菖蒲芋螺不同大小基因组DNA片段的分离制备

使用常规酚/氯仿抽提法(CTAB法)、离心柱法和磁珠吸附法,分别从菖蒲芋螺毒腺、肌肉及肝胰脏中提取基因组DNA,利用普通琼脂糖凝胶电泳与脉冲场电泳(PFGE)对基因组DNA片段进行分离,并检测提取DNA的大小和分布范围,同时,对脉冲场电泳条件进行了优化。结果表明,3种方法均可提取基因组DNA,其中CTAB法和离心柱法提取的毒腺DNA纯度高,产率大,可用于后续实验,而磁珠法产率适中且纯度不高,但离心柱法的DNA片段较小,大多在9 kb以下,而CTAB法提取的DNA片段较大,获得的20 kb以上的大片段量较多,更适合于大片段基因组文库的构建。3种芋螺组织中,肝胰脏产率最高但片段弥散有降解,毒腺DNA片段较大且集中,产率也较高,而肌肉的DNA片段产率最低且片段较小。因此,用CTAB法提取菖蒲芋螺毒腺的DNA更适合构建大片段基因组DNA文库。筛选高质量的菖蒲芋螺的DNA,利用优化的脉冲场电泳条件进行分离回收,获得了不同大小片段的DNA,为后续菖蒲芋螺不同载体系统的基因组文库构建奠定了基础。

Separation and Preparation of Different Size Genomic DNA Fragments of Conus Vexillum

In order to provide basis for construction of the genomic library of Conus vexillum (C.vexillum),different methods were used for isolation and extraction of different size fragments of genomic DNA from C.vexillum native to Hainan.Genomic DNA was isolated from venom glands,hepatopancreas and muscle of C.vexillum using three methods of CTAB,spin column and magnetic bead respectively.The genomic DNA fragments were separated by agarose gel electrophoresis and pulsed-field gel electrophoresis,of which the size and distribution range of extracted DNA was detected.The results showed that the genomic DNA could be extracted by all the three methods.The purity and yield of DNAs isolated by CTAB and centrifugal column methods using venom gland of C.vexillum were good and high,which could meet the following experimental requirements.While the yield of the magnetic bead method is less and the purity is not good.The length of most DNA fragments isolated by the centrifugal column method was less than 9 kb,while most fragments'sizes isolated by CTAB method were more than 20 kb.Among the three kinds of organs,hepatopancreas had highest yield of DNA,but its fragment was dispersed with degradation happened,the DNA yield of venom glands was high but less than hepatopancreas slightly,its integrity was the best;however the DNA yield of muscle was lowest and its fragments was smaller.The high quality genomic DNA fragments with different sizes were separated and prepared successfully by optimized pulsed-field gel electrophoresis.In conclusion,using CTAB method to isolate C.vexillum DNA is better than the other methods for big fragment genomic DNA library construction.Different size genomic DNA fragments recovered by pulsed-field gel electrophoresis would lay a foundation for further genomic library construction of C.vexillum using different gene library vector systems.