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 scaffold 45 (Release ver.3.0) 22472-22550 peptidase M14, carboxy peptidase A [ ] RNA RNA interference : RNAi RNA double-strand RNA : dsrna RNA 21 25 RNA mrna RNAi 1998 mrna RNAi dsrna RNase Dicer 21 23 small interfering RNAs : sirnas 2 sirnas RNA RISC sirna mrna RNAi RNAi Chlamydomonas reinhardtii spectinomycin aada Chlamydomonas P-1030 aada mrna 3
DNA RNAi aada mrna RNAi-37 Tag aph DNA Tag RNAi Tag RNAi RNAi RESDA-PCR Tag Chlamydomonas Chlamydomonas Tag Tag Tag RNAi [ ] Chlamydomonas RNAi Chlamydomonas RNAi C. reinhardtii mt-(cc-124) P-(1030) spectinomycin 19-P(1030) aada DNA DNA zeocin ble zeocin TAP RNAi spectinomycin, zeocin TAP spectinomycin RNAi-37 1 DNA RNAi-37 Tag paromomycin aph paromomycin TAP Tag 96 TAP ampicillin 100 g/ml, zeocin 10 g/ml, spectinomycin 100 g/ml, 200 g/ml, 300 g/ml TAP 1/10 spectinomycin Tag 1 CTAB DNA RESDA-PCR DNA RESDA-PCA Tag Tag RESDA-PCR Gonzalez-Ballester et al.,2006 4
Tag aph [aph1460tail/5 -GGCAAGCCGATAAACACCAGC-3 ],[aph1380tail/5 -AGTGCACGCAAC GCATCGTCC-3 ] hsp70a [hsp70a/tail/r1/5 -GTAAAGTGGAGGTCACCGT-3 ] 4 [Deg Alu /5 -CCAGTGAGCAGAGTGACGIIIIINNSWCA SWCAGCTT-3 ],[Deg Pst /5 -CCAGTGAGCAGAGTGACGIIIIINNSCTGCAGW-3 ],[Deg Sac / 5 -CCAGTGAGCAGAGTGACGIIIIINNSCCGCGGW-3 ],[Deg Taq /5 -CCAGTGAGCAGAGTGACGIIIIINNSWGTCGAA-3 ] [Q0/ 5 -CCAGTGAGCAGAGTGACG-3 ] I=, N A,G,T,C,S G,C,W A,T PCR 1 M 2 0.8 M 1 M 2 0.8 M Taq DNA Polymerase Bio Labs 0.2 ThenmoPol Buffer 2.5 2.5mM dntp mixture 2 DNA 2 25 1 M 4 0.8 M 1 M 4 0.8 M Ex Taq Takara 0.4 10 Ex Taq Buffer 5 2.5mM dntp mixture 4 PCR DNA2 50 [(96,5min) 1 (95,1min ; 60,1min ; 72,3min) 5 (95,1min ; 25,3min ; ramping to 72,over 3min ; 72,3min) 1 (95,1min ; 60,1min ; 72,3min ; 95,1min ; 60,1min ; 72,3min ; 95,1min ; 40,1min ; 72,3min) 20 (72,10min) 1 (4, )] [(96,5min) 1 (95,1min ; 60,1min ; 72,3min) 42 (72,10min) 1 (4, )] [(96,5min) 1 (95,30s ; 60,30s ; 72,3min) 42 (72,10min) 1 (4, )] Ex Taq 5 bp BioLabs Taq DNA Polymerase aph Tag aph Tag PCR PCR [pkf1480f/5 -GCTGGCGTTTTAC CGGCTG-3 ] aph pbluescript KS(-) 5
200 300bp 7 [pkf1700r/5 -CAGCACGAAACGGGGAGC-3 ], [pkf2120r/5 -CTTCCAGGGGGAAACGC-3 ],[pkf2490r/5 -TCAGCAGAGCGCAGATACC-3 ],[pkf2780r/5 -GATCACGCATCTTCCCGAC-3 ],[pkf3030r/5 -CCGACATTATCGCGAGC-3 ],[pkf3330r/5 -ATCGAATGCAACCGGCGC-3 ],[pkf3590r/5 -CTCGTCCAACATCAATACA ACC-3 ] PCR 1 M 2 0.8 M 1 M 2 0.8 M Ex Taq Takara 0.2 10 Ex Taq Buffer 2.5 2.5mM dntp mixture 2 DNA1 25 [(96,5min) 1 (95,30s ; 60,30s ; 72,3min) 42 (72,10min) 1 (4, )] RESDA-PCR Tag Tag aph [pkf3500f/5 -TGCCATTCTCACCGGATTCAG-3 ], [pkf3520f/5 -CAGTCGTCACTCATGGTGATTTCTC-3 ],[pkf3560f/5 -ACTTGATAACCTTA TTTTTGACGAGGGGA-3 ] 3 Tag 4 PCR Tag [pkf1610f/5 -AAATGGAGGCGCTCGTTGATCTG-3 ], [pkf1650f/5 -GGTGGATGGAAGATACTGCTCTC-3 ],[pkf1670f/5 -CTCTCAAGTGCTGAA GCGGTAG-3 ],[pkf2730f/5 -CCATCATCCAGCCAGAAAGTG-3 ],[pkf2750f/5 -AAGTGAG GGAGCCACGCAC-3 ],[pkf2770f/5 -GTCTGCGTTGTCGGGAAGATG-3 ] PCR PCR 0.8 Tag PCR clean up kit Promega 1.5 m Membrane binding Buffer heat block 60 30 Membrane binding Buffer 10mg 10 16,000g (14,000rpm) DNA 700 Membrane Wash Solution 16,000g (14,000rpm) 500 Membrane Wash Solution 16,000g (14,000 rpm) 5 30 16,000g (14,000 rpm) DNA 10 6
DNA Tag [hsp70a/tail/r1/5 -GTAAAGTGGAGGTCACCGT-3 ] Tag Tag Tag Chlamydomonas center BLAST Tag Tag Tag 12kbp [ ] Chlamydomonas Tag 31,000 spectinomycin 114 Tag 1 42 12 Tag Tag Tag RESDA-PCR 12 (26-8D, 92-12C, 96-8H, 96-9H, 107-11B, 173-1C, 176-10G, 191-9F, 293-5E, 295-3F, 307-11H, 310-1G) 11 (23-8D, 92-12C, 96-8H, 96-9H, 107-11B, 173-1C, 176-10G, 191-9F, 293-5E, 295-3F, 310-1G ) Alu,Pst,Sac,Taq PCR 3 Tag Tag 96-9H, 92-12C, 173-1C, 310-1G, 295-3F, 293-5E, 307-11H 7 307-11H, 173-1C 2 aph 96-9H [pkf2730f/5 -CCATCATCCAGCCAGAAAGTG-3 ],[pkf2750f/5 -AAGTGAGGGAGCCACG CAC-3 ],[pkf2770f/5 -GTCTGCGTTGTCGGGAAGATG-3 ] 92-12C,310-1G,295-3F,293-5E [pkf1610f/5 -AAATGGAGGCGCTCGTTGATCTG-3 ],[pkf1650f/5 -GGTGGATGGAAGATA CTGCTCTC-3 ],[pkf1670f/5 -CTCTCAAGTGCTGAAGCGGTAG-3 ] 307-11H, 173-1C aph ( 4) 7
Tag Tag RESDA-PCR 12 (26-8D, 92-12C, 96-8H, 96-9H, 107-11B, 173-1C, 176-10G, 191-9F, 293-5E, 295-3F, 307-11H, 310-1G ) 8 (26-8D, 92-12C, 96-8H, 96-9H, 293-5E, 295-3F, 307-11H, 310-1G ) Alu,Pst,Sac,Taq 5 Tag Tag PCR DNA Chlamydomonas BLAST Tag 1 [ ] RESDA-PCR RESDA-PCR Tag 600 bp 1 kbp Chlamydomonas Tag Tag 200 bp hsp70a Tag Q0 Q0 hsp70a/tail/r1 DNA Tag PCR Tag BLAST Tag 12kbp / M14, A APG5,KOG1474, CP12, MSRA3, RPN6 96-8H, 176-10G Tag scaffold Tag APG5 M14, A scaffold Tag APG5 / 8
RNAi spectinomycin RNAi spectinomycin [ ] [ ] (1)Gonzalez-Ballester D,de Montaiqu A,Galvan A,Fernandez E.Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA. Anal Biochem. 2006 Jun 15;353(2):302. Gregory J. Hannon, RNAi, (2004) (3) /, RNA 2000 (4) / / /, RNAi 2004 [ ] Tag 9
Tag Tag PCR Tag Tag aada psi Not Kpu Xba pbluescript KS(-) aph sp1, sp2, sp3 Tag amp 10 g/ml zeo 10 g/ml spc 100 g/ml spc 300 g/ml 10
RNAi spectinomycin Tag RNAi RNAi Tag spc300 g/ml TAP spectinomycin 96-9H 92-12C A P S T A P S T 1kbp 600bp 200bp Tag 200bp DNA Ladder Alu,Pst,Sac,Taq PCR 96-8H, 92-12C DNA H H 11
Tag 200 bp DNA Ladder 96-8H, 96-9H, 186-2H 96-8H 186-2H 7 96-9H 5 96-9H 293-5E A S T A S T 2 1 600bp 200bp Tag 200bp DNA Ladder Alu, Sac,Taq PCR 96-9H, 293-5E Pst 2 12
13
1 Tag PCR DNA Tag Tag Tag 14