摘 要

本文以凡纳滨对虾（Litopenaeus vannamei）为研究对象，对与其生长代谢密切相关的TOR（Target of Rapamycin）信号通路在基因水平以及蛋白水平上进行了初步的研究，以期能够在未来对虾的生产实践中提供理论依据。

为探究TOR信号通路调节的细胞过程，将雷帕霉素（TOR信号通路抑制剂）直接注射到凡纳滨对虾的肌肉组织，利用转录组测序技术分析了对虾肌肉组织基因表达水平的变化，发现许多差异表达基因都与细胞自噬过程相关，推测TOR信号通路抑制参与调控凡纳滨对虾的细胞自噬过程。这些基因包括自噬相关基因、凋亡相关基因、Ca²⁺调节相关基因、热休克蛋白（hsps）基因、p53基因以及许多溶酶体功能基因。利用Western Blot技术发现在凡纳滨对虾中，雷帕霉素能够有效抑制TOR信号通路，促进细胞自噬。此外，在TOR信号通路受到抑制从而引发自噬的过程中，与自噬起始密切相关的ATG1和ATG13蛋白的磷酸化水平都显著下降。

利用RACE技术首次在凡纳滨对虾中克隆得到了rheb基因，全长898 bp，开放阅读框549 bp，共编码182个氨基酸。利用qRT-PCR技术进行该基因的组织特异性表达分析，发现该基因在所有组织中都表达，在肠道中的表达量最低。分析饥饿、氨基酸注射以及雷帕霉素注射等不同处理条件下基因表达的差异，发现饥饿与雷帕霉素都可以促进rheb的表达，而亮氨酸或精氨酸的注射可以使饥饿引起的rheb表达量变化恢复到正常水平，但是对雷帕霉素引起的rheb表达量变化却没有影响。

分析了TOR信号通路中另一个重要基因raptor在饥饿、氨基酸注射以及雷帕霉素注射等不同的处理方式下的表达变化，发现饥饿与雷帕霉素都可以促进raptor的表达，而氨基酸的注射可以缓解饥饿和雷帕霉素注射对raptor表达量的影响。通过对raptor基因的RNA干扰，发现S6K与4EBP蛋白的磷酸化与Raptor蛋白密切相关，并且4EBP的磷酸化过程要比S6K更加依赖Raptor蛋白。

鉴于目前针对水产动物营养与代谢分子调控机制的研究报道较少，尤其在甲壳动物中的研究更为罕见。上述结果一方面加深了我们对甲壳动物TOR信号通路的认知，有助于了解对虾等经济动物生长与代谢的分子机理以及揭示TOR信号通路在生命进化过程中的衍化痕迹。另外，从分子水平上探索对虾生长代谢调控的机理，为苗种的选育、饲料营养成分添加配比以及优化养殖管理模式提供了理论依据。

关键词：凡纳滨对虾，TOR，雷帕霉素，Rheb，Raptor

ABSTRACT

The study is about the TOR signaling pathway that is closely related to the cell growth and metabolism in Litopenaeus vannamei. The experiment carried out explored the TOR signaling pathway and will provide theoretical support for the future production of prawns.

In order to find the cellular processes regulated by TOR signaling pathway, rapamycin (an inhibitor of TOR signaling pathway) was injected into shrimps, and the gene expression changes analysed by RNAseq. The expression of some genes involved in apoptosis, autophagy, some heat shock protein genes and genes related to function of lysosome and feedback regulation of TOR signaling pathway were found differently expressed. The TOR signaling pathway can be depressed by rapamycin and autophagy were promoted in shrimps. To further explore the autophagy regulatory mechanism, the concentration and phosphorylation of related proteins were analyzed by Western Blot. Phosphorylating of ATG1 can be depressed and dephosphorylating of ATG13 can be promoted by treatment with the rapamycin.

The cDNA sequence of rheb from Litopenaeus vannamei was cloned for the first time by RACE technology. The full lenghth of rheb cDNA sequence is 898 bp, containing an open reading frame of 549 bp that encoded 182 amino acid. It was found that the rheb expressed in all tissues detected, and the expression level of rheb in the intestine was the lowest. Dietary restriction or rapamycin injection could promote the expression of rheb, and the leucine and arginine injection could relieve the accelerated expression of rheb caused by dietary restriction. What’s more, rapamycin could totally hinder the regulation of leucine to rheb in shrimp.

The expression of raptor, another major factor of TOR signaling pathway, was found promoted by dietary restriction or rapamycin injection. The amino acids could relieve the accelerated expression of raptor caused by dietary restriction and rapamycin injection. In addition, we found the phosphorylation of S6K and 4EBP were closely related to Raptor. Compared with S6K, the phosphorylation of 4EBP was more dependent on Raptor.

The reports on the molecular mechanisms of nutrition and metabolism in aquatic animals are rare, especially in crustaceans. The results of the study can deepen our understanding of the important signaling pathway in crustaceans and reveal the evolutionary traces of TOR signaling pathway. What’s more, the mechanism research can provide more reliable way to select the new breed and optimize the feed formula.

Key words: Litopenaeus vannamei, TOR, Rapamycin, Rheb, Raptor