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Table of Content

    07 November 2014, Volume 30 Issue 11
    Research Progress and Prospect on the Genetic Mapping Strategy of Sweet Potato
    Liang Xuelian,Xie Zhenwen
    2014, 30(11):  1-6. 
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    The establishment of the sweet potato molecular genetic map is of great significance on development and application of molecular breeding technology system. At present, while studies of sweet potato molecular genetic mapping have being made certain progress, still there are a lot of technical bottlenecks, such as application and optimization of mapping strategy. The paper firstly summarized the genetic research progress of sweet potato in both classical and molecular aspects;and three methods and strategies of molecular genetic mapping on sweet potato were analyzed in detail;then the approaches of improving efficiency and quality of sweet potato mapping were discussed, including optimizing the quality of mapping groups, overcoming the partial separation of progeny, integrating genetic linkage maps derived from different groups, and choosing appropriate molecular markers;finally, the importance of chromosome association in genetic mapping was emphasized, and what need to be enhanced in the field of molecular breeding was also put forwarded. All of these were in order to establishing a precise molecular graph and providing new ideas on sweet potato molecular breeding using genetic map.
    Review of Arabidopsis 1-Aminocyclopropane-1-Carboxylic Acid Synthases
    Lü Shufang,Jiang Jing
    2014, 30(11):  7-13. 
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    1-aminocyclopropane-1-carboxylic acid(ACC)synthase(ACS)is the key rate-limiting enzyme of ethylene biosynthesis. The activity of ACS enzyme is the basis of ACC or ethylene regulates plant growth and development. This regulation is mainly involved in various levels:transcription, post-transcriptional modification, enzyme structure formation, biochemical characteristics, and so on. Here we briefly review the research progresses of ACS enzymic activity.
    CLE Peptide Signaling Molecules in Plants
    Gao Li
    2014, 30(11):  14-23. 
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    Peptide ligands have long been recognized as signaling molecules which are involved in diverse developmental and physiological processes in animal systems. In recent years, peptide ligands have also well been known signaling molecules in plants, indicating that the function of peptides as singling molecules in inter-cellular communication is evolutionarily conserved. Recently, CLE(CLAVATA3/EMBRYO SURROUNDING REGION)family is well be researched of signaling molecules in plants. Similar to peptide ligands maturation in animals, the CLE peptides are matured by post-translational proteolysis and modification by studying the CLV3 from Arabidopsis and TDIF from Zinnia. In this review, we outline the molecular characteristics, biological functions and the post-translational maturation of CLE gene family members. We also provide prospective vision in this field.
    Research Progress of Gene Silencing by RNA Interfering Technology in Mammal
    Cao Tengwei,Gu Lingyun,Huang He,Gao Zhen,Li Mingfang
    2014, 30(11):  24-31. 
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    Variation of metabolic pathways result from RNA interference and related RNA silencing pathways have revolutionized our understanding of gene regulation. Gene silencing has been used as a research tool to control the expression of specific genes in numerous experimental organisms. Importing the different cells with specific siRNA need different transfection reagent to achieve maximum transfection efficiency, and will not produce larger cell toxicity. In recent years, new nano-material such as carbon nanotubes exert interference effect expanding us the traditional liposomes and virus’ understanding. The rapid progress in the field of RNA interference was summarized, and then compare different RNA interference experiments used several different carriers. Further, the perspectives of future research on RNA interference mediated from traditional liposomes to virus and polymer nano-material in disease treatment and clinic diagnose were proposed.
    Research Progress on Tilapia Immunology
    Gan Zhen,Wang Bei,Lu Yishan,Tang Jufen,Jian Jichang,Wu Zaohe
    2014, 30(11):  32-39. 
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    Tilapia is one of the major fishes farmed in China. In recent years, outbreak of diseases caused great economic losses in tilapia aquaculture industry. It’s generally believed that immunoprophylaxis and immunotherapy have great advantages in water environmental protection and food safety management, so the research about exploring characteristics of immune system and mechanisms of immune response in fish gradually become a hot issue in academic circles. In this review, the non-specific immunity, humoral immunity and cell-mediated immunity of tilapia were discussed in order to provide useful data for further research on immunoprophylaxis and immunotherapy for diseases of tilapia.
    Research Progress of Proteins Associated with Per os Infectivity of the Occlusion-derived Virus
    Xiang Xingwei ,Zhou Yufang
    2014, 30(11):  40-47. 
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    The study of baculovirus proteins associated with per os infectivity is of great importance not only for viral biology but also for the fact that these proteins expose vulnerabilities in the insect immune system and this knowledge is also fundamental for the development of new strategies for insect control. Recent researches show that the proteins associated with per os infectivity of the occlusion-derived virus(ODV)include P74, PIF1, PIF2, PIF3, PIF4, PIF5(ODV-E56), PIF6, ODV-E66, VP91, Ac108, ORF 145 and ORF 150. This paper reviewed recent research achievements about the structure and function of the proteins and analyzed the molecular biology characteristics of these proteins.
    Application Progress of mazF Gene in Genetic Modification System
    Shi Yang,Dong Huina,Zhang Dawei
    2014, 30(11):  48-54. 
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    Toxin-antitoxin system(TA)exists in most of the genetic material of bacteria. mazEF is a kind of toxin-antitoxin system in E. coli. MazF encoded by mazF is an mRNA interferase that specifically cleaves free mRNAs at ACA sequences, resulting in inhibited protein synthesis and cell growth arrest. Recently, some scholars have used mazF as a counter-selection marker in genetic modification systems, and achieved modifications of the genome in various strains. In this article, the research advances of E. coli mazF used as counter-selection marker in bacterial genetic modification were reviewed, followed by application of other toxin gene. Finally, perspective of the possible new pathways for developing new genetic modification methods were addressed.
    Advanced in Microbiological Agent of Straw Degradation Under Low Temperature
    Zhao Xu ,Wang Wenli, Li Juan, Hu He
    2014, 30(11):  55-61. 
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    The components of straw are cellulose, hemicellulose and lignin. Straw degradation microorganisms including bacteria, actinomyces and fungi. The methods of breeding straw degradation microorganisms include screening from nature, mutation breeding, protoplast fusion breeding, gene engineering breeding and so on. At present, the straw degradation strains under low temperature can be screened few in number, the ability of straw degradation is confined, further study need to be done on the mechanism of low temperature straw degradation. Microbiological agent of straw degradation under low temperature was summarized.
    Progress in Studies of Ligninolytic Enzymes and Genes
    Dong Xiuqin,Yuan Hongli,Gao Tongguo
    2014, 30(11):  62-72. 
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    Efficient enzymatic conversion of renewable biomass becomes the focus of intensive research currently throughout the world. Lignocellulose is comprised mainly of cellulose, hemicelluloses and lignin. Removal of lignin from the complex lignocellulosic matrix is considered as the key process of comprehensive lignocellulose utilization, which renders recalcitrant lignocellulosic biomass more accessible to the hydrolytic enzyme system. Biodegradation of lignin by fungi is more environment friendly and less energy intensive, compared to other pretreatment methods. Its mechanism is based principally on the activity of different extracellular enzymes. Here we reviewed the recent progress in characteristics of fungal lignin-degrading enzymes, including lignin peroxidase(LiP), manganese peroxidase(MnP), laccase and versatile peroxidase(VP), and also their applications in genetic engineering and genomics research.
    Function of MicroRNAs During the Differentiation Process of Brown Adipocytes
    Hao Meilin, Huang Ying, Huang Limei, Yang Minghua, Li Qihua ,Jia Junjing ,Zhao Sumei
    2014, 30(11):  73-78. 
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    MicroRNAs(miRNAs)are small, non-coding regulatory RNAs, which negatively regulate post-transcriptionally gene expression. miRNA plays the role in many biological functions including controlling the developmental timing, regulation of cell differentiation and apoptosis, organ development, fat metabolism. Recent studies indicated that the expression levels of transcriptional factors related genes which were important for the development of brown adipocyte tissue was affected by miRNAs directly or indirectly. This article reviewed the recent progress on the function of miRNA during the differentiation process of brown adipocytes.
    The Diversity of Small Non-coding RNA
    Huang Junjun, Wang Huahua, Liang Weihong
    2014, 30(11):  79-83. 
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    RNA is a ubiquitous family of large biological molecules that perform multiple vital roles. There are many RNA types with different structure. RNA has many important biological functions for the diversity and structure. Non-coding RNA(ncRNA)show unprecedented complexity and diversity along with further research on ncRNA. This paper mainly introduced the structure and function of two categories, housekeeping ncRNA and regulation ncRNA, such as tRNA, snRNA, scRNA, rRNA, siRNA, miRNA, piRNA and nat-siRNA, in order to understand the small ncRNA diversity in vivo. It provides accessible reference source of further mining and utilization of ncRNAs and new insights on RNA recognition and status.
    The Research of Genome Editing
    Wu Lu ,Wang Lei ,Ren Yuan, Yuan Hui
    2014, 30(11):  84-90. 
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    Genome Editing can be used to operate artificially almost any gene in cells or organisms. This research method of gene function is useful for fundamental research and clinic treatment. The major component of this tool is artificial nuclease with specific DNA-binding domain and non-specific DNA-modifying domain. This article reviews the characteristics, principle, construction and application of ZFN, TALEN and CRISPR/Cas, which would be useful reference for related research.
    Research Progress in Breeding Technique of Sugarcane Healthy Seedlings or Seedcanes
    Liang Yongjian,Zhang Xiaoqiu,Yang Liu,Yang Litao,Li Yangrui
    2014, 30(11):  91-96. 
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    Sugarcane healthy seedlings or seedcanes are those sterilized by hot water treatment or axillary shoot tip culture, including axillary plantlets, asexual seedcanes for propagation or commercial production, which are produced under strict isolation conditions and meet certain criteria. In recent years, a temporary immersion bioreactors system(TIBs)has been developed and applied, which speeds up the multiplication of the healthy seedlings, improves the multiplication coefficient, and reduces the labor requirement. More recently, a new technique so-called ‘Plene’ has been developed, which substantially reduces the seedcane rate, and is good for mechanical operation so reducing the planting cost.
    Analysis on Genetic Diversity of Hainan Upland Rice Local Varietiesand Establishment of Molecular ID with SRAP Markers
    Yao Yufei,Wang Ying,Zhuang Nansheng,Gao Heqiong
    2014, 30(11):  97-106. 
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    SRAP markers were used to analyze the genetic relationships of 28 upland rice local varieties from Hainan province. Forty-six primer combinations with abundant polymorphism were selected from 255 primer combinations, and 18 primer combinations were picked to amplify the 28 material. A total of 180 bands were scored, and 160(87.75%)out of them were polymorphic, with an average of 10 bands and 8.9 polymorphic bands for each primer combination. Cluster analysis using UPMGA method showed that the genetic similarity coefficient ranged from 0.687 0 to 0.921 2 among the test upland rice varieties. The tested materials were classified into two clusters at the similarity coefficient of 0.687 0, and cluster I included Hainan shanlan upland rice;the cluster Ⅱ involved upland rice poya and varieties developed by our team. The clustering result of Hainan upland rice varieties showed close genetic relationship among Shanlan upland rice varieties. The Hainan upland rice poya might has some genetic similarities with nonglutinous rice germplasms. In addition, we established the molecular identity card of 28 Hainan upland rice varieties with SRAP markers. All these results provided the basis for the Shanlan upland rice heredity relationship and the Shanlan upland rice germplasm resources’ appraisal and the new variety’s breeding.
    Enhanced Oxidative Stress Tolerance of Transgenic Rice Plants Overexpressing OsPHGPx Gene
    Song Jianhui,Li Tian,Wu Xiuyun,Wu Fuqing,Liu Jinyuan
    2014, 30(11):  107-113. 
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    Plants can express phospholipid hydroperoxide glutathione peroxidase(PHGPx)to resist oxidative stress caused by biotic or abiotic stress. But the physiological role of PHGPx in conferring stress defence is still unclear. In this study, agrobacterium genetic transformation technology was successfully used to obtain transgenic rice overexpressing OsPHGPx. PCR, Real-time PCR and Western blot analysis was carried out on the transgenic rice, and all the results showed that OsPHGPx gene was transformed into the rice successfully. In addition, compared with the non-transgenic rice, seedlings of transgenic lines demonstrated enhanced tolerance to oxidative stress caused by paraquat.
    Clone and Function Analysis MiR160f in Common Wild Rice(Oryza rufipogon Griff.)
    Yang Songnan,Wang Jiao,Chen Zongxiang,Tian Xinjie,Zhang Jingwen,Long Yan,Pei Xinwu,Yuan Qianhua
    2014, 30(11):  114-118. 
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    MicroRNAs are a class of non-coding RNAs involved in post- transcriptional control of gene expression. Former study on Arabidopsis thaliana reveals that miR160 involves in root cell division and differentiation by regulating auxin response factors(ARF)thus influence the root development. This study cloned miR160f gene from common wild rice and transferred into Arabidopsis thaliana to identify its function. The results showed that over-expression of miR160f decreased the number of rosette leaves, shortened bloting time, leading to early flowering. RT-PCR showed the expressions of gene ARF10,ARF16 and ARF17 are down-regulation caused by miR160f in transgenic Arabidopsis thaliana, while the deficiency of ARF10 and ARF16 protein can inhibit root cap cell differentiation, lose control of cell division and lead to ectopic expansion of apical stem cell populations Therefore, the result demonstrates that miR160 not only influence on root development, but also may influence flowering time of common wild rice.
    Functions of Potato Tuber Formation-related Gene STI-LIKE in Phenotype of Arabidopsis thaliana|
    Ping Haitao,Wang Dongxia,Zhou Xiaojie
    2014, 30(11):  119-124. 
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    A series of gene’s expression and silencing were involved in potato tuber formation. The full length of potato STI-LIKE gene was cloned by using the RT-PCR(reverse transcription PCR)method from the potato variety “Atlantic”. The expression of STI-LIKE gene in all potato organs were analyses by real time PCR analysis. The results showed that STI-LIKE gene expressed in all potato organs. The bioinformatics analysis indicated that STI-LIKE protein contains three TPR domains, and the STI-LIKE gene was homologous recombination in higher plants. The expression vector(pBI121)and interference vectors(pHANNIBAL and pART27)were constructed and transferred to Arabidopsis thaliana. The STI-LIKE protein expression level was examined by Western blot with specific antibody. The results indicated that STI-LIKE gene might be associated with the growth and development of seedling of Arabidopsis.
    Quantitative Analysis on Feedback of AGPase to Photosynetic Rate in Potato
    Bai Hua,Cui Xueqiong,Bai Shaoxing,Yao Xinling
    2014, 30(11):  125-129. 
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    To reveal feedback regulation of ADP-pyrophosphrolase(AGPase)to photosynthesis, AGPase activities(AA), starch contents(SC)in tuber, photosynthetic rates(PR), chlorophyll contents(CC)and sucrose contents(SUC)in leafs were assayed with potato transgenic lines showing difference on ADP-pyrophosphrolase activities. The data analysis showed that AA, SC and PR in transgenic lines were significantly higher than control line. Significantly differences were found between AA, SC and PR among the lines. Correlation analysis showed that there were positive correlations between AA, SC and PR. Per unit rising of AGPase activities can contributed 2.5% increase of starch contents in sink tissue. AGPase was a factor playing a role in regulation of photosynthetic rates. The result indicated that AGPase not only regulates starch accomulation on the downstream, also feedback to modulate photosynthetic rate.
    Cloning and Expression Analysis of Sugarcane Isoflavone Reductase-like(IRL)Gene
    Xie Xiaona,Zhang Xiaoqiu,Shao Min,Zhu Hui,Yang Litao,Li Yangrui
    2014, 30(11):  130-137. 
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    The SoIRL gene cDNA sequence was cloned from sugarcane variety GT11 using RT-PCR techniques. The bioinformatics methods were used to analyze the putative amino acid sequence, and Real-time PCR method was used to study the expression of SoIRL gene in different tissues and under different stresses. The results showed that the full-length cDNA of SoIRL(GenBank accession number:KF808324)in sugarcane was cloned. The sequence consists of 1 167 bp with an intact open reading frame of 927 bp, encoding a polypeptide of 303 amino acids. Phylogenetic tree analysis indicated that SoIRL was highlg closely related to IRL of Zea mays. Real-time PCR results showed that the SoIRL expressed in root, stalk and leaf. Furthermore, SoIRL transcription level was induced under the treatment of the bacterial of ratoon stuning diaease, low temperature, PEG, NaCl and ABA stresses, but the expression patterns were different. Gene SoIRL was firstly isolated and characterized from sugarcane(GT11), which may participate in sugarcane resistance to RSD, and also play a role in the sugarcane resistance to chilling, drought and salt stress environments.
    Growth Characteristic and Insect-Resistance of Transgenic Populus alba Populus hopeiensis
    Chen Yuchao ,Zhang Li Gong, Lei Gan ,Xiaoyan, Shi Lei ,Song Yuxia
    2014, 30(11):  138-141. 
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    The stability of insect-resistance gene Cry3A, phenophase, growth status and insect-resistance of transgenic poplar hybrid(Populus alba×Populus hopeiensis)was investigated, non- transgenic Populus alba×Populus hopeiensis as control. The results showed that the Cry3A gene in transgenic Populus alba×Populus hopeiensis integrated stably. The Cry3A gene could be expressed at RNA level and protein level in root, shoot and leaf. The Bt protein level was significantly different in different tissues, and was highest in shoot. The phenophase and growth status of transgenic Populus alba×Populus hopeiensis changed indistinctively. But it showed distinct insect-resistance.
    Soluble Expression and Activity Analysis of Mallard IFN-α
    Liu Lanlan,Zhuang Yanna,Yu Xiaohong,Zeng Xiangwei
    2014, 30(11):  142-146. 
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    In order to acquire efficient expression and production of biologically active duck IFN-α, the mature protein gene of duck IFN-α was amplified from pMD18T-MaIFN-α. The fragment was linked to the prokaryotic expression vector pET-32a to construct the recombinant expression plasmids pET-32a(+)-MaIFN-α, and then converted into E.coli BL21 cells. The fusion protein was induced to express in the change temperature condition. SDS-PAGE and western blotting analysis were used to examine the fusion protein. After purified by Ni2+ resin column, the activity of the expression product was determined. The results showed that the recombinant pET-32a(+)-MaIFN-α expressed a soluble protein after being induced by IPTG. Western blotting analysis showed the fusion protein had expected antigenicity. The activity of the expressed duck IFN-α detected by inhibiting cytopathic effect was about 8×104 U/mL. The activity detected by the Real-time PCR showed that the expressed duck IFN-α had a strong inhibition of NDV replication in DEF. This indicated that the expressed duck IFN-α was verified to be of high antiviral activity.
    Cloning and Sequence Analysis of MyoD Gene in Red Tilmpa Oreochromis sp.(O.mossambicus×O.niloticus)
    Guo Yihui ,Fan Sigang, Huang Guiju, Zhu Kecheng, Yu Dahui
    2014, 30(11):  147-151. 
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    As a member of myogenic regulatory factors(MRFs), MyoD play a positive regulation in the process of muscle proliferating.In this study, MyoD cDNA of Red Tilmpa Oreochromis sp.(O.mossambicus×O.niloticus)was obtained using homology cloning strategy and rapid cDNA end amploification. The results showed that the open reading frame(ORF)of MyoD was 903 bp length and encoded 300 amino acids, including basic helix-loop-helix(bHLH)domain which composed of 1st to 114th amino acids and 109th to 165th amino acids, CDK4 combined structure(199th to 213th)and Helix III structure(233th to 249th). The signal peptide was not detected in MyoD. The secondary structure of MyoD protein is HLH dimmer. The phylogenetic trees was constructed based on some fish MyoD ORF and suggested that Oreochromis sp. O. aureaus and O. niloticus were clustered together firstly.
    Cloning and Tissue Expression Analysis of PAX7 Gene in Grass Carp
    Gan Tian ,Leng Xiangjun, Guo Ting, Hu Jing, Wei Jing, Li Xiaoqin
    2014, 30(11):  152-158. 
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    Paired box gene 7(PAX7)is crucially important to the cellular renewal, differentiation and apoptosis, especially in neural crest development, gastrulation, and muscle self-renewal. The PAX7 domains sequence is conserved among several species, such as zebrafish, apteronotidae, and rainbow trout, and the conserved sequence zebrafish was used to design degenerate primers for reverse-transcription PCR(RT-PCR). A partial sequence of PAX7 from grass carp was obtained for a 645 bp segment encoding a 214 amino acid peptide, containing a paired box domain with 128 amino acids. The deduced protein showed homology to zebrafish(Danio rerio), apteronotidae(Sternopygus macrurus), Japanese Medaka(Oryzias latipes), gilthead bream(Sparus aurata), rainbow trout(Oncorhynchus mykiss), Atlantic salmon(Salmo salar), Arctic charr(Salvelinus alpinus), human(Homo sapiens), wild yak(Bos grunniens mutus), brown rat(Rattus norvegicus), and mouse(Mus musculus)with 90%-97% identities. Analysis of the PAX7 phylogenetic tree revealed that the grass carp joined with zebrafish, and there was a confluence of Japanese medaka, Arctic charr, Atlantic salmon, rainbow trout, apteronotidae and gilthead bream. The other branch was consisted of mouse, brown rat, wild yak and human. These results conformed to the traditional species classification evolution status. The expression in tissues was detected by Real-time quantitative PCR, which indicated that the highest level of PAX7 gene expression was found in muscle, a lower expression level was found in foregut and skin, and the lowest detectable level was found in heart, brain, kidney and liver. The results are in agreement with the function of the gene.
    The Expression of Cathepsin B in the Different Ovary Development Stages of Marsupenaeus japonicus
    Cheng Cheng ,Lin Peng
    2014, 30(11):  159-163. 
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    The expression of Cathepsin B is tested in the different ovary development stages of Marsupenaeus japonicus by Real-time PCR. The results show Cathepsin B is in the highest level in the previtellogenic stage(PR), medium level in the endogenous vitellogenic stage(EN)and exogenous vitellogenic stage(EX). However the expression suddenly declines and is in the lowest level in the maturing stage(MA). Cathepsin B of PR has significant difference compared with that of EN, EX and MA respectively(P<0.05). There is no significant difference between EN and EX stages(P>0.05). The expression of Cathepsin B in MA is significantly different from that in other three stages(P<0.05). The results of Real-time PCR indicate that Cathepsin B may play one important role in the ovary development of Marsupenaeus japonicus.
    Cloning and Bioinformatics Analysis of the Phosphoenolpyruvate Carboxylase(Chpepc2)Gene from Marine Chlorella sp.
    Huang Xiwen,Shi Dingji,Jia Xiaohui,Tian Qilin,Jia Rui,He Peimin
    2014, 30(11):  164-173. 
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    To further understand the characteristics of algae PEPCase, marine Chlorella sp. pepc2 gene was cloned and analyzed the structural and functional characteristics of marine Chlorella sp. PEPCase2 by bioinformatics method. The experimental results showed that, the cDNA sequence of marine Chlorella sp. pepc2 gene, compared with the cDNA sequence of Chlamydomonas reinhardtii pepc2 gene, the similarity was 90%, and its amino acid sequence has the same characteristics with C. reinhardtii PEPCase2, so this gene encodes the bacterial-type PEPCase of marine Chlorella sp.;The secondary structures of marine Chlorella sp. PEPCase2 mainly include α-helix, β-turn, random coli and extended strand, and its tertiary structure is a parallel β-barrel structure in the centre with α-helix structures outside;marine Chlorella sp. PEPCase2 has many important physiological functions, mainly related to a variety of important material synthesis.
    Expression of FtsZ Protein from Xanthomonas oryzae in Escherichia coli
    Chen Yang, Huang ,Yunhong, Li Suzhen, Long Zhonger
    2014, 30(11):  174-178. 
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    It was to prepare FtsZ protein using techniques of modern molecular biology. The ftsZ gene was amplified from Xanthomonas oryzae by nested PCR, and recombinant plasmid pET-22b-ftsZ was constructed and transformed to E.coli BL21. The clony fragment was identificatified by PCR screening, Nde I/Xho I digestion and DNA sequencing, the positive clones were induced by IPTG for expression;the fusion protein was purified through Ni-NTA Resin, and identified by SDS-PAGE and Western blotting. Results showed that the recombinant plasmid pET-22b-ftsZ was constructed successfully, the FtsZ-6×His fusion protein was expressed in recombined E. coli BL21 induced by IPTG, and purified through Ni-NTA Resin by electrophoretic purity.
    Lactobacillus pentosus Fermentation Lactic Acid Production and High Yield Strain Mutation Breeding
    Wang Yiqiang,Wang Qiye,Ma Guohui,Lin Liyun
    2014, 30(11):  179-186. 
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    Lactobacillus pentosus is a potential strain which can make use of the lignocellulose hydrolysate fermentation to produce lactic acid, optimization of fermentation conditions and the breeding of high yield strain are the important means to improve lactic acid production. Fermentation medium and fermentation conditions were optimized for strain Lactobacillus pentosus ATCC 8041, through single factor experiment, Plackett-Burman design and response surface experiment. The results showed that the best combination of fermentation medium is 93.11 g/L glucose, 5.19 g/L yeast powder, 29.43 g/L calcium carbonate, 10.00 g/L peptone, 5.00 g/L Na2HPO4?12H2O, 0.20 g/L MgSO4, 50 mg/L MnSO4. The best fermentation conditions is temperature 37℃, pH6.5, quantity of 6%, and fluid amount 80%. The lactic acid production is 54.12 g/L under the optimized fermentation medium and fermentation conditions. And then, Lactobacillus pentosus ATCC 8041 was taken as the original strain, treated with UV mutation ATCC 8041 protoplast, after through multiple screening, finally we have got a mutant which has high lactic acid production, named Lactobacillus pentosus lactic UVC-02 which conserved in China Center for Type Culture Collection, registration number for CCTCC M 2013209. The results for glucose fermentation showed that lactic UVC-02 produced 64.17 g/L lactic acid, which was 18.6% higher than the original strain.
    Isolation, Identification and Enzymatic Characterization of a Cellulase-Producing Bacteria
    Jia Bohan ,Zhou Wei, Zhao Luodi ,Yang Pu ,Gou Min
    2014, 30(11):  187-192. 
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    A cellulase-producing bacteria strain JJ-3 with high activity was isolated from the soil containing rotted leaves, which was identified as Klebsiella oxytoca based on the 16S rRNA sequence analysis. The optimal enzyme-producing conditions of strain JJ-3 was investigated and was as follows:filter paper as carbon source, peptone as nitrogen source, pH8.0, three-day fermentation. And the fermentation culture had higher FPase activity under neutral and alkaline conditions, with 118.7 U/mL(pH7.0), 167.8 U/mL(pH8.0), 120 U/mL(pH9.0). The results of enzymatic characterization showed that the optimal pH and temperature for enzyme reaction was 7.0 and 40℃, respectively. FPase was sensitive to temperature, while it kept good stability during the pH7.0 to 8.0. This study suggested that the cellulase from strain JJ-3 meets the request of neutral and alkaline cellulase.
    Expression, Crystallization and Substrate Binding Studies of Human Histone N-terminal Acetyltransferase Nat11
    Huang Jiaxin ,Li Haitao
    2014, 30(11):  193-200. 
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    The alpha-amino groups of histones H4 and H2A can be acetylated by histone N-terminal acetyltransferase 11(Nat11), which plays an important role in epigenetic regulation. The cDNA of human Nat11 was amplified and cloned into pSUMO vector. The resultant construct was transformed into E.coli strain BL21(DE3)for recombinant protein expression. Homogenous Nat11 was highly purified through a series of purification procedures including nickel column affinity chromatography. Using isothermal titration calorimetry(ITC), we measured micromolar binding constants between Nat11 and histone H4 peptides. MALDI-TOF mass spectrometry analysis revealed that purified Nat11 was pre-bound with acetyl coenzyme A or coenzyme A that was co-purified from E.coli. After ITC titration using unmodified peptide as ligand, N-acetylated product was detected by mass spectrometry, suggesting that the purified Nat11 is active. We performed crystallization screening and successfully obtained single crystal of a truncate form of Nat11 and substrate-enzyme recombinant protein after optimization.
    MTMR2 Interacts with 14-3-3 and Regulates H2O2 Induced Cell Senescence
    Li Chaoqun,Chen Yali,Xiao Dongguang,Pei Huadong
    2014, 30(11):  201-205. 
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    H2O2-induced cell senescence plays an important role in tumorigenesis. To further study the underlying mechanism, we identified five relevant members through a genome wide shRNA screening and took MTMR2(Myotubularin-related protein 2) as an example to figure out its function in H2O2-induced cell senescence. By plasmid construction and expression in combination with purification and mass spectrometry, we found a significant interacting protein of MTMR2:14-3-3 proteins family, and their interaction was then confirmed using biochemical methods.
    The Codon Optimization and Fused Expression of LL-37 and IFN-α2a in Pichia pastoris GS115
    Zhang Mingjie
    2014, 30(11):  206-213. 
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    To express the fussed protein of human LL-37 and IFN-α2a in Pichia pastoris with codon optimization. Recording to the codon preference of P. pastoris, the codons of LL-37 and IFN-α2a were optimized. A flexible linker of GlyGlyGlyGlySer was placed between LL-37 and IFN-α2a. The newly scheming DNA sequence was synthesized chemically. P. pastoris GS115LI was constructed by integrating the new gene into GS115 with help of plasmid pPIC9K. With the scanning of geneticin concentration ladder, two strains, named as GS115LI1 and GS115LI2, were gotten with high copy number. With SDS-PAGE detection, anti-virus activity detection and antimicrobial activity detection of broth, the results proved GS115LI1 and GS115LI2 not only expressed the fused proteins, but also maintained the activities of LL-37 and IFN-α2a respectively. After fermentation and induction of recombinant, the productivity of fusion protein was 819.1 mg/L. With salting out, hydrophobic chromatography and ionic exchange chromatography, the purity of fusion protein was 97%, recovery rate was 46.2% and the potency of IFN was 2.6×108 IU/mg. After codon optimization, the fused protein of human LL-37 and IFN-α2a was expressed effectively in P. pastroris with antimicrobial activity of LL-37 and antifungus activity of IFN-α2a.
    The Process Optimization of Pichia pastoris GS115LI for Producing Fused Protein of LL-37 and IFN-α2a
    Zhang Mingjie
    2014, 30(11):  214-218. 
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    With experiments of single factor, the optimal process of recombinant P. pastoris GS115LI for producing fused protein of LL-37 and IFN-α2a were using BMMY as inducing medium, inoculum as OD600=5.5, temperature as 26℃, pH6.0, inducing chemical(methanol)adding amount as 1.0% every 24 h, incubating shaking speed as 200 r/min, and inducing time as 144 h. Under those optimal conditions, the 27.9 mm of LL-37 antimicrobial activity could be gotten. Compared with the original conditions, the productivity of fused protein of LL-37 and IFN-α2a was increased as 32.29%.
    Preparation and Analysis of Lyophilization Characterization of Human D-dimer
    Wu Jianwei ,Cai Lei ,Wang Jihua, Tang Shixing
    2014, 30(11):  219-224. 
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    D-dimer is formed by sequential action of 3 enzymes from fibrinogen. After producing D-dimer, we did the research on its lyophilization process. To produce cross-linked fibrin, fibrinogen was digested by thrombin and Factor XIIa. Then, fibrin degradation products(FDP)was prepared from cross-linked fibrin being degraded by plasmin. D-dimer was purified from FDP through ultrafiltration and prepared into lyophilized powder. By optimizing the lyophilization program, lyophilized D-dimer was stable for at least 12 days at 37℃, stable for at least 8 days at 25℃ and 30 days at 4℃ after reconstitution, and not different when being dissolved by different solvent.The preparation system established in this research is feasible and efficient. Depend on its stability, lyophilized D-dimer could be provided as biological raw materials for further research.
    Application of Auto-induction System in the Synthesis of 2’-deoxycytidine
    Wang Xi,Duan Shenglin,Xiong Shuli,Zheng Guilan,Zhang Guiyou,Wang Hongzhong
    2014, 30(11):  225-232. 
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    This experiment was carried out in order to synthesize 2’-deoxycytidine(dCyd)more efficiently. DCyd is a significant intermediate for many antiviral and anticancer drugs such as Gemcitabine(dFdC), Dideoxycytidine(ddC), etc. A convenient and efficient bioprocess was reported here to synthesize dCyd by E.coli BL21(DE3)containing gene of N-deoxyribosyltransferaseⅡ(NDT)over-expressed using ZYM-Fe-5052 auto-induction system. The results showed that auto-induction system could make the addition of inducer during cultivation unnecessary and obtain a higher cell density. The recombinant cells from auto-induction system were as efficient as that from LB system on the conversion yields of dCyd. When the ratio of 2’-deoxythymidine(dThd)and cytosine was 1∶3, the conversion yield could be 86.5%. The substrates ratio of 2’-deoxythymidine (dThd) and cytosine as 1∶1.5 and 1 mg/mL recombinant cells which were dissolved in phosphate buffer of pH6.5, then cultured at 30℃ for 1 h were selected as the suitable reaction conditions,and the conversion yield could be 71.9%. This method also has potential for the preparation of other nucleoside analogues.