Genes And Micrornas Related To The Spermatogenesis Of Bactrocera Dorsalis

Bactrocera dorsalis is one of most damaging horticultural insect pests and attacks more than 250 types of fruits and vegetables.Sterile insect technique(SIT)is a genetic pest control method which controls the insect pest population by decreasing the reproductive capacity via delivering mass sterile male into field,and is being considered effective against insect pests e.g.tsetse fly and screw worm.Research achievements of modern molecular techniques such as transgenesis,RNAi and CRISPR Cas9 etc,Recently,genetically enhanced sterile insect technique is being become reality.Screening and functional characterization of genes and mi RNAs related to spermatogenesis will offer targets to construct SIT based on genetic modification and can help to enhance the sterile insect technique.Therefore,genes and mi RNAs related to spermatogenesis are also considered promising candidates to develop genetic based sterile insect technique.In the present study,we have identified and screened the genes and mi RNAs related to spermatogenesis in B.dorsalis,we have found that target genes TF gaga,orb2,tektin1 and tssk1 can effectively decrease male fertility,and revealed that mi RNA-125-3p and mi RNA-276b-3p regulate the spermatogenesis by targeting the orb2 gene.This research results offer target genes and mi RNAs to construct genetic based sterile insect technique without radiation,and helps in developing new ideas for insect pest control.The major results are as follow:In the first part of this study eight testicular genes TF gaga,parkin,orb2,netrin-A,netrin-B,tektin1,Ddx1 and tssk1 were selected on the basis of previous studies.q RT-PCR analysis represented that TF gaga,orb2,tssk1 and tektin1 were differentially expressed in the testis than the other body tissues.On the basis of their highest expression,these four genes(TF gaga,orb2,tssk1 and tektin1)were selected for functional characterization.Their sequence alignment and phylogenetic analysis showed that these genes are conserved genes among different agricultural pests.ORF was selected from their predicted sequence in order to prepare ds RNA,cloned from the testis of males of B.dorsalis.RNAi analysis through feeding the ds RNA to the males of B.dorsalis silenced the expressions of all target genes.Due to effects of silencing on the reproductive capacity of B.dorsalis males TF gaga,orb2,tssk1 and tektin1 exhibited male sterility 64.92%,71.19%,64.49% and 58.99% respectively.In the second part of this study functions of mi RNAs in the reproduction of males of B.dorsalis were investigated.Orb2 gene was selected on the basis of highest impact on male fertility from our functional studies.All mi RNAs targeting the 3′UTR of orb2 were predicted by using bio information tool(RNA hybrid).Six mi RNAs let-7-5p,mi R-9c-3p,mi R-125-3p,mi R-210-5p,mi R-276b-3p and mi R-3931-3p showed complementarity to the 3′UTR of orb2.Further Dual Luciferase Assay(DLR)in HEK293 T cells confirmed that two mi RNAs,mi R-125-3p and mi R-276b-3p target the 3′UTR of orb2 gene.q RTPCR analysis showed that mi R-125-3p and mi R-276b-3p had opposite expression to orb2.Expression of mi R-125-3p and mi R-276b-3p was high in other tissues than testis.Their opposite expressions to the orb2 suggested that these mi RNAs might have any relation with orb2.q RT-PCR analysis after administration of agomi R-125-3p and agomi R-276b-3p to the males of B.dorsalis through feeding exhibited that level of mi R-125-3p and mi R-276b-3p was significantly increased and the expression of orb2 m RNA was significantly reduced.After feeding the agomi R-125-3p and agomi R-276b-3p,reproductive capacity of males was decreased by 67% and 69% respectively,but after feeding the combination of agomi R-125-3p and agomi R-276b-3p sterility reached up to 83%.Functional analysis of genes related to spermatogenesis shows that TF gaga,orb2,tssk1 and tektin1 effect the spermatogenesis of male flies of B.dorsalis and those are necessary for male fertility.Furthermore,it is revealed that mi RNA-125-3p and mi RNA-276b-3p regulate the spermatogenesis by targeting the orb2.Orb2 and its mi RNA are most promising candidates for development of genetic based sterile insect technique(SIT).