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2018 Vol.36, Issue 3 Preview Page

Research Article

Reduced Ethylene Production in Tomato Fruits upon CRSPR/Cas9-mediated LeMADS-RIN Mutagenesis

토마토 과실에서 CRSPR/Cas9 시스템 이용 LeMADS-RIN 유전자 편집에 의한 에틸렌 생산량 감소

Yu Jin Jung1,2
,
Geung-Joo Lee3
,
Sangsu Bae4
,
Kwon Kyoo Kang1,2
정 유진1,2
,
이 긍주3
,
배 상수4
,
강 권규1,2
Department of Horticultural Life Science, Hankyong National University
Institute of Genetic Engineering, Hankyong National University
3Department of Horticulture, Chungnam National University
4Department of Chemistry, Hanyang University
한경대학교 원예생명과학과
한경대학교 유전공학연구소
3충남대학교 원예학과
4한양대학교 화학과
*교신저자. *Corresponding Author. 
June 2018. pp. 396-405
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Abstract

Recent progress in genome editing methods has opened new opportunities for reverse genetics- based studies in plants. The clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genomic region of a variety of organisms including plants. Here, we described a high-frequency targeted mutagenesis utilizing Agrobacterium-delivered CRISPR/Cas9 in tomato. This system consists of an Agrobacterium binary vector and three guide RNAs for single gene targeting. We evaluated the system for its mutagenesis frequency and heritability using LeMADS- RIN gene of tomato. T0 transgenic events carrying mutations in the LeMADS-RIN gene occurred at rates over 10.6% mutants per transgenic event in both ‘Mamirio’ and ‘Golden Bell’ tomato genotypes. Three independent T1 transgenic lines and wild-type (WT) tomato plants were used for ethylene analysis. Compared with WT plants, edited mutants exhibited more incompletely-ripening fruits and lower ethylene contents. Following genetic combination through segregation, null segre-gants carrying only the desired mutant alleles without the CRISPR transgene could be retrieved among the T1 progeny. These Cas9/gRNA transgenic lines, therefore, can be used to convey the CRISPR- based mutagenesis by genetic cross to tomato lines that are not amenable to genetic transformation.

Keywords
Agrobacterium
fruits ripening
gene editing
golden bell
mamirio
single-guide RNA

게놈편집 방법으로 최근 주목 받고 있는 CRISPR/CRISPR-associated 9 endonuclease(CRISPR/Cas9) 시스템은 식물에서 역유전학 기반 연구를 위한 새로운 기회가 되었으며, 특정 게놈 영역에서 돌연변이를 유발하는데 사용되는 새로운 전략으로 자리잡고 있다. 본 연구에서는 Agrobacterium법을 이용하여 CRISPR/Cas9 효소 및 sgRNA가 포함한 식물 벡터를 토마토 게놈에 도입하여 LeMADS-RIN 유전자의 게놈 편집을 유도하였다. 총 17개의 single copy가 도입된 형질전환체(T0)를 선발하여 sgRNA 영역의 PAM 영역 부근에서 T/A로 치환된 변이체, 1bp에서 최대 28bp의 결실 변이체를 선발하여 계통화하였다. 그 중 TG4, TG12와 TG26의 과실에서 에틸렌 생산량의 변화를 살펴본 결과 대조구에 비해 현저히 감소되었으며, 다양한 과색의 변이를 보였다. 따라서 이들 변이체 후대에서 CRISPR/Cas9을 포함한 T-DNA영역이 없는 null segregants을 선발하여 저장성이 좋은 토마토 육성에 이용할 수 있다.

키워드
아그로박테리움
과실숙성
유전자 편집
‘골든벨’
‘마미리오’
단일가이드 RNA
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Information
  • Publisher :KOREAN SOCIETY FOR HORTICULTURAL SCIENCE
  • Publisher(Ko) :한국원예학회
  • Journal Title :Horticultural Science and Technology
  • Journal Title(Ko) :원예과학기술지
  • Volume : 36
  • No :3
  • Pages :396-405
  • Received Date : 2017-09-14
  • Revised Date : 2017-10-10
  • Accepted Date : 2017-10-10
  • DOI :https://doi.org/10.12972/kjhst.20180039
Journal Informaiton Horticultural Science and Technology Horticultural Science and Technology
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