초록 ▼

Ⅲ. 연구개발의 내용 및 범위
분자생물학, 생리학 및 유전공학적 방법을 이용하여, 식물체의 발달과 생장 최적화 개발을 위해 AtSKIP 단백질의 생화학적 특성을 도입한 형질전환체로부터 형태적 특징을 분석 수행한 연구과제의 내용 및 범위는 다음과 같다.
AtSKIP 단백질의 인산화 과정에 따른 식물 발달 및 생장에 대한 분석과 기작을 규명하기 위해 AtSKIP 인산화 변형 단백질 제작 및 형질전환체들을 확보하였고, 확보한 우수 순종 형질전환 식물체로부터 발달 단계에 따른 AtSKIP 및 AtSKIP^DD

Ⅲ. 연구개발의 내용 및 범위
분자생물학, 생리학 및 유전공학적 방법을 이용하여, 식물체의 발달과 생장 최적화 개발을 위해 AtSKIP 단백질의 생화학적 특성을 도입한 형질전환체로부터 형태적 특징을 분석 수행한 연구과제의 내용 및 범위는 다음과 같다.
AtSKIP 단백질의 인산화 과정에 따른 식물 발달 및 생장에 대한 분석과 기작을 규명하기 위해 AtSKIP 인산화 변형 단백질 제작 및 형질전환체들을 확보하였고, 확보한 우수 순종 형질전환 식물체로부터 발달 단계에 따른 AtSKIP 및 AtSKIP^DD 형질전환체의 표현형 비교 분석, 광 상태 및 암 상태에서의 형질전환체들간의 표현형 비교 분석, 서로 다른 광 조건하에서 사이토키닌 반응에 대한 표현형 분석, ARR7 프로모터
-GUS 형질전환체를 이용한 사이토키닌 신호 전달 분석 및 AtSKIP 단백질과의 상호 결합인자들을 탐색하였다.
또한, AtSKIP 단백질의 인산화 과정에 대한 옥신 및 사이토키닌 호르몬 신호 전달 경로 및 발달 제어 시스템 기작을 규명하기 위하여 옥신에 대한 뿌리 생성 및 발달 분석, 옥신 농도에 따른 측근 발생의 변화 분석, 식물 생장 효율성 분석, DR5 프로모터
-GUS 형질전환체를 이용한 옥신 신호 전달 분석, AtSKIP 및 AtSKIP^DD에 의해 조절되어지는 하부 유전자들을 발굴하였으며, AtLPK1 유전자의 고염 및 병원균에 대한 생리적 기능을 규명하였다.

Abstract ▼

Ⅲ. Research contents
Plant materials and growth conditions
Arabidopsis plants were cultured in vitro on Murashige and Skoog (MS) medium under a 16 h light/8 h dark cycle at 22°C. Growth conditions in the chamber were 22°C and 16 h light/8 h dark cycles. To induce AtSKIP or its mutant expressio

Ⅲ. Research contents
Plant materials and growth conditions
Arabidopsis plants were cultured in vitro on Murashige and Skoog (MS) medium under a 16 h light/8 h dark cycle at 22°C. Growth conditions in the chamber were 22°C and 16 h light/8 h dark cycles. To induce AtSKIP or its mutant expression in transgenic seedlings, MS medium was supplemented with DEX (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 30 mM. As the DEX preparation is soluble in ethanol, the control medium was supplemented with the same volume of ethanol but without DEX. All phenotypic characterizations were performed with homozygous progeny obtained by self-fertilization.
Vector construction and Arabidopsis transformation
A SpeI site was added to the 5′ and 3′ -ends of the open reading frame (ORF) of AtSKIP gene and the AtSKIP^DD mutant. The SpeI fragment of the AtSKIP gene and AtSKIPDD mutant were ligated in-frame into a steroid-inducible pTA7002 binary vector. Because both the vector and ORF of the AtSKIP gene and AtSKIP^DD mutants included the XhoI site, we used XhoI to identify the correct orientation of the inserted gene. The 2-kb upstream genomic fragment from the AtSKIP translation start codon was amplified via PCR tog enerate the AtSKIP promoter-driven GUS construct, which was digested by HindIII and PstI. The fragment was cloned into the binary vector pCAMBIA1391Za, resulting in transcriptional fusion of the AtSKIP promoter with the GUS coding region. The transformation of Arabidopsis was conducted via vacuumin filtration (Bechtold and Pelletier 1998) using Agrobacterium tumefaciens GV3101. The resultant T3 homozygous transgenic lines were evaluated for the related phenotypes. The AtSKIP^DD mutation was introduced with the Quick-Changesite-directed mutagenesis kit (Stratagene, LaJolla, CA, USA) and confirmed by sequencing.
Identification of AtSKIP and AtSKIP^DD transgenic plants
Site-specific AtSKIP phosphorylation has been predicted to be located in Ser-235 and Ser-243 of the highly conserved SKIP/SNW domain in Arabidopsis (Bentem et al. 2008). To explore the physiological role of AtSKIP, we constructed AtSKIP and its putative phosphorylated form AtSKIP (AtSKIP^DD; mutated Ser into Asp, respectively, at the Ser-235 and Ser-243 sites of AtSKIP) transgenic plants. We fused the AtSKIP and AtSKIP^DD coding region behind the DEX-inducible promoter region to overcome the difficulties and limitations of seed harvest due to transgene-induced abnormal flower or silique development.
To verify whether the phenotypes of transgenic seedlings were correlated with transgenic AtSKIP or AtSKIP^DD levels, reverse transcription polymerase chain reaction (RT-PCR) was performed using RNA extracted from 14-dag transgenic plants with or without DEX as a template. As mutated nucleotides in AtSKIP^DD destroyed the original BsmI restriction enzyme site of the AtSKIP gene, we utilized BsmI to distinguish the mutated gene AtSKIPDD from the original AtSKIP. RT-PCR products from transgenic plants with or without DEX treatment were employed as substrates. As a result, all other RT-PCR products were digested into 1118 bp and 723 bp by the BsmI restriction enzyme, except the RT-PCR product from DEX-treated AtSKIP^DD seedlings, which fit to the expected fragment sizes of the enzyme-digested AtSKIP PCR products. In contrast, a major amount of RT-PCR product could not be digested by the enzyme, indicating that the mutations destroy the BsmI recognized site in AtSKIP^DD transgenic lines. These results indicate the efficiency of the DEX inducing-system in plants and the occurrence of mutation in the sequence composition of AtSKIP^DD.
GUS staining
Seedlings were taken carefully from the MS plates. The seedlings were placed directly in 50 mM NaHPO4 buffer, pH 7.0, containing 2 mM X-gluc. The seedlings were then incubated at 37°C for 12h or until sufficient staining developed. After clearing in 70% ethanol overnight, the seedlings were photographed through a light microscope.
Leaf development assay
Seedlings were grown in vitro on DEX-containing MS medium. After sterilization, vernalization, and the plates were put in the growth chamber for 1 to 3 weeks, and then pictures were taken for phenotypic evaluations.
Callus induction/greening assay
Arabidopsis seedlings were grown on medium with 30 mM DEX in the dark for 3 d to produce elongated hypocotyls. Hypocotyls were excised and transferred to DEX-containing medium with 300 ng/ml NAA and different concentrations (0– 300 ng/ml) of kinetin. After 2 weeks, a representative callus from each plant line and cytokinin concentration was selected and arranged for a composite photograph.
Extraction of RNA and RT-PCR
Total RNA was extracted from the frozen samples using the Plant RNeasy Extraction kit (Qiagen, Valencia, CA, USA). To remove any residual genomic DNA in the preparation, the RNA was treated with RNase-free DNaseI in accordance with the manufacturer’s instructions (Qiagen). RNA concentration was quantified accurately via spectrophotometric measurements, and 5 mg of total RNA was separated on a 1.2% formaldehyde agarose gel to check the concentration and monitor integrity. Five hundred ng of total RNA was used in the RT-PCR reaction, together with the gene-specific primers.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요 약 문 ... 3
• S U M M A R Y ... 9
• 목 차 ... 19
• 제1장 서 론 ... 20
• 제1절 연구개발의 목적, 필요성 및 범위 ... 20
• 제2장 국내외 기술개발 현황 ... 21
• 제1절 연구개발대상 기술의 국내ㆍ외 현황 ... 21
• 제3장 연구개발수행 내용 및 결과 ... 22
• 제1절 연구수행 내용 및 결과 ... 22
• 제4장 연구개발목표 달성도 및 대외기여도 ... 41
• 제1절 목표대비 달성도 ... 41
• 제2절 정량적 성과 ... 41
• 제5장 연구개발결과의 활용계획 ... 43
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 44
• 제1절 Post-translational 변형에 대한 기술 정보 ... 44
• 제7장 참고문헌 ... 45