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클레마티스에서의 F3'5'H 유전자 분리 및 화색별 발현분석
김소영,천경성,임성민,권오현,유봉식,김미선,이수영 한국화훼학회 2016 화훼연구 Vol.24 No.3
Clematis patens는 미나리아재비과 으아리속에 속하는 다년 생 숙근초로 꽃잎화한 꽃받침을 가지고 있는 점이 특징이며 흰색, 분홍색, 자주색, 보라색, 등 다양한 화색이 존재한다. 본 연구에서는 C. patens ‘The President’에서 flavonoid 생합성 경로에 관여하는 유전자 중 delphinidin 계열 anthocyanin 색 소 합성을 유도하는 F3'5'H(ClF3'5'H)를 분리 동정하였으며, 화색 및 발달 단계별 ClF3'5'H 유전자의 발현 패턴을 분석하 였다. 또한 클레마티스 화색별 anthocyanin의 함량을 알아보 았다. 그 결과, 기존 보고된 Aconitum carmichaelii의 F3'5'H 유전자의 아미노산 서열과 79% 일치하여 높은 상동성을 갖는 것을 확인하였으며, ClF3'5'H 유전자의 발현 패턴은 흰색을 띠 는 클레마티스에서는 ClF3'5'H 유전자의 발현량이 많지는 않 았으나 다른 발달 단계에 비해 완전 개화한 단계에서 발현이 강했다. 분홍색을 띠는 클레마티스에서는 모든 발달 단계에서 ClF3'5'H 유전자의 발현이 검출되지 않았으며, 자주색과 보라 색을 띠는 클레마티스에서는 개화 직전 및 완전 개화 단계에 서 발현하였다. 이 결과로부터 클레마티스의 다양한 화색과 클 레마티스의 F3'5'H 유전자 발현과의 상관관계가 시사되었다. Clematis is a genus of about 300 species within the buttercup family Ranunculaceae. Flower colors in the species are divided into four colors, white, pink, red and purple. Flavonoid 3',5'-hydroxylase is the key enzyme in the syntheses of 3',5'-hydroxylated anthocyanidins, which are usually required for the expression of blue or purple flower color. We isolated homologues of the F3'5'H gene (named ClF3'5'H ) from Clematis patens ‘The President’. We analyzed the gene expression and accumulation of anthocyanins in petal of ten cultivars with different flower colors. As a result, the expression of the gene was detected in fully-opened flower of the white-colored petal. Whereas, the expression of the gene was not detected in the pink-colored petal of C. patens ‘Comtesse de Bouchaud’ and ‘Hagley Hybrid’. Also, the gene expressed strongly in opening flower and fully-opened flower with purple - colored petals. It is suggested that the expression of the ClF3'5'H gene iinvolves different flower colors in C. patens.
강윤임,최윤정,천경성,이수영,곽해련,김동욱,이영란,임기병 한국화훼학회 2016 화훼연구 Vol.24 No.3
나리에 있어 Cucumber mosaic virus(CMV) 바이러스 저항 성 육종을 위해 주요 모본 및 부본으로 이용한 우수 유전자원 에 대해 바이러스 저항성 검정을 실시하였다. 바이러스 접종 원은 CMV-Taean으로 농업과학원에서 분양 받아 담배에 증식 하여 접종하였다. 바이러스 접종 식물체는 조직배양을 이용 하여 ‘Connecticut King’, ‘Party Diamond’, ‘Prato’, ‘Casa Blanca’, ‘Mona’, ‘Dublin’, ‘Rodrigo’, ‘Brunello’, ‘Avocado’ 품 종과 ‘FA08-17’ 계통에 대해 바이러스 무병주를 생산하여 실 험에 이용하였다. 바이러스 접종 효율을 높이기 위하여 접종 횟수를 1회와 2회로 나누어 실시하였으며 인산염 버퍼의 pH 를 6.9, 7.2, 7.5로 달리하여 접종하였다. 2회 접종, 인산염의 pH 6.9에서 접종 성공률이 높았다. ‘Casa Blanca’, ‘Avocado’ 품종의 잎에서 약한 모자이크와 노란색의 mottle 증상이 관측 되었으며 ‘Brunello’, ‘Rodrigo’, ‘FA08-17’에서 Hypersensitive response like 반응이 관찰되었다. ‘Brunello’, ‘Rodrigo’ 품종 에서 RT-PCR 검정 결과 바이러스가 검출되지 않았다. Reverse transcription-polymerase chain reaction(RT-PCR) 결과 바이 러스가 검출되었던 ‘Connecticut King’, ‘Prato’, ‘Casa Blanca’ 의 경우 Enzyme-linked immunosorbent assay(ELISA) 흡광도 가 낮게 나타났다. RT-PCR과 ELISA 검사 결과 잎과 구근에 서 CMV 검출이 다르게 나타남에 따라 나리에 있을 것으로 추측되며 바이러스 감염과 연관된 TOM1, OBE2, DND1 유 전자를 대상으로 발현 분석을 하였으나 유의한 결과를 얻지 못하였다. Ten cultivars of Lilium (‘Connecticut King’, ‘Party Diamond’, ‘Prato’, ‘Casa Blanca’, ‘Mona’, ‘Dublin’, ‘Rodrigo’, ‘Brunello’, ‘Avocado’, and ‘FA08-17’) were screened for resistance to Cucumber mosaic virus (CMV). CMV-Taean which was used as sources was propagated in tobacco plants (Nicotiana tabacum cv. Xanthi-nc) at the National Institute of Agricultural Sciences of Korea. Virus free plantlets made for each variety by apical meristem culture then were planted in a greenhouse covered with net. Virus was inoculated one and two times with different phosphate buffer of pH 6.9, 7.2 and 7.5 in the plantlets of each variety. The twice inoculation and phosphate buffer of pH 6.9 increased the virus infection. Most infected plants did not show any symptoms in all varieties, but leaves of ‘Casa Blanca’ and ‘Avocado’ showed slight mosaic and yellow mottle symptom. Leaves of ‘Brunello’, ‘Rodrigo’, and ‘FA08-17’ showed a hypersensitive response (HR)-like responses to CMV infection. Results from Reverse transcription-polymerase chain reaction (RT-PCR) for CMV detection revealed that ‘Brunello’ and ‘Rodrigo’ were not infected with virus. Although CMV was detected in inoculated leaves of ‘Connecticut King’, ‘Prato’, and ‘Casa Blanca’ by RT-PCR, the absorptions of Enzyme-linked immunosorbent assay (ELISA) were low in leaves and bulbs. TOM1, OBE2, and DND1 genes, which are assumed to exist in respect of the virus infection, were analyzed in leaves and bulbs of all varieties but gene expressions were not significant.
무측지성 국화 형질전환 계통 영양번식 제2세대의 형태적 및 분자생물학적 특성
이수영,김정호,천경성,이은경,김원희,권오현,이혜진 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.4
This study examined the phenotypic and molecular characteristics of the 2nd clone (T0V2) plants of LeLs-antisense gene-transgenic chrysanthemum line (LeLs80) that exhibited non-branching, proving the relevance of these characteristics as a factor for use in environmental risk assessment. Results of the Southern blot analysis showed that three copies of the LeLs-antisense gene were introduced into the transgenic line, and northern analysis showed that the transcripted gene was normally expressed in the transgenic line. A flanking T-DNA sequencing method was used to determine that sequences of 184 and 464 bps flanked the LeLs-antisense gene in the transgenic line. These sequences, respectively, matched the 35S promoter for expression of the nptⅡ gene and the NOS terminator for expression of the LeLs-antisense gene within the pCAMBIA 2300 vector.
장기간 계대배양 된 장미 배발생 캘러스로부터 식물체 재분화 및 비정형체로부터 새로운 배발생캘러스 재생
이수영,도경란,천경성,김원희,권오현,이혜진 한국식물생명공학회 2014 식물생명공학회지 Vol.41 No.2
Long-term subcultured rose embryogenic calluses,which had been maintained for more than 5 to 6 years sincethe first embryogenesis from calluses induced from in vitroroots of rose, were identified as potential material for thedevelopment of transgenic plants. The first embryogeniccalluses from ‘Sweet Yellow’ and two breeding lines (KR056002and KR056006) were obtained in 2007 and 2009, respectively. Subsequently, we found that plants regenerated from longtermembryogenic calluses (LEC). Whereas the LEC from‘Sweet Yellow’ takes 3 to 4 months to regenerate plants,those of the two breeding lines take 4 to 5 months. Thisperiod of time is the same as that taken for plants to regeneratefrom the first embryogenic callus. New embryogenesis wasobserved from atypical bodies (ABs) that appeared duringthe process of long-term subculture. We found that it ispossible to use the AB as a material for new embryogenesis.
Flower color modification through expression of Aquilegia buergeriana F3′5′H in Petunia hybrida
Lee Young Ah,천경성,Shin Ju Young,Kim Jeong Ho,Song Bina,Kim Se Jin,박필만,An Hye Ryun,Kim Yae Jin,이준대,Lee Su Young 한국원예학회 2023 Horticulture, Environment, and Biotechnology Vol.64 No.4
Aquilegia buergeriana is a native plant in Korea with blue fl owers. Flavonoid 3′,5′ hydroxylase ( F3′5′H ) is a key gene involved in the synthesis of delphinidin pigment responsible for the fl ower's blue color. We isolated the F3′5′H from the petals of A. buergeriana ( AbF3′5′H ) and introduced the AbF3′5′H gene into Petunia hybrida using Agrobacterium-mediated transformation. Forty-fi ve plants were acquired from a kanamycin-supplemented medium. Fifteen of these were identifi ed as transgenic plants using polymerase chain reaction (PCR). Quantitative real-time PCR (qRT-PCR) analysis revealed that the AbF3′5′H was expressed in the petal, corolla tube, and stigma of P. hybrida . AbF3′5′H -transgenic plant (T 0 ) fl ower color was darker than that of non-transgenic plants (NTs). Particularly, the stigma color was dramatically changed, from light yellow green (145C) to purple (N77C or N79D). The segregation ratio of the three transgenic (T 1 ) lines was identifi ed as 3:1 by PCR analysis of AbF3 ′5′H and neomycin phosphotransferase-II. The fl ower color change of the transgenic lines (T 1 ) was similar to that of T 0 . qRT-PCR analysis showed that AbF3′5′H -transgenic T 1 lines had a higher AbF3′5′H expression than NT in all fl oral organs. Moreover, delphinidin was confi rmed to be accumulated in both corolla tube and stigma and was enhanced in the petals of AbF3′5′H -transgenic T 1 lines through UPLC analysis. Our fi ndings indicate the role of AbF3′5′H in fl ower color change. These results also indicate the functionality of AbF3′5′H in bluish fl ower modifi cations