| TORC1,as a conserved serine/threonine kinase,is the regulatory center of cell growth and metabolism.Rag GTPases are members of the Ras-associated GTPases subfamily and are localized in lysosomes.Rag GTPases in mammals are heterodimers composed of RagA/B and RagC/D that regulate TORC1 activity.When RagA combined with GTP and RagC combined with GDP,the Rag complex was active and promoted TORC1 activity.However,when RagA combined with GDP and RagC combined with GTP,Rag complex was in a deactivated state and could not promote TORC1 activity.Drosophila Rag GTPases consist of only RagA and RagC:RagA is the RagA/RagB homolog in mammals,and RagC is the RagC/D homolog.In this study,Drosophila was selected as the research object,and relevant RagA transgenic Drosophila was constructed by gene editing technology to explore the function and mechanism of RagA in the growth and development of Drosophila.The main results are as follows:(1)RagA-knockdown Drosophila could not develop into adult worms due to the dysfunction in pupal stage.Based on the degeneracy of codon,pUASp-RagA-wt,an overexpression vector that changed RagA RNA interference target sites,was constructed by fusion PCR and homologous recombinant cloning.The UAS-RagA-wt transgenic Drosophila was screened by microinjection and genetic hybridization.Overexpression of wild-type RagA in the RagA knockdown background by genetic hybridization could completely rescue the lethal effect caused by RagA knockdown.The cause of death caused by RagA knockdown was determined to be RagA protein deficiency.(2)To explore whether RagA regulation of Drosophila development is related to the activity of Rag GTPases,On the basis of pUASp-RagA-wt,RagA overexpression vector pUASp-RagA-Q61L binding to GTP and RagA overexpression vector pUASp-RagA-T16N binding to GDP were constructed by point mutation.Transgenic Drosophila UAS-RagA-Q61L and UAS-RagA-T16N were constructed by microinjection.The effects of RagA in different active states on TORC1 activity were detected by somatic cell cloning.It was found that RagA-Q61L combined with GTP promoted TORC1 activity.RagA-T16N combined with GDP inhibited TORC1 activity.(3)Previous studies have found that the knockdown of Seh1,an upstream regulator of TORC1,will reduce the activity of TORC1 and lead to abnormal ovarian development in Drosophila.Overexpression of wild-type and GTP-binding RagA could rescue the abnormal ovarian development caused by Seh1 knockdown,but overexpression of GDP-binding RagA did not.These results suggest that Rag GTPase activity may play an important role in Seh1 regulation of oocyte development.(4)The effect of RagA activity controlled by GTP/GDP status on the growth and development of Drosophila was determined by crossing the different RagA activity transgenic lines with Tub-Gal4/TM6 Drosophila and counting the emergence rate of offspring.The results showed that overexpression of RagA-Q61L combined with GTP could partially rescue the lethal effect caused by RagA knockdown.Overexpression of RagA-T16N combined with GDP could not rescue the lethal effect caused by RagA knockdown.These results indicated that RagA played an important role in the growth and development of Drosophila,and the combination of RagA with GTP/GDP is needed to maintain dynamic balance.The combination of RagA and GTP or GDP alone would cause the TORC1 activity to be too high or too low,affecting the growth and development of Drosophila.(5)In order to further explore the effects of RagA on the growth and development of Drosophila,RagA knockout Drosophila was constructed using CRISPR-Cas9 technology.Through the statistics of reproductive ability and survival rate of RagA knockout Drosophila at various stages,RagA knockout caused embryo death of Drosophila.Compared with RagA knockdown,knockout leads to earlier death and has a greater impact.When RagA was whole-body knockowned,Drosophila was able to develop to the pupal stage,whereas the knockout resulted in embryonic lethality.To further explore function of RagA at the cellular level,The RagA mutant chimeric Drosophila HS-FLP;Ubi-RFP,FRT82B/RagA--FRT82B was constructed by FLP/FRT system,explores the RagA knockout the effect on regulating TORC1 activity and somatic growth and the results showed that RagA knockout reduced TORC1 activity and slowed down cell growth.In summary,the thesis proved that RagA plays an important role in Drosophila reproduction,growth and development.In addition,the binding of RagA with GTP/GDP needs to maintain a dynamic equilibrium.If RagA is combined with GTP or GDP alone,TORC1 activity will be too high or too low,which will affect the growth and development of Drosophila. |
