[논문]빵과 면의 가공적성 증진을 위한 밀 저장단백질 Glu-1Dy10을 발현하는 마커프리 형질전환 벼 개발

박수권; 신동진; 황운하; 허연재; 김태헌; 오세윤; 조준현; 한상익; 이승식; 남민희; 박동수

doi:10.5352/jls.2014.24.6.618

빵과 면의 가공적성 증진을 위한 밀 저장단백질 Glu-1Dy10을 발현하는 마커프리 형질전환 벼 개발
Development of Marker-free Transgenic Rice Expressing the Wheat Storage Protein, Glu-1Dy10, for Increasing Quality Processing of Bread and Noodles 원문보기

생명과학회지 = Journal of life science, v.24 no.6 = no.170, 2014년, pp.618 - 625

초록
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쌀가루는 많은 식품 가공에 이용된다. 그러나 밀가루 반죽이 빵과 면을 포함한 많은 식품 가공 제품에 적합한 반면에, 쌀로 만든 반죽은 신장성과 탄력성이 부족하다. 고분자 글루테닌 서브유닛(HMW-GS)은 밀의 가공 적성을 결정하는데 중요한 역할을 한다. 본 연구에서, 우리는 아그로박테리움(Agrobacterium) 동시 형질전환법을 이용하여 한국 밀 품종인 '조경'으로부터 HMW-GS를 암호화하는 밀 Glu-1Dy10 유전자를 발현하는 marker-free 형질전환 벼 식물체를 개발하였다. 오직 Glu-1Dy10 유전자와 HPTII (hygromycin phosphotransferase II) 저항성 유전자만을 포함하는 분리된 DNA 조각들로 구성된 두 가지 발현 카셋트(cassettes)를 독립적으로 아그로박테리움(Agrobacterium) EHA105 에 도입하였다. Glu-1Dy10 또는 HPTII를 함유하는 EHA105 를 각각 3:1 비율로 벼 캘러스에 접종하였다. 290개의 하이그로마이신(hygromycin) 저항성 $T_0$ 식물체 중에서 우리는 벼 게놈에 Glu-1Dy10과 HPTII 유전자가 모두 삽입된 29개의 형질전환 라인을 획득하였다. 우리는 Glu-1Dy10 유전자가 벼 게놈 내로 도입된 것을 Southern blot 분석을 통해 다시 확인하였다. 형질전환 벼 종자에서 Glu-1Dy10의 전사(Transcripts)와 단백질을 semi-quantitative RT-PCR과 Western blot 분석을 통해서 확인하였다. 최종적으로, 오직 Glu-1Dy10 유전자를 갖는 marker-free 식물체를 $T_1$ 세대에서 성공적으로 선발할 수 있었다.

Abstract ▼ AI-Helper

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar 'Jokyeong' using Agrobacterium-mediated co-transformation. Two expression cassettes, consisting of separate DNA fragments containing Glu-1Dy10 and hygromycin phosphotransferase II (HPTII) resistance genes, were introduced separately into Agrobacterium tumefaciens EHA105 for co-infection. Each EHA105 strain harboring Glu-1Dy10 or HPTII was infected into rice calli at a 3: 1 ratio of Glu-1Bx7 and HPTII. Among 290 hygromycin-resistant $T_0$ plants, we obtained 29 transgenic lines with both the Glu-1Dy10 and HPTII genes inserted into the rice genome. We reconfirmed the integration of the Glu-1Dy10 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the Glu-1Dy10 in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the Glu-1Dy10 gene were successfully screened in the $T_1$ generation.

주제어

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가설 설정

No protein bands were detected in one copy-inserted lines and muti-copies inserted lines. We guess that the expression levels of Glu-1Dy10 were too low to detect signal in case of the one-copy inserted lines. In case of Multi-copies inserted lines, this phenomenon may be related to homology-dependent gene silencing in plants [2].

제안 방법

tumefaciens EHA105 strain for plant transformation. Each binary vector was rescued from the EHA105 strain harboring Glu-1Dy10 and the HPTII, and then the HPTII and Glu-1Dy10 genes were validated by PCR analysis with specific primers.
We obtained 290 independent hygromycin-resistant T₀ plants through Agrobacterium-mediated co-transformation system. Genomic DNA from 290 independent T₀ plants was extracted and insertion of HPTII and Glu-1Dy10 genes was analyzed using PCR analysis with gene specific primers. As shown in Figure 2, HPTII gene in all of T₀ plants was amplified, but no PCR products in ‘Dongjin’ used as negative controls were detected.
Identification of T₀ plants by gene specific primer sets. Glu-1Dy10 (upper panel) and HPTII (lower panel) genes were amplified using Glu-1Dy10 and HPTII specific primer sets, respectively. SM, molecular marker; ‘Dongjin’ (Korean rice cultivar), non-transgenic plant; 1-23, co-transformed transgenic lines.
PCR was performed with the GeneAmp System 9700 (Applied Biosystems, Foster City, CA, USA) with a gene-specific primer set (Glu-1Dy10; forward 5'-CGCAAGACAATATGAGCAAAC-3', reverse 5'-GTTGCCTTTGTCCTGTGTGCT-3', HPTII; forward 5'-CGCTTCTGCGGGCGATTT-3', reverse 5'-CCCATTCGGACCGCAAGGA -3') and EF Taq DNA polymerase (Solgent Co. Seoul, South Korea).
After klenow enzyme treatment for blunt ligation, the vector was self-ligated. Then, amplified the Glu-1Dy10 gene with the KpnI and XbaI restriction enzyme sites was constructed into pBTEX binary vectors under the control of HMW (high-molecular weight) glutenin promoter (Fig. 1, upper panel). The positive selectable marker cassette for co-transformation was used by an empty pBTEX binary vector (Fig.
We used HMW glutenin promoter to express Glu-1Dy10 because Glu-1Dy10 expression in rice endosperm is important for rice flour quality. Total RNAs from randomly selected two-copy inserted T₁ transgenic seeds (13, 16, 29 lines) were extracted, and Glu-1Dy10 gene transcript level was examined by semi-quantitative RT-PCR. The Glu-1Dy10 transcripts were successively expressed in the T₁ generation transgenic seeds, whereas Glu-1Dy10 expression in ‘Dongjin’ was not detected (Fig.

대상 데이터

The primers were as follows: Glu-1Dy10 forward 5'-CGCAAGACAATATGAGCAAAC3', Glu-1Dy10 reverse 5'-GTTGCCTTTGTCCTGTGTGCT -3'; OsActin primers were used as internal standards for mRNA expression profiling [17, 32].

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