Construction TOR Activity Monitoring System In Vivo And Identification Potential Downstream Substrates By Forward Genetics Screening

Target of Rapamycin（TOR）is a conserved serine/threonine protein kinase in eukaryotes.As a molecular switch,it could response to signals such as nutrition,hormone and stress to regulate protein translation,cell division and differentiation,metabolism,autophagy,aging,organ development and many other biological processes.It is very important for sessile plants that could help plants quickly respond to internal and external signals to survival,so plant TOR kinase has always been a research hotspot in the field of plants.Because the traditional in vitro or semi-in vitro TOR kinase activity detection methods rely on specific and sensitive phosphorylated antibodies and the inability to monitor their spatiotemporal changes in real time has hindered the study of TOR upstream signaling pathway components and mechanisms and cognition of protein biological function.Therefore,in this study,a monitoring system capable of quickly detecting the activity of TOR kinase in plant was established by genetic engineering technology and cell biology methods and two components of the TOR downstream signaling pathway were screened by forward genetics method.Preliminary work found that the change of NIA1 nuclear localization is related to TOR activity.On this basis,the protoplast system and the plant system were used to prove that TOR kinase activity can be characterized by the nuclear-cytoplasmic shuttle of NR protein.In order to construct a pure TOR kinase detection system,genetic engineering technology was used to modify NIA1 key structures:the Mo²⁺and NO₃^-binding sites and nuclear localization sequence that could affect the NR localization.Using cell biology method to verify these modifications in the protoplast system,it was found that the localization change of NIA1 regulated by TOR does not depend on the NIA1 enzyme function.Then we successfully constructed the NLS-NIA1 or NES-NIA1 as positive control.In particular,NLS-NIA1 causes NIA1 to accumulate in the nucleus so that we directly observed localization of NIA1 and we also observed most of the NES-NIA1 distributed in cytoplasm but little in nuclear.In a word,based on the above modifications,in the first part of study we preliminarily constructed a detection system to characterize TOR kinase activity in vivo with the nuclear-plasmid shuttle event of NIA1 and lay the foundation for the establishment of a stable system.We screened two components of TOR downstream signaling pathway by forward genetics and other methods.Using root hair,root,leaf,stem and other phenotypes as the screening model,we screened and re-confirmed EMS mutant library with the background of XVE-tor and finally obtained two mutants torsr9 and torsr11 that could resist to both est and Rap and Torin2 with long root length.Backcrossing experiments showed that torsr9 and torsr11 were recessive mutation and conform to Mendelian inheritance law.SNPs were sequenced and analyzed by whole genome sequencing and biological information methods.torsr9 finally located two candidate genes GLUTAMYL/GLUTAMINYL-TRNA SYNTHETASE and SK11,torsr11 finally located 7 candidate genes,ATVT-1,AIR9,ZW10,GA2OX3,WRKY33,ROS1,LOS2 and DUF827.Then using molecular detecting and biochemical methods to preliminarily identify candidate genes,two candidate genes in the torsr9 mutant were detected,and only four candidate genes,ATVT-1,AIR9,ROS1,and LOS2,were detected in the torsr11 mutant.T-DNA insertion mutants atvt-1,air9,zw10,ga2ox3response to Rap and Torin2 showed that atvt-1 could resist to Rap and Torin2 and root growth was promoted;air9,zw10,and ga2ox3 cannot could resist to Rap and Torin2,and root growth was inhibited.The two results further indicated that ATVT-1 may be candidate gene of torsr11,and at the same time,the phenotypes of candidate genes AIR9,ZW10 and GA2OX3 may be not for gene inactivation.In addition,the biochemical method was used to explore the response of torsr9 and torsr11 to glucose and nitrogen starvation of TOR upstream signals,torsr9 was not sensitive to glucose and nitrogen starvation,and torsr11 is only insensitive to nitrogen starvation.In short,in the second part of study,we identified two new components of TOR downstream signaling pathway,and they may be involved in nutrients（C,N）-TOR pathway and regulate root,shoot growth and other related phenotypes,which will undoubtedly promote our new research and understanding of TOR kinase.