饲料安全 Feed Security

玉米赤霉烯酮降解酶在枯草芽孢杆菌中的表达及其对母猪繁殖性能的影响

展开
  • 1. 甘肃农业大学动物医学院, 兰州 730070;
    2. 河南省农业科学院, 农业部动物免疫学重点实验室, 郑州 450002;
    3. 甘肃农业大学生命科学技术学院, 兰州 730070
王相生(1972-),男,河南濮阳人,博士研究生,从事临床兽医学产科方向的研究。E-mail:wxs201302@163.com

收稿日期: 2017-04-11

  网络出版日期: 2017-10-31

基金资助

“十二五”国家科技支撑计划“畜禽产品安全监控和检测技术研究与示范”(2014BAD13B05)

Expression of Zearalenone Degrading Enzyme in Bacillus subilis and Its Effects on Reproductive Performance of Sows

Expand
  • 1. College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China;
    2. Henan Academy of Agricultural Sciences, Key Laboratory of Animal Immunology of the Ministry of Agriculture, Zhengzhou 450002, China;
    3. College of Life Science and Technology, Gansu Agriculture University, Lanzhou 730070, China

Received date: 2017-04-11

  Online published: 2017-10-31

摘要

本试验旨在建立玉米赤霉烯酮(ZEN)降解酶基因ZEN-jjm在枯草芽孢杆菌中的表达体系,确定其产物的生物降解活性及对母猪繁殖性能的作用。克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切后连接至pHT01表达载体中,构建重组质粒;转化枯草芽孢杆菌,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析ZEN-jjm蛋白表达水平。高效液相色谱法(HPLC)检测表达的ZEN-jjm蛋白降解ZEN的活性。通过对母猪的繁殖性能的评价,确定ZEN降解酶降解ZEN对母猪繁殖性能的影响。双酶切和测序结果表明:ZEN-jjm成功插入pHT01中,SDS-PAGE表明获得1株高效表达目的蛋白的重组枯草芽孢杆菌,其大小约为29 ku。HPLC结果表明表达的ZEN-jjm蛋白能有效地降解ZEN;表达的ZEN-jjm蛋白能显著缓解ZEN对繁殖母猪的毒害作用(P<0.05)。综上:1)本研究成功构建了表达ZEN-jjm在枯草芽孢杆菌中的表达载体,并在枯草芽孢杆菌中得到了表达。2)表达的ZEN-jjm蛋白具有降解ZEN的生物活性。3)在饲粮中添加ZEN-jjm蛋白能显著降低ZEN对母猪能繁殖性能的危害。

本文引用格式

王相生, 孙亚宁, 阮崇美, 张全伟, 张勇, 胡骁飞 . 玉米赤霉烯酮降解酶在枯草芽孢杆菌中的表达及其对母猪繁殖性能的影响[J]. 动物营养学报, 2017 , 29(11) : 4019 -4025 . DOI: 10.3969/j.issn.1006-267x.2017.11.023

Abstract

The purpose of this study was to establish an expressing system of zearalenone (ZEN) degrading enzyme gene (ZEN-jjm) in Bacillus subtilis, and to determine the biodegradation activity and effect of its expression product on reproductive performance of sows. The ZEN-jjm gene was cloned and the recombinant expression plasmid was constructed by ligation cloned ZEN-jjm and pHT01 vector digested with EcoRⅠand NotⅠ enzymes, and then transformed into Bacillus subilis. Expression of ZEN-jjm protein in Bacillus subilis was analyzed by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). And the degrading activity of ZEN-jjm protein was determined by high performance liquid chromatography (HPLC). The effects of ZEN degrading enzyme on reproductive performance of sows were evaluated. The results of double enzyme digestion and DNA sequencing demonstrated that ZEN-jjm was inserted into pHT01. The SDS-PAGE showed that one Bacillus subilis strain with high-level expression was obtained, and the size of the expressed protein was about 29 ku. The HPLC result illustrated that the expressed ZEN-jjm protein could effectively degrade ZEN. The evaluation results of reproductive performance of sows indicated that the expressed ZEN-jjm protein could significantly relieve the toxicity for sows caused by ZEN (P<0.05). In conclusion:1) the ZEN-jjm expression vector is successfully constructed and the ZEN-jjm protein is expressed in Bacillus subtilis in this study. 2) The expressed ZEN-jjm protein has the biological activity of degrading ZEN. 3) ZEN-jjm protein can significantly reduce the harm of ZEN on reproductive performance of sows when it is added in the feed.

参考文献

[1] BENNETT J W,KLICH M.Mycotoxins[J].Clinical Microbiology Reviews,2003,16(3):497-516.  

[2] TAKAHASH-ANDO N,KIMURA M,KAKEYA H,et al.A novel lactonohydrolase responsible for the detoxification of zearalenone:enzyme purification and gene cloning[J].Biochemical Journal,2002,365(1):1-16.  

[3] ZINEDINE A,SORIANO J M,MOLTÓ J C,et al.Review on the toxicity,occurrence,metabolism,detoxification,regulations and intake of zearalenone:an oestrogenic mycotoxin[J].Food and Chemical Toxicology,2007,45(1):1-18.  

[4] GLAVITIS R,VANYI A. More important mycotoxicoses in pigs.Comprehensive clinico-pathological communication[J].Magyar Allatorvosok Lapja,1995,50(7):407-420.

[5] 熊凯华,程波财,胡威,等.玉米赤霉烯酮降解的研究进展[J].中国粮油学报,2010,25(1):138-142.

[6] 计成,赵丽红.黄曲霉毒素生物降解的研究及前景展望[J].动物营养学报,2010,22(2):241-245.

[7] 薛章荣.减少镰刀霉菌毒素污染的分子生物学和生物技术[J].世界农药,2007,29(4):7-10.

[8] BAKUTIS B,BALIUKONIENE V,PADKEVIÈCIUS A.Use of biological method for detoxification of mycotoxins[J].Botanica Lithuanica,2005,7:123-129.

[9] KAKEYA H,TAKAHASHI-ANDO N,KIMURA M,et al.Biotransformation of the mycotoxin,zearalenone,to a non-esrtogenic compound by a fungal strain of Clonostachys sp.[J].Bioscience,Biotechnology,and Biochemistry,2002,66(12):2723-2726.  

[10] 程波财,史文婷,罗洁,等.玉米赤霉烯酮降解酶基因(ZEN-jjm)的克隆、表达及活性分析[J].农业生物技术学报,2010,18(2):225-230.

[11] OLDENBURG E,HÖPPNER F,ELLNER F,et al.Fusarium diseases of maize associated with mycotoxin contamination of agricultural products intended to be used for food and feed[J].Mycotoxin Research,2017,33(3):167-182.  

[12] KOWALSKA K,HABROWSKA-GÓRCZY?SKA D E,DOMI?SKA K,et al.The dose-dependent effect of zearalenone on mitochondrial metabolism,plasma membrane permeabilization and cell cycle in human prostate cancer cell lines[J].Chemosphere,2017,180:455-466.

[13] TAKAHASHI-ANDO N,OHSATO S,SHIBATA T, et al.Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea[J].Applied and Environmental Microbiology,2004,70(6):3239-3245.  

[14] MOLNAR O,SCHATZMAYR G,FUCHS E,et al.Trichosporon mycotoxinivorans sp. nov.,a new yeast species useful in biological detoxification of various mycotoxins[J].Systematic and Applied Microbiology,2004,27(6):661-671.  

[15] MOKOENA M P,CHELULE P K,GQALENI N.Reduction of fumonisin B1 and zearalenone by lactic acid bacteria in fermented maize meal[J].Journal of Food Protection,2005,68(10):2095-2099.  

[16] KARLOVSKY P.Biological detoxification of fungal toxins and its use in plant breeding,feed and food production[J].Natural Toxins,1998,7(1):1-23.

[17] MINERVINI F,DELL'AQUILA M E,MINOIA P,et al.Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17β-estradiol levels in mural granulosa cell cultures[J].Toxicology in Vitro,2001,15(4/5):489-495.

[18] JADAMUS A,SCHNEIDER D.Long-term effect of fusariotoxins on the reproductive performance of sows[J].Feed Magazine,2002,10:396-405.

[19] JAN OBREMSKI A,GAJECKI M,ZWIERZCHOWSKI W,et al.The level of zearalenone and a-azearalenol in the blood of gilts with clinical symptoms of toxicosis, fed diets with a low zearalenone content[J].Journal of Animal and Feed Sciences,2003,12(3):529-538.  

[20] GZYL-MALCHER B,FILEK M,RUDOLPHI-SKÓRSKA E,et al.Studies of lipid monolayers prepared from native and model plant membranes in their interaction with zearalenone and its mixture with selenium ions[J].The Journal of Membrane Biology,2017,250(3):273-284.  
文章导航

/