528 Plant Mol Biol Rep (2011)29:525-532 Table 1 Overall information of Br/CEI,BrCBF,and BrCOR/4 genes Name Genbank cDNA Open reading Amino Isoelectric Molecular Half life (h) Instability accession No length (bp) frame (bp) acids point (pD) weight (kDa) index BrICEl EU374158 1.558 1,491 497 4.98 127.40 4.4 (unstable) 44.13 BrCBF DQ402470 1,003 648 216 5.32 24.05 30 (stable) 36.73 BrCOR14 D0192529 549 387 129 5.61 13.75 30(stable) 23.24 extremely low homology with the proteins deposited in all were 6.13-fold and 117.14-fold higher than the basal, databases respectively.But maximum accumulation of Br/CE/was at 24 h,whereas at 8 h BrCBF showed the highest Real-Time Fluorescence Quantitative PCR Analysis expression.In addition,BrICEl,BrCBF,and BrCOR14 genes'maximum expression appeared all at 4 h under Real-time PCR analysis showed that Br/CE/,BrCBF and drought stress carried out by Mannitol treatment with 2.2- BrCOR/4 generally displayed a trend of increased first and 6.2-13.7-fold higher than the basal(Fig.6). then decreased with the highest expression at 1,4,and 24 h, respectively,under 4C treatment.Comparison of the expression quantity of the three genes at different time Discussion points revealed that BrICEI's expression was more than 9.15-fold higher in 1 h after 4C treatment compared with Bioinformatics analysis revealed that BrICEI,BrCBF,and the basal expression and decreased immediately,followed BrCOR14 genes strongly resemble Arabidopsis ICEl, by accumulation of BrCBF at 4 h and which dramatically CBF3,and COR/5b separately.Simultaneously sequence decreased after that,and BrCOR/4 with the highest analysis showed that BrICEl contains the highly conserved expression at 24 h (49.9-fold more than the basal bHLH domain necessary in Arabidopsis ICE families,as expression)and hold the line till 7 days(Fig.5). well as bZIP and HR1 which strongly suggest that BrICEl While for ABA treatment only under 0.5 h did Br/CE/ is a DNA-binding protein.Whereas BrCBF has CANNTG show 4.3-fold higher expression than the basal.The other core element,AP2 domain as well as conserved amino acid two genes showed no significant difference at different time sequences,specifically YRG and NLS domain.While AP2 points.For salt(NaCl)treatment,the maximum accumula- and NLS have been evolutionarily conserved elements tion of BrICEI was 3.48-fold higher.BrCBF and BrCOR14 necessary for the structure or function of these CBF Table 2 Comparison of the non-heading Chinese cabbage amino acid sequences of BrICEl,BrCBF,and BrCOR14 with other cold-responsive protcins in the NCBI database Enzyme source Number of a.a Identity (% Positives (% Genbank accession No. Brassica campestris ssp.chinensis L.Makino (BrICEl) 498 EU374158 Arabidopsis thaliana (ICE1) 494 89 92 NP189309.2 A.thaliana (ICE2) 828 59 67 NP172746.1 Capsella bursa-pastoris (Cbice53) 492 89 AAS79350 B.campestris ssp.chinensis (BrCBF) 216 DQ402470 A.thaliana (CBF3) 216 73 85 ACI15599.1 A.thaliana (CBF2) 216 72 84 NP567719 A.thaliana (CBF1) 213 72 82 ABV27062.1 B.juncea (DREB1B) 214 84 91 ABX00639.1 B.napus(CBF) 214 6 91 AAD45623.1 B.campestris ssp.chinensis (BrCOR14) 129 DQ192529 A.thaliana (COR15B) 141 76 85 NP181781.1 A.thaliana (COR15A) 139 69 76 NP181782 B.rapa subsp.Pekinensis (COR) 129 97 98 ABF60663.1 B.napus (BN115) 142 78 83 AAA66068.1 Capsella bursa-pastoris (CBCOR15) 139 71 78 AAR99417.1 ≌Springerextremely low homology with the proteins deposited in all databases. Real-Time Fluorescence Quantitative PCR Analysis Real-time PCR analysis showed that BrICE1, BrCBF and BrCOR14 generally displayed a trend of increased first and then decreased with the highest expression at 1, 4, and 24 h, respectively, under 4°C treatment. Comparison of the expression quantity of the three genes at different time points revealed that BrICE1’s expression was more than 9.15-fold higher in 1 h after 4°C treatment compared with the basal expression and decreased immediately, followed by accumulation of BrCBF at 4 h and which dramatically decreased after that, and BrCOR14 with the highest expression at 24 h (49.9-fold more than the basal expression) and hold the line till 7 days (Fig. 5). While for ABA treatment only under 0.5 h did BrICE1 show 4.3-fold higher expression than the basal. The other two genes showed no significant difference at different time points. For salt (NaCl) treatment, the maximum accumula￾tion of BrICE1 was 3.48-fold higher. BrCBF and BrCOR14 were 6.13-fold and 117.14-fold higher than the basal, respectively. But maximum accumulation of BrICE1 was at 24 h, whereas at 8 h BrCBF showed the highest expression. In addition, BrICE1, BrCBF, and BrCOR14 genes’ maximum expression appeared all at 4 h under drought stress carried out by Mannitol treatment with 2.2– 6.2–13.7-fold higher than the basal (Fig. 6). Discussion Bioinformatics analysis revealed that BrICE1, BrCBF, and BrCOR14 genes strongly resemble Arabidopsis ICE1, CBF3, and COR15b separately. Simultaneously sequence analysis showed that BrICE1 contains the highly conserved bHLH domain necessary in Arabidopsis ICE families, as well as bZIP and HR1 which strongly suggest that BrICE1 is a DNA-binding protein. Whereas BrCBF has CANNTG core element, AP2 domain as well as conserved amino acid sequences, specifically YRG and NLS domain. While AP2 and NLS have been evolutionarily conserved elements necessary for the structure or function of these CBF Table 2 Comparison of the non-heading Chinese cabbage amino acid sequences of BrICE1, BrCBF, and BrCOR14 with other cold-responsive proteins in the NCBI database Enzyme source Number of a. a Identity (%) Positives (%) Genbank accession No. Brassica campestris ssp. chinensis L. Makino (BrICE1) 498 – – EU 374158 Arabidopsis thaliana (ICE1) 494 89 92 NP_189309.2 A. thaliana (ICE2) 828 59 67 NP_172746.1 Capsella bursa-pastoris (Cbice53) 492 86 89 AAS79350 B. campestris ssp. chinensis (BrCBF) 216 – – DQ402470 A. thaliana (CBF3) 216 73 85 ACI15599.1 A. thaliana (CBF2) 216 72 84 NP_567719 A. thaliana (CBF1) 213 72 82 ABV27062.1 B. juncea (DREB1B) 214 84 91 ABX00639.1 B. napus (CBF) 214 83 91 AAD45623.1 B. campestris ssp. chinensis (BrCOR14) 129 – – DQ192529 A. thaliana (COR15B) 141 76 85 NP_181781.1 A. thaliana (COR15A) 139 69 76 NP_181782 B. rapa subsp. Pekinensis (COR) 129 97 98 ABF60663.1 B. napus (BN115) 142 78 83 AAA66068.1 Capsella bursa-pastoris (CBCOR15) 139 71 78 AAR99417.1 Table 1 Overall information of BrICE1, BrCBF, and BrCOR14 genes Name Genbank accession No. cDNA length (bp) Open reading frame (bp) Amino acids Isoelectric point (pI) Molecular weight (kDa) Half life (h) Instability index BrICE1 EU374158 1,558 1,491 497 4.98 127.40 4.4 (unstable) 44.13 BrCBF DQ402470 1,003 648 216 5.32 24.05 30 (stable) 36.73 BrCOR14 DQ192529 549 387 129 5.61 13.75 30 (stable) 23.24 528 Plant Mol Biol Rep (2011) 29:525–532