Table of Content

19 June 2015, Volume 31 Issue 6

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Review

Research Progress on Biological Functions of Long Non-coding RNA in Plants

Niu Xulong, Feng Wanjun, Ma Jinhu, Xing Guofang

2015, 31(6):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.001

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Long non-coding RNAs(lncRNAs)as newly discovered regulators for gene expression, participate in a wide range of biological processes in plants, and also could response to environmental stresses. In this review, we summarize the generation and classification of lncRNAs, the discovered lncRNAs in plants, their effects on the development processes of different organs in plants, as well as the relationship between lncRNA and small RNA, research methods, and issues in the current studies of lncRNAs.

Activation and Regulation on the Cold Response Pathway of ICE1-CBF in Plants

Wei Junyan, Zhao Jia, Zhao Shiqi, Zhou Qiying, Yuan Zhengfang, Li Xianwen

2015, 31(6):  8-12.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.016

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Cold stress signal is sensed while changes of cell membrane fluidity in plant cells cause the changes of Ca²⁺ influx. The rising of Ca²⁺ in cytoplasm leads to the changes of activities of various calcium-regulating proteins in plant cells, then cold-responsive genes are activated by cascade reactions, and thus plant resistance to low temperature is enhanced. At present, it is almost certain that the main path of activating cold-responsive genes is ICE1-CBF regulatory pathway. In this paper. We review the recent studies of the cold signal sensing and transduction, expression activation and regulation of cold-responsive genes in plant cells, which lays the theoretical foundation for the further study of cold acclimation of plants.

Research Progress of Transcription Factors in Solanaceae Plants

An Liyu, Wang Zhimin, Tang Qinglin, Wang Yongqing, Yang Yang, Tian Shibing, Song Ming

2015, 31(6):  13-19.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.002

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Transcription factors are of DNA-binding proteins which can interact with eukaryotic gene promoter regions, specifically activate or inhibit gene’s transcription by interacting with other proteins or between them. Transcription factors are almost involved in the entire development process of Solanaceae(vegetative growth, reproductive growth and response to the external environment, etc). The research progress of several main transcription factor families such as MYB, NAC, WRKY, MADS and AP2/ERF in Solanaceae plants were reviewed, which is expected to provide a reference for the research and utilization of Solanaceae plants.

Regulation Mechanism of Anthocyanin Synthesis in Purple Shoots of Tea by Lighting

Jin Qifang, Sun Weijiang, Chen Zhidan

2015, 31(6):  20-27.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.003

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Purple shoots of tea as a specific tea germplasm with the characteristics of high anthocyanin and purplish red have earned the attentions from scholars. Studies on the biosynthesis pathway of anthocyanin and its key genes in plants have become a hot spot；currently the main regulatory genes and structural genes associated with anthocyanin synthesis and metabolism have been cloned and identified from different species. Previous studies have shown that anthocyanin synthesis and metabolism are closely correlated with the environment, and lighting is the major affecting factor, however its regulation mechanism is uncertain yet. Summarizing the regulation of lighting on anthocyanin in other purple plants, and further exploring the regulation of lighting on the genes related to anthocyanin synthesis in the purple shoots of tea provide the basis for the study of specific tea germplasm and breeding biotechnology.

Metabolic Engineering of Seed Oil in Camelina sativa L. Crantz，a New Type of Industrial Oilseed Crop

Yuan Lixia, Mao Xue, Yang Zhirong, Xue Jin’ai, Li Runzhi

2015, 31(6):  28-36.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.006

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Camelina sativa L. Crantz is a new and potential industrial oilseed crop with low input and environment-friendly. The seed yields 43%(W/W)oil with α-linolenic acid content over 35%. The seed oil can be used for edible oil and animal feed, health-promoting food, biofuel oil and high-valued oil chemicals. In this study we characterize the important agronomic traits and the excellent oil properties of camelina. Particularly, we describe the genetic improvements of the seed oil quality by gene engineering, which include pathway assembly of long chain polyunsaturated fatty acids, ω-7 fatty acids, wax esters, medium- and short-chain fatty acids, and enrichment of single fatty acid. We also dissect and discuss trends in camelina functional genomics and the prospects for development of camelina commercialization as a new bioenergy oilseed in China.

Research Advances on Antibacterial Mechanism of Bacillus amyloliquefaciens

Chen Zhe, Huang Jing, Zhao Jia, Wang Changbiao, Liang Hong

2015, 31(6):  37-41.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.004

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Bacillus amyloliquefaciens produces a wide range of antibacterial substances which effectively inhibit fungal and bacterial activities. In recent years, more and more studies have focused on antibacterial mechanism of B. amyloliquefaciens, and most of them already at the molecular level. Hence, we review the research progress on the genome sequences and antibacterial mechanism of B. amyloliquefaciens in this study.

Research Advances in the Regulation Mechanism of Mitophagy

Wang Zhishu, Tan Xiaorong, Liu Huanhuan

2015, 31(6):  42-47.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.005

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Mitochondria provide energy and materials for cells, while a variety of factors can lead to damage, aging and dysfunction of mitochondria, and they are potentially dangerous for the cells and must be cleared promptly. Mitophagy can fulfill above task and maintain cell homeostasis. Under some severe conditions, mitophagy supplies living-essentials by degrading mitochondria and helps the cells survive. Additionally mitophagy may play a role in controlling the quantity and quality of mitochondria through degrading some normal mitochondria. There are different pathways and mechanisms in different organisms. In yeast, mitophagy is mainly regulated by phosphorylation of Atg32. In mammals, mitophagy is protein-mediated by Parkin-PINK1, Nix and FUNDC1 respectively. Research on mitophagy in plants is mainly focused on Arabidopsis thaliana only, and the mechanism is not well understood yet. Here we review the research advances in mitophagy in yeast, mammals and plants, with focus on the mechanisms and factors involved.

Research Advances on Tumor Cell Metabolism Regulated by PKM2

Xu Kun, Wang Ziying

2015, 31(6):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.007

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Tumor cell metabolism is exemplified by high glucose consumption and lactate production. The M2 isoform of pyruvate kinase(PKM2)plays a vital role in this metabolic phenotype, experiments both in vitro and in vivo revealed that over-expression of PKM2 would increase the Warburg effect and promote the tumor growth. However, the mechanisms of how PKM2 regulates tumor cell metabolism are still not completely understood. Current researches have elucidated novel PKM2 regulatory mechanisms. In this review the current understanding is summarized and future directions in this field are explored, highlighting controversies regarding the activity and specificity of PKM2 in tumor. Finally, the potential therapeutic implications and strategies are discussed.

Development Status and Trend Analysis in Aquaculture Vaccines

Wang Zhongliang, Wang Bei, Lu Yishan, Wu Zaohe

2015, 31(6):  55-59.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.008

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Aquaculture vaccines not only boost immunity of aquatic animals and prevent aquaculture diseases, but also reduce the use of drugs, lower the cost of aquaculture reproduction and solve problems of food safety and environment pollutions caused by drug residues, and finally lead aquaculture to develop in the green and sustainable direction. As thus, aquaculture vaccines become one of the research hotspots in the diseases control for aquatic animals. In this paper, the history of vaccine development, vaccine types, and vaccine delivery methods are summarized. The status and trend of key technologies in aquaculture vaccines are introduced.

Method

Multiplex Automated Genome Engineering

Li Dan, Gao Haijun

2015, 31(6):  60-66.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.009

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Genome editing is widely used in genome engineering research and site-specific nuclease technologies and CRISPR/Cas system focus on single gene editing. Owing to the huge size of genome, there are some limitations on the applications of these technologies. Multiplex Automated Genome Engineering(MAGE)is a new, fast and efficient genome editing technology, which can operate multiple genes simultaneously, and be used in knockout and replacement of Escherichia coli genes. This review illustrates the recent advances in the theory, operation protocol and technological innovation of MAGE, its application and development trend were also discussed.

Principle and Application of Microscale Thermophoresis in Studies of Biomolecular Interactions

Ai Qiushi, Cao Xiangyu, Zhao Qian, Niu Yali, Song Shuishan,

2015, 31(6):  67-73.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.010

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Microscale Thermophoresis(MST)is a new technique to study biomolecular interactions in recent years. It is based on thermophoresis, i.e., the directional movements of molecules in a temperature gradient, which results in subsequent changes of molecular properties such as sizes, charges, hydration shell and conformation. MST combines the precise fluorescence detection and sensitive thermophoresis and provides a sensitive, fast and precise detection technique to analyze biomolecular interactions. Wor­king principle, detection process and application of MST in biology researches are reviewed here.

Application of Molecular Markers in Studies of Camellia oleifera

Dong Bin, Li Rongxi, Huang Yongfang, Hong Wenhong, Tan Sha,

2015, 31(6):  74-80.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.011

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Recently the molecular marker techniques develop rapidly and are widely used in studies of Camellia oleifera. Owing to advantages of high-efficiency and reliability with molecular marker technique, its rapid development offers fast and efficient measure in genetic diversity analysis, cultivar identification, valuable gene mapping, molecular marker-assisted selection and the construction of molecular genetic maps. The applications of several molecular marker techniques, i.e., RAPD, AFLP, SSR, ISSR and SRAP etc. in studying C. oleifera are reviewed. The current problems and the focuses in the future researches are also discussed while utilizing the molecular marker in studying C. oleifera.

Application of 3 DNA Fingerprinting Techniques in Strain Identification

Liu Yi, Yao Su, Li Hui, Liu Yang, Liu Yong, Liu Bo, Cheng Chi

2015, 31(6):  81-86.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.012

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Four strains of Geobacillus were analyzed with 3 DNA fingerprinting techniques of ERIC-PCR, BOX-PCR and RAPD for searching the proper strain identification measure for the genus especially Geobacillus stearothermophilus. Each of 4 strains of G. stearothermophilus had different fingerprint. The bands by simple and quick ERIC-PCR were quite clear, and the different strains could be recognized clearly. The bands by BOX-PCR were less, and the strains of the same bacteria could not be recognized clearly. More bands could be obtained by RAPD with higher identification；however the workload of identification and cost increased significantly. Three techniques may effectively identify the strains of Geobacillus, and ERIC-PCR is the most effective and fast method to identify strains of G. stearothermophilus.

Fast Detection of Freshness of Chilled Pork by Real-time Quantitative PCR

Song Yanmin, Shi Limin, Xu Yuancong, Xu Wentao, Huang Lan, Liang Zhihong,

2015, 31(6):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.013

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The current detection techniques for freshness of chilled pork are complex, so exploring fast detection techniques becomes hotspot. In this study Total Volatile Basic Nitrogen(TVB-N)was detected by the method defined in national standard, and Plate Culture Method(PCM)and real-time quantitative PCR were used to count the total amount of microorganisms causing spoilage of chilled pork. With the extension of storage time of the chilled pork at 4℃, the total number of microorganism colony and TVB-N had the linear correlation, and the level of spoilage of chilled pork behaved the positive correlation. The extracted bacterial genome by acetone-chloroform had the clear bands and showed the fine extraction result. Single-factor analysis of variance revealed that there was no significant difference of total number of colony(P=0. 7190)between by real-time quantitative PCR based on SYBER Green I and by counting in PCM, i. e. , consistency was quite solid. The detection time was reduced by real-time quantitative PCR with higher detection sensitivity. The amount of microorganisms is a crucial index to evaluate the freshness of chilled pork, and real-time quantitative PCR can be a new technology for quickly checking the freshness of chilled pork.

Report

Construction of DNA Fingerprinting and Analysis of Genetic Diversity for Wheat Varieties(Lines) in Regional Test of Hebei

Li Hongbo, Pang Binshuang, Liu Lihua, Liu Yangna, Zhao Changping, Chen Jingtang

2015, 31(6):  93-99.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.034

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In order to provide a reference for improving and innovating breeding materials, we used forty two pairs of SSR primers to construct DNA fingerprints and detect genetic diversity among 70 wheat varieties(lines), which participated in regional test of Hebei in 2009-2013. The results showed that 303 allelic were revealed on 42 SSR loci, 7.21 alleles were detected for each SSR locus, and the average PIC(polymorphism information content)value was 0.69. Moreover, the average gene diversity was 0.73, and the genetic similarity varied from 0.05 to 1.00 with the average of 0.28. These results indicated that there is a large genetic difference between the wheat varieties(lines)in regional test of Hebei. The cluster analysis divided the 70 wheat varieties(lines)into four groups and six subgroups by UPGMA method. These results show that the wheat varieties(lines)have similar genetic background in the same breeding institute or region. We should strengthen the introduction and utilization of foreign wheat germplasm resources.

Genetic Analysis and Gene Mapping of a Dominant Rolled Leaf Mutant z2 in Rice

Zhang Longdi, Wang Yanwei, Zhang Zhiguo, Wu Jinxia

2015, 31(6):  100-105.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.014

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Moderate rolled leaf is significant in ideotype breeding of rice. An outward rolled leaf mutant z2 was discovered from the rice T-DNA insertion mutant library, and its leaves became curly during tillering stage. Compared with the wild type, there was no significant difference in its main agronomic traits, and its photosynthetic was more efficient. Paraffin sections showed that the number of bulliform cells(8-9)in the mutant was higher than that in the wild type(4-5). Through successive selfing, phenotype of z2 was stable and was controlled by a pair of dominant nuclear genes. We made the combination of z2/Dular to establish F2 mapping population, and used map-based cloning method to locate the gene on rice chromosome 2 between maker InDel1812 and InDel1870 with 580 kb physical distance.

Construction of Root Expression Vector of Brassinosteroid Gene BAS1 and Genetic Transformation into Tobacco

Shu Hongmei, Guo Shuqiao, Gong Yuanyong, Ni Wanchao

2015, 31(6):  106-110.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.015

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In order to allow brassinosteroid gene BAS1 overexpress in root, the root expression vector pCAMBIA2301-RB7-rbc with root promoter TobRB7 was constructed. Then inactivated gene BAS1 was integrated into the root overexpression vector pCAMBIA2301-RB7-BAS1-rbc, and they were transformed into tobacco by Agrobactrium tumefaciems infection. With the verification by PCR and RT-PCR, the target gene BAS1 was transformed into tobacco genome successfully, and expressed in root；the expression of brassinosteroid receptor kinase gene BRI1 in roots of transgenic plants was affected.

Optimization of Agrobacterium-mediated Transformation System for Brassica oleracea var. acephala with Hypocotyls as Explants

Gao Hang, Gao Yuliang, Li Kuihua

2015, 31(6):  111-115.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.017

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An efficient transformation method for kale(Brassica oleracea var. acephala)cultivar Hongou was developed using hypocotyls as explant tissues with the following 3 steps：firstly optimizing the differentiation system of adventitious buds；secondly investigating the effects of Agrobacterium infection concentration and time on differentiation rate and the selective concentration of Hygromicin B(Hyg)gene during differentiation and rooting of adventitious buds；finally verifying the selective gene Hyg in the resistant plants by PCR. The highest differentiation rate(84.17%)of adventitious buds was found on MS + 6-BA 5.0 mg/L + AgNO₃ 9.0 mg/L medium. An infection concentration OD₆₀₀ of Agrobacterium as 0.5 and infection time for 5 min were favorable for genetic transformation, and the differentiation rate was 69.17%. The selective concentration of Hyg during the differentiation and rooting of adventitious buds were 4.0 mg/L and 30.0 mg/L, respectively. Moreover, the amplified specific gene Hyg bands were observed at the expected 602 bp by PCR analysis, which preliminarily demonstrated that the screened gene Hyg was integrated into the B. oleracea var. acephala genome.

cDNA-AFLP Analysis of Dormancy-related Genes in Seed Hypocotyl of Paeonia lactiflora

Sun Xiaomei, Han Ningning, Yang Hongguang, Yang Panpan

2015, 31(6):  116-121.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.018

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To explore the dormancy mechanism of seed hypocotyl of Paeonia lactiflora and isolate dormancy-related genes from P. lactiflora, a cDNA-AFLP technology was used to identify differentially expressed genes between the stages of seeds with stratification for 0 d and germination. A total of 3 600 differentially expressed transcript-derived fragments(TDFs)were screened by selective PCR amplification using 256 primer combinations, and 1 200 TDFs were recovered successfully. Then selected 500 TDFs were cloned and sequenced, and 42 effective sequences were obtained. Bioinformatics analysis with BLAST showed that 30 TDFs were homologous to the known functional genes, which involved in gene expression regulation, signal transduction, stress responding, material and energy metabolism etc. during the dormancy of seed hypocotyl of P. lactiflora. Nine TDFs were similar to genes of unknown functions and hypothetical proteins, and another 3 TDFs had no sequence homology to GenBank entries and might be new unknown genes, which could help us to clarify the mechanism of seed hypocotyl dormancy of P. lactiflora.

Cloning and Bioinformatics Analysis of euFUL Gene in Panax ginseng

Guo Shuangshuang, Liu Cuijing, Han Mei, Yang Limin

2015, 31(6):  122-128.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.019

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To study the flower development of Panax ginseng, designing specific primers according to gene fragments in the GenBank data, and the 5' end deletion coding sequence of a euFUL(named PgFUL)gene in ginseng flower was cloned by the rapid amplification of cDNA ends(RACE). PgFUL consists of 867 nucleotides, and encoding a protein of 212 amino acids. The bioinformatics analysis shows that PgFUL-encoding protein has isoelectric point(pI)of 5.80 and a calculated molecular weight about 24.64 kD without transmembrane region and signal peptide. In the secondary structure, the percentage of α-helix, extended strand and random coil are 60.19%, 5.21% and 34.60%, respectively. The homologous analysis by sequence alignment and phylogenetic indicates that PgFUL is in 100% similarity with ginseng PgMADS protein3(BAK20018.1)and 95% with Menispermum canadense(AGX01589.1), which belongs to evolutionary euFUL in functional gene A.

Screening，Identification and Potassium-dissolving Characteristics of Potassium-dissolving Actinomycete in Banana Rhizosphere Soil

Chen Yufeng, Ke Chunliang, Zhou Dengbo, Gao Zhufen, Qi Chunlin, Zhang Xiyan

2015, 31(6):  129-137.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.020

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According to the characteristics and mechanism of potassium-dissolving by potassium-dissolving bacteria, strains of the highly efficient and stable potassium-dissolving bacteria were isolated from the banana rhizosphere soil. Firstly by primary screening in potassium-dissolving separation culture medium and screening culture medium, a number of highly efficient potassium-dissolving bacteria were obtained. Then identifying the strain types and determining the potassium-dissolving ratio in laboratory, highly efficient strains of potassium-dissolving actinomycetes were gained. Finally, based on the morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis, the strains were identified, the potassium-dissolving ratios of them were measured under different conditions, and the potassium-dissolving characteristics were studied. After identifying the isolated 16 strains of efficient potassium-dissolving bacteria and measuring their potassium-dissolving ratios, finally a stable potassium-dissolving Streptomycete M3-4 was obtained. The potassium-dissolving ratio reached the highest under the conditions: 120 h, 30℃, pH=6, the medium potassium mineral content was 5 g, shaking speed in 250 r/min, maltose as carbon source, and peptone as nitrogen sources. The strain is Streptomyces shaanxiensis and potassium-dissolving ratio is about 20%.

Screening and Denitrification Characteristics of an Aerobic Denitrifying-Heterotrophic Nitrification Bacterium

Lian Hongmin, Qiu Zhongping, He Kunming, Zhou Wenxiu

2015, 31(6):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.021

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Aiming at the fact that the degradation of NO₃-N and NO₂-N in leachate is difficult, an aerobic denitrifying bacterium HN, preliminary identified as Pseudomonas, was isolated from an aerobic biological reactor landfill. Based on its degradation capacities to different nitrogen sources, we studied the denitrification characteristics of HN and optimized the conditions for denitrification. The results showed that under aerobic conditions and potassium nitrate as sole nitrogen source, the removal efficiency of nitrogen in nitrate was up to 95.44% at 96 h；when using ammonium sulfate as sole nitrogen source, HN could remove 85.14% nitrogen in ammonia at 60 h；when the carbon source was ethanol, C/N was 7:1, initial pH was 7.5, the temperature was 35℃ and inoculation amount was 10%, HN had the highest removal efficiency.

Effects of Galactooligosaccharide on Exopolysaccharide Produced by Intestinal Probiotics

Xin Yueqiang, Liang Rongrong, Wang Ruiming

2015, 31(6):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.023

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The aim of the study is to investigate the effects of galactooligosaccharide on the yield of exopolysaccharides produced by intestinal probiotics. Using galactooligosaccharide, glucose, galactose and lactose as carbon sources, the utilization of carbon sources, the yield and varieties of exopolysaccharides and the adhesion of harmful bacteria were studied. Results indicated that galactooligosaccharide not only promoted reproduction of Bifidobacterium longum and Lactobacillus plantarum, but also improved the yield of exopolysaccharides with 208.78 μg/mL and 192.78 μg/mL respectively. Varieties of exopolysaccharides produced while using galactooligosaccharide as carbon source were more than other 3 ones, and the adhesion to harmful bacteria such as Escherichia coli was obvious. It is proved that galactooligosaccharide could promote B. longum and L. plantarum to produce more exopolysaccharides compared with the other 3 carbon sources.

A Study on the Degradation Capacity of Several Non-model White-rot Fungi

Wu Xuejun, Cui Baokai

2015, 31(6):  151-156.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.024

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Based on testing of guaiacol selective medium, 3 isolates from 11 white-rot fungi were selected, and they are Trametes velutina Dai 10149, T. ochracea Cui 6888 and T. pubescens Cui 7571. Then growth curve of the 3 isolates based on weight method were plotted, and the changes of protein content and two extracellular enzyme activities were monitored. The results showed that T. pubescens Cui 7571 had excellent ability of growth, and stable and outstanding ability of secreting cellulase and laccase. It can significantly decompose the lignocelluloses in conifer, hardwood and monocotyledon plants.

Effects of Nitrogen and Phosphorus Contents on the Oil Degradation Rate

Sun Wanhong, Chen Lihua, Xu Hongwei

2015, 31(6):  157-164.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.025

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Five oil-degradation bacterial strains were cultured, isolated and screened from soil at Xifeng Oilfield of Gansu Province, northwestern China. On-site experiments by adding different levels of urea and ammonium dibasic phosphate to oil-contaminated soil lasted 63 d, the effects of nitrogen and phosphorus contents on the oil degradation rate by mixed bacterial agent from 5 strains were investigated. The results reveal that nitrogen and phosphorus contents in oil-contaminated soil of Xifeng Oilfield are in low level, and it is not conducive for bioremediation；however, artificially adding a certain amount of nitrogen and phosphorus for bioremediation of oil-contaminated soil with mixed bacterial agent have significant promoting effects. The changes of nitrogen and phosphorus show two stages in the 63 d microbial remediation process for 1. 5% and 3% oil-contaminated soil：the contents of nitrogen and phosphorus decrease rapidly in the first stage(0-28 d)；while they fluctuate after 35 d. The microbial remediation effect for the 3% oil-contaminated soil is obvious, and the maximum oil-degradation rate reaches 52. 5%. GS-MC was used to measure and analyze the degradation rate and evolution pattern of mixed bacterial agent for hopane- the major constituent of oil. The results show that, in the optimal ratio of nitrogen to phosphorus, the degradation rate of mixed bacterial agent for hopane-like compounds in the oil-contaminated soil is over 80%, the highest degradation rate(86. 3%)for onocerane.

Polymorphism Analysis of MSTN Gene Exons of Ganzhou Simmental Cattle in Gansu Province

Li Jiyou, Liang Chunhua, Liu Xia, Sun Xuejing, Du Xiaohua

2015, 31(6):  165-169.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.026

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PCR-SSCP(single-strand conformational polymorphism)was used to detect the genetic polymorphism in 3 exons of myostatin(MSTN)gene in 50 individuals of Ganzhou Simmental cattle in Gansu. The alleles in a population were verified by sequencing, which provided the theoretical evidences for investigating the correlation between MSTN gene of Simmental cattle and meat quality. The results showed that the polymorphism in the exon 2 was controlled by B and C alleles, which formed 3 genotypes of BB, CC and BC with the frequencies at 0. 22(11/50), 0. 64(32/50)and 0. 14(7/50)respectively. The polymorphism in the exon 1 and exon 3 was controlled by A and D allele respectively, which formed AA and DD genotypes with frequencies at 1(50/50). Sequence analysis indicated that there was one single nucleotide mutation of C to T at 41^st nucleotide in the exon 2 of Ganzhou Simmental cattle；however this did not lead to the amino acid changed, so it was a synonymous mutation. There were no mutations in the exon 1 and exon 3. The statistical results showed that the exon 2 of MSTN gene in 50 individuals of Ganzhou Simmental cattle had a moderate level of polymorphism, and the exon 1 and exon 3 had no polymorphism.

Isolation，Culture and Characterization of Derived Cells from Neonatal Porcine Bone Marrow Mesenchymal Stem Cells

Ruan Zheng, Wang Lianfang, Hu Xiuzhong, Wu Jianying, Zhang Sihua, Dai Changyun, Hua Juan, Xia Yu, Hu Xiaoming, Li Jie, Huang Haijun

2015, 31(6):  170-176.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.027

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Recently it has become possible to use bone marrow mesenchymal stem cells(BMSCs)in the clinical treatment of certain diseases. However, BMSCs, the seed cells used in transplantation, are limited in vitro proliferation capability and can only be obtained from very few sources. In order to acquire a substitute for BMSCs, the method of differential velocity adherent screening was used in this study to isolate a derived strain of porcine BMSCs, named bone marrow mesenchymal stem-derived cells(BMSDCs). The biological characteristics of BMSDCs and BMSCs cells were compared by continuous morphological imaging by inverted microscope, and the growth curves of both cells were monitored by the MTT method. Furthermore, their characteristics of differentiation in vitro were examined, and flow cytometry was used to measure cell surface markers. Our results suggest that the doubling times of BMSCs and BMSDCs are 31. 3 h and 30. 3 h, respectively, and the average passaging time is 3-5 d and 2-3 d, respectively. Cultured BMSCs and BMSDCs are both positive for CD34 and CD90 and negative for CD44 and CD45. Both BMSCs and BMSDCs can differentiate into adipocytes and myoblasts by induced differentiation in vitro. Concerning their passaging abilities, the former can have 15 to 20 passages in vitro, whereas latter can have a longer period of time(more than 200 passages)while normal karyotypes are still maintained. We conclude that, under certain experimental circumstances, cultured porcine BMSDCs in vitro can survive and proliferate while maintaining multi-directional differentiation potential of BMSCs, and potentially it can be an ideal type of seed cell for tissue engineering.

Isolation，Culture and Identification of Beijing Duck Embryonic Metanephric Mesenchymal Stem Cells

Chen Jia, Wang Guiyan, Zhang Yu

2015, 31(6):  177-182.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.028

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This study aimed to establish in vitro culture system for the poultry metanephric mesenchymal stem cells(MMSCs), and study their biological characteristics and multi-directional differentiation potential. Enzyme digestion method was applied to separate Beijing duck embryo MMSCs and to draw the growth curve. MMSCs were identified by immunofluorescence and RT-PCR. MMSCs were induced to differentiate into adipocytes and islet cells. The results showed that MMSCs had solid proliferative activity and could express MSCs specific markers, and be induced to differentiate into adipocytes and islet cells. In summary, Beijing duck embryo MMSCs have a strong self-renewing ability and multi-directional differentiation potential in vitro, and can be saved as seed cells for tissue engineering.

Cloning and Prokaryotic Expression of Glutathione Reductase Gene in Vibrio harveyi

Zhu Fan, Ding Yu, Lu Yishan, Jian Jichang, Wu Zaohe

2015, 31(6):  183-188.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.029

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The purpose of this study is to clone glutathione reductase(GR)gene from Vibrio harveyi, construct the prokaryotic expression vector of it, and obtain the corresponding expressed protein. Digested GR and pET-32a(+)were cut with the double enzymes BamH I and Xho I, then ligated with T4 ligase to construct the recombinant plasmid pET-GR. Then pET-GR was transformed into Escherichia coli BL21(DE3), which was induced by IPTG, and their expressions were analyzed by SDS-PAGE. An approximately 68.9 kD exogenous protein was observed on the SDS-PAGE. The optimal expression condition for the recombinant plasmid pET-GR was that the recombinant E. coli BL21(DE3)was induced for 4 h at 28℃ by 0. 7 mmol/L of IPTG and it expressed in E. coli as the inclusion bodies. The conclusion of the study is that GR gene from V. harveyi can efficiently express in E. coli.

Prokaryotic Expression，Antibody Preparation of Gonad-specific Protein EsSOX21b-like of the Chinese Mitten Crab Eriocheir sinensis

Yang Guocui, Cui Zheng, Qiu Gaofeng

2015, 31(6):  189-194.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.030

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The Sox(SRY-related HMG-box)is a large family of genes which encode transcription factors with high-mobility-group DNA binding domain related to the SRY(sex determining region Y). In previous studies we isolated and identified EsSox21b-like transcripts exclusively expressed only in gonads of the Chinese mitten crab(Eriocheir sinensis), this study is to verify whether EsSOX21b-like protein is also specifically expressed in the gonad. Gene EsSox21b-like was cloned to expression vector pGEX-2T, the recombinant prokaryotic expression plasmid pGEX-2T-EsSox21b-like was constructed, then transferred into host Escherichia coli BL21, and induced by IPTG. SDS-PAGE analysis revealed that the fusion protein was expressed as inclusion body with the molecular weight of 54 kD. The recombinant proteins were subsequently purified by Ni⁺ affinity chromatography. Then the purified protein was used to immunize rabbits for preparation of the EsSOX21b-like antibody. Western blotting detection showed the antibody specifically recognized the EsSOX21b-like protein in the gonad, with molecular weight of 28 kD, whereas no expression was detected in other tissues. The expression level of the target protein was much higher in testis than that in ovary. This result implied that the EsSOX21b-like protein could have a regulatory role in the gonad development.

Effects of Starvation on Serum Biochemical Indexes in Large Yellow Croaker(Larimichthys crocea)at Low Temperature

Xu Hao, Zhang Dongling, Chen Qingkai, Ye Kun, Wang Zhiyong

2015, 31(6):  195-199.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.031

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The study was conducted to investigate the effects of starvation on serum biochemical indexes in 9-month-old large yellow croaker(Larimichthys crocea)at low temperature(13℃). Four hundred fishes were randomly divided into 2 groups, one group as control group was fed with formula feed, and the other as experimental group was starved. Eleven serum biochemical indexes were examined 30 d after experiment started. The results revealed that the UA, UREA, TC, and ALT of experimental group were extremely significantly higher than control group(P < 0. 01)；while the ALB and AST of experimental group were extremely significantly lower than control group(P < 0. 01). Compared to control group, the TG and Ca²⁺ of experimental group were significantly higher(P < 0. 05). There was no significant difference in both TP and Mg²⁺ between experimental and control group.

Effects of 17β-estradiol on the Sex Differentiation of Zebrafish(Danio rerio)

Li Guochao, Yu Kaimin, Feng Weimin, Liu Lili, Zhang Jiayu, Yan Yanchun

2015, 31(6):  200-208.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.032

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Previous studies have demonstrated that 17β-estradiol(E2)could increase the female proportion in zebrafish population. In order to study mechanisms of feminization caused by E2, quantitative real-time PCR(qRT-PCR)was used to analyze expression levels of genes involved in sexual differentiation(brca2, sox9a, sox9b, dmrt1 and cyp19a1a)under different conditions. The results indicate that E2 can up-regulate the expression levels of the female-predominant genes(brac2 and sox9b), partially down-regulate the expression levels of male-predominant gene(sox9a)and has different effects on sex hormone conversion pathway(cyp19a1a)in different developmental stages of zebrafish.

Construction and Application of RARE-regulated Luciferase Reporter Gene System Stimulated by Retinoid Acid

Yuan Yuan, Chen Yaqiong

2015, 31(6):  209-215.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.033

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Biostearin plays a crucial role in the development of metabolic diseases by target gene regulation of retinoic acid(RA)in metabolically active tissues and interaction with circadian pathway. The luciferase reporter gene system regulated by retinoic acid response element(RARE)was constructed and expressed in 293T and primary hepatocyte of a mouse, which provides the possibility for studying biostearin and circadian, as well as signaling molecules of upstream regulation of target genes in researching metabolism. By PCR-based site-specific mutagenesis, RARE promoter was inserted into multiple clone sites of pGL3-Basic vector. In 293T cells, we detected the gene expression and half maximal effective concentration(EC50)of RARE responding to RA and screened the possible target genes of regulating RARE. The pGL3-RARE-Luc plasmid and pShuttle vector were digested by restriction enzyme and ligated. Through transformation into competent cell BJ518 the recombinant adenovirus vector Ad-Basic-RARE-Luc was obtained and amplified after transfection to MGH cells. The activity of recombinant adenovirus was detected in primary hepatocyte of a mouse with Dual-luciferase Reporter Assay System. The results indicated that luciferase reporter gene vector regulated by RARE in 293T cells was stimulated by RA. RARβ promoted the stimulation of RA in RARE regulation, and CRY1 prohibited the response of RARE-Luc to RA. The adenovirus vector Adeasy-Basic-RARE-Luc responding to RA stimulation in primary hepatocyte of a mouse was constructed successfully.

Expression of Canine Interferon Alpha in Silkworm-baculovirus Expression System and the Antiviral Activity Assay

Li Haoyang, Hu Xiaoyuan, Yi Yongzhu, Yang Xin, Zhang Zhifang, Li Yinü

2015, 31(6):  216-220.  doi:10.13560/j.cnki.biotech.bull.1985.2015.06.022

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Canine interferon alpha(CaIFNα)was widely used in the prophylaxis and cure of canine diseases. The purpose of this study is to develop an efficient method to express CaIFNα. A canine interferon alpha gene in GenBank was optimized according to the codon bias of silkworm and synthesized, and then cloned into the baculovirus transfer vector pVL1393 to construct the recombinant plasmid pVL-CaIFNα. The pVL-CaIFNα was co-transfected with the BmBacmid DNA into silkworm cells and after in vivo recombination. The recombinant virus was gathered and used to infect silkworm larvae. The hemolymph of infected larvae was collected for antiviral activity assay. The antiviral activity of expressed CaIFNα was examined on Madin-Darby canine kidney cells infected with the vesicular stomatitis virus expressing green fluorescent protein(VSV*GFP). The result showed that CaIFNα expressed in silkworm can inhibit the multiplication of VSV*GFP and the antiviral activity was about 1.78×10⁶U/mL.