526 Plant Mol Biol Rep (2011)29:525-532 DREB,a family of AP2-domain,are also transcriptional Isolation of cDNAs Encoding Cold-Response Proteins activators binded to the DRE/CRT element (GCC-box pathogenesis-regulated promoter element)and activated A non-heading Chinese cabbage cDNA fragment encoding transcription(Zhou et al.2007;Buittner and Singh 1997; an ICE-like polypeptide was isolated by reverse Stockinger et al.1997:Thomashow 1999)of the CBF transcription-polymerase chain reaction (RT-PCR)using regulon including genes,specifically corl5 or cor47,thus degenerate primers 5'-ATGGTTCTTGACGGAAACA increasing chilling and freezing tolerance of plants. ACGGTG-3'and 5'-AAAGGGCTTTAGTTCTTCTA Meanwhile,it should be noted that ICE/was expressed ACTCTGCTTC-3'based on the complete open reading constitutively,being only slightly up-regulated by cold, frame (ORF)of Arabidopsis cold-responsive gene ICEI but CBF expression was induced by cold treatment (Genbank accession number AY195621)and C.bursa- (Medina et al.1999;Gao et al.2002;Thomashow 2001; pastoris ICE53 (Genbank accession number AY506804). Chinnusamy et al.2003).Whereas the overexpression of The anticipate product size was 1,558 bp.Total RNA was CBF1 (Jaglo-Ottosen et al.1998)and DREBla/CBF3 isolated from seedlings incubated at 4C for 8 h using (Kasuga et al.1999)in Arabidopsis were shown to be able TaKaRa RNAiso Reagent(Takara,Japan).The first strand to drive expression of COR genes in the absence of low cDNA was reversed using TaKaRa RNA PCR Kit (AMV) temperature and impart constitutive salt and drought Ver.2.1 (TaKaRa,Japan).The PCR mixture contained tolerance,while not abscisic acid (ABA)stress,which 2.5 uL buffer (10xPCR),1.5 uL MgCL2 (25 mM), suggested that it is involved in the expression of cold-, 1.5 uL dNTPs (2.5 mM each),0.25 uL LA-Tag DNA salt-,and drought-regulated genes through an ABA- polymerase (5 UmL/L),10-pmol-specific primers each independent pathway (Kasuga et al.1999;Yamaguchi- 50 ng cDNA,and ddH2O up to 25 uL.Amplification Shinozaki and Shinozaki 1994). profile was 94C for 5 min,35 cycles of 94Cfor 30 s,65C Non-heading Chinese cabbage,like Arabidopsis,cold for 1 min,72C for I min 30 s,and a final extension of 72C acclimates and is a member of the Cruciferae family.We for 10 min.The products were resolved in 1.0%(whv)agarose speculate that non-heading Chinese cabbage may have gel and purified,then cloned into the pGEM-T vector similar cold acclimation process as Arabidopsis.Recently. (Tiangen,China)followed by sequencing. several cold-regulated genes have been cloned from Arabi- cDNAs encoding full-length CBF-like and COR-like dopsis,Capsella bursa-pastoris,and Brassica napus (Jaglo- proteins were isolated by RT-PCR and rapid amplification Ottosen et al.2001;Wang et al.2005).Up till now,there has of cDNA ends(RACE)previously (Jiang et al.2007a,b). been no report on the cloning of cold-regulated genes from The sequences for the entire cDNA insert were determined non-heading Chinese cabbage.In this paper,we reported that and deposited the molecular cloning of BrICEl,BrCBF,and BrCOR14 genes from non-heading Chinese cabbage,bioinformatics Bioinformatics Analysis analysis revealed that these three genes strongly resembled ICE,CBF,and COR genes from other species. Associated molecular information was analyzed using software Clustal W.and other databases listed below: NCBI (http://www.ncbi.nlm.nih.gov/),ProtParam (http:/ Materials and Methods us.expasy.org/tools/protparam.html),and TMHMMv2.0 (http://www.cbs.dtu.dk/services/TMHMM/).Alignment Plant Materials scores of the amino acid sequences of the identified cold- responsive genes with other known homologous proteins A non-heading Chinese cabbage (Brassica campestris ssp. were processed by PROSITE (http://www.Expasy.org/pro chinensis L.Makino)cold-resistant inbred line.043,from site/),InterProScan (http://www.ebi.ac.uk/Tools/InterPro non-heading Chinese cabbage project team in Nanjing Scan/)and WU-Blast2 (http://www.ebi.ac.uk/Tools/blast2 Agricultural University was used in the present study. index.html).Secondary structure analyses were carried out Healthy seeds were grown in controlled environmental by SOMPA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat. chambers at 20C to 22C under continuous cool-white plpage=npsa_sopm%20a.html). fluorescent illumination of 100 to 150 umolm2s light intensity as described by Gilmour et al.(1998)Stress Real-Time Fluorescence Quantitative PCR Analysis treatments were performed with seedlings at three-leaf stage.For cold acclimation,plants were incubated at 4C For cold acclimation.seedlings were transferred to 4C for under continuous cool-white fluorescent illumination at varying lengths of time (0 h,0.5 h,1 h,2 h,4 h,8 h,12 h, approximately 50 umolm2s light intensity for varying 24 h,4 days and 7 days),with Saran Wrap covered to slow lengths of time. evaporation.For ABA,salt and drought stresses,seedlings SpringerDREB, a family of AP2-domain, are also transcriptional activators binded to the DRE/CRT element (GCC-box pathogenesis-regulated promoter element) and activated transcription (Zhou et al. 2007; Büttner and Singh 1997; Stockinger et al. 1997; Thomashow 1999) of the CBF regulon including genes, specifically cor15 or cor47, thus increasing chilling and freezing tolerance of plants. Meanwhile, it should be noted that ICE1 was expressed constitutively, being only slightly up-regulated by cold, but CBF expression was induced by cold treatment (Medina et al. 1999; Gao et al. 2002; Thomashow 2001; Chinnusamy et al. 2003). Whereas the overexpression of CBF1 (Jaglo-Ottosen et al. 1998) and DREB1a/CBF3 (Kasuga et al. 1999) in Arabidopsis were shown to be able to drive expression of COR genes in the absence of low temperature and impart constitutive salt and drought tolerance, while not abscisic acid (ABA) stress, which suggested that it is involved in the expression of cold-, salt-, and drought-regulated genes through an ABA￾independent pathway (Kasuga et al. 1999; Yamaguchi￾Shinozaki and Shinozaki 1994). Non-heading Chinese cabbage, like Arabidopsis, cold acclimates and is a member of the Cruciferae family. We speculate that non-heading Chinese cabbage may have similar cold acclimation process as Arabidopsis. Recently, several cold-regulated genes have been cloned from Arabi￾dopsis, Capsella bursa-pastoris, and Brassica napus (Jaglo￾Ottosen et al. 2001; Wang et al. 2005). Up till now, there has been no report on the cloning of cold-regulated genes from non-heading Chinese cabbage. In this paper, we reported that the molecular cloning of BrICE1, BrCBF, and BrCOR14 genes from non-heading Chinese cabbage, bioinformatics analysis revealed that these three genes strongly resembled ICE, CBF, and COR genes from other species. Materials and Methods Plant Materials A non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino) cold-resistant inbred line, 043, from non-heading Chinese cabbage project team in Nanjing Agricultural University was used in the present study. Healthy seeds were grown in controlled environmental chambers at 20°C to 22°C under continuous cool-white fluorescent illumination of 100 to 150 μmolm−2 s −1 light intensity as described by Gilmour et al. (1998) Stress treatments were performed with seedlings at three-leaf stage. For cold acclimation, plants were incubated at 4°C under continuous cool-white fluorescent illumination at approximately 50 μmolm−2 s −1 light intensity for varying lengths of time. Isolation of cDNAs Encoding Cold-Response Proteins A non-heading Chinese cabbage cDNA fragment encoding an ICE-like polypeptide was isolated by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers 5′-ATGGTTCTTGACGGAAACA ACGGTG-3′ and 5′-AAAGGGCTTTAGTTCTTCTA ACTCTGCTTC-3′ based on the complete open reading frame (ORF) of Arabidopsis cold-responsive gene ICE1 (Genbank accession number AY195621) and C. bursa￾pastoris ICE53 (Genbank accession number AY506804). The anticipate product size was 1,558 bp. Total RNA was isolated from seedlings incubated at 4°C for 8 h using TaKaRa RNAiso Reagent (Takara, Japan). The first strand cDNA was reversed using TaKaRa RNA PCR Kit (AMV) Ver.2.1 (TaKaRa, Japan). The PCR mixture contained 2.5 μL buffer (10×PCR), 1.5 μL MgCL2 (25 mM), 1.5 μL dNTPs (2.5 mM each), 0.25 μL LA-Taq DNA polymerase (5 UmL/L), 10-pmol-specific primers each, 50 ng cDNA, and ddH2O up to 25 μL. Amplification profile was 94°C for 5 min, 35 cycles of 94°Cfor 30 s, 65°C for 1 min, 72°C for 1 min 30 s, and a final extension of 72°C for 10 min. The products were resolved in 1.0% (w/v) agarose gel and purified, then cloned into the pGEM-T vector (Tiangen, China) followed by sequencing. cDNAs encoding full-length CBF-like and COR-like proteins were isolated by RT-PCR and rapid amplification of cDNA ends (RACE) previously (Jiang et al. 2007a, b). The sequences for the entire cDNA insert were determined and deposited. Bioinformatics Analysis Associated molecular information was analyzed using software Clustal W, and other databases listed below: NCBI (http://www.ncbi.nlm.nih.gov/), ProtParam (http:// us.expasy.org/tools/protparam.html), and TMHMMv2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Alignment scores of the amino acid sequences of the identified cold￾responsive genes with other known homologous proteins were processed by PROSITE (http://www.Expasy.org/pro site/), InterProScan (http://www.ebi.ac.uk/Tools/InterPro Scan/) and WU-Blast2 (http://www.ebi.ac.uk/Tools/blast2/ index.html). Secondary structure analyses were carried out by SOMPA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat. pl?page=npsa_sopm%20a.html). Real-Time Fluorescence Quantitative PCR Analysis For cold acclimation, seedlings were transferred to 4°C for varying lengths of time (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 4 days and 7 days), with SaranWrap covered to slow evaporation. For ABA, salt and drought stresses, seedlings 526 Plant Mol Biol Rep (2011) 29:525–532