| Plutella xylostella is a major pest of cruciferous vegetables and crops worldwide,causing economic losses of up to $ 4 billion-5 billion annually.Long-term use of insecticides leads to the rapid evolution of insect resistance to insecticides,which brings great difficulties to pest control.Trehalose is the major blood sugar in insects,which was instant source of energy and played a crucial role in resisting abiotic stresses.The hydrolysis of trehalose is under the control of trehalase.Recently,the trehalase gained increasing attention in insect physiology as it regulated energy metabolism and glucose generation via trehalose catabolism.However,trehalase gene of P.xylostella and the transcriptional regulation mechanism are still unknown.In this study,we cloned and verified two trehalase genes from P.xylostella and compared the variations of trehalase activity and gene expression levels in different strains of P.xylostella.It was found that CCAAT enhanced the ability of binding protein(C/EBPα)to regulate the transcription of trehalase genes,which provides important theoretical data for the potential of trehalase as an insecticidal target.The main findings are as follows:(1)Comparison of trehalase activity and gene expression in different strains of P.xylostella.The trehalase activity of sensitive strains of P.xylostella at different developmental stages and different strains was measured.It was found that the trehalase activity of sensitive strains reached the peak at initial stage old third instar larvae,and the trehalase activity of resistant strains was higher than that of sensitive strains.The trehalose gene expression in resistant strain was higher than that of sensitive one.And the higher expression of trehalase gene enhanced the activity of trehalase and the adaptability of insect pests to adverse environment.(2)Cloning,expression analysis and functional verification of trehalase gene in P.xylostella.Full-length sequences of trehalase genes PxTreh1 and PxTreh2 from sensitive strains of P.xylostella were cloned.Sequence homology comparison showed that PxTreh1 and PxTreh2 proteins had low homology with reported insect Treh1 and Treh2 proteins,and Phylogenetic tree analysis showed that there was significant difference for trehalase genes in varied insects.According real-time quantitative PCR,PxTreh1 and PxTreh2 genes were expressed in all developmental stages of P.xylostella,and the expression level of PxTreh2 was significantly higher than that of PxTreh1,especially in 1st instar larvae and adults.While the highest expression level of PxTreh1 was found in eggs.The high expression level of PxTreh2 can also be found in different tissues,and the expression of both genes was higher in the chest and lower in the midgut.(3)Regulation of transcription factor C/EBPα on PxTreh1 and PxTreh2 in P.xylostella.The transcription starting sites and the promoter sequences of PxTreh1 and PxTreh2 were identified by 5’RACE and further amplification of 5’flanking sequence respectively.And PxTreh1 promoter-487 bp to + 402 bp and PxTreh2 promoter-203 bp to + 196 bp were important regulatory regions,according to dual luciferase gene reporting system.(4)Dual luciferase activity assay coupled with RNA interference(RNAi)technology revealed that the expression of PxTreh1 and PxTreh2 genes was regulated by C/EBPα.Yeast single hybridization(Y1H)experiment showed that there was no interaction between C/EBPα and PxTreh1,but the interaction existed between C/EBPα and PxTreh2 promoters.Coupled with site mutation test,it was found that C/EBPα played an indirect regulatory role on PxTreh1,and PxTreh2 might also be regulated by other transcription factors. |
