漠斑牙鲆（Paralichthys lethostigma）LHβ基因克隆及其在卵巢不同发育期的表达特征

摘要

The role of the endocrine pathway in the regulation of southern flounder Paralichthys lethostigma reproduction has been unclear. Here we aimed to explore the underlying mechanisms by investigating the expression pattern of luteinizing hormoneβ subunit (LHβ). Using samples isolated from pituitary ofP. we obtained the full-length cDNA sequence of LHβ with the RACE method. The spatial and temporal expression patterns ofLHβmRNA in different tissues during an ovarian maturation cycle were analyzed using the qPCR assay. The levels of plasma LH and E2during an ovarian maturation cycle were also measured using the ELISA method. Our results revealed an asynchronous oocyte development and batch spawning in southern flounder. The cDNA sequence of LHβhad a length of 597 bp and it encoded 145 amino acids. LHβ expression was detected in all examined tissues except for muscle, which implied it could be involved in multiple physiological functions in southern flounder. The LHβ mRNA level in pituitary was high through the entire ovarian maturation cycle, and peaked at stage V and VI. In the ovary the LHβ mRNA level peaked at stage V. In the liver the LHβ mRNA level was the highest at stage II and relatively high at stage V. The levels of plasma LH and E2 fluctuated in a similar pattern during the ovarian maturation cycle. These results suggested that LHβmight regulate the ovarian maturation cycle via endocrine, paracrine and autocrine pathways. Our study provided insights into the functions of the endocrine pathway in the reproduction of southern flounder.%利用cDNA末端快速克隆(Rapid Amplification of cDNA Ends, RACE)方法获得了漠斑牙鲆(Paralichthys lethostigma)促黄体素(Luteinizing Hormone, LH)β亚基的cDNA全长序列，检测了LHβ亚基mRNA的组织表达水平，揭示了垂体、肝脏和卵巢中LHβ亚基mRNA在卵巢发育周期中的表达水平变化，利用酶联免疫技术(Enzyme-Linked Immuno Sorbent Assay, ELISA)测定了血浆LH和雌二醇(Estrodiol, E2)表达水平的变化。结果表明，漠斑牙鲆为卵巢非同步发育分批产卵性鱼类， LHβ亚基cDNA序列全长597bp，开放阅读框(Open reading frame, ORF)长438 bp，编码145个氨基酸，LHβ亚基mRNA具有广泛的组织分布特性。与肝脏和卵巢相比，垂体中LHβ亚基mRNA表达水平在卵巢发育各个阶段都有较高表达水平，在IV期表达水平迅速升高至较高水平并保持至VI期。卵巢中LHβ mRNA表达水平在V期达到峰值，而肝脏中LHβ mRNA表达水平在II期时达到峰值，在V期也有相对较高的表达水平。血浆LH和E2表达水平在卵巢发育周期中均呈现趋势一致的规律性变化。研究结果可为认识漠斑牙鲆生殖调控机制提供基础资料。