Table of Content

• 
• Current Biotechnology
  Volume 4 Issue 2 25 March 2014
• Research on the Mechanism of Gamma-aminobutyric Acid Shunt in Plant Response to Adversity Stresses
• YANG Ze-wei1,2, WANG Long-hai1,2, ZHU Li2*, LANG-Zhihong2, LUO Xue-gang1
• 2014, 4(2):  77-84.  DOI: 10.3969/j.issn.2095-2341.2014.02.01
• Abstract ( ) PDF (1362KB)( )
GABA is a non-protein amino acid，which is common in organisms. Its synthesis and metabolism is via of the GABA-shunt which is a bypass of Krebs cycleand. The physiological function of GABA in higher plant is very extensive. It is involved in a series of important life activities in plants, such as growth and development, carbon/nitrogen nutrient balance, adversity stress response, signal transmission and so on. This paper will focus on the research progress on mechanism and biological function of GABA shunt in plant adversity stress response on physiological function, biochemical and molecular level.
• Theoretical Basis of Gene Splitting Technique and its Application on Control of Transgene Flow
• DONG Yu-feng, WANG Xu-jing, TANG Qiao-ling, WANG Zhi-xing*
• 2014, 4(2):  85-89.  DOI: 10.3969/j.issn.2095-2341.2014.02.02
• Abstract ( ) PDF (1510KB)( )
Genetically modified (GM) safety problems caused increasingly debate as the planting area of GM crops improving year by year, in which, gene flow from transgenic crops to wild relatives by pollen spread is one of the focus of attention. Gene split technology provides a new way to limit transgenic wander from GM crops. It is based on the intein-mediated protein splicing. It has been proved that different gene was split and the expression products were re-assembled into functional protein in arabidopsis, tobacco, wheat and other crops by this method until now. These successful cases provide theoretical basis and experimental support for using this method to limit gene flow of GM. In this article， we reviewed the research progress on theoretical basis of gene split and the application of gene split technology on gene flow control.
• Research Progress on GlnR-mediated Regulation
• JIANG Hu1,2, TIAN Jian2, YING Bi1, WU Ning-feng2*, XU Bo1*
• 2014, 4(2):  90-95.  DOI: 10.3969/j.issn.2095-2341.2014.0 .
• Abstract ( ) PDF (1178KB)( )
GlnR is a global transcriptional regulator for nitrogen metabolism that can be found in most Gram-positive bacteria. The GlnR belongs to the OmpR family as well as MerR family. GlnR activates or represses expression of various genes involved in nitrogen metabolism in response to changes in nitrogen availability. It also plays a role in the regulation of phosphate metabolism，antibiotic biosynthesis and bacterial virulence. Here, the differences of GlnR-mediated nitrogen regulation in bacteria and the cross-regulation with other metabolic pathways are detailed. The researches on GlnR-mediated regulation provide further insight into central nitrogen metabolism in bacteria and have important implications for researches on signal transduction in regulation of nitrogen metabolism.
• The Regulation Mechanism of Three Pathways on Carborn Catabolite Repression in Pseudomonas
• GU Wei, ZHAN Yu-hua, DENG Zhi-ping, LU Wei*
• 2014, 4(2):  96-101.  DOI: 10.3969/j.issn.2095-2341.2014.0 .
• Abstract ( ) PDF (1625KB)( )
Bacteria have metabolism regulation systems that allow cells to selectively assimilate a preferred compound among a mixture of several potential carbon sources, at the same time inhibiting the use of non-perferred carbon sources. This process is known as carbon catabolite repression (CCR). Current studies have revealed three different systems to be involved in catabolite repression: CbrAB/Crc, CyoABCDE and PTSNtr. CbrAB/Crc system is composed of two component system CbrA/CbrB, non-coding RNA and Crc protein, which can regulate gene transcription of non-preferred carbon sources through modulating the level of free Crc. CyoABCDE system contains several terminal oxidases, which control non-preferred carbon sources through coordinating the composition of the electron transport chain. PTSNtr system is comprised of PtsP protein, PtsO protein and PtsN protein, which act on CCR through the phosphorylation of PtsN protein. In this paper, we summarize the regulation mechanisms of CCR in Pseudomonas.
• New Opportunities of Aminoglycoside Antibiotics Application
• 2014, 4(2):  102-106.  DOI: 10.3969/j.issn.2095-2341.2014.02.05
• Abstract ( ) PDF (1737KB)( )
Aminoglycoside antibiotics are a kinds of early discovered and applied clinical antibiotics. Although they have not completely disappeared from the market， along with the emergence of broad spectrum antibiotics with fewer side effects，its importance has diminished. At present，due to the infection caused by multidrug resistant (MDR) bacteria increased dramatically, as one of the few treatment options，aminoglycoside antibiotics are back to peoples vision，especially for Gram-negative bacterial infection. Although their resistance mechanism has been basically clear，the understanding of antibacterial mode is far from comprehensive. Facing more and more virtually untreatable infections, this kind of old antibiotics would have application potential to combat multi-drug resistant pathogens.
• Cloning of Whole ORF Coding Genes of FMDV and Construction of Recombinant Adenoviral Vector
• GAO Peng, LI Zhi-yong, LIU Ji-Xing*
• 2014, 4(2):  107-112.  DOI: 10.3969/j.issn.2095-2341.2014.0 .
• Abstract ( ) PDF (4567KB)( )
In order to develop a recombinant adenoviral vaccine, genes of a whole ORF coding genes of foot-and-mouth disease virus (FMDV) were amplified by RT-PCR and subsequently cloned into the vector pAdTrack-CMV to construct the recombinant adenoviral pAdTrack-CMV-ORF. PCR amplification and digestion with restriction endonucleases confirmed that the cloned ORF coding genes shared 99.8% similarity with that of original virulent strain type FMDV. Then the shuttle plasmid was linearized and transformed into competent cells of BJ5183 which containing the adenoviral backbone plasmid pAdeasy-2. The recombinant adenoviral plasmid (pAd-ORF) was identified by PCR amplification and digestion with PacⅠ. In this paper, we cloned the whole ORF coding genes of O type FMDV and successfully constructed the recombinant adenoviral shuttle vector.
• Benzalkonium Chloride's Bacteria-killing Effect on Affinity Chromatography Gel and Residues Detection
• RUAN Qian-kun, HAN Zhi-gang*, YU Jun-yang, DU Yi-da, YANG Da-jun
• 2014, 4(2):  113-117.  DOI: 10.3969/j.issn.2095-2341.2014.0 .
• Abstract ( ) PDF (1574KB)( )
An ideal bactericide benzalkonium chloride which can effectively kill the bacteria infected in affinity chromatography gel and not affect the protein adsorption capacity was eventually selected from several bactericides by quantitative bactericidal test on bacterial suspension and affinity chromatography gel protein adsorption quantity determination. An ion pair reversed phase high performance liquid chromatography (RP-IPHPLC) method was developed for the determination of benzalkonium chloride residue in affinity chromatography gel. Separation was carried out on C18 column, using acetonitrile and 0.2mol/L sodium heptanesulfonate solution as the mobile phase in an isocratic elution．The detection wavelength was 210 nm; the detection limit was 0.000 25 mg/mL; relative standard deviation of benzalkonium chloride determination test in practical samples was 0.63%. This method presents excellent sensitivity and precision.
• Optimization of Fermentation Conditions of Amphomycin-producing Strain Streptomyces canus sp. FIM-0916
• YANG Huang-jian1, ZHANG Zhu-lan1, HUANG Xiao-zhen2, ZHENG Wei1*
• 2014, 4(2):  118-123.  DOI: 10.3969/j.issn.2095-2341.2014.02.08
• Abstract ( ) PDF (1488KB)( )
The fermentation medium and conditions of Streptomyces canus sp. FIM0916 were optimizied for  producing amphomycin by single-factor and orthogonal test design. The optimized medium formula is 1.5% sucrose, 3.5% soybean meal, 0.1% L-histidine, 0.1% KNO₃ and 0.1% CaCO₃；the optimal fermentation conditions are as follows： filling volume is 120 mL for a 500 mL shake flask, shakers rotating speed is 250 r/min， inoculum size is 2%， temperature 28℃， and cultivation time of seed 54 h; the optimal fermentation time are 5 d. Under optimized conditions, the titer of amphomycin was 248% higher than that of control,  which laid the foundation for subsequent development on amphomycin.
• Antimicrobial Activity of Protein from Gracilaria Endophytic Fungi NSS1
• WANG Wei-cai, ZHANG Fu-yun*, LIU Ye, LU Hang, LI Zhen, ZHAO Yu
• 2014, 4(2):  124-128.  DOI: 10.3969/j.issn.2095-2341.2014.02.09
• Abstract ( ) PDF (1364KB)( )
Inorder to study antimicrobail activity of protein from Gracilaria endophytic fungi NSS1, the antimicrobial protein was prepared by ammonium sulfate precipitation, the best ammonium sulfate saturation was 65%. Antibacterial activity to bacterial indicator was detected by filter paper method, the results showed that the antibacterial crude protein could inhibit the growth of five indicator bacteria, and the best inhibitory effect was obtained when the protein concentration reached 750 μg/mL. The scavenging effect of superoxide anion free radical (O-2) from protein was detected by the pyrogallol autoxidation method. The effect of protein on tumor cells was detected by the MTT method. The antibacterial activity was stable under the condition of below 100℃ and neutral pH. The activity of protein was not sensitive to UV irradiation, glycerol, methanol and trypsin. Different concentrations of protein solution all had effect on removing superoxide anion. It had the strongest inhibitory effect on tumor cells, when the protein concentration was 360 μg/mL. Above results indicated that the antibacterial protein had strong antibacterial activity, antioxidant activity and antitumor activity.
• Physicochemical Properties of Jujube Fruit Peel Red Pigment and it's Application in Food Additives
• YU Yan-qin, DU Jing-qi, ZHAO Jun, YANG Wei-min*
• 2014, 4(2):  129-134.  DOI: 10.3969/j.issn.2095-2341.2014.0 .
• Abstract ( ) PDF (2271KB)( )
Using jujube (Ziziphus jujuba Mill var. muzao) fruit peel as raw materials, after enzymatic hydrolysis, extracted by microwave ultrasonic extraction device,  centrifugation,  filtration and vacuum evaporated to get the red pigment product, we identified its physicochemical properties,  quality indicators and food safety. The red pigment product is soluble in water,  formic acid, but insoluble in organic solvents. It has good stability with temperature change,  some metal ions (K+, Mg2+, Zn2+), sucrose and sodium benzoate andcitric acid. Light has a certain impact on physicochemical properties of the red pigment product, absorbance first decreased and then increased as light time continued. The red pigment product is sensitive to pH, unstable in the range of pH1.0~7.0, and more stable under pH7.0~14.0. Af value of the pigment is 6.52,  presents weak tinting strength. Microbiological testing showed total number of colonies was 8×10-5CFU/mL in red pigment product,  coliform MPN value was below 3, showed negative color and no Escherichia coli, which showed that the possibility of bacterial contamination would be very low. The results indicated that the jujube fruit peel red pigment is excellent water-soluble pigment with good stability, safty and non-toxicity, it can be used to develop food additives.
• Extraction Method for Determination of Chlorpyrifos Residue in Soil and Ligusticum chuanxiong
• MU Xiang-rong, MA Yu-ying*, YUAN Mao-hua, MA Ling, JIANG Gui-hua, CAO Yun
• 2014, 4(2):  135-141.  DOI: 10.3969/j.issn.2095-2341.2014.02.11
• Abstract ( ) PDF (1270KB)( )
To establish the optimum extraction method for determination of chlorpyrifos in soil and Ligusticum chuanxiong, chlorpyrifos was determined by GC\|MS method. The extraction solvent, extraction time, extraction times and solvent volume levels was studied by orthogonal design to optimize the extraction method. The optimum chlorpyrifos residue extraction conditions for aerial parts and rhizome of Ligusticum chuanxiong were 3 fold of acetonitrile, ultrasonic extracting 15 min for twice. The optimum chlorpyrifos residue extraction conditions for soil were 10 fold of ethyl acetate, shock extracting 30 min for twice. The optimum extraction methods was validated to be feasible, which meet the requirements of the pesticide residue analysis.
• Detecting Residual Host Genomic DNA in Biotech Drugs by Real-Time Quantitative PCR
• CAO Chen-hua§, LIU Xiao-zhi§, DUAN Yue-jiao, LIU Su-xia, ZHAO Wei, CHANG Liang, JI Li-man*, GAO Jian*
• 2014, 4(2):  142-145.  DOI: 10.3969/j.issn.2095-2341.2014.02.12
• Abstract ( ) PDF (1963KB)( )
We detected residual host DNA in yeast expressing biotech drugs by SYBR GreenⅠ fluorescent based real-time quantitative polymerase chain reaction. The detection sensitivity of this method was up to 1.0 fg/μL. The linear correlation coefficient of standard curve was above 0.99 when DNA concentration was bwtween 1.0 fg/μL to 1.0 ng/μL. Three batch of samples was determined, and the residual host DNA were 8.635×105 fg/μL、6.265×102 fg/μL and 1.436  fg/μL respectively. The results suggested that this easy-handled, rapid and highly sensitive method could be used for the determination of residual yeast DNA in biotech drug products.