Agrobacterium을 이용한 고추의 Transient Expression 시스템
Development of an Agrobacterium-mediated Transient Expression System for Intact Leaves of Chili Pepper
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Agrobacterium을 이용한 GUS 유전자를 효과적으로 발현시키기 위하여 수행되어진 실험 결과를 요약하면 다음과 같다. 박테이라 농도별 실험을 수행한 결과 균의 전처리 배양 농도 OD600nm 0.3일때 원심분리한 후 얻어진 균을 희석한 후의 최종 농도는 OD600nm 0.8로 맞춘 실험 처리구에서 GUS 유전자 발현율이55%로 가장 높게 나타났다. 병원성 유도 배지 내에 Acetosyringone (AS)이 첨가되지 않은 경우 GUS 유전자가 발현된 고추 잎을 얻을 수 없었으나, 200 μM을 첨가했을 때 90%의 가장 높은 GUS 유전자 발현율을 나타내어 많은 수의 GUS spots을 관찰할 수 있었다. Agrobacterium에 의한 고추 잎의 감염 정도를 조사한 바 Agrobacterium으로 감염시킨지 3일째부터는 박테리아에 의한 감염 정도가 심해져서 GUS 유전자 발현 정도가 약해지므로 Agrobacterium으로 감염시킨지 2일째 되었을 때 GUS 유전자 발현이 가장 강하게 나타난 것을 확인하였다. 이 같은 결과는 박테리아에 의한 감염이 일어난지 3일째 부터는 식물체의 감염부위 고사를 일으키는 것과 관련된 것으로 보인다.
Abstract
We established a transient gene expression system in chili pepper leaves based on Agrobacterium- mediated transformation of GUS gene. For the best GUS transient expression, two step culture system was adopted. When the Agrobacterium tumefaciens cell density of pre-culture was A600nm 0.3, the cells were harvested and diluted to A600nm 0.8 with virulence induction medium after cell harvested. The addition of acetosyringone (200 μM) in virulence induction step was a key factor for successful transient expression. Additionally, Younger leaves showed more effective transient expression than older leaves. Temporally, the strongest intensity of GUS expression was detected at 2 days after infiltration. These results demonstrate that Agrobacterium-mediated transient expression can be used for a simple in vivo assays of plant promoters, transcription factors and furthermore provide efficient protocol for chili pepper transformation.서 론
참고문헌 (25)
- Arroyo R, "In vitro plant regeneration from cotyledon and hypocotyls segments in two bell pepper cultivars", 10, pp.414~416 (1991)
- Belarmino MM, "Agrobacterium-mediated genetic transformation of a phalenopsis orchard", 19, pp.435~442 (2000)
- Butos MM, "Transient gene expression in electroporated bean cotyledon protoplasts", 9, pp.322~332 (1991)
- Begum D, "Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter", pp.1469~1479 (1991)
- Diaz I, "Plant regeneration from protoplasts of Capsicum annum", 7, pp.210~212 (1988)
- Dillen W,, "Electroporation-mediated DNA delivery to seedling tissue of Phaseolus vulgaris L. (common bean", 15, pp.119~124 (1995)
- Drake PMW, "Expression of the gusA in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation", 48, pp.151~155 (1997)
- Fari M, "Relationship between position and morphogenetic response of pepper hypocotyls explants cultured in vitro", 15, pp.207~213 (1981)
- Humara JM,, "Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer", 19, pp.51~58 (1999)
- Javier PR,, "Enhanced regeneration of tomato and pepper seedling explants for Agrobacterium-mediated transformation", 67, pp.173~180 (2001)
- Jefferson RA, "beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants", pp.3901~3907 (1987)
- Kapila J, "An Agrobacterium- mediated transient expression system for intact leaves", 122, pp.101~108 (1997)
- Kim SH,, "Constitutive expression of rice MADS box gene using seed explants in hot pepper (Capsicum annuum L.).", 12, pp.221~226 (2001)
- Lee SJ, "In vitro plant regeneration and Agrobacterium-mediated transformation from cotyledon explants of hot pepper", 5, pp.289~294 (1993)
- Lee YH,, "A new selection method for pepper transformation: callus-mediated shoot formation" (2004)
- Leon P, "Transient gene expression in protoplasts of Phaseolus vulgaris isolated from a cell suspension culture", 95, pp.968~972 (1991)
- Li D, "Establishment of highly efficient transformation system for pepper", 21, pp.785~788 (2003)
- Liu W, "Agrobacterium-induced gall formation in bell pepper and formation of shoot-like structures expressing introduced genes", pp.360~364 (1990)
- Mondal TK,, "Transgenic tea Camellia sinensis (L.) O. kuntze cv. Kangra Jat plants obtained by Agrobacterium-mediated transformation of somatic embryos", 20, pp.712~720 (2001)
- Pena L,, "High efficiency Agrobacterium- mediated transformation and regeneration of Citrus", 104, pp.183~191 (1995)
- Phillips GC, "Organogenesis in pepper tissue cultures", 4, pp.261~269 (1985)
- Seong ES, "The effect of Agrobacterium density on transformation efficiency in apple", 30, pp.215~219 (2003)
- Smith RH, "Agrobacterium tumefaciens transformation of monocotyledons", pp.301~309 (1995)
- Schmidt R, "Construction of an intron- containing marker gene splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation", pp.245~250 (1990)
- Zhu YX, "Transgenic sweet pepper plants from Agrobacteirum-mediated transformation", 16, pp.71~95 (1996)