CCoAOMT 유전자 발현 억제에 의한 현사시나무의 화학조성 변화

Abstract

본 연구에서는 리그닌 전구물질 생합성에 관여하는 CCoAOMT (Caffeoyl-CoA-O-methyltransferase)유전자의 발현을 분자생물학적으로 억제시킨 상태와 정상적인 상태에서 생장한 현사시나무를 대상으로 홀로셀룰로 오스, 리그닌과 단당류 조성에 관한 정량 분석을 각각 실시하였다. 그리고 각각의 목질바이오매스로부터 MWL (milled wood lignin)을 추출하여 메톡실기정량, DFRC, Py-GC/MS, GPC, <TEX>$^{13}C$</TEX> NMR 분석 등 리그닌의 화학 구조적 특성을 비교하였다. 정상재와 형질전환체의 홀로셀룰로오스 함량은 각각 81.6%와 82.3%로 큰 차이는 없었지만, 리그닌 함량은 각각 21.7%와 18.3%로 형질전환체가 약 3.4% 정도 낮았다. 정상재와 형질전환체의 구성 단당류 분석결과, 글루코오스는 각각 511.0 mg/g와 584.8 mg/g, 그리고 자일로오스는 각각 217.8 mg/g와 187.5 mg/g로 나타나 CCoAOMT 발현억제는 단당류 조성에 영향을 미치는 것으로 분석되었다. 각 시 료로부터 단리한 리그닌(MWL)의 메톡실기 정량에 따르면 CCoAOMT 발현을 억제한 시료에서 약 7% 정도의 메톡실기가 감소하는 것으로 나타났고, 이는 <TEX>$^{13}C$</TEX>-NMR 분석과 Py-GC/MS 분석을 통하여 확인할 수 있었다. 또한 Py-GC/MS 분석에 의하면 정상재와 형질전환체 리그닌의 G/S 비율이 각각 0.59와 0.44로 나타난점으로 미루어 CCoAOMT 발현 억제는 리그닌 생합성 과정에서 G unit 생합성보다 S unit 생합성에 더 많은 영향을 미치는 것으로 예측할 수 있다. In this study, chemical compositions - holocellulose, lignin and monomeric sugars - were characterized with two poplar wood cell walls, one of which was grown at normal condition (CPW) and the other was genetically modified by antisence suppression of CCoAOMT gene expression (ACPW). Milled wood lignins were isolated from CPW and ACPW and subjected to methoxyl group, DFRC, Py-GC/MS, GPC, <TEX>$^{13}C$</TEX>-NMR analysis, respectively. There were few differences in holocellulose contents in both cell walls, which were determined to 81.6% in CPW and to 82.3% in ACPW. However, lignin contents in ACPW was clearly decreased by the suppression of CCoAOMT gene expression. In CPW 21.7% of lignin contents was determined, while lignin contents in ACPW was lowered to 18.3%. The relative poor solubility of ACPW in alkali solution could be attributed to the reduction of lignin content. The glucose contents of CPW and ACPW were measured to 511.0 mg/g and 584.8 mg/g and xylose contents 217.8 mg/g and 187.5 mg/g, respectively, indicating that suppression of CCoAOMT gene expression could be also influenced to the formation of monomeric sugar compositions. In depth investigation for milled wood lignin (MWL) isolated from both samples revealed that the methoxyl contents at ACPW was decreased by 7% in comparison to that of CPW, which were indirectly evidenced by <TEX>$^{13}C$</TEX>-NMR spectra and Py-GC/MS. According to the data from Py-GC/MS S/G ratios of lignin in CPW and ACPW were determined to 0.59 and 0.44, respectively. As conclusive remark, the biosynthesis of syringyl unit could be further influenced by antisense suppression of CCoAOMT during phenylpropanoid pathway in the plant cell wall rather than that of guaiacyl unit.