Cloning,Expression And Physiological Function Of Glutamine Synthetase Genes From Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)(Diptera: Tephritidae),is a worldwide devastating agricultural pest.There is a severe pesticide resistance in B.dorsalis resulted from the long-term,excessive uses of pesticides.Previous researches mostly focused on the mechanism of insecticide resistance and its improvement methods.In recent years,more efforts have been done to investigate the novel insecticide targets in B.dorsalis.Glutamine synthetase(GS)functioning in most cells catalyzes glutamate to glutamine,which plays a key role in many biological processes,such as cell growth,energy metabolism,protein and nucleotide synthesis,and immune responses.In this study,we cloned two BdGSs and characterized their functions aiming to illustrate their physiological functions in regulation of larval development and adult fecundity to provide new candidate genes controlling the orange fruit fly which is based on RNAi.The main results are as follows: 1 Analysis of BdGSs sequences and phylogenyDepending on the polymerase chain reaction,mitochondrial(BdGS-m)and cytosolic(BdGS-c)isoforms from B.dorsalis were cloned in this study.Based on amino acid sequence,homology analysis showed that BdGS-m shared an 82% amino acid identity with LcGS-m and 80% with DmGS-m.BdGS-c shared a 90% amino acid identity with LcGS-c and DmGS-c.Multiple alignments showed that there were 5 conserved regions in the GSs protein.Based on the deduced amino acid sequences of BdGSs,GS enzymes were predicted to be hydrophilic proteins without transmerabrane domain and signal peptide.2 Spatio-temporal expression patterns of BdGSsThe expression patterns of BdGSs in different developmental stages were analyzed using RT-qPCR,which showed that BdGS-m was highly expressed in egg and adult,while BdGS-c in larva and adult.Both two genes were relatively highly expressed in adult,suggesting a regulation of development and fecundity in B.dorsalis adults.In larval stage,the expression level of BdGS-c was higher than BdGS-m,and even increased markedly with the development of larvae,suggesting a predominant role of BdGS-c in larval development,while BdGS-m potentially modulated the early development of the embryo.The expression patterns in different larval tissues revealed that BdGS-m was highly expressed in the Malpighian tubules(MTs),while BdGS-c was stable among the tested tissues.BdGSs expressed in MTs could likely participate in fluid secretion,while BdGSs expressed in fat body and midgut were likely to function in removing excessive nitrogen and toxic compounds from B.dorsalis.The expression patterns in different adult tissues revealed that BdGS-m highly expressed in fat body possibly regulated the ammonia metabolism and fecundity by modulating the synthesis of vitellogenin protein,while BdGSs expressed in head potentially participated in regulating the repair and regeneration of nervous system and immune response.3 Response to environmental stresses of BdGSsThe expression levels of BdGS-m and BdGS-c increased markedly when larvae were under the high temperature stress,UV-B stress and pesticide stress.The high temperature,UV-B stress and pesticide can lead to the accumulation of reactive oxygen species(ROS).Hence,we predicted that BdGSs played a key role in the anti-oxidant effect in oriental fruit fly.It is noteworthy that,in our study,the mRNA levels of BdGSs showed no significant changes under the low temperature stress,which might result from the slower rates of ammonia metabolism and ROS accumulation.4 Functional analysis of BdGSs involved in larval developmentThe assay of 2-day-old larvae feeding on artificial diet mixed with inhibitor for 5 days showed a smaller size,lower pupation rate,lighter pupal weight,delay in pupation,lower emergence rate,as well as the decrease of GS enzyme activity.These phenotypic changes might result from the excessive ammonia in hemolymph which failed to be removed after larvae feeding with specific inhibitor.B.dorsalis larvae were injected with exogenous dsRNA targeting BdGS-c,which showed that larvae interfered by RNAi displayed a longer developmental duration and lighter pupal weight.Interfering BdGS-c by RNAi potentially led to the defect in the ammonia metabolism and the block of the metabolite secretion in MTs.The expression level of BdSpook,a key Halloween gene,markedly decreased at 24 h after injection of dsBdGS-c,indicating a regulation of the ecdysone synthesis.The influence of GS on the larval development,pupation and emergence would give grounds for the search of new targets controlling the pest of B.dorsalis.5 Functional analysis of BdGSs involved in adult fecundityInhibition of BdGSs expression in B.dorsalis female adults by RNAi led to a decrease of genes expression in mRNA level.We found that silencing BdGSs affetced the ovarian development and the oviposition number,which suggested that GS played essential role in fecundity of B.dorsalis female.