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[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]
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苯吡唑草酮抗性菌株的筛选及抗性HPPD基因的克隆与表达()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
26卷
期数:
2020年05期
页码:
1066-1074
栏目:
土壤与农业微生物应用专栏
出版日期:
2020-10-25

文章信息/Info

Title:
Isolation of topramezone-resistant strain and cloning and expression of its HPPD gene
作者:
彭乾; 刘斌; 刘军委; 盛梦瑶; 史砚; 何健; 贺芹
南京农业大学生命科学学院 南京 210095
Author(s):
PENG Qian;  LIU Bin;  LIU Junwei;  SHENG Mengyao;  SHI Yan;  HE Jian & HE Qin?
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
关键词:
苯吡唑草酮; 对羟苯基丙酮酸双加氧酶; 抗性基因; 基因的克隆与表达
Keywords:
topramezone;  4-hroxyphenylpyruvate dioxygenase;  resistant gene;  gene cloning and expression
DOI:
10.19675/j.cnki.1006-687x.2020.02036
摘要:
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）
Abstract:
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.

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相似文献/References:

[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