Biotechnology Bulletin

Progress on Disease-resistant Breeding of Major Diseases in Sugarcane

Fu Yuhua, Lu Jiaju, Li Xiangyong

2014, 30(2): 1-6.

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Sugarcane production has been severely affected by diseases, which not only cause losses of yield and sugar content but also affect perennial root and even cause variety degeneration. Cultivating disease-resistant variety is the economic and effective way to ensure sugarcane production. The progress on disease-resistant breeding including pathogen detection, germplasm screening and genetic engineering of sugarcane mosaic disease, sugarcane smut, ratoon stunting disease and pokkah boeng disease were discussed.

Advances on the Baculovirus Expression Vector System for Protein Complex Production

Wan Jing, Xiang Xingwei, Jiang Lingli, Zhou Xiangyang,

2014, 30(2): 7-14.

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Most essential functions of eukaryotic cells are catalyzed by complex built of many subunits. To well understand their biological function in the process of health and disease, it is imperative to study these complexes. The low abundance and heterogeneity of many essential complexes have limited their extraction from native source material. The baculovirus expression vector system（BEVS）, specifically tailored for multiprotein complex production, has been proven itself to be uniquely suited for overcoming this impeding bottleneck. Here we highlight recent major achievements in multiprotein complex structure research which were catalyzed by this versatile recombinant complex expression tool.

Research Progress on Heat Shock Protein 90 of Insects

Zhang Ke, Weng Qunfang, Fu Haohao

2014, 30(2): 15-23.

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Heat shock protein 90（Hsp90）is one of the most extensive molecular chaperones in cells, indirect regulation of intracellular multiple and cell proliferation, differentiation, and survival, diapause, apoptosis and related signal transduction pathway. In recent years, awareness of Hsp90 family members at the molecular level, Hsp90 has become immune cells, signal transduction and anti-tumor research frontiers. Insects functional genome research is a worldwide craze, heat shock protein related to insect diapause research also unceasingly thorough. This article summarized the biological characteristics of Hsp90 at home and abroad in recent years, the biological function of Hsp90 and its control research present situation and prospect in insect, in order to provide references information for the research of integrated pest control.

Progress in the Research Regarding to the Function of Protein Rep78/68

Zhan Shenbiao, Lü Yinghui, Li Zhaofa,

2014, 0(2): 24-29.

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Rep78/68, a type of nonstructural proteins encoded by adeno-associated virus（AAV）genome, have a variety of control functions. Rep78/68 promote the integration of AAV DNA site-specific into human chromosome 19, suppress the virus biological activity, such as adenovirus, simian virus 40, adjust the AAV DNA replication and transcription, etc. This paper mainly discusses the mechanism of site-specific integration, the mechanism of Rep78 / AAV ITR/ICP8 complex formation and the characteristics of the oligomerization of Rep68 protein, the limit of the nuclear output of p53 by Rep78 protein, the interaction between AAV Rep78 and human papilloma virus（HPV）E1 protein prompt the combination of Rep78 and ITR DNA, inhibition of the hepatitis B virus（HBV）by Rep78. While the detailed mechanism still remaines to be improved and enriched, further elucidatation of Rep78/68 protein related mechanism may improve the production technology of rAAV and develop gene drug based on rAAV, which has a great significance.

Research Progress of Functional Genes Related with Important Economic Traits for Aquatic Animals

Liu Yuexing, Ma Hongyu, Ma Chunyan, Jiang Wei, Li Shujuan, Ma Lingbo,

2014, 0(2): 30-40.

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With the rapid development of genomics technique, a large number of functional genes have been identified from aquatic animals. Furthermore, many alleles and genotypes of these genes have been found to be associated or linked with economically important traits by constructing the association between genes polymorphisms and traits. Studying on gene resources not only helps to understand the molecular regulatory mechanism on economical traits, but also provides theoretical and technical assistances for molecular marker-assisted selection in aquatic animals. This paper reviewed the progress of functional genes related with economically important traits for in aquatic animals that will be useful to study on gene regulatory network and molecular marker-assisted selection.

Edaphic Factor that Affect the Activity of Entomopathogenic Fungus Metarhizium anisopliae

Li Zhenlun, Li Xinqiang, Yang Shuiying,

2014, 0(2): 41-46.

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Metarhizium anisopliae is an important entomopathogenic fungus which is common and widely distributed in soil throughout the world. M. anisopliae plays an essential role in the regulation and control of soil pests and it is one of the most widely used biological insecticides. However, the efficiency of controlling soil pests is not steady, and one of the reasons is the activity of M. anisopliae affected by the complexity edaphic factors. The present article reviewed 5 edaphic factors that influence the activity of M. anisopliae, such as soil temperature and moisture, soil pH, edaphon and soil texture. New research direction about edaphic factor influencing on the activity of M. anisopliae was also discussed. The review would provide reference for further study of mechanism about the edaphic factors influencing life activities of M. anisopliae, which would be benefic to regulate and control soil pests using the M. anisopliae.

Application of Molecular Biological Methods to Research on the Genetic Diversities of Virioplankton

Gao Ebin, Wang Ziqian

2014, 0(2): 47-55.

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Viruses are recognized as an important component of aquatic microbial communities in marine and freshwater environments, and play a significant role in regulating microbial loop, driving global biogeochemical cycling and maintaining clonal diversities of phytoplankton and bacterioplankton populations. However, the structure and diversity of virioplankton communities have not been extensively and overall investigated using classical viral culture and quantitative methods, and the rapid development and application of molecular-ecological techniques offer a new way. The basic concept of some molecular biological methods including cloning library, PFGE, DNA fingerprinting, DNA chip and metagonemics were described, and their application to address the population composition and genetic diversities of virioplankton in natural aquatic environment, and to investigate the ecological relationship between virioplankton and environmental factors.

Research Progresses of Germplasm Identification Methods in Sturgeons

Guo Xianghe, Dong Ying, Hu Hongxia, Zhao Yanzhen,

2014, 0(2): 56-63.

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Sturgeon is a kind of large economic fish. Due to overharvesting, habitat destruction and their biological characteristics such as sexual maturation at a late age and low survival rate of larvae, sturgeon natural resources in the world were depleting. In order to meet the needs of sturgeon caviar and meat, artificial cultivations were developed and had been the main source of sturgeon products. So far, there was not yet a perfect germplasm identification method in sturgeons at home and abroad. Some common sturgeon germplasm identification methods were summarized. The advantages and disadvantages of each method were analyzed and listed. It was helpful to establish a stable and reliable sturgeon germplasm identification method.

Establishment and Application of Duplex PCR to the Pathogen of “Cracked Head” from Yellow Catfish（Pelteobagrus fulvidraco）

Wei Lili, Wu Huadong, Liu Yi,

2014, 0(2): 64-68.

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“Cracked head disease” is one of the major diseases in cultured yellow catfish（Pelteobagrus fulvidraco）. In this study, two special oligonucleotide primers for duplex PCR amplication were designed to detect the pathogen of“cracked head disease”from yellow catfish（Pelteobagrus fulvidraco）. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of duplex PCR were studied. Finally, the duplex PCR is well developed successfully allowing the detection of the two bacterial pathogens in one PCR tube with relatively equal intensities DAN bands when analyzed in an agarose gel. The result showed the expected DNA fragments of 470 bp and 268 bp were observed, respectively. The sensitivity of duplex PCR was 1.38 ng/μL. When it was applied to detect the bacterial in brain of fish which were naturally infected in Nanchang, Jiangxi Province, Edwardsiella ictaluri was detected in 11 diseased samples of yellow catfish. The result was coherence with that of traditional technology of physiology and biochemistry, which showed that the duplex PCR could be used not only to diagnose the diseased yellow catfish but also to monitor the fish infected by bacteria. It can be concluded that the duplex PCR is specific and sensitive. It is a reliable tool for identification of the Edwardsiellosis with less time and cost, and it can be used in quick diagnose and epidemiology investigation of bacterial of “Cracked head disease” from yellow catfish.

Specific Detection of Genetically Modified Rice Bt63 Using Loop-Mediated Isothermal Amplification

Zhang Mingzhe, Chen Xi, Xu Junfeng, Chen Wujian, Wu Rong, Wu Zhiyi,

2014, 0(2): 69-74.

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It focused on the specific detection of genetically modified rice Bt63 using loop-mediated isothermal amplification（LAMP）. The LAMP could effectively detect genetically modified rice Bt63 with high specificity and the sensitivity limit is 0.01%（W/W）. LAMP is a rapid and accurate method and would be applied to inspection, quarantine and food safety.

Hybrid Purity Identification of Reyan No. 2 Using SSR Markers

Liu Ziji Zhan Yuanfeng Yang Yan

2014, 0(2): 75-78.

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The purity identification of Reyan No.2 was performed by using SSR marker technique. The results showed that 5 SSR markers were polymorphic between the parents of Reyan No.2, among which 4 were co-dominant markers, one was dominant marker. To ensure the accuracy and reliability, 4 co-dominant SSR markers were used for purity identification of Reyan No.2. The hybrid purity was 98.82%, which was completely consistent with field identification. It is feasible to conduct purity identification of Reyan No.2 using SSR markers.

Agrobacterium tumefaciens-mediated Transformation of Shoot Tip of Polygonum cuspidatum

Liu Zhongyu, Zhao Shujin,

2014, 0(2): 79-84.

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In order to establish Agrobacterium tumefaciens -mediated transformation system of Polygonum cuspidatum shoot tip, some key factors that affect transformation frequency of P. cuspidatum shoot tip were studied. The shoot tips of P. cuspidatum was infected by Agrobacterium tumefaciens EHA105 with the plasmid which contains PcRS gene. The superior transformation system was as following, pre-cultured 2 days and then infected 10 minutes in suspension（OD₆₀₀=0.6）, co-cultured 3 days, transgenic tissues and shoots were selected with 8 mg/L hygromycin. Six transgenic plants from 300 explants were obtained and the intergration of transgene was confirmed by PCR and Southern blot analysis.

Isolation and Sequence Analysis of the Paeonia suffruticosa WD40 Transcription Factor Genes PsWD40-1 and PsWD40-2

Zhang Chao, Gao Shulin, Du Danni, Wu Fan, Dong Li

2014, 0(2): 85-90.

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Two Unigene sequences that share high homology with WD40 protein involved in plant anthocyanin biosynthesis were obtained from previous-constructed tree peony（Paeonia suffruticosa ‘Luoyang Hong’）petal transcriptome database and named as PsWD40-1 and PsWD40-2. Sequences of open reading frame（ORF）in PsWD40-1 and PsWD40-2 were amplified with designed specific primers using RT-PCR technology and sequenced. Results showed that PsWD40-1 contains a 1 035 bp ORF encoding 344 amino acid residues, and PsWD40-2 contains a 1 032 bp ORF encoding 343 amino acid residues. The predicted protein sequences of PsWD40-1 and PsWD40-2 genes both contain the typical WD40 structural domain. PsWD40-1 shares high similarity（96%）with VvWDR2 of Vitis vinifera, and PsWD40-2 shares 87% similarity with VvWDR1 of Vitis vinifera. Phylogenetic tree analysis showed that PsWD40-1 is grouped into the MP1 clade with VvWDR2 of Vitis vinifera, AtAN11 of Arabidopsis thaliana, and GhTTG2 of Gossypium hirsutum, while PsWD40-2 is grouped into the other clade, TTG1/PAC1 clade, with VvWDR1 of Vitis vinifera, PhAN11 of Petunia hybrida, and PfWD40 of Perilla frutescens.

Establishment of Agrobacterium-mediated Asiatic Lily‘Cedeazzle’Transformation System and Transfer of GsZFP1 Genes

Zhang Huan, Zheng Jia, Kan Dandan, Fan Jinping,

2014, 0(2): 91-96.

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With small scales of aseptic seedling as genetic transformation, zinc finger transcription factor gene GsZFP1 was transformed into‘Cedeazzle’aseptic small scales by Agrobacterium-mediated, and the genetic transformation system of Asiatic Lily‘Cedeazzle’was established. Fifty putative transgenic plants were obtained, and 20 were identified as positive transgenic plants by PCR assay, PCR transform rate was 40%；and 12 positive plants were obtained by RT-PCR test. The results showed that GsZFP1 was expressed in Asiatic Lily‘Cedeazzle’.

Analyzing Polymorphism of Hezuo Swine SLA-DQA Gene Exon 4

Zhang Guohua, Ma Xiaojun, Lü Weili,

2014, 0(2): 97-101.

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The Hezuo swine SLA-DQA gene adaptive mutation, antigen recognition region（exon 4）by PCR amplification and subsequent single-strand conformation polymorphism（SSCP）and sequence analysis . The results show that 439 Hezuo swine individual SLA -DQA exon 4 detection of three alleles and six genotypes（AA, BB, CC, AB, AC, BC）the highest frequency of the A allele and AA genotype, genotype was the dominant gene and advantages .On a different type of PCR-SSCP pieces with sequencing analysis found that the fourmutated sites（5 068 bp T→C, 5 109 bp and 5 149 bp insert C, 5 131 bp A → G cause serine into glycine, 5 135 bp C → T, at 5 136 bp insert A, the 5 234 bp G → A lead tyrosyl acid becomes cysteine.Genetic analysis：cooperation pig polymorphic information content（PIC）was 0.240 1, belongs to the moderate polymorphism；significant distribution of various genotypes .The findings confirm that cooperation pig SLA - DQA gene the 4 exon has low rich genetic polymorphism .

Separation and Identification of Oviduct Glycoprotein ROGP-III from Rana chensinensis

Li Jiahui, Liu Huimin Zhao Hongyu Liu Jingsheng

2014, 0(2): 102-106.

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To study the function of the single glycoprotein in Rana chensinensis, this research determined the best coarse extraction process by orthogonal test, then purified a 66 kD single protein by salting out, dialysis, ion-exchange column chromatography and gel filtration chromatography. The purified protein identified as glycoprotein by PAS test. By HPLC identification, its purity is 97.2%, named as ROGP-III, after enzymolysis by PNGase F, it speculated that the polysaccharide content is about 17%.

Bioinformatics Analysis of the Gene and Protein of Ubiquitin-conjugated Enzyme from Musca domestic

Guo Guo, Wu Qinyi, Wu Jianwei, Zhao Xuejun, Fu Ping, Zhang Yong

2014, 0(2): 107-111.

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A cDNA sequence of Musca domestic ubiquitin-conjugated enzyme（MD-E2）was obtained by in EST technology. Some characters of the MD-E2 gene and encoded protein sequence were predicted and analyzed by bioinformatics methods in the following aspects, such as general physical and chemical properties, hydrophobicity, signal peptide, secondary structure and localization sites in cells. Results showed that the MD-E2 contains a complete ORF（480bp）encoding 159 amino acid with a predicted molecular weight about 18.117 9 kD and pI 8.64. The protein encoded by MD-E2 gene has no signal peptide, and no transmembrane domains, and it is a hydrophilic protein which is mainly located in cell nucleus. The secondary structures are mainly composed of random coil.

Feasibility of Fluorescent Tag on Takifugu rubripes

Chen Lei, Liu Haiying, Wang Xiuli, Zhang Hong, Zhang Song, Yang Guifu, Zhang Xiuqin,

2014, 0(2): 112-115.

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Visible implant elastomer（VIE）tag was used to tag Takifugu rubripes body weight（13.58±1.19）g, body length（7.15±#br#0.61）cm from 13, July of 2012 to 1, October of 2012. The part of tag was the top half of left caudal peduncle in the group Ⅰ, and it was the lower half in the group Ⅱ.The survival rate and growth were no significant differences in all groups between 30 days to 80 days（P>0.05）. The tag retention rates were 100% in the tagging groups, they fell later, and it was higher under the shine of ultraviolet than perusal. The tag retention rates of tagging groups were more than 93% in the 80 days, so VIE was a suitable tagging method for Takifugu rubripes.

Cloning and Prokaryotic Expression of Secretory Form of Immunoglobulin M（sIgM）Heavy Chain Gene in GIFT Strain of Nile Tilapia（Oreochromis niloticus）Induced by Streptococcus agalactiae

Wang Pei, Lu Yishan, Wang Bei, Tang Jufen, Cai Jia, Wu Zaohe, Jian Jichang,

2014, 0(2): 116-123.

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GIFT strain of Nile tilapia（Oreochromis niloticus）, induced by inactivated Streptococcus agalactiae, was used to construct the head kidney cDNA library. The full-length sIgM cDNA of Nile Tilapia was cloned using homological cloning and rapid amplification of cDNA ends（RACE）methods. Results showed the full-length of sIgM cDNA was 1 921 bp, containing a 5' untranslated region（5'-UTR）of 41 bp, a 3'-UTR of 140 bp and an open reading frame of 1 740 bp encoding 579 amino acids with an estimated molecular weight of 64.26 kD and an estimated isoelectric point of 5.36. In the N-terminal of sIgM exists a signal peptide structure. The phylogenetic trees constructed showed that sIgM of Nile tilapia shared the closest relationship with the corresponding proteins of Paralichthys olivaceus and Rachycentron canadum. The sIgM had four CH domains. The constant segment of sIgM gene was amplified and inserted into the pET-28a（+）vector to construct the prokaryotic expression plasmid pET28a-sIgM. The optimal expression condition was set as 0.05 mmol/ L IPTG, induced temperature 37℃, and induced time 4 hours.The recombinant sIgM fusion proteins was purified by His TrapTM HP column, and then identified by western blot using His-Tag Mouse mAb.

Tgf2 Transposon Multiple Cloning Sites Cloning and Expression

Tao Ran, Chang Yumei, Liang Liqun, Tang Ran, Dou Xinjie, Wang Nan, Li Mingyun,

2014, 0(2): 124-129.

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The goldfish pTgf2-EF1α-EGFP transposon vector was reconstructed by insertion of artificial synthesized multiple colning sites（MCS）. In order to examine its effectiveness, the recombinant vectors of pTgf2-EF1α-MCS-EGFP and pTgf2-EF1α-MCS-MIPS--EGFP containing the exogenous gene, Myo-inositol-3-phosphate synthase（MIPS）were injected into 1-2 cells of zebrafish embryos. Both fluorescence observation of GFP and PCR detection of exogenous gene sizes verified the effectiveness of the reconstructed vector, in spite of slight differences in fluorescent strength of GFP. Moreover, the reconstructed vector has a higher integration efficiency（31.4%）in the genome of zebrafish based on the results. The successful construction of recombinant expression vector displays goldfish Tgf2 transposon can mediate exogenous gene expression in zebrafish and it would be useful to Tgf2 transposon in gene function research of fish.

Fusion Expression of Channel Catfish（Ictalurus punctatus）LEAP2 Mature Peptide in Escherichia coli

Gao Bei, Tao Yan

2014, 0(2): 130-135.

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Antimicrobial peptides generally are some small molecule peptides with strong ability to fight against microbial organisms.They play an important role in the host’s immune system and are considered to be good substitutes for traditional antibiotics. A cDNA fragment（mLEAP2）encoding the LEAP2（liver-expressed antimicrobial peptide 2）mature peptide consisted of 41-amino-acid was cloned from the liver of channel catfish（Ictalurus punctatus）by RT-PCR. When the amino acid sequence of channel catfish mLEAP2 was compared with those of poikilothermal fish and other species, fourteen residues were observed at conserved positions, especially four highly conserved cysteine residues occurred at C-terminal regions of these mLEAP2s, suggesting that two disulfide bridges resulted from these four cysteines possibly related to antimicrobial activity of LEAP2. Subsequently, the mLEAP2 gene was fused with a trxA partner gene with a 6×His-tag and an enterokinase site to construct the recombinant expression plasmid pET32a-mLEAP2. The fusion protein “trxA-mLEAP2” was successfully expressed in E .coli BL21（DE3）at 25℃ after a 16 h induction with 0.7 mmol/L IPTG. Tricine-SDS-PAGE showed that the fusion protein existed in both the supernatant and inclusion body after sonication and centrifugation. The purified fusion protein was successfully obtained by immobilized metal affinity chromatography（IMAC）.

Construction of a Phage-displayed Anti-clenbuterol Antibody Library and Selection of Monoclonal Antibody

Dong Jinhua, Tang Yuhai, Qiao Ning, Han Jinhong, Dong Meihua, Dong Yiyang,

2014, 0(2): 136-142.

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An anti-clenbuterol antibody was developed by immunization of mouse and construction of a phage displaying antibody library. The antibody has a novel sequence and the utilization of gene segments in germline was also identified. With this antibody, a competitive assay was performed and gave a limit of detection of 15.6 ng/mL. The IC₅₀ of phage-antibody with free clenbuterol against immobilized bovine serum albumin conjugated clenbuterol was 0.602 μg/mL. All the results suggested that the antibody is useful in practical applications.

Construction of a Multipurpose M13KE Phage Display System

Fang Yueqin, Guo Juanning, Lu Haojie, Zhu Guoqiang,

2014, 0(2): 143-147.

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It was to engineer a multipurpose M13KE phage display vector from M13KE wild-type phage for the longer peptide or protein display library without helper phage, as well as to expand the scope of targeting high-throughput screening. Based on the relationship of the structure and function of minor coat protein of wild-type M13KE, a truncated gene III encoding minor coat protein from M13KE phage was cloned and a fusion gene fragment containing a lac/tac promoter and the gene III signal peptide sequence was assembled using splice-overlapping-extension polymerase chain reaction. SDS-PAGE and Western blot analysis with anti-M13 pIII monoclonal antibody was employed to detect the expression of the recombinant gene III. Two peptide tags, c-Myc and HA tag sequences were fused to the recombinant gene for expression and analysis, and the following screening steps. The recombinant gene III（tgIII）was inserted into the unessential part of M13KE and the corresponding protein III was detected with SDS-PAGE and Western blot with the anti-pIII antibody. The tgIII containing two tags expressed both c-Myc and HA peptides using methods of SDS-PAGE and Western blot with their specific monoclonal antibodies. We made the construction of a multipurpose M13KE phage display system. In the near future, we hope to apply this engineered phage display vector to carry longer peptide libraries for high-throughput screening without helper phage.

Effect of Animal Endogenous Polypeptide CGA-N46 on Cell Proliferation of Candidas

Yan Xiaohui, Li Ruifang, Zhang Huiru, Yin Yanjie, Lu Yanbo, Lu Yali

2014, 0(2): 148-152.

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CGA-N46 is an N terminal derived peptide of human Chromogranin A (CGA) that has anti-fungal activities. The growth inhibition effect on Candida krusei was measured with MTT assay. The cell wall integrity of Candida krusei was examined by monitoring β-1,3-glucanase activity. The effect of CGA-N46 on the permeability of Candida krusei membrane was examined using Propidium iodide detection. The effect of CGA-N46 on mitochondrial membrane potential was evaluated through Rh-123 assay. The results of showed 0.5 mg/mL CGA-N46 could significantly inhibit the proliferation of the C. krusei cells. CGA-N46 had no obvious effect on the β -1, 3-glucanase activity. There was no significant influence on the permeability of C. krusei cell membrane; CGA-N46 could significantly decrease the mitochondrial membrane potential. A conclusion can therefore be made that CGA-N46 may inhibit the growth of Candidas by affecting the mitochondrial membrane potential.

Construction of Recombinant Saccharomyces cerevisiae Strains Through Expressing the Key Genes in the Xylose Metabolism Under the Control of Different Promoters for Co-fermentation Glucose and Xylose

Jin Yi, Wang Zhen, Mo Chunling, Yang XiuShan, Tian Shen

2014, 0(2): 153-160.

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In order to obtain a recombinant yeast strain which can co-ferment both xylose and glucose, the key genes of enzymes in the xylose metabolism were cloned and transformed into Saccharomyces. cerevisiae Y5. The XYL1 and XYL2 genes from Pichia stipitis CBS6054 were placed under the PGKp, which is a constitutive strong promoter；endogenesis XKS1 gene from S.cerevisiae Y5 was controlled by HXK2p and the native promoter XKS1p, respectively. We measured the activity of XR、XDH and XK and evaluated the xylose-fermenting abilities of the engineered strains. The highest xylulokinase activity was observed in the strain Y5-X3-1 whose XKS1 was controlled by HXK2p. The activity of enzyme ratio was 1∶5∶4 between XR, XDH and XK. Furthermore, the xylose consumption of S.cerevisiae Y5-X3-1 was 5 times as the host strain during co-fermentation, and the highest ethanol concentration was 24.35 g/L, which correspond to 73% of the theoretical value.

Extraction and Purification of Microbial Protein—Glutaminase

Guan Jing, Sun Renqiang, Song Jiawen, Huang Di, Cheng Nan, Ceng yuan Hanfeng, Zhang Yang, Chen Xiyue, Zhao Yishan, Zhu Chendan, Zheng Ye, Kang Li, Gao Hongliang

2014, 0(2): 161-165.

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Protein glutaminase（PG）is an enzyme that catalyzes the specific hydrolysis of glutaminyl residues in proteins and peptide. The metabolic pattern of Chryseobacterium indologenes in PG synthesis and primary purification of PG were studied. The pH value and ammonia concentration increased during the fermentation. The maximal PG activity reached 0.359 U/mL for 14 hours culture. The efficiency of purification and yield of PG were maximal when culture borth was centrifuged and the supernatant was concentrated for four times by ultrafiltration with 3 kD cut-off membrane. Ultrafiltrate was precipitated by 4 times volumes ethanol and the maximal yield of PG was 72.7%. Precipitated PG by ethanol was dissolved in PBS buffer and further purified by SP-Sepharose Fast Flow ion-exchange chromatography. A single peak was got in elution profile. The final yield of PG was 31.72% and the PG was purified 124 times after multiple purification steps. The purified sample contained a single band in SDS-PAGE and corresponding molecular weight was about 20 kD.

Functional Ingredients Analysis of Bacterial Lobster Sauce Fermented from Different Beans

Yang Shengyuan, Wei Jin,

2014, 0(2): 166-170.

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The functional ingredients of bacterial soybean lobster sauce（SLS）, black bean lobster sauce（BBLS）, mung bean lobster sauce（MBLS）, pea lobster sauce（PLS）, foot bean lobster sauce（FBLS）, red bean lobster sauce（RBLS）and red phaseolus bean lobster sauce（RPBLS）were investigated. The results indicated the moisture content of the seven kinds of lobster sauce was range 45% to 55%. SLS, BBLS, PLS and MBLS showed strong fibrinolysin activity and amylase activity. The fibrinolysin activity and amylase activity of SLS was（149.07±6.63）IU/g and（120.07±0.39）U/g respectively, which was the highest of the seven kinds of lobster sauce. All of the seven kinds of lobster sauce have DPPH free radical scavenging activity with the order of SLS> BBLS> MBLS> PLS> FBLS> RBLS> RPBLS. The DPPH free radical scavenging activity of SLS was 98.14±1.01%. There was great difference on the content of acid soluble polypeptides between the different kinds of lobster sauce. The content of acid soluble polypeptides of SLS and PLS was（82.79±3.14）mg/g and（42.63±1.17）mg/g respectively, and the content of acid soluble polypeptides in the other five kinds of lobster sauce was rang 25 mg/g to 29 mg/g. The content of free amino acids of SLS, BBLS, MBLS and PLS was（38.89±2.27）mg/g, （32.91±1.13）mg/g, （27.80±0.79）mg/g and（34.12±1.57）mg/g respectively, however, the content of free amino acids of the other three kinds of lobster sauce was only about half of that of SLS. The functional ingredients analysis showed that soybean was the most suitable raw materials for lobster sauce.

The Isolation，Screening and Identification of CaCO₃ Mineralization Strain from Sand Boon

Hu Weilian, Dai Dehui

2014, 0(2): 171-175.

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Sand bone（named by the research team）is a tube that is agglutinated by sand with cementing material mainly composed of calcium carbonate. It was found in Zhongwei area of Ningxia Autonomous Region. Strain 3# has strong ability of CaCO₃ mineralization among 12 strains isolated from sand bone. Mineral deposit was produced and covered the flask wall after 72 h shake cultivation with 1.0% CaCl₂ liquid medium. To analyze the mineral deposits, XRD and SEM were performed. The result was showed that mineral deposit was made up of calcium carbonate, which contains two crystal forms（calcite and vaterite）. Based on morphological, physiological and biochemical properties and 16S rDNA sequence analysis, strain 3# was identified as Paenibacillus sp., and was named Paenibacillus sp. s3 . It laid a good foundation for reaching formation mechanism of sand boon further.

Effect of pH on Yield of Lactic Acid from Kitchen Wastes Fermentation by Rhizopus Oryzae AS 3.819

Zhou Qun, Sheng Li

2014, 0(2): 176-180.

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In order to realize resource recycling of kitchen waste, the enhancement of fermentative production of lactic acid（LA）by Rhizopus oryzae AS 3.819 was investigated. Batch experiments were carried out to analyze the effect of pH on the yield of total lactic acid and the distribution of L- and D-lactic acid among total lactic acid during the non-sterilized fermentation of kitchen wastes by rhizopus oryzae AS 3.819. The results showed that when rhizopus oryzae was cultured in medium temperature（34℃）, the optimal growth condition was pH 7, the optimal fermentation condition was pH8. The concentration of reduced sugar（calculated as glucose）was low, and its concentration was higher at neutral and alkali conditions（pH6-8）than at acidic conditions（non-controlled pH and pH5）. The L-lactic acid was the predominant isomer form at pH 8. The maximum total lactic acid production rate was 1g/（L?h）, the ratio of L-lactic acid kept at above 0.75 during the whole experimental fermentation time and reached the maximum value（0.99）at 60 h and the maximum L-lactic acid was 59.8 g/L. To obtain high #br#L-lactic acid yield and optical purity simultaneously, it was suggested that PH should be controlled at 8.

High Throughput Drug Screening on Protein PAN of Influenza Virus

Zhang Guofang, Li Zhenzhen, Yao Weili, Wang Lijun, Zhu Xianming,

2014, 0(2): 181-186.

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PAN protein, which is an endonuclease and highly conserved in influenza virus, is a potential target for the discovery and development of anti-influenza drugs. Three inhibitors of PAN protein were obtained from a compounds library. The molecular docking analysis showed that these compounds can interact with the divalent metal ions and those amino acid residues in the active sites, implying that these components might be the lead compounds for the novel anti-influenza drugs.

Clone and Expression of Human ETA

Pan Xiaofei, Shi Lili, Tan Chubing, Lü Chaojun, Xu Weiren, Tang Lida,

2014, 0(2): 187-192.

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It was to facilitate inhibitor screening of endothelin receptor, cloning of human endothelin receptor gene ETA, construction of eukaryotic expression vectors and transient expression of pTag-Lite SNAP-ETA in CHO-K1 cells. Method Human ETA gene fragment were amplified from human lung cancer cell line A549 via RT-PCR and inserted into pTag-Lite SNAP plasmid, the recombinant plasmids were then transiently transfected into CHO-K1 cells using FuGENE? HD, expression of ETA was observed via fluorescence microscope. Result Sequencing result showed pTag-Lite SNAP-ETA expression vector were correctly constructed. Fluorescence microscope images indicated that two vectors were expressed in CHO-K1 cells.

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