Microsoft Word - 修士論文 小松晃明 #3376B5.doc
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1 trna pol RNAi RNAi induction by inverted repeat silencer construct bearing a trna type promoter in Chlamydomonas
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3 RNAi RNA RNA RNAi RNA 2 RNA RNase RNA bp sirna sirna RISC RNP sirna mrna mrna sirna mrna RNA RNA (RdRP) RNA RNAi RNAi RNA RNA RNAi 3 3 RdRP RdRP RNAi RNA RNAi mrna (IR) DNA RNA RNA RNAi IR pol rbcs2 IR RNAi (spc) aada 1
4 rbcs2 aada ORF N (770bp) IR RNAi RNAi37 RNAi IR RNAi CG C 15.3 RNAi 53.4 RNAi RNAi DNA RNAi37 RNA RNA RAN aada sirna IR pol pol RNAi pol RNA RNA pol pol 5SrRNA pol po trna IR RNAi RNA trna trna trna trna Pol TATA box trna Abox Bbox TTTT trna 5 3 trna trna RNA 3 RNA trna trna 3 trna trna 6 trna aada 5 ORF 150bp IR DNA RNAi 2
5 aada 19P-(1030) 1 RNAi 6 RNAi RNAi DNA RNAi DNA RNAi pol DNA pol RNAi37 RNAi 24 RNAi pol trna RNAi pol 3
6 TAP 1 Tris 2.42 g TAP salts 25 ml Phosphate solution ml Hutner trace elements 1.0 ml 1.0 ml N-Free TAP TAP TAP Salts NH4Cl 15g KCl 15g KCl-TAPSalts TAP TAP salts ( 1 ) NH 4 Cl 15 g MgSO 4 7H 2 O 4.0 g CaCl 2 2H 2 O 2.0 g Phosphate solution ( 1 ) K 2 HPO g KH 2 PO g Hutner trace elements( 1 ) EDTA, disodium salt 50 g ZnSO 4 7H2O 22 g H 3 BO g MnCl 2 4H 2 O 5.06 g CoCl 2 6H 2 O 1.61 g CuSO 4 5H 2 O 1.57 g (NH 4 ) 6 Mo 7 O 24 4H 2 O 1.10 g FeSO 4 7H 2 O 4.99 g 4
7 TAP 1l Bacto-aga 10g X ampicillin sodium(amp) (Wako) stock solution : 100 mg/ml TAP/amp:100 _g/ml ampicillin TAP Zeocin zeo (Invitrogen) 100 mg/ml stock solution TAP/zeo X : X _g/ml Zeocin TAP Spectinomycin dihydrochloride spc (sigma) Stock solution : 100 mg/ml TAP/spec X : X _g/ml spectinomycin TAP DNA construct RNAi aada mrna 150bp( A +1 52bp) IR (zeo) ble ble rbcs2 IR trna Chlamydomonas center trna PCR DNA PCR DNA pyrobest 0.2µM DNA PCR PCR pulification kit trna 60bp-MCS- trna 60bp DNAtRNA/fragmentA [5 - TATCGAATTCAAGAGCTCTCATCTCCAGCATCCGCCTCGGTTGCAGAGTGACA ACTCGACTGCGGTCGAGAACGTTGAGTCGGAT-3 ](2µM) MCS (BamH I/Pst 5
8 I/Mlu I/Bgl II) DNA trna/fragmentb [5 - CACCGCGGTGGCGGCCGCTTAAAAAGGACAAAACGGAAGATCTTCCGACGC GTCGCTGCAGTTGGATCCGACTCAACGTTCTCGA-3 ] 2µM UP/MCS/term/Fnew [5 -TATCGAATTCAAGAGCTCTCATCTCCAG-3 ] UP/MCS/term/Rnew [5 -CACCGCGGTGGCGGCC-3 ] PCR trna 60bp EcoR I MCS (BamH I/Pst I/Mlu I/Bgl II) Not I [5 - TATCGAATTCAAGAGCTCTCATCTCCAGCATCCGCCTCGGTTGCAGAGTGACA ACTCGACTGCGGTCGAGAACGTTGAGTCGGATCCAACTGCAGCGACGCGTC GGAAGATCTTCCGTTTTGTCCTTTTTAAGCGGCCGCCACCGCGGTG-3 ] DNA 1 DNA EcoR I Not I ble psp124s(lumbreras et al.,1997(a)) AGT A EcoR I BamH I trna/fragmenta trna trna [5 - GTCGTCATCGTATAGTGGTCAGTATCCCTGCCTGTCACGCAGGAGGCCGGGG TTCGATTCCCCGTGACGGC-3 ] PCR Type I 7 bp GAGAAC trna/asp/f1/new [5 -GAGAACGATGAGTCGTCATCGTATAGTGG-3 ] BAmH I trna/asp/r1[5 -TTGGATCCGCCGTCACGGGGAAT-3 ] PCR Type 7 bp trna/asp/f1/new [5 - GAGAACGATGAGTCGTCATCGTATAGTGG-3 ] trna/asp/nostem/r[5 - TTGGATCCATTATTCGGGGAATCGAACC-3 ] PCR Type 2 bp trna/asp/f1/new [5 - GAGAACGATGAGTCGTCATCGTATAGTGG-3 ] trna/asp/5bpstem/r[5 - TTGGATCCGTGCGTCACGGGGAATCGA-3 ] PCR Type 14 bp trna/asp/f1/new [5-6
9 GAGAACGATGAGTCGTCATCGTATAGTGG-3 ] trna/asp/14bpstem/r[5 - TTGGATCCACGATGAGCCGTCACGGGGAAT-3 ] PCR Type bp trna/asp/m14-2/f1[5 -GATGATGATGACTCGTATAGTGGTCAGTAT-3 ] trna/asp/m14-2/r1[5 -ATGAATATGACCGGGGAATCGAACCC-3 ] 1st PCR trnaasp/m14-2/f2new[5 - AGAACGATGATGATGACTCGTATAGTGGTC-3 ] trna/asp/m14-2/r2[5 - TTGGATCCACGATGAATATGACCGGGGAAT-3 ] 2nd PCR Type val trna trna/asp/val/f1[5 - TTGGTTTCCGTAGTGTCGTATAGTGGTCAG-3 ] trna/asp/val/r1[5 - GTTGGTTTTTGTAGTGCGGGGAATCGAACC-3 ] st PCR trna/asp/val/f2new[5 -AGAACGATGAGTTGGTTTCCGTAGTGTCGT-3 ] trna/asp/val/r2[5 -TTGGATCCGTTGGTTTTTGTAGTGCGGGGA-3 ] 2nd PCR trna trna/fragmenta 3 EcoR I BamH I EcoR I BamH I trna/fragmentb 3 aada 150bp linker GFP 106bp aada PCR aada aada p679(cerutti et al., 1997(a)) 5 BamH I trna aada/sense/f [5 -AAGGATCCGGTAGTTGGCGTCATCG-3 ] 5 GFP 8 bp trna aada/sense/r [5 - GCAGCACGGCTCGCCGCGTTGTTT-3 ] PCR aada p679 5 EcoR I trna aada/anti/f [5 -TTGAATTCGCTCGCCGCGTTGTTT-3 ] 5 MluI KpnI trnaaada/anti/rnew [5 - AAGGTACCACGCGTGTAGTTGGCGTCATCG-3 ] PCR GFP percrgfp Entelechon GmbH aada 8 bp trnalinkergfp/f [5 - CGGCGAGCCGTGCTGCTGCCCGA-3 ] EcoR I trnalinkergfp/r [5 -CGCGGAATTCGGTGACGAACTCCAGC-3 ] PCR aada GFP PCR aada EcoR I (Ligation kit (TAKARA)) aada GFP aada IR trna Asp 60bp-MCS- 7
10 MCS BamH I Mlu I IR 6 trna RNAi 5,6,7, 19P(1030) (cc-124) p-(1030) aada 300µg/ml Chlamydomonas genetic center GLE GLE CC-620mt+ CC-621mt- GLE CC-620mt+ CC-621mt- 500ml TAP 200ml 200ml (4,000rpm 8 ) 400ml N-Free TAP 20 (4,000rpm 8 ) 20ml N-Free TAP CC-620mt+ CC-621mt- 500ml 1 50ml (5,000rpm 10 ) (5,000rpm 10 ) (MILLIPORE Millex 0.45µm) GLE 19P(1030) DNA Chlamydomonas center text cell/ml 19P(1030) 50ml (3,000rpm-5 ) TAP 600µl 8
11 3.5ml GLE TAP 2 TAP 500µl 0.5mm 300mg PEG100µl Kpn I DNA 1µg 20 TAP 5ml 6 PEG TAP 2 PEG 10µg/ml 1 PCR DNA DNA SDS-phenol 10ml 8000rpm-3 SDS-EB(2% SDS, 400mM NaCl, 40mM EDTA ph8.0, 100mM Tris-HCl ph8.0)800µl proteinase-k(invitrogen)4.8µl 65 1 PCI( : )0.8ml rpm-5 2 (15,000rpm-20 ) DNA 70% TE (10mM Tris-HCl ph mM EDTA ph8.0) PCR DNA PCR ble ble ble F [5 -GATGGCCAAGCTGACCAGCGCCGTTC- 3 ] ble R [5 -TTAGTCCTGCTCCTCGGCCACGAAGT-3 ] 528bp trnalinkergfp/f [5 - CGGCGAGCCGTGCTGCTGCCCGA-3 ] trnalinkergfp/r [5 - CGCGGAATTCGGTGACGAACTCCAGC-3 ] 118 bp DNA DNA Current Protocols in Molecular Biology PREPARATION OF PLANT DNA USING CTAB 9
12 TAP 50ml 3,000rpm-5 TEN (10mM Tris-HCl 10mM EDTA 150mM NaCl ph8.0) SDS-EB 800µl proteinase-k 4.8µl PCI 30 (10,000rpm-5 ) CIA ( 24 1) 30 (10,000rpm-5 ) 65 1/7vol 5M NaCl 65 CTAB/NaCl(10%(w/v) CTAB 0.7M NaCl) 1/10vol CIA 5 (5,000rpm-5 ) CTAB precipitation (1%(w/v)CTAB 50mM Tris-HCl ph8.0 10mM EDTA ph8.0) 65 NaCl 0.5M CTAB DNA 500µl High-salt TE (100mM Tris-HCl ph mM EDTA ph8.0 1M NaC) 10mg/ml RNaseA(QIAGEN)2µl /10 TE DNA DNA PCR DIG Probe Synthesis Kit Roche aada p679 aada probe /F [5 - CAGTGATATTGATTTGCTGGTT-3 ] aada probe /R [5 - TGGACAAATTCTTCCAACTGAT-3 ] aada 618bp PCR ble ble F [5 -GATGGCCAAGCTGACCAGCGCCGTTC-3 ] ble R [5 - TTAGTCCTGCTCCTCGGCCACGAAGT-3 ] 2nd exon 3rd exon 528bp PCR rbcs2 DNA rbcs2 DNA probe/f [5 -GCAAGCTTGTACTACGACAACCGCTAC-3 ] rbcs2 DNA probe/f [5 -ATGAATTCCACGGAGCGCTTGTTGG-3 ] 4 exon 224bp PCR 10
13 ble RNAi DNA 15µg TE 10µl DNA 10 (TAKARA )2µl EtBr 4 100v BPB / 0.4M NaOH 1M NaCl 200ml 15 DNA 3MM Whatman Chr 3MM ( + (Amersham )) 3MM 500g 16 3MM 2 SSC(300mM NaCl 30mM C 6 H 5 O 7 Na 3 2H 2 O) DNA 2 SSC ml 20 SSC 62.5ml H g Na g SDS 17.5g 10 50ml 500ml 3ml DNA 2µl PCR ml DNA SSC 0.1 SDS SSC 10 SDS M 150mM NaCl ph7.5 Tween blocking stock solution 2g 10 Tween20 20ml 10 PBS-20ml 160ml 200ml 20ml 30 blocking 20ml Anti-Digoxigenin-AP, Fab 11
14 fragments(roche ) 2µl 30 40ml,Tween µl 15 2 (0.1M Tris-HCl 0.1M NaCl ph9.5)10ml CDP-Star, ready-to-use(roche ) 0.5ml CCD (FujiFilm Las-1000) RNAi amp 30ml TAP 500µl TAP 1/400 20µl amp 100µg/ml [ 1/10 1/100 1/1000 1/10 1/50 1/250 ] 8µl RNA RNA 50mlTAP 100ml 50ml 50ml 8,000rpm 5min TRIzol 1ml 8,000rpm 5 2ml 0.2ml CIA ,000rpm ml 10 12,000rpm
15 RNA ble mrna RNA 15µg rbcs2 mrna 3µg 7µl [( 25µl 37% 8.3µl 20 MOPS 2.5µl 10 (TAKARA ) 10µl H2O 4.2µl (Total 50µl)]5µl MOPS 1 MOPS v BPB / 20 SSC (3M NaCl 300mM C 6 H 5 O 7 Na 3 2H2O)200ml SSC 3MM 3MM,, 3MM,, 500g 16 3MM 20 SSC 2 SSC 3MM UV 4 RNA SSC ml DNA 2µl PCR ml DNA SSC 0.1 SDS SSC 10 SDS Tween-20 3 blocking 20ml 30 blocking 20ml 2µl 30min 40ml Tween µl ml CDP-star0.5ml CCD ] 13
16 RNAi 1 trna RNAi aada 19P(1030) DNA PCR ble 8 Type 37, Type 52 Type 30, Type 51, Type 53, Type IR aada 1 DNA Apa I Bgl ble (1770bp) Apa I ble DNA Apa I Bgl aada RNAi aada 19P(1030) aada 8 Eco T14 I aada aada IR RNAi Type 6 Type 6 Type 6 Type 8 Type 4 Type amp 100_g/ml zeo 10_g/ml spc 100,200,300_g/ml 1/10 spc Type spc 6 RNAi 9 spc Type 1 amp zeo spc 1/10 1/100 1/1000 zeo spc 10 zeo spc ble IR RNAi 14
17 RNAi pol RNAi RNAi 8 6 zeo spc 11 Type zeo spc Type strain3 zeo spc IR PCR aada aada probe /F aada probe /R trnalinkergfp/f trnalinkergfp/r ble ble F bler PCR PCR Type 8 Type strain3 ble DNA ble mrna zeo pol ble mrna ble zeo spc IR ble pol pol DNA spc µg/ml 9 spc 300µg 3 200µg 2 80µg 1 type type Type Type Type Type Type Type type RNAi 12 Type RNAi 15
18 Type RNAi Type Type 3 RNA Type trna RNA RNA Type 14bp pol RNAi RNA pol RNAi pol rbcs2 aada IR RNAi RNAi37 spc80 7 RNAi pol 13 Type strain4 RNAi /250 spc80µg/ml 10 Type strain4 RNAi 14 Type strain4 RNAi 16
19 pol IR RNAi pol Type RNAi37 RNAi pol trna RNAi RNAi RNAi RNAi sirna RNAi RNAi DNA ) Ran-on trna RNAi Dicer RNA 17
20 Adeniyi-Jones S, H.Romeo P and Zasloff M, Generation of long read-through transcripts in vivo and in vitro by deletion of 3' termination and processing sequences in the human trnamet gene, Nucleic Acids Res. 1984, 12, Ebbs ML, Bartee L, and Bender J. H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases. Mol Cell Biol. 2005, 25, Kawasaki H, and Taira K, Short hairpin type of dsrnas that are controlled by trnaval promoter signifcantly induce RNAi-mediated gene silencing in the cytoplasm of human cells, Nucleic Acids Res Jan 15;31(2): Koblenz B, Schoppmeier J, Grunow A and Karl-Ferdinand Lechtreck Centrin deficiency in Chlamydomonas causes defectsin basal body replication, segregation and maturation, Cell Science 2003,116, Kuwabara T, Warashina M, Koseki S, Sano M, Ohkawa J, Nakayama K, and Taira K, Significant higher activity of a cytoplasmic hammerheard ribozyme than a corresponding nuclear counterpart: engineered trnas with an extended 3 end can be exported efficiently and specifically to the cytoplasm in mammalian cell. Nucleic Acids Res.2001, 29,, Lu S, Shi R, Tsao1 C.C, Yi X, Li L and Vincent L. Chiang RNA silencing in plants by the expression of sirna duplexes, Nucleic Acids Res Dec 2;32(21):e171. Molnar A, Schwach F, Studholme DJ, Thuenemann EC, and Baulcombe DC. mirnas control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature. 2007, 447, Sprague K.U, Transcription of Eukaryotic trna Genes, Intstitue of- Molucular Biology, University of Oregon, Eugene, Oregon
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Chlamydomonas reinhardtii trna RNAi Comparison of RNAi effect stability induced by the inverted repeat construct driven by modified trna promoters in Chlamydomonas reinhardtii 1125008 1 3 4 12 12 13 13
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2006 Chlamydomonas reinhardtii RNA 1070010 2007 3 20 p.3 p.3 p.4 p.7 p.8 p.8 p.9 p.9 2 [ ] RNA RNA RNA aada RNA RNAi-37 aph DNA Tag RNA Tag RESDA-PCR Tag Chlamydomonas reinhardtii Tag RNAi 176-10G Tag
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