凡纳滨对虾TOR蛋白下游信号传导因子eIF4E2和eIF4E1A基因克隆与功能分析

为研究mTOR信号通路在凡纳滨对虾(Litopenaeus vannamei)氨基酸代谢中的调控作用,本研究利用RACE技术首次克隆获得了凡纳滨对虾肌肉组织中eif4e2和eif4e1a基因的全长c DNA序列。eif4e2基因c DNA序列全长1 069 bp,开放阅读框699 bp,编码232个氨基酸;eif4e1a基因c DNA序列全长3579bp,开放阅读框627bp,编码208个氨基酸。氨基酸序列比对和进化分析均证实eIF4E2和eIF4E1A为目前已知的eIF4E家族蛋白的同源蛋白。利用实时荧光定量PCR的方法研究了eif4e2和eif4e1a基因在凡纳滨对虾不同组织中的表达差异以及注射雷帕霉素或氨基酸后肌肉中eif4e2和eif4e1a基因表达量的变化情况,探讨了eif4e2和eif4e1a基因在细胞生长中的调控作用。结果表明,凡纳滨对虾eif4e2和eif4e1a基因在眼柄、肝胰脏、肠道、胃、鳃丝和肌肉等组织中均有表达;其中eif4e2基因在肌肉中的表达量显著高于其他组织(P0.05),eif4e1a基因在肠道和肌肉中都有较高表达量,且在肠道中的表达量显著高于其在肌肉中的表达量(P0.05);注射雷帕霉素后2 h内肌肉中eif4e2和eif4e1a基因表达量都出现显著下降(P0.05);肌肉中eif4e1a基因的表达量在单独注射亮氨酸或精氨酸4 h内均未出现变化,但在同时注射亮氨酸和精氨酸后表达量显著增加(P0.05);肌肉中eif4e2基因在注射亮氨酸、精氨酸及亮氨酸加精氨酸后表达量都明显提高(P0.05),且同时注射亮氨酸和精氨酸后,基因表达变量明显比单独注射亮氨酸或精氨酸后变化量大。本研究从动物分子营养学角度出发,克隆了凡纳滨对虾mTOR信号通路中eif4e2和eif4e1a两个基因,并证明其能够通过mTOR信号通路接收不同氨基酸信号来调控细胞生长,对于深入了解对虾的生长和代谢调控机制、饲料配方的优化以及建立合理的养殖管理模式都具有重要的意义。

Cloning and functional analysis of TOR downstream signaling factors eIF4E2 and eIF4E1A of Litopenaeus vannamei

The aim of this study was to explore the regulation function of mTOR signaling in the amino acid metabolism of Litopenaeus vannamei. We cloned the full length cDNA of eif4e2 and eif4e1a genes from the muscle of L. vannamei for the first time. The cDNA of eif4e2 was 1069 bp and contained a 699 bp open reading frame that encoded 208 amino acids. The cDNA of eif4e1a was 3579 bp and contained a 627 bp open reading frame that encoded 232 amino acids. We individually compared the deduced eIF4E2 and eIF4E1A amino acid sequences of L. vannamei with other known sequences. The results confirmed their homology. We used a real-time polymerase chain reaction (PCR) to detect the tissue-specific expressions of eif4e2 and eif4e1a in L. vannamei and the responses of eif4e2 and eif4e1a in muscle to the injection of rapamcyin or amino acid (leucine or arginine). We also investigated the functions of eif4e2 and eif4e1a in the cell growth of L. vannamei. The results showed that the eif4e2 and eif4e1a genes were expressed in all the detected tissues, including the eyestalk, hepatopancreas, stomach, intestine, gill, and muscle. The relative expression level of eif4e2 was significantly higher than that of other tissues (P < 0.05). The relative expression levels of eif4e1a in intestine and muscle were both higher than those in other tissues, and that of the intestine was higher than that of the muscle (P < 0.05). The relative expression levels of eif4e2 and eif4e1a in muscle decreased after the injection of rapamcyin (P < 0.05). The relative expression level of eif4e1a was unchanged four hours after the injection of unitary leucine or arginine, while it increased significantly after the simultaneous injections of leucine and arginine (P < 0.05). The relative expression level of eif4e2 increased significantly after the injection of unitary leucine, unitary arginine, or both leucine and arginine (P < 0.05), and the relative expression level of eif4e2 after the simultaneous injections of leucine and arginine was significantly higher than that after injection of unitary leucine or arginine. In conclusion, with respect to animal molecular nutriology, we cloned full-length cDNA of eif4e2 and eif4e1a genes from the muscle of L. vannamei, and the results indicate that eif4e2 and eif4e1a can accept the amino acid signal and regulate cell growth in L. vannamei muscle. This study represents a significant contribution to our understanding of the mechanisms of growth and metabolism regulation, the optimization of feed formulas, and the establishment of a reasonable management model for shrimp culture development.