| 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(12041N),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 foxtailUsing 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 were 1.4468?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 AHED-3?JCJT-1?JCJT-2 and JZJR-1 were 2.2658?1.8916?2.0143 ? 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-1 mg-1 pro,the activities of resistance populations AHFD-3?JCJT-1 and JCJT-2 were 0.3496.0.5293 and 0.5338 nmol· min-1 mg-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-1 pro,respectively increased 2.07?1.32 and 1.82 times.NADPH-cytochrome P450 reductase activity of sensitive population was 0,0551 nmol ·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-1 mg-1 pro 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-1 mg-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 foxtailThe 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. |
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