Plant Mol Biol Rep (2011)29:525-532 527 were raised on the water saturated cotton in the growth illustrated that BrICEl was more likely an ortholog of chamber at 20C to 22C.ABA stress was carried out by ICEl.BrCBF was 73%identical to A.thaliana CBF3 and 200 uM Abscisic acid,Salt stress was induced by 200 mM 72%to A.thaliana CBF2 and CBFI which suggest that NaCl and drought stress was imposed by 300 mM mannitol BrCBF was an ortholog of CBF3,while BrCOR14 was for 0 h,0.5 h,2 h,4 h,8 h,24 h and 4 days,respectively. with 76%identity to A.thaliana COR15B and 69%identity Total RNAs were isolated separately.Gene-specific primers to A.thaliana COR15A which demonstrated that BrCOR14 for BrICEI,BrCBF,and BrCOR14 were 5'-ACAACAA was more like an orthology of COR15B.Alignment scores CGCAACACCCT-3'and 5-ACGACGCCAACA CCTCT-3'. of the amino acid sequences of the three genes with other 5'-GTGTGTGAAGTGAGGGAACCAAAC-3'and 5'-CC known cold-responsive proteins in Cruciferous family also AAGCCGAGTCCGCATAAT-3',5'-TTCTTCTT show high concordance (Table 2). TCCCCAGCG-3'and 5'-TTCCATCACCTTTCTCGG-3' The results also suggested that the BrICEl was highly designed based on non-heading Chinese cabbage BrICEl, conservative to the ICEl in possessing a bipartite nuclear BrCBF,and BrCOR/4 genes.Primers for Actins were localization signal (NLS)domain from K304 to K353 and a 5'-ACAACTCCATCATGAAGTGT-3'and 5'-GAGATC bHLH domain from R316 to L352,as well as potential CACATCTGTTGGAA-3'used as control.Real-time PCR recognition sites,for instance basic-leucine zipper (bZIP) was carried out by One Step SYBR PrimescriptTM that contains a basic region mediating sequence-specific RT-PCR Kit (Takara,Japan).Mixtures contained 12.5 ul DNA binding followed by a leucine zipper region(required SYBR,10 pmol adaptor primers each,50 ng cDNA,ddH2O for dimerization)transcription factors,HRI (shown to bind up to 25 ul.Amplification profile was 95C for 120 s,35 the small G protein rho or to activate PKN in its GTP-bound cycles of 95C for 10 s,55C for 20 s,and 72C for 20 s on form)and Pfam domain (Fig.2). Corbett Rotor-Gene 3000A.Each reaction was carried out Besides BrCBF gene contained a consensus sequence, in a separate PCR system with two replicates and was CANNTG (CACCTG).Alignment of the BrCBF proteins repeated three times.The data were analyzed using data indicated that it contained a highly conserved AP2 DNA- analysis software that comes with the machine. binding domain of 60 amino acid residues from /s2 to A109 as well as three potential strikingly conserved elements, YRG (consisted of 23 amino acids containing the con- Results served YRG amino acid motif and was functionally important for DNA binding),NLS domain (from amino Isolation of cDNAs Encoding Cold-Response Proteins acid R42 to Rs9)and ALA-RICH domain (from amino acid Ts4 to K128).Besides,the prediction also revealed that RNA quality was determined by 1%agarose gel electro- BrCBF contains a dehydration-responsive element(Fig.3). phoresis.28S,18S,and 5S RNA in total extracted RNA In addition,the DNA sequence at the presumed transcrip- had clean bands and proper proportion (Fig.1).BrICE/, tion site of BrCOR/4.CCG.is identified as a common core BrCBF,and BrCOR/4 were obtained by RT-PCR and sequence. RACE.Overall information of the putative proteins was shown in Table 1.The BrICEl protein was estimated to Secondary Structure Analysis have a half life of about 4.4 h and instability index of 44.13. thus being classified as unstable,while BrCBF and Secondary structure analyses indicated(Fig.4)that BrICEl BrCOR14 were classified as stable.Furthermore,alignment consisted of 158 a-helices,40 B-turns jointed by 83 scores of the amino acid sequences of the identified cold- extended strands,and 216 random coils which resembled responsive genes with other known homologous proteins the secondary structure of Arabidopsis ICE1 (NP 189309.2) predicted that BrICEl was 89%identity to Arabidopsis and CbICE53 (C.bursa-pastoris AAS79350). ICEl and 59%to Arabidopsis thaliana ICE2 which BrCBF consisted of 84 a-helices,nine B-turns jointed by 33 extended strands and 90 random coils which commendably Fig.1 RNA quality resembled the secondary structure of CBF3(AF074602)and determination CbCBF (AY391121.1).It was also note worthy that a-helices occurred predominantly in the structure of BrCBF Moreover,the BrCOR14 consisted of 78 a-helices,seven B-turns jointed by 17 extended strands,and 26 random coils, 28S 18S which did not resemble the secondary structure of COR15B (A.thaliana NM_129814)and CbCOR15B (AY437888.1) 5S that much.Subsequently,homology modeling analysis was carried out,while no suitable target was found due to the 鱼Springerwere raised on the water saturated cotton in the growth chamber at 20°C to 22°C. ABA stress was carried out by 200 μM Abscisic acid, Salt stress was induced by 200 mM NaCl and drought stress was imposed by 300 mM mannitol for 0 h, 0.5 h, 2 h, 4 h, 8 h, 24 h and 4 days, respectively. Total RNAs were isolated separately. Gene-specific primers for BrICE1, BrCBF, and BrCOR14 were 5′-ACAACAA CGCAACACCCT-3′ and 5′-ACGACGCCAACA CCTCT-3′, 5′-GTGTGTGAAGTGAGGGAACCAAAC-3′ and 5′-CC AAGCCGAGTCCGCATAAT- 3 ′, 5′-TTCTTCTT TCCCCAGCG-3′ and 5′-TTCCATCACCTTTCTCGG-3′ designed based on non-heading Chinese cabbage BrICE1, BrCBF, and BrCOR14 genes. Primers for Actins were 5′-ACAACTCCATCATGAAGTGT-3′ and 5′-GAGATC CACATCTGTTGGAA-3′ used as control. Real-time PCR was carried out by One Step SYBR® Primescript™ RT-PCR Kit (Takara, Japan). Mixtures contained 12.5 μl SYBR, 10 pmol adaptor primers each, 50 ng cDNA, ddH2O up to 25 μl. Amplification profile was 95°C for 120 s, 35 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s on Corbett Rotor-Gene 3000A. Each reaction was carried out in a separate PCR system with two replicates and was repeated three times. The data were analyzed using data analysis software that comes with the machine. Results Isolation of cDNAs Encoding Cold-Response Proteins RNA quality was determined by 1% agarose gel electro￾phoresis. 28S, 18S, and 5S RNA in total extracted RNA had clean bands and proper proportion (Fig. 1). BrICE1, BrCBF, and BrCOR14 were obtained by RT-PCR and RACE. Overall information of the putative proteins was shown in Table 1. The BrICE1 protein was estimated to have a half life of about 4.4 h and instability index of 44.13, thus being classified as unstable, while BrCBF and BrCOR14 were classified as stable. Furthermore, alignment scores of the amino acid sequences of the identified cold￾responsive genes with other known homologous proteins predicted that BrICE1 was 89% identity to Arabidopsis ICE1 and 59% to Arabidopsis thaliana ICE2 which illustrated that BrICE1 was more likely an ortholog of ICE1, BrCBF was 73% identical to A. thaliana CBF3 and 72% to A. thaliana CBF2 and CBF1 which suggest that BrCBF was an ortholog of CBF3, while BrCOR14 was with 76% identity to A. thaliana COR15B and 69% identity to A. thaliana COR15A which demonstrated that BrCOR14 was more like an orthology of COR15B. Alignment scores of the amino acid sequences of the three genes with other known cold-responsive proteins in Cruciferous family also show high concordance (Table 2). The results also suggested that the BrICE1 was highly conservative to the ICE1 in possessing a bipartite nuclear localization signal (NLS) domain from K304 to K353 and a bHLH domain from R316 to L352, as well as potential recognition sites, for instance basic-leucine zipper (bZIP) that contains a basic region mediating sequence-specific DNA binding followed by a leucine zipper region (required for dimerization) transcription factors, HR1 (shown to bind the small G protein rho or to activate PKN in its GTP-bound form) and Pfam domain (Fig. 2). Besides BrCBF gene contained a consensus sequence, CANNTG (CACCTG). Alignment of the BrCBF proteins indicated that it contained a highly conserved AP2 DNA￾binding domain of 60 amino acid residues from I52 to A109 as well as three potential strikingly conserved elements, YRG (consisted of 23 amino acids containing the con￾served YRG amino acid motif and was functionally important for DNA binding), NLS domain (from amino acid R42 to R59) and ALA-RICH domain (from amino acid T84 to K128). Besides, the prediction also revealed that BrCBF contains a dehydration-responsive element (Fig. 3). In addition, the DNA sequence at the presumed transcrip￾tion site of BrCOR14, CCG, is identified as a common core sequence. Secondary Structure Analysis Secondary structure analyses indicated (Fig. 4) that BrICE1 consisted of 158 α-helices, 40 β-turns jointed by 83 extended strands, and 216 random coils which resembled the secondary structure of Arabidopsis ICE1 (NP_189309.2) and CbICE53 (C. bursa-pastoris AAS79350). BrCBF consisted of 84 a-helices, nine β-turns jointed by 33 extended strands and 90 random coils which commendably resembled the secondary structure of CBF3 (AF074602) and CbCBF (AY391121.1). It was also note worthy that a-helices occurred predominantly in the structure of BrCBF. Moreover, the BrCOR14 consisted of 78 α-helices, seven β-turns jointed by 17 extended strands, and 26 random coils, which did not resemble the secondary structure of COR15B (A. thaliana NM_129814) and CbCOR15B (AY437888.1) that much. Subsequently, homology modeling analysis was carried out, while no suitable target was found due to the Fig. 1 RNA quality determination Plant Mol Biol Rep (2011) 29:525–532 527