Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Molecular cloning and characterization of β-1,3-glucanase gene from Zoysia japonica steud

들잔디로부터 β-1,3-glucanase 유전자의 클로닝 및 특성분석

  • Kang, So-Mi (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Kang, Hong-Gyu (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Sun, Hyeon-Jin (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Yang, Dae-Hwa (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Kwon, Yong-Ik (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Ko, Suk-Min (BVC, Korea Research Institute of Bioscience & Biotechnology) ;
  • Lee, Hyo-Yeon (Subtropical Horticulture Research Institute, Jeju National University, Faculty of Biotechnology, Jeju National University)
  • 강소미 (제주대학교 아열대원예산업연구소) ;
  • 강홍규 (제주대학교 아열대원예산업연구소) ;
  • 선현진 (제주대학교 아열대원예산업연구소) ;
  • 양대화 (제주대학교 아열대원예산업연구소) ;
  • 권용익 (제주대학교 아열대원예산업연구소) ;
  • 고석민 ;
  • 이효연 (제주대학교 아열대원예산업연구소, 제주대학교 분자생명공학전공)
  • Received : 2016.12.12
  • Accepted : 2016.12.20
  • Published : 2016.12.31
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Abstract

Rhizoctonia leaf blight (large patch) has become a serious problem in Korean lawn grass, which is extremely hard to treat and develops mostly from the roots of lawn grass to wither it away. Rhizoctonia leaf blight (large patch) is caused by Rhizoctonia solani AG2-2 (IV). To develop zoysia japonica with strong disease tolerance against this pathogenic bacterium, ${\beta}-1,3-glucanase$ was cloned from zoysia japonica, which is one of the PR-Proteins known to play a critical role in plant defense reaction. ${\beta}-1,3-glucanase$ is known to be generated within the cells when plant tissues have a hypersensitive reaction due to virus or bacterium infection and secreted outside the cells to play mainly the function of resistance against pathogenic bacteria in the space between the cells. This study utilized the commonly preserved part in the sequence of corn, wheat, barley, and rice which had been researched for their disease tolerance among the ${\beta}-1,3-glucanase$ monocotyledonous plants. Based on the part, degenerate PCR was performed to find out the sequence and full-length cDNA was cloned. E.coli over-expression was conducted in this study to mass purify target protein and implement in vitro activation measurement and antibacterial test. In addition, to interpret the functions of ZjGlu1 gene, each gene-incorporating plant transformation vectors were produced to make lawn grass transformant. Based on ZjGlu1 protein, antibacterial activity test was conducted on 9 strains. As a result, R. cerealis, F. culmorum, R.solani AG-1 (1B), and T. atroviride were found to have antibacterial activity. The gene-specific expression amount in each organ showed no huge difference in the organs based upon the transformant and against 18s gene expression amount.

한국형 잔디에서는 다른 병에 비해 진전 속도가 빠르고 주로 뿌리에서부터 발병하여 잔디를 고사시키고 발병 후 구제하기 매우 어려운 라이족토니아잎마름병(라지패취)이 큰 문제로 대두되고 있다. 라이족토니아잎마름병(라지패취)은 Rhizoctonia solani AG2-2 (IV)병원균에 의해 발생하는데, 이 병원균에 강한 내병성 들잔디를 개발하기 위해 식물방어반응에 중요한 역할을 하는 것으로 알려진 PR-Protein 중 하나인 ${\beta}-1,3-glucanase$를 들잔디로부터 클로닝 하였다. ${\beta}-1,3-glucanase$는 바이러스나 균의 감염으로 인해 식물조직이 과민반응을 일으킬 때 세포내에서 생성되고 세포 외로 분비되어 세포 사이 공간에서 주로 병원균 저항성기능을 하는 것으로 알려져 있다. ${\beta}-1,3-glucanase$ 단자엽식물 중 내병성에 대한 연구가 되어있는 옥수수, 밀, 보리, 벼의 염기서열에서 공통으로 보존되어 있는 부분을 이용해 degenerate PCR을 수행하고 얻어낸 sequence를 통해 Full-length의 cDNA를 클로닝 하였다. E.coli overexpression을 수행하여 목표 단백질을 대량 정제하여 in vitro 활성 측정 및 항균테스트를 진행하였다. 또한, ZjGlu1 유전자의 기능을 해석하기 위해 각각의 유전자를 도입한 식물형질전환용 벡터를 제작하여 잔디 형질전환체 제작을 하였다. ZjGlu1 단백질을 이용하여 9개의 균주에 대해 항균활성 테스트를 진행 한 결과 R. cerealis, F. culmorum, R.solani AG-1 (1B), T. atroviride 에서 항균활성을 보였으며, 형질전환체를 이용해 18s 유전자의 발현량을 상대로 한 각 유전자의 기관별 발현량은 크게 차이없이 모든기관에 발현되는 것을 확인할 수 있었다.

Keywords

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