[논문]벼에서 밀 고분자 글루테닌 단백질(TaGlu-Ax1) 발현을 통하여 쌀가루 가공적성 증진을 위한 마커프리(marker-free) 형질전환 벼의 개발

정남희; 전승호; 김둘이; 이춘석; 옥현충; 박기도; 홍하철; 이승식; 문중경; 박수권

doi:10.5352/jls.2016.26.10.1121

벼에서 밀 고분자 글루테닌 단백질(TaGlu-Ax1) 발현을 통하여 쌀가루 가공적성 증진을 위한 마커프리(marker-free) 형질전환 벼의 개발
Development of Marker-free TaGlu-Ax1 Transgenic Rice Harboring a Wheat High-molecular-weight Glutenin Subunit (HMW-GS) Protein 원문보기

생명과학회지 = Journal of life science, v.26 no.10 = no.198, 2016년, pp.1121 - 1129

정남희 (국립식량과학원 작물기초기반과) , 전승호 (경남과학기술대학교 농학한약자원학과) , 김둘이 (국립식량과학원 작물기초기반과) , 이춘석 (국립식량과학원 작물기초기반과) , 옥현충 (국립식량과학원 작물기초기반과) , 박기도 (국립식량과학원 작물기초기반과) , 홍하철 (국립농업과학원 기획조정과) , 이승식 (한국원자력원구원 첨단방사선 연구소) , 문중경 (국립농업과학원 기획조정과) , 박수권 (국립식량과학원 작물기초기반과)

초록
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밀의 고분자 글루테닌 서브유닛[high molecular-weight glutenin subunit (HMW-GS)]은 밀가루의 성질을 결정하는데 가장 중요한 요소이며 가공적성을 나타내는데 중요한 역할을 수행한다. 우리는 Agrobacterium 동시 형질전환법을 이용하여 한국 밀 품종인 ‘조경’으로부터 밀 HMW-GS을 암호화하는 TaGlu-Ax1 유전자를 가지는 marker-free 형질전환 벼를 생산하였다. TaGlu-Ax1 유전자의 종자 특이적 발현을 위하여 밀에서 존재하는 TaGlu-Bx7 유전자의 자체 프로모터를 벡터 내에 삽입하였다. 동시 접종을 위해서 오직 TaGlu-Ax1 유전자와 hygromycin phosphotransferase II (HPTII) 저항성 유전자만으로 구성된 두 종류의 발현 카세트를 독립적으로 Agrobacterium EHA105에 도입하였고, TaGlu-Ax1와 HPTII가 도입된 각각의 EHA105 Agrobacterium을 3:1 비율로 혼합하여 벼 캘러스에 접종하였다. 210개의 HPTII 저항성 형질전환체 중에서 벼 게놈에 TaGlu-Ax1과 HPTII가 모두 삽입된 20개의 형질전환 라인을 획득하였다. TaGlu-Ax1와 HPTII가 벼 게놈에 도입된 것을 Southern blot을 통해서 다시 확인하였다. 형질전환 벼 T₁ 세대의 종자에서 밀 TaGlu-Ax1 유전자가 전사와 번역되어 오직 TaGlu-Ax1만을 가지는 marker-free 식물체를 T₁세대에서 성공적으로 선발할 수 있었다. TaGlu-Ax1 유전자가 발현되는 marker-free 형질전환 식물체는 야생형(wild type)과의 표현형 차이는 없었다. 형질전환 벼의 쌀가루의 제빵적성을 비교하였을 때 TaGlu-Ax1 유전자만이 발현되어서는 제빵적성이 더 나아지지 않았다. 그러므로 더 많은 밀 고분자 및 저분자 글루테닌, 글리아딘의 유전자의 집적과 조합이 쌀가루 가공적성을 증진시키는데 필요하다. 결론적으로 TaGlu-Ax1 marker-free 형질전환 벼는 쌀가루 가공적성을 증진시키는데 좋은 재료로 사용될 것이다.

Abstract ▼ AI-Helper

High-molecular-weight glutenin subunits (HMW-GSs) are extremely important determinants of the functional properties of wheat dough. Transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding a HMG-GS were produced from the Korean wheat cultivar ‘Jokyeong’ and used to enhance the bread-making quality of rice dough using the Agrobacterium-mediated co-transformation method. Two expression cassettes with separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately into the Agrobacterium tumefaciens EHA105 strain for co-infection. Rice calli were infected with each EHA105 strain harboring TaGlu-Ax1 or HPTII at a 3:1 ratio of TaGlu-Ax1 and HPTII. Among 210 hygromycin-resistant T0 plants, 20 transgenic lines harboring both the TaGlu-Ax1 and HPTII genes in the rice genome were obtained. The integration of the TaGlu-Ax1 gene into the rice genome was reconfirmed by Southern blot analysis. The transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened in the T1 generation. There were no morphological differences between the wild-type and marker-free transgenic plants. The quality of only one HMW-GS (TaGlu-Ax1) was unsuitable for bread making using transgenic rice dough. Greater numbers and combinations of HMW and LMW-GSs and gliadins of wheat are required to further improve the processing qualities of rice dough. TaGlu-Ax1 marker-free transgenic plants could provide good materials to make transgenic rice with improved bread-making qualities.

주제어

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문제 정의

rice. The final objective of this study is to develop transgenic rice of increased processing quality than non- transgenic rice. Therefore, we first measured swollen ratio of dough in TaGlu-Ax1 transgenic rice (line 6), with Jokyeong (Korea wheat variety), Dongjin and Goami (Korea rice varie- ty) which used for material of rice noodle after first fermentation.

제안 방법

7. Analysis of bread-making quality of Jokeong (Korea wheat variety), Goami (Korea rice variety), Dongjin (non-transgenic rice) and marker-free transgenic rice (T₂ seeds in line 6). Heights of swollen doughs of Jokyeong and rice flours were measured after baking procedure.
In this study, we cloned TaGlu-Ax1, which is one of HMW-GS genes, and validated the insertion of the TaGluAx1 gene in T₀ plants through PCR analysis with gene specif- ic primers and Southern blot analysis (Fig. 2, Fig. 3). The TaGlu- Bx7-own promoter was introduced for seed specific expression of TaGlu-Ax1 gene.
The bread-making procedure used the straight dough method with 100 g of wheat and rice flours (Goami, Dongjin and TaGlu-Ax1 transgenic rice), 6 g of sugar, 1.5 g of salt, 3 g of dry milk, 6 g of butter, 3 g of yeast and 60 g of fresh water at 30℃, 90% moisture conditions for 60 min first fermentation. After punching, second fermentation and bak- ing were carried out at 30℃ for 40 min, and 210℃ for 25 min, respectively.

이론/모형

In this study, we generated marker-free transgenic rice expressing the wheat HMW-GS protein without any herbi- cide or antibiotic resistance marker genes using the co-trans- formation method. The marker-free transgenic plant express- ing TaGlu-Ax1 gene is critical material for generating trans- genic plant advanced quality processing of bread and noodle without antibiotic markers.

성능/효과

7). This results indicated that only one HMW-GS (TaGlu-Ax1) insufficient for increasing processing quality (especially bread-making quality) of transgenic rice dough. Finally, we think that accu- mulation and combination of more HMW-GSs and LMW- GSs and gliadins of wheat is required for improved process- ing quality of rice dough.

후속연구

As a result, the marker-free TaGlu-Ax1 transgenic rice de- veloped in this study is valuable to be utilized in breeding programs as a good material for improved processing quality.
The marker-free transgenic plant express- ing TaGlu-Ax1 gene is critical material for generating trans- genic plant advanced quality processing of bread and noodle without antibiotic markers. Moreover, the marker-free trans- genic rice developed in this study should provide useful for improving processing quality in rice breeding program.

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