The Function Of CsCYP6A2 And CsCYP306A1 Genes In The Lethal Process Of Vetiver Zizanioideis To Chilo Suppressalis

Striped stem borers,Chilo suppressalis(Walker)is one of the most important pest of rice in China,which is a threaten to rice production.At present,the control of C.suppressalis is still primarily dependent on chemical control measures.However,unreasonable and long-term use of chemical pesticides has been led to many negative environmental effects,such as the reduction of biodiversity and natural biological control,high insecticide resistance in pests,environmental pollution and ecological imbalance.In recent years,the incidence of C.suppressalis on the rice is urgently seek safe and effective control measures.While planting vetiver(Vetiveria zizanioideis)in the rice field can effectively attract the female adults of C.suppressalis to lay the eggs,the results is that the larvae can’t complete their life cycles by feeding on it.It means that feeding on vetiver can lead the C.suppressalis larvae to be death.Previous study have been indicated that the lethal effect is probably related to the decrease of cytochrome P450 enzyme activity in C.suppressalis after feeding on vetiver,however,the lethal mechanism is not clear at present.In this study,the CYP6A2 and CYP306A1 genes of C.suppressalis(CsCYP6A2 and CsCYP306A1)were systematically studied at the molecular level.The expression of two cytochrome P450 was analyzed at different developmental stages,different tisues and the effect of feeding on vetiver.The detoxification metabolic mechanism of CsCYP6A2 and CsCYP306A1 genes on vetiver was studied by RNAi technology.The main findings are as follows:1.Two cytochrome P450 genes of C.suppressalis were obtained by transcriptome method.The full-length sequences of two cytochrome P450 genes were cloned.The sequence analysis showed that the open reading frame(ORF)of CsCYP6A2 was 1518 bp,encoding a protein of 505 amino acids.The molecular weight of CsCYP6A2 was 58.10 kDa and the isoelectric point was 8.55.Four characteristic sequences of cytochrome P450 genes were predicted,including the C helix conserved sequence WxxxR,the K helix conserved sequence ExLR,the PERF motif PxxFxPE/DR and the P450 marker sequence PFxxGxxxCxG located in the heme binding region.The ORF of CsCYP306A1 gene was 1437 bp,encoding a protein of 478 amino acids.The molecular weight of CsCYP306A1 was 54.78 kDa and the isoelectric point was 6.58.Two characteristic sequences of cytochrome P450 genes were predicted that they will be,including the C helix conserved sequence WxxxR and I helical conserved sequence GxE/DTT/S.2.Real-time PCR was used to detect the expression of CsCYP6A2 and CsCYP306A1 at different developmental stages.It was found that CsCYP6A2 and CsCYP306A1 expressed in all developmental stages of C.suppressalis.However,CsCYP6A2 had the highest expression at the 5th instar,followed by the 3rd instar,the 4th instar and the 6th instar,while the relative expression of CsCYP6A2 was lower at other stage.The expression level of CsCYP306A1 in the egg was the highest,followed by the 5th instar and the female adults,and the lowest in the male adults.3.The expression results of CsCYP6A2 and CsCYP306A1 in different tissues of 3rd instar larvae of C.suppressalis showed that two cytochrome P450 genes were expressed in all tissues,however,the expression of CsCYP6A2 was highest in fat body,followed by epidermis and midgut.The expression of CsCYP306A1 was highest in midgut,followed by fat body and epidermis.Both CsCYP6A2 and CsCYP306A1 genes had the lowest expression in head.4.The expression of two P450 genes in 3rd instar larvae of C.suppressalis after feeding on rice and vetiver for different days was determined by real-time PCR.Compared to the control(larvae feeding on rice),the expression of CsCYP6A2 in different tissues was down-regulated in the larvae feeding on vetiver,which indicate that vetiver may contain some substances that inhibit the expression of CsCYP6A2.The expression of CsCYP306A1 in the larvae feeding on vetiver was higher than that in the larvae 1 day and 2 days after feeding on rice,and then the expression level in the vetiver treatment group was lower than that in the rice control group with the feeding time prolonged.In addition,the expression of CsCYP306A1 was highest in midgut,which indicated that CsCYP306A1 was involved in the interpretation of toxic substances in the metabolic vetiver,but the expression of this gene was inhibited as the feeding time was prolonged.5.Our RNAi study of RNAi which compared with control group of buffer injection was found that the pupation rate was decreased in the larvae injected by dsCYP6A2 and dsCYP306A1.The larvae after injecting were fed with rice and vetiver,respectively,and then we found the mortality was gradually increased with the feeding time extended,and the mortality of vetiver treatment group was higher than that in the rice control group.The expression of CsCYP6A2 and CsCYP306A1 in midgut and fat body from the larvae feeding on vetiver was also significantly lower than that in the larvae feeding on rice.The results showed that the two cytochrome P450 genes obtained in this study probably involved the lethal mechanism of trap plant vetiver against the larvae of C.suppressalis.In summary,two cytochrome P450 genes were obtained from C.suppressalis by genome sequencing and cloned their full-length sequences,which laid the foundation of further research on the lethal mechanism of vetiver against the larvae of C.suppressalis.The expression characteristics of two cytochrome P450 genes at different developmental stages and tissues of C.suppressalis were analyzed by Real-time RCR technique.The effects of vetiver on their expression were also analyzed.The relationship between CsCYP6A2 and CsCYP306A1 and the toxic metabolisms of vetiver was also discussed based on RNAi.The results will be provided in further information of the biological and physiological characteristics of cytochrome P450 in C.suppressalis,and it is also important for revealing the lethal mechanism of vetiver on C.suppressalis.Our results showed that the useful scientific basis technology for the development of sustainable control method of C.suppressalis based on the trap plant vetiver in rice ecosystem.