日本看麦娘（Alopecurus japonicus）是麦田恶性杂草之一，广泛分布在长江中下游地区，严重影响小麦的产量和品质。在过去很多年，乙酰辅酶A羧化酶（Acetyl coenzyme A carboxylase, ACCase）抑制剂类除草剂精噁唑禾草灵（fenoxaprop-P-ethyl）一直是小麦田禾本科杂草防除的主要药剂，随着该药剂的长期使用，日本看麦娘对其产生了抗药性。国内已有较多研究表明ACCase氨基酸突变是抗性日本看麦娘对精噁唑禾草灵产生抗性的重要靶标机理，但是否有代谢酶的解毒作用存在未见报道，对于已存在靶标抗性的日本看麦娘种群是否还存在代谢抗性仍不清楚，为了明确主要代谢酶与日本看麦娘抗精噁唑禾草灵的关系，本文以AHFD-1（色氨酸-2027-半胱氨酸）、AHFD-3（天冬氨酸-2078-甘氨酸）、JCJT-1（异亮氨酸-1781-亮氨酸）、JCJT-2（异亮氨酸-1781-亮氨酸）和JZJR-1（异亮氨酸-2041-天冬酰胺）五个明确靶标突变的抗性种群及敏感种群JNXW-1为研究对象，对细胞色素P450氧化酶系和谷胱甘肽-S-转移酶（GST）两大重要代谢酶家族的含量、活性及基因表达量进行了研究，得到如下的结果与结论。 1、日本看麦娘细胞色素P450氧化酶系与抗精噁唑禾草灵关系的研究 采用差光度法测定细胞色素P450氧化酶系的组分细胞色素P450和细胞色素b5<下标!>含量。结果显示，精噁唑禾草灵处理前，AHFD-1、AHFD-3、JCJT-1、JCJT-2和JZJR-1 抗性种群P450含量分别为0.8213、1.4468、1.2200、1.2216和1.4208 nmol·mg-1pro，JNXW-1敏感种群细胞色素P450含量为1.1545 nmol·mg-1pro，药后抗性种群AHFD-3、JCJT-1、JCJT-2和JZJR-1细胞色素P450含量上升，最大值显著（P<0.05）高于敏感种群1.1926 nmol·mg-1pro，含量分别为2.2658、1.8916、2.0143和1.8975 nmol·mg-1pro。AHFD-1种群药剂处理前显著低于敏感种群，药剂处理后变化不明显。药剂处理前后，敏感种群细胞色素b5含量均高于各抗性种群。 采用模式底物分光光度法测定日本看麦娘细胞色素P450氧化酶系介导的对硝基苯甲醚-O-脱甲基酶（PNOD）和NADPH-细胞色素P450还原酶活性。结果表明，精噁唑禾草灵处理前，抗性种群AHFD-1、AHFD-3、JCJT-1和JCJT-2种群细胞色素P450氧化酶系介导的PNOD的活性分别为0.4414、0.3496、0.5293和0.5338 nmol·min-1mg-1pro，敏感种群细胞色素活性为0.5241nmol·min-1mg-1pro；施药后抗性种群 AHFD-3、JCJT-1和JCJT-2活性最大值分别为0.7238、0.6969和0.9595 nmol·min-1mg-1pro，分别升高2.07、1.32和1.82倍，AHFD-1种群药剂处理前后无显著性变化，而JNXW-1敏感日本看麦娘PNOD的活性逐渐下降，7d时下降到0.2746 nmol·min-1mg-1pro。施药前抗性种群AHFD-1、AHFD-3、JCJT-1和JCJT-2种群中NADPH-细胞色素P450还原酶活性分别为0.0447、0.0588、0.0601和0.0975 nmol·min-1mg-1pro，敏感种群活性为0.0551nmol·min-1mg-1pro；施药后抗性种群AHFD-3、JCJT-1和JCJT-2中该酶的活性显著（P<0.05）高于敏感种群（0.0734），分别为0.1061、0.0941和0.1475 nmol·min-1mg-1pro，而AHFD-1种群活性与敏感种群无显著性差异。 采用整株生测法对日本看麦娘细胞色素P450氧化酶系抑制剂的作用进行了测定，所用抑制剂为胡椒基丁醚（PBO）和1-氨基苯并三唑（ABT）。结果表明:细胞色素P450氧化酶系抑制剂PBO和ABT可提高精噁唑禾草灵对抗性种群AHFD-3、JCJT-1、JCJT-2和JZJR-1种群的抑制效果。其中施用ABT和精噁唑禾草灵后，AHFD-3和JCJT-2种群ED50下降最为明显，分别由320.25和336.00 g a.i.·ha–1下降到194.25和147.00 g a.i.·ha–1。抑制剂PBO和ABT无法提高精噁唑禾草灵对AHFD-1种群的抑制效果。 依据植物P450的保守区（motif），设计简并引物克隆出10条基因，分别属于不同的P450家族，将其进行命名编号Alo-1~10。对其进行表达量实验，结果表明药剂处理后，AHFD-3和JCJT-2两个种群的Alo-2、Alo-3和Alo-9三个基因表达量上调，是敏感种群的2倍以上；JCJT-1种群的Alo-2和Alo-3两个基因表达量上调，是敏感种群的2倍以上；JZJR-1种群的Alo-3基因表达量比敏感种群高2倍以上；AHFD-1种群上调基因的表达量中均未高于敏感种群2倍以上。 2、日本看麦娘谷胱甘肽-S-转移酶与抗精噁唑禾草灵关系的研究 对谷胱甘肽-S-转移酶（glutathione S-transferases, GST）进行研究，明确GST是否在日本看麦娘对精噁唑禾草灵抗性中起作用。采用模式底物分光光度法测定药剂处理前后抗精噁唑禾草灵日本看麦娘GST活性的动态变化。结果表明：精噁唑禾草灵处理后，GST活性总体先上升，后下降，且抗性种群GST活性并不显著高于敏感种群。依据大穗看麦娘（Alopecurus myosuroides）、菵草（Beckmannia. syzigachne）及小麦的GST基因，设计引物，克隆出3条GST家族基因，对三条基因进行表达量分析。结果表明：药剂处理前后，抗性与敏感种群GST基因均无显著差异。 综上所述， 在5个已经产生靶标抗性的种群中，AHFD-3、JCJT-1、JCJT-2和JZJR-1这4个种群存在代谢抗性。其中AHFD-3、JCJT-1和JCJT-2种群涉及的代谢抗性与细胞色素P450含量、细胞色素P450介导的PNOD和NADPH-P450还原酶活性及细胞色素P450基因表达量有关，JZJR-1种群涉及的代谢抗性与细胞色素P450含量及细胞色素P450基因表达量有关。AHFD-3、JCJT-1、JCJT-2和JZJR-1种群涉及的代谢抗性与GST无关。AHFD-1种群不涉及细胞色素P450氧化酶系和GST方面的代谢抗性。

Janpanese foxtail (Alopecurus japonicas) is one of execrable weed in wheat field, widely distributed in the middle and lower reaches of the Yangtze river region, seriously affect the yield and quality of wheat. The early control of janpanese foxtail depends on fenoxaprop-P-ethyl, a kind of ACCase-inhibitor herbicide. The long-term use of this herbicide lead to the resistance. Preliminary studies of our laboratory have found that the amino acid mutation of ACCase was important target mechanism to fenoxaprop-P-ethyl resistance in janpanese foxtail population, but did not study the mechanism of non-target mechanism which metabolic resistance taking apart in. This article selects populations AHFD-1 (W2027C), AHFD-3 (D2078G), JCJT-1 (I1781L), JCJT-2 (I1781L) and JZJR-1 (I2041N), which the mutation of ACCase have been confirmed, and JNXW-1 as the sensitive population, study the two important metabolic enzyme family, cytochrome P450 and glutathione S-transferases. 1. The study of relationship between Cytochrome P450 and fenoxaprop-P-ethyl resistance in janpanese foxtail Using differential spectrophotometry to measure the content of cytochrome P450 and cytochrome b5, which were components of cytochrome P450 enzyme system. Results show that before treatment of fenoxaprop-P-ethyl, the cytochrome P450 content of sensitive population was 1.1545 nmol·mg-1pro, the content of resistance populations AHFD-3、JCJT-1、JCJT-2 and JZJR-1 were1.4468、1.2200、1.2216 and 1.4208 nmol·mg-1pro. After treatment of fenoxaprop-P-ethyl, the cytochrome P450 content of sensitive population had a modest rise, the maximum value was 1.1926 nmol·mg-1pro. The cytochrome P450 content of AHFD-3、JCJT-1、JCJT-2 and JZJR-1 were 2.2658、1.8916、2.0143和1.8975 nmol·mg-1pro after treatment of fenoxaprop-P-ethyl, significantly higher than sensitive population. Before and after treatment, the cytochrome P450 content of sensitive population were higher than resistant populations. Using the model substrate fluorescence spectrophotometry to measure the activities of PNOD and NADPH-cytochrome P450 reductase mediated by cytochrome P450 enzyme system. Results show that before treatment of fenoxaprop-P-ethyl, the PNOD activity mediated by cytochrome P450 enzyme system of sensitive population was 0.5241nmol·min-1mg-1pro, the activities of resistance populations AHFD-3、JCJT-1 and JCJT-2 were 0.3496、0.5293 and 0.5338 nmol·min-1mg-1pro. After treatment of fenoxaprop-P-ethyl, the PNOD activity of sensitive population decreased gradually to 0.2746 nmol·min-1mg-1pro in the seventh day. The maximum values of resistance populations AHFD-3、JCJT-1 and JCJT-2 were 0.7238、0.6969 and 0.9595 nmol·min-1mg-1pro, respectively increased 2.07、1.32 and 1.82 times. NADPH-cytochrome P450 reductase activity of sensitive population was 0.0551nmol·min-1mg-1pro before treatment, this reductase activities of resistance populations AHFD-3、JCJT-1 and JCJT-2 were 0.0588、0.0601 and 0.0975 nmol·min-1mg-1pro. NADPH-cytochrome P450 reductase activity of sensitive population was 0.0734nmol ·min-1mg-1pro after treatment, the activities of this reductase were significantly increased in resistance populations AHFD-3、JCJT-1 and JCJT-2, respectively were 0.1061、0.0941 and 0.1475 nmol·min-1mg-1pro. Using the whole-plant measurement method to study the effect of cytochrome P450 enzyme system inhibitor for janpanese foxtail, the inhibitors used were piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The results showed that PBO and ABT can improve the inhibiting effect of fenoxaprop-P-ethyl to resistance populations AHFD-3、JCJT-1、JCJT-2 and JZJR-1. After the use of PBO and fenoxaprop-P-ethyl, the ED50 of AHFD-3 and JCJT-2 significantly decreased, respectively from 320.25 and 336.00 g a.i.·ha–1 to 194.25 and 147.00 g a.i.·ha–1. On the basis of plant P450 conservative district (motif), we designed the degenerate primers and cloned eighteen genes, they belong to different families, named respectively Alo-1~18. Expression quantity experiments were produced with ten genes, the results showed that the expression quantity of Alo-5、Alo-6 and Alo-13 of AHFD-3 and JCJT-2 were significantly increased after fenoxaprop-P-ethyl treatment, the expression quantity of Alo-5 and Alo-6 of JCJT-1 were significantly increased, the expression quantity of Alo-6 and Alo-13 of JZJR-1 significantly higher than sensitive population. 2. The study of relationship between glutathione S-transferases and fenoxaprop-P-ethyl resistance in janpanese foxtail The function of glutathione S-transferases (GST) to fenoxaprop-P-ethyl resistance in janpanese foxtail have been studied. Using model substrate fluorescence spectrophotometry to study the dynamic changes of the GST activity before and after fenoxaprop-P-ethyl treatment. The results show that the GST activity of sensitive population was rised in the first stage and then decreased after fenoxaprop-P-ethyl treatment, the change trends was higher than resistant populations. On the basis of the GST gene of Alopecurus myosuroides, Beckmannia syzigachne and wheat, primers were designed and three genes of GST families were cloned, their expression quantity were studied. The results show that there was no significant difference in the expression quantity of GST genes of resistance and sensitive populations before and after fenoxaprop-P-ethyl treatment. In conclusion, in five populations which have already produced target resistance, the metabolic resistance was exist in AHFD-3、JCJT-1、JCJT-2 and JZJR-1. The metabolism resistance was related to the content of cytochrome P450, the activities of PNOD and NADPH-cytochrome P450 reductase mediated by cytochrome P450 enzyme system and gene expression quantities in AHFD-3、JCJT-1 and JCJT-2 populations. The metabolism resistance was related to the content of cytochrome P450 and gene expression quantities in JZJR-1 population. The metabolism resistance was irrelevant to GST in AHFD-3、JCJT-1、JCJT-2 and JZJR-1 populations. AHFD-1 population showed no significant difference with sensitive population in all experiments, show that there was no metabolic resistance of cytochrome P450 enzyme system and GST in this population.