Helicoverpa Armigera (hübner) Insecticide Resistance Monitoring In Shihezi And Cloning Of Cyp6ae14 And Ak Gene

Helicoverpa armigera(Hubner) belongs to Lepidoptera Noctuidae Helicoverpa armigera Heliothinae is, is a major pest of cotton production, cotton bollworm resistance to pesticides is a long time been closely important issues. Therefore, after investigation, In this study, C Rust P, cypermethrin, emamectin benzoate and endosulfan insecticides on four kinds of agricultural areas Shihezi earnest examination centers, peach town, the town springs to three test points such as cotton bollworm Helicoverpa armigera(Hubner) populations recommended by FAO Measurement of resistance of micro-drip method. The results showed that:Shihezi three populations of cotton bollworm were produced on low-level resistance to cypermethrin, multiple resistance were 5.38 times,7.47 times,7.88 times; of the game Dan, profenofos and emamectin benzoate three kinds of agents did not produce significant resistance levels. Meanwhile, Shihezi and susceptible strains of Helicoverpa armigera(Hubner) in vivo some of the major detoxification enzyme of the enzyme protein, and the specific activity measured for comparison with the susceptible strain of Helicoverpa armigera(Hubner) Shihezi enzyme protein content and activity of Helicoverpa armigera(Hubner) differences The results showed that cotton bollworm Shihezi the body's pro MFO content and activity were higher than susceptible strains of cotton bollworm and the difference is significant, and carboxylesterase, phosphate and glutathione-S-transferase The zymogen content and specific activity differences were not significant.Cytochrome P450-dependent monooxygenases are a very important enzymatic system, which has been found in all living organisms systems examined.These monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds.It has been regarded as a key object in the biology field because of its diversity in structures and functions.Then, In order to know more about the structures and functions of individual P450s in H.armigera,efforts were given to identify the individual P450 and to express it in heterologous expression systems.Using two pairs of primers designed according to sequence of CYP6AE14, we cloned full length of CYP6AE14 from midgut and fat body tissue in cotton bollworm by PCR. The resultant sequence is 1836bp in length, encoding a protein of 505 amino acid residues. Chen Xiaoya, measured with the teachers of the gene sequence comparisons showed that cotton bollworm Shihezi base sequence of the gene in vivo the first base position 74,1059,1086,1113 and 1545 measured with the same teacher Chen Xiaoya, the location of bases are different, and in the translation of amino acid sequences, only the first 74 base pairs of amino acids encoding an impact, because the degeneracy of the genetic code of the other four amino acid bases did not affect. Using prokaryotic expression vector PET28-a expressed in E. coli Cytochrome P450 CYP6AE14 gene, and verified by polyacrylamide gel electrophoresis, the expression of P450 protein in approximately 50 kD band appeared to induce the target for further gene function laid the foundation for verification.In the process of insect life activities, arginine kinase (Arginine kinase, AK) is the body of the main phosphagen kinase, which through the catalytic arginine and reversible reaction between ATP, the energy stored in the phosphoric acid arginine high-energy phosphate bond, or the phosphoric acid arginine decomposition of ATP, the body's energy metabolism of insects, storage and use of a key regulatory role. Because arginine kinase exists only invertebrates, and the energy of the original enzyme and the phosphate metabolism pathway in mammalian cells is completely different, so arginine kinase as a good control of insects sensitive target. Therefore, this study was cloned cotton arginine kinase expression. AK gene sequence length of the 1068bp, encoding 355 amino acids, molecular weight of about 37 kD. The arginine kinase genes connected to the prokaryotic expression vector pET-28a, on and into E. coli for prokaryotic expression. At the same time by real time quantitative PCR, that AK gene expression in vivo Shihezi area than cotton bollworm Helicoverpa armigera susceptible strain AK gene expression in vivo high 77.36%. Can be learned through the above, the enzyme is the major metabolic enzymes in vivo, if the arginine kinase (AK) gene in this study is experimental in vivo overexpression of cotton bollworm, Helicoverpa armigera it will show the life of metabolic activity is very strong, followed by outside sources will bollworm in incentives, the enhancement of their metabolic capacity, thus enhancing its resistance to bollworm. Therefore, this study bollworm arginine kinase was cloned and expressed, is designed to develop in order to arginine kinase inhibitors that target pest provide the basis for a new theoretical basis.