| 摘要: |
| 在企鹅珍珠贝遗传育种研究中, 为保证在贝类存活的条件下获得其DNA信息, 采用企鹅珍珠贝(Pteria penguin)贝壳边缘和足丝研究非致死性DNA提取方法。不同于贝壳获取过程中会对实验贝体产生一定损伤, 足丝是无生命的细胞外纤维束, 其获取方法简单且属于非损伤性取样, 对实验贝几乎无伤害, 是潜在的获取具足丝贝类基因组DNA的优良材料。本研究采用脱钙与未脱钙两种处理方式, 并结合海洋动物组织基因组DNA提取试剂盒法及有机溶剂萃取法(OSE)提取贝壳及足丝DNA, 对获得的贝壳DNA及足丝DNA进行PCR扩增。结果显示, 足丝有机溶剂法(OSE法)提取效率显著高于贝壳DNA提取方法, 脱钙足丝与未脱钙足丝的DNA提取效率无显著差异, 分别为(0.016 7±0.002 9) μg/mg和(0.016 1±0.003 1) μg/mg。未脱钙足丝的OSE法获得的DNA产物纯度最优, A260/280值为1.400 0±0.040 0, A260/230值显著高于贝壳DNA及足丝DNA的其他提取方法, 为0.910 0±0.080 0。除脱钙贝壳OSE法外, 其他所有DNA提取方法均可用于后续PCR扩增, 测序结果表明产物DNA来源于企鹅珍珠贝, 且贝壳及足丝取样方式均未导致企鹅珍珠贝死亡。综上,未脱钙足丝的OSE法优于其他方法, 研究结果可为非致死性DNA提取提供参考, 也为企鹅珍珠贝的选择育种研究及珍稀贝类保护奠定前期基础。 |
| 关键词: 企鹅珍珠贝 非致死性 贝壳DNA提取 足丝DNA提取 |
| DOI：10.11759/hykx20220315004 |
| 分类号:S917.4 |
| 基金项目:国家自然科学基金项目(31860727) |
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| Nonlethal DNA extraction methods for Pteria penguin |
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CHEN Yi, LIU Er-tian, ZHAN Xin, GU Zhi-feng, WANG Ai-min
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College of Ocean, State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China
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| Abstract: |
| It is necessary to obtain DNA from live shellfish for genetic breeding research on Pteria penguin (P. penguin). Unlike shell sampling, which can damage the shellfish, byssi are nonliving extracellular fiber bundles, which are simple to obtain and nonlethal to the shellfish, making them a potentially good material for obtaining genomic DNA. In this study, the shell edge and byssus of P. penguin were used to study nonlethal DNA extraction methods. Shell and byssus samples were treated with decalcifying and undecalcifying methods. The DNA was extracted using a marine animal tissue genomic DNA extraction kit method and the organic solvent extraction (OSE) method. The shell and byssus DNA were amplified by polymerase chain reaction (PCR) analysis. The results showed that the DNA extraction efficiency of the byssus OSE method was significantly higher than that of the shell DNA extraction method. No significant difference was observed between the DNA extraction efficiencies of the decalcified and undecalcified byssus, which were 0.016 7±0.002 9 μg/mg and 0.016 1±0.003 1 μg/mg, respectively. DNA obtained by the OSE method for undecalcified byssus had optimal purity, with an A260/280 value of 1.400 0±0.004 8. The A260/230value was significantly higher than the other method for the shell DNA and byssus DNA, at 0.910 0±0.080 0. The DNA extraction methods, except the OSE method with decalcified shells, could be used for subsequent PCR amplification, and the sequencing results indicated that the DNA product was derived from P. penguin. Additionally, neither the shell nor the byssus sampling methods resulted in mortality of P. penguin. In summary, the OSE method for the undecalcified byssus was superior to the other methods. These results provide a novel nonlethal method for extracting DNA and lay the foundation for selective breeding of P. penguin and the conservation genetics of this rare shellfish species. |
| Key words: Pteria penguin non-lethal shell DNA extraction byssus DNA extraction |
