| 张晓静,张婵婵,马得友,梁佳怡,姚美竹,李子骥.Ajcaspase-8基因在仿刺参早期发育过程中的表达分析.渔业科学进展,2025,46(3):108-118 |
| Ajcaspase-8基因在仿刺参早期发育过程中的表达分析 |
| Cloning of the Ajcaspase-8 gene and its temporal expression and distribution analyses during embryonic development in the sea cucumber Apostichopus japonicus |
| 投稿时间：2024-12-23 修订日期：2025-02-03 |
| DOI：10.19663/j.issn2095-9869.20241223001 |
| 中文关键词: 仿刺参 caspase-8 基因克隆 时空表达 变态发育 凋亡 |
| 英文关键词: Apostichopus japonicus caspase-8 Gene cloning Spatiotemporal expression Metamorphosis Apoptosis |
| 基金项目:国家重点研发计划(2023YFD2401103)和国家自然科学基金(41706180)共同资助 |
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| 中文摘要: |
| 变态是仿刺参(Apostichopus japonicus)等重要水产养殖动物发育的关键时期，涉及幼体形态习性的重大转变。caspase-8作为半胱氨酸蛋白酶家族中最重要的凋亡启动子，参与了鱼、贝等许多水生动物的胚胎发育以及免疫防御过程。为探究caspase-8基因在仿刺参早期发育过程中的作用，本研究采用RACE技术克隆获得了其cDNA全长并对序列特征进行分析，利用实时荧光定量PCR方法(RT-qPCR)检测了该基因在刺参成体组织和幼体发育过程中的相对表达量，通过整体胚胎杂交技术和TUNEL技术在变态期间幼体中分别展示了Ajcaspase-8基因的表达和细胞凋亡情况。结果显示，Ajcaspase-8基因cDNA全长为2 790 bp，共编码671个氨基酸；推导蛋白具有caspase家族保守的CASc结构域以及五肽序列QACQG，另有DED结构域和P20、P10大小亚基，它们是行使凋亡启动caspase的主要元件；Ajcaspase-8与玉足海参(Holothuria leucospilota)聚为一支，与青鳉(Oryzias latipes)的遗传距离更近，显示了其较高的进化地位。RT-qPCR分析发现，Ajcaspase-8基因在成年刺参组织中呈持家型表达，分别于体腔细胞中最高、在性腺中最低(P<0.05)，表明其参与免疫过程；从受精卵到稚参的早期发育过程中，Ajcaspase-8均有表达，到樽形幼体期达到峰值，显著高于其他发育阶段(P<0.05)；在稚参内的表达较樽形幼体有所降低(P<0.05)，但仍显著高于其他阶段(P<0.05)，说明Ajcaspase-8在刺参幼体变态发育中发挥潜在作用。整体原位杂交研究显示，变态期间Ajcaspase-8阳性信号仅定位于大耳幼体的胃部，集中在樽形幼体球状体周边，大量出现在稚参肠内，暗示了该基因在刺参变态幼体的冗余组织和消化道中活跃表达。TUNEL可视化分析显示，幼体凋亡细胞的数量随变态进程呈递增趋势，暗示细胞凋亡是刺参变态期间维持机体内稳态的重要途径。综合上述研究结果，推测Ajcaspase-8基因在仿刺参变态附着过程中发挥重要作用，其可能是以启动细胞凋亡的方式参与幼体形态的重构和免疫防御。 |
| 英文摘要: |
| Metamorphosis is an essential stage during the ontogenesis of several important aquaculture animals, such as Apostichopus japonicus, and it covers a wide range of extensive alterations in the morphology and ecological behavior of larvae. Caspase-8 has been shown as the foremost apoptotic promoter, involved in embryonic development and immune defense processes in various aquatic animals, including fish and shellfish. To elucidate the function of the caspase-8 gene in the larval growth of the sea cucumber A. japonicus, RACE technology was employed to obtain its full-length cDNA, followed by analyses of its sequence characteristics. The expression levels of Ajcaspase-8 were assessed in adult tissues and during various larval development periods using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Moreover, the expression localization of Ajcaspase-8 and the occurrence of cell apoptosis in metamorphosizing larvae were determined via whole embryo hybridization and TUNEL techniques, respectively. The results indicated that the full-length cDNA of the Ajcaspase-8 gene was 2,790 bp, encoding 671 amino acids that included the hydrolytic active CASc domain and conserved pentapeptide QACQG characteristic of the Caspase family. Furthermore, a death receptor domain and two subunits (P20 and P10) were present in Ajcaspase-8 deduced protein, which acted as key motifs to perform the function of an initiator caspase in the apoptosis pathway. Ajcaspase-8 was clustered into a branch with Holothuria leucospilota, occupying a closer genetic distance to Oryzias latipes, indicating the advanced evolutionary status of this molecule. The RT-qPCR results showed that the Ajcaspase-8 gene in adult sea cucumbers exhibited housekeeping expression, with peak expression in coelomocytes and minimal expression in gonads (P<0.05), indicating its involvement in immunity. During the entire developmental process of A. japonicus larvae, the expression of this gene was consistently detected, showing a peak value during the doliolarial stage and second-highest value during the juvenile stage (P<0.05), both of which were significantly higher than those of other developmental stages (P<0.05). This finding suggests that Ajcaspase-8 may play an important role in the morphological reshaping of larvae during metamorphosis. The in situ hybridization analysis showed that Ajcaspase-8 positive signals were exclusively localized in the stomach of late-stage auricularia, concentrated near the hyaline sphere of doliolaria, and greatly intensified in the intestines of juveniles. This indicated that this gene was actively expressed in the redundant and digestive tissues of larvae during metamorphosis. TUNEL visualization showed that the number of apoptotic cells increased with metamorphosis, suggesting that apoptosis was an important mechanism for maintaining the homeostasis of metamorphic larvae. Taken together, these results show that the Ajcaspase-8 gene is likely to play important roles in the metamorphosis and A. japonicus attachment, suggesting that it could initiate cell apoptosis to participate in morphological reconstruction and immune defense during the crucial process of ontogenesis in this species. This study provides fundamental data for further investigation of the regulatory mechanisms underlying metamorphosis in sea cucumbers. |
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