Identification Of Cytochrome P450s And Characterization Of CYP3310B1 In Pardosa Pseudoannulata

Cytochrome P450 monooxygenase(P450)is a huge and diverse superfamily,encoding enzymes with broad-spectrum monooxygenases and related activities.P450 is one of the two most important monooxygenase systems in an organism,and play important roles in the growth and development of different species.In arthropods,it is involved in the biosynthesis of endogenous compounds such as juvenile hormones,ecdysteroids,long-chain hydrocarbons,as well as the metabolism of pheromones,fatty acids,lipids,and exogenous compounds such as pesticides.Pardosa pseudoannulata is the dominant predator in rice fields in the south of China,and plays an important role in the control of major insect pests such as rice planthoppers.At present,the systematic research on arthropod P450 is mainly focus on the insecta,but less on the arachnid.The entire genome identification and analysis of P450s in Pardosa pseudoannulata provides a basis for studying the evolutionary relationship and gene function of arthropod P450s.1.Identification and phylogenetic analysis of cytochrome P450s in Pardosa pseudoannulataBased on the genome and different tissue transcriptomes of Pardosa pseudoannulata,128 P450s were identified.According to the official nomenclature and phylogenetic analysis,the 128 predicted Pardosa pseudoannulata cytochrome P450s are divided into 4 clades,namely CYP2,CYP3,CYP4,and mitochondrial clade,and further divided into 21 families and 47 subfamilies.In addition,based on the comparative genomic analysis of 18 species,it was found that the P450 gene family of Pardosa pseudoannulata had an expansion phenomenon,and all the expanded P450 genes were classified into a new family.namely the CYP3310 family of CYP4 clade.KEGG annotations indicated that this gene family might be involved in the fatty acid metabolism or cuticular hydrocarbon synthesis.2.Spatiotemporal expression profile of CYP33 10s in Pardosa pseudoannulataThe expression abundance(FPKM value)in the transcriptomes from different tissues indicated that CYP3310s mainly expressed in spider’s appendages.Furthermore,the mRNA levels in different body parts by qPCR revealed that CYP3310s specifically expressed in the cuticle of spiders.The expression levels in different sexes and developmental stages illustrated that CYP3310s fluctuated significantly with the growth and development of spiders,but there is no uniform pattern between different genes.For example,CYP3310A1 shows a higher expression level during female spiders’ negative development and CYP3310B1 after male spider mating.The spatiotemporal expression profile of CYP3310s was consistent with the functional annotation by KEGG,however further study was necessary.3.The effects of CYP3310B1 on the arachidonic acid metabolismArachidonic acid is one of the most important fatty acid in organisms.The crude enzymes from showed metabolic activity against arachidonic acid.Additionally,this activity was inhibited by PBO,a specific inhibiter of P450s.These results indicated that arachidonic acid was directly metabolized by P450s.In order to detect whether CYP3310s were involved in the arachidonic acid metabolism,CYP3310B1 was selected as the target,which showed relatively high expression level among CYP3310s.Compared to the control,the mRNA level of CYP3310B1 was 66.67%decreased by RNAi.Although the crude enzymes from both RNAi-treated spiders and control had metabolic activity against arachidonic acid,HPLC-MS/MS analysis revealed a number of different metabolic products.In further study,identification of these products would give more information about the function of CYP3310s.