High throughput VIGS (Virus-Induced Gene Silencing) 기술을 이용하여 식물기능유전체 연구를 수행하였다. 3000개의 Nicotiana benthamiana sequenced 중에서 식물조직발달, 호르몬작용, 식호전달 등에 관련된 유전자 492개와 unknow/hypothetical 유전자 725개 등 총 1217개를 선택하여 pTV00 vector로 cloning하고 Agrobacterium으로 transformation 한 후 각 유전자 당 6개의 Nicotiana benthamiana 식물
High throughput VIGS (Virus-Induced Gene Silencing) 기술을 이용하여 식물기능유전체 연구를 수행하였다. 3000개의 Nicotiana benthamiana sequenced 중에서 식물조직발달, 호르몬작용, 식호전달 등에 관련된 유전자 492개와 unknow/hypothetical 유전자 725개 등 총 1217개를 선택하여 pTV00 vector로 cloning하고 Agrobacterium으로 transformation 한 후 각 유전자 당 6개의 Nicotiana benthamiana 식물체를 사용하여 Agrobacterium infiltration으로 VIGS를 수행하였다. Infiltration 후 2-3주 후 식물체의 형질 변화를 control과 비교하여 조사하였다. phenotype은 cell death, leaf yellowing, growth arrest, abnormal organs, premature senescence, flower abnormality, apical dominance breakdown등으로 나누어 분석하였다. 1217개의 유전자 중 136개 유전자에서 식물체 형질변화가 유도되었다. 나타나는 형질에 대한 결과는 sequence DB 및 유전자 expression profile DB와 연계하여 phenotype DB로 구축 하였다. 이 연구를 통하여 식물발달 과정을 콘트롤하는 새로운 유전자들을 발굴하였고 그들 유전자작용 기작을 탐구하므로서 식물발달조절의 전체 네트워크를 이해하고자 하였다. 주요한 유전자의 기능을 여러 분자생물학적, 생화학적, 세포생물학적인 테크닉을 이용하여 심층분석하였다. 이렇게 발굴된 기능성유전자는 고부가가치 형질전환 식물개발에 활용하고자 한다.
Abstract▼
The purpose of this study is to carry out plant functional genomics using virus-indued gens silencing to understand plant developmental reguatory pathways. We used TRV (tobacco rattle virus)-based VIGS system using Nicotiana benthamiana as a host. Three thousand N. benthamiana cDNAs were random-wque
The purpose of this study is to carry out plant functional genomics using virus-indued gens silencing to understand plant developmental reguatory pathways. We used TRV (tobacco rattle virus)-based VIGS system using Nicotiana benthamiana as a host. Three thousand N. benthamiana cDNAs were random-wquenced, and target genes were selected for VIGS-mediated fuction analysis. The target genes include plant developmental genes, signal transduction genes, and hormone-related genes. Total 1,217 cDNAs were cloned into pTV00 (vigs VECTOR), and the recombinant plasmids were transformed into Agrobacterium. Then Agrobacterium suspension was infiltrated into N. benthamiana plants at 3 weeks after germination. After 2-3 weeks after infiltration, the visible phenotypes appeared in the newly emerged plant tissues. Among 1,217 cDNAs, 136 cDNAs showed visible morphological phenotypes, including stunted growth, cell death, changes in leaf color, wrinkled leaves, changes in plant architecture, and flower abnormality. Thus using VIGS, we identified many novel genes that modulate plant developmental porcesses and chracterized their action mechanisms in plant development using various moleular and biochemical tools. We also probed the protein-protein interaction in order to understand the global picture of developmental regulatory networks. The detailed results obtained in this project were summarized below. We identified NtRpn3, a regulatory subunit of 26S proteasome, as a interacting protein of NtCDPK1 calcium-dependent protein kinase in Nicotiana tabacum using yeast two hybrid system. Rpn3 in yeast is an essential protein involved in proteolysis of cell cycle regulatory proteins, and the carrot homologue of Rpn3 was previously isolated as a nuclear antigen that is mainly expressed in the meristem. NtCDPK1 physically interacts with NtRpn3 in vitro in a Ca2+ as a cofactor. NtCDPK1 and NtRpn3 are colocalized in the nucleus, unclear periphery and around plasma membrane in vivo. Both NtCDPK1 and AtRpn3, and NtRpn3 homolog of Arabidopsis, are mainly expressed in the rapidly proliferating tissues including shoot and root meristems, and developing floral buds. Virus-induced gene silencing of either NtRpn3 or NtCDPK1 resulted in the phenotypes of abnormal cell morphology and premature cell death in newly emerged leaves. Finally, NtCDPK1 interacts with NtRpn3 in vivo as shown by co-immunoprecipitation. Based on these results, we propose that NtCDPK1 and NtRpn3 are interacting in a common signal transduction pathway possibly for regulation of cell division, differentiation, and cell death in tobacco.
목차 Contents
제1장 연구개발과제의 개요...18
제2장 국내외 기술개발 현황...19
제3장 연구개발수행 내용 및 결과...22
가. High throughput VIGS system...22
나. Virus-induced gene silencing 을 이용한 식물기능유전체연구...25
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