| 摘要: |
| DNA纤维-荧光原位杂交(FISH)技术具有分辨率高的优点,藻类细胞遗传学领域至今仍没有相应的研究报道。鉴于此,本研究在分离并获得海带配子体细胞核的基础上,通过核裂解时间及DNA纤维拉伸方法的优化,以制备高质量基因组DNA纤维。利用荧光标记的海带18S rDNA为探针,对制备的DNA纤维进行FISH分析。结果显示,在海带伸展的基因组DNA纤维上显示出线性排列的“串珠状”荧光信号;这与含有海带45S rDNA序列的细菌人工染色体(BAC)克隆质粒的纤维-FISH结果一致。从而表明我们已利用海带基因组DNA纤维构建了45S rRNA基因的高分辨率可视图谱;其分辨率约为2.09 kb,与高等植物的研究结果相当。本研究所获得的海带45S rDNA高分辨率物理图谱为其基因组的正确组装提供了重要的细胞学证据,也可供其他藻类基因组中串联重复序列及多拷贝基因的组装等研究来参考。 |
| 关键词: 海带 荧光原位杂交(FISH) DNA纤维 18S rDNA |
| DOI：10.11759/hykx20250917002 |
| 分类号:Q2-33 |
| 基金项目:国家自然科学基金(32172963,41376136) |
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| High-resolution visual mapping of 45S rRNA genes by fluorescence in situ hybridization of the extended DNA fibers in Saccharina japonica |
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CHEN Siqi1, CAI Yiyan1, ZHANG Cheng1, BI Yanhui1, ZHOU Zhigang2
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1.Shanghai Ocean University, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai 201306, China;2.Shanghai Ocean University, International Joint Research Center for Marine Biology, Shanghai 201306, China
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| Abstract: |
| The DNA fiber-fluorescence in situ hybridization (FISH) technique offers high spatial resolution. However, so far, it has not been reported in algal cytogenetics research. To address this gap, the present study optimized the nuclear lysis time and conditions for the DNA fiber extension method to obtain high-quality genomic DNA from isolated nuclei of Saccharina japonica gametophytes. Fiber-FISH was subsequently conducted on the extended DNA fibers using fluorophore-labeled 18S rDNA as a probe. The resulting FISH signals exhibited a linear, beads-on-a-string arrangement along the genomic DNA fibers. This pattern was consistent with that obtained using a bacterial artificial chromosome clone containing the kelp 45S rDNA sequence. Based on these extended genomic DNA fibers, a high-resolution physical map of 45S rDNA genes was constructed, achieving a resolution of ≤2.09 kb, comparable to that reported in higher plants. This map provides crucial cytological evidence for accurate kelp genome assembly. In addition, it may also serve as a reference approach for assembling tandem repeat sequences and multi-copy genes in other algal genomes. |
| Key words: Saccharina japonica fluorescence in situ hybridization DNA fiber 18S rDNA |
