조사료로서 소화율을 향상시킨 신품종 형질 전환 오차드그래스를 개발할 목적으로 lignin 합성경로에 있어서 중요한 효소 유전자 중의 하나인 COMT 유전자를 cloning하여 그 특성을 해명하였다. 오차드그래스의 COMT 유전자는 식물체의 전 조직에서 발현되고 있었으며, 특히 줄기와 뿌리조직에서 높은 발현량을 나타냄으로서 목질화에 크게 관여하는 lignin 생합성 유전자일 것으로 판단되었다. Dgcomt 유전자의 발현을 억제시킨 형질전환 오차드그래스를

Background : Miscanthus sinensis is a diploid hybrid and a temperate, perennial, cross-pollinating grass used as bioenergy plant, biomass production and high quality cellulose and ethanol production. This study was to carried out to investigate the expression of MsCOMT gene and the variation of lignocellulosic component and phenolic compounds contents in transgenic plants. Methods and Results : Multiple bands were detected from the homologous region of the COMT gene in PCR analysis. In order to obtain more detailed results, putative transgenic lines were estimated by RT-PCR analysis to confirm the expression of mRNA. Also, analysis of the lignin, cellulose, hemicellulose, and phenolic compound contents of transgenic Miscanthus plants were performed. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose contents in transgenic plants were not increased. Variation in cellulose and hemicellulose contents had no correlation with variation in lignin content of transgenic plants. Conclusion : In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.

Background : Miscanthus is a diploid hybrid and a temperate, perennial, cross-pollinating grass used as bioenergy plant, biomass production and high quality cellulose and ethanol production. This study was to determine an efficient transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of MsCOMT–AS gene. Methods and Results : An efficient transformation of callus from M. sinensis was established using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish stable transformation system, we obtained high transformation rate from callus by various transformation factor explant type, strain, co-culture periods, acetosyringone concentration, and selective marker. Finally, in this study, seven putative transgenic plants were obtained. Through various tests including PCR analysis and southern blot were to detect antisense of COMT digested Xba I and Sac I restriction enzymes. The biomass of the control plant was superior than transgenic plants. Conclusion : This study was to develop transgenic Miscanthus sinensis by Agrobacterium tumerfeciens mediated transformation to produce high bioethanols and to reduce the lignin content of transgenic plants. Detailed characterization of the transgenic plants revealed interesting finding about COMT gene expression in the segregates

Caffeic acid O-methyltransferase (COMT) methylates N-acetylserotonin into melatonin; that is, it has N-acetylserotonin O-methyltransferase (ASMT) activity. The ASMT activity of COMT was first detected in Arabidopsis thaliana COMT (AtCOMT). To confirm the ASMT activity of COMT in other plant species, we evaluated the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT). Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The Km and Vmax values for the ASMT activity of OsCOMT were 243 μm and 2,400 pmol/min/mg protein, which were similar to those of AtCOMT. Similar to AtCOMT, OsCOMT was localized in the cytoplasm. In vitro ASMT activity was significantly inhibited by either caffeic acid or quercetin in a dose-dependent manner. Analogously, in vivo production of melatonin was significantly inhibited by quercetin in 4-week-old detached rice leaves, suggestive of a positive role of COMT in melatonin biosynthesis in plants.