Study On The Target Enzyme And Metabolizing Enzymes Resistant Mechanism Of Japanese Foxtail (Alopecurus Japonicus) To Haloxyfop-P-Methyl

The Japanese foxtail(Alopecurus japonicus) was a serious weed in oilseed rape in the Yangtze River basin. It distributes widely and seriously and restrains the production of oilseed rape. The haloxyfop-P-methyl is a kind of aryloxyphenoxypropionate herbicide. Its mode of action is inhibiting the activity of acetyl coenzyme A carboxylase (ACCase) of grass weeds, then blocking the normal fatty acid synthesis in plants. As a highly effective herbicide, it was widely used in oilseed rape to prevent grass weeds such as Alopecurus japonicus. But the long-term and single use of haloxyfop-P-methyl in oilseed rape has caused resistance to Alopecurus japonicus. After the preliminary study in our laboratory, we have firstly found the resistance of Alopecurus japonicus to haloxyfop-P-methyl, and then firstly report its resistant level and cross-resistance. In this paper, the resistance mechanism on target and metabolizing enzymes would be further explored based on previous studies.In this paper, the resistant and sensitive populations which have been reported by our team were used as research materials. Enzymes activity, enzymes gene sequences and the expression of enzymes gene were analysed both resistant and sensitive biotypes.The study of target enzyme shows that, the activity of ACCase in sensitive plants was significantly higher than that in resistant ones without herbicide treatment. At different herbicide concentration, the activity of ACCase between the resistant and sensitive populations were different, when the concentration was lower than0.1μmol·L-1, the activity of sensitive populations was significantly higher than that in resistant populations; while if the concentration was higher than0.1μmol·L-1, the result was completely different. With the change of inhibitor concentration, the ACCase activity in two groups shows differeft trends, we found that the sensitive populations’ACCase activity significantly decreased with the increasing of inhibitor concentration, but the decline of resistant populations were not very significant. Overall, the IC50inhibiting ACCase activity of sensitive populations is0.0026μmol·L-1, and the resistant populations is0.028μmol·L-1, which is10.78-foldhigher than the sensitive populations.In the study of ACCase gene sequences, part full-length genomic DNA sequence of ACCase was12151bp with an opened reading frame (ORF) of6960bp which encoded2320amino acid residues. The comparasion of ACCase sequences between the resistant and sensitive populations showed that there were11different bases in gDNA,8different bases in cDNA and4different amino acids in cDNA. The four amino acids mutations were: Arg-1734-Gly, Met-1738-Leu, Thr-1739-Ser, Ile-2041-Asn. The amino acid mutation (Ile2041-Asn2041) found in resistant had been reported to confer resistance of Alopecurus. myosuroides and Lolium rigidum to aryloxy-phenoxypropionate (AOPP), and this mutation can not produce cross-resistant to CHD herbicide was also been verificated in this study. So the mutation of Ile-2041-Asn was one of resistant mechanism, which conferred resiatance of Japanese foxtail to haloxyfop-P-methyl. The correlation between other mutation sites with resistance is unknown.The expression level of ACCase gene between resistant and sensitive populations have no significant difference. So the resistant plants were minor increase in the expression level of target enzyme.The study of activity and contents of metabolizing enzymes shows that, after herbicide treatment, the content of cytochrome P450in resistant and sensitive populations shows different trends. With the extension of herbicide treatment, the content of sensitive populations showed slowly increased firstly and then slowly decreased, but the change in resistant populations were obvious, and the maximum value in resistant populations were significantly higher than the sensitive ones. The trend of the content of cytochrome b5was basically consistent with cytochrome P450. With the herbicide treatment, the trend of NADPH-P450reductase was basically consistent with cytochrome P450too, but the activity between resistant and sensitive populations always have no significant differences. The study of GST (glutathione S-transferases) activity shows that before the herbicide treatment, the resistant populations were significant higher than sensitive ones, but after treatment with the extrent of time the resistant populations showed decreased firstly and then increased. The activity of sensitive populations showed declining trend, which has not significantly change, but in the entire treatment time, the activity of resistant populations was always higher than sensitive populations.The expression level of metabolizing enzymes gene shows that the expression level of CYP71C6v1between resistant and sensitive populations have no significant difference, at the same time, GST gene have the same result with P450gene. So, the resistant plants have no significant difference in the expression of metabolizing enzymes gene.