|本期目录/Table of Contents|

[1]彭乾,刘斌,刘军委,等.苯吡唑草酮抗性菌株的筛选及抗性HPPD基因的克隆与表达[J].应用与环境生物学报,2020,26(05):1066-1074.[doi: 10.19675/j.cnki.1006-687x.2020.02036]
 PENG Qian,LIU Bin,LIU Junwei,et al.Isolation of topramezone-resistant strain and cloning and expression of its HPPD gene[J].Chinese Journal of Applied & Environmental Biology,2020,26(05):1066-1074.[doi: 10.19675/j.cnki.1006-687x.2020.02036]





Isolation of topramezone-resistant strain and cloning and expression of its HPPD gene
南京农业大学生命科学学院 南京 210095
PENG Qian LIU Bin LIU Junwei SHENG Mengyao SHI Yan HE Jian & HE Qin?
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
topramezone 4-hroxyphenylpyruvate dioxygenase resistant gene gene cloning and expression
筛选分离对苯吡唑草酮具有高抗性的菌株,并从中克隆抗除草剂苯吡唑草酮的4-羟苯基丙酮酸双加氧酶(HPPD)基因,可为研发抗苯吡唑草酮转基因作物提供基因资源. 在加入苯吡唑草酮的TMSM平板上筛选到抗苯吡唑草酮的菌株,通过16S rRNA分析确定其分类地位,用PCR扩增出抗性菌株的HPPD基因,构建高效表达载体pET-Enhppd并在大肠杆菌Escherichia coli BL21(DE3)中表达,利用Co2+亲和层析对表达产物进行纯化,通过IC50分析菌株对各种HPPD抑制剂类除草剂的抗性情况. 在加入5 600 μmol/L的苯吡唑草酮平板上筛选到一株高抗苯吡唑草酮的革兰氏阴性细菌,命名为Ace-21,并初步鉴定为剑菌属(Ensifer sp.). 从菌株Ace-21中克隆到编码HPPD的基因,命名为Enhppd. 该基因全长为1 113 bp,编码370个氨基酸,蛋白分子量(Mr)为41.4 × 103. EnHPPD与已报道HPPD同源性较低,仅有33.9%-54.6%. 在大肠杆菌表达的EnHPPD通过Co2+亲和层析纯化至单一的条带. 纯化的EnHPPD具有对羟苯基丙酮酸双加氧酶活性,比酶活为10.2 U/mg. 抗性分析结果表明EnHPPD对苯吡唑草酮具有较高的抗性,其IC50为12.7 μmol/L. 此外,EnHPPD还对其他HPPD抑制剂类除草剂如硝磺草酮和环磺酮有较强的抗性,其IC50分别达到35.1 μmol/L和23.6 μmol/L,显示EnHPPD对其他HPPD抑制剂类除草剂也具有较强的抗性. 本研究分离筛选到高抗苯吡唑草酮的细菌Ensifer sp. Ace-21,并从该菌克隆获得其HPPD基因Enhppd;表达纯化的EnHPPD能高抗苯吡唑草酮、硝磺草酮和环磺酮等多种HPPD抑制剂类除草剂,是一个性能比较优良的广谱HPPD抑制剂类除草剂抗性基因资源,在构建HPPD抑制剂类除草剂转基因作物中有很好的应用前景. (图8 表1 参35)
The aim of this study was to screen the topramezone-resistant strain and cloning expression of the HPPD gene, which is a candidate gene resource for the construction of genetically modified (GM) herbicide-resistant crops. The strains that were found to be resistant to topramezone were screened on TMSM agar plates with topramezone added as a selection pressure. The isolated strains were identified based on phylogenetic analysis of the 16S rRNA gene sequence. The hppd gene sequence was amplified from the topramezone-resistant strain by PCR and connected to the expression vector pET29a (+) by homologous recombination. The recombinant vector was introduced into Escherichia coli BL21(DE3) and the expressed product was purified by Co2+ affinity chromatography. The resistance of the purified HPPD to various HPPD inhibitor herbicides was analyzed by IC50. One bacterial strain that could grow on TMSM, which had been treated with the addition of μmol/L of topramezone, was isolated and named as Ace-21. Strain Ace-21 was identified as Ensifer sp. The hppd gene of strain Ace-21 was successfully amplified and named Enhppd. The Enhppd gene was 1 113 bp in length, coding for 370 amino acids. EnHPPD shared 33.9%-54.6% sequence identity with previously reported HPPDs. The purified EnHPPD migrated as a single band on SDS-PAGE with an approximate molecular mass of 41.4 × 103, which was consistent with the theoretical molecular mass. The purified EnHPPD showed HPPD activity, with the specific activity being 10.2 U/mg. The IC50 values of topramezone, mesotrione, and isoxazolidone against EnHPPD were 12.7, 35.1, and 23.6 μmol/L, respectively, which indicated that EnHPPD was highly resistant to the HPPD inhibitor herbicide. In this study, we isolated a bacteria Ensifer sp. Ace-21 that was highly resistant to topramezone and cloned a HPPD gene Enhppd. EnHPPD showed high resistance to HPPD inhibitor herbicides such as topramezone, mesotrione, and isoxazolidone, which indicated that this gene has a potentially useful application value in herbicide-resistant transgenic engineering.


1 韩天富. 转基因和非转基因大豆生产体系除草成本和生产效益的估算[J]. 作物杂志, 2008 (2): 1-3 [Han TF. Estimation of weeding costs and production benefits of GM and non-GM soybean production systems [J]. Crops, 2008 (2): 1-3]
2 Heap I, Duke SO. Overview of glyphosate-resistant weeds worldwide [J]. Pest Manage Sci, 2018, 74 (5): 1040-1049
3 Lederer B, Boger P. Recombinant p-hydroxyphenylpyruvate dioxygenase of high activity [J]. Z Naturforsch C, 2005, 60 (50): 549-556
4 王园园, 王敏, 相世刚, 刘琪, 强胜, 宋小玲. 全球抗除草剂转基因作物转化事件分析[J]. 农业生物技术学报, 2018, 26 (1): 167-175 [Wang YY, Wang M, Xiang SG, Liu Q, Qiang S, Song XL. Analysis of transformation events of global herbicide-resistant transgenic crops [J]. Chin J Agric Biotechol, 2018, 26 (1): 167-175]
5 Ahrens H, Lange G, Muller T, Rosinger C, Willms L, van Almsick A. 4-Hydroxyphenylpyruvate dioxygenase inhibitors in combination with safeners: solutions for modern and sustainable agriculture [J]. Angew Chem Int Edit, 2013, 52 (36): 9388-9398
6 Zhao N, Zuo L, Li W, Guo W, Liu W, Wang J. Publisher correction: greenhouse and field evaluation of isoxaflutole for weed control in maize in China [J]. Sci Rep, 2017, 7 (1): 16519
7 Mitchell G, Bartlett DW, Fraser TE, Hawkes TR, Holt DC, Townson JK, Wichert RA. Mesotrione: a new selective herbicide for use in maize [J]. Pest Manage Sci, 2001, 57 (2): 120-128
8 Grossmann K, Ehrhardt T. On the mechanism of action and selectivity of the corn herbicide topramezone: a new inhibitor of 4‐hydroxyphenylpyruvate dioxygenase [J]. Pest Manage Sci, 2007, 63 (5): 429-439
9 Ahrens H, Lange G, Muller T, Rosinger C, Willms L, Van Almsick A. 4-Hydroxyphenylpyruvate dioxygenase inhibitors in combination with safeners: solutions for modern and sustainable agriculture [J]. Angew Chem Int Edit, 2013, 52 (36): 9388-9398
10 Gitsopoulos TK, Melidis V, Evgenidis G. Response of maize (Zea mays L.) to post-emergence applications of topramezone [J]. Crop Prot, 2010, 29 (10): 1091-1093
11 Buck JD. Nonstaining (KOH) method for determination of gram reactions of marine bacteria [J]. Appl Environ Microbiol, 1982, 44 (4): 992-993
12 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells [J]. Nucleic Acids Res, 1988, 16 (3): 1215
13 Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes [J]. Appl Environ Microbiol, 2008, 74: 2461-2470
14 Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies [J] Int J Syst Evol Microbiol, 2017, 67: 613-1617
15 Saitou N. The neighbor-joining method: a new method for reconstructing phylogenetic tree [J]. Mol Biol Evol, 1987, 4: 406-425
16 Yao SG, Chen L, Yang Z, Yao L, Zhu JC, Qiu JG, Wang GX He J. The properties of 5-methyltetrahydrofolate dehydrogenase (MetF1) and its role in the tetrahydrofolate (THF)-dependent dicamba demethylation system in Rhizorhabdus dicambivorans Ndbn-20 [J]. J Bacteriol, 2019, 201 (17): e00096-19
17 Wang DW, Lin HY, Cao RJ, Yang SG, Chen Q.; Hao GF, Yang WC, Yang GF. Synthesis and herbicidal evaluation of triketone- containing quinazoline-2, 4-diones [J]. J Agric Food Chem, 2014, 62 (49): 11786-11796
18 Arias-Barrau E, Olivera ER, Luengo JM., Fernández C, Galán B, García JL, Díaz E, Minambres B. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida [J]. J Bacteriol, 2004, 186 (15): 5062-5077
19 Sparnins VL, Chapman PJ. Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria [J]. J Bacteriol, 1976, 127 (1): 362-366
20 Chen CY, Wu KM, Chang YC, Chang CH, Tsai HC, Liao TL, Liu YM, Chen HJ, Shen ABT, Li JC. Comparative genome analysis of Vibrio vulnificus, a marine pathogen [J]. Gent Res, 2003, 13 (12): 2577-2587
21 Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FSL, Hufnagle WO, Kowalik DJ, Lagrou M. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen [J]. Nature, 2000, 406 (6799): 959
22 Steinert M, Flügel M, Schuppler M, Helbig JH, Supriyono A, Proksch P, Lück PC. The Lly Protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila [J]. FEMS Microbiol Lett, 2001, 203 (1): 41-47
23 Rüetschi U, Odelh?g B, Lindsted S, Barros‐S?derling J, Persson B, J?rnvall H. Characterization of 4-hydroxyphenylpyruvate dioxygenase: primary structure of the Pseudomonas enzyme [J]. Eur J Biochem, 1992, 205 (2): 459-466
24 Denoya CD, Skinner DD, Morgenstern MR. A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli [J]. J Bacteriol, 1994, 176 (17): 5312-5319
25 黄彦, 夏冰洁, 崔中利. 硝磺草酮抗性菌株的筛选及抗性基因的克隆表达[J]. 微生物学通报, 2015, 42 (10): 1895-1902 [Huang Y, Xia BJ, Cui ZL. Isolation of mesotrione-resistant strain and cloning and expression of HPPD [J]. Microbiol China, 2015, 42 (10): 1895-1902]
26 Liu B, Peng Q, Sheng MY, Ni HY, Xiao X, Tao Q, He Q, He J. Isolation and characterization of a topramezone-resistant 4-hydroxyphenylpyruvate dioxygenase (HPPD) from Sphingobium sp. TPM-19 [J]. J Agric Food Chem, 2020, 68 (4): 1022-1029
27 Liang Y, Minami H, Sato F. Isolation of herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase from cultured Coptis japonica cells [J]. Biosci Biotechnol Biochem, 2008, 72 (11): 3058-3062
28 Lee CM, Yeo YS, Lee JH, Kim SJ, Kim JB, Han NS, Koo BS, Yoon SH. Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome [J]. Biochem Biophys Res Commun, 2008, 370 (2): 322-326
29 Dreesen R, Capt A, Oberdoerfer R, Coats I, Pallett KE. Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FG?72-2 soybean [J]. Regul Toxicol Pharm, 2018, 97: 170-185
30 Hawkes TR, Langford MP, Viner R, Blain RE, Callaghan FM, Mackay EA, Hogg BV, Singh S, Dale RP. Characterization of 4-hydroxyphenylpyruvate dioxygenases, inhibition by herbicides and engineering for herbicide tolerance in crops [J]. Pestic Biochem Phys, 2019, 156: 9-28
31 Fu Y, Sun YN, Yi KH, Li MQ, Cao HF, Li JZ, Ye F. Combination of virtual screening protocol by in silico toward the discovery of novel 4-hydroxyphenylpyruvate dioxygenase inhibitors [J]. Front Chem, 2018, 6 (6): 1-15
32 Schindler CEM, Hollenbach E, Mietzner T, Schleifer K, Zacharias M. Free energy calculations elucidate substrate binding, gating mechanism, and tolerance‐promoting mutations in herbicide target 4-hydroxyphenylpyruvate dioxygenase [J]. Protein Sci, 2019, 28 (6): 1048-1058
33 Sharma B, Ranganathan SV, Belfort G. Weaker N-terminal interactions for the protective over the causative Aβ peptide dimer Mutants [J]. ACS Chem Neurosci, 2018, 9 (6): 1247-1253
34 Fritze IM, Linden L, Freigang J, Auerbach G, Huber R, Steinbacher S. The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase [J]. Plant Physiol, 2004, 134 (4): 1388-1400
35 Yang C, Pflugrath JW, Camper DL, Foster ML, Pernich DJ, Walsh TA. Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases [J]. Biochemistry, 2004, 43 (32): 10414-10423


[1]彭乾 刘斌 刘军委 盛梦瑶 史砚 何健 贺芹**.苯吡唑草酮抗性菌株的筛选及抗性HPPD基因的克隆表达[J].应用与环境生物学报,2021,27(01):1.[doi:10.19675/j.cnki.1006-687x.2020.02036]
 PENG Qian,LIU Bin,Liu Junwei,et al.Isolation of topramezone-resistant strain and cloning and expression of its HPPD[J].Chinese Journal of Applied & Environmental Biology,2021,27(05):1.[doi:10.19675/j.cnki.1006-687x.2020.02036]

更新日期/Last Update: 2020-10-25