The Molecular Mechanism Of PSMB3 Regulating Female Reproductive Development In Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis(Hendel),one of the important quarantine pests,is highly invasive and has caused a great lost to the horticultural crops in China.Due to global warming and changes in farming system,the areas infested by B.dorsalis become wider,thus makes a greater challenge to control it.The newly emerged female B.dorsalis adults need to take in nutrients for the ovaries matured,only then can they mate and lay eggs.So the regulation of female adult reproduction may be an important entry point for pest control.By functional analysis on key genes of female reproduction of B.dorsalis,we can reveal the molecular mechanism on reproductive regulation which may provide new methods for pest control.Our previous study indicated that proteasome β3 subunit(PSMB3)plays an important role in female reproductive development.In this study,RNAi was carried out that knockdown the PSMB3 of female adults would lead to the obstruction of yolk deposition,suppression of ovarian development,and a significant decrease the egg production.However,the molecular mechanism that regulates reproductive development was still uncleared.In this study,we aimed to unravel the molecular mechanism of it,and help to understand female B.dorsalis reproductive development better.Here,we use RNAi,RNA-seq,q RT-PCR to identify the differential expression genes and ecdysone rescue experiments were carried out to show how PSMB3 regulates female reproduction in B.dorsalis.Here are the main results:1.B.dorsalis PSMB3 regulates the deposition of yolk protein and ovarian development by controling the ecdysone biosynthesis.First,RNAi was used to knockdown PSMB3 in female B.dorsalis on 4d post eclosion,and we found that ovary development was significantly inhibited on 9d after injection.Then RNA-seq was performed on RNA samples from ovatian and fat body of female B.dorsalis that PSMB3 was successfully knockdown.Results from KEGG pathway enrichment analysis of the differentially expressed genes showed that the pathways such as insect hormone biosynthesis,fat digestion and absorption and ovarian steroidogenesis were significantly enriched in ovary.The results of cluster analysis of the differentially expressed genes showed that the expression of genes in ecdysone synthesis pathway genes like Cholesterol 7-desaturase(Neverland),CYP307A1(Spook),CYP302A1(Disembodied),CYP315A1(Shadow)in ovary were significantly reduced.In addition,the expression of vitellogenin genes Vg1 and Vg2 in fat body were also reduced.The q RT-PCR verificantion results are consistent with the RNA-seq results.In addition,q RT-PCR detection found that the expression of Ec R,USP,E74,E75,HR3 genes that related to ecdysone signal pathway were significantly reduced.Besides,we found that after PSMB3 RNAi,females appear a red abdomen appearance under brighfield light,indicated the expression of the cofactor of CYP enzyme family may be suppressed.Further q RT-PCR detection revealed that the expression of Updo,Coprox and Ppox that related to heme synthesis were reduced.Meanwhile,ecdysone titer in hemolymph detected by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)was significantly decreased in PSMB3-knockdown female of B.dorsalis.The above results indicate that PSMB3 was requlired for ovarian development,by influence ecdysone synthesis,regulating ecdysone signal transduction and vitellogenin synthesis.2.The deposition of yolk protein and the ovarian development of female B.dorsalis can be rescued by ecdysone.The ecdysone rescue experiment to the RNAi-females with a knockdown of PSMB3 was performed.After ecdysone injection,the relative expression of Vg1 and Vg2 in fat body were increased to normal levels as in the Egfp group.In fertility,ds PSMB3 injection females,the length of ovaries was signficantly increased,the probability of poor egg yolk deposition was significantly reduce and the total number of eggs laid and the hatching rate were slightly increase without significant difference.These results shows that the injection of ecdysone into the RNAi-females with knockdown of PSMB3 could rescue the yolk deposition and ovarian development,partially rescue the total number of eggs laid and the hatching rate.In summary,PSMB3 plays an important role in the reproduction of the female B.dorsalis,via the regulation of ecdysone synthesis in the ovary,thereby regulating the ecdysone signal pathway and vitellogenin in fat body,ultimately affects the female reproductive development.This study not only clarified the molecular machanism of proteasome in regulation of females B.dorsalis,furthering our understanding of the post-translational regulatory mechanism in insect reproductive development,but also providing new ideas for green prevention of B.dorsalis.