Protocorm-like body를 이용한 호접란 형질전환 연구

허연재(Yeon-Jae Hur),김은영(Eun-Young Kim),양원태(Won-Tae Yang),이영병(Young-Byoung Lee),이재헌(Jae-Hun Lee),정영수(Young-Soo Jung),남재성(Jae-Sung Nam),윤대진(Dae-Jin Yun),이기환(Ki-Hwan Yi),김도훈(Doh-Ho Kim) 한국생명과학회 2009 생명과학회지 Vol.19 No.3

본 연구는 Agrobacterium을 이용한 효율적인 호접란 형질전환 시스템의 확립을 위해 실시하였고, 호접란 PLB의 대량증식을 위한 배지 조성은 하이포넥스 기본 배지에 활성탄 1g/l, 티아민 0.1 mg/l 및 sucrose 30 g/l을 배지에 첨가한 경우 PLB가 안정적으로 증식되었다. 계대 배양시 PLB 절단 방법이 PLB의 생존과 증식에 큰 영향을 미치며, PLB 위쪽으로부터 1/3 부위를 절단하여 계대 배양한 경우 90% 이상의 가장 높은 증식률을 나타났다. Agrobacterium 접종 시 균이 묻어 있는 침으로 PLB를 찔러 상처를 내는 dipping 방법을 이용하여 형질전환 효율을 높일 수 있었다. Agrobacterium 배양액의 흡광도 값이 0.8일 때 형질전환 효율이 가장 높았고, 형질전환된 PLB의 선발은 1 ㎎/l 정도의 저농도 hygromycin을 함유한 배지에 계대배양 하는 것이 효율적이었다. 호접란 형질전환체를 먼저 GUS assay를 통하여 선발하였고, 선발된 개체로부터 genomic DNA를 추출하여 GUS 특이적 primer와 probe로 PCR과 Southern blot 분석을 수행하고 유전자의 도입여부를 조사한 결과 GUS 염색된 형질전환체에서 정상적으로 GUS 유전자가 도입된 것을 확인할 수 있었다. Agrobacterium tumefaciens-mediated transformation procedure for the phalaenopsis orchid, established by using Protocormlike bodies (PLBs), was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. PLBs obtained from the axillary bud of a peduncle were maintained on a hyponex medium supplemented with 1 g/l of activated charcoal, 30 g/l of sucrose and 0.1 mg/l thiamine. The multiplication rate of PLBs was about 90% in case of subculture PLBs to be cut transversely into 1/3 part from top position. The PLBs were inoculated with Agrobacterium strain EHA105 harboring both β-glucuronidase (GUS) and hygromycin-resistant genes for 20 minutes after dipping treatment. Transformation efficiency was the highest with a Agrobacterium culture medium and dipping treatment of O.D. 0.8. Newly induced PLBs were put on selection medium containing 1 ㎎/l hygromycin for 2 months. Hygromycin-resistant phalaenopsis plants that regenerated after the selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by PCR and Southern blot using GUS specific primers and probe.

벼 키다리병 저항성 유전자원 탐색

허연재(Yeon-Jae Hur),이샛별(Sais-Beul Lee),신동진(Dongjin Shin),김태헌(Tae-Heon Kim),조준현(Jun-Hyeon Cho),한상익(Sang-Ik Han),오성환(Seong-Hwan Oh),이지윤(Ji-Yoon Lee),손영보(Young-Bo Son),이종희(Jong-Hee Lee),권택민(Tackmin Kwon),박 한국육종학회 2016 한국육종학회지 Vol.48 No.1

Bakanae (foolish seedling) disease caused by Gibberella fujikuroi creates serious problems in the foremost rice growing countries. This study was conducted to identify new resistance genetic sources to Bakanae disease. Bioassay showed that 11 varieties including Gwangmyeongbyeo, Hawn, Wonseadaesoo, Erguailai etc. were resistant to bakanae disease among 254 rice germplasm. Mismatch ratio between phenotype on bakanae disease bioassay and allele type of RM9, a SSR marker closely linked the bakanae disease resistant QTL, qBK1, were 38.3%. These results suggest that RM9 might be used for selecting qBK1, but it cannot be used for wide range of rice germplasm. Resistant germplasm in this study might be have resistant genes different from qBK1. The eleven varieties resistant to selected in this study will be used to identify new resistant alleles or genes to improve bakanae disease resistance in rice.

흑미 수확시기에 따른 이화학적 특성 및 항산화 활성 변화

박지영(Ji-Young Park),한상익(Sang-Ik Han),허연재(Yeon-jae Hur),이유영(Yu-Young Lee),이병원(Byung-Won Lee),심은영(Eun-Yeong Sim),함현미(Hyeonmi Ham),김병주(Byeong-Ju Kim),이춘우(Chun-Woo Lee),이성준(Sung-Joon Lee),오성환(Sung-Hwan Oh) 한국식품영양과학회 2015 한국식품영양과학회지 Vol.44 No.11

본 연구는 흑미 수확시기에 따른 종실의 이화학적 특성 및 항산화 활성 변이를 검토하였는데, 실험에 이용된 흑미 5개 품종(눈큰흑찰, 신농흑찰, 신토흑미, 조생흑찰, 흑진주)의 출수일로부터 각각 35, 40, 45, 50, 55일에 수확을 하여 출수 후 수확일수에 따른 특성을 비교한 결과, 무기성분(칼륨, 칼슘, 마그네슘)은 수확시기에 따른 유의성이 인정되지 않았다. 기능성 성분인 폴리페놀의 경우 흑진주를 제외하고 4개 품종에서 출수 후 35일 이후 시간이 지날수록 감소하는 경향이었고, 플라보노이드도 조생흑찰 및 흑진주를 제외하고 수확시기에 따라 함량이 감소하는 경향이었다. 수확시기와 안토시아닌 함량과의 관계를 분석한 결과 모든 품종이부의 상관관계를 보였고, 흑진주벼를 제외하고는 R<SUP>2</SUP> 값이 0.8~0.9 정도로 유의성이 인정되었다. 항산화 활성 또한 흑진주 벼를 제외하고는 출수 후 35일경이 항산화 활성이 가장 높았고, 이후 점점 감소하는 경향이었다. This study compared physicochemical properties and antioxidant activities according to different harvest times in five varieties of black rice (BR). Five cultivars of BR, Nunkeunheukchal (NKHC), Sinnongheugchal (SNHC), Sintoheugmi (STHM), Josaengheugchal (JSHC), and Heugjinju (HJJ), were selected for the study. Cultivars were transplanted on June 20th and cultivated by the Department of Southern Area Crop Science, NICS in Miryang, Korea. Heading dates of NKHC, SNHC, STHM, JSHC, and HJJ were Aug. 17th, Aug. 30th, Sep. 1st, Aug. 20th, and Aug. 12th, respectively. The five different harvest times were as follows; 35, 40, 45, 50, and 55 days after heading date. The highest level of anthocyanin was 35 days after heading date in all varieties. Levels of anthocyanin in NKHC, SNHC, STHM, JSHC, and HJJ were 164, 203, 251, 67, and 210 mg/100 g, respectively. Content of anthocyanin significantly decreased 35 days after heading in four varieties, excluding JSHC. Highest DPPH IC50 values were 57, 29, 10, and 27 μg/mL in NKHC, SN HC, STHM, and JSHC extracts, respectively. Highest ABTS IC50 values were 24, 13, 9, and 11 μg/mL in NKHC, SNHC, STHM, and JSHC extracts, respectively. The antioxidant activities were highest 35 days after the heading date and significantly decreased in four varieties, excluding HJJ. The antioxidant activity of HJJ showed no significant difference, according to harvest time. This study suggests that the content of anthocyanin and antioxidant activity could be considered as factors affecting optimal harvest period to produce highly qualified black rice.

Development of Marker-free Transgenic Rice Expressing the Wheat Storage Protein, Glu-1Dy10, for Increasing Quality Processing of Bread and Noodles

Soo-Kwon Park(박수권),DongJin Shin(신동진),Woon-Ha Hwang(황운하),Yeon-Jae Hur(허연재),Tae-Heon Kim(김태헌),Se-Yun Oh(오세윤),Jun-Hyun Cho(조준현),Sang-Ik Han(한상익),Seung-Sik Lee(이승식),Min-Hee Nam(남민희),Dong-Soo Park(박동수) 한국생명과학회 2014 생명과학회지 Vol.24 No.6

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 T0 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 T1 세대에서 성공적으로 선발할 수 있었다. 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 T0 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 T1 generation.

남부 평야지에서 생산된 유색미 품종별 항산화 성분 및 활성 비교

박지영(Ji-Young Park),함현미(Hyeonmi Ham),한상익(Sang-Ik Han),오성환(Sung-Hwan Oh),송유천(You Chun Song),조준현(Jun Hyeon Cho),허연재(Yeon-jae Hur),이유영(Yu-Young Lee),이병원(Byung-Won Lee),최용환(Yong Hwan Choi) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.8

남부 평야지에서 생산된 유색미의 품종별 항산화 성분으로 총폴리페놀, 총플라보노이드 및 안토시아닌을 분석하고 항산화 활성을 측정한 결과를 비교하였다. 총폴리페놀 함량은 흑미> 적미> 녹미 순이었고, 흑미 중에서도 흑진주, 조은흑미, 신토흑미 등이 높았다. 총플라보노이드 함량은 유색미 종류에 따른 차이보다는 품종별로 차이가 있었다. 흑진주와 홍진주, 건강홍미 등이 높은 결과를 나타내었다. 안토시아닌 함량은 적미와 녹미에서는 나타나지 않았고, 흑미에서는 신토흑미, 흑진주, 청풍흑찰 순으로 높은 결과를 보였다. ABTS 라디칼 소거능은 흑진주의 IC50 값이 낮아 항산화 활성이 높았고, 신토흑미, 신농흑찰, 홍진주, 적진주, 조생흑찰 등이 높았다. DPPH 라디칼 소거능에서는 흑진주의 IC50 값이 가장 낮았고, 건강홍미, 신토흑미가 다음으로 낮은 IC50 값을 보여 항산화 활성이 높았다. 총폴리페놀, 총플라보노이드, 안토시아닌 함량이 높은 흑미인 흑진주는 가장 높은 항산화 활성을 나타내었다. 총폴리페놀과 총플라보노이드 함량이 낮은 녹미는 흑미와 적미에 비해 유의하게 항산화 활성이 낮게 나타났다. 본 연구에서 분석한 총폴리페놀, 총플라보노이드, 안토시아닌과 ABTS・DPPH 라디칼 소거능에서 측정한 IC50 값과는 높은 부의 상관관계를 보이므로 항산화 성분과 항산화 활성은 상관관계가 높았다. This study compared antioxidant compounds and antioxidant activities according to various varieties of 18 colored rice. Selected rice cultivars for this study were as follows: black rice (12 cultivars)-Heugjinjubyeo, Nunkeunheugchal, Joeunheugmi, Josaengheugchal, Heuggwang, Heugseol, Shinmyeongheugchal, Shinongheugchal, Shintoheugmi, Cheongpungheukchal, Heughyang, Boseogheugchal, red rice (four cultivars)-Jeogjinju, Jeogjinjuchal, Hongjinju, Geonganghongmi, and green rice (two cultivars)-Nogwonchal and Jungmo1020. These were transplanted on June 30th and cultivated from the Department of Southern Area Crop Science, National Institute of Crop Science in Miryang, Korea. Total polyphenol (TP) content of black rice cultivars was higher than that of red rice varieties. Heugjinjubyeo exhibited the highest TP, which was 9.55±0.05 mg gallic acid equivalent/g sample. There were no significant differences between black rice cultivars and red rice cultivars in terms of total flavonoid (TF) level. Heugjinjubyeo and Hongjinju showed the higher TF content than any other variety. Shintoheugmi revealed the highest anthocyanin content (194.26±0.65 mg/100 g). Methanol extract of Heugjinjubyeo had the highest antioxidant activities. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities of Heugjinjubyeo extract were 14.70±0.62 and 30.68±2.22 μg/mL, respectively. There were significant correlations between antioxidant compound content and antioxidant activities.

백진주의 저아밀로스 유전분석 및 분자지도 작성

이종희(Jong-Hee Lee),조준현(Jun-Hyeon Cho),이지윤(Ji-Yoon Lee),손영보(Young-Bo Sohn),한상익(Sang-Ik Han),신동진(Dongjin Shin),송유천(You-Chun Song),박동수(Dong-Soo Park),여운상(Un-Sang Yeo),오명규(Myeong- Gyu Oh),허연재(Yeon-Jae Hur) 한국육종학회 2016 한국육종학회지 Vol.48 No.3

The amylose contents of rice determine eating quality which is one of the major traits in rice breeding program. To identify the low-amylose gene of the japonica rice cultivar Baegjinju, genetic analysis was conducted using 200 F2 population derived from a cross between the japonica cultivars, Saeilmi and Baegjinju. Individual F2 plants were classified as wild type (translucent grain) and mutant type (dull grain) based on the grain appearance of brown rice. Two hundred F2 plants were segregated into 155 wild type plants and 45 mutant type plants, which fit the 3:1 ratio (x2 = 0.667, df = 1, p = 0.414) and this result indicated the low-amylose gene of Baegjinju is a single recessive gene which controls the amylose contents. Linkage analysis was conducted to localize the low-amylose gene of Baegjinju and fine mapped within an 800-kb interval between 17.5 to 18.8Mb on short arm of chromosome 10. Co-segregated SSR marker, RM25648 was developed and it could be useful for marker-assisted selection and determination of the genetic resource related with amylose contents in rice breeding.