| 摘要: |
| 为获得高效抗除草剂的基因，提高转基因作物的应用能力，本研究使用含硝磺草酮的酪氨酸无机盐培养基(TMSM)平板筛选法，筛选得到一株能够耐受5 000 μmol/L硝磺草酮的革兰氏阴性细菌MST-5，经过16S rRNA序列分析鉴定为嗜麦芽窄食单胞菌属(Stenotrophomonas sp.)。根据4-羟基苯丙酮酸双加氧酶(4-hydroxyphenylpyruvate dioxygenase，HPPD)同源基因设计引物，从MST-5中克隆到编码HPPD的基因Sthppd，序列分析发现Sthppd序列长度为1 071 bp，编码356个氨基酸。同源比对发现，StHPPD与已报道的HPPD相似性仅为47%~51%。在大肠杆菌中进行原核表达Sthppd并通过Co2+亲和层析纯化获得StHPPD纯蛋白，活性检测表明此蛋白具有4-羟苯基丙酮酸双加氧酶活性，抗性分析显示其对硝磺草酮具有较高的抗性，IC50为15.5 μmol/L，同时对其它HPPD抑制剂类除草剂如苯吡唑草酮和二酮腈也有较强抗性，IC50分别达到10.8 和28.2 μmol/L。StHPPD的最适反应温度为30 ℃，最适pH为7.0，大部分金属离子和某些化学添加剂严重抑制StHPPD的活性，Fe3+、Mg2+和Urea对StHPPD酶活基本没有影响。综上，StHPPD具备较高硝磺草酮抗性，在构建抗除草剂转基因作物、与HPPD抑制剂类除草剂搭配在相关转基因作物的田间应用中，具有一定价值。 |
| 关键词: 硝磺草酮 HPPD 抗性菌株 基因克隆 除草剂抗性 |
| DOI：10.11841/j.issn.1007-4333.2021.09.08 |
| 投稿时间：2020-11-17 |
| 基金项目:国家自然科学基金项目(32070371) |
|
| Isolation of mesotrione-resistant strain and cloning and expression of its hppd |
|
ZHANG Lei1,2,LI Lin1*
|
| (1.Life Science College, China West Normal University, Nanchong 637000, China;2.Nanjing Risun Biotechnology Ltd., Nanjing 210000, China) |
| Abstract: |
| To improve the traits of transgenic plants through efficient herbicide-resistant genes, a Gram-stain-negative bacterial strain MST-5, which was highly resistant to mesotrione, was isolated from herbicide contaminated soil. 16S rRNA gene sequence analysis showed that strain MST-5 belongs to the genus Stenotrophomonas sp. Subsequently, Sthppd, encoding a 4-hydroxyphenylpyruvate dioxygenase StHPPD, was cloned from MST-5. Its length was 1 071 bp, encoding 356 amino-acid residues. The sequence identity between StHPPD and other HPPDs was only 47%-51%. The purified StHPPD displayed high resistance to HPPD inhibitor herbicides. The IC50 values of mesotrione, topramezone and diketonitrile against StHPPD were 15. 5, 10. 8, 28. 2 μmol/L, respectively. The optimum temperature and optimum pH of StHPPD were 30 ℃ and 7. 0, respectively. The enzymatic activity of StHPPD was severely inhibited by most metal ion and some chemical additives, while was not affected by Fe3+, Mg2+ and Urea. In conclusion, Sthppd showed high resistance to hervicide and it was suggested that Sthppd might have a potential value in transgenic engineering of mesotrione-resistant crops. |
| Key words: mesotrione HPPD resistant strain gene cloning herbicide resistance |
