[1]朱子薇,潘丽婷,周义东,等.玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库的构建[J].江苏农业学报,2017,(02):456-462.[doi:doi:10.3969/j.issn.1000-4440.2017.02.034]
 ZHU Zi-wei,PAN Li-ting,ZHOU Yi-dong,et al.Construction of a mutant library for zearalenone-degrading bacterium Bacillus amyloliquefaciens MQ01[J].,2017,(02):456-462.[doi:doi:10.3969/j.issn.1000-4440.2017.02.034]
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玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库的构建()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年02期
页码:
456-462
栏目:
加工贮藏·质量安全
出版日期:
2017-04-30

文章信息/Info

Title:
Construction of a mutant library for zearalenone-degrading bacterium Bacillus amyloliquefaciens MQ01
作者:
朱子薇12潘丽婷12周义东12王燕霞2史建荣2洪青1徐剑宏12
(1.南京农业大学生命科学学院/农业部农业环境微生物工程重点开放实验室,江苏南京210095;2. 江苏省农业科学院食品质量安全与检测研究所/江苏省食品质量安全重点实验室——省部共建国家重点实验室培育基地/农业部农产品质量安全风险评估实验室(南京) /江苏省转基因安全评价公共服务中心/江苏省现代粮食流通与安全协同创新中心,江苏南京210014)
Author(s):
ZHU Zi-wei12PAN Li-ting12ZHOU Yi-dong12WANG Yan-xia2SHI Jian-rong2HONG Qing1XU Jian-hong12
(1.The Key Laboratory of Agricultural Environmental Microbial Engineering of Ministry of Agriculture, College of Life Science, Nanjing Agricultural University, Nanjing 210095, China;2.Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Cultivation Base/Key Laboratory of Control Technology and Standard for Agro-product Quality and Safety, Ministry of Agriculture/Collaborative Innovation Center for Modern Grain Circulation and Safety/Institute of Food Quality and Safety, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
玉米赤霉烯酮Bacillus amyloliquefaciens MQ01转座子突变突变体文库
Keywords:
zearalenoneBacillus amyloliquefaciens MQ01transposon mutantsmutant library
分类号:
Q785
DOI:
doi:10.3969/j.issn.1000-4440.2017.02.034
文献标志码:
A
摘要:
构建玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01突变体文库,以期获得丢失玉米赤霉烯酮降解功能的突变子,克隆玉米赤霉烯酮降解酶基因,阐明MQ01降解玉米赤霉烯酮的分子机制。将携带转座子TnYLB-1的穿梭载体pMarA电转化至玉米赤霉烯酮降解菌Bacillus amyloliquefaciens MQ01中,50 ℃ 高温条件下,把穿梭载体pMarA上转座子TnYLB-1随机插入到菌株MQ01基因组中,获得转座子插入突变的阳性克隆,构建MQ01菌株的突变体文库,随机挑选突变子采用PCR和Southern杂交方法进行验证。本研究成功获得了3 000多个TnYLB-1转座子插入突变的阳性克隆,构建了B. amyloliquefaciens MQ01的突变子文库,结果显示TnYLB-1转座子以单拷贝的形式随机插入到B. amyloliquefaciens MQ01的基因组DNA 中,从而可以从转座子突变文库中筛选丢失玉米赤霉烯酮降解功能的转座突变子,从菌株MQ01中克隆玉米赤霉烯酮降解酶基因。
Abstract:
To construct a mutant library for zearalenone-degrading bacterium Bacillus amyloliquefaciens MQ01 for cloning zearalenone degrading enzyme gene and defining the molecular mechanisms of zearalenone degradation by MQ01. The shuttle vector pMarA carrying transposon TnYLB-1 was electroporatred into zearalenone-degrading bacterium B. amyloliquefaciens MQ01. The transposon TnYLB-1 was then inserted randomly into the genomic DNA of MQ01 at 50℃. A total of 3 000 positive clones of TnYLB-1 transposon inserted mutation were obtained, and a mutant library for MQ01 was constructed there by. PCR and Southern hybridization revealed that the TnYLB-1 transposon was inserted randomly into the genomic DNA of B. amyloliquefaciens MQ01 in the form of single copy, enabling the screening of transposon mutants which lose the zearalenone-degrading ability from the mutant library, and the cloning of zearalenone-degrading enzyme gene.

参考文献/References:

[1]ALMEIDA-FERREIRA G C, BARBOSA-TESSMANN I P, SEGA R, et al. Occurrence of zearalenone in wheat- and corn-based products commercialized in the State of Parana [J]. Brazil J Microbiol, 2013, 44(2):371-375.
[2]BERTUZZI T, CAMARDO LEGGIERI M, BATTILANI P, et al. Co-occurrence of type A and B trichothecenes and zearalenone in wheat grown in northern Italy over the years 2009-2011 [J]. Food Addit Contam Part B Surveill, 2014,7(4): 273-281.
[3]KLARIC M S, CVETNIC Z, PEPELJNJAK S, et al. Co-occurrence of aflatoxins, ochratoxin A, fumonisins, and zearalenone in cereals and feed, determined by competitive direct enzyme-linked immunosorbent assay and thin-layer chromatography [J]. Arh Hig Rada Toksikol, 2009, 60(4): 427-434.
[4]RASHEDI M, SOHRABI H R, ASHJAAZADEH M A, et al. Zearalenone contamination in barley, corn, silage and wheat bran [J]. Toxicol Ind Health, 2012, 28(9):779-782.
[5]LU J, YU J Y, LIM S, et al. Cellular mechanisms of the cytotoxic effects of the zearalenone metabolites alpha-zearalenol and beta-zearalenol on RAW264.7 macrophages [J]. Toxicol In Vitro, 2013, 27(3):1007-1017.
[6]PISTOL G C, BRAICU C, MOTIU M, et al. Zearalenone mycotoxin affects immune mediators, MAPK signalling molecules, nuclear receptors and genome-wide gene expression in pig spleen [J]. PLoS One, 2015,10(5): e0127503.
[7]CHOI B K, CHO J H, JEONG S H, et al. Zearalenone affects immune-related parameters in lymphoid organs and serum of rats vaccinated with porcine parvovirus vaccine [J]. Toxicol Res, 2012, 28(4): 279-288.
[8]ZATECKA E, DED L, ELZEINOVA F, et al. Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice [J]. Reprod Toxicol,2014, 45:20-30.
[9]BELHASSEN H, JIMENEZ-DIAZ I, ARREBOLA J P, et al. Zearalenone and its metabolites in urine and breast cancer risk: a case-control study in Tunisia [J]. Chemosphere, 2015, 128:1-6.
[10]NOGOWSKI L, NOWAK K W, KACZMAREK P, et al. The influence of coumestrol, zearalenone, and genistein administration on insulin receptors and insulin secretion in ovariectomized rats [J]. J Recept Signal Transduct Res, 2002, 22(1-4): 449-457.
[11]AVANTAGGIATO G, HAVENAAR R, & VISCONTI A. Assessing the zearalenone-binding activity of adsorbent materials during passage through a dynamic in vitro gastrointestinal model [J]. Food Chem Toxicol, 2003, 41(10): 1283-1290.
[12]SASAKI R, TAKAHASHI N, SAKAO K, et al. Adsorption of zearalenone to Japanese acid clay and influencing factors [J]. Mycotoxin Res, 2014,30(1):33-41.
[13]LEMKE S L, MAYURA K, OTTINGER S E, et al. Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay [J]. J Toxicol Environ Health A, 1999,56(4): 283-295.
[14]KOSAWANG C, KARLSSON M, VELEZ H, et al. Zearalenone detoxification by zearalenone hydrolase is important for the antagonistic ability of Clonostachys rosea against mycotoxigenic Fusarium graminearum [J]. Fungal Biol, 2014,118(4):364-373.
[15]VEKIRU E, HAMETNER C, MITTERBAUER R, et al. Cleavage of zearalenone by Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite [J]. Appl Environ Microbiol, 2010,76(7):2353-2359.
[16]TAN H, ZHANG Z, HU Y, et al. Isolation and characterization of Pseudomonas otitidis TH-N1 capable of degrading Zearalenone [J]. Food Control, 2015, 47:285-290.
[17]KARLOVSKY P. Biological detoxification of fungal toxins and its use in plant breeding, feed and food production [J]. Natural Toxins,1999, 7(1):1-23.
[18]TAKAHASHI-ANDO N, KIMURA M, KAKEYA OSADA, et al. A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning [J]. Biochemistry, 2002, 365(Pt1): 1-6.
[19]TAKAHASHI-ANDO N , TOKAI T, HAMAMOTO H, et al. Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene [J]. Applied Microbiology and Biotechnology, 2005, 67(6): 838-844.
[20]YU Y S, QIU L P, WU H, et al. Oxidation of zearalenone by extracellular enzymes from Acinetobacter sp. SM04 into smaller estrogenic products [J]. World Journal of Microbiology and Biotechnology, 2011,27(11):2675-2681.
[21]YU Y S, WU H, TANG Y Q, et al. Cloning, expression of a peroxiredoxin gene from Acinetobacter sp. SM04 and characterization of its recombinant protein for zearalenone detoxification [J]. Microbiol Res, 2012, 167(3): 121-126.
[22]LE BRETON Y, MOHAPATRA N P, HALDENWANG W G. In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon [J]. Appl Environ Microbiol,2006, 72(1): 327-333.
[23]NATHALIE T, CHRISTIAN L, JIM H E J, et al. Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579 [J]. Journal of Microbiological Methods, 2006, 67(3): 543-548.
[24]SPIZIZEN J. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate [J]. Proc Natl Acad Sci USA , 1958, 44(10): 1072-1078.
[25]LIU J, HE D, LI X Z. γ-Polyglutamic acid (γ-PGA) produced by Bacillus amyloliquefaciens co6 promoting its colonization on fruit surface [J]. Int J Food Microbiol, 2010, 142(1/2): 190-197.
[26]SOUTHERN E M. Detection of specific sequences among DNA fragments separated by gel electrophoresis[J]. Mol Biol, 1975, 98(3): 503-517.
[27]LAMPE D J, AKERLEY B J , RUBIN E J, et al. Hyperactive transposase mutants of the Himar1 mariner transposon [J]. Proc Natl Acad Sci USA , 1999, 96(20): 11428-11433.

备注/Memo

备注/Memo:
收稿日期:2016-08-04 基金项目:国家自然科学基金项目(31471662、U1604234);公益性行业(农业)科研专项(201303088);江苏省农业科技创新基金项目[CX(16)1059] 作者简介:朱子薇(1990-),女,江苏南京人,硕士研究生,研究方向为环境微生物。(E-mail)731632896@qq.com 通讯作者:徐剑宏, (Tel) 84392001;(E-mail)xujianhongnj@126.com
更新日期/Last Update: 2017-05-02