Genome-wide Identification Of Glutathione S-transferase And The Role Of ZcGSTe1 Regulating The Oxidative Level During Vitellogenesis In Zeugodacus Cucurbitae

Glutathione S-transferases（GSTs）are classical detoxifying metabolic enzymes.In addition to detoxification,GSTs are also involved in antioxidant enzyme activities in various physiological processes.The melon fly,Zeugodacus cucurbitae（Coquillett）,is an important quarantine pest in the family Diptera Tephritidae,with a high reproductive capacity.Adult females lay eggs in fruits and vegetables,and the eggs hatch into larvae that feed on the crop,causing serious economic losses.Therefore,concerning this biological characteristic of egg-laying by adult females,we focused on the Zeugodacus cucurbitae female adult and identified the GSTs at the genome-wide level,analyzed the spatiotemporal expression of these GSTs.Finally,we identified one GST highly expressed in the ovary,ZcGSTe1.RT-q PCR,fluorescence in situ hybridization tissue localization,CRISPR/Cas9 gene editing,and heterologous expression were used to explore its role in regulating the ovarian development process,we finally found that this gene is involved in the dynamic balance of reactive oxygen species（ROS）in ovary.This dissertation aims to investigate the role of ZcGSTe1 in regulating female fertility in Z.cucurbitae,which will enrich the functional understanding of insect GSTs,and provide new ideas to study insect reproductive physiology.The main research content is as follows:1.Identification and spatiotemporal profiles analysis of GSTs in Z.cucurbitaeBased on Z.cucurbitae genome data,a total of 34 GST genes were identified,including 32 cytosolic GSTs and 2 microsomal GSTs.Cytosolic GSTs were classified into6 families,including 11 delta,13 epsilon,three theta,one sigma,two zeta,and two omega.GSTs of each family were clustered with Drosophila melanogaster GSTs family.GSTs in melon fly encode proteins with molecular weights ranging from 20.15 to 29.06 k Da and isoelectric points ranging from 4.31 to 10.52.The spatiotemporal expression analysis based on transcriptome data showed that most of the GSTs were dynamically expressed at different developmental stages,and several were expressed at a specific stage.The expression profile among adult tissues showed they were highly expressed in anti-stress-related tissues（midgut,Malpighian tubule,fat body）.One of the epsilon family genes,ZcGSTe1,was specifically highly expressed in ovarian tissues,and its expression gradually increased with sexual maturation.This expression profile is aligned with the vitellogenic stages during the sexual maturation of Z.cucurbitae females,suggesting an involvement in ovarian development.2.Cloning,prokaryotic expression and in situ hybridization analysis of the ZcGSTe1 of Z.cucurbitaeThe full-length sequence of ZcGSTe1 was successfully amplified,with an open reading frame length of 666 bp,encoding 221 aa and a molecular protein weight of 25.18k Da.Multiple sequence alignment and structural analysis revealed that ZcGSTe1 is highly conserved and has a similar protein structure model to the housefly GST6B.To verify the spatiotemporal expression profile of ZcGSTe1 in Z.cucurbitae,RT-q PCR was used to analyze its expression in various developmental stages and adult tissues of melon fly,and the results were consistent with the quantitative transcriptome expression.Meanwhile,the expression profile of this gene during the sexual maturation of Z.cucurbitae females showed an increasing expression from 5-to 9-day-old,suggesting that this gene was associated with the sexual maturation of females.The expression of ZcGSTe1 was found to be highest in early embryonic development（1 h after egg laying）,suggesting that this gene is also involved in early embryonic development.To further investigate the function of ZcGSTe1,in vitro expression of recombinant protein using E.coli heterologous expression system was successful and confirmed by SDS-PAGE and Western blotting.Enzymatic analysis revealed that the activity of ZcGSTe1 catalyzing the substrates GSH and CDNB was 113.09±27.02 U/mg prot,and the peroxidase activity was 45.87±4.22U/mg prot.A gene-specific probe was prepared to analyze the expression site of ZcGSTe1in the ovary of Z.cucurbitae,and its fluorescent signal was concentrated in the oocytes.3.Construction of ZcGSTe1 homozygous mutant based on CRISPR/Cas9 system to analyze its roles in Z.cucurbitaeThe sgRNA was designed in the upstream region of the ZcGSTe1 exon,and the off-target effect of the mutation was assessed using Cas OT,which revealed that there isn’t a potential off-target site.After embryo injection,the feathered G₀ mutation site and genotype were confirmed and backcrossed with the wild type for multiple generations.Finally,a ZcGSTe1^-/-homozygous mutant with a 28 bp deletion was obtained.The homozygous mutation was detected by RT-q PCR,and ZcGSTe1 did not express,which demonstrated that the gene was successfully knocked out.Compared with wild-type females,the mating rate of the homozygous mutant was decreased by 20.57%,and the egg production per female was significantly reduced by 62%on the first day after mating,but the egg production in the following days was not affected.No significant changes were observed in the egg-hatching rate.The ovarian area of female mutants was 15.65%smaller than that of the wild type on 6-day-old and returned to normal size on 9-day-old.The expression of ZcVg1,an important gene for ovarian development,was significantly decreased by 22.36%on 6-day-old,and the mutants were enriched with more ROS than wild-type oocytes,suggesting that ZcGSTe1 may participate in the ovarian development process by playing an antioxidant role in regulating ROS level.