脊尾白虾UCHL5的基因克隆及其在卵巢发育中的功能研究

Molecular Cloning of the Ubiquitin Carboxyl-Terminal Hydrolase Isozyme L5 and Its Functional Analysis During Ovarian Development in Exopalaemon carinicauda

关键词:  脊尾白虾  UCHL5  卵巢  实时荧光定量PCR  原核表达

Key words:  Exopalaemon carinicauda  UCHL5 gene  Ovary  Real-time quantitative PCR  Prokaryotic expression

摘要:

为了解泛素羧基端水解酶5基因(ubiquitin carboxyl-terminal hydrolase isozyme L5, UCHL5)在脊尾白虾(Exopalaemon carinicauda)卵巢发育中的作用，本研究采用RACE技术克隆了脊尾白虾UCHL5全长cDNA，并对其在脊尾白虾不同组织和不同卵巢发育时期的表达情况进行分析，在此基础上构建了pET32a-UCHL5原核表达载体，进行诱导表达研究。结果显示，UCHL5 cDNA全长为1440 bp，共编码329个氨基酸，预测其相对分子量为37.57 kDa，理论等电点为5.51。系统进化分析表明，脊尾白虾UCHL5氨基酸序列与凡纳滨对虾(Litopenaeus vannamei)同源性最高(83%)，与同为甲壳纲的凡纳滨对虾、拟穴青蟹(Scylla paramamosain)和三疣梭子蟹(Portunus trituberculatus)聚为一支。荧光定量PCR分析结果显示，UCHL5在鳃中的表达量最高，其次是卵巢，且在以上 2种组织中的表达量均显著高于其他组织；在卵巢不同发育时期，UCHL5的表达呈先上升后下降的趋势，在产后恢复期(Ⅴ期)急剧下降至最低水平。构建的UCHL5原核表达载体在大肠杆菌(Escherichia coli)中成功表达UCHL5融合蛋白。本研究结果表明，UCHL5可能与脊尾白虾卵巢发育有密切关系，为研究甲壳动物卵巢发育的分子调控机制提供了理论基础。

Abstract:

The ridgetail white prawn Exopalaemon carinicauda belongs to the suborder Pleocyemata, in the family Palaemonidae, and is one of the most economically important pond-reared shrimp. At present, most of the egg-carrying broodstock are harvested from natural sea areas, which considerably restricts the development of E. carinicauda aquaculture. Thus, artificial propagation methods are required to enhance the productivity of E. carinicauda in aquaculture systems. To develop these systems, there is a need for a greater knowledge base encompassing the mechanism of ovarian and embryonic development of E. carinicauda. Ubiquitin C-terminal hydrolases (UCHLs) are a subset of deubiquitinating enzymes that are involved in numerous physiological processes. Many studies have shown that the UCH family of deubiquitinating enzymes plays an important role in reproduction. In this experiment, the full-length cDNA of UCHL5 was identified and characterized using an approach that combines transcriptome data and rapid amplification of cDNA ends (RACE). The expression profile of UCHL5 in different developmental stages of the ovary and various tissues was determined using real-time quantitative PCR. The pET32a-UCHL5 prokaryotic expression recombinant plasmid was constructed and induction expression was carried out. The cloned UCHL5 gene was 1440 bp in length, encoding 329 amino acids. Isoelectric point (pI) of UCHL5 protein was 5.51, and molecular weight was 37.57 kDa Homology and phylogenetic analysis showed that the deduced amino acid sequence of UCHL5 shared high homology in different species and the highest conservation with Litopenaeus vannamei (83%). Real-time quantitative PCR results indicated that the expression quantity of UCHL5 was the highest in the gill tissue of E. carinicauda, followed by that in the ovary. The expression quantities of UCHL5 at different ovary developmental stages (Ⅰ~Ⅳ) were progressively upregulated, and the expression of UCHL5 was the lowest in stageⅤ. The fusion protein UCHL5 obtained through prokaryotic expression was 52 kDa. From the results of this study, we may conclude that UCHL5 is possibly playing an important role in the ovarian development of E. carinicauda.