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수분ㆍ수정 시기를 이용한 Bialaphos 저항성 형질전환 담배의 개발
이효연,노일섭,김진호,유장렬,이종석,김학진,Toshiaki KAMEYA 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.2
비선택성 제초제인 bialaphos는 고등식물에 있어서 glutamine 합성을 억제하여 식물체를 고사 시키는 능력을 갖고 있다. 본 연구에서 acetylteansferase에 의해 encoding된 bialaphos 저항성 유전자(bar gene)는 세균(Pseudomonas sryngae pv tabaci)의 genomic DNA로부터 cloning된 것을 사용하였다. 수분시킨 담배의 화계에 일정한 시간별로 bar 유전자를 처리한 결과 수분 후 30-40시간 사이의 처리구 에서 형질전환 식물체가 가장 많이 얻어 졌다. 그러한 형질전환 식물체의 kanamycin과 bialaphos 저항성 형질은 자식후대(T$_1$, T$_2$)에 있어서도 우성형질로 유전되었으나 wild type의 담배는 상기의 약제를 처리 하였을때 전부 고사하였다. 그리고, T$_1$세대의 형질전환 식물체로부터 전 염색체 DNA를 추출하여 Southern 분석한 결과 bar 유전자가 식물의 염색체상에 안정하게 존재하는 것을 확인하였다. 이상의 결과로부터 담배의 수분, 수정 시기에 외부유전자인 bar를 화주에 처리함으로써 bialaphos 저항성 식물을 만들어낼 수 있었다. The herbicide bialaphos is a potent inhibitor of glutamine synthetase in higher plants. A bialaphos resistance (bar) gene encoding for an acetyltransferase was isolated from genomic DNA of Pseudomonas syringae pv tabaci. The bar gene was ligated to the binary vector pBI121. Pistils of tobacco plane were heated with the bar gene containing plasmid DNA at various times after pollination. When the treatment was applied at 30 and 40 h after pollination, a number of transgenic plants were obtained. Premary transformation (T$_{0}$ generation) and their progenies (T$_1$T$_2$) were resistant to both bialaphos and kanamycin at a dosage lathal to untransformed control plants. Stable integration of bar gene into chromosomal DNA was proven by Southern blot analysis of genomic DNA isolated from T$_1$progenies. These results show that the bialaphos resistant plane could be obtained by treatment to pistils with the exgenous bar gene through the fertilization cycle of tobacco.o.
Expression Analysis of an APETALA1/FRUITFULL-like Gene in Phalaenopsis sp. ‘Hatsuyuki’ (Orchidaceae)
송인자,Tatsuya Fukuda,고석민,Takuro Ito,Jun Yokoyama,Hiroyuki Ichikawa,Yoh Horikawa,Toshiaki Kameya,Akira Kanno,이효연 한국원예학회 2011 Horticulture, Environment, and Biotechnology Vol.52 No.2
Members of the APETALA1 (AP1)/FRUITFULL (FUL)-like gene family of MADS-box genes play important roles in controlling the development of floral organs. To understand the molecular mechanisms of floral development in orchid, we isolated and characterized a Phalaenopsis AP1/FUL-like gene, PhalFUL. The results of phylogenetic analysis indicated that PhalFUL is in the monocots group of AP1/FUL-like gene. PhalFUL transcripts were detected in the flower buds, but not in vegetative organs. Moreover, in situ hybridization experiments revealed PhalFUL hybridization signals in all floral organ primordia at a very early stage of floral development, and continued expression in the column of whorls 3 and 4 until late developmental stages. These expression patterns were similar to those of the FUL-like genes in Arabidopsis (FUL) and Antirrhinum (DEFH28), suggesting that the PhalFUL is similar in function to FUL and DEFH28.
Young Ho Kim,Young Ok Lee,Ill Sup Nou,Hee Wan Kang,Toshiaki Kameya,Takashi Saito,Kwon Kyoo Kang 한국자원식물학회 1998 Plant Resources Vol.1 No.1
A cDNA fragment encoding iron storage protein generated by polymerase chain reaction(PCR) using highly conserved regions of ferritin related genes were used to screen a red pepper cDNA library, cDNA clone was designated as Fpl. Fpl clone contained a 5 nontranslated region of 51bp containing stop codons. Down stream from 5 UTR, an open reading frame of 750bp was observed, followed by a 3 UTR of 272bp. The deduced amino acid sequence of red pepper protein(Fpl) showed 84%, 48% and 36% identity with soybean(SolC), human(HuL-H) and horse spleen (HOS-L) ferritin respectively. Northern blot analysis of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 12h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.
Young Ho Kim,Young Ok Lee,Ill Sup Nou,Ill Yong Shim,Toshiaki Kameya,Takashi Saito,Kwon Kyoo Kang 한국자원식물학회 1998 Plant Resources Vol.1 No.1
Total iron content and ferritin distribution have been determined in red pepperi Cupsicum annum L.) during development stage under conditions of iron nutritional status from hydroponic culture. Color of the leaves become chlorotic on iron deficient and high concentration. The plant height on each iron concentration had retarding effect at concentration lower than 254M and greater than 1254M. In normal green leaves, total iron content was almost constant with a mean value of 2.5pmole of iron/mg of dry matter, except at 63day, for which it increases slightly to tumole. However, iron content of chlorotic plants grew on iron free medium was not almost detectable. Also, In post chlorotic leaves(++Fe), iron content was evidently increased until 7days after transfer on liquid medium, but decreased from after 14days. Also, ferritin protein analysed total protein extracts prepared from leaves of different ages using antibodies raised against ferritin protein. Ferritin protein decreased progressively during the first week of germination and was not detectable in vegetative tissues. Ferritin protein in post chlorotic leaves was evidently strongly enhanced until lidays afier transfer on liquid medium but decreased until the leves hecame chlorotic.
Young Ho Kim,Young Ok Lee,Ill Sup Nou,Ill Yong Shim,Toshiaki Kameya,Takashi Saito,Kwon Kyoo Kang 한국자원식물학회 1998 Plant Resources Vol.1 No.1
The Fipl gene, originally isolated from red pepper secdlings, encode the iron storage protein, and have a high homology with ferritin genes at DNA and amino acid level. In order to determine ferritin protein expression in vegetative tissue, Fpl gene was constructed in plant expression vector(PIG121 Hm) and introduced in red pepper{ var. Bukang, Chungyang and Kalay-Kimjang 2) via Agrobacterium tumefaciensinediated transformation. After selection on MS media containing kanamycin(Km), putatively selecied transformants were confirmed by amplification of selectable marker genet Fpl and NPTII) by polymerase chain reaction. Northern blot showed that transcripts of Fp1 gene were detected in mature leaves of the plants. In A6, A7 and A8 and Al4 of transgenic plants, transcript of Fpl gene was increased seven-fold to eight-fold than other transgenic plants. Also the proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbil anti-ferritin polyclonal antibody. The expression protein appeared as strong band of apparent mass of 23.5kDa, suggesting the iron accumulation in transgenic red pepper plants.