한국콩연구회

항암 및 항산화 기능성 형질전환 벼, 콩의 실용화 연구

Development of a functional crops producing isoflavone and tocotrienol

사업명 : 작물시험연구

주관연구기관 :국립식량과학원

연구책임자 : 박향미

발행년월 : 2012-01

주관부처 : 농촌진흥청

원문

http://www.ndsl.kr/ndsl/commons/util/ndslOriginalView.do?dbt=TRKO&cn=TRKO201200009804&rn=&url=&pageCode=PG18

초록

Isoflavonoids are a diverse group of plant natural products synthesized from phenylpropanoid pathway which play important roles in plant growth and development. There have been considerable attentions as health-promoting nutraceuticals because of their antioxidant and estrogenic anti-cancer activity. In our study, we attempted to develope genistein-enriched rice for recommendable daily consumption through engineering the isoflavone pathway. Both overexpression and RNAi suppression strategies were used to manipulate the expression of several genes encoding key enzymes in the flavonoids/isoflavonoids pathway in transgenic rice. Rice plants were transformed with two soybean (Glycine max L) isoflavone synthase (GmIFS1, 2) genomic DNA under the control of the seed specific rice globulin promoter. HPLC-MS analysis demonstrated the presence of genistein as the major isoflavone metabolite in the transgenic plants. Substantial amounts of genistein (up to 87.0 μg/g FW) were found in seeds. However, the amounts of genistein showed annual variation in the field condition. For producing transgenic rice seeds with high level of genistein in a stable manner, we transformed maize C1 and R-S genes that together activate most structural genes in the flavonoid biosynthetic pathway. Furthermore, we constructed a RNAi vector to suppress the flavanone 3-hydroxylase (F3H) gene expression for blocking anthocyanin synthetic pathway. We obtained transgenic rice plants harboring RNAi-F3H vector or C1 and R-S overexpression vector and then we pyramided these three genes(IFS1/ /C1R-S/RNAiF3H) by crossing. These plants will be used for further analysis of isoflavone detection and molecular characterization.

The amylose content of starch is a major factor in the texture of cooked cereal grains. Therefore, down-regulation of amylose synthesis is one of the alternative method to improve eating quality of rice. We developed transgenic rice plants designed to suppress granule-bound starch synthase I (GBSSI) gene using RNA interference (RNAi) technology. Transgenic plants with RNAi vector containing the 3'-UTR region of GBSSI showed a lower amylose content in rice endosperm than that of wild type. The range of amylose content was 5.9~9.0% in the transgenic plants, whereas that of wild type was 17.7~18.0%. Transgenic rices showed the decrease of short chain and the increase of long chain by analyzing chain length distribution of amylopectin in the endosperm. In the SEM micrographs, we found that compound starch granules in whole grains of the wild type rice were readily split during fracturing, while the starch granules in RNAi-transgenic lines showed small voluminous, non-angular rounded bodies.

토코트리에놀 합성 형질전환체 작성 및 계통 선발에서 동진벼로부터 OsHGGT 유전자 분리하고, 종자발현 및 항시발현 프로모터에 도입 유전자가 발현되도록 연결된 형질전환운반체 제작하였다. 제작된 형질전환 운반체를 이용하여 콩 형질전환 식물체를 작성하고 우수 계통을 선발하였다. 형질전환체의 분자생물학적 분석 및 토코트리에놀 합성 확인에서는 Southern blot 분석을 통해 도입 유전자 수를 확인하고 RT-PCR과 real-time PCR 분석으로 유전자 발현 확인하였다. 토코트리에놀 성분은 HPLC로 분석하였다. 토코트리에놀 합성 형질전환 콩의 농업적 형질평가 및 고함유 계통선발에서는 경장 등 농업적 형질 평가와 토코트리에놀 합성량이 높은 계통을 선발하였다.