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rge amounts in the thylakoid membranes of chloroplasts and play a function in defending chlorophylls from active oxygen and peroxides. Therefore, the lower in carotenoids causes the loss of their protective impact against the generation250 S. Yamamoto et al.Journal of Pesticide Scienceof active oxygen by light in the plant, resulting in bleaching and top to death.4) Fenquinotrione is assumed to become an HPPD inhibitor because its chemical structure and herbicidal symptoms are extremely equivalent to these of HPPD inhibitors. In this study, we examined the mode of action of fenquinotrione by examining its inhibitory effects on HPPD activity. The aspects accountable for the fantastic rice selectivity of fenquinotrione are also discussed.have been purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Rice plants (Oryza sativa L. var. Kinmaze) and Arabidopsis plants (Arabidopsis thaliana, ecotype Columbia-0) were used in this study. two. Bioresource for construction from the HPPD enzyme assay Pseudomonas aeruginosa strain PAO1 for isolation on the homogentisate dioxygenase (HGD) gene was obtained from the Biological Resource Center, NITE (NBRC, Tokyo, Japan). 3. Cloning and expression of Arabidopsis HPPD (AtHPPD) The AtHPPD gene (At1g06570) was amplified from Arabidopsis cDNA making use of the Phusion Hot Begin II DNA Polymerase (ACAT Inhibitor list Thermo Fisher Scientific, MA, USA). The primers utilized for amplification of the AtHPPD gene had been 5-TCG AAG GTC GTC ATA TGG GC C ACC AAA ACG CCG CC-3 (forward primer) and 5-GTT AG C AGC CGG ATC CTC ATC CCA CTA ACT GTT TG-3 (reverse primer). The PCR product was ligated in to the Escherichia coli expression pET-16b vector (MNK1 Compound Novagen, WI, USA) digested with Nde I and BamH I employing an In-Fusion HD Cloning Kit (TaKaRa Bio Inc., Shiga, Japan). The resultant vector was introduced into the E. coli BL21 star (DE3) strain (Thermo Fisher Scientific) using the heat shock approach and after that plated on Luria ertani (LB) agar medium supplemented with 100 /mL ampicillin for transformant selection. The transformed E. coli cells had been picked out and grown to OD600=0.5.6 in 2 T medium supplemented with 100 /mL ampicillin at 37 . The expression of N-terminal His-tagged AtHPPD was induced by 1 mM IPTG and cultured at 16 for 24 hr. Escherichia coli cells had been har-Materials and methods1. Chemical compounds and plants Fenquinotrione and its derivatives and metabolites have been synthesized by the Kumiai Chemical Business Co., Ltd. (Shizuoka, Japan). The structure of fenquinotrione, nuclear magnetic resonance (NMR) information, and mass spectrometry (MS) data for genuine standards are shown in Table 1. Three 14C-labeled compounds of fenquinotrione were utilized in the metabolic study: a 1-position label of a cyclohexenyl moiety (certain activity 4.94 MBq/mg, radiochemical purity 98.three , abbreviated as [Cy-14C] FQ) synthesized by the Institute of Isotopes Co., Ltd. (Budapest, Hungary); the uniform label of a chlorophenyl ring (certain activity five.63 MBq/mg, radiochemical purity 99.two , abbreviated as [Qu-14C] FQ); along with the uniform label of a phenyl ring (precise activity 5.29 MBq/mg, radiochemical purity 99.6 , abbreviated as [Bz-14C] FQ) synthesized by the Sekisui Health-related Co., Ltd. (Ibaraki, Japan). The active kind of benzobicyclon was synthesized by the Kumiai Chemical Sector Co., Ltd. Tefuryltrione, HPP, L(+)-ascorbic acid, iron(II) sulfate heptahydrate (FeSO4 H2O), and isopropylthio–galactoside (IPTG)Table 1. Compounds Fenquinotrione StructureH NMR data and MS data of authe