白斑狗鱼新基因lincRNA-Abcc10的克隆、表达及敲除模型构建
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王顺哲(1998-),男,硕士研究生,研究方向为鱼类遗传育种.E-mail:1458226514@qq.com

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S917

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国家自然科学基金项目(32260915); 新疆自然科学基金项目(2022D01A66); 新疆农业大学大学生创新项目(S202210758036).


Cloning, expression and knockout model construction of a novel lincRNA-Abcc10 gene in northern pike (Esox lucius)
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    摘要:

    长非编码 RNA (lncRNAs)作为一种新的表观遗传因子, 在研究鱼类性别决定和性别分化过程中具有重要的作用。本研究利用 RACE 技术, 对白斑狗鱼(Esox Lucius)性别特异性 lncRNAs 基因 Novel01784 进行克隆, 获得了全长共 1328 bp 的序列。在线预测工具 CPC2 和 CPAT 的分析结果显示, 该基因不具有编码蛋白质的能力。通过 Blast 比对分析将其定位在 9 号染色体, 且位于 sp2 Abcc10 基因之间, 根据其位置关系将其命名为 lincRNAAbcc10。从共定位分析结果显示, lincRNA-Abcc10 可能参与调控离子运输、卵泡细胞发育和线粒体活性, 其表达模式与部分邻近基因相似。其次, 对白斑狗鱼 lincRNA-Abcc10 3 个时期在 8 个组织中的 qRT-PCR 结果显示, 该基因在卵巢中特异性表达, 随着时间增长表达量逐渐下降, 说明其可能在卵巢早期分化过程中起着重要的作用。此外, 通过对白斑狗鱼 126 d 卵巢核质 RNA 分离, qRT-PCR 结果显示, lincRNA-Abcc10 分布于细胞质内, 可能以顺式或反式方式调控邻近基因的翻译水平。最后, 利用 CRISPR/Cas9 技术建立了 lincRNA-Abcc10 突变模型, 为进一步研究其基因功能奠定了基础, 本研究为 lncRNA 在鱼类性别分化的作用提供了新的思路。

    Abstract:

    Long non-coding RNA (lncRNAs) are involved in various biological processes, including sex differentiation. However, most studies on lncRNAs are limited to mammals, and a few studies are available on their functions in teleost fish. In this study, the sex-specific gene Novel01784 was identified using 126-day-old gonadal transcriptome data from northern pike (Esox lucius), and the full-length 1328 bp sequence was cloned using RACE. The results of CPC2 and CPAT analyses showed that the gene did not encode proteins. BLAST alignment analysis revealed that it was located on chromosome 9, between the sp2 and Abcc10 genes. Based on its location, it was named lincRNA-Abcc10. Transcriptome analysis was used to analyze the expression profiles of lincRNA-Abcc10 and its adjacent 11 genes in the ovaries of northern pikes at 126, 188, and 320 d. The results showed that lincRNA-Abcc10 was downregulated over time, and Mrpl4 and Mrpl45 were downregulated at all time points, similar to the expression pattern of lincRNA-Abcc10. According to the gene annotations for Mrpl4 and Mrpl45, lincRNA-Abcc10 may be involved in the regulation of ion transport, follicular cell development, and mitochondrial activity. Second, the qRT-PCR results of lincRNA-Abcc10 in eight northern pike tissues at three stages of showed that on day 126, the expression of lincRNA-Abcc10 in the female ovary was significantly higher than that in the testis, whereas in other tissues, it was only slightly expressed in the male muscle. On day 188, the expression of lincRNA-Abcc10 in the ovaries was significantly higher than that in the testes. In other tissues, it was slightly expressed in the liver, kidney, and muscle of female fish and in the head kidney and muscle of male fish. At 320 d, the expression of copyright Abcc10 in the ovaries was significantly higher than that in the testes. Other tissues included the brain, intestines, muscles, and kidneys of male fish. According to the comparative analysis of the relative expression in gonads at different developmental stages, the results showed that with the continuous development of the northern pike, the expression of lincRNA-Abcc10 at each stage decreased significantly compared to the previous period. It has also been suggested that it plays an important role in early ovarian differentiation. Additionally, through the isolation of nuclear RNA from 126-day-old northern pike ovaries, qRT-PCR results showed that lincRNA-Abcc10 was distributed in the cytoplasm and might regulate the translation level of adjacent genes in a cis or trans manner. Finally, a lincRNA-Abcc10 mutation model was established using the CRISPR/Cas9 technology, which laid the foundation for further studies on its gene function. This study provides new insights into the roles of lncRNAs in fish sex differentiation.

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王顺哲,杨倩,刘璎慧,刘祎,汪泳昌,张俊杰.白斑狗鱼新基因lincRNA-Abcc10的克隆、表达及敲除模型构建[J].中国水产科学,2024,31(1):59-69
WANG Shunzhe, YANG Qian, LIU Yinghui, LIU Yi, WANG Yongchang, ZHANG Junjie. Cloning, expression and knockout model construction of a novel lincRNA-Abcc10 gene in northern pike (Esox lucius)[J]. Journal of Fishery Sciences of China,2024,31(1):59-69

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  • 收稿日期:2023-11-23
  • 最后修改日期:2023-12-19
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  • 在线发布日期: 2024-04-29
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