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1、作物学报 ACTA AGRONOMICA SINICA 2015, 41(1): 6671 http:/zwxb.chinacrops.org/ ISSN 0496-3490; CODEN TSHPA9 E-mail: nS= , CTAB E4|yF DNAb BrRUB1 cDNA , s2 !9+s BrRUB1-D1-F: 5-ATGCAGAT CTTCGTCAAAACCCTGAC-3aBrRUB1-D1-R: 5-T CCCCACCAAAACCAGAAGAGATC-3, LA Taq DNA (TaKaRa) PCR 9bQHq94M 30 s, 55| 30 s, 72% 2 m
2、in, 35 , 72% 7 minbPCR l , peasy-T18 , v) Top10 , 4XHb 1.6 BrRUB1ySV% FMVcy DNA9 , lyb Sal I Spe I M PCR pA7-GFP vector, laaa4| DNAkb b1iZE|yzF (HVr8 )SV% , | 1.5 cm 1.5 cm SV , !Br , !SV%H , ! 16 h, Zeiss 510 nete;A4b ?o 488 nm505550 nmm , P Zeiss LSMq| , iP Adobe Photoshop q)d , Moun-tain View, CA
3、11b 1.7 BH BrRUB1 HF UV-A)HVr 7500 Real time PCR System (Applied Biosys-tems, S )_ BrRUB1 yHvaa=aa# UV-A ) 68 T 41 HWHVrb BrRUB1 BrRUB1-F: 5-ATTATCCCTTCAAGCCACC-3; BrRUB1-R: 5-AACGAGCAGATCGACAGC-3b=SBrActin BrActin-F: 5-GCTCAGTCCAAGAG AGGTATTC-3; BrActin-R: 5-GCTCGTTGTAGAAA GTGTGATC-3bk4 ABI High- C
4、apacity cDNA Reverse Transcription Kits# Power SYBR Green PCR Master Mixb20 LQ8 10 L Power SYBR Green PCR Master Mix, 8.4 L cDNA(d ), _Q_ 0.8 L (10 mol L1)b Real-time PCR 95 30 sM , 409 (95 10 s, 60 34 s), V 60F 90Vb BrActin cVr= , yMcVr 2Ct, Ct=Ct(y ) Ct(Actin)b! 3 k , 3 3 Qb 2 结果与分析 2.1 BH BrRUB1y
5、 cDNAXH#s XH BrRUB1y cDNA 650 bp, ORF u 471 bp, I 156 , yF945 bp, c 3 A0 2 =c0 , =c0174 bp (intron1) 121 bp (intron2) (m 1)bs (ProtParam)sAU , BrRUBIs0 17.413 kD; (pI) 5.77, 25 , 23 , s0T C771H1283N211O240S2, 09 2507; 38.15, ; VT GRAVY 0.383; V BrRUBIT ; AU , BrRUB1 UBQ-E2W , Wa NEDD8M , UBQEBb V NC
6、BIVE2 aw0l 9 10 RUBy , sV , BrRUB1 10 RUB , r90%b BrRUB10=aE3SE2 At RUB1(NP_564379.2)Kr96%, w0 Al RUB1 (XP_002891151.1) 95%, E2 At RUB2(NP_565812.1) 94%, 4 RUB1Bsb BrRUB1S RUB1 9 , 73 (XP_006660632.1)a!0 (XP_ 004965723.1)av (NP_001276242.1)aIN (XP_ 004248366.1) 95%, l (EMS64626.1)a (NP_001148606.1)
7、94%, 6 RUB156Bs (m 2)b 2.2 BrRUB1 % BrRUB1| , 图1 津田芜菁BrRUB1基因核苷酸序列及其推导的氨基酸序列(框内为内含子) Fig. 1 cDNA and deduced protein sequence of BrRUB1 from Tsuda (introns are boxed) 图2 BrRUB1进化树分析 Fig. 2 BrRUB1 phylogenetic tree 1000 BootStrap_ , VV 25%Sb The numbers next to the nods indicate bootstrap values of m
8、ore than 25% support from 1000 replicates. 1 ZY : BHWM1y BrRUB1XH#Vrs 69 TAU|byBrRUB1-GFPHVr8 (m 3), |SV% ,iWVrb;A_V , s;s% , BrRUB1% (m 4)b 图3 pBrRUB1-GFP表达载体示意图 Fig. 3 Schematic diagram of pBrRUB1-GFP construct 图4 BrRUB1的亚细胞定位 Fig. 4 Subcellular localization of BrRUB1 GFP BrRUB1-GFP SV%bS 100 mb S
9、ubcellular localization of GFP and BrRUB1-GFP in onion epidermal cells. Bar=100 m. 2.3 BrRUB1 yVrs 2.3.1 BH BrRUB1yF+sVr 4|HF9 RNA, HActinyT=v , BrRUB1y qRT-PCRsbAU (m 5), BH BrRUB1 VrK , QbVr , =VrKb BrRUB1 yHVrFVr+sb 2.3.2 UV-A;Hc_ i 60 dBH , | 图5 qRT-PCR分析芜菁不同组织中BrRUB1基因的表达 Fig. 5 Expression of BrRUB1 gene in various Tsuda tissues by qRT-PCR UV-A;v , sY|v 0a1a3a6 24 h_b UV-A;v 1 hcnA6 , 3 h7S , iO 6 h
