Functional Analysis Of GSTe1 And GSTe10 Involved In Ovarian Development In Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)(Diptera: Tephritidae),is an important worldwide quarantine pest.It harms for the edible and economic value of the fruit with strong reproduction ability.Due to the long-term overuse of pesticides,the fruit fly has a severe resistance to multiple pesticides.The ovary is an important tissue of female reproductive system and it has a direct relationship with the reproduction.The related fields have been hot research topics in insect development and an important way to explore new methods for pest control.Glutathione S-transferases(GSTs)is an important supergene family.At present,insects GST functional research mainly focused on detoxification and pesticide resistance.Some other physiological functions are relatively limited.In this thesis,according to the transcriptomic and genomic databases of B.dorsalis,we reported a genome-wide identification of the GST genes in this insect pest.Two GSTs(BdGSTe1 and BdGSTe10)that are highly expressed in the ovary were identified using tissue expression profiles.Furthermore,their open reading frames were cloned by PCR.The sequence analysis of BdGSTe1 and BdGSTe10 were reported.Moreover,the recombinant proteins of Bd GSTe1 and BdGSTe10 were expressed by constructing pET-28a(+)prokaryotic expression vector.Besides,the temporal and spatial distributions of BdGSTe1 and BdGSTe10 transcripts were investigated by RT-qPCR.Finally,we revealed the physiological function of BdGSTe1 and BdGSTe10 in the development of the ovary of Bactrocera dorsalis by RNAi technology.The results of this study will enrich the physiological functions and functional polymorphisms of GSTs to provide new candidate genes to control the oriental fruit fly based on RNAi.The main results are as follows.1 Identification of BdGSTe1 and BdGSTe10Based on the genome and transcriptomes of developmental stages and tissues of B.dorsalis,we identified 34 GSTs including 3 microsomal GSTs and 31 cytosolic GSTs which 9 GSTs were newly discovered.Based on the expression profiles of BdGSTs in the different tissues,we identified BdGSTe1 and BdGSTe10 which are highly expressed,especially BdGSTe1 with high tissue specificity.2 Molecular cloning and heterologous expression of BdGSTe1 and BdGSTe10BdGSTe10 were cloned from B.dorsalis By RT-PCR.BdGSTe10 was 669 bp in length and encode 222 amino acids.Multiple alignments showed that there were 5 Gsides(GSH binding sites)and 9 H-sides(hydrophobic substance binding sites)in BdGSTe1 and BdGSTe10 amino acid sequences.The conserved Ser12 suggested that these two genes are active GSTs.BdGSTe1 selected BamHI and EcoRI as restriction sites,BdGSTe10 selected BamHI and SalI as restriction sites,used DNA recombination technology to constructed BdGSTe1-pET-28a(+)and BdGSTe10-pET-28a(+)vector.After induction by IPTG,BdGSTe1 and Bd GSTe10 recombinant proteins were expressed.SDS-PAG analysis showed only one specific protein band at 25 kDa.The purified BdGSTe1 and BdGSTe10 recombinant proteins had GST activity.3 Spatio-temporal expression patterns and localization in ovary of BdGSTe1 and BdGSTe10The expression patterns of BdGSTe1 and BdGSTe10 in different developmental stages,maturation of virgin female adults and postnatal embryos were analyzed using RT-qPCR.BdGSTe1 and BdGSTe10 were highly expressed in eggs and female adults.The increase during the middle to late stages of sexual maturity in the ovary suggested that they may be involved in the ovarian development of female adults of B.dorsalis.These two genes were highly expressed in the early embryo,suggested that they play an important role in embryonic development.4 BdGSTe1 and BdGSTe10 participate in ovarian development of B.dorsalisBdGSTe1 and BdGSTe10 were silenced by 41% and 65% respectively using RNAi by feeding dsRNA.We found that silencing BdGSTe1 and BdGSTe10 affected the ovarian development and the oviposition number,and the expression of two vitellogenin genes,BdVg1 and BdVg3 were significantly reduced which suggested that BdGSTe1 and BdGSTe10 participate in ovarian development of B.dorsalis.The level of reactive oxygen species(ROS)in the ovary of B.dorsalis was increased after silencing BdGSTe1 and BdGSTe10.BdGSTe1 and BdGSTe10 may have antioxidant effects and participate in maintaining ovarian ROS homeostasis.