Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
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Comparison of RNA Interference-mediated Gene Silencing and T-DNA Integration Techniques for Gene Function Analysis in Chinese Cabbage

RNA Interference 및 T-DNA Integration 방법에 의한 배추 기능유전자 Silencing 효과 비교

  • Yu, Jae-Gyeong (Department of Horticultural Biotechnology, Kyung Hee University) ;
  • Lee, Gi-Ho (Department of Horticultural Biotechnology, Kyung Hee University) ;
  • Park, Young-Doo (Department of Horticultural Biotechnology, Kyung Hee University)
  • 유재경 (경희대학교 생명과학대학 원예생명공학과) ;
  • 이기호 (경희대학교 생명과학대학 원예생명공학과) ;
  • 박영두 (경희대학교 생명과학대학 원예생명공학과)
  • Received : 2012.05.09
  • Accepted : 2012.07.18
  • Published : 2012.12.31
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Abstract

To compare RNA interference-mediated gene silencing technique and T-DNA integration for gene function analysis in Chinese cabbage, BrSAMS-knockout (KO) line and BrSAMS-knockdown (KD) line were used. The KO line had lost the function of a Brassica rapa S-adenosylmethionine synthetase (BrSAMS) gene by T-DNA insertion and the KD line had shown down-regulated BrSAMS genes' expression by dsRNA cleavage. From microarray results of the KO and KD lines, genes linked to SAMS such as sterol, sucrose, homogalacturonan biosynthesis and glutaredoxin-related protein, serine/threonine protein kinase, and gibberellin-responsive protein showed distinct differences in their expression levels. Even though one BrSAMS gene in the KO line was broken by T-DNA insertion, gene expression pattern of that line did not show remarkable differences compared to wild type control. However, the KD line obtained by RNAi technique showed prominent difference in its gene expression. Besides, change of polyamine and ethylene synthesis genes directly associated with BrSAMS was displayed much more in the KD line. In the microarray analysis of the KO line, BrSAMS function could not be clearly defined because of BrSAMS redundancy due to the genome triplication events in Brassicaceae. In conclusion, we supposed that gene knock-down method by RNAi silencing is more effective than knock-out method by T-DNA insertion for gene function analysis of polyploidy crops such as Chinese cabbage.

본 연구는 배추의 유전자 기능분석을 위한 RNAi 유전자 침묵 기법과 T-DNA 삽입 기법을 비교하기 위해 수행하였다. 두 종류의 형질전환 계통이 이용되었으며 BrSAMS-knockout(KO) 계통은 T-DNA 삽입으로 한 개의 Brassica rapa S-adenosylmethionine synthetase(BrSAMS) 유전자가 기능을 상실한 계통이었으며 BrSAMS-knockdown(KD) 계통은 RNAi 방법을 통해 BrSAMS 유전자들의 발현이 억제된 계통이었다. KO 계통과 KD 계통의 microarray 분석 결과에서는 SAMS 유전자와 관련된 sterol, 자당, homogalacturonan 생합성 및 glutaredoxin-related protein, serine/threonine protein kinase, 그리고 gibberellin-responsive protein 유전자들의 발현 수준이 뚜렷한 차이를 보여 주었다. 그러나 KO 계통의 유전자 발현 양상은 하나의 BrSAMS 유전자가 기능을 상실하였음에도 불구하고 대조 계통과 비교하여 RNAi기법을 적용한 KD 계통에 비해 큰 차이를 보여주지 못했다. 또한 직접적으로 SAMS 유전자와 관련된 폴리아민과 에틸렌 합성 유전자들의 발현 변화도 KD 계통에서 더 잘 나타났다. 본 연구에서 microarray 결과를 이용한 KO 계통의 BrSAMS 기능분석은 배추과식물의 게놈 triplication 발생으로 인하여 다수로 존재하는 SAMS 유전자들 때문에 명확한 결론을 얻을 수 없었다. 결론적으로 배추와 같은 배수체 작물의 유전자 기능 분석은 RNAi silencing에 의한 유전자 knock-down 기법이 T-DNA 삽입에 의한 knock-out 기법보다 더욱 효율적인 것으로 나타났다.

Keywords

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