제 9 장 유전자 발현과 전사체의 분석

1. 제 9 장유전자 발현과 전사체의 분석

3. 유전자 발현조절 부위의 분석 -gelretardation assay

4. -Foot printing analysis

5. -Report 유전자를 이용한 발현부위의 분석

7. -Luciferase

8. -Green Fluorescence protein  해파리에서 분리 Bright DAPI GFP Merge (DAPI+GFP) Merge (Bright + DAPI+GFP)

9. Control BA ACC ABA GA 2.4D IAA NAA NPA -Gus  Beta-glucuronidase

12. -NorthernHybridization

13. -RNase Protection assay

14. -RT-PCR

15. -Real time PCR TaqMAN probe를이용한 방법

16. -Differential Display Differential Display was invented in 1992 by Drs. Arthur Pardee and Peng Liang to allow rapid, accurate and sensitive detection of altered gene expression (Science. 1992, 257:967; U.S. Patent 5,262,311).

22. 번역은 역전사 RNA (Antisense RNA) 에 의해 조절될 수 있다. 과잉의 철원자를 저장하기 위한 단백질 Fur (철 농도를 감지하는 단백질)

24. RNA interference (RNA 방해) dsRNA를 인식 분해 세포내에는 dsDNA와 dsRNA는 존재하나dsRNA는 존재하지 않는다. 그러나 바이러스에 감염되는 경우 바이러스의 게놈은 dsRNA를 거쳐 이동되어진다. 이를 감지하여 분쇄하는 기능. RISC

25. RNA interference (RNA 방해)의 증폭과 확산 RNA-dependent RNA polymerase

26. 식물의 RNA interference (RNA 방해)에 의한 Post-transcriptional gene silencing

27. Starch modification genes From gene study to biotechnology • Starch-branching enzyme I gene (IbSBEI) (Biotechnology Letters, 2006, Hamada et al.) • High amylose content of sweetpotato starch by RNA interference of the IbSBEII(Plant Biotechnology, 2006, Shimada et al.) • Cloning and characterization of sweetpotato Isoamylasegene (Breeding Science, 2005, Kim et al.) • Amylose free by RNA interference of the GBSSI (Submitted to Molecular Breeding, 2006, Shimada et al.) • Further cloning of Starch synthase I, II, III genes are under study in University of Seoul

28. 1. Amylose free starch in sweetpotato tuber GBSSI (Granule-Bound Starch Synthase I) x RNAi x Amylose ADP-glucose Amylopectin

29. Wild-type plant Transgenic plant

30. Iodine staining pattern of root and starch granules Kokei-14 GR-1 GR-2 GR-3 GR-4 GR-5 Kokei 14 GR-1

31. Apparent amylose contents of starches * : Apparent amylose content was determined by the iodine-colorimetry. **: Attached phosphate content was determined as inorganic phosphate after washing the defatted starch.

32. 2. Increase of amylose contents in starch Amylose ADP-glucose x Amylopectin x RNAi SBEII (Starch Branching Enzyme II)

33. Diagrammatic representation of silencing construct 35S-P hpt T RB LB MCS pCAMBIA1300 HindIII BamHI XbaI SacI EcoRI NOS-T CaMV35S-P 1st exon GUS 1st exon SBEII Splicing sense orientation SBEII dsRNA antisense orientation

34. Knockdown of SBEII function by RNA interference C 1 2 3 4 10.25 17.9 23.3 24.23 0 (%) Amylose content Iodine staining pattern of non-transgenic Kokei 14 (c), transgenic lines with dsRNA of SBEII (1 - 3) and a transgenic line with dsRNA of GBSSI (4)

35. Sample Total fresh Starch Amylose weight (g) yield (g) content (%) ± Control 281.7 5.8 15.05 0.4 ± ASGS - 1 259.6 5.2 17.90 0.7 ± ASGS - 2 181.8 4.7 23.33 0.3 ± ASGS - 3 241.3 4.1 15.67 0.5 ± ASGS - 4 144.5 5.9 24.23 0. 3 ± ASIS - 1 277.0 4.5 20.00 0.1 ± ASIS - 2 244.4 4.9 23.35 0.8 ± ASIS - 3 173.5 4.4 24.85 0.6 ± ASIS - 5 205.2 4.2 23.85 0.1 The yield and amylose content in starch • Dry weight of starch in 30g fresh weight tuber • Average  S.D. (3-6) (Ref. : Plant Biotechnology, 2006, Shimada et al.)

36. Unit-chain distributions of representative starches by GPC (Gel-permeation chromatography) Amylose-free Control Disappearance of amylose fraction GBSSI-RNAi GR-1 High-amylose Increase of amylose fraction Decrease of short chains 30 42 54 66 78 SBEII-RNAi ASGS1 * Provided by Dr. Kitahara at University of Kagoshima Elution time (min)

37. Unit-chain distributions of transgenic sweetpotato starches by Gel-permeation chromatography Amylose-free Control Disappearance of amylose fraction GBSSI-RNAi GR-1 High-amylose Increase of amylose fraction Decrease of short chains Otani et al. (2007) Plant Cell Reports26: 1801-1907 30 42 54 66 78 SBEII-RNAi ASGS1 Elution time (min)