Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Iron fortification of grains by introducing a recombinant gene of ferritin with seed promoters in rice

종자 특이 프로모터와 대두 Ferritin 유전자에 의한 벼 종실의 철분강화

  • Cho, Yong-Gu (Department of Crop Science, Chungbuk National University) ;
  • Kim, Hyung-Keun (Department of Crop Science, Chungbuk National University) ;
  • Choi, Jang-Sun (Department of Horticulture, Hankyong National University) ;
  • Jung, Yu-Jin (Department of Horticulture, Hankyong National University) ;
  • Kang, Kwon-Kyoo (Department of Horticulture, Hankyong National University)
  • Published : 2009.03.31
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Abstract

The recombinant DNAs, pGBF, pGTF, and pZ4F, using soybean ferritin gene have constructed with the promoters derived from seed proteins, glutelin, globulin, and zein. The recombinant ferritin genes were transformed into rice plant by Agrobacterium-mediated transformation. Iron contents and agronomic traits have been evaluated in the transgenic progenies. The embryogenic calli survived from second selection medium were regenerated at the rates of 19.2% with pGBF, 15.0% with pGTF, and 18.4% with pZ4F in Donganbyeo and 6.7% with pGBF, 11.7% with pGTF, and 3.4% with pZ4F in Hwashinbyeo. The introduction of ferritin gene in putative transgenic rice plants was confirmed by PCR and Southern blot analysis and also the expression of ferritin gene was identified by Northern blot and Western blot analysis. The iron accumulation in transgenic rice grains of the transgenic rice plant, T1-2, with zein promoter and ferritin gene contained 171.4 ppm showing 6.4 times higher than 26.7 ppm of Hwashinbyeo seed as wild type rice, but the transgenic plants with globulin and glutelin showed a bit higher iron contents with a range from 2.1 to 3.0 times compare to wild type grain. The growth responses of transgenic plants showed the large variances in plant height and number of tillers. However, there were some transgenic plants having similar phenotype to wild type plants. In the T1 generation of transgenic plants, plant height, culm length, panicle length, and number of tillers were similar to those of wild type plants, but ripened grain ratio ranged from 53.3% to 82.2% with relatively high variation. The transgenic rice plants would be useful for developing rice varieties with high iron content in rice grains.

Ferritin 유전자를 벼의 저장기관인 배유에 특이적으로 발현시킬 수 있는 glutelin, gGlobulin 및 zein 프로모터를 활용하여 쌀알에 최대로 발현시켜, 고부가가치를 가진 가공용 벼 품종을 육성하여 천연의 철 성분이 강화된 유아용 이유식 생산에 이용할 수 있으므로 유아들에게 천연의 철분을 안정적으로 공급할 수 있는 형질전환체를 육성하였다. 종자 저장단백질인 glutelin, globulin 및 zein의 프로모터와 ferritin 유전자를 pMJ21 vector에 pGBF, pGTF 및 pZ4F 등의 Ti-plasmid를 Agrobacterium에 도입하여 동안벼와 화신벼에 형질전환 하였다. 동안벼 종자를 사용하였을 때 pGBF 재조합 유전자는 19.2%, pGTF는 15.0%, pZ4F는 18.4%가 재분화되었고, 화신벼 종자를 사용하였을 때에는 pGBF 재조합 유전자는 6.7%, pGTF는 11.7%, pZ4F는 3.4%가 재분화되었다. 형질전환 벼의 ferritin 유전자의 도입여부는 PCR 분석과 Southern 분석으로 확인하였으며 ferritin 유전자의 유전자 발현은 Norihern 및 Western 분석에 의해 확인하였다. Southern blot 분석 결과로부터 각각의 배유특이 프로모터 유래 형질전환체 중에서 single copy로 도입된 개체를 선발 할 수 있었다. 또한 이들 형질전환 계통들에서 도입유전자의 발현량은 wild type 벼에 비하여 매우 높게 나타났다. 또한 철 단백질의 철분 축적 정도를 분석한 결과 Zein 프로모터를 사용한 형질전환 계통 (T1-2)에서 171.4 ppm으로 wild type과 비교하여 6.4 배의 철분함량 증가를 보였다. 그러나 globulin 및 glutelin 프로모터 유래 형질전환체에서는 wild type과 비교하여 $2.1{\sim}3.0$ 배의 철분함량 증가를 보였다. 벼 형질전환체들의 생육상황을 조사한 결과 초장은 변이 폭이 매우 크게 나타났으며, 대조품종과 비교하여 50%정도 감소한 왜성 및 이형 식물체도 출현되었다. 따라서 본 연구에서는 형질전환체 중에서 표현형 적으로 대조품종과 거의 같은 식물체를 선발하여 후대를 육성하였다. 육성한 T1 세대에서 형질전환체의 초장, 간장, 수장, 분얼수 및 등숙률을 조사한 결과 초장, 간장, 수장, 분얼수에 있어서는 대조 품종과 큰 변이를 보이지 않았으나 등숙률에 있어서는 $53.3{\sim}82.2%$의 비교적 큰 변이를 나타내었다.

Keywords

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