| Leek maggot is a major harm to a variety of vegetables in many provinces in China are reported,is the enemy of vegetable production.Leek maggots also known as root maggots mainly harm leek,due to dwelling in the roots of leek,prevention and control is extremely difficult.With the vegetable farmers on the abuse of chemical pesticides and over-reliance,leek maggots selection pressure,its resistance to a variety of chemicals gradually increased,making the prevention and control of leek maggots by the daunting challenge.The molecular biology research has not been reported,the insect detoxification system in its resistance to external environmental stress plays an extremely important role.It is helpful to provide a scientific basis for the prevention and control of insects at the molecular level,especially from the point of view of ecological friendliness,to understand the expression level of different target enzymes in the detoxification enzyme mechanism.In this paper,the primers of the different samples were screened by highthroughput Illumina Hiseq 2000,and the cytoplasmic P450 oxidase(CPY)(GSTs)and non-specific esterase(ESTs)were used to clone the four detoxification enzymes in phoxim,chlorpyrifos and imidacloprid,respectively.Under the transcription level of the study,the specific results are as follows:1.Nine candidate candidate genes of RPL10,EF1?,G6 PDH,UBC,ACT,RPS3,TUB,GAPDH,EF2? were screened in 23 different samples by Real-Time PCR.The results showed that the optimum internal parameters were ACT1 and EF2? in different developmental stages.The most suitable parameters were EF1? and EF1-? and ACT1? in the different tissues.The optimal internal reference genes were RPL10,EF1-? and RPL10.The optimal internal reference gene was RPL10 under different pesticide stress,and the double internal reference genes were RPL10 and RPS3.2.The genes of four detoxification enzymes,ABC transporter,cytochrome P450 oxidase system(CPY),glutathione-S-transferase(GSTs)and non-specific esterase (ESTs)were studied The nucleotide sequences of the clones were 1275,1733,609 and 801 bases,respectively.The results of phylogenetic analysis showed that the CPYP gene was similar to that of the Diptera family,and the homology of the CYP gene was 97%,which was more homologous than that of other insect families.The GST gene was similar to that of the housefly,The homology of Drosophila melanogaster was 97% and 96%,which was more homologous than that of other insect families.The homology of EST gene was 98% with that of Aedes albopictus and Aedes albopictus Which is more homologous than the other insect families.The homology of the ABC transporter and the Anopheles gambiae is 95%,and the kinship is more homologous than the other insect families.The expression level of four genes was studied by Real-Time PCR technique in different treatment time of different pesticides.The results showed that the expression of glutathione-S-transferase(GST)was not significant in the treatment of phoxim,and the expression of ABC transporter and EST was significantly increased.In the detoxification of phoxim,the glutathione-S-transferase system does not play the corresponding detoxification function,and the main role is ABC transporter and esterase(EST)enzyme system.Under the control of chlorpyrifos,the four kinds of detoxification enzymes increased significantly with time,especially the expression of ABC transporter gene was the most significant and the duration was longer,which indicated that ABC transporters played the main detoxification under chlorpyrifos stress(GST)was less affected by the time of nicotine pesticide(imidacloprid)stress,and the other three genes showed a decreasing trend with time,Lower than the control group expression level.Under the stress of nicotine pesticide(imidacloprid),the ABC transporter,EST and GST genes showed a downward trend on the second day,and the effect of fluoxifen was slowed down and did not affect the expression level of GST gene. |
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