硝磺草酮抗性菌株的筛选及抗性hppd基因的克隆表达

Isolation of mesotrione-resistant strain and cloning and expression of its hppd

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5000μmol/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1071bp,编码356个氨基酸。同源比对发现,St HPPD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St HPPD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,IC_(50)为15.5μmol/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,IC_(50)分别达到10.8和28.2μmol/L。St HPPD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St HPPD的活性,Fe^(3+)、Mg^(2+)和Urea对St HPPD酶活基本没有影响。综上,St HPPD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。

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.16 S 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 IC_(50) values of mesotrione,topramezone and diketonitrile against StHPPD were 15.5,10.8,28.2μmol/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 Fe^(3+)、Mg^(2+)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.