Identification and characterization of TOR in Macrobrachium rosenbergii and its role in muscle protein and lipid production

Abstract

The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation.

Figure 1

(A) Nucleotide and predicted amino acid sequences of MrTOR. Purple boxes indicate start (ATG) and stop codons (TGA). Red region represents DUF3385 structural domain; gray region, FAT structural domain; pink region, rapamycin-binding structural domain; blue region, PI3Kc structural domain; yellow region, FATC structural domain. (B) Predicted TOR protein functional domains.

Figure 2

(A) Amino acid sequence comparison of MrTOR with other species. Red box indicates DUF3385 structural domain; black box, FAT structural domain; blue box, rapamycin-binding structural domain; green box, PI3Kc structural domain; orange box, FATC structural domain. GenBank accession numbers: Homo sapiens (NP_001373429.1); Mus musculus (NP_064393.2); Xenopus laevis (XP_018081150.1); Danio rerio (NP_001070679.2; P. monodon (XP_037804060.1); Penaeus vannamei (QHT73480.1); P. japonicus (UYO77156.1); P. clarkii (XP_045619169.1); E. sinensis (XP_050711953.1); Portunus trituberculatus (XP_045121188.1); P. chinensis (AHX84170.1). (B) Similarity of MrTOR amino acid sequence to TOR amino acid sequences of other species. (C) MrTOR phylogenetic analysis. The number on the node indicates the confidence value of the test for 1000 bootstrap repetitions.

Figure 3

(A) MrTOR expression levels in each tissue. Different letters indicate significant differences (P < 0.05). Results after hybridization with sense probes (negative control). (B) and antisense probes (experimental group) (C). Arrows indicate positive signals.

Figure 4

Relative expression levels of MrTOR in muscle after interference. Interference chain 1, 2 and 3 indicate G1, G2 and G3, respectively. Different letters indicate significant differences (P < 0.05).

Figure 5

Relative expression levels of (A) eIF4E, (B) eIF4B, (C) S6K, and (D) FAS genes after interference. (E) TG and (F) T-CHO contents in tissue after interference. (G) LPS activity and (H) FAS activity after interference. Different letters indicate significant differences (P < 0.05).

Figure 6

MrTOR expression levels after (A) Arg injection, (B) Leu injection, and (C) Leu and Arg injection. (D) Expression levels of different genes after amino acid injection. (E) TG and (F) T-CHO contents, and (G) LPS activity and (H) FAS activities at different times after amino acid injection. Different letters indicate significant differences (P < 0.05).

Figure 7

Expression levels of (A) MrTOR and (B) amino acid-related genes after starvation and post-starvation amino acid injection. (C) AMPK, (D) LPS, and (E) FAS activities after starvation and post-starvation amino acid injection. Different letters indicate significant differences (P < 0.05).

Figure 8

Predicted TOR signaling pathway in M. rosenbergii.