들잔디로부터 β-1,3-glucanase 유전자의 클로닝 및 특성분석
Molecular cloning and characterization of β-1,3-glucanase gene from Zoysia japonica steud
0
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
한국형 잔디에서는 다른 병에 비해 진전 속도가 빠르고주로 뿌리에서부터 발병하여 잔디를 고사시키고 발병 후구제하기 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 개발하기위해 식물방어반응에 중요한 역할을 하는 것으로 알려진PR-Protein 중 하나인 β-1,3-glucanase를 들잔디로부터 클로닝 하였다. β-1,3-glucanase는 바이러스나 균의 감염으로 인해 식물조직이 과민반응을 일으킬 때 세포내에서 생성되고 세포 외로 분비되어 세포 사이 공간에서 주로 병원균 저항성기능을 하는 것으로 알려져 있다. β-1,3-glucanase 단자엽식물 중 내병성에 대한 연구가 되어있는 옥수수, 밀, 보리, 벼의 염기서열에서 공통으로 보존되어 있는 부분을 이용해 degenerate PCR을 수행하고 얻어낸 sequence 를 통해 Full-length의 cDNA를 클로닝 하였다. E.coli overexpression을수행하여 목표 단백질을 대량 정제하여 in vitro 활성 측정 및 항균테스트를 진행하였다. 또한, ZjGlu1 유전자의 기능을 해석하기 위해 각각의 유전자를 도입한식물형질전환용 벡터를 제작하여 잔디 형질전환체 제작을 하였다. ZjGlu1 단백질을 이용하여 9개의 균주에 대해항균활성 테스트를 진행 한 결과 R. cerealis, F. culmorum, R.solani AG-1 (1B), T. atroviride 에서 항균활성을 보였으며, 형질전환체를 이용해 18s 유전자의 발현량을 상대로한 각 유전자의 기관별 발현량은 크게 차이없이 모든기관에 발현되는 것을 확인할 수 있었다.
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, β-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. β-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 β-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.
참고문헌 (27)
- 배은지, "우리나라 잔디 생산과 재배 관리실태", Weed & Turfgrass Science, 2(1), pp.95~99 (2013), 한국잔디학회
- Liu, "Cloning and characterization of a wheat β-1,3-glucanase gene induced by the stripe rust pathogen pucinia striiformis F. sp", Tritici. Mol Biol Rep, 37, pp.1045~1052 (2010)
- Cheong YH, "Molecular cloning of a soybean class III beta-1,3-glucanase gene that is regulated both developmentally and in response to pathogen infection", Plant Sci, 154(1), pp.71~81 (2000)
- Choi HW, "The pepper Extracelluar Xyloglucan-Specific Endo-1,4-Glucanase Inhibitor Protein Gene, CaXEGIP1, Is Required for Plant Cell Death and Defense Reponses", Plant Physiol, 161, pp.384~396 (2013)
- Claude P, "Antifungal Proteins", Appl Environ Microb, 67, pp.2883~2894 (2001)
- Deannna L. Funnell, "Expression of the tovacco β-1,3-glucanase gene, PR-2d, following induction of SAR with peronospora tabacina", Physiol Mol Plant P, 65, pp.285~296 (2004)
- Ganapathi Sridevi, "Combined expression of chitinase and β-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani", Plant Sci, 275, pp.283~290 (2008)
- Ganesan M, "Overexpression of phytochrome A and its hyperactive mutant improves shade tolerance and turf quality in creeping bentgrass and zoysiagrass", Planta, 236, pp.1135~1150 (2012)
- Gerhard Leubner-metzger, "Fuctions and regulation of plant β-1,3-glucanase (PR-2)", ~ (1999)
- Irene Romero, "Functionality of a classⅠbeta-1,3-glucanase from skin of table grapes berries", Plant Sci, 174, pp.641~648 (2008)
- Janice Lisboa de Marco, "Purification and Chracterization of a β-Glucanase Produced by Trichoderma harzianim showing Biocontrol Potential", Braz Arch Biol Techn, 50, pp.21~29 (2007)
- 강재영, "잔디 병해 방제를 위한 약용식물의 항균작용 탐색", Weed & Turfgrass Science, 2(1), pp.70~75 (2013), 한국잔디학회
- Kim DS, "Disease and weed occurring in zoysiagrass", National institute of forest science, 517 (2013)
- "Korea forest service 2012", ~
- Lee JP, "Contribution of turfgrass industry to the economy in Florida state and present and future of Korea turfgrass industry", Korean Turfgrass Science, 15, pp.187~198 (2001)
- 이상명, "Rhizoctonia solani AG2-2 IV에 대한 Helicosporium sp. KCTC 0635BP의 항균활성", Weed & Turfgrass Science, 2(2), pp.202~206 (2013), 한국잔디학회
- Mahmoud WF, "Cold-Active Winter Rye Glucanases with Ice-Binding Capacity1,2", Plant Physiol, 141, pp.459~1472 (2006)
- P. B. Kirti, "Cloning, Characterization and Antifungal Activity of Defensin Tfgd1 fromTrigonella foenum-graecum L.", BMB reports, 39(3), pp.278~283 (2006), 한국생화학분자생물학회
- Qiao LX, "Chracterization of the β-1,3-glucanase gene in peanut(Archis hypogaea L.) by cloning and genetic transformation", Genetics and Molecular Research, 13, pp.1893~1904 (2014)
- Robert Leah, "Biochemical and Molecular Characterization of Three Barley Seed Proteins with Antifungal Properties", J Biol Chem, 266(3), pp.1564~1573 (1991)
- Rui Cheng, "Recombinant expression and characterization of an acid-, alkali- and slat-tolerant β-1,3-1,4-glucanase from Paenibacillus sp. S09", Biotechnol Lett, 36, pp.797~803 (2014)
- Saboki Ebrahim, "Science against microbial pathogens; communicating current research and technological advanced" (2011)
- Shi Y, "Cloning and expression analysis of two β-1,2-glucanase genes from strawberry", J Plant physiol, 163, pp.956~967 (2006)
- 이효연, "Production of Herbicide-tolerant Zoysiagrass by Agrobacterium-mediated Transformation", Molecules and Cells, 16(1), pp.19~27 (2003), 한국분자세포생물학회
- Vaiyapuri Balansubramanian, "Plant β-1,3-glucanase: their biology functions and transgenic expression against phytopathogenic fungi", Biotechnol Lett, 34, pp.1983~1990 (2012)
- Vivek Dogra, "Cloning and functional characterization of β-1,3-glucanase gene from Podophyllum hexandrum – A high altitude Himalayan plant", Gene, 554, pp.25~31 (2015)
- Xie YR, "ZmGns, a maize class I β-1,3-glucanase, is induced by biotic stresses and possesses strong antimicrobial activity", J Integr Plant Biol, 57(3), pp.271~283 (2015)