中华鳖Gper基因克隆、表达及其在精巢中的功能

G蛋白偶联雌激素受体（Gper）是一种膜雌激素受体，介导雌激素的非基因组途径。为研究G蛋白偶联雌激素受体基因（ cDNA全长序列，利用qRT-PCR分析其在不同组织及胚胎不同发育阶段的性腺中的表达模式，并通过来曲唑（letrozole）和Gper抑制剂G-15处理雄鳖初步分析Gper在精巢中的作用。结果显示，中华鳖 cDNA序列全长2023 bp，包含705 bp 5''非编码区、241 bp 3''非编码区和1077 bp开放阅读框，编码358个氨基酸，其氨基酸序列上有7个跨膜结构域和Asp-Arg-Tyr（DRY）三联体结构，基因编码蛋白分子量为41.084 kD，等电点为6.844。表达量变化呈相同趋势：16期表达量最高，随着性腺分化过程表达量显著降低。Letrozole处理组中2表达量明显升高；G-15处理组精巢中，精子发生与促细胞凋亡相关基因表达量显著升高。结果表明，可能参与中华鳖性腺分化早期过程，并调控雄性生殖细胞增殖。

Molecular cloning and initial function analysis of Gper in male Pelodiscus sinensis

Estrogens play a crucial role in the normal function of postnatal ovaries and testes in vertebrates. Estrogens have been reported to induce proliferation of spermatogonium stem cells and spermatogonia in both birds and mammals. Previous studies have showed that estrogens bind either to classical intracellular estrogen receptors (Esr1 and Esr2) or to a membrane estrogen receptor such as G protein-coupled estrogen receptor (Gper) and can therefore trigger both genomic or non-genomic signaling pathways. In comparison with the genomic pathway, non-genomic signaling occurs rapidly (within seconds to minutes) and involves the production of secondary messengers, the activation of protein kinases, and the modulation of ion-channels. Gper is a 7-transmembrane protein that belongs to the G protein-coupled receptor (Gpcr) superfamily, the members of which participate in various endocrine and metabolic processes. Gper is widely found in vertebrates such as mammals, birds, reptiles, and fish and is involved in many signaling pathways regulating cell and nervous system repair and other important physiological functions. Gper is very important in germ cell proliferation, in females in particular. is also expressed in germ cells in males, but little information on the role of in gonadal reproduction is available. The Chinese soft-shelled turtle () is a member of the Reptilia family of Chelonia Trionychidae and is an economically important aquaculture species in China. To improve the economic benefits of , it is important to study the characteristics and the mechanism of gonadal differentiation and reproduction of this species. However, limited studies on the reproduction of the turtle exist. The aims of this study were to identify the sequence information and function of Gper in the male turtle. The full-length cDNA sequence was cloned using rapid amplification of cDNA ends technology. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression level of mRNA in different tissues and different developmental stages of the gonad. Finally, the role of was analyzed by injecting letrozole and G-15 to inhibit aromatizing enzyme and Gper was 2023 bp long, including 705 bp at the 5''-UTR, 241 bp at the 3''-UTR, and a 1077 bp open reading frame (ORF) encoding a peptide of 358 amino acids. One exon of the ORF was the same as other reptiles. The putative peptide contained 7-transmembrane domains and an Asp-Arg-Tyr (DRY) structure, and Gper therefore belongs to the Rhodopsin subfamily. Multiple sequence comparisons of vertebrate Gper proteins indicated that Gper has the highest similarity with the corresponding protein in Chelonia while it has the lowest similarity with the corresponding protein in fish. Quantitative real-time PCR detected , and the maximum level was detected in the brain, followed by the ovary. Moreover, under different incubation temperatures, expression in the critical stage of gonadal differentiation showed the same trend:the highest expression level was found in stage 16 of embryonic development, and significantly decreased with gonadal differentiation. After injection of letrozole and G-15, the expression of was significantly decreased in the letrozole treatment group, but the expression of 2 were significantly increased. After inhibiting Gper protein activity, the expression of 2 obviously increased. More than that, the expression of genes related to spermatogenesis and apoptosis significantly increased after G-15 treatment. From the above, the role of Gper in the testes of is likely to be involved in early gonadal differentiation and regulates the proliferation of male germ cells. This study provides new insights into the role of Gper in the gonads of