Cloning And Functional Analysis Of The Molting Gene CYP302A1 In Daphnia Sinensis

Molting is one of the important physiological processes in cladocera,and its growth and molting alternate.The molting of cladocera is mainly regulated by juvenile hormone and ecdysone together.The CYP302 Al gene is one of the key genes for ecdysone synthesis in cladocera,but its specific function is still unclear.At present,some investigations have been reported on the genes of the ecdysone synthesis pathway of cladoceran,but the molecular mechanisms of the ecdysone synthesis pathway and the ecdysone signal transduction pathway are still insufficient.The study on molecular biology of Daphnia species has become a hot spot after the genome of Daphnia pulex and Daphnia magna were successively reported.In this study,D.sinensis was used as the research object.Based on the transcriptome,q PCR and RNA interference technologies,the sequence characteristics of CYP302A1 gene,and the changes on expression levels of downstream response genes and individual phenotypic characteristics after knock-down of CYP302A1 gene were analyzed.Moreover,the expression sites of CYP302A1 gene were determined by whole mount in situ hybridization technique.The functional study of the CYP302A1 gene will help to clarify the ecdysone synthesis pathway of Daphnia(cladocera),and provide a reference for the future study of ecdysis-related signaling pathways.The main results are as follows:1.The CYP302A1 gene of D.sinensis belongs to the cytochrome P450 family,with a total length of 5962 bp.Its open reading frame(ORF)is 1596 bp in length,which there are 5 characteristic conserved domains in the 531 amino acids encoded,namely helix-C(WHRLR)conserved domain,helix-I(AGIDT)conserved domain,helix-K(ESFRL)conservative domain,PERF(QFVPERW)conservative domain,and heme binding(PFGHGPRACIA)conserved domain.Through the phylogenetic tree constructed,D.sinensis was the most closely related to other Daphnia species,followed by Tetranychus cinnabarinus(Acari).Analysis on the expression levels of Halloween family genes in the ecdysone synthesis pathway showed that the expression levels of CYP307A1 and CYP306A1 and CYP302A1 and CYP315A1 genes were significantly higher in adult D.sinensis individuals than in larvae,whereas the CYP314A1 gene were the opposite.This is consistent with the results of other arthropod,which proves the high conservatism of cytochrome P450 family.2.Through the feeding experiment of Escherichia coli HT115(carrying two different recombinant plasmids),in the 10% E.coli concentration treatment,there was less toxicity to D.sinensis,and the molting time of offspring at first reproduction was longer than that in the other E.coli concentration treatments.In ds RNA mediated study,the expression level of the CYP302A1 gene dropped significantly(knock-down of 68.34%)in the 5% E.coli concentration treatment.In addition,the expression levels of Ec R,HR3 and USP downstream genes decreased significantly,whereas the expression level of the FTZ-F1 gene increased significantly.On the phenotype,the molting time of D.sinensis prolonged,and even the embryos disintegrated or died in the incubation capsule.It implied that the CYP302A1 gene in D.sinensis played an important role in the ecdysone synthesis pathway,and knock-down of the CYP302A1 gene had a negative impact on the growth,development and reproduction of D.sinensis.3.Whole mount in situ hybridization showed that the CYP302A1 gene of D.sinensis had mainly six expression sites before RNA interference,which were respectively located in the first antennal ganglion,ovary,gastric coeca,olfactory hair,thoracic limb and tail spines.After the RNA interference,the expression signal of the CYP302A1 gene was not detected in the first antennal ganglion of D.sinensis,and the expression signal in the ovary was obviously attenuated,which was consistent with the disintegration or death of D.sinensis embryo in the incubation capsule.During molting activities,the CYP302A1 gene in the thoracic limbs and tail spines of D.sinensis began to be expressed.In conclusion,the CYP302A1 gene is the main gene related to ecdysone synthesis and plays an important role on the molting,growth and reproduction in cladocera.