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벼종자 미랑 단백질의 프로테오믹스 연구를 위한 글루테린 저장 단백질의 제거방법
우선희,김세영,김태선,조성우,조건,정근욱,김선림,조용구,김홍식,송범헌,이철원,정승근,박영목,Woo, Sun-Hee,Kim, Se-Young,Kim, Tae-Seon,Cho, Seong-Woo,Cho, Kun,Chung, Keun-Yook,Kim, Sun-Lim,Cho, Yong-Gu,Kim, Hong-Sig,Song, Boem-Heon,Lee, Chul-W 한국작물학회 2006 한국작물학회지 Vol.51 No.suppl1
본 연구는 벼종자 미량 단백질의 프로테오믹스 연구를 위하여 벼종자에 고 함량으로 존재하는 벼종자 글루테린 저장 단백질을 제거하는 방법에 관한 것이다. 따라서 본 연구는, (A) 벼종자에 액체 질소를 가하고 분쇄하여 벼종자 가루를 만드는 분쇄단계; (B) 상기 분쇄된 벼종자 가루를 물에 현탁하여 현탁액을 만드는 현탁단계; (C)상기 현탁액 중 미용해 물질을 제거하는 분리단계를 포함하는, 벼종자 미량 단백질의 프로테오믹스 연구를 위한 벼종자 글루테린 저장 단백질의 제거방법에 관하여 검토하였다. 본 연구의 결과, 단순하고 신속하며 저렴하고 효율적인 방법으로 미량 비글루테린 단백질들을 용이하게 동정할 수 있을 것으로 판단되었다. Abundant proteins often cause problems in proteome study. Glutelin family proteins (hereafter referred to glutelin) are present in rice proteome sample as over-whelming constituents with very high abundance. In order to increase the number of identified proteins in rice proteome study, we developed a newly improved method for sample preparation through the removal of glutelin. When the protein samples from rice seed were extracted by the conventional trichloroacetic acid (TCA) extraction method, glutelin accounts for about 60% of total rice seed proteins in SDS gels. Using our new water extraction method, glutelin consists of only about 10% of total proteins. After analyzing on a two-dimensional gel electrophoresis (2-DE), 937 protein spots were detected using the conventional TCA extraction method. On the other hand, 1240 proteins could be seen using the new water extraction method. The selectivity for non-glutelin and less abundant protein by the water extraction method was also confirmed by ESI-Q/TOF mass spectrometry analysis. Thus, the new water extraction method developed here can be efficiently used to study the proteome analysis of rice storage seed.
우선희,김홍식,송범헌,이철원,박영목,정승근,조용구,Woo, Sun-Hee,Kim, Hong-Sig,Song, Berm-Heun,Lee, Chul-Won,Park, Young-Mok,Jong, Seung-Keun,Cho, Yong-Gu 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.3
In this review, we described the catalogues of the rice proteome which were constructed in our program, and functional characterization of some of these proteins was discussed. Mass-spectrometry is the most prevalent technique to rapidly identify a large number of proteome analysis. However, the conventional Western blotting/sequencing technique has been used in many laboratories. As a first step to efficiently construct protein cata-file in proteome analysis of major cereals, we have analyzed the N-terminal sequences of 100 rice embryo proteins and 70 wheat spike proteins separated by two-dimensional electrophoresis. Edman degradation revealed the N-terminal peptide sequences of only 31 rice proteins and 47 wheat proteins, suggesting that the rest of separated protein sports are N-terminally blocked. To efficiently determine the internal sequence of blocked proteins, we have developed a modified Cleveland peptide mapping method. Using this above method, the internal sequences of all blocked rice proteins(i, e., 69 proteins) were determined. Among these 100 rice proteins, thirty were proteins for which homologous sequence in the rice genome database could be identified. However, the rest of the proteins lacked homologous proteins. This appears to be consistent with the fact that about 45% of total rice cDNA have been deposited in the EMBL database. Also, the major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that tuned out to be calreticulin, gibberellin-binding protein, which is ribulose-1.5-bisphosphate carboxylase/oxygense active in rice, and leginsulin-binding protein in soybean have functions in the signal transduction pathway. Proteomics is well suited not only to determine interaction between pairs of proteins, but also to identify multisubunit complexes. Currently, a protein-protein interaction database for plant proteins(http://genome.c.kanazawa-u.ac.jp/Y2H)could be a very useful tool for the plant research community. Also, the information thus obtained from the plant proteome would be helpful in predicting the function of the unknown proteins and would be useful be in the plant molecular breeding.
In vitro culture of hybrid ovule between Fagopyrum esculentum Moench and F. homotropicum Ohnishi
Sun Hee Woo(禹仙熙),Taiji Adachi(足立泰二),Seung Keun Jong(鄭丞根) 한국육종학회 1997 한국육종학회지 Vol.29 No.3
Interspecific hybridization in buckwheat is of great importance to transfer desirable genes from wild species to cultivars as well as to broaden genetic variation in cultivars. In vitro ovule culture following the bud pollination was made to overcome postzygotic cross/self-incompatibility in interspecific hybridization between Fagopyrum esculentum Moench (common heterostylous buckwheat) and F. homotropicum Ohnishi (homostylous buckwheat). The percentage of fertilized ovaries of F. esculentum flowers (thrum-type) pollinated with F. homotropicum was 50%, while that of the reciprocal cross was as low as 10% and their ovaries were smaller. Vigorous pollen tube elongation following the bud pollination indicated the possibility for overcoming of breeding barriers in interspecific crosses between buckwheat species. Pre-embryo formation was observed 48 hours after pollination and ovules cultured up to 5 days after pollination tended to develop callus. But ovules isolated 11 days after pollination had apparently lost their viability. Hybrid plantlets were produced from only the ovules dissected 5 days after bud pollination. Regeneration rate was better in ovule culture on White medium than other mediums tested. Interspecific hybrid plants between F. esculentum and F. homotropicum were regenerated with the rate of 24.2% in average.
전단 자극에 의한 심방 근세포 칼슘 웨이브의 발생 : Phospholipase C-이노시톨 1,4,5-삼인산 수용체 신호전달의 역할
김준철(Joon-Chul Kim),우선희(Sun-Hee Woo) 大韓藥學會 2015 약학회지 Vol.59 No.4
Cardiac myocytes are subjected to fluid shear stress during each contraction and relaxation. Under pathological conditions, such as valve disease, heart failure or hypertension, shear stress in cardiac chamber increases due to high blood volume and pressure. The shear stress induces proarrhythmic longitudinal global Ca2+ waves in atrial myocytes. In the present study, we further explored underlying cellular mechanism for the shear stress-induced longitudinal global Ca2+ wave in isolated rat atrial myocytes. A shear stress of ~16 dyn/cm2 was applied onto entire single myocyte using pressurized fluid puffing. Confocal Ca2+ imaging was performed to measure local and global Ca2+ signals. Shear stress elicited longitudinally propagating global Ca2+ wave (~80 μm/s). The occurrence of shear stress-induced atrial Ca2+ wave was eliminated by the inhibition of ryanodine receptors (RyRs) or inositol 1,4,5-trisphosphate receptors (IP3Rs). In addition, pretreatment of phospholipase C (PLC) inhibitor U73122, but not its inactive analogue U73343, abolished the generation of longitudinal Ca2+ wave under shear stress. Our data suggest that shear-induced longitudinal Ca2+ wave may be induced by Ca2+-induced Ca2+ release through the RyRs which is triggered by PLC-IP3R signaling in atrial myocytes.