Biotechnology Bulletin

LEA Protein and Its Application in Improvement of Stress Tolerance in Plants

Wang Yanrong, Zhang Zhiguo, Wu Jinxia

2015, 31(3): 1-9. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.001

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Adverse conditions, including drought, salinity and extreme temperatures, often restrict the growth and development of plants. In plants, there exist a group of highly hydrophilic proteins, known as LEA(late embryogenesis abundant)proteins, which generally accumulate at the last stage of embryogenesis under natural conditions. It has a strong resistance to various abiotic stresses, and can respond to drought, cold, high salt and ABA signals. LEA proteins can maintain normal metabolic reactions of plants by maintaining cellular osmotic pressure, protecting the cell membrane structure and functioning as a molecular chaperone to protect other proteins. In this paper, the classification, structure, stress tolerance mechanisms of LEA protein and its application in improvement of stress tolerance in plant were reviewed.

Progress on Study of CYC-like Genes During the Floral Development of Angiosperm

Xu Yunru, Li Yueying, Pang Hongbo, Zhang Yuxin, Liu Yulian

2015, 31(3): 10-16. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.002

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CYCLOIDEA(CYC)-like genes are members of TCP family and play an important role during floral development. CYC-like genes underwent gene duplication deep within angiosperm phylogeny, they diversified into three major clades, CYC1, CYC2, CYC3, and CYC2 clade genes are main regulators in floral symmetry. But the studies on CYC1 and CYC3 are limited. In this paper, recent insights into CYC-like genes and the prospective of the study on CYC genes were reviewed.

Research Advance on the Biosynthesis of Volatile Organic Compounds in Plant

Li Fangfang, Tao Shutian, Zhang Huping

2015, 31(3): 17-24. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.003

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Plant volatile organic compounds(VOCs)are closely correlative to production and life of human beings. In agriculture, plant volatile organic compounds play important roles in attracting pollinators, defending against biotic and abiotic stress, mediating the exchange of information between plants, giving fruit special scents. In this review, the biosynthesis of terpenoids, phenylpropanoids/benzenoids, fatty acid derivatives and amino acid derivatives were introduced, works and methods in the future were put forward, in an attempt to providing useful information for further studies in the field.

Progresses of Microbial Synthesis of Poly-γ-Glutamic Acid of Related Genes，Synthesis Mechanism and Fermentation

Yan Tao, Xi Hongsheng

2015, 31(3): 25-34. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.004

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γ-polyglutamic acid(γ-PGA)is naturally occurring poly amino acids with characteristics of water solubility, biodegradability, edible and non-toxicity towards human, animals and the environment. Therefore, γ-Poly(glutamic acid)and its derivatives have been of interest in a broad range of industrial fields such as environment, medicine, food, cosmetics and feed additives. This paper focuses on the microbial synthesis of γ-PGA of related genes, synthesis mechanism and fermentation.

Advance in Antitumor Mechanism of Bioactive Compounds in Edible Mushrooms

Chen Kaixu, Wang Weilan, Liu Jun, Zhang Fuchun, Zheng Xiufen

2015, 31(3): 35-42. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.005

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Edible mushrooms have been globally consumed for centuries to promote human health, prevent and treat human diseases. In different types of edible mushrooms, there are distinct bioactive compounds, mainly consisted of polysaccharide, protein, terpenoids, alkaloids and others substances. They possess many medicinal effects such as anti-cancer, anti-cardiovascular diseases, and anti-diabetes, etc. Mushroom bioactive constituents have been found to have anti-cancer effects against several major cancer types. It is reported that edible mushrooms exert an anti-cancer effect through regulating the expression level of relevant factors in cell signaling pathway, which leads to inhibition of cell proliferation and induction of cellular apoptosis. These findings may provide theoretical basis for utilizing edible mushrooms as potential natural and non-toxic antitumor agents. This paper provided a brief review on the research progress in antitumor mechanism of different bioactive compounds in edible mushrooms..

Advance of the ApplicationofSSRMolecularMarkersinTobaccoResearch

Chen Jie, Yang Jing, Long Shengxian, Xiao Ciping,Yang Changyi, Huang Qingzhong, Wang Wei

2015, 31(3): 43-48. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.006

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SSR molecular marker technology as one of the most commonly used molecular markers, the marker technique result is reliable and good repeatability. In recent years, SSR marker shows a broad application prospect in tobacco genetic breeding, is a large potential for application of molecular markers. This paper introduced the principle and distribution characteristics of SSR molecular marker, and summarized the study of gene location and molecular marker-assisted selection, germplasm research, the construction of molecular genetic maps and the seed purity identification and authenticity of tobacco, and analysed the application prospect of molecular markers in tobacco genetic breeding. The main aim is to supply useful information so as to promote the development of SSR molecular marker technology research in tobacco.

Research Progress of Agricultural Crop Active Component Extraction

Xia Rizhao, Liao Xiaolan

2015, 31(3): 49-56. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.007

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The development of new pesticide by analogy synthetic and rational design of biolo-gical is a research focus of pesticide toxicology and pesticide chemistry. Conventional extraction such as Solvent extraction and Tissue crushing were summaried in this review. In addition, Ultrasonic wave extraction, Microwave extraction, Enzymatic method, Superitical fluid extraction, Ultrahigh-press extraction, Subcritical water extraction, Ultrasonic and Combined extraction such as Microwave assisted extraction, Mechanochemical assisted extraction. Accelerated solvent extraction, High-voltage pulsed electric field extraction, Ultrafiltration extraction also were referred. At the same time, author prospected the characteristics, application examples and trends of botanical active component, provide reference and basis for the extraction, development and application for agricultural crop active component.

Progress of Research and Application of the RNA Interference Technology in Crustacean

Qiu Xier, Zhu Dongfa, Zhou Yanqi, Liu Zhiye, Xie Xi

2015, 31(3): 57-63. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.008

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RNA interference(RNAi)is a phenomenon that the gene is post-transcriptionally inhibited by using specific double-stranded RNA(dsRNA), gene silencing will be happened eventually. As a new technology, RNAi contributes to the understanding of the the functions and mechanisms of genes. RNAi is widely used in insects, fungus, plants and mammals, however, the reseachs on the crustacean are relatively less than the former. This review highlights the advancements of RNAi in crustaceans, including the study of methods, gene functions about CHH family, mechanisms of molting and gonadal regulation. In addition, RNAi plays an important role in an antiviral defense in crustacean.

Evaluating the Development of Small Interfering RNA Expression Vector from Its Functional Elements

Hou Ying, Sun Xikui, Tang Mingqing

2015, 31(3): 64-69. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.009

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RNA interference as a potent gene silencing approach plays important parts in gene regulation, functional study and gene therapy. The small interfering RNA expression vector achieves a sustainable, stable and controllable application of the RNAi technology, and becomes a promising way for gene silencing. Although the interfering RNA expression vector has progressed to the second generation, and many commercial products were availabe, the discrepancies among efficiency, safety and controllability of these vectors still exist. The development of the interfering RNA expression vector seems get into a lag phase. Therefore, this article analysed the history and development of these small interference RNA expression vectors on the basis of their functional blocks, providing a theoretical fundation for the vector’s optimization and selection.

Research Advances of Genetic Engineering of Microalgae for Improving Lipid Production

Li Yi, Wang Chaogang, Hu Zhangli

2015, 31(3): 70-81. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.010

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In recent years, microalgae oil biodiesel has become a hot spot because of its strategic importance. Microalgae are a promising feedstock for biodiesel due to their short growth cycle, easy to mass culture, ability to absorb CO₂ and no taking farmland, etc. However, large-scale development and utilization of microalgae biomass energy is limited by less knowledge about metabolic mechanisms of lipid synthesis and lagging research of genome in microalgae. With the development of modern biotechnology, improving lipid content and biomass of microalgae may achieve through genetic engineering and metabolic engineering methods. This review describes recent efforts to metabolic mechanisms on lipid biosynthesis and genetic engineering techniques on increasing lipid content, which provide technical reserves for attaining high lipid transgenic microalgae and producing microalgae biodiesel.

Research Advances of Biological Treatment of Uranium-containing Wastewater

Tan Wenfa, Lü Junwen, Tang Dongshan

2015, 31(3): 82-87. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.011

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With the rapid development and application of nuclear energy, more and more uranium-containing wastewater is generated, urgently needing to be solved. Biotechnological method has good prospect because of its low cost and high efficiency. The status and progress of biological treatment of uranium-containing wastewater are briefly reviewed in this paper. Different types of degradation manner of uranium are introduced in the article together with their degradation mechanism, working principles and an analysis to their merits and demerits. Last but not least, this dissertation points out the further research area of biological treatment of uranium-containing wastewater and its developing trendency.

The Expression of Rice OsSHAT1 and OsRSR1 in Response to Hormones and Abiotic Stresses

Li Zhe, Zhang Shaoxuan, Huang Rongfeng,

2015, 31(3): 88-95. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.012

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It was to research whether AP2 family factors involved in the stress response process. In this study, sequence alignment revealed that OsSHAT1 and OsRSR1 proteins share 41.92% sequence identity, indicating that OsSHAT1 and OsRSR1 are homologous members of AP2 family. And the promoter sequences of OsSHAT1 and OsRSR1 genes contain multiple stress and hormone responsible cis-acting elements, such as ABRE, DRE, MYB, MYC, WRKY, GCC-box and ERE. Transcript analysis showed that the expression of OsSHAT1 was suppressed by low temperature, NaCl, ABA, ACC, but induced by drought；while the transcripts of OsRSR1 was inhibited by low temperature, drought, ABA and ACC, but enhanced by NaCl. It is speculated that OsSHAT1 and OsRSR1 may influence the expression of stress related genes by transcriptional regulation in differential ways, and then adjust the distinctive stress response of rice.

Cloning and Characterization Analysis of ZmJAZ4，a JAZ Family Gene in Maize(Zea mays L.)

Yan Shengwei, Sun Cheng, Zhou Xiaojin, Chen Rumei

2015, 31(3): 96-101. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.013

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Jasmonate ZIM-domain(JAZ)proteins are a group of plant-specific transcription factors, which have important function in plant growing development and abiotic stress by inhibiting the expression of genes associated with JA regulation. In this study, a novel JAZ family gene, named ZmJAZ4, was isolated from maize(Zea mays L.)inbred lines B73. The ZmJAZ4 cDNA has a total length of 651 bp and encodes a protein of 216 amino acids with a molecular weight of about 23.1 kD and a isoelectric points of 10.78, belonging to the basic protein. Real-time RT-PCR showed that ZmJAZ4 was mainly expressed in shoot apex meristem, tassel, developing seeds and endosperm. Phylogenetic analysis revealed that ZmJAZ4 was related to AtJAZ10 from Arabidopsis. Subcellular localization experiment showed that ZmJAZ4 was localized to the nucleus. ZmJAZ4 possessed no transcriptional activating activity in yeast cells. Various treatments were performed to detect the expression level of ZmJAZ4 in response to phytohormones or abiotic stresses. The transcripts of ZmJAZ4 was induced by PEG, NaCl, SA, GA and ABA treatment in shoots and that was induced by ABA and GA in roots. The above results showed that ZmJAZ4 may be an important transcriptional regulator, which participates in many hormones signaling pathways and abiotic stress.

Cloning and Expression Analysis of a Pollen Development Gene MF21 in Broccoli

Pei Xuli, Jing Zan’ge, Tang Zheng, Wang Yan, Wang Zhen, Chen Zhongwen, Luo Tiankuan, Zhang Xiaoling

2015, 31(3): 102-107. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.014

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In this study, the MF21 gene were cloned from a broccoli maintenance line ‘WN12-95B’. Bioinformatic analysis showed that the full length of MF21 gene was 468 bp and encoded 151 amino acid peptides. The relative molecular weight and pI of MF21 was 16.70 kD and 8.56, respectively. Meanwhile, the MF21 protein was speculated as a hydrophobic protein. The MF21 protein include a 22 amino acid length signal peptide, and also contain two CK2phosphorylation sites, four N-myristoylaton sites and one tyrosine kinase phosphorylation site. Random coils and β-strands were main components of the two-dimension structure. Based on the molecular evolution, we found that the MF21 gene had approximate evolution relationship between broccoli and Brassica carinata. Theexpression analysis of MF21 gene by qRT-PCR indicated that this gene was mainly expressed in bud of broccoli and had tissue specificity. Theexpressive abundance of bud in different development stage had greatdifferences between the Ogu CMS line and its maintenance line.

Cloning and Expression Analysis of AmDREB2.1 in Ammopiptanthus mongolicus

Li Zhanglei, Gao Fei, Cao Yuzhen, Zhang Zhiwei, Wang Ning, Li Huayun, Zhou Yijun

2015, 31(3): 108-114. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.015

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A new dehydration responsive element binding protein(DREB)transcription factor gene, which was named as AmDREB2.1, was isolated from Ammopiptanthus mongolicus(Masxim.)(Cheng f.)root transcriptome database, that it had been established in our previous work. The full length of the AmDREB2.1 cDNA was 978 bp, including a single 531 bp opening reading frame which encoded a 176-amino acid peptide with a conserved AP2 domain. Quantitative real-time PCR analysis revealed that the AmDREB2.1 expressed in leaf and root of A. mongolicus, but there were different expression patterns under drought or low temperature stress respectively, and it could be mainly induced by drought in root.

The Differential Expression of Monosaccharide Transporter Genes in Disease-free Sugarcane Plants

Wang Jungang, Zhao Tingting, Yang Benpeng, Cai Wenwei, Zhang Shuzhen

2015, 31(3): 115-120. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.016

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The sugarcane yield can be increased by planting disease-free sugarcane seedlings. However, the physiological and molecular mechanisms of this yield increasing are still not clear now. Monosaccharide transporters are one type of key regulatory proteins involved in carbon accumulation and partitioning processes, and play an important role in plants defense process. The differential expression of three monosaccharide transporter genes(SGT1, SGT2 and PST2a)in disease-free and untreated sugarcane plants was studied to clarify the potential function of these genes on sugarcane yields. The expression levels of SGT1, SGT2 and PST2a in leaves of disease-free plants at seedling and tillering stages were obviously higher than untreated plants. Furthermore, the expression levels of these genes were increased in immature internodes of disease-free plants at elongation and mature stages, but there were no difference in mature internodes. The sugarcane leaves(at seedling and tillering stages)and the immature internodes(at elongation and mature stages)grow rapidly and demand for more monosaccharide to provide energy and the precursor of biosynthesis. The up-regulations of these monosaccharide transporter genes might accelerate the growth and biomass accumulation of disease-free sugarcane plants by increasing the transporting and uptake of monosaccharide.

Construction a Metabolic Engineering Strain to Produce 1，3-propanediol from Klebsiella pneumoniae by ldhA Gene Deletion Mutation

Chen Lifei, Li Meng, Ma Chunling, Yang Jianlou

2015, 31(3): 121-126. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.017

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Production of 1, 3-propanediol from glycerol by Klebsiella pneumoniae is restrained by lactate formation. The increase of lactate in the broth can inhibit cell growth and reduce 1, 3-propanediol conversion rate. The lactate dehydrogenase gene(ldhA)of production lactate by Klebsiella pneumoniae was modified by λRed recombination system. One 300 bp homologous recombinant fragment：ldhAl-Cm-ldhA2 was constructed for the gene knockout. After resistance experiments, PCR determination, K. pneumonia2-1ΔldhA with ldhA gene knocked out obtained by λRed recombination. After 24 h fermentation, the maximum yield of lactate was reduced to 0.49 g/L from 10.16 g/L and the maximum threonine yield of 1, 3- propanediol was increased to 85.76 g/L from 78.83 g/L, comparing with that of the original strain K. pneumonia2-1. The conversation rate of glycerol was improved to 65.97% from 60.64%, increasing by 5.33%.

Screening and Breeding of a Strain for High Yield of Succinic Acid

Wang Le, Yu Guanghai, Yang Shuoye, Wang Weiwei, Hui Ming

2015, 31(3): 127-134. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.018

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It was to improve the production of succinic acid. After the primary and secondary screening, a strain with high-yield of succinic acid was isolated from the soil. The succinic acid production of 34.45% was determined by the high performance liquid chromatography(HPLC)after the fermentation. Later, the UV mutagenic breeding of strains has been performed, resulting in the succinic acid production of 50.30% which increased by 46% in the substrate of glucose concentration of 100 g/L with the residual sugar lower than 10 g/L. After the determinations of morphological characteristics, physiological and biochemical index and 16S rDNA sequence analysis, the strain was identified as actinobacillus.

Expression of the Extracellular Domain of SLA-1 Derived from Topigs Pig in Escheichia coli

Zhai Xiaoxin, Zhang Xiujuan, Yang Jie, Gao Fengshan

2015, 31(3): 135-139. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.019

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In order to construct the recombinant prokaryotic expressed vector of the heavy chain of SLA-1 derived from Topigs pig and to study its expression in pET-21a(+), a pair of primers was designed to amplify the extracellular domain of SLA-1-TPK gene(named SLA-1-TPKe)followed by sub-cloning the gene into pMD19-T Simple Vector. After identification by cleavage with Nde I and Xho I, the SLA-1-TPKe was ligated to pET-21a(+)and transformed into BL21(Rosseta)to be induced to express followed by analysis of the expressing products by SDS-PAGE. Finally, the inclusion bodies of the SLA-I-TPKe was isolated and detected by SDS-PAGE. The PCR result was shown that the length of the SLA-1-TPKe was about 851 bp which was consistent with the calculated value. Then, the SLA-1-TPKe was successfully cloned into the pMD19-T Simple Vector and identified by cleavage with Nde I and Xho I and the inserted fragment was about 831 bp. In succession, the gene was successfully inserted into pET-21a(+)followed by transforming into Escherichia coli BL21(Rosseta). After induction and expression, the SLA-1-TPKe was successfully expressed with the interest of protein about 31 kD. The protein was expressed mainly as inclusion body protein with the purity of 90%.

Isolation，Identification and Algicidal Activity of Acinetobacter calcoaceticus

Wang Yun, Zhang Yemeng, Li Peipei

2015, 31(3): 140-145. doi:10.13560/j.cnki.biotech.bull 1985.2015.04.020

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The microcystis bloom not only result in the death of the aquatic animals due to hypoxia, but also produce mycrocystins which is harmful to the human beings and wild animals. One algicidal bacterium against toxic Microcystis aeruginosa PCC 7806 was isolated from the Baiguishan Reservoir in Pingdingshan of Henan province using liquid infection technology and was named algicidal bacterium 5. The 16S rDNA sequence analysis indicated that this bacterium belongs to Acinetobacter calcoaceticus. It specificallyremovesPCC 7806bycelllysis, but has no effect on FACHB-930 and Scenedesmus obliquus. Interestingly, the growth of haematococcus and Chlamydomonas reinhardtii are promoted. The algicidal effect of bacterium against PCC 7806 is better when the volume ratio of 1∶1. The thalli and cell-free filtrate shows the same lytic effect. There are no bacteria adhering to the surface of the PCC 7806. And no bacterial biofilm were observed. These results may suggest that The A. calcoaceticus may secrete substance and compete for nutrients to remove PCC 7806.

The Comparsion of Bacillus Species Classification Based on Fatty Acid and 16S rRNA Gene

Liu Guohong, Liu Bo, Lin Yingzhi, Tang Jianyang

2015, 31(3): 146-153. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.021

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In order to explore the application of fatty acid composition in taxonomy of Bacillus species, the fatty acid constitutions of 25 strains were detected by Microbial Identification System(MIDI). The clusters of fatty acid profiles and 16S rRNA gene sequences were analyzed by SPPS16.0 and Mega4, respectively. The results showed that phylogeny analysis based on the fatty acid biomarkers could not only fully reflect the relationships among the Bacillus species, but also group the Bacillus species according to the biological characteristics. However, 16S rRNA phylogeny only perfectly showed the relationships among the species. For example, four species growing well under the alkaline conditions and three species round spore-forming were clustered together, respectively. Result showed that Bacillus species can be clustered together not only according to the related ship, but also classified by the biological characteristics.

Screening and Identification of Antagonistic Endophytes Against Drug-resistant Bacteria from Medicinal Plants

Liu Xiaoyu, Ma Yuchao

2015, 31(3): 154-160. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.022

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Methicillin-resistant Staphylococcus aureus(MRSA), Cephalosporin-resistant Escherichia coli and Imipenem-resistant Pseudomonas aeruginosa have caused severe infection in clinical therapy and greatly endanger human health. The aim of this study is to isolate and screen highly active antagonistic endophytes against drug-resistant bacteria from medicinal plant. After two-step screening, 18 strains have antagonistic effect on MRSA were obtained from 197 strains isolated from 22 medicinal plants, none of the strains has antagonistic effect on Cephalosporin-resistant Escherichia coli and Imipenem-resistant Pseudomonas aeruginosa；16S rRNA sequence analysis showed that eight strains belong to Streptomyces, six strains belong to Bacillus, and four strains belong to Pseudomonas. Five strains which have strong anti bacterial activity were chosen for fermentation, and the fermented liquid of QN1 have strong inhibition effect on MRSA.

Screening of Nattokinase Producing Strain and Characterazation of Nattokinase

Zhao Zhonglin, Li Shuying, Nie Ying, Li Yan, Yuan Chao, Tang Xuanming

2015, 31(3): 161-164. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.023

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To develop new kinds of natto health care products with specific physiological activity, a nattokinase high-yield strain was isolated and used for natto production. The characteristics and nattokinase activities of the isolated strain were compared with other 3 kinds of natto strains. Results showed that the natto products of munber 5 strain have golden color and soy sauce flavor, and have the best length of natto wire drawing. The result of fermentation time showed that nattokinase content reached the maximum value at 17 h.

Recombinant Expression of Rhizopus chinensis Lipase in Aspergillus niger

Zhang Qian, Wang Jianying, Lin Zhi, Jia Jia, Guo Hongtao

2015, 31(3): 165-170. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.024

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This article carried out the recombinant expression of Rhizopus chinensis lipase(RCL)gene in Aspergillus niger. Promoter of Pgpd from A. niger and RCL-TtrpC fusing fragment were respectively obtained by PCR and further cloned to plasmid pCHAMBIA2302 to generate the RCL over-expression vector pCHAMBIA2302∷Pgpd-RCL-TtrpC. The resulting vector was introduced into Agrobacterium tumefaciens EHA105, and then transformed into A. niger through the mediation of A. tumefaciens. Seven randomly selected transformants were analyzed by PCR and Southern blot. As a result, all the transformants were found to be positive. Then, the seven transformants were subjected to fermentation and lipase assay, and the results showed that all the transformants secreted recombinant RCL, among which transformant T6 produced the highest lipase specific activity, reaching up to approximately 71 U/mL. The fermentation broth was further analyzed by SDS-PAGE, which revealed the molecular weight of the recombinant RCL was 37 kD.

Preparation of Protoplast for Efficient DNA Transformation of Citric Acid Hyper-producing Aspergillus niger Industrial Strain

Zhang Xiaoli, Zheng Xiaomei, Man Yun, Luo Hu, Yu Jiandong, Zheng Ping, Liu Hao, Sun Jibin

2015, 31(3): 171-177. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.025

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Aspergillus niger is the major industrial strain for citric acid production. In spite of many successes of modern molecular biology approaches in engineering laboratory or protein-producing strains of A. niger, there is few positive report on its application for citric acid industrial strains mainly due to the hard-to-transform nature of these strains. In this study, the protoplast-PEG mediated genetic transformation system for citric acid industrial strain was extensively studied, suggesting an optimized protocol for protoplast preparation, regeneration and DNA transformation. The concentration of protoplasts reached up to 10⁶ /mL by lysing younger mycelia for 2.5 h after 48 h incubation of a proper amount of conidia spores in enrichment medium. The optimal lysing enzyme mixtures comprised of 1.5% lysing enzyme, 0.5% snail enzyme and 0.2% lysozyme. Concentration of protoplast influenced the protoplast-PEG mediated transformation efficiency, which reached the maximal when the concentration of protoplast was higher than 10⁶/mL. The genetic transformation system established in this study should pave the way to molecular biology study of the citric acid hyper-producing strains, for further understanding its acid-tolerant physiology and for rational design of the industrial strain for further improvement of the citric acid production process as well as creation of new organic acid-producing cell factories.

Surface Display of Methyl Parathion Hydrolase on Saccharomyces cerevisiae and Its Application in Degradation of Methyl Parathion

Wang Xingxing, Chi Zhe

2015, 31(3): 178-184. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.026

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The methyl parathion hydrolase(MPH)gene of Pseudomonas sp.WBC-3 was amplified by PCR and cloned into the multiple cloning site of the surface display vector pYD1 to construct a recombinant plasmid pYD1-MPH. Then plasmid pYD1-MPH was transformed into Saccharomyces cerevisiae EBY100. The 2% galactose was used to induce the expression of MPH on the cell surface of EBYl00, and the display of MPH on the cell surface of S.cerevisiae was confirmed by immunofluorescence. The characteristic of the displayed MPH and degradation effect of methyl parathion in water by the engineered yeast were also investigated. The result showed that the engineered yeast strain, which have a whole cell catalytic activity of MPH, was successfully constructed. The activity of MPH displayed on the yeast cells was 18.2 U/mg of cell dry cells by the induction of 2% galactose for 48 h. The displayed MPH had the optimal pH of 9.5 and the optimal temperature of 30℃, respectively and was stable in the pH range of 4.0-10.5 and up to 45℃. The displayed MPH was stimulated by Mn²⁺，Co²⁺，Zn²⁺，Ca²⁺，Hg²⁺，K⁺，Ni²⁺, and was inhibited by Na⁺，Fe³⁺，Ag⁺. The engineered yeast strain could hydrolyze over 80% of 20.0 mg/L methyl parathion in tap water in 1 h.

Optimization of Extract Condition of Safflower Oil Body and Analysis of Stability

Yang Jing, Han Gaoqiang, Liu Zhongliang, Lu Zhen, Wu Zhibang, Wang Qingman, Zou Deyi,Qiang Weidong, Chen Wei

2015, 31(3): 185-190. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.027

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In order to achieve a wide range of applications in many fields, especially on oil body as a substrate carrier system of the foundation, extraction method of safflower oil and characteristics of safflower oil body lotion was researched. Extraction efficiency of safflower oil body was compared, including its value at different pH, NaCl concentration conditions, an average particle size of safflower oil and stability, as well as its viscosity to conduct investigations. Results showed that the safflower oil body dispersed more evenly, at pH≥6, the average particle size of 1.75-2.05 μm and pH ≤ 6 conditions, the average particle size of 1.50-1.75 μm; while NaCl concentration of 0.2 and 0.4 mg/mL, NaCl concentration 1.2 and 2.0 mg/mL, the safflower oil bodies appeared aggregation. When sucrose concentration between 0.1 and 0.2 mg/mL, safflower oil dispersion is uniform; when the sugar concentration of 0.4-1.0 mg/mL, safflower body intensive, with increasing concentration of sucrose, safflower oil body size gradually began to heterogeneity. The optimum extraction condition for safflower oil body is pH7, NaCl 0.2 mg/mL, sucrose concentration of 0.1 mg/mL. The study proved that safflower oil body without adding detergent or without physical approach, the saved is unstable.

Cloning and Bioinformatics Analysis of GABA A Receptor γ2 Subunit Gene in Carassais auratus gibebiol

Zhao Yini, Hu Kun, Sun Qi, Yang Xianle, Ruan Jiming, Zhou Ailing

2015, 31(3): 191-198. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.028

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The application of the specificity of GABA receptor subunits in drug screening and drug development got widely attention recently, of which three function subunits α1, β2 and γ2 studied deeply. Furthermore, Carassais auratus gibebiol as its good growth and reproduction had been widely farmed in china. In the present study, we cloned and characterized GABA A receptor γ2 subunit full-length gene. The full-length Carassais auratus gibebiol GABA A receptor γ2 subunit cDNA was 2 763 bp in length, contained a 1437 bp open reading frame(ORF), and encoded 477 amino acids which constituting a 55.3 kD protein molecule with an isoelectric point of 9.13. The γ2 subunit protein was hydrophilic. The protein sequence had one signal peptide, consisting of 35 amino acid residue. The sequence of amino acids contained four transmembrane regions, which length of 23，20，23 and 23 aa, involving in electronic transfer catalysis between the internal and external membrane. We found three N-glycosylation sites, two O- glycosylation sites and an extracellular domain which had obvious ligand-gated ion channels’ characteristics. Sequence comparison revealed that the similarity of the γ2 subunit protein all above 89% with other aquatic animals showed that it belongs to GABA A receptor subunits’ family. We conducted a phylogenetic analysis using the neighbor joining(NJ)method. The evolutionary tree showed that the γ2 subunit protein was clustered with the zebra fish which indicated that they are the two most closely related species.

Songjiang Sea Bass(Trachidermus fasciatus)Analysis of Gene Cloning and Expression of Coagulation Factor XI

Qi Qi, Qi Yunyue, Yang Hui, Bi Caihong, Han Xiaodi

2015, 31(3): 199-206. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.029

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It was to clone the full length cDNA encoding coagulation factor XI(FXI)of Trachidermus fasciatus. A TfXI gene from Trachidermus fasciatus(TfXI)was cloned and characterized by RACE technology. The TfXI cDNA composed of 1 287 bp with a 13 bp of 5'-UTR, 1 143 bp open reading frame(ORF)and 131 bp 3'-UTR, encoded a polypeptide of 280 amino acids. Sequence alignment of TfXI showed the highest similarity of 63% with Haplochromis burtoni FXI protein. After LPS stimulation, transcripts of TfXI were significantly increased and reached to peak at 96 h p.i. It indicated that TfXI may play an important role in immune response of T. fasciatus during pathogen challenge.

Studies on Genetic Features of Sex Reversal in Cynoglossus semilaevis

Song Chao, Jiang Li, Wang Jingwei, Li Xiaofang, Li Geng, Zhang Xiaohui, Wang Shu, Liu Zhe, Li Hengde

2015, 31(3): 207-212. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.030

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The remarkable individual size differences of half-smooth tongue(Cynoglossus semilavi.)between female and male fishes exist. However, the lower ratio of female in cultured populations arise from sex reversal of the females leads to lower production efficiency. In sex-determination of some fishes and amphibians, sex reversal is interesting biology question and its molecular genetic mechanisms are rarely explored. In this study, 10 half-sib families are set up by utilizing two types of male parents：genetically males and pseudo males which are genetically females. The results showed that the females were all reversed into physiologically male fishes in two families with pseudo male parent. In the other 8 families with normal male parent, the ratio of sex reversal in individual populations presents continuous distribution, which fits for features of QTLs(Quantitative Trait Loci)typically；the heredity of sex reversal is lower, only 0.058. All of these results showed that, the pseudo males as parents, which demonstrate full paternal-effects, the female progenies were all reversed into pseudo males；interfamily selection for improving the genetic advances of sex ratio or genetic evaluation of sex reversal by using genetic markers are advantageous over intrafamily selection for lower heredity of sex-reversal；the continuous distribution of ratio of sex reversal implies the sex-determination for half-smooth tongue is depended on the interactions among multiple QTLs.

Expression of Chicken Neurexophilin 1 Gene in Pichia pastoris

Chen Meiling, Nie Bin, Jiang Xunping, Liu Guiqiong

2015, 31(3): 213-217. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.030

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Neurexophilin 1 is the differentially expressed gene at the chicken utero-vaginal junction. According to the mRNA sequence of chicken nxph1 and codon bias of Pichia pastoris, the modified DNA sequence for chicken nxph1 which encodes the same amino acid sequence as that of chicken nxph1were synthesized. Specific primers were designed to amplify part of the synthesized DNA sequence whose expression product has no signal peptide. The two sequences were inserted into pPICZαA vector to construct the recombinant pPICZαA/nxph1 and pPICZαA/△nxph1(without signal peptide), and then the recombinant plasmids were transfected into Pichia pastoris GS115 by electroporation. The positive recombinant strains were inducted to express protein by addition of 1 % methanol. The expression protein was displayed on the Tricine-SDS- PAGE electrophoresis and Western blot. The result showed that chicken NXPH1 protein and △NXPH1 protein were successfully expressed, and the molecular weight of the protein was approximately 29 kD, which provided foundations for analyzing functions of NXPH1 in chicken reproductive tract.

Preparation of Prokaryotic Expression Construct of Human FGF21 cDNA and Its Recombinant Protein Expression

Zhang Lilin, Tang Qinglan, Xu Qingzhong, Lei Tingwen, Li Hongmei

2015, 31(3): 218-222. doi:10.13560/j.cnki.biotech.bull.1985.2015.04.032

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Preparation of prokaryotic expression constructs of human FGF21(fibroblast growth factor，FGF)cDNA and induction of recombinant hFGF21 protein expression. Total RNA was extracted from human liver, and the target cDNA fragment was obtained using RT-PCR. The amplified cDNA fragment was cloned into pMD19-T for preservation. Then the expression construct pET-28a(+)-hFGF21 was successfully constructed and expressed with IPTG induction, and the his-hFGF21 protein was purified with histide-selective nickel affinity gel and identified by Western blot. Western blot analysis showed that the fusion protein had specific binding with a FGF21 antibody.

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