Analysis Of Expression Patterns Of Three Carboxylesterase Genes From Helicoverpa Armigera

Helicoverpa armigera（Hübne）is a kind of important agricultural pests in Noctuidae around the world.Carboxylesterase is important detoxification enzyme in insects and involved in the metabolic detoxification of insecticides such as organophosphorus,pyrethroids and carbamates.Based on the preliminary studies of our research group,carboxylesterase genes such as WH001G were isolated.The relative expression levels of carboxylesterase WH001G,WH001A and WH001H in different developmental stages and tissues of Helicoverpa armigera were determined and analyzed by fluorescence quantitative PCR with double internal parameters.To clarify the effect of pyrethroid insecticides on the expression level of carboxylesterase in Helicoverpa armigera,the expression levels of three carboxylesterase genes in the third instar of Helicoverpa armigera at 0,4,8,12,24,48 and 72 hours after fenvalerate insecticide treatment(LD₃₀),were analyzed based on the toxicity curve of fenvalerate in laboratory..The above results are intended to lay a foundation for defining the function of carboxylesterase in the metabolic capacity in Helicoverpa armigera.The main results are as follows:1.Analysis of relative expression levels of carboxylesterase genes:WH001G,WH001A and WH001H in different developmentalstages and tissues of Helicoverpa armigeraAmong the eight different stages of growth and development:eggs,1-5 instars,pupae and adults of Helicoverpa armigera,the expression level of carboxylesterase WH001G was the highest at the 1st and 5th instars,about 5 times higher compared with that of the control（EGG）;the relative expression level of the 2nd instar（N2）was a little lower than the 1st and 5th instars,and the relative expression level of 3rd and 4th instars（N4）was obviousely lower than that of the 2nd instar（N2）,about only 2-4 times higher than that of eggs;however,the expression level of WH001G in pupae（P）and adult（AD）were lowest.The relative expression level of WH001G in the midgut（MG）was the highest in different tissues,about 76 times higher than that in egg and was relatively high in the foregut（FG）,but the expression level of WH001G was lower in other tissues.The expression of WH001A in eggs,1-5 instars and adults of Helicoverpa armigera increased gradually reached the highest level at the fifth instar（N5）,which was 30 times higher than egg with the exception of both the third instar（N3）and the adult stage（AD）The expression of WH001A in the fourth instar（N4）and pupa stage（P）was 15 and 18 times higher than egg,respectively.The expression in the second instar（N2）was lower than that in the fourth instar（N4）.The expression of WH001A was slightly higher in the pupae stage（P）and three stages with the exception of eggs.In different tissues,the highest expression of WH001A was in fat bodies（FB）about 5.7 times as much as that in brain,followed by foregut（FG）and midgut（MG）,which were 2.59 and 1.17 times as much as that in brain,respectively.WH001A was not expressed in the other two parts except the head（BR）.From egg（EGG）to 5th instar larval（N5）,the expression of WH001H increased gradually,reaching the highest level at 5th instar（N5）,which was 52 times higher than that in egg.The expression of WH001H in pupae（P）and adult（AD）was very low.The highest expression of WH001H was found in the midgut（MG）,which was 160 times higher than that in head（BR）and was slightly expressed in the other four parts except brain.2.Toxicity of Helicoverpa armigera and the effect of fenvalerate on the expression of carboxylesterase WH001G and WH001A from Helicoverpa armigeraWhen fenvalerate at the dose of LD₃₀ was used to treat the 3rd instar larvae of Helicoverpa armigera,the expression of WH001G increased significantly in 0-4h and declined in 4h-72h.Compared with the control group,the expression of WH001G was higher at 0-12h,but the relative expression level of WH001G was lower than that of the control group within12-72h,particularly at 24 h.The relative expression of WH001G in the experimental group was only one third of that of the control group.After fenvalerate treatment,the expression of WH001A gradually decreased within 24 hours,increased from 24 hours to 48 hours,and then decreased after 48 hours.In addition,the expression of WH001A in the experimental group was higher than that in the control group except at 24 and 72 hours.In conclusion,in different developmental stages of Helicoverpa armigera,the expression level of carboxylesterase WH001G was the highest in the 1st and 5th instars of Helicoverpa armigera,the highest level of WH001A was in the 4th and 5th instars;in different tissues,the highest levels of WH001G and WH001H were in the midgut.and the highest level of WH001A was in the fat body,followed by the foregut.The expression of WH001G was higher at 4h,8h and 12h and lower at 24h and 48h than that of the control at the dose of LD₃₀for fenvalerate₀,indicating that the expression of Helicoverpa armigera was up-regulated at the early stage of fenvalerate treatment,however,WH001A was up-regulated slightly within 12h of fenvalerate treatment and the change was not obvious.