Table of Content

09 January 2016, Volume 32 Issue 1

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Research Progress on Endophytes in Medicinal Plant Artemisia annua

SONG Chun-zhu, CHEN Xin-wen, CHEN Dong-hong

2016, 32(1):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.002

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As the most effective anti-malaria Chinese medicinal herb, Artemisia annua has other broad-spectrum of application in medicine and agriculture fields. Furthermore, endophyte resources closely related to it have also become the hot spot for investigation at present. Here, we summarize and sort the research progress on endophytic microbes in sweet wormwood, which are mainly applied on anti-tumor, anti-oxidant, anti-microbe and the promotion of artemisinin biosynthesis.

Research Advances on Plant Aspartic Proteinase

GE Wei-na, LI Chao, ZHANG Jia-qi, ZHANG Lan, WANG Zhen-yi

2016, 32(1):  8-14.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.003

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Aspartic proteinases constitute one of four super-families of proteolytic enzymes, and are widely distributed in varied living organisms and involved in many biological processes. Compared with other organisms, the research on plant aspartic proteinases is relatively lagging behind. Since the end of last century, significant progress has been made in this area. Except conserved proteinase domain, typical plant aspartic proteinases have a specific insert only in plant. After the separation of plant and animal, the gene family of aspartic proteinases has expanded largely;these proteinases play important role in whole progress of plant growth and development, especially in stress response, sexual reproduction, senescence and programmed cell death, and the processing and degradation of protein. In this context, the research advances in plant aspartic protease is reviewed and its future development is prospected.

Research Advance on the Effect of Food Functional Components on Animal Genomic DNA Methylation

ZHAO Jing, LI Nan, WU Ru, YANG Zhan-wei, HU Wen-bing, WANG Wen-jun

2016, 32(1):  15-19.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.004

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DNA methylation is one of the major epigenetic modifications in eukaryotic genomes, which can be influenced by certain food functional components, such as polyphenols, flavonoids, vitamin, n-3 polyunsaturated fatty acid etc. The effects of food functional components on DNA methylation are two-fold, either modulating the methyltransferase’s activity, and/or changing the number of methyl groups. Based upon the recent progress on the ongoing research, this paper expounds the effects and the possible mechanisms of a variety of functional components, i. e. , polyphenols, flavonoids, vitamin(folic acid, VB12, VB6), n-3 polyunsaturated fatty acid etc. , on the DNA methylation, which is expected to provide new ideas on exploring the molecular mechanisms of food functional components on DNA methylation modifications.

Research Progress on a Long Non-coding RNA MALAT1

SONG Tie-feng, YUAN Ying, WANG Hui-qin, ZHUANG Chun-yu, WANG Nan, ZHANG Tong-cun

2016, 32(1):  20-28.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.005

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Long non-coding RNAs(lncRNAs)are the ones with transcripts that are longer than 200 nt. They do not encode proteins, but regulate the expression levels of the gene as a RNA molecular at transcriptional, post-transcriptional and epigenetic modification. Metastasis-associated lung adenocarcinoma transcript 1(MALAT1)was firstly discovered in non-small cell lung cancer, and has caused the concerns from scholars. MALAT1 located on chromosome 11q13.1 and was highly conserved. In recent years, studies have shown that MALAT1 specifically recruited SR protein family members, was involved in epigenetic regulation and cell cycle regulation. MALAT1, which is highly expressed in many human tumors, promotes tumor proliferation, invasion and metastasis. In addition, recently it is found that MALAT1 plays an important role in angiogenesis. This article systematically reviewed the characteristics and mechanism of MALAT1, as well as its biological function in tumor and vascular systems.

The Biological Characteristics and Potential Applications of Epidermal Stem Cells

LIU Xue-ting, BAI Chun-yu, GUAN Wei-jun, MA Yue-hui

2016, 32(1):  29-32.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.006

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The epidermis is a crucial barrier for people to sustain life and maintain our organs in their functions. Epidermal stem cells(ESCs)play an important role in the process of maintaining epidermis balance and repairing the damaged ones, as they can differentiate into the layers of skin tissue, so that the epidermis is always in the state of continuous proliferation, differentiation and apoptosis. With the rapid development of cell biology, people growingly realize the vital role of ESCs. In this paper, research progress of epidermal stem cells, with the emphasis on the classification of epidermal stem cells, orientation, characteristics, functions and application prospect, purport for related researchers of epidermal stem cells biological characteristics and application have more comprehensive understanding.

Small Non-coding RNA and Male Sterility

LIAO Ke, CHAI Zhi-xin, ZHONG Jin-cheng

2016, 32(1):  33-40.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.007

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Small non-coding RNA(sncRNA)is a kind of RNA with 20-30 nucleotides, and does not have the function of encoding proteins, but has extensive biological functions in terms of regulatory mechanism. In the process of animal sperm formation there are two types of sncRNAs, namely miRNAs and piRNAs playing a critical regulatory role. When they are expressed abnormally in testicular tissues of animals, which causes the production of sperms in obstacle, so as male sterility would appear. In this paper, we summarize sncRNA’s structure and function, and effects on male fertility in order to offer the references for the further researches of the sncRNA regulatory mechanisms.

The Function and Role of Microbial Agents in Composting Technology of Garden Waste

ZHAO Kai-ning, ZHAO Guo-zhu, GUO Hui, WANG Xiao-xu, ZHU Sheng-nan, XU Rui

2016, 32(1):  41-48.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.008

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The increase of garden waste causes severe pressure to environment. Microbial agents inoculated in garden waste compost can accelerate the maturity of composting, enhance the fertilizer’s efficiency and improve the utilization ratio of resources, which has shown favorable application prospect in terms of garden waste reuse. Starting from the current processing methods and applications of microbial agents in composting technology, the function and role of microorganisms in the composting of garden waste and fermented microbial fertilizer were introduced, and the important parameters of monitoring compost maturity and the technical methods of detecting the changes of microorganism’s community in fermentation process were described. Finally, the application of microbial agents inoculated in the composting technology of garden waste was prospected, which is expected to provide a reference for the research and development of related areas.

The Application of Nanobiotechnology in Medicine

YANG Hui, DING Liang, YUE Zhi-lian

2016, 32(1):  49-57.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.009

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In recent years, nanomaterials and nanobiotechnology have been growingly applied in the clinical treatment and diagnosis, and the development of nanopharmaceuticals, nanomedicial materials, nanochip technology for diagnosis in vitro, have been going on gradually and progressing significantly. This paper discusses the status and developing prospects of nanomaterials and nanobiotechnology mainly from two aspects of nanomedicine and nanodiagnostics.

Research Progress on Production of Tetramethylpyrazine by Fermentation

HOU Xiao-yuan, GU Ru-lin, LIANG Wen-long, XIAO Zi-jun

2016, 32(1):  58-64.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.010

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2, 3, 5, 6-tetramethylpyrazine(TTMP)as an important food flavor additive, it has additional medicinal values, such as in cardiovascular and cerebrovascular health, respiratory system diseases and glomerular disease. In this paper, we reviewed the research progress on fermentation production of TTMP, analyzed the synthesis mechanism of TTMP, and summarized the effective strategies to increase the yield of TTMP, including the screening of endogenous high-yield strains, the optimization of fermentation process, the feeding of ammonium salt, and multi-step fermentation control. We concluded that the production of TTMP can be divided into two stages, i. e. the enzymatic synthesis of precursor acetoin and then the spontaneous condensation of TTMP from acetoin and ammonium. Further, the fermentation strategy with multiple-step temperature control effectively increased the production yield. The extraction and purification of the product were introduced as well. The future prospects of fermentation production of TTMP are discussed in light of the current progress, challenges, and trends in this field.

Research Progress on the Extraction and Purification of Phycocyanin from Spirulina

FU Li-li, NA Ri, GUO Jiu-feng, JIN Jing

2016, 32(1):  65-68.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.011

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Phycocyanin has been widely used in food, cosmetics, and medicine due to its physiological properties such as anti-oxidant, improving the body’s immunity, anti-cancer, anti-inflammatory and protecting hepatocyte. Its extraction and purification technology has attracted wide attentions. This work lists several extraction and purification technologies of phycocyanin in recent years, and compares the merits and drawbacks of the major methods.

Synchronously Detecting the Allergenic Ingredients of Soybean and Celery in Food by Real-time Fluorescent PCR

WANG Yong-xin, CHENG Xiao, AN Hong, NIE Lei, ZHANG Bo, LIU Juan-juan

2016, 32(1):  69-73.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.012

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Soybean and celery as food ingredients are allergen for some special groups of people. Designing the specific primers based on atp A gene of soybean and mtd gene of celery and utilizing TaqMan probes with different fluorescents, we set up a fluorescent real-time PCR method allowing the simultaneous detection of allergenic constituents of soybean and celery in food. The specificity of the method was evaluated using template DNAs from soybean or celery and other 20 species such as rice, wheat, barley, peanut, sesame, maize, murphy, tomato, walnut, pistachio, cashew nut, sunflower seeds, almond, apple, pear, strawberry, pork, beef, mutton and fish, only the specific amplifications of soybean and celery were observed. The limit of quantification was 0. 01% through sensitivity tests. In conclusion, the developed real-time PCR method is a specific, sensitive and efficacious assay for the simultaneous detection of allergenic soybean and celery in food.

The Extraction of Metagenom DNA in Branch Bark

ZHAO Ling-yun, FAN Dong-ying, LI Yan-fang, YAN Xia, HUANG Li-li

2016, 32(1):  74-79.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.018

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A suitable method capable of extracting high-quality metagenom DNA from a variety of branch barks was developed based on previous experiences of plant DNA extraction. The method consisted of several steps：adding PVP while grinding materials, washing bark powder with bufferⅠ, simultaneously using SDS and CTAB, low-temperature frozen and then melting samples after adding high concentration of potassium acetate(KAc), precipitating DNA using isopropanol under high concentration of NaCl, and adding trace RNase A after DNA completely dissolved. Finally, the concentrations of genomic DNA obtained by this method ranged from 190 to 970 ng/μL with all in high purity. All DNA can be digested by restriction endonuclease and used as templates for bacterial 16S rRNA gene amplification. The DNA of apple bark as the template was amplified into gene BIP and PDI, then the genome DNA of apple bark was detected by a sequencing firm, and the quality satisfied the requirements of environmental metagenome for studying microbial diversity by high-throughput sequencing.

An Analysis of Phylogenetic Relationship of the Genus Disanthus Distributed Disjunctively in China and Japan Based on cpDNA Sequences

LI Li-ka, LI Xiang-qin, XIE Guo-wen, LI Hai-sheng, ZHENG Yi-sheng, TENG Jie-hua, GAO Jin-wei

2016, 32(1):  80-87.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.013

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Disanthus is an endemic and endangered genus in East Asia and distributed disjunctively in China and Japan. In this project, we conducted the phylogenetic study of 164 individual plants in 16 natural populations of Disanthus based on the 3 fragments(psbA-trnH, rps16F-R2, and psaA-ycf3)of chloroplast DNA(cpDNA). The results of the phylogenetic relationship confirmed that Disanthus gercidifolius in Japan and Disanthus cercidifolius subsp. Longipes in China were monophyletic groups, and they had no share of haploid type. Furthermore, Japanese populations could be divided into 2 geographical branches, one from the center of Honshu(population QE and CY), and the other from the southwestern Honshu(population ga, gd and JH). PERMUT analysis showed that total genetic diversity(Ht = 0.725)of Disanthus between populations was higher than the average genetic diversity(Hs = 0.148)within populations. Regarding the genetic difference between populations, Nst(0.905)was larger than Gst(0.976;P < 0.01), demonstrating that there were significantly geographical structure of pedigree. AMOVA results further revealed that genetic variation occurred mainly between populations(53.08%), while 24.11% among populations within a group and 22.80% among individuals within a population. These results indicated that the genetic differentiation within Disanthus between Chinese and Japanese subspecies were obvious.

The Regulation of 6-BA, IAA, GA₃ and ABA on the Ripening and Senescence of Mu Jujube’s Fruits(Ziziphus jujuba L.)

YANG Wei-min, DU Jing-qi, ZHAO Jun

2016, 32(1):  88-91.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.014

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In white mature period of Mu jujube, treating the fruits with different plant hormones 6-BA, IAA, GA₃ and ABA, the regulatory effect of them on the ripening and senescence of fruits were studied through the detection of physiological and biochemical changes. Results showed that： the contents of DNA in jujube fruit after 6-BA, IAA, GA₃ and ABA treatment varied significantly, and the effects with ABA and 6-BA treatment were the most;the contents of MDA in jujube tissue after GA₃ and IAA treatment were significantly lower than those in the control group, and the results after 6-BA and ABA treatment were contrary;the contents of proline in jujube tissue were significantly lower than those in the control group except ABA treatment, and the contents of Vc were in the opposite; the CAT activity differed greatly, the CAT activities with ABA and 6-BA treatment were significantly higher than those in the control group, and those with IAA and GA₃ were contrary. In conclusion, the physiological role of 6-BA, IAA, GA₃ and ABA presented as the mutual promotion and antagonists in many ways, and showed their regulations on ripening and senescence in Mu jujube fruit.

The Establishment of High-frequency Plant Regeneration System from the Radicle Tip of Chinese Chive(Allium tuberosum Rottler)

WANG Ting-ting, GAO Xing-ying, LI Mei-lan, SONG Hong-xia, HOU Lei-ping

2016, 32(1):  92-96.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.015

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In order to establish the high frequencey regeneration system of Allium tuberosum, using the radicle as the explant, the paper examined callus and bud induction from its various parts. The results showed that the radicle tip was the best explant for inducing callus with the induction rate of 81.4%, and the bud induction rate reached 73.3%. Comparing the induction of callus and differentiation of bud in several media formulas, the regeneration rate was the highest in medium of MS+1 mg/L BA+0.5 mg/L NAA+0.5 mg/L KT, with induction rate over 80% and the differentiation rate of adventitious bud reaching 78.8%, respectively, while the propagation coefficient was 24.7. The percentage of adventitious buds elongating to be independent plant was 85.5% on elongation medium of MS+1 mg/L GA+0.5 mg/L KT at 30 d. The rooting rate and root system for the independent shoots were the best in the medium of 1/2 MS+0.3 mg/L IAA, and in which a single plant from the differentiation of radicle was generated at approximate 130 d.

The Study of Regeneration System from Different Sea Island Cotton (Gossypium barbadense L.)Cultivars

DING Xi-lian, QU Yan-ying, LI Qiong, GUO Jia-yan, CHEN Quan-jia

2016, 32(1):  97-102.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.016

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The hypocotyls of 6 sea island cotton cultivars, i. e. , DJ-1(373), Tianchang 2(376), 5917(380), Achang-599(381), Ta 07-152(383), and S0717(408)were used as explant materials;calli were successfully induced with two optimized combinations of plant growth regulator(PGR)screened by the method of double factors randomized block, and the plants were regenerated successfully. The results showed that, the best PGR combination to induce callus for 380, 383, and 381 was 0.1 mg/L 2, 4-D + 0.1 mg/L KT, while that for 373, 376, and 408 was 0.3 mg/L NAA + 0.1 mg/L KT. The induced calli inoculated into the medium of double KNO₃ generated embryogenic ones, and inoculating them into the medium of 0.1 mg/L KT and 0.05 mg/L IBA promoted the somatic embryogenesis and their maturing. The culture of malformed seedlings shortened the cycle of regeneration system, and the regenerated plants were all obtained from 6 cultivars in 6 to 8 months. A stable and efficient regeneration system without genotype limit was established.

Multiple Gene Transformation in Sugarcane and Multiple PCR Detection

WANG Wen-zhi, YANG Ben-peng, CAI Wen-wei, XIONG Guo-ru, WANG Jun-gang, FENG Cui-lian, ZHANG Shu-zhen

2016, 32(1):  103-108.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.017

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Multiple traits of crops such as rice and corn had been improved in single time of transformation by the strategy of multiple gene transformation. For exploring the feasibility of multiple gene transformation of sugarcane in this research, a multiple gene expression vector was used and transformed to sugarcane and many transformed plants were produced. Multiple PCR method was used to analyze the integration and deficiency of multiple genes in the transformed plants. Results showed that multiple gene transformation of sugarcane was feasible. But due to the insertion of large DNA fragments some genes were lost during the integration. Thus it is a need to have sufficient transformed plants then valuable transformed plants can be selected for further research.

The Construction of Expression Vector of Gene CBF from Vitis amurensis and the Effects of It on the Root Growth of Arabidopsis

WANG Fa-wei, DONG Jin-ye, LI Xiao-wei, LIU Yang, WU Xue-yan, WANG Nan, DONG Yuan-yuan, CHEN Huan, LI Hai-yan

2016, 32(1):  109-114.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.019

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In order to investigate the response mechanism of CBF gene in the regulation of plant to the salt stress, the plant over-expression vectors of VaCBF1, VaCBF2 and VaCBF3 from Vitis amurensis were constructed. The results by restriction enzyme digestion and agarose gel electrophoresis showed that 3 genes were correctly inserted into pBASTA, indicating that the expression vectors were successfully constructed. Then, 3 over-expressed vectors were transformed into Agrobacterimu tumefaciens EHA105, and Arabidopsis thaliana was leached using floral dip method. The transgenic plants of A. thaliana with over-expression of 3 genes were screened by herbicide Basta. Furthermore, wild and transgenic plants were treated with salt stress. The results revealed that the elongation length of primary root of OE-CBF2 transgenic plant was longer than others, and the lateral roots in 3 genes transgenic plants were longer than wild ones. These results indicate that the CBF genes of V. amurensis play the critical regulation role in the root development during salt stress.

Cloning and Expression Profile of DREB2.2 Gene from Zoysia japonica var. pallida cv Jiaodong

KE Xiang NONG, Jun-xiu, SHI Da-lin, MA Li-peng, LI Jing, WEI Shan-jun

2016, 32(1):  115-123.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.020

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DREB(dehydration responsive element binding protein)is a type of transcription factor commonly existing in higher plants, and involves in abiotic stress response and the process of growth and development. In this study, using Zoysia japonica var. pallida cv Jiaodong as material, the coding sequence of gene DREB2.2 was cloned, then the bioinformatics were analyzed, and the expression profile of the gene in the stress environment was detected by semi-quantitative PCR technique. Sequencing analysis indicated that DREB2.2 had 2 transcripts of long and short. The long transcript, DREB2.2-L, was 1 067 bp in length, with a premature ORF because of a 50 bp insertion sequence, and putatively encoding 65 amino acids. The short one, DREB2.2-S, was 1 017 bp in length, encoding 338 amino acids;the putative protein was 37.3 kD, pI was 4.86, and it belonged to the A-2 group of DREB subfamily, containing an AP2 conservative structure domain and nuclear localization sequence. The results of semi-quantitative PCR showed that both DREB2.2-S and DREB2.2-L were expressed in normal condition, the expressions were up-regulated under low temperature stress with the highest level at 2 h exposure, and were slightly up-regulated during the 2 h and 24 h of drought stress, but showed no response to high salt stress. In both normal and stress conditions, the mRNA amount of DREB2.2-L was slightly higher than that of DREB2.2-S.

The Differential Expression of Genes for Key Enzymes in Gibberellin Metabolism in Potato Mutant

SHI Jian-bin, YANG Yong-zhi, WANG Jian

2016, 32(1):  124-130.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.021

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In order to reveal the correlation between the mutation of plant height and genes of key enzymes in gibberellin(GA)metabolic pathway, using the mutant strain 4P2-9 of potato as materials, we established double standard curve by relative quantitative RT-PCR, and tested the expression of GA metabolism-related genes in the “Plateau 4” and its mutant “4P2-9” using Actin gene as reference gene. The results showed that the plant height and the internode number of 4P2-9 significantly were lower than the control material(P<0.05);however, the internode length was significantly higher than the control(P<0.05), moreover the expressions of gene KS, KO and GID1 for key enzymes in GA metabolism were significantly down-regulated, and were 0.725, 0.657 and 0.890 times the control, respectively. The expressions of GA20ox1 and GA2ox1 genes were increased, and were 1.425 and 1.557 times the control, respectively. The results revealed that the expression changes of genes for key enzymes in the GA metabolism was one reason of the variation of 4P2-9 plant height, and the up-regulation of gene GA20ox1 and GA2ox1 expression was the main factor for the decrease of plant height.

The Study of Immune Activities of Extracts from Wild and Cultivated Cistanche deserticola in Xinjiang

YANG Xiu-mei, YANG Yu, WANG Dan-yang, WU Dao-cheng, ZHANG Ai-lian

2016, 32(1):  131-137.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.022

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This work is to compare the differences of major active components in the extracts between wild and cultivated Cistanche deserticola in Xinjiang, detect their capacities to induce the maturation of dendritic cells(DCs)in mice, and evaluate their immune activities. The extracts were prepared by ultrasonic extraction, and the contents of polysaccharides and phenylethanoid glycosides were measured by ultraviolet-visible spectrophotometer method. The immune to BALB/c mice with extracts was through foot pad, and the expressions of CD86(cell differentiation antigens 86)and MHCII(major histocompatibility complex II)on the surface of CD11c⁺DCs inside mouse were detected by flow cytometry. The polysaccharides contents in the aqueous extracts from wild and cultivated C. deserticola were 59.58% and 76.82% respectively, and the contents of phenylethanoid glycosides in the alcohol extracts were 17.12% and 8.48% respectively. The immune tests of mice showed that the extracts from both wild and cultivated C. deserticola enhanced the expression level of CD86 and MHCII on the surface of CD11c⁺DCs inside mice(P<0.01);and the effect was similar to that of the positive control group(LPS). The effect of the same dosage of wild and cultivated ethanol extracts on the CD86 was significantly different(P<0.05). The effect of the other extracts on MHCII and CD86 was not significantly different(P>0.05). In conclusion, there were differences in the main components between Xinjiang wild and cultivated C. deserticola, and in appropriate concentration they both could significantly promoted DCs maturation in mice, and the difference was not significant.

Prokaryotic Expression of i-type Lysozyme from Periplaneta americana and Preparation of Its Polyclonal Antibodies

WANG Yun, ZHAI Su-zhen, ZHANG Chun-lin, WANG Ji-ping

2016, 32(1):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.023

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The objective of this work is to express i-type lysozyme(PaI)from Periplaneta americana in Escherichia coli and prepare its polyclonal antibodies of anti-PaI in mice. The coding gene of mature peptide region was amplified by PCR and prokaryotic expression vector pET28a-PaI was constructed. PaI-His fusion protein was expressed with IPTG induction and purified by Ni-NTA affinity chromatography. Then, Balb/c mice were immunized with the purified recombinant protein, and the titers of antiserum and the specificity of polyclonal antibodies were detected by indirect ELISA and Western blotting respectively. Results were as below. The length of nucleotide sequence encoding the mature peptide was 414 bp that encoded a putative protein with 137 amino acids. The constructed prokaryotic expression vector pET28a-PaI was successfully expressed in Escherichia coli. SDS-PAGE detection indicated that the PaI-His fusion protein was about 18 kD. Indirect ELISA and Western blotting analysis showed that the antiserum from immunized mice had high titer and specificity. In conclusion, the prokaryotic expression of PaI protein was successfully realized, and the anti-PaI polyclonal antibody with high efficiency and specificity were prepared, which laid the foundation for the further researches on the biological function of PaI protein.

Expression and Antiviral Activity Detection of Porcine Interferon -gamma in Silkworm

LIU Xing-jian, LI Hao-yang, HU Xiao-yuan, ZHANG Zhi-fang, YI Yong-zhu, LI Yi-nv

2016, 32(1):  144-148.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.024

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Interferon is used for threating viral infectious diseases and enhancing vaccine efficacy in stockbreeding. In this study, recombinant porcine interferon-gamma(PoIFNγ)was expressed in silkworm by using Baculovirus expression vector system. Based on the published sequences, the gene of PoIFNγ was artificially synthesized after codon optimization. The optimized gene was firstly cloned into pVL1393 transfer vector of Baculovirus, with virus BmBacmid DNA which was co-transfected into BmN cell line, and recombinant BmNPV was obtained. The PoIFNγ gene was in the downstream of polyhedrosis gene promoter in BmNPV. The silkworm larva infected with recombinant BmNPV gained the expression of PoIFNγ that was detected by Western blotting. The activity of interferon was measured by using micro-cytopathic method that uses interferon to inhibit the VSV-GFP infecting VERO cells. The results showed that the titer of interferon was over 6×10⁵ IU/mL. The activated PoIFNγ was successfully expressed in silkworm larvae.

Cloning and Tissue-specific Expression of CAPN3 Gene in Yak

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

2016, 32(1):  149-155.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.025

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Using the longissimus of yak(Bos grunniens)back as material, the CDs sequence of yak CAPN3 gene was cloned by RT-PCR, and it was analyzed by bioinformatics. The results indicated that the length of CDs in yak CAPN3 gene was 2 469 bp and encoding 822 amino acid residues. Bioinformatics analysis showed that the protein encoded by CAPN3 was the non-secretory surface protein, containing 35 phosphorylation site, and mainly played a biological role in the cytoplasm and nuclei. The secondary structure was mainly composed of α-helices, random coil, extended chain and β-turn, having the structure domains of CysPc, calpain-Ⅲ and EFh families and no signal peptide. The CAPN3 of yak was the most similar with those of Bos taurus, Ovis aries and Sus scrofa in phylogenetic tree. Real-time PCR analysis revealed the expressions of CAPN3 in varied tissues. There were expressions in all 7 tissues, and they were high in the muscle and pancreas.

The Development and Application of Multiplex PCR Panels of Microsatellites in Hyriopsis cumingii

YIN Hao, BAI Zhi-yi, HAN Xue-kai, LI Jia-le

2016, 32(1):  156-162.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.026

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In order to increase the analysis efficiency of microsatellite in Hyriopsis cumingii, 3 groups of 4 multiplex PCRs panels were established from the developed microsatellite labels. The stable multiplex PCR panels were used to analyze the genetic diversities of 56 purple breeding lines of H. cumingii. The results showed that the average number of alleles(A)was 18.75, the effective number of alleles(An)was 9.010, the average of polymorphism information content(PIC)was 0.857, the average observed heterozygosity(Ho)and expected heterozygosity(He)were 0.809 and 0.876, respectively, and Shannon information index(I)was 2.422. The Cervus v3.0 software was used to have parentage assignment for 114 individuals in 3 full-sib families, and the results revealed that accuracy of parentage assignment by this 3 groups of multiplex PCR panels was 100%. Therefore, this multiplex panels can be applied in genetic diversity studies of H. cumingii population, parentage assignment and family management with increased detection efficiency and reduced test cost.

Prokaryotic Expression and Purification of Zebrafish SFPQ

YANG Chuan, HU Min

2016, 32(1):  163-168.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.027

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Splicing Factor Proline and Glutamine Rich(SFPQ)is a protein that expresses widely in vertebrates. As a tumor suppressor gene, it plays the vital regulation role in tumorigenesis of living organisms. So far the researches of SFPQ were focused on human being and mice, little has been known about the SFPQ of zebrafish. In order to acquire the prokaryotic-expressed SFPQ protein for further research, recombinant pET28a-SFPQ plasmid of zebrafish was constructed and transformed into the chemical competent cells of BL21. The optimal condition was explored while IPTG was applied to induce the expression of zebrafish SFPQ protein in low-temperature, and the SFPQ protein was purified by Ni-NTA agarose nickel column. The results demonstrated that the expression of the protein was the most while inducing for 10 h by 0.75 mmol/L IPTG at 27℃. The results of Western blot revealed that the purified SFPQ protein specifically bound with the antibody, indicating that the purified zebrafish SFPQ protein may be utilized in the further experiments.

The Deleterious Effects of Norfloxacin on Zebrafish Embryonic Development and TGF-β1 Expression

WANG Dong-mei, GU Cong-you, LIU Tong, ZHANG Qiong-yu, LI Pei, HU Xiao-jun

2016, 32(1):  169-173.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.028

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The aim of this work is to investigate the deleterious effects of norfloxacin on zebrafish embryonic development in different periods, and determine its effects on TGF-β1 expression. Zebrafish embryos were exposed to different concentrations of norfloxacin(0, 10, 20, and 40 μmol/L). The effects of norfloxacin on spinal curvature, pericardial cyst, yolk edema and mortality of zebrafishes in the different developmental periods were observed. Furthermore, the mRNA level of TGF-β1 was examined by qPCR. The results showed that norfloxacin had significantly deleterious effects on zebrafish embryonic development, as mainly evidenced by spinal curvature, pericardial cyst and yolk edema. With the increase of concentration of norfloxacin, embryonic development was delayed, and the hatch period and mortality increased. The mortality rate of zebrafish embryos at 96 hpf re ached 76.46% after exposure to 40 μmol/L norfloxacin. Moreover, comparing with control group, the increase trend on mRNA level of TGF-β1 in zebrafish embryo exposing to different dose of norfloxacin slowed down with the prolonging of development time. Conclusively, norfloxacin has teratogenic and lethal effects on zebrafish embryonic development, suggesting norfloxacin residue in water system has serious latent dangers to the reproduction and development of fish.

Breeding of a Phenazine-1-carboxamid-producing Strain by ARTP Mutation and Its Optimization of Fermentation

TAN Jian, XIONG Xin, LIANG Wan-li, PENG Hua-song, ZHANG Xue-hong

2016, 32(1):  174-179.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.029

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Using an atmospheric and room temperature plasma(ARTP)jet to mutagenize a phenazine-1-carboxamide-producting strain Pseudomonas Chlororaphis P3, the mutant strain P3-9 with the highest phenazine-1-carboxamide(PCN)yield of more than 2 093 mg/L was obtained from the primary-screened 20 mutant ones, and it was 125% that by the original strain. Furthermore, single factor experiment was used to investigate the effects of varied nutrient factors on the PCN synthesis of P3-9. The results showed that the best carbon source and nitrogen source were glycerol and tryptone respectively. Adding Fe³⁺ or Fe²⁺ had a significant effect on PCN production, and no measurable effect while adding aromatic amino acids of phenylalanine, tryptophan and tyrosine. After the optimization, the PCN yield of strain P3-9 reached 2 810 mg/L, which was the highest yield of PCN by mutation breeding in the world so far.

The Effect of Kar2p Overexpression on Plectasin Expression in Pichia pastoris

WANG Nan, LI Gang-qiang, GUO Wen-fan, LIU De-hu

2016, 32(1):  180-186.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.001

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Kar2p is one of the molecular chaperones. In order to investigate its effect on expression of heterologous proteins in Pichia pastoris, the gene encoding the protein was cloned from P. pastoris GS115 and transformed into the P. pastoris transformant P-Ple. The twice transformant P-PleKar that simultaneously over-expressed plectasin and Kar2p was obtained. The transcription levels of Kar2p in P-Ple and P-PleKar were analyzed by real-time PCR. Kar2p transcription in P-PleKar was up-regulated about 18 folds as in P-Ple. Comparing the growth curves between P-Ple and P-PleKar, the overexpression of Kar2p had no effect on cell growth of host, but inhibited the expression of heterologous protein plectasin. Duo to the Kar2p’s effect, the highest antibacterial activity of plectasin significantly decreased at the 48 h of induction. This did not only inhibit the expression of plectasin, but also the expression and secretion of protease in P. pastoris. It was evidenced that plectasin secreted into the culture medium maintained high antibacterial activity in the last stage of fermentation, and were not rapidly degraded by protease accumulated in culture. Compared with P-Ple, the overexpression of Kar2p slightly reduced the expression level of plectasin in P-PleKar, but kept the expression level stable for a longer time.

Identification and Analysis of a Nattokinase-producing Strain

WANG Xue-yan, SUN Yu-fei, FENG Fei, CHEN Xiao-fei, LI Feng, ZHOU Fu-zhong

2016, 32(1):  187-194.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.030

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The objective of this work is to identify an isolate Td of yielding high nattokinase, and to analyze the molecular characteristics of its nattokinase. The morphologic, biochemical and physiological characterizations, and molecular biological methods were used to identify the taxonomic position of isolate Td;mass spectrometric determination and analysis of MALDI-TOF, SDS-PAGE and fibrinolytic activity were employed to measure the characteristics of nattokinase, and PCR method was adopted to clone the full length of nattokinase gene. The bacterial strain Td was identified as Bacillus subtilis subsp. subtilis according to the results of morphologic, biochemical and physiological characterizations, as well as 16S rDNA, the sequences of 16S rDNA gyrA, hybrid rate of DNA-DNA. The yield of nattokinase produced by strain Td reached 300 mg/L, accounted for over 40% of the total protein. Fibrinolytic activity was over 230 U/mL. Amino acid sequence of nattokinase produced by Td showed the highest similarity to the subtilisin E. and the full length of the gene was 1143 bp. Conclusively, as a high-yield and high-activity strain for nattokinase, B. subtilis subsp. subtilis Td is a promising candidate for industrial development and utilization.

Isolation and Identification of a Deep-sea-derived Fungus FS86 and Its Biological Activities

WU Hou-lv, LI Hao-hua, TAN Guo-hui, CHEN Yu-chan, LIU Yun-mei, ZHANG Wei-min

2016, 32(1):  195-200.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.031

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A strain of fungus FS86 was isolated from deep-sea sediment of South China Sea by the spread plate method. According to the culture features, morphological characteristics and ITS sequence analysis, the strain was identified as Cladosporium sphaerospermum. Bioassay results showed that the fermentation extract of the strain FS86 demonstrated significant inhibitory effects against 8 pathogenic fungi and 4 tumor cell lines with MICs of 0.1-4 mg/mL and IC₅₀ values of 0.86-2.13 μg/mL, respectively.

Construction of Pichia pastoris Surface Display Technology as Whole -cell Biocatalyst for Thymidine Phosphorylase

WANG Jie, YU Lei, YANG Dong, LI Jie, WANG Hong-zhong

2016, 32(1):  201-206.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.032

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Thymidine phosphorylase(TP)extensively involves in nucleoside metabolism and catalyzes the formation of many nucleoside analogs(NA). A yeast cell surface display system for TP was firstly constructed in this study as whole-cell biocatalyst. A deoA gene encoding TP was cloned from Escherichia coli K12 strain and inserted into the yeast expression vector pKFS. Recombinant vector was linearized and electro-transformed into Pichia pastoris GS115 cells. High copy transformant was induced by methanol for 96 h, and the results of immunofluorescence indicated that TP successfully displayed on the surface of P. pastoris. β-thymidine was used as substrate and recombinant GS115 cells as whole-cell biocatalyst, HPLC analysis demonstrated that TP on the surface of P. pastoris had catalytic activity, and catalyzed the production from β-thymidine to thymine.

The Effect of Scaffold Combined with Salidroside on Rat Bone Mesenchymal Stem Cells

WANG Jiu-na, XIANG Da-wei, TANG Jun-jie, LI Gen, ZHAO Ling, QIN Wen, ZHAO Hong-bin

2016, 32(1):  207-212.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.033

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The objective of this work is to investigate the effect of collagen scaffolds combined with salidroside microspheres on rat bone mesenchymal stem cells(rBMSCs). The salidroside was made into microspheres that combined into the collagen. The surface character of material was observed by scanning electron microscopy(SEM). The material was implanted with rBMSCs and observed for the cell adhesion on the material by SEM. The cell proliferation on the material was measured by CCK-8, and the cell proliferation and morphology on the material were detected by HE staining. S100 immunofluorescence was used to detect the differentiation of stem cells into nerve cells. Cells were seeded onto the scaffold, adhered and grew well on materials. While the time increased, the cells on the same material were significant proliferation(P < 0.05). The expression of nerve cell marker protein S100 was positive. Conclusively, collagen combined with salidroside microspheres had good biocompatibility, salidroside microspheres did not affect cell proliferation on the material, and it could induce effectively rBMSCs into nerve cells.

The Affecting Mechanism of 5-aza-2'-deoxycytidine on the NG108-15 Cells in Hypoxia

ZHANG Zhu-xia, SUN Ming-ying, YANG Jie, JIANG Shu-yan, YAN Shao-chun, SHAO Guo

2016, 32(1):  213-219.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.034

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The aim of this study is to explore the mechanisms of 5-aza-2'deoxycytidine(5-Aza-CdR)and/or hypoxia and/or hypoxic preconditioning on the proliferation and cycle of NG108-15 cells while using the inhibitor(5-Aza-CdR)of DNA methyltransferases(DNMTs)to interfere the nerve cell line NG108-15. The 10.0 μmol/L 5-Aza-CdR was added to cell culture medium, and then cells were treated in hypoxia and/or hypoxic preconditioning. The cell morphology was observed under inverted microscope. The proliferation of NG108-15 was measured using the RTCA. The cell apoptosis and cell cycle were analyzed through flow cytometry analysis. The mRNA levels of DNMTs and cell cycle-associated gene were analyzed by real-time PCR. 5-Aza-CdR and hypoxia inhibited NG108-15 cells proliferation, promoted cell early and late apoptosis, and hypoxic preconditioning promoted cell proliferation. However, as cells were treated by hypoxic or hypoxic preconditioning after the cell treated with 5-Aza-CdR, DNMTs and mRNA transcription of cell cycle-associated genes increased. Conclusively, the jointed treatment of 10.0 μM 5-Aza-CdR with hypoxic inhibited the proliferation of nerve cell line NG108-15, and hypoxic preconditioning had a promoting effect on the cell proliferation.

A Novel Method of Unit Assembly and Activity Assay for Transcription Activator-like Effector Nucleases

GAO Jing, WEI Di, CHI Zhen-fen, ZHANG Gui-rong, ZHANG Wan-ming, NIE Ling-yun

2016, 32(1):  220-228.  doi:10.13560/j.cnki.biotech.bull.1985.2016.01.035

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A novel method of unit assembly(UA)and activity assay based on transcription activator-like effector nucleases(TALENs)are described. An online tool TALE-NT 2.0 was used to design recognition and splice sites on mitochondrial DNA(mtDNA)of TALENs. By means of pEGFP-N1 plasmid, the segments of target sequence were randomly integrated in the nuclear genome of HEK293F. The constructed cell line HEK293F-T1 was for activity assay of TALENs. Based on transcription activator-like effector(TALE)natural repeats, a new type of artificial TALEs single-unit repeats were designed. According to the recognition sites of TALENs, appropriate single-unit repeats were selected and assembled by UA. TALEs series unit sequence containing the corresponding double enzyme cutting sites and TALENs vector sequence were designed, which was directionally ligated and transiently transfected into HEK293F-T1 cell line. The results showed that the TALES units were directionally assembled by new method, and it had a high cloning efficiency;moreover, the nested peaks clearly appeared after transient transfection. In conclusion, the novel method of unit assembly improves cloning efficiency of constructing TALLENs, even is not limited by repeat-variable residue(RVD)in the last 0.5 unit, and increases the flexibility of designing the target sequences.

Cover

2016, 32(1):  300. 

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Content

2016, 32(1):  400. 

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Copyright

2016, 32(1):  500. 

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