress on Amplified Methods to Clone Flanking Sequence

PU Yuan-bo, GUO Cui, PING Shu-zhen

2016, 32(11): 72-79. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.010

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Amplifying the flanking sequence of genes plays an important role in molecular biology research. Until so far,the methods used to clone flanking sequence can been categorized into three groups：I-PCR,LA-PCR and TAIL-PCR. This paper briefly introduces the principle and present applications of these methods,which provides basis for researchers to choose more reliable and reasonable method.

Current Situation Analysis and Detection Techniques of Pathogenic Escherichia coli

WEI Yu-jun, WANG Zi-ting, XU Yuan-cong, XU Wen-tao

2016, 32(11): 80-92. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.011

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Pathogenic Escherichia coli is one of the most common pathogenic microorganisms. This kind of pathogen spreads through a variety of ways and its infection has caused many food safety accidents in the world. Though the emergence of its antibacterial resistance has raised concern,it also facilitates institutions and researchers to design new bacteriostatic methods. As direct pathogenic factor,E. coli risk virulence factor has drawn much attention by researchers. Also the technology of detecting E. colideveloped rapidly too. Compared to traditional methods,advanced detection techniques for pathogens are fast,specific and accurate in determination of the pathogen. Based on the research status in domestic and abroad,this article introduces the research progress on assessments of E. coli virulence and techniques of detecting E. coli from several aspects such as the situation of E. coli's contamination,infection route,symptoms,antibacterial resistance and its risk virulence factors,as well as detection techniques.

Methods of Measuring Telomere Length and Its Application

ZHANG Bin, WANG Chang-li

2016, 32(11): 93-98. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.012

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Aging and tumor are the two main factors influencing human longevity. Many studies have proved that telomere play important roles in these progresses. Thus, it is critical to familiarize with assay methods of telomere length. The most commonly used methods include telomere restriction fragment（TRF）length analysis, quantitative PCR（qPCR）, fluorescent in situ hybridization（FISH）and single telomere length analysis（STELA）. However, each method has the scope of application, in the process of research and application, we have to understand the principle of each method, its advantages and disadvantages, then we may choose the appropriate detection method to achieve the aim of our study and solve specific issues. The commonly used detection methods of telomere length are summarized here.

Screening of Reference Genes in Bacillus pumilus by Real-time Fluorescence Quantitative PCR

HE Ting-ting, SONG Ting, WANG Chao, ZHANG Chang-bin, WANG Hai-yan

2016, 32(11): 99-106. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.028

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In order to study the gene expression in Bacillus pumilus through real-time fluorescence quantitative PCR,we need to determine the appropriate reference gene firstly. The expressions of five candidate reference genes（16S rRNA,mecA,cadR,rpoB,andsphP）under different fermentation phases of B. pumilus were analyzed by real-time fluorescence quantitative PCR,and their expression stabilities were evaluated by geNorm and NormFinder software. According to the analysis by geNorm software,16S rRNA and mecA were the most stable genes and the number of optimal reference genes were 2. Analysis by NormFinder software revealed that mecA was the most stable gene. The differential expressions of 3 functional genes of B. pumilus indicated that 16S rRNA and mecA were appropriate reference genes,moreover,expression abundance of mecA was moderate,thus it was more suitable to be used as a reference gene in studying structural gene. Additionally,2 reference genes can also be used for calibration in order to acquire more accurate results.

New Probe and Primer for Molecular Beacon-TaqMan Real Time Fluorescent Quantitative PCR

JIANG Wen-can, YUE Su-wen, JIANG Hong, GE Su-jun, WANG Cheng-bin

2016, 32(11): 107-114. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.029

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Our purpose is to eliminate the false positive problem caused by primer-probe aggregation and extension,as well as the false negative problem caused by primer dimer（PD）. First,the primer and probe aggregation results were tested for different probes;then the appropriate internal control gene（IC）was selected based on the competitive interference experiments of duplex PCR,simultaneously,the practicability of central-home primer excluding the inference of PD was verified on the plasmid of the IC;and finally,the sensitivities of different systems for TaqMan method were compared. The probe HBVP4,containing an antisense base,did not produce false positive results in the repeated trials;the concentration difference at which interferences for the templates of competitive and non-competitive duplex PCR were noticed was 20 times and 100 times,respectively. The dilution that could detected by central-home primer and common primer was 10^-9 and 10^-8,respectively. And for 3 HBV gene detection systems,the detection could be until Ct33 if using common primer,and Ct35 if using central-home primer and by the accession of IC. Based on adjustments of TaqMan-Molecular Beacon and introducing antisense,the false positive problem caused by primer-probe aggregation and extension was excluded. In probe method,the use of central-home primer and IC could both reduced the influences caused by PD,and therefore increase the sensitivity of detection.

Construction of Acid-tolerant and Flocculating Saccharomyces cerevisiae Strain for Ethanol Production by Protoplast Fusion

GOU Min, YANG Bai-xue, TANG Yue-qin, Kida Kenji

2016, 32(11): 115-123. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.013

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In order to obtain an excellent strain with multi traits for ethanol production,using flocculating strain RHZ-1 and acid-tolerant strain SEB2 as the parents,auxotrophic strains were obtained by UV mutagenesis induction. Saccharomyces cerevisiae strain RS-1 with excellent acid-tolerant and flocculating ability was selected by screening fusants with the technology of protoplast fusion. When fermenting 15% YPD at 30℃ and pH2.2 for 24 h,the ethanol yield was 47.87 g/L,the glucose consumption rate and ethanol yield of strain RS-1 was greatly improved compared to the original strain RHZ-1,increased 19.5% and 9.8%,respectively. When fermenting 15% YPD at 35℃ and pH2.2 for 48 h,the ethanol yield was 38 g/L,the glucose consumption rate and ethanol yield of strain RS-1 increased 46.8% and 21.6%,respectively compared to the original strain RHZ-1. The results suggest that the strain obtained in this study could be a potential microorganism for ethanol industry.

Effects of the Latex Total RNA Precipitated with Different Methods on Amplification Efficiency

WU Chun-tai, LI Yu, FENG Wei-yu, ZENG Ri-zhong

2016, 32(11): 124-129. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.014

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This work is to find out the effects of different precipitation methods on the latex total RNA and amplification efficiency of PCR as well as to seek the better precipitation method. According to the characteristics that Herva brasiliensis latex was rich in proteins,polysaccharides and rubber particles,on the basis of SDS method modified in our laboratory,the total RNA in the latex of H. brasiliensis was precipitated respectively with LiCl,NaCl + isopropanol,NaCl + NaAc + absolute ethanol,and NaAc + absolute ethanol,and then the integrity of the RNA,purity,content,and RT-PCR amplification efficiency were compared. The results indicated that complete RNA was obtained by any of 4 methods. But the differences in purity and content of RNA and amplification efficiency of RT-PCR among 4 methods were also observed. In which the purity and yield of total RNA precipitated with NaCl + NaAc + absolute ethanol and 18S and REF gene amplification efficiency of latex tissues in rubber tree were higher,indicating that NaCl + NaAc + absolute ethanol was effective method to precipitate total RNA.

Establishment of Extraction Methods of Membrane Proteins from Extremely Thermoacidophilic Acidianus manzaensis and Its Preliminary Application

YANG Yun, LIU Hong-chang, XIA Jin-lan, MA Ya-long, NIE Zhen-yuan

2016, 32(11): 130-136. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.015

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Using Acidianus manzaensis as research object,the methods of extracting the membrane proteins were explored and optimized,then the optimized methods were used to exact the membrane proteins of the bacterium cultured on elemental sulfur（S⁰）or ferrous iron（Fe²⁺）as energy substrates,and the differentiated expressions of the extracted membrane proteins from 2 substrates were analyzed by SDS-polyacrylamide gel electrophoresis（SDS-PAGE）. The uncorrelated extracellular proteins were firstly removed by incubating the cells in 80℃ for 60-70 min. Then the extraction effects of different extraction reagents（Triton X-110,SDS and Triton X-114）on the membrane proteins were comparatively studied,and the results indicated that Triton X-114 was better than others and the optimal concentration of Triton X-114 was 10%（w/v）. Similarly,the precipitation effects of different protein precipitation reagents（trichloroacetic acid（TCA）,acetone,TCA/acetone,methyl alcohol and ethyl alcohol）on the proteins were comparatively studied,and the results indicated that the precipitation by TCA/acetone was the worst,as TCA/acetone caused the loss of low-molecular weight proteins;while there were no significant differences by others. Compared comprehensively,the most commonly used acetone was chosen as the precipitation reagent in the extraction of membrane. Finally,the membrane proteins of A. manzaensis cultured on S⁰ and Fe²⁺ were extracted via the optimized traction method and comparatively analyzed by SDS-PAGE. The results showed that the protein bands with 35.6 kD and 16.8 kD only appeared in A. manzaensis cultured on S⁰,which was probably related to sulfur oxidation of this strain. The protein bands with 72 kD and 26 kD were significantly up-regulated in A. manzaensis cultured on Fe²⁺comparedwith on S⁰,indicating that the protein may be related to iron oxidation of this strain.

Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics

YAO Xue, LIU Wen-li, PEI Guang-qian, TONG Yi-gang, LUO Ya-ping

2016, 32(11): 137-143. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.016

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Based on the experimental design and data analysis, we screened a high-throughput sequencing method which may measure the community of microorganisms in a low level on the human skin as real as possible. By completely random design of the experiments with multi-factor multi-level, we analyzed the effects of varied factors in the DNA extraction, PCR amplification, sample collection and sequencing on the composition characteristics of microbial community. The results showed that regarding the sample collection, the optimal collection swab and extraction liquid were screened out. In the aspect of DNA, the optimal pretreatment method and reagent box type were selected; on PCR reaction, the optimal template concentration and annealing temperature were determined; and on the sequencing aspect, the suitable sequencing depth was also chosen. In conclusion, through above series of experiments to screen the conditions, the appropriate method for analyzing the composition characteristics of community structure of bacteria in a low level was determined, and successfully applied to human palm skin.

Differential Expression Analysis of Gene Expression Profiles During Fruit Development and Ripening of Lycium ruthenicum

PENG Yong, CHEN Shang-wu, MA Hui-qin

2016, 32(11): 144-151. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.017

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The purpose of this study was to sequence the transcriptome at 3 different developmental stages of Lycium ruthenicum Murr. fruit,as well as analyze and compare the variations of their gene expression profiles. The RNAs of the fruits were extracted,and sequenced by Illumina high-throughput,then the their functions were annotated and classified using public database such as GO,KEGG,etc.,further their genes were compared and analyzed by digital gene expression profiles. Enrichment analysis results by KEGG pathway showed that in the “leucine biosynthesis pathway”,there were 7 genes differentially expressed in color changing vs early light green skin stage,while 35 differentially expressed genes in full ripening stage vs color changing stage,and all were up-regulated. The gene expressions of 3 rate-limiting enzymes in the non-reversible reactions of glycolysis all were up-regulated in the full ripening stage. Moreover,the functions and metabolic pathways of all genes involved in leucine biosynthesis and glycolysis of L. ruthenicum fruit were analyzed,aiming at providing the theoretical basis for exploring the germplasm of L. ruthenicum.

Development of Transgenic Maize Plants Tolerant to Glyphosate via Pollen-mediated Transformation and Their Glyphosate Tolerance

YANG Hui-zhen, REN Zhi-qiang, XIAO Jian-hong, BU Hua-hu, LIU Hui-min

2016, 32(11): 152-161. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.018

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In order to develop transgenic maize plants tolerant to glyphosate and further study their herbicide tolerance and the genetic characteristics of exogenous gene in transgenic plants,plasmid pBI101-aroA-M12 harboring aroA-M12 gene was transformed into maize inbred Chang ‘7-2' plants by pollen-mediated transformation method. Fifty-four transgenic plants were obtained by PCR detection and Southern blot analysis. The detection results of molecular analysis and bioassay in the field showed that target aroA-M12gene was integrated into the genome of transgenic plants and could stably inherit to next generation. Moreover,the aroA-M12 gene conferred the glyphosate-tolerance to transgenic plants. The result of genetic rule of target gene in transgenic plants suggested that the target gene was segregated according to the ratio of 3∶1 in T₂ generation transgenic plants（lines）and it was consistent with the Mendel law of single-factor heredity. The results of ELISA analysis and protein concentration determination of transgenic plants confirmed that the target gene was expressed in transgenic plants,and the protein amount of gene expression were 40.5-112.6 ng/g leaf fresh weight,there was a significant correlation between protein amount of expression of each transgenic plant and corresponding glyphosate-tolerance of that plant,correlation coefficients r = 0.942 3（P<0.01）. Ultimately,4 pure lines of transgene with high glyphosate-tolerance were obtained from this experiment.

Screening and Identification of New Genetic Factors Interacting with GL2 During Plant Trichome Development

SHI Shuang-yue, CHEN Zi-yu, AN Li-jun

2016, 32(11): 162-169. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.019

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Trichomes widely exist on aboveground parts of terrestrial plants,exhibiting as natural barriers between plants and the environment,and also servering many biological functions. The transcription factor GLABRA 2（GL2）is a key factor regulating trichome initiation and the following developmental processes. Screening and identification of new GL2 interactors will lay a foundation for further uncovering the regulatory molecular mechanisms of trichome development. Through large-scale genetic screening and cloning,a new mutant M12-01 without trichome in the leaves was found,and genetic analysis indicated that the phenotype of M12-01single mutant was controlled by a recessive single nuclear gene. The phenotype of M12-01 single mutant was similar to that of functional deletion mutant of Arabidopsis TTG1 gene. Sequencing TTG1 gene revealed that a single nucleotide replacement from guanine to adenine in +445 of TTG1 gene occurred,which resulted in the encoded glycine changed to arginine. This work confirmed that mutation of TTG1 enhanced the phenotype of gl2-3 mutant,and genetic interaction between gene GL2 and TTG1,which provides new genetic materials for further studying the molecular mechanism of GL2 regulation on the development of plant trichome.

Cloning,Expression Analysis and Vector Construction of MYB21 Gene in Kenaf

QIAN Jing-hua, ZHOU Bu-jin, KONG Xiang-jun, LI Zeng-qiang, SHI Qi-qi, LIAO Xiao-fang, ZHOU Rui-yang, CHEN Peng

2016, 32(11): 170-179. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.020

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This work is to clone MYB21 gene and analyze its expression levels in different organs of kenaf for both cytoplasmic male sterility（CMS）and its maintainer,to construct over-expression vector and RNAi vector,and to lay the foundation for further study on the function of MYB21 gene in kenaf. The MYB21 gene was cloned using a homology method. The expression patterns of MYB21 in different organs for CMS and its maintainer were analyzed by quantitative real-time PCR. Using enzyme digestion-ligation method,the expression vector and RNAi vector were constructed. As results,there was no difference on gene sequence between CMS and its maintainer. The full cDNA sequence of MYB21 gene was 922 bp,including an 843 bp open reading frame. The full DNA sequence ofMYB21 gene was 1 108 bp,containing 3 exons and 2 introns（the NCBI GenBank accession number：KT898146）. MYB21 gene was predominantly expressed in anther,the expression level of MYB21 gene in anther between CMS and its maintainer was highly significant difference. Also the over-expression vector and RNAi vector were constructed successfully. In conclusion,the full-length sequence of MYB21 gene in kenaf is obtained,MYB21 gene is mainly expressed in anther of kenaf,and the over-expression vector PBI121-MYB21and RNAi vector pART27-PK-R1-F2 can be used for function study of MYB21 gene.

Cloning and Prokaryotic Expression Analysis of Tyrosine Kinase Gene in Large Yellow Croaker

ZHANG Zai-peng, LIN Peng, GUO Song-lin, WANG Yi-lei, ZHANG Zi-ping, FENG Jian-jun

2016, 32(11): 180-187. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.021

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To identify the structure and function of the lymphocyte-specific protein tyrosine kinase（LCK）gene（LcLCK）in large yellow croaker（Larimichthys crocea）,a full length cDNA of LcLCK was cloned from large yellow croaker by RT-PCR and RACE. The expressions of LcLCK in various tissues of adult male and female large yellow croaker as well as at different stages of the embryonic development were analyzed and examined via qRT-PCR. The recombinant prokaryotic vector was also constructed and transferred intoEscherichia coli for efficient expression. The results were as following：Its full-length cDNA sequence was 2 334 bp,with a 1 503 bp open reading frame encoding a protein of 501 amino acids. The predicted protein contained the typical domains of SH3,SH2 and TyrKc in the Src kinase family of non-receptor tyrosine. Two motifs of GCXCS and CXXC were found in the N-terminal region of LCK,while the two conserved Tyr sites in accordance with the Tyr³⁹⁴ and Tyr⁵⁰⁵ of LCK from human were present in C-terminal. Phylogenetic analysis showed that the deduced LCK clustered with Japanese pufferfish（Takifugu rubripes）. qRT-PCR revealed that the expressions of LcLCK were detected in all adult tissues examined,with higher expression in the main immune tissues such as spleen,head kidney,and gills of both sexes;and the expression level of LcLCK gene in these tissues from female was higher than that of the male. Early in the embryonic development,the high levels of LcLCK were observed during the multiple cells,blastula,and gastrula stages with the expression peak in blastula stage. The significant decrease of LcLCK expression was found at formation of yolk plug stage,and remained at the low expression level from formation of eye lens stage to pre-hatching stage,whereas an increase of gene expression was observed at alevin stage. The constructed prokaryotic vector pGEX-4T-2-LCK was expressed successfully in E. coli. In SDS-PAGE analysis,the new recombinant LcLCK protein band was about 84 kD that was in accordance with the expected. In conclusion,LcLCK is closely related to the cellular immune responses of adult male and female large yellow croaker as well as the mature of immune organs producing T-cell during embryonic development.

On the Involvement of CKS1B in Gonad Development of Mud Crab Scylla paramamosain

HAN Kun-huang, DAI Yan-bin, ZOU Zhi-hua, ZHANG Zi-ping, WANG Yi-lei

2016, 32(11): 188-193. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.030

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CDC28 protein kinase regulatory subunit 1B（CKS1B）is a member of the highly conserved cyclin kinase subunit 1（CKS1）protein family,and involves in cell division of eukaryotic organisms as a regulatory subunit of cyclin-dependent kinases. Therefore,it is of significance to study the function of CKS1 family genes involved in the molecular mechanism of regulating crustacean's gonad development. In the present study,the fragment of CKS1B gene was selected from the gonad EST library of S. paramamosain,and the full-length cDNA sequence of Sp-CKS1B was cloned,further the expressions of Sp-CKS1B in different tissues and different development stages of gonad were examined using qRT-PCR. As results,the full length cDNA of Sp-CKS1B was 722 bp,including a 5'untranslated region（5' UTR）of 82 bp,a 3'UTR of 379 bp,and an open reading frame of 261 bp encoding a protein of 86 amino acid. The protein contained a typical CKS conservative sequence belonging to the CKS family. Its expression level in the ovary of O5 stage female crabs was the highest,and presented extremely significant difference from other tissues（P<0.01）. Meanwhile,the expression level of Sp-CKS1B gene in T1 was the highest among the other stages of gonad development,followed by O5 stage,both of them were significantly higher than O1-O4 stage of female with significant difference（P<0.05）. Its lowest expression level was in the T3 stage,which was significantly lower than in T1,T2,O4,and O5 stages（P<0.05）. These results suggest thatSp-CKS1B may play a critical role in the gonad development of the mud crab.

Prokaryotic Expression and Purification of Single-chain Variable Fragment（scFv）Against Staphylococcus aureus

LI Jing-quan, XU Yong-ping, WANG Xi-tao, LI Yuan, WANG Li-li, LI Xiao-yu

2016, 32(11): 194-201. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.022

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This work aims to clone and express single-chain variable region fragment（scFv）of Staphylococcus aureus in Escherichia coli,and to obtain the target protein with scFv activity. A plasmid containing target scFv gene was constructed,then transformed into a prokaryotic expression strain to be induced,and the activity of the harvested protein was identified. As results：（1）Recombinant plasmid pCold I-scFv was successfully constructed,and transformed into the expression strain of Escherichia coli.（2）After induction,the target proteins mainly exist in pellet in the form of inclusion bodies. （3）Inclusion body was dissolved successfully using 4 mol/L urea. （4）Purified and renatured target protein by column chromatography and dialysis was favorable. （5）The refolded protein showed specific binding activity with S. aureus in ELISA experiment. Conclusively,the target protein with S. aureus antibody activity was successfully acquired by plasmid construction and prokaryotic expression,inclusion body dissolving and refolding.

Soluble Expression,Purification and Activity Assay of Recombinant Human βNGF Expressed in Escherichia coli

BAI Yang, ZHANG Yong-lei, ZOU You-tu, HUANG Fen-fei, CHEN Sheng-liang, RUAN Ka, GE Ping-hui, MA Yan-ling, WANG Ming-zao, CHEN Xing

2016, 32(11): 202-207. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.023

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The goal of this work is to express the recombinant human nerve growth factor（rhβNGF）in Escherichia coli in soluble form,to separate and purify the expressed products,and to determine the biological activity. First,hβNGF gene was amplified and then inserted into expression vector pMAL-c2X,then E. coli expression system of hβNGF-MBP was constructed and induced for expression. Further,the MBP in purified expressed products was cleaved by Factor Xa enzyme,after identified by Western blot,the biological activity was examined by TF-1 method. The results showed that enzyme digestion and sequencing confirmed that the recombinant plasmid pMAL-c2X-hβNGF was constructed correctly,hβNGF-MBP was secretory expressed under 2℃,180 r/min and 0.5 mmol/L IPTG induction. MBP tag in hβNGF-MBP was removed by Factor Xa digestion,and the purified hβNGF via SDS-PAGE was about 13 kD with the purity over 95%. The protein was identified as hβNGF by Western blot. The biological activity test showed that the specific activity was about 1×10⁶U/mg. Overall,recombinant hβNGF was successfully expressed in E. coli in soluble form with high biological activity.

Expression and Cellular Localization of Dendrolimus puntatus Cytoplasmic Polyhedrosis Virus NSP1 in Insects

JIN Liang, XU Cui-ping, WANG Jin-chang, GUAN Li-mei, ZHANG Wen-chao, HUANG Chao, GAO Xue-mei, WANG Hong-xiu

2016, 32(11): 208-214. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.024

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Genome segment 5 of Dendrolimus puntatus cytoplasmic polyhedrosis virus（DpCPV）was predicted to encode a protein of 881 amino acids with a molecular mass of 101 kD non-structural protein（NSP1）. In order to study the function of the DpCPV NSP1 protein,PCR primers were designed according to the S8 segment genome sequence. The antigen of DpCPV 1 genome S5-1 piece's area（1-600 bp）was in prokaryotic expression,and the polyclonal antibodies against the expressed proteins were raised in rabbits. The diluted virus was used to infect Autographa californica,midgut anatomical samples were taken in each day after infection,and the synthesis curve of NSP1 protein vs time was measured by Western blot. The results of Western blot indicated that the synthesized protein expressed by S5 segment was observed in the first day of infection;in addition to full length protein（101 kD）,20 kD and 80 kD protein bands also were detected,indicating that the expression of NSP1 was initiated in early stage and cleaving of NSP1 protein occurred. Moreover,for the first time,the DpCPV 1 S5 fragment was expressed in insect cells and subcellular localization of insects was observed. The results revealed that the NSP1 protein was present in the cytoplasm of the cells.

Cloning and Characterization of a Novel Cold-active Bifunctional Xylosidase/Arabinfuranosidease AX543

ZHANG Ming-hui, LI Zhong-yuan, QIU Hai-yan, WANG Cui-qiong, WANG Hui, ZHANG Tong-cun

2016, 32(11): 215-223. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.025

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This work aims to obtain the cold-active xylosidase with high catalytic activity at low temperature,and to study its heterologous expression,as well as analyze its enzymatic characteristics. A novel psychrophilic bifunctional xylosidase/arabinofuranosidase gene ax543 in GH43 family was cloned using Touch down PCR and TAIL PCR,and was successfully expressed in Pichia pastoris. Analysis of biochemical characterization suggested that recombinant enzyme AX543 exhibited optimal activity at 25℃,and remained 54% and 21% relative activities at 15℃ and 4℃. Moreover,AX543 with xylosidase and arabinofuranosidase activity hydrolyzed xylobiose,xylotriose,birchwood xylan,beechwood xylan,and wheat arabinoxylan,and showed a 1.46 fold synergic effect with xylanase Xyn11-1. AX543 also presented xylose and arabinose tolerance with K_i 84.78 mmol/L and 54.01 mmol/L.

Isolation and Identification of Alicyclobacillus A1 and Its Bio-solubilization of Middle- and Low-grade Rock Phosphates

LI Ling-ling, LÜ Zao-sheng, YANG Zhong-hua, ZUO Zhen-yu, LI Yao-yi

2016, 32(11): 224-234. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.026

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Bio-solubilization of phosphorous has pioneered a novel approach to make full use of rich but refractory middle- and low-grade phosphate ores in China,and this work aims to screen high-efficiency phosphorous-solubilizing bacteria for utilization of middle- and low-grade phosphate ores. A facultative heterotrophic strain A1 was isolated from the acid mine drainage of Yichang phosphate mines of Hubei province,and was identified as Alicyclobacillus aeris according to its morphological,physiological and biochemical characteristics,and 16S rDNA sequence. For strain A1,YSG medium was optimal among four media regarding bacterial growth and its bio-solubilization of phosphorous from phosphate ores;the fraction of phosphorous leached by strain A1 reached 77.22%,which increased about 62% compared to chemical leaching of abiotic control. According to X-ray diffraction（XRD）analyses of raw phosphate ores and bioleached residues in YSG medium as well as high performance liquid chromatography of the leachate,soluble phosphates were released by dissolving fluorapatite and carbonate-fluorapatite in phosphate ores via organic acid mixture（mainly oxilic acid and citric acid）generated during the fermentation of strain A1 with organic carbon sources,and simultaneously gypsum was formed.

Optimizing the Enzyme-producing Conditions of Chitinase-producing Strain S68-CM5

HU Ji-hua, CAO Xu, MENG Li-qiang, CHEN Jing-yu, JIANG Wei, LIU Yu-shuai, ZHANG Shu-mei, LI Jing

2016, 32(11): 235-240. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.027

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In order to improve the capacity of Serratia marcescens strain S68-CM5 producing chitinase,the fermentation conditions of enzyme production were optimized. Using Plackett-Burman design and response surface method,the conditions of medium and fermentation were explored. The results showed that the optimal enzyme production medium：1.5% colloidal chitin,beef extract 7 g/L,yeast extract 2 g/L,glucose 8 g/L,NaCl 3.5 g/L,peptone 2 g/L,and hydrogen phosphate dehydrate potassium 3.5 g/L;the optimal culture conditions for enzyme production：pH6.88,temperature 27.32℃,shakers revolutions 155.82 r/min,incubation time 60 h,and inoculums size 1% liquid volume 50 mL/250 mL. After optimization,the enzyme production reached 7.131 U/mL,and 1.43 folds of that before optimization.

Organic Fertilizer Production with Bacillus natto Fermenting Fish Viscera and Its Application in Inhibiting Rhizoctonia solani

YE Ri-ying, SUN Li-jun, WANG Ya-ling, JI Hong-wu, XU De-feng, LIAO Jian-meng

2016, 32(11): 241-247. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.031

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In order to explore the effect of organic fertilizer with Bacillus natto on crop growth and increase the resistance of crops on basal stem rot microbe Rhizoctonia solani,organic fertilizer with fish viscera was produced using Bacillus natto as fermental and functional microbe. The final fertilizer was utilized in the experiment of testing crop' s resistance to R. solani：seeds of tomato and pepper were respectively sowed in garden soil and garden soil with infected R. solani,and the growth of which were compared. B. naato and R. solani were cultivated in petri dish for their confrontation. The results revealed that there was no significant effects of the final organic fertilizer on the germination of both seeds. In two groups of garden soil with fertilizer,both of tomato and pepper growth were nearly 3 times of that in garden soil without fertilizer. In the infected garden soil without fertilizer,the percentage of basal stem rot for tomato and pepper seedlings were 55% and 50% respectively;while in the infected garden soil with with fertilizer,the percentage of basal stem rot for them were respectively 11% and 17%. Bacterial colony on confrontation of B. naato and of R. solani in petri dish showed that R. solani was inhibited. Overall,the above result indicated：R. solani was inhibited by B. natto,and the growth of tomato and pepper was enhanced by the organic fertilizer produced with B. natto fermentation,and the percentages of basal stem rot for tomato and pepper seedlings decreased.

Effects of Major Enzymes in the Biosynthetic Pathway of Vitamin K₂on MK-7 Production

XU Jian-zhong, WANG Ying-yu, YAN Wei-liu, ZHANG Wei-guo

2016, 32(11): 248-254. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.032

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This paper focused on understanding the effects of different enzyme genes on MK-7 production by cloning and expressing the genes of 6 enzymes in the biosynthetic pathway of vitamin K₂ with strain Y-2. Firstly,the recombinant plasmids with single gene of the 6 genes were constructed according to the description of Guide to Molecular Cloning Experiment. Subsequently,the recombinant plasmids were successfully transformed into the strain Y-2 via the improved “spizizen” transformation method. Results showed that respective overexpression of the 6 genes involved in vitamin K₂ biosynthetic pathway all was beneficial to raise MK-7 production. It should be noted that overexpression of gene hep S had the greatest effects on MK-7 production among these 6 genes,which resulted in an increase by 38% in shaking group and by 48% in static group. However,the effects of other genes on MK-7 production varied depending on the culture mode.

Expression of CydDC in Escherichia coli and the Effect of It on Glutathione Transportation

QUAN Cong, ZHANG Jing, WU Hui, LI Zhi-min, YE Qin

2016, 32(11): 255-260. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.033

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This study is to co-express the CydDC and GshF in the recombinant Escherichia coli,and to increases the glutathione production by enhancing glutathione transportation. As the results from the experiments,the target protein CydDC and GshF were successfully expressed,the glutathione production of MG1655（pTrc99a-as/pBAD33-cydDC）reached 0.22 mmol/L,and the content of glutathione in supernatant significantly increased and reached 81.9%,which was 1.11-fold and 1.29-fold of control strain MG1655（pTrc99a-as/pBAD33）,respectively.

Preparation of Crosslinked Enzyme Aggregates of Glutathione Synthetases and Its Application

TIAN Hui, YANG Feng-chen, LU Hong-yu, XU Qi, YANG Yong

2016, 32(11): 261-270. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.034

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In order to reduce the cost in synthesis of glutathione（GSH）,and concurrently to realize the enzyme storage and the commercialization,the glutathione synthetase was precipitated through appropriate precipitant,and immobilized crosslinked enzyme aggregates（CLEAs）were prepared by glutaraldehyde crosslinking. By optimizing the precipitation and crosslinking conditions,as well as the uses of protein concentration and additives,finally the optimal preparation condition was determined as：4℃,30 g/L protein concentration,adding PEI-600（additive W∶enzyme protein w = 1∶5）fully mixed with the enzyme solution,then 60% saturated ammonium sulfate added for precipitation for 4 h,and finally crosslinked by 0.5% glutaraldehyde. The final protein precipitation rate was 94.9% and the recovery of enzyme activity was 48.2%,enzyme activity per CLEAs was 4.9 U/g,respectively,compared to the control group 28.7% and 2.57 U/g,and increased by 19.5% and 2.33 U/g. The highest yield of GSH reached 11.26 g/L,GSH yield was 9.03 g/L in the 12th batches of conversion,and the relative enzyme activity was 78.4%;after 33 d storage at 4℃,the relative enzyme activity remained 92.12%. In conclusion,after optimizing the preparation conditions,the CLEAs of glutathione synthetases presented higher initial enzyme activity,which could be stably converted more than 10 batches,with favorable storage stability and solid industrialization prospects.

Effect and Mechanism of Selenium Nanoparticle Against Islet β Cell Apoptosis

RAO Lei, RAO Min, QIU Hong-hong, LI Hong-hui, LIU Jing, ZHUANG Man-jiao

2016, 32(11): 271-277. doi:10.13560/j.cnki.biotech.bull.1985.2016.11.035

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The purpose of this research was to study the effect and molecular mechanism of selenium nanoparticles（SeNPs）in the treatment of islet β cell apoptosis in type 2 diabetes mellitus. Electron microscope and nanoparticle size analyzer were used to identify SeNPs surface morphology,and relevant molecular biological techniques were used to validate the protection effect and mechanism of SeNPs on islet β cells apoptotic model. Results showed that the surface charge of selenium nanoparticle decorated by chitosan（CS）increased to 24.8 mv,and its storage time was extended to over 100 days. Cytology results showed that SeNPs under 2 μmol/L had no toxicity. Meanwhile,SeNPs in 38.1 nmol/L activated TrxR activity and reduced intracellular ROS level,which led to apoptotic model cells survival rate significantly increased 31.75%. Furthermore,the animal experiments also verified that SeNPs could resist the apoptosis of islet cell β. In conclusion,SeNPs may eliminate the apoptosis of islet cell β in type 2 diabetes mellitus,which shows a potential therapeutic activity in the treatment of type 2 diabetes.

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2016, 32(11): 300.

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