水稻对羟苯基丙酮酸双氧化酶基因(OsHPPD)位点突变对OsHPPD活性以及硝磺草酮抗性的影响  

Influention of p-Hydroxyphenylpyruvate Dioxygenase from Oryza sativa(OsHPPD)Gene on Activity of OsHPPD and Resistance of Mesotrione

作　　者：金曼 陈雷 刘志宏 陈敏 唐晓艳 杨晓怀 JIN Man;CHEN Lei;LIU Zhihong;CHEN Min;TANG Xiaoyan;YANG Xiaohuai(Shenzhen Agricultural Technology Promotion Center,Shenzhen 518040,China;Shenzhen Institute of Molecular Crop Design,Shenzhen 518110,China)

机构地区：[1]深圳市农业科技促进中心,广东深圳518040 [2]深圳市作物分子设计育种研究院,广东深圳518110

出　　处：《杂草学报》2024年第3期42-50,共9页Journal of Weed Science

基　　金：深圳市科技计划(编号:JCYJ20230807145759008);国家自然科学基金(编号:31901532)。

摘　　要：为明确水稻对羟苯基丙酮酸双氧化酶基因(OsHPPD)位点突变对OsHPPD活性及硝磺草酮抗性的影响,以日本晴水稻为材料,克隆获得OsHPPD,并与玉米、拟南芥、荧光假单胞菌以及燕麦的HPPD进行BLAST比对,分析不同来源的HPPD蛋白序列,确定拟突变的氨基酸位点。再利用重叠PCR对OsHPPD上的12个氨基酸进行定点突变,获得14个OsHPPD突变体。对OsHPPD以及突变体蛋白进行体外原核表达,利用比色法测定突变体蛋白酶的相对活性以及对硝磺草酮的相对抗性。结果表明,V224I位点突变后较OsHPPD相对抗性增强14百分点,V224I位点突变后OsHPPD催化位点发生改变,导致OsHPPD的相对活性和对硝磺草酮的抗性增强,这对研制耐HPPD除草剂的种质资源、培育抗除草剂水稻新品种均具有重要作用。This study aimed to clarify influence of p-hydroxyphenylpyruvate dioxygenase from Oryza sativa(OsHPPD)gene on activity of OsHPPD and resistance of mesotrione.OsHPPD was cloned from O.sativa‘Nipponbar’,and then OsHPPD was compared with HPPD of Zea mays,Arabidopsis thaliana,Pseudomonas fluorescein and Avena sativa by BLAST.We analyzed HPPD protein sequences from different species to determine the amino acid sites to be mutated.Using overlapping PCR,12 amino acids in OsHPPD were subjected to site directed mutagenesis,resulting in 14 OsHPPD mutants.OsHPPD and its mutants were expressed in prokaryotic cells in vitro.Then relative activity of HPPD enzyme and relative resistance to mesotrione were determined by colorimetry.The results showed that relative resistance was enhanced 14 percentage points after V224I site mutation.The site mutation changed the catalytic site of OsHPPD,which resulted in the enhanced relative activity of OsHPPD and relative resistance to mesotrione.This site mutation is of great significance for the development of HPPD-tolerant germplasm resources and new herbicide-resistant rice variety.

关 键 词：OsHPPD 位点突变 相对活性 硝磺草酮 相对抗性 

分 类 号：S451.21[农业科学—植物保护]