Improved lignin contents of phragmites communis with molecular approach

국문 초록 (Abstract)

본 연구는 최근 바이오에너지의 원료로 각광받고 있는 갈대의 조직배양 기술 및 대량생산 기술과, 리그닌 생합성에 관련하는 갈대 특이적 COMT 와 AOMT유전자를 분리, 그리고 갈대의 형질전환법 확립과 더불어 리그닌의 함량을 낮추어 보다 바이오에너지 원료로서의 이용성을 증대시킨 갈대 형질전환체의 획득에 관한 것이다.
갈대의 callus 유기는 아직 갈대의 연구에서 보고된 바 없는 4-FA 함유배지에서 가장 좋은 결과를 보였으며, 갈대의 재분화에서는 NAA와 BAP의 차이가 클 때 (NAA 0.5mg/L, BA 2mg/L) 좋은 재분화율을 보였다. 습지식물인 갈대는 고체배지보다 액체배지에서의 성장이 월등히 좋았으며, 대량생산 적용 시 최적 sucrose의 농도는 60g/L로 확인되었다.
리그닌 생합성에 관련하는 Phenylpropanoid pathway 상의 중요 효소인 COMT와 AOMT의 유전자를 분리하였다. 조직별 COMT유전자와 AOMT 유전자의 발현을 mRNA레벨에서 조사한 결과, 뿌리와 잎에서 높은 발현량을 보였으며, 줄기와 뿌리줄기에서 낮은 발현량을 보였다. 또한 시기별 분석에서 AOMT는 노화여부와 상관없이 거의 일정한 발현을 보였지만, COMT는 노화가 진행될수록 크게 증가함을 보였다. 또한 COMT와 AOMT는 호르몬 등의 생장조절물질의 처리에서 2,4-D 처리와 자스몬산, 살리실산에 크게 반응하였다.
한 식물의 이용성 증대를 위해서는 형질전환법의 확립이 우선되는 문제라 할 수 있다. 하지만 아직 갈대에 대한 형질전환법은 보고된 바가 없다. Part4에서는 Agrobacterium tumefaciens를 이용한 갈대의 형질전환에 성공하여, GUS gene의 발현을 RNA 레벨과 단백질 레벨에서 확인하였고, callus 단계에서의 염색에도 성공하였다. 또한 part4의 주요목적인 COMT 와 AOMT의 RNA interferencing을 통한 lignin 저하 역시 RNA 레벨에서 확인하였다.

본 실험에서는 갈대 작물에서 처음으로 lignin 생합성 관련유전자인 COMT 와 AOMT를 분리하였고, 이는 바이오에너지 작물로 각광받는 갈대의 연구에 이용될 수 있으리라 생각한다. 또한, 아직 보고된 바 없는 갈대의 형질전환법 역시 앞으로의 갈대 작물의 이용성 증대에 작은 도움이 되리라 생각된다.

다국어 초록 (Multilingual Abstract)

Reed grass (Pragmites communis Trin.) is one of the widely distributed perennial grass species in the world living in a wide variant environment, especially in wet land. Recently it has been expected to be the raw material of bio-energy which can be supplied stably with lingo-cellulose without effects on food resources. However, there is seldom methodical research on reed.
An in vitro culture system for the large-scale propagation of Phragmites communis Trin. (common reed) was established in part I by optimizing culturing conditions for callus induction and differentiation along with plant propagation using regenerated plantlets. Callus formation was induced from stem segments with callus induction medium containing auxin 4-fluorophenoxyacetic acid (4-FA) or 2,4-dichlorophenoxy acetic acid (2,4-D). A high frequency of callus induction was observed when applied for the first time on 4-FA 1.0 mg L-1 medium (up to 93%).
Regeneration system for reed was studied. The highest regeneration capacity was associated with the medium containing 0.05 mg L-1 NAA and 2 mg L-1 BA, and the combination of 0.2 mg L-1 NAA and 2 mg L-1 BA. These regeneration and propagation systems could be used for mass propagation or an in vitro culture system.
The Caffeic acid O-methyltransferase (COMT) and Caffeoyl CoA O-methyltransferase (CCoAOMT) of Phragmites communis Trin. (common reed) were studied in part Ⅱ. COMT and CCoAOMT are important genes for lignin biosynthesis. COMT and CCoAOMT were found and showed high affinity with amino acid sequences of other gramineous plant’s COMT and CCoAOMT. The deduced protein sequence from cDNA shares homology with the reported gramineous plants. Southern analysis indicates that multi gene codes for both two enzyme in reed. COMT and CCoAOMT exist in distinct organs, and the highest expression level of two enzymes was observed in root and leaf. Temporal expression of these enzyme analysis showed that the expression level of CCoAOMT was gradually increased expression level while COMT was ascended steeply as developmental stage proceeded in reed.

Agrobacterium tumefiaciens mediated transformation is a widely used transformation method in various plants. Despite the recent attention on reed as a bio-energy resource, transformation of reed has been not reported yet. Transformation system for reed was reported in part Ⅳ. Callus originated from Phragmites communis Trin. was used for transformation. The CaMV 35S promoter inserted pCAMBIA 1381 vector was transformed in callus mediated by Agrobacterium tumefaciens. Generated green embryos in selection medium were stained for verifying expressed GUS gene. The GUS transgenic plants were ascertained on RNA expression level and protein expression level also.
Caffeic acid O-methyltransferase (COMT) and Caffeoyl CoA O-methyltransferase (CCoAOMT) involved in phenylpropanoid pathway play a crucial role in lignin biosynthesis. After establishing transformation procedure of reed, COMT and CCoAOMT deficient transgenic plants were produced. The transgenic plants were showed severely decreased lignin level and some of transformants showed dwarfism caused by insufficient lignin.

목차 (Table of Contents)

• Literature review 1
• Part I: Induction of somatic embryogenesis and plant regeneration in the reed grass (Phragmites communis Trin.)
• Ⅰ.Introduction 9
• Ⅱ. Materials and methods 11
• Ⅲ. Results and discussion 14
• Part Ⅱ: Characterization and expression pattern of COMT and CCoAOMT genes from Phragmites communis Trin.
• Ⅰ. Introduction 27
• Ⅱ. Materials and methods 28
• Ⅲ. Results and discussion 32
• Part Ⅲ: Inhibition of the gene expression for COMT and CCoAOMT by RNA interference in Phragmites communis Trin.
• Ⅰ. Introduction 46
• Ⅱ. Materials and methods 47
• Ⅲ. Results and discussion 51
• Part Ⅳ: General discussion 60
• References 66
• 국문초록 77