JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.2 P.113-122

Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application

Author(s): Hai-ping Lu, Martin Edwards, Qi-zhao Wang, Hai-jun Zhao, Hao-wei Fu, Jian-zhong Huang, Angharad Gatehouse, Qing-yao Shu
Affiliation(s): State Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310029, China; more
Corresponding email(s): qyshu@zju.edu.cn
Key Words: CYP81A6, Bentazon, Metsulfuron-methyl, Expression induction, Xenobiotics

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Abstract: The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides.

细胞色素P450CYP81A6基因在水稻中的表达

目的：分析CYP81A6基因在苯达松及甲磺隆处理下的诱导表达模式，解释该基因与两种除草剂代谢相关的可能原因。
创新点：从两种除草剂降解途径中产生的小分子物质的结构相似性出发，通过基因诱导表达的特点分析，解释CYP81A6和两种除草剂降解相关的原因。
方法：通过实时定量聚合酶链反应（PCR）来分析基因表达的特点；利用CYP81A6启动子与GUS报告基因构建的载体来分析组织特异性表达；通过亚细胞定位来确定CYP81A6发挥功能的场所。
结论：CYP81A6基因受苯达松及甲磺隆诱导，在不同的时间点开始上调，说明了甲磺隆的降解中间产物可以诱导这个基因的表达；CYP81A6是组成型表达，在根、茎、叶中均有表达；亚细胞定位结果证明CYP81A6是一个内质网上的蛋白。

关键词：CYP81A6；苯达松；甲磺隆；诱导表达；异源物

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