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| 企鹅珍珠贝非致死性DNA提取方法的研究 |
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陈一1,2, 刘二田2,1, 战 欣2,1, 顾志峰2,1, 王爱民2,1
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1.海南大学海洋学院;2.海南大学南海海洋资源利用国家重点实验室
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| 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，不对实验贝产生几乎无伤害，是潜在的获取具足丝贝类基因组DNA的良好优良资源材料。本研究贝壳及足丝样品采用脱钙与未脱钙两种处理方式，并结合有机溶剂萃取法(OSE法)及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）提取贝壳及足丝其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0.0167 ± 0.0029 μg/mg和0.0161 ± 0.0031μg/mg。未脱钙足丝的OSE法获得的DNA产物纯度最高优，A260/280值为1.4000 ± 0.0400，A260/230值为0.9100 ± 0.0800，显著高于贝壳DNA及足丝DNA的其他处理提取方法，为0.9100 ± 0.0800，为0.9100 ± 0.0800。除脱钙贝壳的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： |
| 分类号:S917.4 |
| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目） |
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| Study on Non-lethal DNA Extraction Methods of Pteria penguin |
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Chen Yi1,2, Liu Er-tian1,2, Zhan Xin1,2, Gu Zhi-Feng1,2, Wang Ai-min1,2
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1.College of Marine Science, Hainan University;2.tate Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University
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| Abstract: |
| In Pteria penguin genetic breeding research, it is necessary to obtain the DNA information of the shellfish under the condition of ensuring the survival of the shellfish. Unlike shell sampling, which may cause damage to the experimental individuals, byssi are nonliving extracellular fiber bundles, which are simple to obtain and non-lethal to the shellfish, making them a potentially good material for obtaining genomic DNA from shellfish. In this study, the shell edge and byssus of the P. penguin were used to study the non-lethal DNA extraction method. Shell and byssus samples were treated with decalcified and undecalcified method, DNA was extracted by marine animal tissue genomic DNA extraction kit method and organic solvent extraction method (OSE method) and marine animal tissue genomic DNA extraction kit method, then the obtained shell DNA and byssus DNA were amplified by PCR. The results showed that the DNA extraction efficiency of the byssus OSE method was significantly higher than that of shell DNA extraction method. There was no significant difference between the DNA extraction efficiency of decalcified and undecalcified byssus, which were 0.0167±0.0029 μg/mg and 0.0161±0.0031 μg/mg, respectively. DNA obtained by the OSE method for undecalcified byssus had the optimal highest purity, with an A260/280 value of 1.4000±0.00480, and A260/230 value was significantly higher than the other method for shell DNA and byssus DNA, at 0.9100±0.0800. All DNA extraction methods except the OSE method with decalcified shells were available for subsequent PCR amplification, and the sequencing results indicated that the DNA product was derived from P. penguin. In addition, neither shell nor byssus sampling methods resulted in mortality of P. penguin. In summary, the OSE method of undecalcified byssus is superior to other methods. The results of the study can provide a novel method for the extraction of non-lethal DNA, and lay a foundation for the selection and breeding of P. penguin and the conservation genetics of rare shellfish. |
| Key words: Pteria penguin non-lethal shell DNA extraction byssus DNA extraction |