从绿滨蟹（Carcinus maenas）中克隆一氧化氮合酶：切除眼柄对绿滨蟹和褐斑滨蟹（Gecarcinus lateralis）蜕皮腺（Y 器官）中表达的影响比较研究。
Cloning of a nitric oxide synthase from green shore crab, Carcinus maenas: a comparative study of the effects of eyestalk ablation on expression in the molting glands (Y-organs) of C. maenas, and blackback land crab, Gecarcinus lateralis.
机构信息
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
出版信息
Comp Biochem Physiol A Mol Integr Physiol. 2011 Jan;158(1):150-62. doi: 10.1016/j.cbpa.2010.10.013. Epub 2010 Oct 16.
Molting in decapod crustaceans is regulated by ecdysteroids produced by a pair of Y-organs (YOs) located in the cephalothorax. YO ecdysteroidogenesis is suppressed by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ of the eyestalk (ES) ganglia. MIH signaling may involve nitric oxide synthase (NOS) and NO-sensitive guanylyl cyclase (GC-I). A full-length cDNA encoding Carcinus maenas NOS (Cm-NOS; 3836 base pairs) of 1164 amino acid residues (estimated mass 131,833 Da) was cloned with 88% identity to Gecarcinus lateralis NOS (Gl-NOS). End-point reverse transcription-polymerase chain reaction (RT-PCR) showed that Cm-NOS was expressed at varying levels in the YO, testis, ovary, hepatopancreas, midgut, hindgut, heart, thoracic ganglion, and skeletal muscle and was not detected in the gill. Immunofluorescence microscopy showed localization of NOS and cGMP in the steroidogenic cells and the surrounding connective tissue layer of the C. maenas YO. ES ablation (ESA) induced molting in G. lateralis; hemolymph ecdysteroid titers increased during premolt and reached a peak of about 400 pg/μL at 20 days and 24 days post-ESA. By contrast, ESA did not induce molting in C. maenas; hemolymph ecdysteroid titers increased about 2-fold (53 to 121 pg/μL) by 3 days post-ESA and remained at that level at 7 days post-ESA. Real time PCR was used to quantify the effects of ESA on the expression of NOS in C. maenas and G. lateralis YOs. ESA caused 32-fold and 5-fold increases in Gl-NOS and Cm-NOS transcripts by 24 days and 7 days post-ESA, respectively, which were correlated with hemolymph ecdysteroid levels. In addition, GC-I catalytic subunit (Gl-GC-Iβ) mRNA level increased 7.4-fold by 24 days post-ESA, but there was no significant effect of ESA on membrane GC (Gl-GC-II) mRNA level. These data indicate that the YO up-regulates NO signaling components in response to withdrawal of ES neuropeptides.
十足目甲壳动物的蜕皮受一对位于头胸部的 Y 器官（YO）产生的蜕皮甾酮调节。YO 蜕皮甾酮的产生受到由眼柄神经节 X 器官（ES）产生的蜕皮抑制激素（MIH）的抑制。MIH 信号可能涉及一氧化氮合酶（NOS）和一氧化氮敏感型鸟苷酸环化酶（GC-I）。我们克隆了编码十足目美洲巨螯蟹 NOS（Cm-NOS；3836 个碱基对，1164 个氨基酸残基（估计质量 131833 Da）的全长 cDNA，与加利福尼亚蓝蟹 NOS（Gl-NOS）具有 88%的同源性。终点逆转录聚合酶链反应（RT-PCR）显示，Cm-NOS 在 YO、睾丸、卵巢、肝胰腺、中肠、后肠、心脏、胸神经节和骨骼肌中以不同水平表达，在鳃中未检测到。免疫荧光显微镜显示，NOS 和 cGMP 定位于美洲巨螯蟹 YO 的类固醇生成细胞及其周围的结缔组织层。ES 切除（ESA）诱导加利福尼亚蓝蟹蜕皮；蜕皮前血液中蜕皮甾酮的浓度增加，在 ESA 后 20 天和 24 天达到约 400 pg/μL 的峰值。相比之下，ESA 不能诱导美洲巨螯蟹蜕皮；ESA 后 3 天血液中蜕皮甾酮的浓度增加约 2 倍（53 至 121 pg/μL），ESA 后 7 天仍保持该水平。实时 PCR 用于定量 ESA 对美洲巨螯蟹和加利福尼亚蓝蟹 YO 中 NOS 表达的影响。ESA 分别在 ESA 后 24 天和 7 天导致 Gl-NOS 和 Cm-NOS 转录物增加 32 倍和 5 倍，这与血液中蜕皮甾酮水平相关。此外，ESA 后 24 天，Gl-GC-IβmRNA 水平增加 7.4 倍，但 ESA 对膜 GC（Gl-GC-II）mRNA 水平没有显著影响。这些数据表明，YO 上调 NO 信号传导成分以响应 ES 神经肽的撤出。
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