Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Development of a Highly Efficient Isolation Protocol for Mitochondrial DNA and RNA Using Small Scale Plant Tissues

식물의 초경량 조직을 이용한 미토콘드리아의 DNA와 RNA 정제

  • Kim Kyung-Min (Department of Environmental Horticulture, Sangju National University) ;
  • Lim Yong-Suk (Department of Biotechnology, Daegu University of Foreign Studies) ;
  • Shin Dong-Ill (Department of Plant Genetic Engineering, Catholic University of Daegu) ;
  • Sul Ill-Whan (Department of Biotechnology, Daegu University of Foreign Studies)
  • 김경민 (상주대학교 생명자원과학대학 환경원예학과) ;
  • 임용숙 (대구외국어대학교 생명공학부) ;
  • 신동일 (대구가톨릭대학교 자연대학 생명자원학부) ;
  • 설일환 (대구외국어대학교 생명공학부)
  • Published : 2006.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

We present a fast and simple protocol for purification of mitochondria, mitochondrial DNA, and RNA from small amounts of tomato leaves. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. Mitochondria was confirmed by MitoTracker. The mitochondrial DNA was not contaminated by plastid DNA, was successfully used for PCR. Similarly, the isolated mitochondrial RNA was not contaminated only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for RT-PCR analysis

본 실험에서는 토마토의 종자를 기내 배양하여 얻어진 1g 이하의 무균 잎 조직을 이용하여 미토콘드리아를 분리 정제하여 MitoTracker를 이용하여 세포생물학적으로 확인하였고, 이들의 mt를 이용하여 미토콘드리아 DNA와 RNA를 추출과 검정을 하였다. 또한 고농도의 이온성을 이용하여 미토콘드리아와 mtDNA 및 mtRNA을 추출할 수 있었으며, 식물의 여러 종류에도 사용되어질 수 있을 것이다. mtDNA는 PCR 분석에 의하여 plastid DNA와 혼재되어 있지 않음을 확인하였다. mtRNA는 RT-PCR 분석을 통하여 plastid RNA와 흔재되어 있지 않음을 확인할 수 있었다.

Keywords

References

  1. Bookjans, G., B. M. Stummann and K. W. Henningsen. 1984. Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength. Anal. Biochem. 141, 244-247 https://doi.org/10.1016/0003-2697(84)90452-4
  2. Chomeczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction. Anal. Bichem. 162, 156-159
  3. Douce, R., J. Bourguignon, R. Brouquisse and M. Neuburger. 1987. Isolation of plant mitochondria: general principles and criteria of integrity. Meth. Enzymol. 148, 403-415 https://doi.org/10.1016/0076-6879(87)48039-7
  4. Hsu, C. I. and B. C. Mullin. 1988. A new protocol for isolated of mitochondria DNA from cotton seedlings. Plant Cell Reports 7, 356-360 https://doi.org/10.1007/BF00269937
  5. Kemble, R. J. 1987. A rapid single leaf, nucleic acid and assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species. Theor. Appl. Genet. 73, 451-458 https://doi.org/10.1007/BF00262515
  6. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  7. Pehu, E. 1991. RFLP analysis of organellar genomes in somatic bybrids. In: Plant Tissue Culture Manual D6, pp. 1-8, Kluwer Academic Publishers, The Netherlands
  8. Perez, C., J. F. Bonavent and A. Berville. 1990. Preparation of mitochondrial DNAs from sunflowers (Helianthus annuus L.) using a medium with high ionic strength. Plant Mol. Biol. Reptr. 8, 104-113 https://doi.org/10.1007/BF02669764
  9. Scotti, N., T. Cardi and L. Marechal-Drouard. 2001. Mitochondrial DNA and RNA isolation from small amounts of potato tissue. Plant Molecular Biology Reports 19, 67a-67h https://doi.org/10.1007/BF02824080
  10. Triboush, S., N. G. Danilenko and O. G. Davydenko. 1998. A method for isolation of chloroplast DNA and mitochondrial DNA from sunflower. Plant Mol. Biol. Reptr. 16, 183-189 https://doi.org/10.1023/A:1007487806583
  11. Wilson, A. J. and P. S. Chourey. 1984. A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources. Plant Cell Rep. 3, 237-242 https://doi.org/10.1007/BF00269301