생태와환경 (Korean Journal of Ecology and Environment)

  • 제56권1호
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  • Pages.94-103
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  • 2023
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  • 2288-1115(pISSN)
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  • 2288-1123(eISSN)
한국수생태학회 (The Korean Society of Limnology)
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퇴적층 남조류 휴면세포의 생리적-분자생물학적 연구를 위한 Ludox 처리법

A Protocol of Ludox Treatment for Physiological and Molecular Biological Research of Freshwater Cyanobacteria

  • 김건희 (건국대학교 휴먼앤에코케어센터) ;
  • 유경은 (건국대학교 상허생명과학대학 환경보건과학과) ;
  • 호혜인 (건국대학교 상허생명과학대학 환경보건과학과) ;
  • 박채홍 (건국대학교 휴먼앤에코케어센터) ;
  • 김현진 (주식회사 평화엔지니어링) ;
  • 황순진 (건국대학교 상허생명과학대학 환경보건과학과)
  • Keonhee Kim (Human and Ecocare center, Konkuk University) ;
  • Kyeong-eun Yoo (Environmental Health Science, Sanghuh Life Science College, Konkuk University) ;
  • Hye-in Ho (Environmental Health Science, Sanghuh Life Science College, Konkuk University) ;
  • Chaehong Park (Human and Ecocare center, Konkuk University) ;
  • Hyunjin Kim (Pyunghwa Engineering Consultants) ;
  • Soon-Jin Hwang (Environmental Health Science, Sanghuh Life Science College, Konkuk University)
  • 투고 : 2023.03.13
  • 심사 : 2023.03.21
  • 발행 : 2023.03.31
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초록

휴면포자와 같은 남조류의 휴면세포는 남조류의 초기발생 및 대발생의 중요한 씨앗세포이다. 이러한 중요성으로 인해 퇴적층에 존재하는 휴면세포를 분리하기 위해서 다양한 방법들이 시도되었다. Ludox는 해양퇴적물의 세포분리에 주로 활용되는 용액이지만 담수에서는 정확한 사용법을 찾기 어렵다. 본 연구에서는 가장 많이 사용되는 두 가지 Ludox방법(퇴적물 직접처리, 퇴적물 증류수 현탁처리)을 비교하고, 담수 퇴적물에서 남조류 휴면세포의 분리 및 유전자 증폭 효율이 높은 방법을 제안하였다. 퇴적물에서 발견된 휴면세포는 대부분 염주말목의 휴면포자로써 Dolichospermum, Cylindrospermum, Aphanizomenon의 휴면포자 형태와 유사하였다. 퇴적물을 증류수에 현탁하여 처리한 시료보다 퇴적물 그대로 사용한 시료에서 20배 더 많은 휴면포자가 발견되었으며 증류수로 현탁된 퇴적물에서는 분리되지 않은 세포가 대부분 pellet 퇴적물 표층에서 발견되었다. Ludox를 통해 층 분리된 휴면포자는 수층의 특정 깊이에서 밀집하기보다는 주로 상층과 하층에 넓게 퍼져있었다. 퇴적물을 그대로 사용한 시료에서 mibC, Geo, 16S rDNA 유전자 모두 증폭산물이 확인되었으나 퇴적물을 증류수로 현탁한 시료에서는 모든 유전자의 증폭산물이 발견되지 않았다. 따라서 담수 퇴적물에서 남조류의 휴면세포를 분리하는 경우에는 5~10 g의 퇴적물을 전처리 없이 그대로 사용하며, 퇴적물량 4배 부피의 Ludox를 첨가할 때 세포 분리 및 유전자 증폭 효율이 높았다. 본 연구에서 제시하는 Ludox 처리방법은 담수퇴적층에 존재하는 남조류 휴면세포를 분리하기 위한 방법으로써 다른 생물군에서는 동일한 효율이 나타나지 않을 수 있다. 따라서 다른 생물군의 분리에 Ludox를 적용하기 위해서는 퇴적물 전처리 방법 및 대상생물이 존재하는 수층을 파악하는 사전실험이 반드시 필요하다.

Cyanobacterial resting cells, such as akinetes, are important seed cells for cyanobacteria's early development and bloom. Due to their importance, various methods have been attempted to isolate resting cells present in the sediment. Ludox is a solution mainly used for cell separation in marine sediments, but finding an accurate method for use in freshwater is difficult. This study compared the two most commonly used Ludox methods (direct sediment treatment and sediment distilled water suspension treatment). Furthermore, we proposed a highly efficient method for isolating cyanobacterial resting cells and eDNA amplification from freshwater sediments. Most of the resting cells found in the sediment were akinete to the Nostocale and were similar to those of Dolichospermum, Cylindrospermum, and Aphanizomenon. Twenty times more akinetes were found in the conical tube column using the sediment that had no treatment than in the sample treated by suspending the sediment in distilled water. Akinete separated through Ludox were mainly spread over the upper and lower layers in the column rather than concentrated at a specific depth in the column layer. The mibC, Geo, and 16S rDNA genes were successfully amplified using the sediment directly in the sample. However, the amplification products of all genes were not found in the sample in which the sediment was suspended in distilled water. Therefore, 5 g to 10 g of sediment is used without pretreatment when isolating cyanobacterial resting cells from freshwater sediment. Cell isolation and gene amplification efficiency are high when four times the volume of Ludox is added. The Ludox treatment method presented in this study isolates cyanobacterial resting cells in freshwater sediment, and the same efficiency may not appear in other biotas. Therefore, to apply Ludox to the separation of other biotas, it is necessary to conduct a pre-experiment to determine the sediment pretreatment method and the water layer where the target organism exists.

키워드

과제정보

본 연구는 한국환경산업기술원의 '수생태계 건강성 확보 기술개발사업'인 '최첨단 위치기반 USBL ROV 로봇을 탑재한 휴면포자 함유 퇴적물 준설 기술 개발' 과제에 의해 수행되었습니다 (2022003040003).

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