海南产桶形芋螺线粒体基因组DNA提取条件的优化

提取海南产桶形芋螺线粒体基因组完整DNA (mtDNA),并对提取条件进行优化。以桶形芋螺腹足肌肉、毒腺和肝胰脏三个不同组织为材料,分别采用改进高盐沉淀法、细胞器/磁珠法和试剂盒提取三种方法,提取桶形芋螺mtDNA,并利用琼脂糖凝胶电泳和紫外分光光度计对提取mtDNA的纯度和浓度进行测定。以coxⅠ-rRNA小亚基基因和α-芋螺毒素基因设计引物,通过PCR反应来确证所提取的DNA确实是mtDNA。试剂盒法提取肝胰脏、高盐沉淀法提取肝胰脏和腹足肌肉组织这三种方法的产率很高,分别为44.4μg/mg、43.3μg/mg和32.6μg/mg。A260/280比值表明,改进高盐沉淀法提取毒腺和腹足肌肉组织,细胞器磁珠法提取腹足肌肉组织的mtDNA纯度很高。综合比较,采用改进高盐沉淀法,利用桶形芋螺腹足肌肉组织所提取的mtDNA产率高、质量好、纯度高。高质量芋螺mtDNA的获取为利用分子生物学方法对芋螺进行遗传进化分析和系统分类提供了基础。

Optimization of Extraction Conditions in Mitochondrial Genomes DNA of Conus betulinus Linnaeus from Hainan

To extract complete mitochondrial genome DNA(mtDNA) of Conus betulinus linnaeus from Hainan and to optimize the extraction conditions, This study took three tissues of C. betulinus, foot muscle, venom gland and hepatopancreas as materials, and extracted mtDNA of Conus betulinus by three different methods, namely,improved high-concentration-salt precipitation method, organelle/magnetic bead method and kit extraction method respectively. The purity and concentration of each mtDNA were determined by agarose gel electrophoresis and ultraviolet spectrophotometer. The primers was designed with coxⅠ-rRNA small subunit gene and α-conotoxin gene, and the extracted DNA was confirmed to be mtDNA by PCR reaction. The three methods of extraction of hepatopancreas by kit extraction method, hepatopancreas by improved high-concentration-salt precipitation method and foot muscle tissue by improved high-concentration-salt precipitation method had high yields of 44.4 μg/mg,43.3 μg/mg and 32.6 μg/mg, respectively. The A260/280 ratio showed that the purity of mtDNA extracted from venom gland and foot muscle tissue by improved high salt precipitation method and from foot muscle tissue by organellemagnetic beads method was high. Comparing comprehensively, the mtDNA extracted from foot muscle tissue of Conus betulinus by improved high-concentration-salt precipitation method had high yield, good quality and high purity. The acquisition of high quality Conus mtDNA would provide a basis for the genetic evolutionary analysis and systematic classification of conus using molecular biology methods.