Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
권호 표지

Isolation of Myrosinase and Glutathione S-transferase Genes and Transformation of These Genes to Develop Phenylethylisothiocyanate Enriching Chinese Cabbage

배추에서 항암물질 phenylethylisothiocyanate의 다량 합성을 위한 myrosinase와 glutathione S-transferase 유전자 분리 및 이를 이용한 형질전환체 육성

  • Park, Ji-Hyun (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Lee, Su-Jin (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Kim, Bo-Ryung (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Woo, Eun-Teak (Breeding Research Institute, Carrotop Seed Co.) ;
  • Lee, Ji-Sun (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Han, Eun-Hyang (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Lee, Youn-Hyung (Department of Horticultural Biotechnology, Kyunghee University) ;
  • Park, Young-Doo (Department of Horticultural Biotechnology, Kyunghee University)
  • 박지현 (경희대학교 생명과학대학 원예생명공학과) ;
  • 이수진 (경희대학교 생명과학대학 원예생명공학과) ;
  • 김보령 (경희대학교 생명과학대학 원예생명공학과) ;
  • 우은택 (캐로톱씨드 육종연구소) ;
  • 이지선 (경희대학교 생명과학대학 원예생명공학과) ;
  • 한은향 (경희대학교 생명과학대학 원예생명공학과) ;
  • 이윤형 (경희대학교 생명과학대학 원예생명공학과) ;
  • 박영두 (경희대학교 생명과학대학 원예생명공학과)
  • Received : 2011.08.17
  • Accepted : 2011.09.27
  • Published : 2011.12.31
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Abstract

To increase the anti-carcinogens phenylethylisothiocyanate (PEITC), myrosinase (MYR), and glutathione S-transferase (GST), genes related to PEITC pathway were isolated and the gene expressions were regulated by Agrobacterium transformation. Isolated cDNAs, MYR, and GST genes were 1,647 bp and 624 bp, respectively, and the protein expression was confirmed through pET system. Thereafter, we constructed a sense-oriented over-expressing myrosinase (pBMY) and RNAi down-regulated GST (pJJGST) binary vectors for the Chinese cabbage transformation. After the transformation, thirteen over-expressing transgenic Chinese cabbage plants (IMS) with pBMY and five down-regulated ones (IGA) with pJJGST were selected by PCR analysis. Selected $T_0$ transgenic plants were generated to $T_1$ plants by self-pollination. Based on the Southern blot analysis on these $T_1$ transgenic plants, 1-4 copies of T-DNA were transferred to Chinese cabbage genome. Thereafter, RNA expression level of myrosinase gene or GST gene was analyzed through real-time RT PCR of IMS, IGA, and non-transgenic inbred lines. In case of IMS lines, myrosinase gene was increased 1.03-4.25 fold and, in IGA lines, GST gene was decreased by 26.42-42.22 fold compared to non-transgenic ones, respectively. Analysis of PEITC concentrations using GC-MS it showed that some IMS lines and some IGA lines increased concentrations of PEITC up to 4.86 fold and up to 3.89 fold respectively compared to wild type. Finally in this study IMS 1, 3, 5, 12, and 15 and IGA 1, 2, and 4 were selected as developed transgenic lines with increasing quantities of anti-carcinogen PEITC.

본 연구는 배추에서 항암물질 PEITC의 함량을 높이기 위하여 PEITC 대사과정에서 관련 유전자인 myrosinase (MYR)와 Glutathione S-transferase(GST) 유전자를 분리하고 Agrobacterium tumefacien 형질전환 방법을 통하여 유전자 발현을 조절하였다. 분리된 MYR과 GST의 cDNA는 각각 1647bp와 624bp임을 확인하였고 pET system으로 단백질의 발현을 확인하였다. 형질전환을 위해서 MYR-과발현 벡터와 GST-발현억제 벡터를 제작하였으며 이를 이용하여 배추에 형질전환한 후 PCR 검정을 통해 MYR-과발현 벡터로 형질전환된 개체(IMS) 13개체를 GST-발현억제 벡터로 형질전환된 개체(IGA) 5개체를 선발하였다. 선발된 $T_0$ 개체는 $T_1$ 세대로 진전시켰으며 $T_1$ 형질전환 계통의 서던분석 결과 배추 genome내로 1-4 copy의 T-DNA가 삽입된 것을 확인하였다. 유전자 발현양을 real-time RT PCR로 조사한 결과 IMS는 발현량이 1.03-4.25배 증가하였고 IGA는 26.42-42.22배 감소하였다. IMS와 IGA의 각 계통에서 PEITC의 농도를 GC-MS 방법을 이용하여 확인한 결과 IMS는 PEITC 함량이 형질전환이 되지 않은 대조군에 비해 최대 4.86배까지 증가한 계통을 확인하였고 IGA는 최대 3.89배까지 증가된 계통을 확인하였다. 최종적으로 본 연구를 통하여 항암물질 PEITC량의 증가를 보인 형질전환계통 IMS 1, 3, 5, 12, 15 및 IGA 1, 2, 4를 선발하였다.

Keywords

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