Table of Content

29 April 2014, Volume 30 Issue 4

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Review

Advances in Induction of Cassava FEC and Agrobacterium-mediated Transformation

Cheng Qin, Jin Gang, Li Huimin, Peng Xinyi, Li Ping, Wang Liping

2014, 30(4):  1-5. 

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Production of transgenic plants is gradually becoming routine in cassava biotechnology. It is primarily through organogenesis and somatic embryogenesis, the key technology of cassava genetic transformation is friable embryogenic calli(FEC)induction and formation, Agrobacterium-mediated transformation of FEC is the most widely used method to generate transgenic cassava plants.This article provides an overview of cassava regeneration way including embryo and FEC preparation, induction and appreciation, the Agrobacterium-mediated transformation and its application in cassava were summarized.

The Flower Development of Arabidopsis thaliana Affected by Floral Meristem Identity Gene AGL24

Jiang Wei, Gu Huiying, Wang Zhimin, Song Ming, Tang Qinglin

2014, 30(4):  6-13. 

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AGAMOUS-LIKE24(AGL24)encodes a typical MADS-domain protein that plays a very important role at different stages of flower development. This paper reviews how AGL24 interacts with other floral meristem identity genes to affect flower development of Arabidopsis thaliana, then regulate flowering time, which will help us to have a better understanding of flowering gene regulatory network and to regulate effectively the flowering time in production. It can provide reference for plant breeding.

Progress in Endophyte Improving Plant Salt and Alkali Resistance

Li Jiao, Zhang Baolong, Zhao Ying, Xin Shigang, Chen Qiang, Song Ningning, Li Xuemei,

2014, 30(4):  14-18. 

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Soil salinization is one of the main factors of restricting crop yields. How to enhance crop resistance to salt and alkali, has become a major issue of sustainable development of agriculture in our country. Endophytes exist within the plant and have important impact on stress resistance, such as disease resistance, insect resistance, drought resistance. The phenomenon of the symbiotic relationship between endophyte and plant has been paid great attention. This paper mainly summarizes the diversity of endophytes and illustrates endophyte improving salt and alkali resistance by ROS scavenging system, osmotic regulation substances, mineral elements absorption and photosynthesis of plant.

Progress in Study of Foodborne Pathogens in Fresh Fruit and Vegetables

Liu Yiqian, Yi Xinxin

2014, 30(4):  19-24. 

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The number of foodborne illness events caused by eating fresh vegetables and fruit grows rapidly, malignant events have occurred sometimes. This article focuses on the research progress of pathogens associated with vegetables and fruit abroad, vegetables and fruit were introduced microbial quality investigation, analyzed the distribution rule of total bacterial count, coliform group and pathogenic bacteria, the important pathogenic bacteria pollution sources and the influence factors of the pollution rate, the survival rule of pathogenic bacteria in vegetables and fruit, the important factors are sorted. About the methods of removing pathogenic bacteria, this paper introduces the general methods and the methods having potential application value；On the attachments of pathogenic bacteria to vegetables and fruit, this article introduces three adhesions of E. coli O157：H7. In addition, views of study trends are put forward.

Progress on Antimicrobial Peptides’Gene Endogenous Expression Regulation and Clinical Application

Chen Hongwei, Li Yinglun, Liu Juan, Wu Junwei, Huang Qingzhou,

2014, 30(4):  25-29. 

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Antimicrobial peptides are a group of small molecular proteins with antibacterial activities, which are key components of animal and vegetation defense system and play an important role in protecting body from invasion of pathogens. These peptides have many characters such as low molecular weight, thermostability. And they have the wide range of antibacterial, antiviral spectrum and inhibiting growth of tumor cells. However, the development of antimicmbial peptides as antibiotic substitutes is far from easy. The recent warm spots about antimicrobial peptides, especially gene expression regulation and clinical application were reviewed.

Review on the Immunogenicity of Pathogenic Vibrio Outer Membrane Proteins

Lu Panpan, Guo Songlin, Guan Ruizhang, Feng Jianjun

2014, 30(4):  30-35. 

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Vibrio is a common opportunistic pathogen in aquaculture environment, which can lead to an outbreak of mass mortality of fish and shellfish farming. Traditional chemical control methods because of drug resistance, drug residues and other issues make Vibriosis prevention face new challenges, and thus how to get safe and effective prevention and treatment of diseases caused by Vibrio becomes important and difficult. Vibrio is Gram-negative bacteria, the outer membrane is unique to the cell wall of this type of bacteria, which consist of protein, sugar and lipid. The outer membrane protein is easy to be recongnized as foreign by the host immune system then stimulate the humoral and cellular immunity, which may be used as an active ingredient of Vibrio vaccines. A review on the immunogenicity of pathogenic Vibrio outer membrane protein is to provide a new solution for the prevention and treatment of Vibriosis.

Studies on Diversity of Sponges-associated Fungi and Their Secondary Metabolites

Song Kai, Hu Jie, Lin Wenhan, Ji Yubin,

2014, 30(4):  36-42. 

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Because of the unique physiological structure and digest system, marine sponges enrich large numbers of microorganisms. Lots of novel bioactive compounds were isolated and identified from sponges-associated fungi, which is highlight in marine drug discovery. The development on the distribution of fungi, the application of new technique, novel bioactive secondary metabolites and biological activities were reviewed.

Technique

Evaluation of Five DNA Extraction Methods for Different Processed Fish Samples

Li Jinbo, Sheng Jing, Li Xiang, Pan Liangwen, Lü Rong, Yang Jielin,

2014, 30(4):  43-49. 

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It was to find more effective method for DNA extraction, five methodologies for DNA extraction from eleven different processed fish samples were compared. Five methods, including CTAB method and four commercial DNA extraction kits were employed and compared on the extraction of DNA from 11 processed fish samples, such as boiled, oil immersed, fumed, roasted, dried and salt-dried fish etc. The quality and quantity of the extracted DNA were evaluated by measurement of the absorbance at 230, 260 and 280 nm, and by qualitative PCR. The result showed that five methods can all be used to extract from the most processed fish samples, but the quality and quantity of the extracted DNA were different. So every method has its benefits and disadvantages.

Study of Gene Silencing by Artificial miRNA

Zhang Xianhe, Kong Wenwen, Li Yong, Li Jing

2014, 30(4):  50-56. 

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Artificial miRNA(amiRNA)is an effective gene silencing technology. The basic principle of amiRNA is based on its biogenesis mechanism. Using endogenous miRNA precursor as a basic framework, miRNA/miRNA* is then replaced by amiRNA/amiRNA*. The produced amiRNA is expected to silence the target gene. In this paper, the basic principle and method of amiRNA construction was introduced. AmiRNAs were designed to the target gene MYB28 as an example. Three amiRNAs respectively target to the 3' terminal region, 5' region and middle region of MYB28 coding sequence were designed. Precursor of miRNA 319a in Arabidopsis thaliana was taken as a framework and miRNA319a sequence was replaced by amiRNAs targeting to MYB28 gene. The expression level of MYB28 in transgenic plants decreased 86%. Based on our experiments, the unique characteristics of amiRNA and its further applications were discussed.

Optimization of Components for Laccase Production by Panus rudis FG-35 Using Response Surface Methodology

Liu Jian, Liu Jun’ang, Zhou Guoying, Li He, Yang Jing

2014, 30(4):  57-63. 

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We optimized the liquid medium components to produce the laccase activity of Panus rudis FG-35 by using Plackett-Burman design and response surface methodology. The results of single-factor test showed that soluble starch and peptone were the best sources of carbon and nitrogen. Then the Plackett-Burman design was applied to determine the specific medium components affecting laccase activity and found that soluble starch, Ca²⁺and Tween-40 were the key factors. Based on these results, steepest ascent experiments were applied to find central points of laccase activity. These significant parameters were further optimized using Box-Behnken design, response surface methodology and regression analysis. Finally, the optimal medium was：soluble starch 10.040 4 g/L, peptone 0.2 g/L, K₂HPO₄ 1.00 g/L, ZnSO₄?7H₂O 0.008 g/L, MgSO₄?7H₂O 0.5 g/L, CuSO₄?7H₂O 0.007 g/L, FeSO₄?7H₂O 0.005 g/L, MnSO₄ 0.035 g/L, CaCl₂ 0.081 6 g/L, VB₁ 0.1 g/L, Tween-40 0.428%. Under these optimal conditions, the activity of laccase increased from 263.31 U/mL to 127.194 U/mL(1.0-fold increase in total yield), similar to the predictions. And the results of lignin degradation experiments indicated that the optimal medium made contribution to the degradation rate of lignin which about 14.34% improvement over before.

Report

The Cloning and Expression Analysis of PwARP-1 in Picea wilsonii

Sun Fan, Luo Chaobing, Zhou Yanni, Zhang Lingyu

2014, 30(4):  64-70. 

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An auxin repressed protein gene PwARP-1 is cloned from Picea wilsonii. The full length cDNA of PwARP-1 is obtained by RACE-PCR assays based on the cDNA library of Picea wilsonii and EST fragment of PwARP-1. Bioinformatic tools are applied for the prediction of PwARP-1. PwARP-1 encodes a protein with 155 amino acids. The molecular weight is 17.1 kD and theoretical pI is 10.07 and the protein is rich in random coil. The tissue-specific expressions and the response to phytohormones of PwARP-1 were investigated by RT-qPCR assays. The tissue-specific expression assays suggested that the expression level in pollen was the highest among all tissues and the expression level in seed was the lowest. During seed germination, the expression of PwARP-1 was increased, peaked at the 10th day and then decreased. Furthermore, the expression of PwARP-1 was significantly inhibited by Auxin and Gibberellin. However, the expression level of PwARP-1 is up-regulated by Brassinolide and drought stress treatment. The expression level of PwARP-1 declined and then up-regulated under salt stress, methyl jasmonate, and abscisic acid treatments. These results indicate that PwARP-1 has a potential function in seed germination and responding to stresses and phytohormone treatments.

Expression Analysis and Subcellular Localization of the MaBTB Gene in Banana

Chai Wenlong, Jin Zhiqiang, Jia Caihong, Liu Juhua, Miao Hongxia, Zhang Jianbin, Xu Biyu,

2014, 30(4):  71-76. 

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Real-time quantitative PCR of postharvest banana revealed that MaBTB exhibited differential expression patterns that are associated with ethylene biosynthesis. In naturally ripened bananas, expression of MaBTB was in accordance with ethylene biosynthesis. In contrast, for 1-methylcyclopropene-treated bananas, MaBTB expression levels remained constant. Following treatment with ethylene, MaBTB expression in banana fruit significantly increased with ethylene biosynthesis and peaked 3 d after harvest, which was 11 d earlier than that for naturally ripened banana fruits. These results suggested that MaBTB expression was induced by ethylene for regulation of postharvest banana ripening. Finally, subcellular localization assays showed that the MaBTB protein localizes to the nucleus.

The Vector Construction of Anthocyanins as a Visual Marker Gene and Its Transient Expression in Maize Immature Embryos

Zhao Xinmei, Wu Shubiao, Wang Yixue, Cui Guimei, Wang Xiaoqing, Du Jianzhong, Wang Xiaoli, Shang Yongjin, Sun Yi,

2014, 30(4):  77-82. 

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Anthocyanin regulatory factor C1/Bperu genes are constructed respectively with a maize embryo specific promoter GLB1 and a constitutive promoter CaMV35S into plant transformation vectors pGlb1CB and p35SCB, and the recombinant expression vectors were introduced into maize embryos by particle bombardment. Microscopic observation confirmed that the two vectors were actively expressed in maize immature embryo cells. The use of anthocyanins as a marker gene not only could reduce the safety concerns from the public over GMO substantially, but could also be used as a visual indicator for the selection of transgenic plants, which could greatly simplify the screening procedures, improve the efficiency and reduce the costs.

Study on Antibacterial Effects of Fat-soluble Compositions in Nostoc commune

DiaoYi, Yang Zujun,

2014, 30(4):  83-86. 

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The antibacterial effects of fat-soluble compositions from Nostoc commune Vauch. were studied in order to contribute to diverse nutritional and medicinal values of N. commune. Soxhlet extraction method was used to extract crude fats from N. commune, and antimicrobial activities of the liposoluble compounds and fractions isolated by column chromatography were assayed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas stutzeri, Asparagillus niger and Saccharomyces cerevisiae. The results showed fat-soluble compositions were about 2.62% in N. commune. Ethanol elution fractions showed most greatly antimicrobial activities against six bacterial stains while its contents was 32% of crube fats, the benzene fraction with the yeild of 14% in crube fats had worse antimicrobial activities than ethanol elution fractions, and the petroleum ether extract was not inhibitory to six bacterial stains with the yeild of 54% in crube fats. Fat-soluble compositions had most strongly inhibitory effect on E.coli with IC₅₀ 38.75 mg/mL and MIC 64.0 mg/mL and they had least inhibitory effect on Pseudomonas stutzeri with IC₅₀ 89.58 mg/mL and MIC 106.5 mg/mL. Fat-soluble compositions showed antibacterial effects against six bacterial stains：Escherichia coli > Staphylococcus aureus > Bacillus subtilis > Asparagillus niger > Saccharomyces cerevisiae > Pseudomonas stutzeri.

The Polymorphism of MC1R Gene in Yak

Chen Si, Chai Zhixin, Wang Yong, Zhong Jincheng,

2014, 30(4):  87-90. 

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It was to explore the relationship between MC1R genotype and the coat color formation in yak, polymorphism of MC1R gene was detected by PCR-SSCP and DNA sequencing techniques in 64 yaks(33 white coat color and 31 black coat color). The results showed that there were polymorphisms in the amplified region for the Tianzhu yak and Jiulong yak. In Tianzhu yak and Jiulong yak, three genotypes(AA, BB and AB)were detected. The result of χ², three yak breeds were not fit for Hardy-Weinberg law. Sequencing revealed on single nucleotide mutation(C→A)at 179bp and(T→C)at 214 bp of the amplified region of Tianzhu yak and Jiulong yak.

Analyzing Polymorphism of SLA-DQA Gene Exon 2 in the Hezuopig

Zhang Guohua, Ma Xiaojun, Lü Weili,

2014, 30(4):  91-95. 

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The polymorphism, allele number, nucleotide polymorphism sites, amino acid polymorphism sites, the genetic relationship and evolutionary significance of the alleles of the SLA-DQA gene exon 2 in Hezuo pigs were studied. Variation of SLA-DQA gene in the 439 Hezuopigwas investigated by amplification of exon 2 using PCR, followed by single-strand conformational polymorphism(SSCP), DNA sequencing and sequence data analysis. Result showed that seven alleles were found in the SLA-DQA geneexon 2. Eighteen nucleotide polymorphism sites and ten amino acid polymorphism sites were identified in seven SLA-DQA gene exon 2 haplotypes. The study indicated that a high level of sequence polymorphism existed in SLA-DQA gene exon 2 of Hezuo pigs, and the population might have more genetic resources. SLA-DQA gene exon 2 in Hezuo pigs might be differentiated into two major categories alleles from one mutant alleles at first. SLA-DQA gene exon 2 in Hezuo pigs and other pig had high homology, which indicated that SLA-DQA gene exon 2 in pig might come from primordial sequences that were present in a common ancestor and persisted in the pig populations since their divergence. Seven new alleles that had near genetic relationship, may came from the two near allele by mutation.

Phylogenetic Analysis of Three Domestic Pig Breeds in Guizhou Province

Ding Mei, Chen Xiang, Xu Houqiang, Wu Tonggui, Feng Wenwu, Bai Lin,

2014, 30(4):  96-101. 

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PCR product of the complete mitochondrial control regions of 57 pigs from 3 Chinese pig breeds in Guizhou(Baixi pig, Congjiangxiang pig and Qianbeihei pig)were directly sequenced, the genetic distance between the mtDNA of three breeds of pig with other 25 pig breeds was calculated, and the phylogenetic tree was built. The results revealed that, the sequences of the mtDNA D-loop of the three pig breeds were 1 254-1 314 bp, the genetic distance between the three breeds was 0.002 9-0.007 2, comparing Baixi pig with Qianbei Black pig and Congjiang xiang pig, the genetic distance of Baixi pig was great, all of 0.007 2. The results of phylogenetic anslysis supported the view that the Chinese pig breeds respectively originated from the Southern and the Northern, and there were gentic exchanging among the Chinses pig breeds.

Induced Expression and Real-time PCR Detection of IFN-γ from PBMC in Red Jungle Fowl

Sun Tingting, Ji Bin, Ma Zhiliang, Hu Wenli, Lu Qiongfen, Zhang Xiongwei, Chen Peifu,

2014, 30(4):  102-108. 

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In order to establish the method for detection of interferon-gamma(IFN-γ)mRNA expression in PBMC of red jungle fowl(RJF), the induction conditions of IFN-γ expression were optimized. IFN-γ gene and 3-phosphoric acid glycerol dehydrogenase(GAPDH)gene as an internal control were respectively amplified from the total RNA by RT-PCR and cloned into a pMD18-T vector, to obtain the recombinant plasmids(pMD-IFN-γ and pMD-GAPDH)which served as templates to map the standard curve of real-time PCR with SYBR Green I staining. This method was then applied to 34 RJF. The results showed that IFN-γ was expressed at high levels when PMBC were stimulated with 20 μg/mL of Con A for 12 - 25 h. A good linear correlation(R²>0.99)was observed as the templates ranged from 6.72×10²-6.72×10⁹ copies for IFN-γ and 3.94×10²-3.94×10⁹ copies for GAPDH, and the melting curves presented single peaks, respectively. Based on the optimized condition of Con A-induced IFN-γ expression in PBMC, real-time PCR method was successfully established for quantitative analysis of RJF IFN-γ mRNA.

Construction of a Bait Vector for VSV Glycoprotein and Evaluation of Its Self-activation in Yeast Two-hybrid System

Han Yanyan, Song Deguang,

2014, 30(4):  109-114. 

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It was to construct a bait vector for vesicular stomatitis virus glycoprotein(VSV-G)and detect its protein expression and self-activation activity in yeast cells, as a basis for further study of screening and VSV-G protein interaction in two hybrid system. Fragment of VSV-G gene were amplified using RT-PCR and directly ligated to pSos vector, insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The self-activation and toxicity of the bait plasmid transformed into the yeast cells cdcH25(α)was observed in selective culture medium, and the bait protein was confirmed by Western blot. Results showed that VSV-G gene was found in the reconstructed plasmid pSos-VSV-G by sequencing. Yeast two-hybrid tests showed that yeast cells cdcH25(α)transfected with pSos showed that VSV-G had no self-activation and toxicity, the expression of bait protein was confirmed by Western blot. The yeast two-hybrid system can be applied to screen the interacting proteins of VSV-G.

Cloning，Expression and Purification of FK506 Binding Protein Gene from Helicoverpa armigera

Liu Xiaoning, Li Fen, Zhao Wenbo, Zhu Yan, Zhao Jie

2014, 30(4):  115-120. 

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In order to better understand the role of FK506-binding protein gene and its regulatory mechanism in the CYP6B6 expression of Helicoverpa armigera, the FK506-binding protein cDNA sequence from midgut of H. armigera by RT-PCR was cloned on the basis of result of yeast one-hybrid. The fragment digested by double enzymes was linked to a prokaryotic expression vector pET32a to construct the recombinant expression plasmid pET32a-FK506BP, and then converted into competent Escherichia coli BL21 cells. The fusion protein was induced to express by isopropyl-3-D-thiogalactoside(IPTG), and purified by immobilized metal-chelating affinity chromatography(IMAC)using a Ni²⁺ matrix column. SDS-polyaerylamide gel electrophoresis(SDS-PAGE)and Western blotting analysis were used to examine the fusion protein. The results of sequencing and sequence analysis showed that the open reading frame of the FK506 binding protein gene was 327 bp, encoding 108 amino acid residues with the predicted molecular weight and isoelectric point of 11.78 kD and 7.87, respectively. The predicted protein had no signal peptide. The recombinant containing recombinant pET32a-FK506BP expressed a soluble protein after being induced by IPTG. SDS-PAGE and Western blot analysis indicated that the fusion protein, purified using Ni²⁺ affinity chromatography, had the predicted size and higher purity.

Cloning L-lactate Dehydrogenase Gene from Pediococcus acidilactici and Overexpression of It in Recombination Strain E.coli JH12

Luo Xuan, Xu Liyuan, Wang Yongze, Zhao Jinfang, Zhao xiao, Wang Jinhua,

2014, 30(4):  121-126. 

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Gene ldhL encoding L-lactate dehydrogenase was amplified by PCR technique using genome DNA of Escherichia coli SZ85 as temple. The PCR product was cloned into pUcm-T vector and double digested with restriction endonucleases, and then the DNA fragment of ldhL was inserted into pET-28a(+). The recombinants expression plasmid pET-ldhL was transformed into high purity L-lactate production of E. coli. JH12, which inserted ldhL of P. acidilactici into chromosome. The overexpression system of ldhL of Pediococcus acidilactici gene E. coli JH12(pET-28a-ldhL)could product L-lactic acid using 7% xylose. Overexpression of ldhL led to L-lactic acid yield of 64.86 g/L, which is 10 g/L higher than control without Overexpression of ldhL. Meanwhile, high sugar-acid ratio of 96% was achieved by overexpression of ldhL.

Isolation and Identification of a Halotolerant—Vibrio sp. K1-L

Wang Fei, Liu Kang, Fan Jiatong, Cheng Shouliang, Wang Ziyuan

2014, 30(4):  127-131. 

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Strain K1-L, isolated from Yuncheng Salt Lake, Shanxi Province, could swim on the 2%-10% MGM medium. The strain K1-L was Gram-negative with polar flagellum and was 0.4 μm ×(1-2)μm under the scanning electron microscope. Strain K1-L appeared grey, rounded colony. Its optimum salt concentration, temperature and pH ranges were 2%-6%, 30-40℃ and 6.0-8.0. The GC content is 47.06%. After comparing the traditional characteristics of morphology, physiological biochemistry and 16S rRNA sequence analysis, we discovered that the strain K1-L was highly homologous to the Vibrio diabolicus HE800^T and Vibrio rotiferianus LMG 21460^T. The maximum similarity was 99%. It was asserted that K1-L was belonged to the genus Vibrio, we proposed the name Vibrio sp. K1-L for this strain.

Gene Cloning. Expression and Characterization of the Lipase from Bacillus pumilus S6

Su Erzheng, Wu Xiangping, Gao Bei, Wei Dongzhi,

2014, 30(4):  132-138. 

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A lipase-producing strain was screened from the soil samples. It was identified as Bacillus pumilus by the 16S rRNA method. According to the reported lipase genes from Bacillus, the primers were derived and the full-length gene was cloned, named “lipS6”, which had the size of 648 bp, encoding 215 amino acids. “lipS6” held 96% homology with the reported lipase gene B26. Recombinant plasmid pET32a-lipS6 was construced and expressed in E. coli. “lipS6”could be expressed in soluble form, and the lipase activity reached 2 000 U/L. The optimum temperature of recombinant lipase LipS6 was 30℃, and the optimum pH was 9. Low concentrations of Ca²⁺ and Mn²⁺ could activate the activity of lipase LipS6. The harmful effect of hydrophobic organic solvent on the lipase LipS6 was little. LipS6 could catalyze the esterification reaction in the n-hexane system, with the myristic acid and n-butanol as the optimum substrates. These properties show that LipS6 can be used in the fields of pulp and paper, leather, textiles and bioenergy.

Integrative Expression of Stichopus japonicus Lysozyme Gene in Bacillus subtilis

Li Dan, Li Cheng, Sun Lu, Zou Dan, Liu Zhiwen, Cong Lina

2014, 30(4):  139-146. 

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In this study, we cloned promoter P43 of B. subtilis in the front of Lysozyme from Stichopus japonicus(SjLys) gene by constructing the recombinant plasmid pMD18-P43-SjLys. Then we obtained the integration plasmid pDG-P43-SjLys of B. subtilis by subcloning the P43-SjLys fragment into the plasmid of pDG1730. We transformed the linear integration plasmid digested by Xho I into B. subtilis WB600, and selected the re-combinant strain by Spc resistance screening and amylase activity negative screening. After incubation at 37℃, 220 r/min in LB medium, the cellular lysate of this strain was detected by the SDS-PAGE assay. The results demonstrated that B. subtilis WB600/P43-SjLys successfully expressed soluble SjLys after incubated for 60 h. The cellular lysate of B. subtilis WB600/P43-SjLys displayed inhibitive effect on the growth of the Micrococcus lysodeikticus and Staphylococcus aureus. In this study, we expressed SjLys gene integratedly in B. subtilis for the first time, and proposed a potentially new way of producing SjLys protein.

Isolation and Characterization of Endophytic Strains Producing Exopolysaccharide

Huang Yicheng, Liu Yang, Pang Xin, Ma Zhongrui, Han Donglei, Chen Min

2014, 30(4):  147-151. 

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In this study, 24 strains were isolated from carrot, aloe, potato, sweet potato and purple potato. As screening by phenol-sulfuric acid assay, three strains, 25-Z-1, 25-Z-3 and 12-L-6, were proved to be as high exopolysaccharide producing strains. The results of 16S rDNA sequence determination showed that 25-Z-1 and 25-Z-3 isolated from purple potato was about 100% identity compared with Bacillus pumilus strain ST277 and 99% identity compared with Bacillus cereus strain Se05, respectively. Meanwhile the 12-L-6 isolated from aloe identified as Bacillus sp. GZT, with the similarity is of 99%. Culture in LB medium, 30℃ and 170 r/min for 7 d, yield of EPS could reach to 30.3 mg/L, 29.1 mg/L and 28.2 mg/L, respectively.

Optimization of Submerged Culture Requirements for the Cellulase Activity by Phlebiopsis gigantea and Screening the Most Effective Strain

Li Xingchun, He Shuanghui, Dai Yucheng

2014, 30(4):  152-158. 

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The activity of extracellular cellulase could be used for screening the effective biocontrol strain. We could acquire the most effective Phlebiopsis gigantea isolate associated with biocontrol conifer root and butt rots by determining cellulase activity of them. Based on single factor and orthogonal experiment, the optimum medium compositions for the highest cellulase activity were(g/L)：glucose 30.00, peptone 5.00, KH₂PO₄ 3.00, MgSO₄?7H₂O 1.50, the optimal liquid medium volume was 120 mL/250 mL, the optimal inoculum volume was 5%(V/V), initial pH4.0. By determining cellulase activity of P. gigantea isolates, we concluded that P. gigantea 08077 had the highest cellulase activity and 13025 was the lowest. P. gigantea 08077 is better to control the Chinese Heterobasidion parviporum than Rotstop-F that had been registered in Finland.

Screening，Identification and Characterization of an Acid q-amylase Producing Strain and Optimization of Its Fermentable Condition

Wang Jianling, Chen Zhixin, Liu Yihan, Lu Fuping

2014, 30(4):  159-163. 

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Multiple new acid α-amylase producing strains were isolated from soil which is rich in starch. A strain which has the highest enzyme activity was identified as Bacillus subtilis A-7 through analysis of 16S rDNA gene. The optimum temperature and pH was 60℃ and 4.5. The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium. The composition of the best medium was 2% soluble starch, 2% peptone, 0.07% CaCl₂, 0.8% Na₂HPO₄. Under this optimal condition, the activity of acid α-amylase was 221 U/mL and 2.3-fold more than that of no optimization.

Exploiting Cell-free System of Recombinant E. coli to Synthesize Pyrroloquinoline Quinone

Sun Jiguo, Han Zengye, Ge Xizhen, Tian Pingfang,

2014, 30(4):  164-168. 

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In this study, cell-free system was developed to overproduce pyrroloquinoline quinone(PQQ). The PQQ gene cluster from Klebsiella pneumoniae was subcloned into vector harboring lac promoter and a recombinant Escherichia coli was acquired. Subsequently cell homogenate was prepared which showed ~30% increase of PQQ yield compared with in vivo biosynthesis. Not only revealing the presence of intracellular velocity-limiting reaction(s)that retards PQQ biosynthesis, this study also suggests that cell-free system can address this issue. Hence, this study provides basis for further enhancement of PQQ production.

The Study of the Structure of Perchlorate(ClO^4-)-degrading Bacterial Communities Under Autotrophic Conditions

Xie Yuxuan, Guan Xiangyu, Yu Lisha, Liu Fei,

2014, 30(4):  169-175. 

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For the purpose of further investigating the biological degradation under an autotrophic condition and well understanding the microbial community structures in a complex environment, hydrogen was used as an electron donor to completely reduce perchlorate(ClO_{4-)in this study. The composition of microbial communities after degradation was analyzed via the construction of a cloning library by using the High-Throughput Sequencing method(HiSeq 2000). 71 days were needed to completely degrade 10 mg/L ClO4-. Microbial phylogenic analysis of HD(hydrogen degradation)after degradation indicated that the relative abundance of total bacteria in the HD was 84.96% whereas the relative abundance of Proteobacteria was 68.11%, whose percentage accounting for the total bacteria reached to 80.16%. The relative abundance of Dechloromonas which is representative in PRB was 2.7% in the HD. Simultaneously, the relative abundance of Azospira was 3.1%. KEGG was used to analyze the function of bacteria in HD. The relative abundance of genes which engaged in carbohydrate metabolism was 4.75%, and the genes included in energy metabolism was 3.35%, whereas the genes participated in nitrogen cycle was 0.72%, and the genes involving chloride transformation was 0.83%. It was demonstrated that degradation of ClO4- in a complicated condition was achieved by various kinds of microbes rather than a single one. Adding hydrogen as an electron donor to change microbial community played a role in the purification or selection process in the system, which allowed the complex systems to have the specific capacity to remove given contaminates.}

Prokaryotic Expression and Purification of DsRab from Dunaliella salina

Li Xiujuan, Chai Xiaojie, Tao Xiaoying, Zhao Huan, Cong Yuting

2014, 30(4):  176-180. 

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Previous studies indicated that the DsRab transcript could be increased by salt stress. In order to study the functions of DsRab in salinity tolerance, the open reading frame(ORF)of DsRab gene was obtained through PCR. The target fragment was cloned in pGS-21a, and the recombinant plasmid pGS-21a-DsRab was transformed into E. coli BL21(DE3). The recombinant protein was induced with IPTG. Then the prokaryotic expression condition was optiminzed to harvested more supernatant recombinant protein. The products were purified by GST-Sefinose^TM Kit, and identified by SDS-PAGE and Western blot. The results showed that the recombinant expression vector pGS-21a-DsRab was constructed successfully, the molecular weight of the recombinant protein was in the expected line. Western blot analysis showed that the recombinant protein can be identified specifically by the anti-GST antibody.

Effects of Fe³⁺ on the Growth and Oil Content of Chlorella vulgaris for Biodiesel Production

Sun Yuan, Liu Wenbin, Zhou Tiezhu, Xie Tonghui, Liang Bin, Zhang Yongkui

2014, 30(4):  181-186. 

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Biodiesel is a clean renewable energy as the high quality substitute for traditional diesel. Chlorella vulgaris, used for biodiesel production, has been gaining the attention of the society because of its fast growth and high oil content. Iron ion could improve the growth and oil content of C. vulgaris. Effects of the concentration of Fe³⁺and the time to add Fe³⁺on the growth and oil content of autotrophic and heterotrophic C. vulgaris were explored in this study. The best conditions of Fe³⁺ were as follow：(1)10^-3 g/L Fe³⁺ added in the lag phase of autotrophic C. vulgaris, biomass of 2.80 g/L and oil content of 30.90% were obtained, (2)10^-5 g/L Fe³⁺ added in the exponential phase of heterotrophic C. vulgaris, biomass of 3.30 g/L and oil content of 29.05% were obtained. And the oil produced by C. vulgaris under autotrophic and heterotrophic conditions was an ideal feedstock for biodiesel production.

The Establishment and Application of a High Throughput Screening Model for Inhibitors of STAT3

Pan Li, Zhang Ning, Niu Guojun, Meng Jing, Liu Xiang, Yang Cheng

2014, 30(4):  187-192. 

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It was to establish a viable high-throughput drug screening model for discovering inhibitors of Signal Transducer and Activator of Transcription3, and applied the model to screen potential anti-cancer drugs. The STAT3 gene was amplified with PCR and cloned into the expression vector pET-28a. The recombinant STAT3 was over-expressed and purified, and then was used to bind with specific phosphotyrosine peptides. We established a high-throughput drug screening model based on ELISA to obtain some effective compounds. Further, we tested the effect of them on the growth ability and proliferation of the Hepatoma cells using MTT assay. Results showed that it was not only successfully constructed the expression vector pET-28a-STAT3, but also established a reliable and stable model for drug screening. Besides, 8 248 samples were screened, and a positive sample MDC6 was finally obtained. When the drug concentration was less than 500 μmol/L, the inhibition rate of MDC6 reached 92%. The minimum value of IC₅₀ was 3.37 μmol/L, while at the cellular level it was 15.92 μmol/L. In this work, we developed a repeatable and reliable assay for screening of STAT3 inhibitors.