Biotechnology Bulletin

The Research and Regulatory Status of Novel Plant Breeding Techniques in Europe

YANG Yan-ping, DONG Yu, XING Ying, YUAN Jian-xia

2016, 32(2): 1-6. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.032

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Recently, a number of novel plant breeding techniques(NPBTs)with great application prospect, such as oligonucleotide directed mutagenesis(ODM), zinc finger nuclease(ZFN)technology, cisgenesis/intragenesis, RNA-dependent DNA methylation(RdDM), reverse breeding, grafting(on GM rootstock), and agro-infiltration, have been developed rapidly in Europe.These techniques are more specific and targeted than conventional breeding methods, and thereby can provide more precise, rapid and efficient methods for breeders.The results from literature metrology showed that agro-infiltration and RdDM were the most commonly used techniques in EU, and Germany and the UK were the leading countries on the research of NPBTs.To date, it is being discussed whether the plants produced by these new techniques are captured by the EU’s GMO legislation, in particular directive 2001/18/EC.In the abundant reports published by relevant organizations, the classification and supervision of plants and products generated by NPBTs were extensively discussed.The risk assessment of plants by ZFN-3 and Cisgenesis/intragenesis was carried out by the European Food Safety Authority(EFSA)respectively.It was concluded that plants by cisgenesis had similar hazards with that produced by conventional breeding, whereas novel hazards might arise with intragenic and transgenic plants.In addition, the panel suggested that there would be fewer hazards for plants of ZFN-3 than conventional GMOs.

Research Progress on Plant Heat Shock Protein

LI Zhen-yi, LONG Rui-cai, ZHANG Tie-jun, YANG Qing-chuan, KANG Jun-mei

2016, 32(2): 7-13. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.003

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Heat shock protein(HSP)is a kind of ubiquitous protective protein in plant.HSP mainly acts as a molecular chaperone to maintain protein in steady state and repair denatured proteins through binding with target proteins, which keep the stability of plant internal environment.Therefore, HSP plays a key role in plant development and stress-resistance process.These proteins can be classified as 5 families of HSP100, HSP90, HSP70, HSP60 and small molecular HSP, and each family consists of many different HSPs.In this review, based on the reported HSPs of varied species in the abundant literatures, we summarize the structure, function and regulatory mechanism of plant HSPs, which aims at providing reference information for understanding plant HSP and its mechanism.

Research Progress of Acidobacteria Ecology in Soils

WANG Guang-hua, LIU Jun-jie, YU Zhen-hua, WANG Xin-zhen, JIN Jian, LIU Xiao-bing

2016, 32(2): 14-20. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.002

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The phylum Acidobacteria is one of the most important bacterial groups in soils.Based on analysis of 16S rRNA gene sequences, the number of Acidobacteria generally represents about 20% of total soil bacterial communities, some of them even account for more than 50%, which suggests that Acidobacteria play important roles in soil ecological process.In this paper, we reviewed the research progress of Acidobacteria ecology in soils from several aspects, such as effects of plant types, altitude, nitrogen fertilizer and CO₂ enrichment on the distribution of Acidobacteria, as well as rhizosphere effects of Acidobacteria, etc.The relationships between distributions of different taxonomic level Acidobacteria and environmental factors were summarized.The potential ecological functions of Acidobacteria were also presented in this paper.In the end of this paper, the importance for future study of Acidobacteria, such as reinforcement of isolation and pure culture, refinement of molecular ecological research, and adoption of metagenomics and single-cell sequencing approaches were also addressed.

Differences in Regulation of Photosynthetic Gene Expression Between Different Purple Bacteria

DENG Chun-hao, SUN Liang-yu, CHEN Kun-ming

2016, 32(2): 21-29. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.005

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The purple bacteria are prokaryotes that can proceed photosynthesis without oxygen release.They use light energy to produce ATP for their own growth and metabolism.The photosystem of a purple bacterium consists of a series of complex of pigments and proteins encoded by photosynthetic genes like puc, puf, puh, bch, crt and so on.The expressions of their photosynthetic genes mainly are influenced by light and external redox signals；however, the expression and regulation mode of photosynthetic genes are greatly different between different kinds of purple bacteria.This review introduces the research advances on several expression and regulation systems of photosynthetic genes, such as PpsR/CrtJ like regulator, two-component phosphorylation regulation system and CRP-FNR like regulator in purple bacteria while focusing on Rhodobacter sphaeroides and Rhodopseudomonas palustris.The results from the functional and characteristic analysis of the regulation mechanism in different purple bacteria suggest that, each regulation system existing in different bacterial strain shows similar functions but with somewhat unique characteristics.This study gives us further insights for understanding the mechanisms of expression and regulation of photosynthetic genes in the purple bacteria, and provides important references for application of these purple bacteria in the future.

The Research Progress of PGF_2α Induced-Early Response Genes Involved in Luteal Regression

MAO Da-gan, GUO Nan-nan, KUANG Mei-qian

2016, 32(2): 30-37. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.001

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The remaining mammalian corpus luteum plays an important role in female reproductive activities.Prostaglandin F2 alpha(PGF2 alpha), as a main luteolytic factor of the corpus luteum, through binding to its receptor, activates series of early response genes and induces luteal functional(a decrease in the level of progesterone)and structural degradation (programmed cell death).Although PGF_2αhas been widely used in the control of animal synchronization, delivery control and reproductive barrier, the molecular mechanism in the luteal regression is not yet clear.In this review, the advanced progress of PGF_2α receptor and its induced early response genes in ovarian luteal regression is summarized, which provides a theoretical basis to study the molecular mechanism of PGF_2α-induced luteal regression.

Research Progress on Bio-ethylene

SUN Meng-ting, FAN Xiao-lei, GUO Rong-bo, QIU Yan-ling, ZHAO Xiao-xian

2016, 32(2): 38-45. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.004

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Ethylene is the chemical raw material of largest demand in the world, and is widely utilized for producing plastics, textiles and other chemical products.Currently, the most common method of producing ethylene is steam cracking of fossil oil；however, there exist drawbacks of high cost and non-friendly to environment, which bring the opportunity for the development of bio-ethylene.Bio-ethylene is produced from renewable resources of CO₂ and biomass , which contributes to saving resources and protecting environment.In this review, we introduce two pathways of bio-ethylene production—direct and indirect synthesis.Here we focus on the direct synthesis pathway of microorganisms via an ethylene forming enzyme(EFE), describing the structure, sequence and catalytic mechanism of EFE, illustrating several successful cases of engineered heterologous hosts synthetizing ethylene, presenting the strategies for improving yields, and prospecting the development of producing ethylene by microorganisms.

The Role and Mechanism of Polyamine in Cancer Therapy

MA Rong, CHEN Zi-yu, JIANG Dong-mei, KANG Bo

2016, 32(2): 46-50. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.006

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Polyamine is a necessary substance in eukaryotic cells growth and development, the disorder of polyamine metabolism is closely related to the occurrence of cancer.Research shows that enzyme ornithine decarboxylase and S-adenosylmethionine decarboxylase inhibiting polyamine biosynthesis effectively relieve the development of cancer.In addition, using the specificity of transmembrane transport system of polyamine, synthesized polyamine analogues and conjugates are transported to the cells, then it plays the role in anti-cancer therapy by decreasing the levels of polyamine, regulating the acetylation and methylation of histone proteins, and effectively promoting the apoptosis of tumor cells.In order to provide reference for the research of targeted therapy to cancer by using metabolic pathway of polyamine in the future, the research progress on the inhibition of polyamine synthase and using polyamine analogues and conjugates for treating cancer are reviewed in this paper.

Research Progress on Bioremediation Techniques for Mercury-contaminated Soil

WANG Li-hui, YAN Chao-yu, WANG Hao, ZHANG Xiang-yu

2016, 32(2): 51-58. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.007

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Mercury as a sole liquid metal at room temperature, is widely distributed in the environment, which easily results in the soil contamination.Bioremediation technique, as an important technique to remediate the mercury-contaminated soil, has captured attentions due to its low cost and environmental-friendly.This paper sums up the bioremediation techniques for mercury-contaminated soil, mainly on the researches and applications of phytoremediation, microbial remediation and animal remediation.In phytoremediation technology the metabolism mechanism and pattern of mercury in plants mainly are investigated；using exogenous microorganisms to degrade mercury in soil is the focus in the study of microbial remediation；and very little is studied in animal remediation, only enrichment of mercury by earthworms was reported.What is learned from the analysis in this review is that current bioremediation technique has developed to be a multidisciplinary integration technology, covering soil chemistry, plant physiology, ecology, soil microbiology, analytical chemistry and molecular biology.By means of those subjects, we may understand profoundly the rhizosphere micro-interface process in plant extracting mercury, plant internal micro-interface process, and the mechanisms of mercury enrichment by microorganism and animal.Finally, we summarize and induce the current main research trend of bioremediation techniques for mercury-contaminated soil, and clarify several main issues and research directions for the issues in this field.

Research Advance on miRNA qPCR Methods and Its Application

FENG Shi-peng

2016, 32(2): 59-69. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.008

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miRNAis one of thehottestresearch fields in the world.The detection of miRNA expression is the fundamental content in miRNA research.Due to the feature of miRNAs being short and small, varied methods were developed, and quantitative miRNA qPCR isthe most widely used one.This paper reviews thedevelopments of miRNA extraction method, quantitative detection principle of miRNAqPCR, primer design, the reaction condition, and reference gene selection.The developments ofmiRNA qPCR methods in the future are also discussed.All of these may provide a reference for researchers in the field when they detect miRNA by qPCR method.

The Establishment of PMA-qPCR for Detecting Bacterial Angular Leaf Spot on Cucumber and Its Preliminary Application

KONG Wei-wen, LI Yun-long, WANG Jing-qi, LIU Ting-ting, CHEN Nan, JIN Yi, HE Xiao-qing

2016, 32(2): 70-75. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.009

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Bacterial angular leaf spot on cucumber is a common bacterial disease for cucumber, and its causative pathogen is Pseudomonas syringae pv.lachrymans.Currently this disease has caused a worldwide serious economic loss. Duo to the feature of PMA dyes that may distinguish whether the bacterial cells are alive or dead, PMA-qPCR was established by combining it with fluorescence quantitative PCR(qPCR)technology.(1)We compared the sequences of gene gap1 in different strains of the bacteria in GenBank, and found out the conservative area of gene gap1, and designed a pair of specific primers Dxf1 and Dxr1 for this bacterium according to the sequences of conservative area of gene gap1.Then we applied the qPCR method to do amplification using the genome of 5 non-Pseudomonas syringae pv.Lachrymans as template, and only P. syringae pv.lachrymans showed the positive result, proving that primers had specificity.(2)We used the gene gap1 as the target gene to construct a clone vector that was inserted into the competent cells of Escherichia coli.Then the E.coli cells were cultured to have a lot of copies.Using the extracted plasmids as the standard sample, and it was diluted to seven gradients of 5×10¹-5×10⁷ and then drew a standard curve.The amplification efficiency obtained from the standard curve was 96.6%, and the coefficients of variation both within groups and between groups were less than 2%, showing a fine repeatability of the standard curve.(3)We applied the PMA-qPCR method to detect the actual samples and gained a Ct value of 27.99.Based the linear correlation between initial template from the standard curve and Ct values, we measured 7.69×10² copies viable cells per 100 mg sick leaves.This study showed that PMA-qPCR method could quickly identify and quantify the number of living bacteria in actual samples, and provided a technical support for the prevention and control of bacterial angular leaf spot on cucumber.

The Comparison of Three Methods of Monitoring Endogenous Gene Modification

LI Wei-jie, YANG Jiao, HE Gao-ming, WANG Li-min, PI Wen-hui, ZHOU Ping

2016, 32(2): 76-83. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.010

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In orderto obtain accurate mutation information, 3 methods of monitoring the biological activities of artificial nuclease are preliminary determined excluding direct sequencing.The 3 methods of Surveyor nuclease, T7E1(T7 Endonuclease 1)and HRM(High resolution melting)are all based on the principle of forming the twisted duplex DNA from denaturized annealing mutant and wild type DNA sequence, and whether or not target sites were mutated were determined.The results showed that using the 3 detection methods, the mutations at the target sites of exon 1's CRISPR/Cas9 and exon 3's TALEN in ovine gene MNST were successfully detected.Analyzing and comparingthe results and characteristics of the 3 methods, the advantages and disadvantages of them were obtained, which provided a reference for the analysis and identification of results while the cells uses non-homologous ends to join and repair DNA double strand breaks.In conclusion, the results by Surveyor, T7E1 and HRM show that CRISPR/Cas9 and TALEN’s target sites are mutated.

The Construction of Prokaryotic Expression Vector for Human Gene IL-24 and Expression and Purification of Its Protein

YU Fang, YANG Zhong-hua, FAN Han-dong, ZUO Zhen-yu

2016, 32(2): 84-89. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.011

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This work aims to construct a prokaryotic expression vector for human gene IL-24, and express and purify soluble IL-24 protein via ELP-Intein system.The human gene IL-24 without signal peptide was amplified by PCR and cloned into vector pET-ELP-Intein, and the recombinant expression vector pET-ELP-Intein-IL-24 was constructed.The recombinant plasmid was transformed to Escherichia coli BLR(DE3), and the expression was induced at 20℃ by IPTG.The soluble IL-24 protein was purified based on the transformation of ELP protein at different temperatures and the self-cleavage reaction of Intein protein.The purified protein was identified by Western blot.The bioactivity of IL-24 protein was measured by Annexin V-FITC/PI apoptosis detection kit.The recombinant expression vector pET-ELP-Intein-IL-24 was successfully constructed, and the soluble IL-24 protein was expressed for the first time by prokaryotic vector and purified.Western blot analysis showed the target protein specifically bound with IL-24 antibody, which proved that the purified protein was indeed IL-24 protein.Apoptosis detection experiment confirmed that IL-24 protein significantly induced the apoptosis of hepG2 cells.

Cloning and Function Analysis of Gene GhPYR1 in Gossypium hirsutum L.

LIU Yan, MENG Zhi-gang, SUN Guo-qing, WANG Yuan, ZHOU Tao, GUO San-dui, ZHANG Rui

2016, 32(2): 90-99. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.012

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The phytohormone abscisic acid(ABA)plays an important role in response to drought, salinity and other abiotic stress.It is also a key regulator involved in different plant developmental stages such as seed germination, root elongation, and bud dormancy.The PYR/PYL/RCAR protein family is the ABA receptor, which can initiate the ABA signaling pathway and induce the expression of ABA response genes.In this study, gene GhPYR1 was cloned from Gossypium hirsutum L.with electronic cloning and RT-PCR technology.GhPYR1 protein exhibited 73% sequence identity with Arabidopsis AtPYR1 protein.Protein sequence alignment among GhPYR1 and 14 family members of PYR/PYL/RCAR in Arabidopsis as well as the construction of phylogenetic tree revealed that gene GhPYR1 was most closely related to subfamily III of PYR/PYL/RCAR protein in Arabidopsis.T₃ generation Arabidopsis over-expressing gene GhPYR1were more sensitive to exogenous ABA, as its seed germination and early root growth lagged behind than wild type.The germination of transgenic seeds was more strongly inhibited by high salt and drought stress, however, the transgenic lines were significantly better than the wild ones in the seeding stage.Moreover, the ABA-responsive gene RD29A and RAB18 in transgenic Arabidopsis were significantly induced by ABA compared to the wild type.The above results show that the protein encoded by gene GhPYR1 is the receptor of ABA and over-expression of GhPYR1 can improve the sensitivity of plant to ABA and enhance the ability to respond to stress in plants.

Cloning, Expression Analysis and miRNA Study of Gene GDF9 and BMP15 in Yak

LI Ming-xia, PAN He-ping, YAN Ping, WU Xiao-yun, WANG Ying-jie

2016, 32(2): 100-108. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.013

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Gene GDF9(growth differentiation factor 9)and BMP15(bone morphogenetic protein 15)of yak were used as research subject；then 2 exon fragments of GDF9 and BMP15 were cloned by PCR, and the expressions of GDF9 and BMP15 as well as targeted miRNA were analyzed by RT-PCR.The results were as follows.The cloned and amplified exon fragments were analyzed by bioinformatics；the exons of yak gene GDF9 were 411 bp and 1 082 bp, encoding 453 amino acids；the lengths of exons in yak gene BMP15 were 323 bp and 802 bp separately, encoding 372 amino acids.The protein encoded by GDF9 was hydrophilic, containing one signal peptide and 15 potential phosphorylation sites；the protein encoded by BMP15 was also hydrophilic, and owing no signal peptide and 6 potential phosphorylation sites.The expressions of GDF9 and BMP15 in different tissues were examined by RT-PCR.The results showed that GDF9 expressed relatively high in ovary and uterus, and higher in ovary than others tissues, and no expression in heart, kidney, pancreas, and spleen；BMP15 was only detected in ovary.With TargetScan, the target miRNA of yak gene GDF9 and BMP15 were predicted and the expressions of target gene bta-miR-193a-3p and bta-miR-193b of GDF9 in heart, spleen, lung, kidney and uterus were analyzed.miRNAs genes were examined with RT-PCR；the results indicated that bta-miR-193a-3p in the spleen expressed significantly higher than in others tissues, and no expression in heart, lung and uterus；bta-miR-193b expressed lower in lung than spleen and uterus, and no expression was detected in heart and kidney.

cDNA Cloning of Arginase II Gene in Ayu(Plecoglossus altivelis)and the Correlation Between Its Expression and Vibrio anguillarum Infection

DING Fei-fei, LI Chang-hong, CHEN Jiong, ZHANG Qi

2016, 32(2): 109-115. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.014

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Arginase II(Arg-II)is one kind of arginases.Not only can it participates in the urea cycle, but also has close correlation with pathological process.Here, we determined the whole cDNA sequence of Arg-II gene of ayu(Plecoglossus altivelis)by de-novo transcriptome sequencing of ayu monocytes/macrophages.The results showed that the cDNA sequence of the Arg-II was 4707 nucleotides, containing a large open reading frame that encoded a protein of 348 amino acids with a deduced molecular mass of 38.09 kD and a theoretical isoelectric point(pI)of 6.15.Multiple sequence alignment of complete amino acid sequences showed that ayu Arg-II had the typical characters of Arg-II in animals.Sequence comparison showed that ayu Arg-II shared the highest amino acid sequence identity(85.3%)with that of rainbow trout(Oncorhynchus mykiss).Phylogenetic tree analysis also confirmed that fish Arg-II formed a cluster, and ayu Arg-II was most closely related to that of rainbow trout.Quantitative real-time PCR(qRT-PCR)analysis showed that the mRNA of ayu Arg-II mainly had the expression in liver, brain, head kidney and monocytes/macrophages.Upon Vibrio anguillarum infection, the mRNA expression of gene Arg-II significantly up-regulated in the liver, brain, head kidney and monocytes/macrophages.In summary, our present study revealed a tight relationship between the ayu Arg-II expression and V.anguillarum infection, suggesting that ayu Arg-II plays an important role in fish immune response against bacterial infection.This study provides a theoretical basis for studying the functions of fish Arg-II, and its molecular mechanism in regulating fish immune response to pathogens.

The Relationship Between Characteristics of Yielding Carotenoid and Expression of Carotenogenic Genes in Phaffia rhodozyma

ZHENG Chen-hua, DU Xi-ping, LI Li-jun, LI Tian-li, CAO Ying, NI Hui

2016, 32(2): 123-130. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.016

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In order to study the metabolic regulation mechanism of carotenoid synthesis in Phaffia rhodozyma, the carotenoid biosynthesis characteristics and the expression differences of carotenogenic genes in different strains of P.rhodozyma, JMU-VDL668 and JMU-N3 were analyzed by HPLC and real-time qPCR, respectively.The effects of adding β-carotene during the culture of strain JMU-VDL668 to the yield of astaxanthin were also analyzed.The results showed that, the expression levels of the carotenogenic genes crtE, crtI, crtYB and crtS of key enzymes in the metabolic pathway of carotenoid in P.rhodozyma were lower in JMU-VDL668 than JMU-N3, even the expression of crtR was not detected in JMU-N3.The synthesis of astaxanthin increased when β-carotene was added at 72 h and 96 h of culturing JMU-VDL668, and the astaxanthin increased 83% while adding β-carotene at 96 h compared to the control group.The results indicated that the characteristics of yielding carotenoid was positively correlated to the expression level of carotenogenic genes.Moreover, the productivity of cells for total carotenoid in JMU-VDL668 was in a certain range, suggesting that there was a feedback regulation.

The Identification and Analysis of Sulfate-assimilation Related Genes in Acidithiobacillus ferrooxidans

ZHENG Chun-li, WANG Dan, ZHANG Li, CHEN Min-jie, MA Hong-wei, JIANG Zhi-yan

2016, 32(2): 131-139. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.017

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This work aims to identify and analyze the related genes of sulfate-assimilation in Acidithiobacillusferrooxidans ATCC 23270. Firstly, the genes involved in sulfate-assimilation in the genome of A.ferrooxidans ATCC 23270 were analyzed using bioinformatics.Then we co-transcripted and identified the candidate genes cys JI, cys H, cys D-2, cys N, cys D-1, and cys NC probably involving in encoding cysteine(Cys)operon by reverse transcription and gel electrophoresis.Further we cloned in vitro, expressed, purified and recombined the key genes, and confirmed their functions and measured their enzyme activities.Results showed that among these candidate genes of Cys operon, cys D-1 and cys NC were co-transcripted, and Cys JI, cys H, cys D-2 and cys N were co-transcripted.The possible basic metabolic pathway of sulfate-assimilation in A.ferrooxidans ATCC 23270 was determined preliminarily, and the regulatory mechanism of Cys operon was explored.

Isolation and Identification of a High-temperature Resistant Bacterium CICC 10853

DU Hai-bo, ZHANG Xin, LI Zan, LIU Yi, LIU Bo, LI Jin-xia, YAO Su, CHENG Chi

2016, 32(2): 140-145. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.018

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In this study, we identified the high-temperature resistant bacterium CICC 10853 isolated from slant culture.The strain was identified and characterized via morphological features, physiological and biochemical characteristics, 16S rRNA and recN gene sequence analysis.According to the results, strain CICC 10853 was identified as Geobacillus thermoleovorans, which lays the foundation for the further study of biological function of the bacterium.

Isolation, Identification and Decolonization Characteristics of a New Methyl Red Degrading Bacterial Strain Paracoccus sp.L-4

CHEN Liang, WANG Li

2016, 32(2): 146-151. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.019

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The strain Paracoccus sp.L-4 was isolated from Paracoccussp.at first time, and it had the strong ability to degrade methyl red.In LB medium, methyl red in the concentration of 100 mg/L was degraded 91.74% in 16 h, and approximately 100% in 2 d.Furthermore, the optimal conditions for degrading methyl red were 25-30℃ and pH6.0-7.0, respectively.And Zn²⁺, Co²⁺, Cu²⁺, Ag⁺, Al³⁺, Fe³⁺ andFe²⁺ had an significant inhibitory effects on the degradation of methyl red.The degradation and decolorization of methyl red by strain L-4 was performed well in both aerobic and anoxic conditions.When the volume of liquid was ≤ 100 mL, the ratio of decolorization decreased with the increase of volume of liquid.However, when the amount of liquid was ≥ 125 mL, the degradationrate of methyl red was still over 40% at 12 h, indicating that strain L-4 hasa potential application value inbiological treatmentof dye wastewater.

The Optimizationof FermentationCondition for Cellulase Production by Penicillium oxalicum

LI Yang, GAO Xiao-rong, ZHANG Jian

2016, 32(2): 152-157. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.020

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Cellulase plays an important role in the biological energy, textile, feed, paper industry, et al.Screening efficient strains and optimizing the fermentation condition are the effective way of obtaining cellulase.A cellulose-degrading strain JG was isolated from the soil containing rotten wood and straw.Morphology and molecular phylogenetic studies were used for its identification.Some factors during its fermentation were optimized, including initial pH, carbon source, nitrogen source and surfactants.The strain JG was identified as Penicillium oxalicum by analyzing its morphology and ITS(Internal Transcribesd Spacer)gene sequence phylogenetic systematics.Its optimal cultural conditions for the highest cellulase production were as following：initial pH values 2-3, carbon and nitrogen source were CMC-Na and soybean meal.Studying the effects of adding different kinds of surfactant on the fermentation process demonstrated that the lecithin of wide source and relatively low price had an obviously high effects on the cellulase production in JG, and the enzyme activities of FPA, CMCase, and BGL increased by 72.9%, 20.8% and 33.6% respectively.In conclusion, JG produced cellulase under lower initial pH, and lecithin enhanced its cellulase activity significantly.

The Optimization of Ultrasound-assisted Mesothermal Degreasing Process of Silkworm Pupa

ZUO Zhen-yu, YU Fang, HUANG Yan-hong, WANG Jia-yi, LI Ling-ling

2016, 32(2): 158-164. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.021

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Degreasing of silkworm pupa is a vital step prior to others in the refining process, throughout which it is necessary to maintain the protein activity of silkworm pupa.Technology of ultrasound-assisted mesothermal degreasing silkworm pupa was investigated, and response surface methodology was used to optimize the operating parameters and the corresponding prediction models were established.The optimal degreasing conditions were acquired by single-factor experiments and response surface methodology, and listed as follows：petroleum ether：acetone(3∶7)as solvent, liquid-solid ratio 11 mL/g, ultrasound power 125 W, ultrasonic treating time 27 min, and temperature 40℃.The degreasing ratio of silkworm pupa reached(96.8 ± 0.8)％ under these conditions, and the results showed that the degreasing ratio of silkworm pupa was affected in decreasing order by liquid-solid ratio, ultrasonic treating time and ultrasound power.The effects of solvent types and liquid-solid ratio were the decisive factors resulting in the variation of degreasing efficiency of silkworm pupa.Overall, the ultrasound-assisted degreasing process owns the advantages of low temperature, short time, no damage to the activity of product, and high degreasing ratio.

A Research of L-theanine Synthesis with Two Feeding Methods of L-glutamine Using γ-glutamyltranspeptidase

FU Jia-yi, CHEN Sheng, WU Jing

2016, 32(2): 165-171. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.022

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The optimization of L-theanine production by γ-GGT(gamma-glutamyltranspeptidase)was investigated.The constant speed feeding strategy was performed using 200 mmol/L L-glutamine and 2 mol/L ethylamine hydrochloride as the initial substrates，under the condition of 37℃，pH10.0，100 mmol/L L-glutamine was added every 2 hours and the reaction sustained for 14 hours.Eventually the total concentration of L-glutamine and L-theanine were 900 mmol/L and 573.2 mmol/L respectively.The conversion rate of L-theanine reached 63.7%，and the production intensity was 40.9 mmol/(h·L).The variable speed feeding process conducted for 15 hours with the same initial conditions，and the adding L-glutamine to the initial concentration of 200 mmol/L in every 2 hours.After 15 hours，600 mmol/L of L-glutamine was converted to L-theanine with a final concentration of 445.8 mmol/L and the conversion rate and production intensity reached 74.3% and 29.7 mmol/(h·L).

The Influence of Exogenous Nitric Oxide on Microtubulin of Brassica pekinensis Under Drought Stress

MA Xiao-li, JI Rui-ping

2016, 32(2): 172-177. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.023

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In order to study the effect of nitricoxide(NO)on microtubulin in drought-stressed Brassica pekinensis, using B.pekinensis cultivar Jinyu-11 as experimental material, and the drought situation was simulated by treating 7-day seedlings with 15, 18, 21, 24, 27 g/mL of PEG6000 concentrations.Ultraviolet spectrophotometry and SDS-PAGE were used to investigate the differences of microtubulin content in cabbage seedling leaves, and the optimal treatment concentration of PEG6000 was determined to be 27 g/mL.Then the pretreated seedlings were fumigated with various concentrations of NO：50, 100, and 150 μmol/L respectively, and the variations of microtubulin content in the treated cabbages were observed.Results showed that the contents of microtubulin in cabbage seedling leaves demonstrated the trend of increasing with PEG6000 pre-treatments compared to control, but decreased after NO treatment.Moreover, both the content of endogenous NO and the expression of NO-related genes increased significantly under drought stress.Therefore, physiological concentration of NO alleviated the disaggregation of microtubulin caused from drought stress at certain level, which eliminated the damages to plants from the stress.

Systematical Identification and Primer Screening of EST-SSR Marker in Transcriptome of Caragana intermedia

WANG Guang-xia, YANG Qi, WANG Rui-gang, LI Guo-jing

2016, 32(2): 178-184. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.025

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The aim of the study is to systematically identify and preliminary validate the SSR(simple sequence repeats)of EST(expr-essed sequence tags)in transcriptome database of Caragana intermedia for providing the basis in the further development of SSR molecular marker.Searching the SSR loci from Unigenes of the C.intermedia transcriptome by HiSeq²⁰⁰⁰ sequencing technology, total 45 706 SSR were obtained, accounting for 10.38% of the total Unigenes, averagely one SSR per 4.30 kb.Mononucleotide repeats were dominant in SSR with the ratio of 56.47%, bi- and tri-nucleotide repeats were 20.56% and 21.04%, and others were only 1.9%.Among all polynucleotide motifs, bi-nucleotide AG/CT were the most, second most was tri-nucleotide AAG/CTT.Totally 150 SSR primer pairs were randomly selected according to EST-SSR loci, PCR was verified by agarose gel electrophoresis, and 79 primer pairs showed clear amplified DNA fragments.While 21 out of the 79 primer pairs amplified single band, with a ratio of 14.0%.

The Application of the PiggyBac Transposon System in mCherry-transgenic Danio rerio

CAO Shou-ying, BAI Chang-cun

2016, 32(2): 185-191. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.027

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The red fluorescent protein gene(mCherry)was inserted into the modified PiggyBac transposon donor plasmid 5'-PTK-3', and the vector plasmid for gene trap, PTK-mCherry was constructed.PTK-mCherry and PiggyBac transposase mRNA were co-injected into fertilized eggs by microinjection, and the zebrafishes expressing mCherry in different temporally and spatially patterns were acquired.The statistics revealed that the expression rate of mCherry in F0 was 8.2%, indicating that PiggyBac transposon efficiently translocated and captured the different genes in zebrafish, and the efficient screening of mutants was achieved.

Genetic Structure Analysis of Macrobrachiu rosenbergii with Different Developmental Rate at Larval Stage

DAI Xi-lin, GAO Xiang, WANG Hai-yang, MING Lei, JIANG Zong-bing, DING Fu-jiang

2016, 32(2): 192-197. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.026

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In order to investigate the causes of asynchronization of development rate and the difference of growth for Macrobrachium rosenbergii larvae from the perspective of molecular biology, the genetic diversity and variation of M. rosenbergii with different larval development rates of 5 groups were analyzed.The results showed that 57 alleles among 5 groups were detected with 8 pairs of microsatellite primers.Numbers of alleles(Na)of each microsatellite ranged from 5 to 10, the genetic diversity of M.rosenbergii population among 5 groups had no significant differences.Meanwhile, the genetic differentiation coefficient and genetic distance between two populations were very close, indicating that genetic differentiation of M.rosenbergii with different larval development rate among 5 groups mainly resulted from the genetic differences among individuals.Combining with the prior researches, this study revealed that the causes of asynchronization of development rate and the difference of growth for M.rosenbergii larvae were not genetic variation and environmental differences.

Roles of Proline and Soluble Sugar in the Cold-adaptation of Antarctic Ice Microalgae

WANG Yi-bin, MIAO Jin-lai, JIANG Ying-hui, LIU Fang-ming, ZHENG Zhou, LI Guang-you

2016, 32(2): 198-202. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.024

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The content changes of proline and soluble sugar in two Antarctic ice microalgae under low temperature stress were measured and analyzed for understanding their cold-adaptation mechanisms.Measuring the proline and soluble sugar in two microalgae at different low temperatures and culturing time, the changing trend of them with the temperature and stress time at low temperature was obtained, and the reasons of it was analyzed.The lower the temperature was, the more significantly the contents of proline and soluble sugar in Pyramidomonas sp.L-1 increased.The amount of proline was 6.5 times of initial one and the soluble sugar increased 60% after 48 h at -5℃.The content of proline in Chlamydomonas sp.L-4 was in the same way as Pyramidomonas sp.L-1, i.e., the lower the temperature was, the higher the content of proline was.However, the changing trend of the content with the time was in contrast, decreasing with the culturing time.The proline was almost unchanged after 48 hours at -5℃, while soluble sugar increased 1.9 times.In conclusion, proline and soluble sugar play an important role in the cold-adaptation mechanism of Antarctic ice microalgae, however, the action mechanism is different in the different algae species.

Isolation Culture and Identification of Bone Marrow-derived Mesenchymal Stem Cells from Arbas Cashmere Goat

MA Su-ri-gu-ga, PENG Hang, LIU Jia-jia, ZHANG Yi-ting, GUI Hua, LIU Peng-xia

2016, 32(2): 203-210. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.028

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Here we isolated and cultured Arbas cashmere goat bone marrow-derived mesenchymal stem cells(gBM-MSCs)in vitro, identified the biological characteristics of gBM-MSCs, and analyzed the dynamic expressions of differentiation genes while induced in vitro.The primary cultured gBM-MSCs were spindle cell-based, showing radial colony arrangement, and stably and continuously passed down for over 15 generations.The detections by immunofluorescence and flow cytometry showed that gBM-MSCs were positive for mesenchymal specific markers, such as CD13, CD29, CD44, CD106, CD90 and CD166, but negative for hematopoietic stem cell surface markers CD45 and CD34.The induction in vitro confirmed that gBM-MSCs owned the multiple differentiation potential, and differentiated into adipocytes, osteoblasts and chondrocyte.Real-time PCR further indicated that the expressions of differentiation genes increased throughout the culture period.Our results confirmed that the isolated and cultured cells from the bone marrow of Arbas cashmere goat possessed the biological characteristics of gBM-MSCs.Our findings promoted the basic research of MSCs in livestock and laid a theoretical foundation for the further application of gBM-MSCs in transgenic breeding and tissue engineering.

The Regulation of Leptin-mediated AMPK Signal Pathway on Lipid Metabolism in Porcine Subcutaneous Preadipocyte

HUANG Ying, YANG Ming-hua, KONG Ling-fu, HAO Mei-lin, GAO Shi-zheng, LI Yong-neng, ZHAO Su-mei

2016, 32(2): 211-218. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.029

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Using in vitro cultured porcine subcutaneous preadipocytes as the study material, the expression levels of related genes were measured for investigating the molecular regulation mechanism of leptin-mediated AMPK signal pathway on lipid metabolism.The subcutaneous adipocytes were treated with 0 and 100 ng/mL leptin for 48 h, and the adipocytes were identified by oil red O dyeing, and the contents of triglyceride and free fatty acids in the cells were measured by kit, and the mRNA expression levels of related genes in the AMPK signal pathway of subcutaneous preadipocytes were detected using Real-time PCR technology.The results showed that in leptin group, the content of the triglyceride of the subcutaneous preadipocytes was significantly lower(P<0.05)compared with the control group, indicating that the leptin reduced the contents of triglyceride and free fatty acids in the cells.The expression levels of gene lepR, AMPK, CPT-1 in AMPK signal pathway were significantly higher in leptin group than control group(P<0.05), while the expression level of gene ACC in leptin group was significantly lower than control group(P<0.05), implying that leptin initiated the signal transmission in the AMPK signal pathway by regulating the mRNA expression of related genes.The expression levels of gene lepR, AMPK, and CPT-1 were significantly negatively correlated with the content of triglyceride and free fatty acid(P<0.05)；however, the expression level of gene ACC was significantly positively correlated with the content of triglyceride and free fatty acid(P<0.05).In conclusion, the results suggested that leptin activated AMPK signal pathway, reduced the expression of ACC, and increased the expression of CPT-1, which therefore promoted the decomposition of triglyceride and oxidation of fatty acid, and finally decreased the contents of the triglycerides and free fatty acid in the cells.

Auto-inducing Expression of GST-SUMO-MT Fusion Protein in Escherichia coli

PENG Dong,YANG Wei,WANG Zhao,WANG Xi,LIU Zhong-yu

2016, 32(2): 219-224. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.030

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The auto-inducing expression of GST-SUMO-MT fusion protein in Escherichia coli BL21(DE3)was studied.In order to increase the protein yield, we optimized both the auto-induction fermentation conditions and medium.Recombinant E.coli was cultured in shake flask, of which the culture condition(time and temperature for induction)and medium composition(tryptone, yeast extract, glycerol, glucose and lactose)were optimized by single factor and orthogonal tests, and bacterial concentration, expression level of soluble target protein were determined.Result showed that the optimal carbon and nitrogen composition ratio in auto induction medium was obtained as followed：2% tryptone, 2% yeast extract, 0.3% glycerol, 0.05% glucose, 0.3% lactose.The optimum fermentation conditions were：cultivating in 37℃ for 3 h firstly, then culturing in 25℃ for 13 h.Under the optimal condition, the expression level of soluble target protein and bacterial concentration were 2.2 and 2.3 times of those in LB medium under induction of IPTG respectively.

The Establishment of HepG2 Cell Line with TALEN-mediated Knockout of CXCR4

ZHANG Wen-mei, DING Yan, GUO Xing-rong, LI Dong-sheng, ZHAO Wan-hong, WANG Xiao-li

2016, 32(2): 225-228. doi:10.13560/j.cnki.biotech.bull.1985.2016.02.031

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It was to research on the effect of CXCR4 on HepG2 cells, we established one cell line which could down-regulate CXCR4 expression stably by transcription activaor-like effector nuclease (TALEN) gene targeting.The hepatocellular carcinoma cell line HepG2 was chosen for this study, and the expression of CXCR4 was interfered by transcription activator-like effector nuclease (TALEN).The constructed CXCR4 TALEN plasmids were transfected into HepG2 cells, and the targeting efficiency was determined as 40% by enzyme digestion of T7E1.The single clone cell of CXCR4 knockout was screened by gene sequencing, and the significant decrease of gene CXCR4 expression was confirmed by immunofluorescence and Western blot.The hepatocellular carcinoma cell line with stably down-regulating CXCR4 expression was established successfully by TALEN gene targeting.

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2016, 32(2): 300.

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