The Functional Analysis Of TOR From Arabidopsis Thaliana And Thellungiella Halophila

Target of rapamycin (TOR) was first found in yeast. The proteins are large, highly conserved protein kinases encoded in almost every eukaryotic genome. All TOR proteins identified, from the basal eukaryote Giardia lamblia to yeast and metazoans, share the same set of structural motifs. Rapamycin acts by forming an inhibitory complex with its intracellular receptor, the FK506-binding protein, FKBP12, which binds a region in the C terminus of TOR proteins termed FRB (FKBP12-rapamycin binding), thereby inhibiting TOR activity. The immunosuppressant and antibiotic rapamycin potently inhibits growth in several evolutionarily diverse cells, suggesting that TOR has a conserved role in controlling cell growth. Within the last few years, yeast and mammalian TOR has been demonstrated to control several additional readouts, all of which are related to cell growth. These readouts include organization of the actin cytoskeleton, membrane traffic and protein degradation, PKC signaling, ribosome biogenesis, and transcription. Genetic studies in yeast demonstrated that rapamycin-sensitive TOR signaling regulates when a cell grows, and rapamycin-insensitive TOR signaling regulates where a cell grows. Two distinct TOR protein complexes, TORC1 and TORC2, account for the differential sensitivity of TOR signaling to rapamycin. The mTOR signaling network consists of two major branches, each mediated by a specific major branches, each mediated by a specific mTOR complex (mTORC). Rapamycin-sensitive mTORCl controls several pathways that collectively determine the mass of the cell. Rapamycin-insensitive mTORC2 controls the actin cytoskeleton and thereby determines the shape of the cell. mTORCl and possibly mTORC2 respond to growth factors, energy status of the cell, nutrients, and stress.Plant growth, far more than metazoan growth, is intricately linked to nutrient sensing. In plants, nutrients and stress affect not only the growth rate but also the number of organs produced and the timing of developmental transitions. Because of this, the role of TOR in plant growth become more and more important. The Arabidopsis TOR gene spans 17555 bp of genomic DNA and 8007 bp of cDNA. The comparison of the genomic and the cDNA sequences revealed that AtTOR contain 56 exons and 55 introns. The AtTOR protein sequence contains 2481 amino acid residues with a predicted molecular mass of 279 kDa. Some research showed that disruption of AtTOR leads to the premature arrest of endosperm and embryo development. As described above, AtTOR plays a role in plant growth as important as possible. Cell growth is intimately connected with ribosome biogenesis and protein translation. AtTOR may control this pathway.Arabidopsis is not sensitive to rapamycin. In yeast three-hybrid assays and in vitro, AtTOR and rapamycin form a complex with human FKBP12 but not with AtFKBP12. Zea mays is sensitive to rapamycin, which may be as the result of the structure of FKBP12.There is only one TOR complex in Arabidopsis. AtRaptorlB interact with the N-terminal HEAT repeats of AtTOR. This interaction is consistent with TOR-Raptor interactions in other systems, and is the only direct biochemical evidence to date for a plant TORC1 complex. The homolog of Rictor in plant was not found.Salt cress is very tolerant to cold, drought, salt and low nitrogen. It has emerged as a new model system for abiotic stress study.In this research, many methods of molecular biology were used to analysis AtTOR. The ThTOR gene was cloned.The important methods and results were shown as follows:1)The cDNA sequence of AtTOR was analyzed and two specific sequences which located in HEAT domain area of AtTOR 5'-end and the 3'-end kinase were chosen. AtTOR-RNAi plant vector were constructed using these two fragments and transformed into Arabidopsis thaliana. The transgenic plants were identified using molecular methods and the lines where the expression of AtTOR were down-regulated were chosen for the phenotypic analysis. The results showed that the transgenic plants grew slower than the wild type. The 5'RNAi transgenic plants have other characters, such as seed abortion, stem profusion and so on. The callus growth of transgenic lines is also slower than wild type.2) We analyzed AtTOR promoter sequence, and used the PCR method to got the full sequence of AtTOR promoter. PlantCARE database was used to predict the motifs in promoter sequence. The AtTOR promoter contains a TATA-box, a GAAT-box, seven ABA response elements ABREs, a drought responsive element DRE, a thermal response element HSE, and many plant development related elements. We constructed AtTOR promoter::GUS vector with different lengths of promoter, and transformes the vectors into plants, respectively. The result of GUS staining showed that AtTOR gene specific expressed in embryonic axes and leaf primordial, and the location and staining intensity is changed with the different length of promoter. The longest promoter-GUS did not express, and the shortest promoter-GUS had a high expression. From this analysis, it is predicted that there are many new motifs in the promoter sequence.3) The subcellular localization of AtTOR protein was predicted using the Software TargetP 1.1. The result showed that the AtTOR protein is located in chloroplast. Two transmembrane domains of AtTOR protein were predicted by DNAMAN software. The signal peptides always located in N-terminus or C-terminus of AtTOR protein, so the AtTOR 5' end,3' end, and two transmembrane regions were selected to construct the AtTOR-GFP vector, respectively. Analyzing transgenic lines by confocal, the result shows that there is no specific localization signal in roots of transgenic lines, while in leaf AtTOR would be located in chloroplast. However, the result did not show the full-length protein localization, so localization signal is not accurate. The correct result should be gained using other methods, such as immunohistochemistry and the antibody-specific binding to determine the true subcellular localization of AtTOR.4) The expression of AtTOR will change after different stress treatments. The method is following as below:150 mM NaCl,29.1 mM PEG6000,300 mM mannitol,75μM ABA,37℃, or 4℃treated for 0h,3h,6h,12h,24h,48h; 20μM Pb(Ac)2 or 20μM CuCl2 treated for 12h, 24h,48h; 0 mM,50 mM,100 mM,150 mM or 200 mM of NaCl treated for 24h. In this experiment, untreated wild-type Arabidopsis was used as control The expression of AtTOR is very low that AtTOR signal can not be detected using Northern hybridization, so we used Real-time PCR to analyze the expression of AtTOR. The result showed that osmotic stress, cold, heat, ABA could significantly raise the expression of AtTOR, while NaCl and heavy metals reduced the expression of AtTOR.The expression of AtTOR has quite a difference in different organs. Compared with rosette leaf, stem has the lowest expression; the highest expression is in the stem leaf, following is in flowers and siliques, the expression in roots and seedlings was also higher than in the rosette leaves.5) Using RT-PCR,3'-RACE and 5'-RACE methods to clone the full sequence of cDNA from thellungiella halophila, the full length cDNA of ThTOR is 7836 bp, the sequence of open reading frame is 7437 bp,5'UTR is 196 bp, and 3'UTR is 203 bp. ThTOR encodes 2479 amino acids and its molecular mass is about 279 kDa. Compared with AtTOR, the similarity between the cDNA sequences of ThTOR and AtTOR is about 95%. The homology of protein sequencs among the species is as high as 97.5%. ThTOR also has the four basic domains of TOR:FAT domain,1309-1887 Aa; FRB domain,1920-2023 Aa; kinase domain,2090-2340 Aa; FATC domain,2447-2479 Aa.The full-length genomic DNA of ThTOR was amplified using the primers designed according to the cDNA sequence of ThTOR and its genomic DNA sequence is 19162 bp, containing 55 introns and 56 exons.In order to study the functions of ThTOR and AtTOR, the ThTOR genomic DNA was used to construct overexpression vector, and then the construct was transformed into arabidosis and AtTOR-RNAi transgenic lines, respectively. 6) The cDNA sequence of ThTOR was analyzed and one specific fragment which located in FAT domain was chosen to construct a ThTOR-RNAi vector. We transformed this vector into salt cress and got the ThTOR-RNAi transgenic lines. After molecular identification of the transgenic plants, We chose two lines where the expression of ThTOR were down-regulated for the phenotypic analysis. The results showed that the transgenic plants grew slower and their seeds abortion.7) Similar to arabidosis, the expression of ThTOR will change after different stress treatments. The method is following as below:250 mM NaCl,34.9 mM PEG6000,500 mM mannitol,75μM ABA,37℃, or 4℃treated for 0h,3h,6h,12h,24h,48h; 20μM Pb(Ac)2 and 20μM CuCl2 treated separately 12h,24h,48h; 0 mM,100 mM,200 mM,300 mM and 400 mM of NaCl treated 24h. The expression of ThTOR is very low, used Northern hybridization can not detect ThTOR signal. In this experiment, untreated wild-type salt cress as control, using the method of Realtime-PCR to analyzing the expression of ThTOR. The result shown that salt, osmotic, cold, heat, ABA and heavy metals could significantly raise the expression of AtTOR.The expression of ThTOR has quite a difference in different organs. Compared with rosette leaf, stem has the lowest expression; the second lower are root and seedlings; the highest is in the stem leaf, following is flower and siliques, the expression of vernalization roots and rosette leaf was also higher than the rosette leaves.8) Some researches found that the growth of Arabidopsis thaliana is insensitive to rapamycin, whereas corn is sensitive. After the treatment of Arabidopsis and Thellungiella with rapamycin, We observed their germination and growth on MSo media plus rapamycin.. The results showed that the growth of Arabidopsis thaliana and salt cress was not affected 6 days after transferring 4-days old seedlings to MSo medium containing rapamycin. We detected the expression of TOR gene using real-time PCR and found that rapamycin did not change the expression of AtTOR and ThTOR.Seeds of salt cress sown on MSo medium with rapamycin had almost no germination.7 days after the end of stratification, the germination rate of salt cress was less than 10%, whereas Arabidopsis germination was suppressed only in the first several days, which indicated the germination slowed.7 days after stratification, Arabidopsis seeds sown on MSo medium with rapamycin achieved the same level of germination rate as the control..The main innovations of this research and significance:1) It was the first time to clone and analyze the full length of AtTOR promoter sequence. Combined with AtTOR expression patterns, we confirmed the role of regulatory factors in the promoter, identified expression area of AtTOR in Arabidopsis thaliana and explained phenotype of AtTOR-RNAi transgenic lines.2) It was the first time to study the subcellular localization of AtTOR, which formed basis for in-depth study of the function of AtTOR gene.3) It was the first time to clone the cDNA and the genomic DNA of ThTOR. Its protein structure and the genomic DNA sequence were analyzed detailedly. We constructed the overexpression vector using the genomic DNA of ThTOR and the ThTOR-RNAi vector,respectively. The ThTOR-RNAi vector was transformed in salt cress, and phenotypes of transgenic lines were observed.4) It was the first time to find that Although the growth of salt cress, same as Arabidopsis, is insensitive to rapamycin, rapamycin could affect the germination of Arabidopsis thaliana and salt cress. It will be an important research point about the role of TOR in plant in the future.