Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (16): 3369-3376.doi: 10.3864/j.issn.0578-1752.2013.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Prokaryotic Expression and Polyclonal Antibody Preparation of Trichothecene 3-O-Acetyltransferase Gene Tri101

 DONG  Fei, JI  Fang, WU  Ji-Rong, YIN  Xian-Chao, XU  Jian-Hong, SHI  Jian-Rong   

  1. Institute of Food Safety and Detection, Jiangsu Academy of Agricultural Sciences/Jiangsu Center for GMO Evaluation and Detection, Nanjing 210014
  • Received:2013-01-22 Online:2013-08-15 Published:2013-04-15

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Abstract: 【Objective】Tri101 was cloned and the purified protein was obtained by prokaryotic expression. Polyclonal antibody of high titer and high sensitivity was obtained by SPF rabbits immunization, thus providing a basis for the establishment of the transgenic plants and products.【Method】Tri101 was obtained from Fusarium graminearium Schwabe F3210 by PCR and connected to the expression vector pET29a (+), the recombinant vector was transformed into E. coli BL21(DE3) and induced by IPTG. The results indicated that the recombination expression plasmid was constructed and transformed into E. coli BL21 (DE3). The Tri101 protein was induced by IPTG and used as the antigen to immune the SPF rabbits. 【Result】The result of SDS-PAGE showed that the molecular weight of expressed protein was about 50 kD, existing in the form of inclusion body. The expressed protein was purified by HisTrap, and the best imidazole concentration was 200 mmol•L-1. His-tag present in recombinant protein was removed by TEV protease and the digestion product was purified by HisTrap. The purified protein was used to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-Tri101 antiserum was 1﹕12 000，the detection sensitivity was 0.05×10-6 µg•mL-1. The result of Western blotting demonstrated that polyclonal antibody had a very good specificity.【Conclusion】Through Tri101 cloning and prokaryotic expression, the purified fusion protein was prepared by immunizing SPF rabbits. Polyclonal antibody was obtained and used for the establishment for the transgenic wheat and products.

Key words: trichothecene 3-O-acetyltransferase , Tri101 , prokaryotic expression , polyclonal antibody , Fusarium head blight (FHB)

[1]苏锐, 熊嫣, 庆宏, 邓玉林. 转基因作物检测新技术研究进展. 化学通报, 2012, 75(2): 121-125.

Su R, Xiong Y, Qing H, Deng Y L. Advances of the new transgenic crops detection technology. Chemistry, 2012, 75(2): 121-125. (in Chinese)

[2]McMullen M, Jones R, Gallenberg D. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Disease, 1997, 81(12): 1340-1348.

[3]姚金保, 任丽娟, 张平平, 杨学明, 马鸿翔, 姚国才, 张鹏, 周淼平. 小麦赤霉病的抗性遗传分析. 麦类作物学报, 2011, 31(2): 370-375.

Yao J B, Ren L J, Zhang P P, Yang X M, Ma H X, Yao G C, Zhang P, Zhou M P. Genetic analysis of resistance to Fusarium head blight in wheat. Journal of Triticeae Crops, 2011, 31(2): 370-375. (in Chinese)

[4]韩青梅, 曹丽华. 小麦赤霉病的生物防治研究进展. 麦类作物学报, 2003, 23(3): 128-131.

Han Q M, Cao L H. Progress of the biological control on Fusarium graminearum Schw. of wheat. Journal of Triticeae Crops, 2003, 23(3): 128-131. (in Chinese)

[5]白耀博, 刘恒, 李强, 王芳, 王保通. 陕西省小麦赤霉病菌对多菌灵敏感性研究. 植物病理学报, 2012, 42(1): 97-100.

Bai Y B, Liu H, Li Q, Wang F, Wang B T. The sensitivity of Fusarium graminearum to carbendazim in Shaanxi Province. Acta Phytopathologica Sinica, 2012, 42(1): 97-100. (in Chinese)

[6]关二旗, 崔莉, 卞科, 李萌萌, 崔贵金, 陈飞. 赤霉病麦粒中脱氧雪腐镰刀菌烯醇消解方法研究进展. 麦类作物学报, 2012, 32(3): 585-588.

Guan E Q, Cui L, Bian K, Li M M, Cui G J, Chen F. Study on methods to reduce the deoxynivalenol toxin in wheat seeds infected by Fusarium head blight. Journal of Triticeae Crops, 2012, 32(3): 585-588. (in Chinese)

[7]石志琦, 史建荣, 陈怀谷, 林玲, 王裕中. 小麦赤霉病菌对多菌灵的抗药性研究. 农药学学报, 2000, 2(4): 22-27.

Shi Z Q, Shi J R, Chen H G, Lin L, Wang Y Z. Study on resistance of Fusarium graminearum to carbendazim. Chinese Journal of Pesticide Science, 2000, 2(4): 22-27. (in Chinese)

[8]Desjardins A E, Proctor R H. Molecular biology of Fusarium mycotoxins. International Journal of Food Microbiology, 2007, 119(1): 47-50.

[9]Zinedine A, Soriano J M, Molto J C, Manes J. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: An oestrogenic mycotoxin. Food and Chemical Toxicology, 2007, 45(1): 1-18.

[10]Cuomo C A, Güldener U , J R Xu, Trail F, Turgeon B G, Di Pietro A, Walton J D, Ma L J, Baker S E, Rep M, Adam G, Antoniw J, Baldwin T, Calvo S, Chang Y L, DeCaprio D, Gale L R, Gnerre S, Goswami R S, Hammond-Kosack K, Harris L J, Hilburn K, Kennell J C, Kroken S, Magnuson J K, Mannhaupt G, Mauceli E, Mewes H W, Mitterbauer R, Muehlbauer G, Münsterkötter M, Nelson D, O’Donnell K, Ouellet T, Qi W, Quesneville H, Roncero M I G, Seong K Y, Tetko I V, Urban M, Waalwijk C, Ward T J, Yao J, Birren B W, Kistler H C. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science, 2007, 317: 1400-1402.

[11]Butchko R A E, Plattner R D, Proctor R H. Deletion analysis of FUM genes involved in tricarballylic ester formation during fumonisin biosynthesis. Journal of Agricultural and Food Chemistry, 2006, 54(25): 9398-9404.

[12]张岳平. 镰刀菌真菌毒素产生与调控机制研究进展. 生命科学, 2011, 22(3): 311-316.

Zhang Y P. The research advance of biosynthesis and regulation mechanism on Fusarium mycotoxins. Chinese Bulletin of Life Sciences, 2011, 22(3): 311-316. (in Chinese)

[13]Kimura M, Kaneko I, Komiyama M, Takatsuki A, Koshinoi H, Yoneyama K, Yamaguchi I. Trichothecene 3-O-Acetyltransferase protests both the producing organism and transformed yeast from related mycotoxins. The Journal of Biological Chemistry, 1998, 273(3): 1654-1661.

[14]Alexander N J. The Tri101 story: engineering wheat and barley to resist Fusarium head blight. World Mycotoxin Journal, 2008, 1(1): 31-37.

[15]Ohsato S, Ochiai-Fukuda T, Nishiuchi T, Takahashi-Ando N, Koizumi S, Hamamoto H, Kudo T, Yamaguchi I, Kimura M. Transgenic rice plants expressing trichothecene 3-O-acetyltransferase show resistance to the Fusarium phytotoxin deoxynivalenol. Plant Cell Reports, 2007, 26(4): 531-538.

[16]Roda A, Mirasoli M, Guardigli M, Michelini E, Simoni P, Magliulo M. Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize. Analytical and Bioanalytical Chemistry, 2006, 384(6): 1269-1275.

[17]Crespo A L B,  Spencer T A, Nekl E, Pusztai-Carey M, Moar W J, Siegfried B D. Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays. Applied and Environmental Microbiology, 2008, 74(1): 130-135.

[18]朱晓雷, 巴吐尔. 双抗夹心荧光免疫吸附测定法 (sFLISA) 定量检测转基因玉米中Bt蛋白. 新疆农业大学学报, 2009, 32(2): 54-57.

Zhu X L, Batuer. Application of indirect sandwich fluorescence- linked immunosorbent assay (sFLISA) method in detection of Bt protein in transgenic maize. Journal of Xinjiang Agricultural University, 2009, 32(2): 54-57. (in Chinese)

[19]李红兵, 康振生. 适于小麦叶片蛋白质组分析的样品提取方法研究. 西北植物学报, 2011, 31(3): 632-638.

Li H B, Kang Z S. Sample preparation methods suitable for wheat leaf proteome analysis. Acta Botanica Boreali-Occidentalia Sinica, 2011, 31(3): 632-638. (in Chinese) 

[20]邢继红, 徐淑华, 董金皋. 玉米大斑病菌菌丝蛋白电泳分析及抗体检测. 植物病理学报, 2003, 33(4): 383-384.

Xing J H, Xu S H, Dong J G. Electrophoresis of mycelial proteins of Setosphaeria turcica and antibody preparation. Acta Phytopathologica Sinica, 2003, 33(4): 383-384. (in Chinese)

[21]Garvey G S, McCormick S P, Rayment L. Structural and functional characterization of the TRI101 trichothecene 3-O-acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum. The Journal of Biological Chemistry, 2008, 283(3): 1660-1669.

[22]Kimura M, Shingu Y, Yoneyama K, Yamaguchi I. Features of Tri101, the trichothecene 3-O-acetylt-ransferase gene, related to the self-defense mechanism in Fusarium graminearum. Bioscience, Biotechnology, and Biochemistry, 1998, 62(5): 1033-1036.

[23]Kimura M, Matsumoto G, Shingu Y, Yoneyama K, Yamaguchi I. The mystery of the trichothecene 3-O-acetylt ransferase gene analysis of the region around Tri101 and characterization of its homologue from Fusarium sporotrichioides. FERS Letters, 1998, 435(2): 163-168.

[24]闫海霞, 杨丽荣, 薛保国, 段立清, 孙虎, 全鑫. 降解赤霉病菌毒素Tril01基因的原核表达及抗体制备. 植物病理学报, 2010, 40(5): 469-474.

Yan H X, Yang L R, Xue B G, Duan L Q, Sun H , Quan X. Prokaryotic expression and antiserum preparation of toxicity reduction gene Tril01 from Fusarium graminearum Schwabe. Acta Phytopathologica Sinica, 2010, 40(5): 469-474. (in Chinese)

[25]刘伟霞, 潘映红. 适用于小麦叶片蛋白质组分析的样品制备方法. 中国农业科学, 2007, 40(10): 2169-2176.

Liu W X, Pan Y H. Sample preparation methods suitable for wheat leaf proteome analysis. Scientia Agricultura Sinica, 2007, 40(10): 2169-2176. (in Chinese)
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