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한국 콩(Glycine max (L.) Merr) 품종의 효율적인 원형질체 분리 및 유전자 도입
조철오(Chuloh Cho),김둘이(Dool-Yi Kim),최만수(Man-Soo Choi),진민아(Mina Jin),서미숙(Mi-Suk Seo) 한국육종학회 2021 한국육종학회지 Vol.53 No.3
Soybean (Glycine max (L.) Merr.) is one of the most important crops with economic value as a source of protein and vegetableoil for human food and animal feed. In recent years, rapidly developed genome editing techniques have shown widespread application prospectsfor gene function studies and for improving important agronomic traits in many crops. Therefore, it is important to establish a highly efficientmethod for protoplast isolation and transient expression systems in soybeans. In this study, we established an efficient method for protoplastisolation and its application to transient gene expression in Korean soybean cultivars. The protoplasts were isolated from leaves, epicotyls,hypocotyls, cotyledons, and etiolated hypocotyls using various combinations of enzyme mixtures. We found that high-quality and large amountsof protoplasts were isolated from the etiolated hypocotyls when incubated for 8 h under conditions of 0.5% cellulase, 0.5% pectinase, and1% viscozyme. In addition, we observed a high transfection efficiency of green fluorescent protein using etiolated hypocotyl protoplasts. Takentogether, our protoplast isolation and transfection method is highly efficient and can be used for gene function and molecular analysis tobetter understand the biological and physiological processes in soybean.
다양한 계통의 옥수수 미성숙배로부터 캘러스 유도와 식물체 재분화
홍준기,박기진,이강섭,김둘이,김주곤,이승범,서은정,이연희,Hong, Joon Ki,Park, Ki Jin,Lee, Gang-Seob,Kim, Dool Yi,Kim, Ju-Kon,Lee, Seung Bum,Suh, Eun Jung,Lee, Yeon-Hee 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.1
옥수수의 최적 조직 배양 조건을 확립하기 위하여 옥수수 국내 5 계통과 국외 11 계통 총 16 계통을 포트와 포장 재배하여 미성숙 배를 분리하여 배발생 캘러스 유도 및 식물체 재분화율을 조사하였다. MS 배지에 auxin으로 1.5 mg/L Dicamba와 0.5 mg/L 2,4-D 가 첨가된 배지에서 캘러스 형성은 본 실험에 사용된 옥수수 계통 모두에서 높은 빈도로 유도되었으며, 캘러스로부터 식물체 재분화는 5mg/L zeatin이 첨가된 재분화 배지에서 높은 재분화율을 보였다. 또한 포장에서 재배된 옥수수로부터 미성숙 배를 분리하여 사용하였을 때 캘러스 유기 및 식물체 재분화 효율이 높았던 것으로 보아 미성숙 배를 분리하기 위한 옥수수 상태 및 genotype이 중요한 영향을 준다는 것을 알 수 있었다. 본 실험을 통하여 배 발생 캘러스 형성 및 식물체 재분화 효율이 조사된 옥수수 계통들은 생명공학 기술을 활용한 신품종 개발을 위한 형질전환 시스템 개발에 유전자원으로 활용될 수 있는 정보를 제공할 것으로 사료된다. We investigated the callus induction and plant regeneration ability of 16 maize genotypes, including the Korean inbred lines, using 9 to 15 day-old immature zygotic embryos from maize grown in pots and from field cultures. Immature zygotic embryos placed on MS medium supplemented with L-proline 0.7 g/L, MES 0.5 g/L, Dicamba 1.5 mg/L, 2,4-D 0.5 mg/L, $AgNO_3$ 4 mg/L, and sucrose 20 g/L, showed the highest frequency of callus induction. The highest number of shoots regenerated when the embryogenic callus were transferred to MS medium supplemented with 5 mg/L zeatin. The root formation was observed when shoots were grown on MS medium supplemented with 0.2 mg/L indole-3-butyric acid (IBA). Additionally, under the same culture conditions, immature zygotic embryos from maize grown in the field also had a high frequency of plant regeneration. Except one genotype, 15 genotypes showed callus induction and shoot regeneration. Among the 16 genotypes tested, H99, B98, HW3, and B73 yielded the best plant regeneration. H99 showed maximum shoot formation from the primary embryogenic callus. The results suggest that genotypes and growth conditions of the maize plant plays very important roles for enhancing the embryogenesis competence of immature zygotic embryos. The successful regeneration from immature zygotic embryos of maize inbred lines provides a basis for molecular breeding of new cultivars by genetic transformation.
Namhee Jeong(정남희),Seung-Ho Jeon(전승호),Dool-Yi Kim(김둘이),Choonseok Lee(이춘석),Hyun-Choong Ok(옥현충),Ki-Do Park(박기도),Ha-Cheol Hong(홍하철),Seung-Sik Lee(이승식),Jung-Kyung Moon(문중경),Soo-Kwon Park(박수권) 한국생명과학회 2016 생명과학회지 Vol.26 No.10
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 벼캘러스에 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. 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.
EMS 유도 내염성 증진 사료용 옥수수 돌연변이체 선발 및 특성 분석
조철오,김경화,서미숙,최만수,전재범,진민아,김둘이,Cho, Chuloh,Kim, Kyung Hwa,Seo, Mi-Suk,Choi, Man-Soo,Chun, Jaebuhm,Jin, Mina,Kim, Dool-Yi 한국작물학회 2020 한국작물학회지 Vol.65 No.4
Maize (Zea mays L.) is one of the most valuable agricultural crops and is grown under a wide spectrum of environmental conditions. However, maize is moderately sensitive to salt stress, and soil salinity is a serious threat to its production worldwide. In this study, we used ethyl methane sulfonate (EMS) to generate salt-tolerant silage maize mutants. We screened salt-tolerant lines from 203 M<sub>3</sub> mutant populations by evaluating the morphological phenotype after salt stress treatment and selected the 140ES91 line. The 140ES91 mutant showed improved plant growth as well as higher proline content and leaf photosynthetic capacity compared with those of wild-type plants under salt stress conditions. Using whole-genome re-sequencing analysis, 1,103 single nucleotide polymorphisms and 71 insertions or deletions were identified as common variants between KS140 and 140ES91 in comparison with the reference genome B73. Furthermore, the expression patterns of three genes, which are involved in salt stress responses, were increased in the 140ES91 mutant under salt stress. Taken together, the mutant line identified in our study could be used as an improved breeding material for transferring salt tolerance traits in maize varieties.
연구보문 : 형질전환체에서 도입유전자 구조와 메틸화가 유전자 발현에 미치는 영향
이연희 ( Yeon Hee Lee ),손성한 ( Seong Han Sohn ),서석철 ( Seok Chul Suh ),윤인선 ( In Sun Yoon ),김둘이 ( Dool Yi Kim ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.2
35S::hpt-35S::gus-mUbi1::bar 유전자로 형질전환된 벼에서 세대진전을 통한 유전자 구조 및 발현양상을 분석하였다. 1. 하이그로마이신 저항성을 나타낸 형질전환 벼(T0)에서 제초제 감수성을 나타낸 개체는 bar 유전자가 존재하지 않았거나 mUbi1 프로모터의 불완전한 삽입으로 인한 것을 알 수 있었다. GUS 발현은 도입 유전자의 copy 수에 따라 상당한 변이가 있었으며 3 copy 이상 도입된 형질전환체에서는 발현이 약하거나 silencing 현상이 일어났다. 2. 도입 유전자의 구조 및 상태를 알아보기 위하여 Hpa II와 Msp I 으로 genomic DNA를 절단 한 후 Southern 분석을 한 결과 여러 copy가 도입된 형질전환체에서 gus 및 35S 프로모터의 재배열 현상이 뚜렷하였다. 또한 일부 계통에서는 methylation 된 유전자를 확인하였고 demethylation 물질인 5-azaC 처리 후에 GUS 발현이 회복되었다. 3. T0에서 여러 copy의 유전자가 도입되어 발현이 없었던 형질전환체의 경우 후대 개체들에서 1 copy를 포함하고 유전자 발현이 정상적인 개체를 선발할 수 있었다. Transgene expression was analyzed in transgenic rice plants carrying multiple copies of TDNA(RB-35S::hpt-35S::gus-mUbi1::bar-LB) insertions. Only transgenic plants survived on the medium containing hygromycin were used for gus and bar gene expression. Herbicide susceptibility of transgenic lines was caused by incomplete integration of mUbi1::bar gene. Transgenic lines with more than three copies of T-DNA showed partial and complete silencing of gus transgene. In order to investigate the organization and methylation status of T-DNA, Southern blot analysis was carried out using two methylation sensitive enzymes, Hpa II and Msp I. Transgenic lines with multiple copies of T-DNA appeared to have some rearranged and methylated fragments of gus and 35S promoter, but not in transgenic lines with single copy. These results suggested that the methylation derived from these complicated structures of T-DNA induced high variable transgene expression among T2 and T3 progeny sublines. Therefore, in order to produce the GM crops with stable expression of target genes, the molecular characterization of T-DNA insertions should be carefully analyzed across the generation.