다양한 새싹 보리 품종의 사포나린 생합성 유전자의 RNA 발현 및 DNA 다형성 분석

다국어 초록 (Multilingual Abstract)

Flavonoids found in many plant species are known as secondary metabolites with important biological and pharmacological functions. Especially, saponarin, one of the flavonoids found in barley (Hordeum vulgare L.), accumulates at a high level in young leaves.
Among the various barley varieties, the Keunal, which contains high levels of saponarin, is being considered for a patent, supporting technology transfer at the National Institute of Crop Science. Therefore, in this study, we analyzed the RNA expression levels of known saponarin biosynthesis pathway genes [chalcone synthase (CHS), chalcone isomerase (CHI), and UDP-Glc:isovitexin 7-O-glucosyltransferase (OGT)] and saponarin contents during the early developmental stages of Keunal. In addition, RNA expression levels of three saponarin biosynthesis genes during the developmental stage in various barley varieties were analyzed, and DNA polymorphism of the HvCHS1 gene was confirmed.
Firstly, I searched NCBI and JGI databases using the nucleotide sequences of the known saponarin biosynthesis genes (HvCHS, HvCHI, and HvOGT) in barley. I found three HvCHSs (HORVU2Hr1G116390.2, HORVU2Hr1G004170.4, and HORV-U2Hr1G005220.3), one HvCHI (HORVU5Hr1G112670.5), and two HvOGTs (HORVU7Hr1G031800.2 and HORVU3Hr-1G110110.2) in the barley genome, and based on the sequences observed, we designed the primers for reverse transcription-polymerase chain reaction (RT-PCR) and real time-quantitative PCR (RT-qPCR) analyses. In Keunal, RNA expression was observed only for HvCHS1, HvCHI, and HvOGT1, and hence, RNA expression levels of these genes were primarily investigated in subsequent analyses. In Keunal, RNA expression of HvCHS1 and HvOGT1 was highest on day 3, and that of HvCHI was highest on day 1, while the saponarin content showed the greatest accumulation between day 3 and 6. Various abiotic stress treatments (light/dark transfer, drought, and low and high temperatures) significantly affected RNA expression levels of HvCHS1 and HvCHI, however, there was no significant difference in the RNA expression of HvOGT1 and the saponarin content under same conditions. Furthermore, the levels of RNA expression of HvCHS1 and HvCHI were observed only in the stem, while that of HvOGT1 was similar in both root and stem.
Secondly, I investigated RNA expression levels of three saponarin biosynthesis genes in a variety of barley seedlings. According to their expression patterns, I classified three groups as follows: In group 1 (Keunal, Nuri, and Saessal), RNA expression decreased gradually, while in group 2 (Heugdahyang and Huinchal), RNA expression exhibited a sharp decrease, following the high initial expression. In group 3 (18CPB4, 10, 11), the decrease in RNA expression after the observed high expression was lower than in other varieties.
Lastly, I examined the DNA polymorphism of HvCHS1 genes in a variety of barley. As a result, no significant DNA and protein polymorphisms were observed in the HvCHS1 nucleotide and deduced amino acid sequences of eight barley varieties.
Taken together, this study provides a molecular basis for metabolic engineering research that can increase the saponarin content in sprouting barley. However, saponarin biosynthesis genes still need to be discovered, and also biochemical studies such as their enzyme activities are required.

목차 (Table of Contents)

• List of Tablesⅰ
• List of Figures ⅱ
• ABSTRACT ⅴ
• Ⅰ. 서론 1
• Ⅱ. 재료 및 방법 4
• 1. 보리샘플 준비와 성장조건 4
• 2. 발달단계별 샘플 수집과 비생물학적 스트레스 처리 4
• 3. RNA 발현분석 5
• 4. 초고성능 액체 크로마토 그래피(Ultra-High Performance Liquid Chromatography, UHPLC)분석 6
• 5. HvCHS1 유전자 서열 분석 7
• Ⅲ. 결과 9
• 1. 보리게놈에서 HvCHS, HvCHI, HvOGT 염기 서열분석 9
• 2. 큰알보리의 초기 발달 단계에서 사포나린 축적 및 사포나린 생합성 유전자의 RNA발현 22
• 3. 빛이 없을 때 큰알보리 새싹의 사포나린 축적 및 사포나린 생합성 유전자의 RNA 발현 24
• 4. 비생물학적 스트레스에 의한 큰알보리 사포나린 축적 및 사포나린 생합성 유전자의 RNA 발현 27
• (1)가뭄 스트레스 27
• (2)저온 및 고온 스트레스 29
• 5. 큰알보리 줄기, 뿌리 사포나린 합성 유전자 발현비교 32
• 6. 8가지 보리품종에서 사포나린 생합성 유전자의 RNA 발현 34
• 7. 8가지 보리품종의 HvCHS1 유전자의 염기 서열 비교 38
• Ⅳ. 고찰 46
• Ⅴ. 요약 51
• Ⅵ. 참고문헌 54