Gene Cloning And Functional Study On The Important Genes Of MTOR Signalingin Litopenaeus Vannamei

On the basis of transcriptome sequencing of Litopenaeus vannamei and the existing research results of mTOR signaling in model organisms, 33 gene fragments which posablelywere involved in mTOR signalingand had regulation effect in L. vannameiwere obtained.The full length cDNA sequencesof gene raptoreif4e2 and eif4e1 afrom the muscle of L. vannamei were cloned. The cDNA sequence of raptor was 5020 bp, containing a 3804 bp open reading frame, which encoded 1267 amino acid residues.The cDNA of eif4e2 was 1069 bp, containing a 699 bp open reading frame, which encoded 232 amino acid residues. The cDNA of eif4e1 a was 3579 bp, containing a 627 bp open reading frame, which encoded 208 amino acid residues. The deduced Raptor, e IF4E2 and eIF4E1 A amino acids sequences of L. vannamei were separately compared with other known sequences. The results confirmed their homology.Real-time PCR was used to detect the tissue-specific expression of tor, raptor, eif4 ebp, eif4e2 and eif4e1 a in L. vannamei. The results showed that:(1) tor, raptor, eif4 ebp,eif4e2 and eif4e1 a genes were expressed in all detected tissues including eyestalk, hepatopancreas, stomach, intestine, gill and muscle, which indicated that these genes were wildly expressed.(2) The relative expression level of torin hepatopancreas, muscle and eyestalk were significantly higher than that in stomach, intestine and gill; and there was no significantly difference in the relative expression level of torin stomach, intestine and gill.(3) The relative expression level of raptorin hepatopancreas was higherthan that in other tissues(p<0.05).(4) The relative expression level of eif4 ebpin muscle was significantly higher than that in other tissues(p<0.01), and the relative expression level in gill was second(p<0.05).There was no significantly difference in the relative expression level of eif4 ebpin eyestalk, hepatopancreas, stomach, intestine(p<0.05).(5) The relative expression level of eif4e2 in muscle was significantly higher than that in other tissues(p<0.05). The relative expression level of eif4e1 a in intestine and muscle were both higher than other tissues, and that in intestine was higher than in muscle(p<0.05).Real-time PCR was used to detect the responses ofgene tor, raptor, eif4 ebp,eif4e2 and eif4e1 a in the muscle of L. vannamei to injection of rapamcyin and amino acids(leucine or arginine).We discussed these five genes involved in mTOR signaling and genes eif4 ebp,eif4e2 and eif4e1 a involved in the regulation of cell growth. The results showed that:(1) The relative expression levels of tor, raptor, eif4e2 and eif4e1 a in muscle were reduced after injection of rapamcyin, however the relative expression levels of eif4 ebp was incresed(p<0.05).It is indicated that the mTOR signaling in L. vannamei was sensitive to rapamycin.(2) The relative expression levels of tor and raptor unchanged during 4 hours after injection of unitary leucine,while which increased significantly after simultaneous injection of leucine and arginine or unitary injection of arginine during half an hour(p<0.05).(3) The relative expression level of eif4e1 a unchanged during 4 hours after injection of unitary leucine or arginine, while which increased significantly after simultaneous injection of leucine and arginine(p<0.05). The relative expression level of eif4e2 increased significantly after injection of unitary leucine, unitary arginine or both leucine and arginine(p<0.05), and the relative expression level of eif4e2 after simultaneous injection of leucine and arginine was significantly higher than that after injection of unitary leucine or arginine. It is indicated that eif4e2 and eif4e1 a can accept the signal of amino acid and regulate the growth of cell in muscle of L. vannamei, which is similar with that in model organisms, such as mice and zebrafish.In conclusion, the mTOR signaling in L. vannamei was sensitive to rapamycin and could receive the signals from amino acids to regulate the growth of cell in animals. It was the first time to clone the full-length cDNA sequences of several key genes in the mTOR signaling of L. vannamei. These key genes were preliminaryly studied in this study. That provided a reference to the mTOR signaling system research. It is also very meaningful to understand the shrimp growth and metabolic regulation mechanism at the molecular level, which can help to optimize the feed formula of shrimp.