Production of Useful Secondary Metabolites in Plants : Functional Genomics Approaches

Liu, Jang Ryol,Choi, Dong-Woong,Chung, Hwa-Jee,Woo, Sung-Sick 한국식물학회 2002 Journal of Plant Biology Vol.45 No.1

The paradigm of biological research has been changed by recent developments in genomics, high-throughput biology, and bioinformatics. Conventional biology often was based on empirical, labor-intensive, and time-consuming methods. In the new paradigm, biological research e is driven by a holistic approach on the basis of rational, automatic, and high-throughput methods. New functional compounds can be discovered by using high-throughput screening systems. Secondary metabolite pathways and the genes involved in those pathways are then determined by studying functional genomics in conjunction with the data-mining tools of bioinformatics. In addition, these advances in metabolic engineering enable researchers to confer new secondary metabolic pathways to crops by transferring three to five, or more, heterologous genes taken from various other species. In the future, engineering for the production of useful compounds will be designed by a set of software tools that allows the user to specify a cell's genes, proteins, and other molecules, as well as their individual interactions.

Molecular heterogeneity of small subunit ADPglucose Pyrophosphorylase cDNAs Isolated from Tuberous Roots of Sweet Potatoes

Bae, Jung Myung,Liu, Jang Ryol Korean Society of Molecular Biology 1996 Molecules and cells Vol.6 No.5

Genetic Transformation of Chlamydomonas reinhardtii with the RNAi suppressor p19 gene of Tombus Virus

Won Joong Jeong,Jang Ryol Liu,Heriberto Cerutti 한국육종학회 2007 한국육종학회 학술발표회 발표요지 Vol.39 No.-

Stock(Matthiola incana R. Br.)로부터 색소유전자의 분리 및 분석

오승철,유장렬,김석원,민병환 충북대학교 한국과학재단 지정 첨단원예기술개발 연구센터 1997 연구보고서 Vol.2 No.-

색소유전자의 전이를 통하여 새로운 색소발현체계를 가진 품종을 육종하기 위한 기초연구로 stock (Matthiola incana R. Br.)의 꽃봉오리로부터 cDNA-library를 합성하였고 screening을 통하여 anthocyanin 합성경로의 중요효소의 하나인 DFR (dihydroflavonol 4-reductase) 유전자를 분리하였다. 염기서열분석을 수행하여 분리유전자의 크기가 1450 bp 이며 이 중 coding region은 1029 bp임을 확인하였다. 이미 밝혀진 다른 식물체의 DFR 유전자와 서로 염기서열의 유사성을 비교해 본 결과 외떡잎식물인 옥수수와 보리와는 각각 61%를 보였으며, 쌍떡잎식물인 페튜니아, 금어초, 거베라, 과꽃 그리고 카네이션 등 과는 66%-67%의 유사성을 나타내었다. 아울러 염기서열의 G/C 함량분석을 통하여 쌍떡잎식물의 G/C 함량은 외떡잎식물의 그것에 비해 매우 낮은 수치를 나타내었다. 분리유전자의 발현을 확인하기 위하여 인위적으로 기내에서의 전사와 해석을 수행한 결과 42-44 kd 크기의 단백질을 확인하였다. Southern blot 분석의 결과 DFR 유전자는 stock의 genome에 다른 대부분의 식물체와 유사하게 한 개가 존재하며 야생종과 돌연변이종의 stock을 분리 DFR 유전자를 probe 로 Northern blot 분석을 수행하여 돌연변이종인 line 17b+가 DFR 돌연변이임을 확인하였다. In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol 4-reductase (DFR) in Matthiola incana R. Br. A heterologous cDNA probe from Zea mays was used to isolate full-size DFR cDNA clone from a corolla-specific cDNA library. Comparison of the coding region of this DFR cDNA sequence including the sequences of Zea mays, Anthirrinum majus, Petunia hybrida, Callistephus chinensis, Dianthus caryophyllus and Rosa hybrida reveals a similarity higher than 61% at the nucleotide level. The DFR transcript is G/C rich in monocotyledonous plants show a strong codon bias preferring codons with a G or C in the third position. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants and by in vitro expression yealding an enzymatically active reductase. Genomic southern blot analysis showed the presence of one gene for DFR in Matthiola incana. Northern blot analysis of the DFR wild type and mutant lines showed that the lack of DFR activity in the stable acyanic mutant k17b+is clearly by a transcriptional block of the DFR gene.

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Min, Sung Ran,Liu, Jang Ryol,Kim, Suk Weon Springer-Verlag 2007 Plant biotechnology reports Vol.1 No.1

21세기 식물생명공학과 생물산업의 전망 : 유전체 연구에 의한 Paradigm Shift

유장렬,최동욱,정화지,Liu, Jang-Ryol,Choi, Dong-Woog,Chung, Hwa-Jee 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.3

Biotechnology in the 21st century will be driven by three emerging technologies: genomics, high-throughput biology, and bioinformatics. These technologies are complementary to one another. A large number of economically important crops are currently subjected to whole genome sequencing. Functional genomics for determining the functions of the genes comprising the given plant genome is under progress by using various means including phenotyping data from transgenic mutants, gene expression profiling data from DNA microarrays, and metabolic profiling data from LC/mass analysis. The aim of plant molecular breeding is shifting from introducing agronomic traits such as herbicide and insect resistance to introducing quality traits such as healthful oils and proteins, which will lead to improved and nutritional food and feed products. Plant molecular breeding is also expected to aim to develop crops for producing human therapeutic and industrial proteins.

Enhanced Production of Shikonin by Using Polyurethane-entrapped Lithospermum erythrorhizon Cells

Taek, Seo-Weon,Liu, Jang-Ryol,Park, Young-Hoon 한국미생물 · 생명공학회 1989 한국미생물·생명공학회지 Vol.17 No.4

L. erythrorhizon 세포를 polyurethane foam과 함께 증식시킬 경우 shikonin 유도체가 polyurethane에 효과적으로 흡착됨과 동시에 polyurethane을 사용하지 않은 경우와 비교하여 shikonin 생산량이 현저히 증가하였다. 이 같은 증가는 세포를 polyurethane pore에 고정하여 증식시킴으로써 원활한 세포간 접촉을 유지하고 세포 내에 shikonin 농도를 저하시켜shikonin 생성에 좋은 조건을 제공함에 기인한 것으로 생각되었다. 공정의 생산성을 높이기 위하여 여러가지 배양시스템이 검토되었는데, indole-3-acetic acid(1.75mg/ι)와 kinetin(0.1mg/ι)을 함유하는 Schenk-Hildebrandt 배지 (SHIK 배지) 시스템이 가장 효과적이었다. p-Chlorophenoxyacetic acid (2.0 mg/ι)와 kinetin (0.1 mg/ι)를 함유하는 Schenk-Hildebrandt 배지 (SHND 배지) 시스템에 비교하여 SHIK 배지 시스템에서 Shikonin 생성량은 약 4.5배 증가하였다. Polyurethane을 세포를 고정화하는 지지체로 사용할 경우에는 현재 행하여지고 있는 2단계 배양보다 1단계 배양이 더욱 효과적이며 경제적으로도 매우 유리할 것으로 판단되었다. Production of shikonin derivatives by Lithospermum erythrorhizon cells by using polyurethane foam was invesliigated. Shikonin derivatives were effectively adsorbed mostly by phase distribution to polyurethane matrices and their production increased significantly compared to the suspension culture. The enhanced production of shikonin was probably due to more facilitated cell to cell con-tact and lowered intracellular shikonin concentration, both of which are known to be favorable for plant secondary metabolite production. In order to improve the process productivity, tell culture was conducted under various culture conditions: Of them, Schenk and Hildebrandt medium containing indole-3-acetic acid (1.75mg/ι) and kinetin (0.1mg/ι) was considered most appropriate for shikonin production. Production of shikonin increased about 4.5 times in the Schenk and Hildebrandt medium containing indole-3-acetic acid (1.15mg/ι) and kinetin (0.1mg/ι) when compared to the same medium containing p-chlorophenoxyacetic acid (2.0mg/ι) and kinetin (0.1mg/ι). When poly-urethane was used as the support material, a single-stage system was more preferred to the conventional two-stage culture system in terms of shikonin productivity.

Plant regeneration through somatic embryogenesis of floral stem of Nymphoidesindica(L.)O.Kuntze

Suk Weon Kim,Jang Ryol Liu 한국육종학회 2007 한국육종학회 학술발표회 발표요지 Vol.38 No.1

Plant regeneration from zygotic embryo-derived cell suspension cultures and somatic embryogenesis of Ranunculus kazusensis

Suk Weon Kim,Jang Ryol Liu 한국육종학회 2007 한국육종학회 학술발표회 발표요지 Vol.38 No.1

참나리(Lilium lancifolium Thunb.) 접합자배로부터 소자구 형성을 통한 식물체 재생

김경희,유장렬,김석원,Kim, Kyung-Hee,Liu, Jang-Ryol,Kim, Suk-Weon 한국식물생명공학회 2007 식물생명공학회지 Vol.34 No.1

참나리(Lilium lancifolrium)의 접합자배(2n=24)로부터 체세포배발생 및 소자구 형성을 통해 식물체 재분화 체계를 확립하였다. 참나리의 접합자배는 2,4-D가 첨가된 MS 배지에 배양하게되면 소자구 및 체세포배가 동시에 발생하였다. 접합자배로부터 소자구 및 체세포배 형성빈도는 0.1mg/L 2,4-D 첨가 배지에서 각각 66.7% 와 56.7%로 가장 높았다. 그러나 1mg/L 이상의 고농도 2,4-D 처리구에서는 소자구 및 체세포배 형성빈도가 크게 감소하였다. 접합자배에서 형성된 체세포배로부터 식물체를 재생하기 위하여 체세포배를 생장조절제가 첨가되지 않은 MS 기본배지로 옮겨 명배양하였으나 식물체의 발달이 이루어지지 않았으며 신장된 체세포배의 기저부에서 이차적으로 다수의 소자구가 발달하였다. 형성된 소자구를 MS 기본배지로 옮겨 4주간 배양한 결과 정상적인 소식물체로 발달이 이루어졌다. 참나리의 접합자배로부터 직접적인 소자구 형성 및 체세포배로부터 이차적인 소자구 형성을 통해 재생된 식물체는 공히 염색체수가 2n=24 로 2,4-D가 첨가된 배양과정에서 염색체의 안정성이 유지됨을 알 수 있었다. Plant regeneration system from zygotic embryos (2n=24) of Lilium lancifolium Thunb. via bulblet formation was estabished. Zygotic embryos of Lilium lancifolium formed bulblets and somatic embryos simultaneously when they cultured on MS medium supplemented with low concentration of 2,4-D. The highest frequency of bulblet and somatic embryo formation from zygotic embryos of Lilium lancifolium was 66.7% and 56.7%, respectively. The frequency of bulblet and somatic embryo formation was decreased when they cultured on MS medium over than 1 mg/L of 2,4-D. To regenerate whole plants, somatic embryos formed on zygotic embryos were transferred to MS basal medium. However somatic embryos did not fully converted into plantlets. Further incubation in the light, elongated somatic embryos formed numerous bulblets at the base of somatic embryos. Upon transfer to MS basal medium, bulblets were successfully converted into plantlets after further 4 weeks of culture in the light. After acclimatization, plantets from bulblets were transferred to soil and grown to normal plants in growth chamber (approximately $30\;{\mu}mol\;m^{-2}s^{-1}$, 16/8h photo period, $25^{\circ}C$) The chromosome analysis revealed that plants regenerated from zygotic embryos showed 2n=24. These results indicate that chromosome stability of source tissue is maintained during plant regeneration via bulblet formation.