Flavonoid 생합성:생화학과 대사공학적 응용

박종석,박종범,김경환,하선화,한범수,김용환,Park, Jong-Sug,Kim, Jong-Bum,Kim, Kyung-Hwan,Ha, Sun-Hwa,Han, Bum-Soo,Kim, Yong-Hwan 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.4

주요 농작물에서 건강-방어용 flavonoids 생성, phytoalexin (isoflavonoid, flavanol, proanthocyanidin)의 생성 및 소절을 통한 식물의 저항력 증대, 색소 (flavonol, anthocyanin)의 합성에 의한 자외선 방어, nod 유전자 inducer (flavones, isoflavones)의 대량 발현에 의한 혹 형성 (nodulation) 효율증대 등은 대사공학 적으로 향상 가능한 부분들이다. 파란 꽃을 개화하는 품종이 카네이션, 국화, 장미 등 중요 장식용 화훼작물들에는 결핍되어 있는데,이는 F3'5'H 유전자가 없어서 파란색 delphinidin 색소를 생산할 수 없기 때문으로 추정된다. 따라서 F3'5'H 유전자를 형질전환 하여 이러한 제한을 극복하고 delphinidin 유도체 생산이 가능하게 되면 파란색 꽃의 생산 가능성을 증대시킬 수 있게 된다. 또한 영양학적인 측면에서 이미 중요한 생리적 기능이 밝혀진 catechin을 비롯한 proanthocyanidin 과 anthocyanin은 의약품 및 식품첨가제 등 다양한 분야에서 크게 시장성을 넓히고 있어 상업적 측면에서 대사공학의 유망한 목표가 되고 있다. 최근의 대사공학 분야에서의 많은 성공에도 불구하고, flavonoid에 대한 고도의 대사공학 조절을 이용하여 원하는 flavonoid 화합물을 생성하거나, 원치 않는 flavonoid 화합물을 억제하도록 하는 데는 여전히 기술적 문제점들이 남아있다. 예를 들면 IFS와 FLS 등의 유전자 분리 그리고 조직 및 시기 특이적인 promoter 개발 등이 동시에 이루어져야 하며, co-pigmentation 및 액포 pH와 관련된 메카니즘에 대한 이해, 화훼작물들의 형질전환 기술 개발 등이 이루어져야 원하는 꽃의 착색 조절이 가능하게 될 것이다. 최근 나팔꽃에서 액포의 $Na^{+}$H$^{+}$ exchanger를 파괴하여 화색을 변경시킨 mutants 연구를 통하여 조만간 액포 pH의 조절을 이용한 식물 대사공학이 가능할 것으로 기대되고 있다 (Yamaguchi et al. 2001). 아직 자연계에서 기본적인 골격의 변경만으로 수천 종류의 flavonoid가 생성 가능한가는 여전히 의문점으로 남아 있으나, 분명한 것은 다양한 식물 체계에서의 노력으로 농업, 원예, 그리고 영양분 증대를 위한 flavonoid 대사를 어떻게 조절할 것인가에 대한 정보를 얻을 수 있고, 또한 flavonoid 생합성 연구로부터 얻어진 정보들을 통하여 세포질 대사와 기본적인 생물학적 현상에 대한 이해를 넓힐 수 있게 될 것이다. Flavonoid biosynthesis is one of the most extensively studied areas in the secondary metabolism. Due to the study of flavonoid metabolism in diverse plant system, the pathways become the best characterized secondary metabolites and can be excellent targets for metabolic engineering. These flavonoid-derived secondary metabolites have been considerably divergent functional roles: floral pigment, anticancer, antiviral, antitoxin, and hepatoprotective. Three species have been significant for elucidating the flavonoid metabolism and isolating the genes controlling the flavonoid genes: maize (Zea mays), snapdragon (Antirrhinum majus) and petunia (Prtunia hybrida). Recently, many genes involved in biosynthesis of flavonoid have been isolated and characterized using mutation and recombinant DNA technologies including transposon tagging and T-DNA tagging which are novel approaches for the discovery of uncharacterized genes. Metabolic engineering of flavonoid biosynthesis was approached by sense or antisense manipulation of the genes related with flavonoid pathway, or by modified expression of regulatory genes. So, the use of a variety of experimental tools and metabolic engineering facilitated the characterization of the flavonoid metabolism. Here we review recent progresses in flavonoid metabolism: confirmation of genes, metabolic engineering, and applications in the industrial use.

흑조위축병 바이러스 RNA 를 절단하는 망치머리형 라이보자임의 제작

박종석(Jong Sug Park),김주곤(Ju Kon Kim),손성한(Seong Han Sohn),이석순(Sug Soon Lee),황영수(Young Soo Hwang) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.6

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with T₃ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at 55℃, while the cleavage was not detected at 37℃. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at 55℃. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.

벼 흑조위축병 바이러스의 분자생물학적 연구

박종석(Jong Sug Park),배신철(Shin Chyul Bae),김영민(Young Min Kim),백융기(Young Ki Paik),김주곤(Ju Kon Kim),황영수(Young Soo Hwang) 한국응용생명화학회 1994 Applied Biological Chemistry (Appl Biol Chem) Vol.37 No.3

Rice black-streaked dwarf virus (RBSDV), a member of the plant reoviridae fijivirus group, causes a serious damage for rice production in Korea. To characterize the RBSDV genome, virus particles were produced by feeding of planthopper (Laodelphax striatellus F.) earring RBSDV to maize plants for 2 days. In 30∼40 days after feeding, the viral particles were purified from the infected maize roots by using 10∼40% sucrose gradient centrifugation. After treatment of 10% SDS to remove the viral coat proteins, ten viral double-stranded RNAs were resolved in agrose gel electrophoresis. Total dsRNA was then used to synthesize cDNA by reverse transcriptase and a cDNA library was constructed in the λgt11 vector. The phages that contain RBSDV cDNA fragments were selected by hybridizing with the random-primed probe prepared from RBSDV dsRNAs. After subcloning of several cDNA fragments into the pUC19 plasmid mector, one clone (pRV3) was chosen for sequencing. The pRV3 clone was shown to be Located on the RBSDV genome fragment No.3 by RNA gel-blot analysis. Sequence analysis of the clone revealed that the pRV3 contains two partial open reading frames.

해충저항성 Bacillus thuringiensis (Bt) 벼의 환경위해성 평가: 해충저항성 Bt벼가 물벼룩(Daphnia magna)에 미치는 영향

이기종 ( Ki Jong Lee ),김효진 ( Hyo Jin Kim ),이장용 ( Jang Yong Lee ),류태훈 ( Tae Hun Ryu ),박종석 ( Jong Sug Park ),박범석 ( Beom Seok Park ),권순종 ( Soon Jong Kweon ),손수인 ( Soo In Sohn ),서석철 ( Seok Cheol Suh ),오성덕 ( 한국응용생명화학회(구 한국농화학회) 2011 Journal of Applied Biological Chemistry (J. Appl. Vol.54 No.4

해충저항성 Bacillus thuringiensis (Bt) 벼와 낙동벼의 비표적생물체인 물벼룩(Daphniamagna)에 대한 급성독성시험을 실시한 결과, 해충저항성 Bt벼의 48시간-EC50은 4,429.13 mg/L (95% 신뢰한계는 3908.130~5020.363 mg/L), 무영향 농도(NOEC)는 1,800 mg/L이었고, 낙동벼는 48시간-EC50은 2,889.56 mg/L (95% 신뢰한계는 1,073.407~6,854.321 mg/L), 무영향농도는 1,000 mg/L이었다. 처리기간 중 해충저항성 Bt벼와 낙동벼간의 급성독성에 영향을 미칠 수 있는 요인은 발생하지 않았다. Insect-resistant transgenic rice was developed by inserting the mCry1Ac1 a modified gene from the soil bacterium Bacillus thuringiensis (Bt). For biosafety assessment, we studied the effects on survival of cantor Daphnia magna, a commonly used as a model organism in ecotoxicological studies. D. magna fed on Bt rice and its near non-genetically modified (GM) counterparts (Nakdong) grown in the same environment (100% ground rice suspension). The Bt rice was comfirmed to have the insertion of T-DNA and protein expression by the polymerase chain reaction and ELISA analysis. Feeding study showed similar cumulative immobility and abnormal response of D. magna between Bt rice and non-GM counterparts. 48 h-EC50 values of Bt rice and non-GM rice showed 4,429 and 2,889 mg/L respectively. The rice no observed effect concentration (NOEC) values for D. magna was suggested 1,000 mg/L. We conclude that the tested Bt-rice and Nakdong similar cumulative immobility for D. magna the widely used model organism. We found out that there is strong possibility that the growth of Bt rice didn`t affect to non-target insects.

바이러스 질병 예방을 위한 식물 경구 백신 연구 동향

한범수,박종석,김형국,하선화,조강진,김용환,김종범,Hahn, Bum-Soo,Park, Jong-Sug,Kim, Hyeong-Kuk,Ha, Sun-Hwa,Cho, Kang-Jin,Kim, Yong-Hwan,Kim, Jong-Bum 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.2

Transgenic plants have been studied as delivery system for edible vaccine against various diseases. Edible plant vaccines have several potential advantages as follows: an inexpensive source of antigen, easy administration, reduced need for medical personnel, economical to mass produce and easy transport, heat-stable vaccine without refrigerator, generation of systemic and mucosal immunity and safe antigen without fetal animal-virus contaminants. The amount of recombinant antigens in transgenic plants ranged from 0.002 to 0.8% in total soluble protein, depending on promoters for the expression of interested genes and plants to be used for transformation. Throughout the last decade, edible plant vaccine made notable progresses that protect from challenges against virus or bacteria. However edible plant vaccines have still problems that could be solved. First, the strong promoter or inducible promoter or strategy of protein targeting could be solved to improve the low expression of antigens in transgenic plants. Second, the transformation technique of target plant should be developed to be able to eat uncooked. Third, marker-free vector could be constructed to be more safety. In this review we describe advances of edible plant vaccines, focusing on the yields depending on plants/promoters employed and the results of animal/clinical trials, and consider further research for the development of a new plant-derived vaccine.

유전자변형 작물이 토양 미생물상에 미치는 영향

이기종 ( Ki Jong Lee ),오성덕 ( Sung Dug Oh ),손수인 ( Soo In Sohn ),류태훈 ( Tae Hun Ryu ),박종석 ( Jong Sug Park ),이장용 ( Jang Yong Lee ),조현석 ( Hyun Suk Cho ),안병옥 ( Byung Ohg Ahn ) 한국환경농학회 2012 한국환경농학회지 Vol.31 No.2

유전자변형 작물을 종자로 판매하거나 식품, 사료 혹은 가공용으로 이용하기 위해서는 반드시 관련 기관의 승인을 받아야 한다. 관련부처에서는 유전자변형 작물의 승인에 앞서 환경위해성 평가 자료가 과학적으로 타당한지 검토한다. 환경위해성 평가 중 유전자변형 작물이 토양 미생물 군집에 미치는 영향은 충분히 연구되지 못한 분야이다. 최근 토양 환경내 미생물 군집의 특성을 연구하기 위한 발전된 방법들이 개발되고 있다. 배양에 의존적인 또는 비의존적인 기술에 의한토양 미생물의 군집 특성을 조사한 연구와 유전자변형 작물의 환경위해성 평가 적용 가능성을 고찰하였다. 유전자변형 작물의 토양미생물 영향 평가는 사안별 평가 원칙에 의해 이루어져야 한다. 신뢰할 수 있고 상세한 토양 미생물 평가가 이루어지기 위해서는 다양한 분석 방법의 조합이 필요하다. BACKGROUND: Genetically modified (GM) crops must receive relevant regulator`s authorization before they can be sold as seed or used food, feed and processing. Before approving any GM crop, the relevant government ministries are required to examine environmental risk assessment to make scientifically sound and socially acceptable decisions. But one of the least studied and understood areas in the environmental risk assessment of GM crops are their impact on soil microbial community. METHODS AND RESULTS: Recently, advanced methods have been developed to characterize the soil microbial community in various environments. In this study, the culture-dependent and culture-independent technical approaches for profiling soil microbial communities are summarized and their applicability to assess GM crops are discussed. CONCLUSION(S): We concluded that the effect of GM crops on soil microbial community need to be assessed on a case by case basis. The combination of culture-dependent and culture-independent method was necessary for reliable and detailed assessment of effect of GM crops on soil microbial community.

식물의 산업용 지방산 생산을 위한 오일합성 유전자의 기능과 이용 전망

김현욱,이경렬,박종석,노경희,김순희,김종범,Kim, Hyun-Uk,Lee, Kyeong-Ryeol,Park, Jong-Sug,Roh, Kyung-Hee,Kim, Sun-Hee,Kim, Jong-Bum 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.2

식물 종자오일의 지방산은 인간에 필수 지방산을 공급하는 식용 및 생필품 생산에 필요한 다양한 산업원료로 사용된다. 식물 오일의 식용 및 산업용 적합성과 경제성을 극대화하기 위해 유전공학에 의한 종자오일의 양과 지방산 조성 변형을 위한 대사조절연구가 계속 진행되고 있다. 하지만 식물에 일반적으로 존재하지 않는, 산업적으로 유용한 특이지방산의 합성과 종자오일로의 축적은 한계가 있음이 알려져 있다. 그 이유는 재배가 용이하며 생산성이 높은 오일식물의 acyltransferase가 특이지방산에 대한 기질특이성이 떨어지며 또한 특이지방산에 대한 세포막지질에서 종자오일로 전환시키는 편집기작 (editing mechanism)이 없기 때문으로 사료된다. 최근에 모델식물의 종자오일의 축적에 관여하는 acyltransferase에 관한 유전자들이 클로닝되었고, 특이지방산이 합성되는 인지질에서의 편집기작에 관여하는 acyltransferase 유전자들이 밝혀져 이들 유전자들의 정보를 이용하여 특이지방산을 효과적으로 생산.증진할 수 있는 기술이 개발될 수 있을 것으로 기대한다. 피마자오일의 주성분인 산업용 특이지방산인 리시놀레인 지방산을 오일식물에서 생산하기 위해 이에 관여할 것으로 추정되는 11개의 acyltransferase 유전자를 피마자 유전체 데이터베이스에서 존재함을 확인하였다. 이들 유전자들의 도입에 의해 형질전환 식물이 갖고 있지 않은 리시놀레인산에 대한 기질 특이성을 부여하여 종자오일 내의 특이지방산의 생산을 증가시킬 것으로 기대된다. Fatty acids in seed oil from plants are essential for human nutrients and have been used for industrial purpose. The growing demands of seed oil as food resources and feedstocks for industrial uses have attempted to modify fatty acid composition and to increase oil content in transgenic plants. However, production of unusual fatty acids in transgenic plants are limited, which is not synthesized the level same as original plants. This bottleneck was common for production of several unusual fatty acids in transgenic plants and suggests that there is different for substrate preference in oil metabolic pathway enzymes between host oil plants and original wild plants. Review of acyltransferases involved in acyl-editing and seed oil accumulation of oil plant and wild-plant producing unusual fatty acids will design strategies to maximize the production of unusual fatty acids in transgenic plants. In here, we identified eleven acyltransferase genes in castor based on sequence homology, which will be useful to increase hydroxy unusual fatty acids in transgenic plants.

세균성 질병 예방을 위한 식물 경구 백신 연구 동향

한범수,정영재,노경희,박종석,조강진,김용환,김종범,Hahn Bum-Soo,Jeong Young-Jae,Roh Kyung-Hee,Park Jong-Sug,Cho Kang-Jin,Kim Yong-Hwan,Kim Jong-Bum 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.4

Plants have considerable advantages for the production of antigenic proteins because they provide an inexpensive source of protein and an easy administration of vaccine. Since a publication describing edible plant vaccine of HBsAg in 1992, a number of laboratories around the world have studied the use of plants as the bioreactor to produce antigenic proteins of human or animal pathogens. Over the last ten years, these works have been mainly focused on three major strategies for the production of antigenic proteins in plants: stable genetic transformation of either the nuclear or plastid genome, or transient expression in plants using viral vectors. As many antigenic proteins have been expressed in tobacco, also several laboratories have succeeded to express genes encoding antigenic proteins in other crop plants: potato, tomato, maize, carrot, soybean and spinach. At present many works for the production of edible plant vaccine against bacteria-mediated diseases have mostly performed the studies of enterotoxins and adhesion proteins. Also the development of new-type antigens (pili, flagella, surface protein, other enterotoxin and exotoxin etc.) is required for various targets and more efficacy to immunize against microorganism pathogens. Many works mostly studied in experimental animals had good results, and phase I clinical trial of LTB clearly indicated its immunogenic ability. On the other hand, edible plant vaccines have still problems remained to be solved. In addition to the accumulation of sufficient antigen in plants, human health, environment and agriculture regulation should be proven. Also oral tolerance, the physiological response to food antigens and commensal flora is the induction of a state of specific immunological unresponsiveness, needs to be addressed before plant-derived vaccine becomes a therapeutic option.

Microarray 분석을 이용한 유채 종자성숙단계별 유전자 발현 양상

노경희 ( Kyung Hee Roh ),박종석 ( Jong Sug Park ),김종범 ( Jong Bum Kim ),김현욱 ( Hyun Uk Kim ),이경렬 ( Kyeong Ryeol Lee ),김순희 ( Sun Hee Kim ) 한국응용생명화학회(구 한국농화학회) 2012 Journal of Applied Biological Chemistry (J. Appl. Vol.55 No.4

We observed that oil began to accumulate at 25 seed days after flowering (DAF) and reached the maximum potential at 35 seed DAF of oilseed rape, and the greatest weight of 100 seeds was obtained at 35 seed DAF. To survey a broad analysis of gene expression in developing embryos of Brassica napus, the Bn 300k microarray have been constructed. The Bn 300k Microarrary was designed from 80,696 unigenes clustered from 543,448 ESTs and 780 cDNA at NCBI. These arrays have been hybridized in a series of experiments with probes derived from seeds and leaf of B. napus. Approximately 8.5% of the 7,000 genes were expressed as ratios 2-fold higher in seed (25 DAF) than leaves and 0.4% at ratios 10. Also we observed that storage and cell differentiationrelated genes were highly expressed at 10 DAF, whereas energyrelated genes including fatty acid metabolism were increased up depending on seed maturation using Microarray, which was confirmed by reverse transcriptase polymerase chain reaction. These results suggest that B. napus arrays provide a very useful data set of seed-specific expression that can be further analyzed by examination of the promoter regions of these genes and help our understanding of the complex regulatory network in developing seeds.

총설 : 바이오디젤 원료 작물 품종 개량과 생명공학기술 응용

노경희 ( Kyung Hee Rob ),박종석 ( Jong Sug Park ) 한국응용생명화학회(구 한국농화학회) 2007 Applied Biological Chemistry (Appl Biol Chem) Vol.50 No.3