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| 军曹鱼dnd基因cDNA克隆及其在性腺周年发育过程中的表达 |
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邝杰华1, 马骞1,2, 陈刚1,2, 毛非凡1, 周启苓1, 黄建盛1,2, 施钢1, 张健东1,2
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1.广东海洋大学水产学院 广东 湛江 524025;2.南方海洋科学与工程广东省实验室(湛江) 广东 湛江 524025
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| 摘要: |
| 本研究通过cDNA末端快速扩增技术(RACE)克隆了军曹鱼(Rachycentron canadum) dnd基因(Rcdnd)的cDNA序列，全长1339 bp，其中，5′非编码区59 bp，3′非编码区173 bp，开放阅读框(ORF) 1107 bp，共编码368个氨基酸。Rcdnd氨基酸序列含有1段RNA识别保守基序(RRM)以及4个C端保守结构域(CR1~4)，与高体 (Seriola dumerili) dnd的一致性最高(72.3%)。系统进化分析表明，Rcdnd与高体 的同源蛋白亲缘关系最接近。基于半定量RT-PCR的组织表达分布分析结果显示，Rcdnd在性腺中特异表达，在其他组织中均无表达。实时荧光定量PCR(qRT-PCR)结果显示，在精巢发育过程(Ⅱ~Ⅴ期)，Rcdnd的表达水平呈逐渐上升趋势；在卵巢发育过程(Ⅰ~Ⅲ期)，Rcdnd的表达水平先显著升高后趋于稳定，在150 dph (Ⅱ期)时的表达量最高。原位杂交结果显示，不同发育时期性腺组织中，Rcdnd mRNA主要在生殖细胞中表达。在精巢中，Rcdnd mRNA集中表达于精原细胞和初级精母细胞的周缘，次级精母细胞中的杂交信号明显减弱，而精细胞和成熟精子中几乎检测不到杂交信号。在卵巢中，Rcdnd mRNA在卵原细胞中的表达最强，在初级卵母细胞中的表达较弱，其中，Ⅰ、Ⅱ和Ⅲ时相卵母细胞检测到的杂交信号强度无显著差异，且杂交信号均匀分布于细胞质中。研究结果表明，Rcdnd基因可能参与军曹鱼性腺发育过程，上述结果可为揭示军曹鱼两性生殖细胞发生发育的调控机理提供理论依据。 |
| 关键词: 军曹鱼 dnd 基因克隆 性腺发育 表达分析 |
| DOI：10.19663/j.issn2095-9869.20210107002 |
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| Cloning and Expression Analysis of dnd During Annual Gonadal Development of Cobia (Rachycentron canadum) |
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KUANG Jiehua1, MA Qian1,2, CHEN Gang1,2, MAO Feifan1, ZHOU Qiling1, HUANG Jiansheng1,2, SHI Gang1, ZHANG Jiandong1,2
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1.College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong 524025, China;2.Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang, Guangdong 524025, China
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| Abstract: |
| In this study, the full length cDNA sequence of Rachycentron canadum dnd (Rcdnd) was cloned using RACE technology for the first time. In total, the sequence comprises 1339 bp, including a 5′-UTR of 59 bp, a 3′-UTR of 173 bp, and an open reading frame of 1107 bp, encoding a protein of 368 amino acids. The deduced amino acid sequence contains a conserved RNA recognition motif and four conserved regions (CR1~4). Comparisons of the deduced amino acid sequence with those of other teleosts revealed the highest percentage identity (72.3%) with Seriola dumerili. Phylogenetic tree analysis also showed that the dnd of R. canadum was most closely related to the homologous proteins of S. dumerili. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that Rcdnd was specifically expressed in the gonads, but not in other tissues. The results of real-time quantitative PCR (qRT-PCR) revealed that Rcdnd expression tended to gradually increase as the testis developed (Stages Ⅱ to Ⅴ). During the development of the ovary (Stage Ⅰ to Ⅲ), Rcdnd expression first increased substantially and then stabilized; the highest expression level was found at 150 days post hatching (dph) (Stage Ⅱ). Furthermore, the results of chemical in situ hybridization revealed that Rcdnd mRNA was mainly expressed in germ cells but barely detected in somatic cells. In the testis, Rcdnd mRNA signals were concentrated in the periphery of spermatogonia and primary spermatocytes; they were only weakly detected in secondary spermatocytes and barely detected in spermatids and spermatozoa. In the ovary, Rcdnd mRNA was highly expressed in oogonia, and the signals became weak in primary oocytes dispersed in the perinuclear cytoplasm. There were no significant differences in Rcdnd mRNA signals detected in oocytes in phases Ⅰ, Ⅱ, and Ⅲ. In conclusion, these findings suggest that the Rcdnd gene may play an important role in gonadal development and provide a theoretical reference for revealing the regulatory mechanism of germ cell differentiation during gametogenesis in R. canadum. |
| Key words: Rachycentron canadum dnd Gene cloning Gonadal development Expression analysis |