Molecular Cloning Of Target Of Rapamycin Gene From Fenneropenaeus Chinensis And Fundamental Research On TOR Signaling Pathway

This study is about the TOR signal transduction pathway in important economic animal breeding Fenneropenaeus chinensis,which is related to cell growth and nutritional regulation. The full length cDNA of TOR (Target of Rapamycin) gene from Fenneropenaeus chinensis was cloned for the first time by the homology cloning and the RACE technique.The cDNA was 7638 bp containing the 7389 bp open reading frame,which encoded 2462 amino acid. The deduced TOR amino acid sequences of Fenneropenaeus chinensis was compared with other known sequences and domains of TOR, and the results confirmed their homology. Real-time PCR was used to detect the tissue-specific expression of TOR in Fenneropenaeus chinensis. Experimental results show that the TOR mRNA in the muscle tissue is highest and that in the blood cell is the lowest. To research the function of TOR signaling pathway on nutritional regulation, mRNA amounts of TOR gene in muscle and phosphorylation of protein factors in TOR signaling pathways was studied by real-time PCR and western blot after injection of leucine, arginine and rapamycin. Rapamycin is the specific inhibitor of TOR. Real-time PCR was used to detect mRNA amounts of TOR in response to leucine and arginine, and the results showed that the TOR mRNA was significantly increased 24 hours after injection of leucine and arginine. Western blot was used to detect the phosphorylation of protein in TOR signaling pathway, and the results showed that the expression of p-TOR and p-S6K1 protein was significantly higher 30 minutes after injection of leucine and arginine, and recovered after 1 hour. These results indicated that leucine and arginine can stimulate the expression of TOR and the activity of TOR signaling pathway, and response of gene transcription lagged behind protein phosphorylation. Results of western blot also showed that expression of p-TOR and p-S6K1 protein was decreased 30 minutes after injection of rapamycin, and recovered after 2 hours, which indicated that rapamycin can inhibit the activity of TOR and TOR-dependent phosphorylation.