감자의 Sucrose Synthase 유전자를 이용한 전분함량조절

목차

목 차
Ⅰ. 서 론
Ⅱ. 연 구 사
1. 감자의 중요성 및 식물학적 특징
2. 감자의 기내소괴경 형성
3. 감자의 당, 전분대사 과정 및 관련 효소
4. 유전자 클로닝을 통한 감자의 전분 함량 조절
5. 식물체 형질전환
III. 재료 및 방법
1. 전분생합성과정 분석
1.1. 기내소괴경의 형성유도
1.2. 기내소괴경의 분석
2. 식물체 형질전환
3. 유전자 클로닝
3.1. 실험재료
3.2. 실험방법
3.3. 클로닝 벡터의 제조
4. 형질전환체의 분석
4.1. 형질전환체의 polymerase chain reaction에 의한 선발
4.2. Genomic DNA 준비
4.3. mRNA의 준비
4.4. 5′/3′ RACE
4.5. DIG-표지에 의한 Southern 분석
4.6. Sucrose synthase 유전자의 발현 분석
4.7. 포장재배 검사
4.8. 실험의 전체 개요
IV. 결과 및 고찰
1. 전분생합성과정 분석
1.1. 소괴경의 생장 변화
1.2. 당 함량의 변화
1.3. 전분과 sucrose synthase의 변화
2. 감자의 형질전환
2.1. 식물체 재생
2.2. 형질전환
3. 유전자의 클로닝 및 형질전환
3.1. Patatin promoter를 사용한 유전자의 형질전환
3.2. Sucrose synthase 유전자 클로닝 및 형질전환
4. 전분 함량이 변화된 형질전환체의 검정 및 생산
4.1. Sucrose synthase의 활성 및 관련 물질 분석
4.2. Sucrose synthase 유전자의 발현
4.3. 유전자 분석
4.4. 포장재배 검사
V. 종합 고찰
VI. 참고문헌
Abstracts

본문내용

se synthase, most likely responsible for the entire sucrose cleavage in sink tubers, during in vitro tuberization of potato, and then we tried to obtain the transgenic plant altered starch contents through the gene cloning of sucrose synthase.
In vitro tuberization patterns of potatoes depends on genetic characteristics of the cultivars and were maintained in in vitro culture. The levels of sucrose and starch linearly increased, glucose and fructose remained consistently low during microtuber bulking. Sucrose synthase activity and sucrose, starch contents in microtuber increased until the harvested date. There was a closely related between the sucrose, starch content of the microtubers and sucrose synthase activity. The high concentration of sucrose synthase in developing microtubers may be the supporting evidence about a key role for this enzyme in starch biosynthesis in potato.
To develop the tuber-specific expression vector, we constructed vectors of pBI/PAT containing β-glucuronidase under the control of patatin promoter, a NOS-NPTⅡ as marker gene. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBI/PAT from potato cv. Desiree. We obtained 7 transformed lines through NPTⅡ sequences analysis by PCR. In the comparison of GUS staining test in microtuber, patatin promoter represents more powerful than CaMV 35S promoter.
To alter the starch contents in microtuber, we constructed vectors of pBI/PSS and pBI/PSAS containing and sense and antisense potato sucrose synthase under the control of patatin promoter from pBI/PAT cutted β-glucuronidase gene. At the stage of selection for transformant, 50 mg/l kanamycin medium was effectively used without reducing regeneration capability of transformed potato. We obtained 20 transformed lines with pBI/PSS and 7 lines with pBI/PSAS. The quantitative analysis of soluble sugars, starch and sucrose synthase activity from transgenic microtubers showed that sucrose synthase activity and starch content were increased in pBI/PSS lines 8, 22 and 30, but were decreased in pBI/PSAS lines 3 and 10. RT-PCR analysis demonstrated that sucrose synthase gene expression increased in pBI/PSS lines and decreased in pBI/PSAS lines. To investigate the relation between sucrose synthase and other key enzymes in starch biosynthesis, such as AGPase, we performed RT-PCR analysis from different transgenic lines pBI/PSS 8 and pBI/PSAS 3. Expression of AGPase subunit B and S was increased in line pBI/PSS 8, but decreased in line pBI/PSAS 3. It is interesting that in transgenic lines the levels of increase in starch content is declined in tubers harvested grown from pot than that of microtubers. We obtained the transgenic lines pBI/PSS 8, 22 and 30 that increased about 5∼10% starch content and transgenic lines pBI/PSAS 3 and 10 that decreased about 10% starch content.
Key words : Solanum tuberosum, in vitro tuberization, starch, sucrose
synthase, patatin promoter, transgenic potato,

키워드

감자,   기내소괴경 형성,   전분