초록 ▼

IV. 연구개발 결과 및 활용에 대한 건의
1. 육종학자와 연계된 추가 연구의 필요성
3년으로 계획되었던 본 연구가 성공적으로 이루어지면 육종 학자들과 연계된 2년동안의 확장 연구를 수행할 것을 연구 계획서 제출시 명시한 바가 있다. 지금까지의 연구 결과로 볼때, 옥수수의 리폭시지나아제 유전자가 도입된 형질전환 벼는 2년의 확장 연구를 통하여 도열병에 저항성을 지니는 안정적인 벼품종으로의 개발이 가능하리라고 판단된다. 따라서 종묘회사 및 육종학자와 연계된 2 년동안의 추가 연구를 통하여 도열병 저상성 벼를 안정적으로 육성

IV. 연구개발 결과 및 활용에 대한 건의
1. 육종학자와 연계된 추가 연구의 필요성
3년으로 계획되었던 본 연구가 성공적으로 이루어지면 육종 학자들과 연계된 2년동안의 확장 연구를 수행할 것을 연구 계획서 제출시 명시한 바가 있다. 지금까지의 연구 결과로 볼때, 옥수수의 리폭시지나아제 유전자가 도입된 형질전환 벼는 2년의 확장 연구를 통하여 도열병에 저항성을 지니는 안정적인 벼품종으로의 개발이 가능하리라고 판단된다. 따라서 종묘회사 및 육종학자와 연계된 2 년동안의 추가 연구를 통하여 도열병 저상성 벼를 안정적으로 육성하는 연구가 필요하다고 사료된다.
2. 포장 실험으로 확대의 필요성
본 연구에서 얻어진 형질 전환벼의 스트레스 저항성은 실험실과 온실 단위에서 이루어진 결과이므로 농업 현장에서 이용하기 위해서는 포장 실험이 필수적이다. 그러나, 벼는 세대가 비교적 길어서 개발된 형질 벼를 포장에서 육성하기 위해서는 많은 시간이 요구되므로 포장시험을 포함한 추가 연구가 필요하다고 판단된다.

Abstract ▼

RESULTS AND DISCUSSION
1. Characterization of lipoxygenase gene from maize and allene oxide synthase gene from Arabidopsis Sequences of lipoxygcnasc gene from maize and allcnc oxide synthasc genes from Arabidopsis, rice, flax seed were analyzed. Maize lipoxygenase cDNA was classified as a Type I

RESULTS AND DISCUSSION
1. Characterization of lipoxygenase gene from maize and allene oxide synthase gene from Arabidopsis Sequences of lipoxygcnasc gene from maize and allcnc oxide synthasc genes from Arabidopsis, rice, flax seed were analyzed. Maize lipoxygenase cDNA was classified as a Type I LOX and Lox 1 gene family without chloroplast targeting sequence. The promoter region of lipoxygenase contained putative wound and methyl jasmonte responsive cis-element. Allene oxide synthase genes were cytocmomeP450 and Arabidopsis AOS contained chloroplast targeLing sequence. This result indicated that lipoxygenase from maize and allene oxide synthase gene from ArabidolJsis were good candidates for developing transgenic rice with stress-resist2nt transgenic rice plants.
2. Gene expressions of lipoxygcnasc and allcnc oxide synthase
Gene expression kinetics of lipoxygenase and allene oxide synthase were analyrzed. Expression of lipoxygenase gene was promptly induced by wound and biphasic expression kinetic patterm was observed in methyl jasmonate induced expression. Expression of allene oxide synLhase gene was also induced by wound and methyl jasmonate. This result implied that gene expressions of lipoxygenase and allene oxide synthase were induced by wound, and expression of lipoxygenase gene might be regulated by feed-forward activation.
3. Heterologous expression of lipoxygenase and allene oxide synthase genes
Lipoxygenase from maize and allene oxide synthase from Arabidopsis and rice were heterologously expressed in E. coli, and tlleir enzyme funtions were confirmed. These enzynmes were purified and characLerued. The fusion protein of lipoxygenase and allene oxide synthase was also heterologously expressed in E. coli. however, large amounts of inclusion body was formed. This results rationalized the utilization of lipoxygenase and allene oxide synthase for the regulation of jasmonic acid biosynthetic pathway.
4. CharacLerization of lipoxygenase reacLion
The positional specificity of lipoxygenase was deLelTnined. The lipoxygenase catalyzed hydroperoxidations of linolenic acid at C-13 and C-9 with 6:4 ratio. This dual positional specifcity of lipoxygenasc is consistant with results of phylogenetic tree analysis and amino acid residue in the putative active-siLe of lipoxygenase. Lipoxygenase with dual Positional specificity is an ideal choice for development of broad-spectrum stress-resistant transgenic plants since dual positional specific lipoxygenase can not only regulate the biosynthesis of jasmonic acid through its 13-LOX activity but also produce variety of oxylin molecules through it 9-LOX activity.
5. ProducLion and analysis of transgenic rice expressing lipoxygenase and allene oxide synLhase genes
Lipoxygenase with dual positional specificity was utilized for producing transgenic rice plants. Transgenic rice plants overexpressmg allene oxide synthase gene with or without chloroplast targeting sequence Vlere also produced. Transgenic rice plants were analyzed by PCR, souHern, northern, SDS-PAGE and correctly transformed transgenic lines were selected. Transgenic seeds of TO and T1 transgenic lines expressing lipoxygenase or allene oxide synthase were harvested.
6. Effect of jasmonic acid on Lhe growLh of rice seedling
Effect of jasmonic acid treated in the nUhient solution on the growth of rice seedling: Rice seedlings were grown in the nutrient solution containing several concentrations of jasmonic acid ranged from 0 to 100㎛ during a week.
The reduction rale of plant height, number of roots and fresh weighls were increased in the high concentrations of jasmonic acid. However, the degree of reduction rate was different in the carieties. Rice seedlings were grown in the nutrient solution containing several concentrations of jasmonic acid ranged from 0 to 30㎛ during a week. At the seven days after treatment the reduction rales of shoot were increased according to the increasing concenLrations, but those of root were higher in all concentrations than that of control. The contents of chlorophyll and carotenoid by jasmonic acid were the same as that of control regardless the concentration. Electratics of shoot in the treatments of above 20㎛ of jasmonic acid were greatly decreased compared to that of the control and tissue viabilities in the all concentations of jasmonic acid were below 60% of that of conlrol.
Effect of jasmonic acid treated spraymg on leave on the growth of rice
seedling: Rice seedlings were sprayed on the leaves by several concentrations of jamonic acid ranged from 0 to 30㎛. At seven days after spraying shoot lengths and number of tillers in the treatment of above 20㎛ were reduced compared to that of control. Dry weights of main stem treated by jasmonic acid were the same as that of control, however, dry weights of tillers were reduced.
It was same at the 14 days after treatment of jasmonic acid. The content of chlorophyll and carotenoid of the all concentrations of jasmonic acid were higher than that of the control and there were not different among concenLrations of jasmonic acid.
7. Effect of jasmonic acid on the infection of blast and bactelial blight of rice seedling
Effect of jasmonic acid on the infection of blast of rice seedling: Rice seedlings were sprayed on the leaves with several concenLrations of jasmonic acid ranged from 0 to 30㎛ and placed in the field of nmsery test for blast in order to be infested natmally. They were scored 0 to 9 according to the degrec of infection. The infection score of seedlings sprayed with jasmonic acid were lower than that of control. The score of 30㎛ of jasmonic acid was 4.4, the lowest, compared to thal of 6.l of control. The result revealed that jasmonic acid enhanced resistance to blast dcscasc.
Effect of jasmonic acid on the infection of bacterial blight of rice seedling: Seedlings of seven rice varieties Vlere sprayed on the leaves with several concenLration of jasmonic acid ranged from 0 to 30㎛ and inoculated with bactclial blight with scissor cutting method. The average lesion lengths of the 10 and 15㎛ of jasmonic acid wer 82% and 77%, respctivdy, of that of the control. However. The average of lesion lengths of above 20㎛ of jasmonic acid were the same as lhat of the control.
8. Effect of jasmonic acid on the mechanically wounded rice seedling
Several concentrations of jasmonic acid were sprayed on the rice leaves wounded and not wounded mechanically. Plant heights, number of stems, and dry weights in the not wounded plants were the same as those of the control in the below 15㎛ of jasmonic acid, however, in the wounded planls were the same as those of the conlrol in the below 20㎛ at the 7 and 14 days alter treatment with jasmonic acid. The values of F₁₀, F_m , and qN were higher in the
wounded plants than in the not wounded plants at 72 hours after treatment with jasmonic acid. However, the values of F_v/F_m of wounded plants were the same as those of not wounded plants.
9. Effect of jasmonic acid and ABA on rice(Oryza sativa L.) germination and growth of seedling between salt-tnlerance and salt-sensitivity varieties under the salt stress
Jasmontate (TA) and abscisic acid (ABA), plant hormones related with stress-tolerance, have been reported as irnproving salt tolerance of plant. Petri dish bioassay was conducted Lo determine effects of JA and ABA on salt tolerance of tolerant and sllsceptible varieties. Tolerant varieties were germinated at NaCl 23.3 ds/m while germination of susceptible varieties was inhibited. JA alone or combination of NaCl and JA did not affect germination of tolerant varieties whereas salt tolerance of susceptible varietes was significantly improved with increasing of JA concenhation. However, ABA inhibited both tolerant and susceptible varieties by 95% compared with untreated control. In the early growth response of rice varieties to NaCl, plant height of susceptible varieties was 30 to 50% more reduced than was tolerant varieties. Single and combination with JA or ABA exhibited complete inhibition in shoot growth of tolerant and susceptible varieties. Effects of JA and ABA on salt tolerance of rice root growth tended varieties to be similar to those of germination or shoot growth. ABA inhibited root growth of both completely at all concentrations.
The degree of inhibition greater in susceptible varieties than in tolerant varieties as the concentration progressively increased.
10. Resistance of transgenic rice plants expressing lipoxygenase and allene oxide synthase to disease and brown plant hopper.
Resistance of transgenic rice plants to blast and bacterial blight:
Transgenic rice plants containing lox gene were inoculated with Maganporthe grisea(KI-409). Nine plant among 34 transgenic plants were not infected.
Number of lesion, lesion length, lesion width and lesion area of infected transgenic plants except several plants were smaller than that of control. The same transgenic rice plants tested for resistance to blast were inoculated with Xanthomonas oryzae pv. oryzae(HB 01015 K₃ 111). The lesion length of all transgenic plants were shorter than that of control plant. This results indiacted that the transgenic plants expressing lipoxygenase were enhanced resistance to blast and bacterial blight.
Resostamce pf tramsgenic rice plant to brown plant hopper: Transgenic rice plants containing lox and aos gene were inoculated with brown plant hopper. Most of transgenic plants did not reveal resistance to brown plant hopper.

목차 Contents

• 제출문 ... 1
• 요약문 ... 2
• SUMMARY ... 8
• CONTENTS ... 14
• 목차 ... 17
• 제 1 장 연구개발과제의 개요 ... 20
• 제 1 절 연구개발의 목적 및 필요성 ... 21
• 1. 연구개발의 목적 ... 21
• 2. 연구개발의 필요성 ... 22
• 제 2 절 연구개발의 목표 및 내용 ... 24
• 1. 연구개발 목표와 내용 ... 24
• 2. 연구개발 방법 및 설계 ... 29
• 제 2 장 국내외 기술개발 현황 ... 32
• 제 1 절 국외 관련 기술의 현황 ... 33
• 제 2 절 국내 관련 기술위 현황 ... 36
• 제 3 장 연구개발수행 내용 및 결과 ... 37
• 제 1 절 리폭시지나아제 유전자의 구조 분석 ... 38
• 1. 리폭시지나아제 cDNA (6CO2E12) 의 구조 ... 38
• 2. 리폭시지나아제 genomic DNA 및 5 -untranslated region의 구조 ... 43
• 제 2 절 알렌옥사이드 합성효소 유전자의 구조 분석 ... 49
• 1. 알렌 옥사이드 합성효소 유전자의 구조 ... 49
• 제 3 절 리폭시지나아제와 알렌옥사이드 합성효소 유전자의 발현 기구 ... 55
• 1. 상처에 대한 리폭시지나아제 유전자의 발현 기구 ... 55
• 2. 메틸자스몬산 처리에 대한 리폭시지나아제 유전자의 발현 기구 ... 56
• 3. 알렌옥사이드 합성효소 유전자의 발현 기구 ... 57
• 제 4 절 리폭시지나아제 유전자와 알렌 옥사이드 합성효소 유전자의 연결 ... 59
• 1. 리폭시지나아제 유전자와 알렌 옥사이드 합성효소 유전자의 연결 ... 59
• 제 5 절 유전자의 이종발현 ... 63
• 1. 리폭시지나아제의 이종 발현 ... 63
• 2. 벼 알렌 옥사이드 합성효소의 이종 발현 및 생산 ... 65
• 3. Arabidopsis 알렌 옥사이드 합성 효소의 이종 발현 및 생산 ... 67
• 제 6 절 리폭시지나아제의 반응 특성 규명 ... 69
• 1. 리폭시지나아제의 정제 및 활성 검정 ... 69
• 2. 리폭시지나아제의 positional specificity ... 70
• 3. 리폭시지나아제의 반응산물 구조 분석 ... 72
• 제 7 절 형질 전환체의 생산 ... 74
• 1. 옥수수의 리폭시지나아제와 Arabidopsis의 알렌옥사이드 합성효소 유전자를 이용한 형질전환 벼의 생산 ... 74
• 제 8 절 형질전환 벼의 분석 ... 77
• 1. southern, nothern, SDS-PAGE에 의한 형질 전환 벼의 분석 ... 77
• 제 9 절 자스몬산 분석 ... 87
• 1. 자스몬산 분석 방법의 확립 및 상처 스트레스에 따른 자스몬산 함량의 변화 ... 87
• 제 10 절 자스몬산 처리가 벼 생육에 미친 영향 ... 90
• 1. 자스몬산의 뿌리처리에서 벼 유묘의 생육 변화 (실험 1) ... 90
• 2. 자스몬산의 뿌리처리에서 벼 유묘의 생육 변화 (실험 2) ... 100
• 3. 자스몬산의 엽면 처리에서 벼 유묘의 생육 변화 ... 105
• 제 11 절 자스몬산 엽면 처리가 도열병과 흰잎마름병 발병에 미친 영향 ... 110
• 1. 자스몬산 처리가 도열병 발병 정도에 미친 영향 ... 110
• 2. 자스몬산 처리가 흰잎마름병 발병 정도에 미친 영향 ... 114
• 제 12 절 자스몬산 처리가 상처 치유에 미친 영향 ... 116
• 제 13 절 염 스트레스에서 벼(Oryza sativa L.) 내염성 품종과 감수성 품종의 발아와 유묘 생육에 대한 자스몬산과 ABA 처리 효과 ... 125
• 제 14 절 형질전환체의 병해충 저항성 검정 ... 133
• 1. 형질전환벼의 도열병과 흰잎마름병에 대한 저항성 검정 ... 133
• 2. 형질전환체의 벼멸구에 대한 저항성 검정 ... 145
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 151
• 제 1 절 목표 달성도 ... 152
• 1. 제 1 차년도의 연구개발 목표와 평가의 착안점 및 달성도 ... 152
• 2. 제 2 차년도의 연구개발 목표와 평가의 착안점 및 달성도 ... 153
• 3. 제 3 차년도의 연구개발 목표와 평가의 착안점 및 달성도 ... 154
• 4. 최종평가의 착안점 및 달성도 ... 154
• 제 2 절 관련 분야에의 기여도 ... 155
• 1. 기술적 측면에서의 기여도 ... 155
• 2. 학문 발전에의 기여도 ... 155
• 3. 경제, 산업적 측면에서의 기여도 ... 156
• 제 5 장 연구개발결과의 활용계획 ... 158
• 제 1 절 추가 연구의 필요성 ... 159
• 1. 육종학자와 연계된 추가 연구의 필요성 ... 159
• 2. 포장실험으로 확대의 필요성 ... 159
• 제 2 절 타 연구에의 응용 ... 159
• 1. 리폭시지나아제 경로와 관련된 생화학적 연구에의 응용 ... 159
• 2. 자스몬산 생합성과 관련된 분자생물학적 연구에의 응용 ... 160
• 제 3 절 기업화 추진 방안 ... 160
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 161
• 제 1 절 자스몬산 생합성과 스트레스 관련 기술 정보 ... 162
• 제 2 절 스트레스 저항성 작물 생산을 위한 기술 정보 ... 162
• 제 7 장 참고문헌 ... 163
• 끝페이지 ... 168