Identification Of Myrosinase-like Genes And Functional Study Of A Larval Midgut-specific Candidate In Plutella Xylostella

Plant myrosinases(β-thioglucosidases)are members of the glucosidase hydrolase 1(GH1)family.Together with glucosinolates(GS),they constitute a chemical defense system of plants against herbivorous insects.Certain cruciferous vegetable pests have self-encoded myrosinase genes mimicking a myrosinase-GS defense system similar to that of the plants.Plutella xylostella is an important pest on cruciferous vegetables and has a special pathway for metabolically detoxifying GS.However,it is still unclear whether P.xylostella also has the ability to utilize the chemical defense system of the host plant.Based on the genomic information of P.xylostella,this study searched for and characterized the homologous sequences of plant myrosinase genes in this insect.The candidate genes that were preferentially expressed in the midgut of larvae and may be regulated by plant-derived substances were identified to conduct both in vitro and in vivo enzyme activity assays.The aim of this study was to reveal the function and role of homologous sequences of myrosinase genes in the adaptation of P.xylostella to host plants.1.Characterization and expression profiling of the homologous sequences of myrosinase genes in P.xylostellaBased on the genomic information of P.xylostella,13 homologous sequences annotated as myrosinase genes were identified.All of them have conserved domains of the GH1 family.A phylogenetic tree was used to carry out evolutionary analysis.It was found that the myrosinase genes of P.xylostella diverged from each other and are far from the counterparts of Arabidopsis thaliana,indicating there is no horizontal gene transfer between them.The gene expression patterns of the 13 myrosinase genes at different developmental stages of P.xylostella were investigated by qRT-PCR.The results showed that two myrosinase genes were highly and specifically expressed in larvae,five specifically in female adults,one specifically in male adults and one specifically in pupae with a relatively higher expression in female pupae than in male pupae.However,the expression patterns of the remaining four myrosinase genes were irregular,and the expression levels of the 13 myrosinase genes in eggs were very low.In addition,the gene expression patterns of the 13 myrosinase genes in different tissues of larvae were investigated by qRT-PCR.The results showed that five myrosinase genes were specifically expressed in the midgut,2 specifically in the fat body,and 2 specifically in the Malpighian tubules,while the expression patterns of the remaining four myrosinase genes were irregular.2.Radish cotyledon and glucosinolate feeding assays and myrosinase expressionTo investigate whether the homologous sequences of myrosinase genes in P.xylostella were affected by plant gradients,radish cotyledons were used to feed the larvae of the artificial diet(AD)strain with short-and long-term treatment.The results of qRT-PCR showed that the expressions of Px008848,Px009428 and Px006054 genes were significantly induced in the midgut tissues after a food-source shift,and the expression level of the Px008848 gene increased gradually with the increase in feeding time.The Western blot results showed that Px008848 also showed significant upregulation at the protein level.Compared with the artificial diet,the individuals of the AD strain fed radish cotyledons had a longer larval duration and a lower pupal weight.Based on the glucosinolate(GS)content in crucifers,artificial diets with different concentrations of GS were used to feed the larvae of the AD strain.and expression patterns of myrosinase genes were detected by qRT-PCR.The results showed that the expression of myrosinase genes in the AD strain were affected by both the concentration of GS and feeding time.The protein expression level of Px008848 was significantly higher in the group fed a diet with a high concentration of GS than in the control group3.Myrosinase activity verification in P.xylostellaTotal protein was extracted from samples at different developmental stages or from different tissues of larvae in the FZ strain to react with substrates of GS.The myrosinase activity,namely,the ability to hydrolyze GS to produce volatile isothiocyanate(AITC),was detected by gas chromatography-tandem mass spectrometry(GC-MS).The results showed that the homologous sequences of myrosinase genes in P.xylostella do not encode myrosinase,or the produced myrosinase activity is too low to detect.4.The prokaryotic expression and enzymatic activity of Px008848The result of prokaryotic expression of the Px008848 showed that the major form of protein was inclusion bodies.The renatured Px008848 protein did not have myrosinase activity as detected by GC-MS.However,the β-glucosidase(β-GC)activity assay of the Px008848 protein showed a positive result.Considering thatβ-GC plays an important role in hydrolyzing cellulose in plants and detoxification metabolism,it was speculated that the homologous saquences of myrosinase genes in P.xylostella were involved in the hydrolysis of plant cell wall and detoxification metabolism.In this study,the homologous sequences of myrosinase genes in P.xylostella were systematically identified,and the expression and activity assays for genes/proteins were performed.It was found that although there are homologous myrosinase sequences in different insects,not all of them have myrosinase activity.However,the present study also found that the homologous sequences of myrosinase genes in P.xylostella were affected by plant-derived materials and GS,which laid the foundation for the further study of their functions in insects.