초록 ▼

○ 연구결과
- 벼에서 병원균에 반응하는 OsBIMK1를 인산화 시켜주는 OsMEK3을 분리하여 재조합 단백질을 순수 정제한 후 in vitro protein phosphorylation 실험을 통해 병저항성에 관여하는 벼의 MAPK cascade를 부분적으로 밝힘
- 담배의 NtMEK2DD 유전자가 형질전환된 벼에서 하위 MAPK가 활성화된 후 병저항성에 관련된 방어 유전자들의 발현을 촉진되어 벼흰잎마름병에 대한 저항성을 보임
- 담배의 NtMEK2DD와 NtMEK2KR 유전자를 벼에 형질전환 시켜 쌍자엽식물(담배

○ 연구결과
- 벼에서 병원균에 반응하는 OsBIMK1를 인산화 시켜주는 OsMEK3을 분리하여 재조합 단백질을 순수 정제한 후 in vitro protein phosphorylation 실험을 통해 병저항성에 관여하는 벼의 MAPK cascade를 부분적으로 밝힘
- 담배의 NtMEK2DD 유전자가 형질전환된 벼에서 하위 MAPK가 활성화된 후 병저항성에 관련된 방어 유전자들의 발현을 촉진되어 벼흰잎마름병에 대한 저항성을 보임
- 담배의 NtMEK2DD와 NtMEK2KR 유전자를 벼에 형질전환 시켜 쌍자엽식물(담배)과 단자엽식물(벼)에서의 MAPK cascade가 보존되어 있으며 벼에서도 병저항성 관련 MAPK pathway가 과산화수소의 과다생산에 관련되어 있으며 최종적으로 과민감 반응을 보인다는 사실을 밝힘 - Yeast screening을 통해 발굴된 염해 내성 유전자 SAT79 클론은 현재까지 밝혀지지 않은 애기장대 AtSAT79 유전자와 상동 유전자임을 알았으며, RNA gel blot 실험 분석에 의해 AtSAT79 유전자는 염해 스트레스에 대해 반응하는 유전자임을 알 수 있었다. AtSAT79 과다발현 형질전환체들의 염해 내성을 분석하기 위하여 염해 처리 후 뿌리 생장 길이를 조사한 결과 35S::AtSAT79 형질전환체들이 100 mM NaCl 농도 염해 스트레스 하에서 적응 능력이 뛰어남을 알 수 있었다. 또한 AtSAT79 유전자가 염해스트레스 뿐만 아니라 sucrose 스트레스에 대해서도 반응하는 유전자들을 조절함을 알 수 있었다. 또한 본 연구를 통해 AtSAT79 유전자가 sucrose-triggered developmental root growth 과정에 필요한 인자임을 알 수 있었다. 이러한 데이터들을 분석해 볼 때, 세포질에 존재하는 AtSAT79 유전자는 sucrose-triggered pathway에 관련하여 염해에 대한 저항성 작물 개발에 사용할 수 있는 유전자임을 시사함
- 조밀하고 잘 건조된 최상의 캘러스를 선별하는 방법을 통해 재현성이 높고 고효율의 안정적인 벼 형질전환 시스템을 확립하였고 병저항성 기능을 가진 형질전환체를 선발 육성함
- 내병성 및 내염성을 목적으로 창출되어진 형질전환 벼를 유묘상태에서 포장과 실내에서 그 능력을 평가하여 실질적인 분자육종 자원으로 이용될 수 있는지 여부를 확인함

Abstract ▼

I. Title
Functional analysis of useful genes for stress-tolerant rice
II. Importance and objectives of the research
The mitogen-activated protein kinase (MAPK) cascade is a major and evolutionally conserved signaling pathway by which extracellular stimuli are transduced into intracellular r

I. Title
Functional analysis of useful genes for stress-tolerant rice
II. Importance and objectives of the research
The mitogen-activated protein kinase (MAPK) cascade is a major and evolutionally conserved signaling pathway by which extracellular stimuli are transduced into intracellular responses in eukaryotic cells (Widmann et al., 1999). NtMEK2, a tobacco (Nicotiana tabacum) MAPKK, is known to activate both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Ectopic expression of NtMEK2^DD, a constitutively active mutant of NtMEK2, was found to induce HR-like cell death and defense-related gene expression, as well as to promote the generation of H₂O₂ in chloroplasts in tobacco (Yang et al., 2001 Liu et al., 2007). Extensive studies have been conducted to evaluate the functional understandings of many MAPK genes and their upstream kinases in dicotyledonous plants such as Arabidopsis, alfafa, tobacco, tomato and parsely (Jonak et al., 2002; Mishra et al., 2006). To date, a total of 17 MAPK genes have been identified, and several of these have been characterized in rice (Agrawal et al., 2003 Rohila and Yang, 2007). However, little is known about the interactions between components of MAPK and upstream MAPK kinase in rice. Therefore, a more detailed study is necessary to identify upstream kinases and downstream target proteins to better understand the MAPK-mediated pathways in rice.
To understand the MAPK cascade associated with disease resistance in rice, we transformed rice plants with the tobacco NtMEK2^DD or NtMEK2KR, which encodes the constitutively active mutant or the inactive mutant of NtMEK2, respectively, under the control of a steroid inducible promoter. In NtMEK2^DD transgenic rice, activation of the endogenous 48-kDa MBP kinase leads to HR-like cell death,which is preceded by the generation of hydrogen peroxide. Furthermore, expression of the NtMEK2^DD gene in transgenic rice plants induced the expression of several downstream MAPKs and defense-related genes. Taken together, these results suggest that NtMEK2 is functionally interchangeable with a rice MAPKK and also a useful tool for evaluating the MAPK pathway involved in signaling pathways that lead to defense responses in rice.
As our largest single primary food source, the cereal endosperm is among the most economically important structures in biology. The product of double fertilization, it is also among the most novel. The unique value of the endosperm of cereal grains as a food source can be traced directly to the subsequent evolution of the major grain-filling pathways, namely starch and storage protein biosynthesis, within this novel structure. The endosperm displays the basic themes of organ formation in plants, but with a simpler structure than other major organs. Hence, the central issues in endosperm development are directly relevant to other plant organs. Key issues includes regulation of the cell cycle, partitioning of growth between cell division and cell expansion, regulation of cell expansion and terminal differentiation, cell-to-cell signalling, and determination of cell fate. The high growth rate and grain filling processes place extraordinary demands on cellular metabolism and metabolic regulation making the endosperm an excellent model for biochemical genetics. However the basis of the biological and biochemical significance in endosperm is not well understood. The endosperm study will be discovered a number of crop genes that are a key determinant for environment stress tolerance and crop productivity.
III. Research content and scope
1. Functional analysis of pathogen-responsive MAPK cascade in rice
The mitogen-activated protein kinase (MAPK) cascade is a major and evolutionally conserved signaling pathway by which extracellular stimuli are transduced into intracellular responses in eukaryotic cells. MAPK cascades are composed of three protein kinase modules. MAPK, the last kinase in the three-kinase cascade, is activated by dual phosphorylation of Thr and Tyr residues in a TXY motif by MAPK kinase (MAPKK). MAPKK, in turn, is activated by MAPKK kinase (MAPKKK). Extensive studies have been conducted to evaluate the functional understandings of many MAPK genes and their upstream kinases in dicotyledonous plants such as Arabidopsis, alfafa, tobacco, tomato and parsely. So far, a total of 17 MAPK genes have been identified, and several of these have been characterized in rice. However, little is known about the interactions between components of MAPK and upstream MAPK kinase in rice. The OsBWMK1 and OsBIMK1 were pathogen-responsive MAPK genes in rice but no study in MAPK cascade with those genes so far. OsBWMK1 has been induced by infection and wounding of blast pathogen (Magnaporthe grisea), while OsBIMK1 were induced by BTH, chemical inducer of systemic resistance. In this study, upstream MAPK kinase genes related to phosphorylation of OsBWMK1 and OsBIMK1 were isolated and cloned. Expressed MAPK kinase protein was purified to detect in vitro protein phosphorylation and its relationship with the disease resistance of rice. In addition, OsMEK3 transgenic rice plants will be a useful tool for the identification of downstream substrates utilized by rice MAPK, which will help understand disease resistance in rice.
2. Expression of an active tobacco mitogen-activated protein kinase kinase is involved in multiple defense responses in transgenic rice plants
It is known that NtMEK2, a tobacco MAPK kinase, is the upstream kinase of both salicylic acid-induced protein kinase and wounding-induced protein kinase. Expression of NtMEK2^DD, a constitutively active mutant of NtMEK2, induced multiple defense responses in tobacco. To determine if a similar MAPK cascade was involved in disease resistance in rice, transgenic rice plants that contained an active or inactive mutant of NtMEK2 under the control of a steroid inducible promoter were generated. The expression of NtMEK2^DD in transgenic rice plants resulted in HR-like cell death, which was preceded by the activation of endogenous 48-kDa MBP kinase. That MAPK is also activated by Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice. In addition, prolonged activation of the MAPK induced the generation of hydrogen peroxide and up-regulated the expression of subset of MAPKs and defense-related genes including the pathogenesis-related genes, peroxidase and glutathione S-transferase. These results demonstrate that NtMEK2 is functionally interchangeable with rice MAPK kinase in inducing the activation of the downstream MAPK, which in turn induces multiple defense responses in rice.
3. Mitogen-activated protein kinase cascade in signaling polyamine biosynthesis in plants
Expression of NtMEK2^DD, a constitutively active mutant of NtMEK2, activates endogenous SIPK and WIPK and leads to several stress/defense responses in tobacco. In this study, we used ACP (annealing control primer)based differential display RT-PCR to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade. Arginine decarboxylase gene (ADC), which is involved in plant polyamine biosynthesis, was one of 10 differentially expressed genes. When compared with NtMEK2^KR plants, NtMEK2^DD transgenic plants exhibited a significant increase in ADC, ODC and MPO1 transcript levels, as well as in theputrescine and spermine content following SIPK/WIPK activation. Taken together, these results suggest that the NtMEK2-SIPK/WIPK cascade regulates polyamine synthesis, especially putresine synthesis, through transcriptional regulation of the biosynthetic genes in plants.
4. Isolation of the SAT79 gene by yeast screening
It has been suggested that a single salt tolerance mechanism is common to all eukaryotes. We therefore screened a maize kernel cDNA library for salt tolerance by functionally overexpressing it in yeast . The cDNA library was generated from maize kernels 15 - 25 days after pollination using the phagemid vector pAD-GAL4-2.1 (Stratagene, LaJolla, CA, USA). After transformation with the maize cDNA library, yeast cells were selected for their ability to tolerate salt stress. We isolated SAT79 salt-tolerance (SAT) clone, which, based on previous publications, included not only nutrient stress responsive gene but also factor involved in UV stress.
5. Salt tolerance in Arabidopsis overexpressing AtSAT79
To compare salt tolerance in Arabidopsis and AtSAT79-overexpressing yeast cells, we constructed AtSAT79-overexpressing transgenic Arabidopsis lines. Using Kanamycin-resistance segregation and Northern analysis, we selected plants homozygous for AtSAT79. The AtSAT79-overexpressing line seeds were exposed to 100 mM NaCl and primary root length was measured after 7 d. Seeds expressing AtSAT79 showed a higher salt tolerance than wild-type (Col-O) seeds. These data suggest that AtSAT79 seems to confer very effective salt tolerance in plant cells. To determine AtSAT79 protein localization, a CaMV35S promoter-driven fusion gene (35S-AtSAT79-GFP) of the AtSAT79 cDNA and GFP was constitutively expressed in Arabidopsis. In the root cells of the transgenic seedlings, the GFP signal appeared as diffused structure that might be cytosol. To understand the salt tolerance molecular mechanism of AtSAT79 in plants, we compared RNA expression of stress response genes during salt stress. We found that ERD15 expression was not significantly different.
Transcripts of RD29A, RD22 and RD29B, however, accumulated more in AtSAT79-overexpressing lines than in wild-type in response to salt stress.
6. Production of transgenic rice with agronomically useful genes
It has been known that rice tissue culture is very difficult because of low regeneration rate, long cultivation period, complicated cultivation method. Therefore, we try to find out to selection method of the best callus with high density and well-dried, in order to establish effective rice transformation system with good repeatability.
7. Evaluation of disease resistance and salinity tolerance in transgenic rice
High rice yield is required to develop novel crop, resistant to various environmental stresses. Although crop carrying stress-resistant gene is produced, its function is effective at green house but not in field with hard environmental condition. Thus, transformed rice carrying disease or salt resistant gene was studied its function in both green house and field with the raising seedling and confirmed whether it could be the practical olecular breeding resource or not.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 8
• CONTENTS ... 14
• 목 차 ... 16
• 제 1 장 연구개발의 개요 ... 18
• 제 1 절 연구개발의 목적 및 필요성 ... 19
• 제 2 절 연구개발의 범위 ... 21
• 제 2 장 국내외 기술개발 현황 ... 24
• 제 1 절 국내ㆍ외 연구동향 ... 25
• 제 2 절 연구결과의 국내ㆍ외 기술개발현황 위치 ... 27
• 제 3 장 연구개발 수행 내용 및 결과 ... 28
• 제 1 절 벼에서 병원균에 반응하는 MAPK 신호전달체계의 내병성 기능분석 ... 29
• 제 2절 Expression of an active tobacco mitogen-activated protein kinase kinase is involved in multiple defense responses in transgenic rice plants ... 37
• 제 3절 Mitogen-activated protein kinase cascade in signaling polyamine biosynthesis in plant ... 52
• 제 4 절 염해 내성관련 유전자의 분리 및 기능분석 ... 65
• 제 5 절 내재해성 유전자의 벼 형질전환 개체 육성 ... 76
• 제 6 절 형질전환 벼의 병해 포장저항성 및 내염성 평가 ... 84
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 89
• 제 1 절 연도별 연구목표 달성도 ... 90
• 제 2 절 관련분야의 기술발전에의 기여도 ... 93
• 제 5 장 연구개발결과의 활용계획 ... 96
• 제 1 절 추가 연구의 필요성 ... 97
• 제 2 절 타 연구에의 응용 ... 98
• 제 3 절 기업화 추진 방안 ... 98
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 99
• 제 1 절 병원균에 대한 식물 방어 신호 전달 기작에 대한 기술정보 ... 100
• 제 2 절 Mu-TAIL에 의한 기능 유전체 응용에 대한 기술 정보 ... 101
• 제 7 장 참고문헌 ... 102
• 끝페이지 ... 109