KR100496026B1 - Iron fortified potato - Google Patents

Iron fortified potato Download PDF

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KR100496026B1
KR100496026B1 KR10-2002-0079315A KR20020079315A KR100496026B1 KR 100496026 B1 KR100496026 B1 KR 100496026B1 KR 20020079315 A KR20020079315 A KR 20020079315A KR 100496026 B1 KR100496026 B1 KR 100496026B1
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potato
iron
gene
ferritin
ferritin gene
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KR20040051389A (en
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배신철
황덕주
변명옥
고승주
강권규
권순종
김호일
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대한민국
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

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Abstract

본 발명은 철분강화 감자에 관한 것으로서, ferritin 유전자를 감자에서 증강시켜 같은 환경조건하에서 철분 함량을 많이 보유하는 감자 보급종 품종 또는 유종모본을 개발함으로서, 향후 음식물의 철분 결핍문제를 해결할 수 있도록 하기위한 것이다.The present invention relates to an iron-enriched potato, and to enhance the ferritin gene in potatoes to develop a potato dissemination species or seedlings having a high iron content under the same environmental conditions, to solve the iron deficiency problem of food in the future will be.

Description

철분강화 감자{IRON FORTIFIED POTATO}Iron-Reinforced Potatoes {IRON FORTIFIED POTATO}

본 발명은 철분강화 감자에 관한 것으로서, 더욱 상세하게는 음식물의 철분 결핍 문제를 해소할 수 있는 일환으로 철분결합 단백질 유전자를 감자에서 과다 발현시킴으로 같은 환경조건하에서 철분을 많이 보유하는 감자를 개발하기 위한 것이다.The present invention relates to an iron-enhanced potato, and more particularly, to develop a potato having a large amount of iron under the same environmental conditions by overexpressing the iron-binding protein gene in the potato as part of solving the iron deficiency problem of food. will be.

종래에는 철분에 의해서만 철분결합단백질 유전자가 식물체내에서 유도되는 것으로 알려져 있었다.Conventionally, iron binding protein genes are known to be induced in plants only by iron.

본 발명은 35S CaMV 형질전환 벡터에 철분결합단백질 유전자(ferritin gene)를 연결하고 수미감자 품종에 형질전환함으로서, 철분함량에 있어서는 일반 수미감자보다 증가되나, 형태적으로는 차이가 없는 감자품종을 개발토록 하는데 목적이 있다.The present invention connects the iron-binding protein gene (ferritin gene) to the 35S CaMV transforming vector and transforms the rice cultivars into potato varieties. The purpose is to make it.

상기 목적은, 35S CaMV 형질전환 벡터에 철분결합 단백질 유전자(ferritin gene)[논문출처:Goto F, Yoshihara T, Shigemoto N, Toki S, Takawa F(1999) iron fortification of rice seed by the soybean ferritin gene. Nature Biotechnology 17:282-286)를 연결하여 감자 수미품종에 형질전환시킨 것임을 특징으로 하는 형질전환 감자를 통해 이룰 수 있게된다.The aim is to provide a ferritin gene for 35S CaMV transfection vector (Goto F, Yoshihara T, Shigemoto N, Toki S, Takawa F (1999) iron fortification of rice seed by the soybean ferritin gene. Nature Biotechnology 17: 282-286) can be achieved through the transgenic potato, characterized in that transformed to the potato varieties varieties.

즉, 본 발명에서는 콩(soybean) 유래 Ferritin 유전자를 35S CaMV(Cauliflower mosaic virus)벡터에 연결한 후 Agrobacterium tumefaciens LBA4404를 이용하여 수미감자품종에 형질전환하였다. 감자형질전환체 중에서 정상적으로 ferritin 유전자가 발현되는지를 확인하기 위하여 잎의 total RNA를 대상으로 Northern 분석을 실시하였다.That is, in the present invention, the soybean-derived Ferritin gene was linked to 35S CaMV (Cauliflower mosaic virus) vector, and then transformed into Sumi potato varieties using Agrobacterium tumefaciens LBA4404. Northern analysis was performed on total RNA of leaves to confirm whether ferritin gene is normally expressed in potato transformants.

상기 35S CaMV벡터는 이미 식물체 모든 조직에서 발현되는 것으로 알려져 있기 때문에 형질전환체의 철분함량 측정은 감자괴경을 대상으로 조사하였다.Since the 35S CaMV vector is already known to be expressed in all tissues of plants, the iron content of the transformant was investigated in potato tubers.

이하, 본 발명의 발현분석 및 측정과정을 실시예 과정을 통해 보다 구체적으로 살펴보기로 한다.Hereinafter, the expression analysis and measurement process of the present invention will be described in more detail through an example process.

<제1과정> Ferritin 유전자 감자 형질전환용 재조합 DNA작성 및 형질전환<Step 1> Preparation and transformation of recombinant DNA for ferritin gene potato transformation

형질전환 유전자는 콩 Ferritin 유전자이며, Ferritin 유전자를 35S CaMV에 연결하고 terminator로는 PinⅡ terminator를 부착한 후 pCAMBIA3300 binary 벡터에 옮겨 형질전환용 재조합 DNA로 사용하였다.(도 1) The transforming gene is a soybean Ferritin gene, and the ferritin gene was linked to 35S CaMV, and the terminator was attached to a Pin II terminator, which was transferred to pCAMBIA3300 binary vector and used as a recombinant DNA for transformation.

감자형질전환 균주는 Agrobacterium tumefaciens LBA4404를 이용하였다. 즉, MS 배지에서 자란 감자 줄기 절간을 1㎝ 크기로 자른 후, 2일간 28℃ 배양된 Agrobacterium에 200㎕ 배양액을 15㎖ MS 배지에 희석하였으며, 균 희석액에 10분간 감자줄지조직을 감염시킨 후 MSO(MS기본배지+3%sourose)배지에서 48시간 암상태에서 공동 배양하였다. Agrobacterium tumefaciens LBA4404 was used as a potato transformant strain. That is, after cutting the potato stem section grown in MS medium to 1 cm size, 200 μl culture solution was diluted in 15 ml MS medium in Agrobacterium cultured at 28 ° C. for 2 days, and infected with potato stem tissue for 10 minutes in bacterial dilution solution, followed by MSO. (MS base medium + 3% sourose) was co-cultured in the dark for 48 hours.

상기 Agrobacterium에 감염된 줄기조직을 캘러스 유도배지(BA 5mg/L, NAA 0.1mg/L, GA3, PPT(phosphinothricin)1.0mg/L)에서 캘러스를 유기한 후 발근 유도를 위하여 BA 5mg/L, NAA 0.1mg/L, GA3 1.0mg/L, PPT 1.0mg/L이 각각 첨가된 MS배지를 배양하여 뿌리를 형성하였다. Ferritin유전자 형질전환 감자는 5계통을 선발하였다.Stem tissues infected with the Agrobacterium were inoculated with callus in callus induction medium (BA 5mg / L, NAA 0.1mg / L, GA 3 , PPT (phosphinothricin) 1.0mg / L) and BA 5mg / L, NAA for rooting induction. Roots were formed by culturing MS medium containing 0.1 mg / L, GA 3 1.0 mg / L, and PPT 1.0 mg / L, respectively. Ferritin transgenic potatoes were selected from five strains.

<제2과정>Ferritin 유전자의 Northern 발현분석<Step 2> Northern expression analysis of Ferritin gene

재조합 DNA 형질전환 식물체에 대하여 전사체 수준에서 유전자가 발현되는지를 확인하기 위하여 포트로 이식하여 2개월 후 감자잎을 대상으로 전사체를 확인하였다. In order to confirm whether the gene is expressed at the transcript level with respect to the recombinant DNA transgenic plant, two months after transplantation into the pot, the transcript was identified in the potato leaf.

Northern 분석에 사용될 total RNA 분리는 TRIzol reagent(BRL)를 이용하였고, 분리된 10㎍ total RNA를 전기영동에 전개하고 32P로 라벨된 ferritin 유전자를 probe로 이용하였다. 형질전환체 5계통 중에서 Ca/Fe-4를 제외한 4계통 형질전환 감자에서는 도 2의 발현 분석도에 나타내어진 바와같이 전사체가 발현되는 것을 확인할 수 있었다.TRIzol reagent (BRL) was used as the total RNA to be used for Northern analysis. The isolated 10 µg total RNA was subjected to electrophoresis and the ferritin gene labeled 32 P was used as a probe. It was confirmed that the transcript was expressed in the transgenic potatoes except Ca / Fe-4 among the five transformants, as shown in the expression analysis diagram of FIG. 2.

<제3과정>Ferritin유전자의 철분 함량 측정<Step 3> Determination of Iron Content in Ferritin Gene

Northern 분석 결과로 확인된 형질전환체 감자괴경에 대한 철분함량을 측정하는것은 ICP 분석기기를 이용하여 3번을 반복 실시하였다. 그 결과, 도 3의 측정결과그래프에 나타내어진 바와같이 35S CaMV와 ferritin 유전자 도입 형질전환체의 철분함량은 751 ±156(ppm)인 반면, 대조구(control)는 292 ±30(ppm)으로 측정되었다. 형질전환체의 철분함량 증가가 대조구에 비해 2.5배 정도 증가함을 확인할 수 있었다.The iron content of the transformant potato tubers identified by the Northern analysis was repeated three times using an ICP analyzer. As a result, the iron content of the 35S CaMV and ferritin transgenic transformants was 751 ± 156 (ppm), while the control was measured at 292 ± 30 (ppm), as shown in the measurement result graph of FIG. 3. . The iron content of the transformant was confirmed to increase by 2.5 times compared to the control.

따라서, 본 발명에서는 철분결합 단백질 유전자를 감자에서 과다 발현시킴으로서 음식물의 철분결핍 문제를 해소할 수 있게됨을 알 수 있다.Therefore, it can be seen that in the present invention, the iron-binding protein gene is overexpressed in potatoes to solve the iron deficiency problem of food.

이상에서 살펴본 바와같은 본 발명에서는, ferritin 유전자를 감자에서 증강시켜 같은 환경조건하에서 철분 함량을 많이 보유하는 감자 보급종 품종 또는 유종모본을 개발함으로서, 향후 음식물의 철분 결핍문제를 해결하는 효과를 나타내게 된다.In the present invention as described above, by developing the ferritin gene varieties or seedlings having a high iron content under the same environmental conditions by enhancing the ferritin gene in potatoes, it will have the effect of solving the iron deficiency problem of food in the future .

도 1은 Ferritin 유전자 감자 형질전환용 재조합 DNA.1 is a recombinant DNA for ferritin gene potato transformation.

도 2는 Ferritin 유전자의 Northern 발현 분석도.2 is a Northern expression analysis of Ferritin gene.

도 3은 Ferritin 유전자의 철분 함량 측정결과 그래프.3 is a graph showing the iron content of Ferritin gene.

Claims (1)

35S CaMV 형질전환 벡터에 철분결합 단백질 유전자(ferritin gene)를 연결하여 감자 수미품종에 형질전환시킨 철분강화 감자.An iron-enriched potato transformed into a potato Sumi varieties by connecting a ferritin gene to a 35S CaMV transformation vector.
KR10-2002-0079315A 2002-12-12 2002-12-12 Iron fortified potato KR100496026B1 (en)

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