Cloning，subcellular localization and expression analysis of BobHLH121 gene from Brassica oleracea L.

【Objective】The whole length of Brassica oleracea L. bHLH transcription（BobHLH121 gene）was cloned by homologous cloning method，and its expression pattern under organ/tissue and low temperature stress was analyzed， which provided theoretical reference for studying the mechanism of bHLH transcription factor in low temperature response. 【Method】The sequence of the coding region（CDS）of BobHLH121 gene was cloned，the prediction analysis was performed using bioinformatics software，and the pCAMBIA1300-BobHLH121-GFP fusion expression vector was constructed through the transfection of tobacco epidermal cells，and the fluorescent signal was observed to determine the subcellular localization of the protein. The relative expression of the BobHLH121 gene under low temperature stress was measured using real-time fluorescence quantitative PCR（qRT-PCR）.【Result】The CDS length of BobHLH121 gene was 1367 bp， localized on chromosome C06，encoding 311 amino acids，protein molecular weight of 34.89 kD，theoretical isoelectric point（pI）was 5.42，the instability coefficient was 52.29，and the average hydrophobicity was -0.733，indicating that the protein was an unstable hydrophilic protein，which was acidic and localized in the nucleus. The secondary structure of BobHLH121 protein mainly consisted of α-helix（30.87%），extended chain（9.32%），β-turn（2.89%）and random coil （56.91%），with a conserved HLH domain. BobHLH121 protein was closely related to bHLH proteins in Arabidopsis thaliana（NP_191768.2），A. lyrata（EFH52923.1），Camelina sativa （XP_01909381.1），Capsella brusa-pastoris（XP_ 023638997.1），B. napus（CDY65446.1），Chinese cabbage（XP_009104362.1）and radish（XP_018442860.1），and had amino acid similarities of 55.6%，54.8%，52.0%，50.3%，76.4%，73.9% and 61.1%，respectively. Phylogenetic tree analysis showed that BobHLH121 was on the same small clade as the A. thaliana（NP_191768.2）and A. lyrata（EFH52923.1） bHLH protein. A large number of cis-acting elements for plant growth and development，abiotic stress，hormone response，and light response existed in the promoter region of the BobHLH121 gene. The relative expression of the BobHLH121 gene was significantly higher in leaves than in other tissues（P<0.05，the same below）and lower in all other tissues. The relative expression of BobHLH121 gene was significantly decreased under low temperature treatment for 6-12 h under low temperature treatment at 0 h（control），and increased sharply at 24 h under low temperature treatment，which was significantly higher than that of other treatments，and decreased significantly at 48 h under low temperature treatment. It indicated that the gene might be delayed in expression under low temperature stress.【Conclusion】BobHLH121 has a conserved HLH domain typical of the bHLH family，has obvious tissue expression specificity，may participate in the regulation of growth and development of cabbage leaves，and respond to low temperature stress，may play a regulatory role in cabbage cold tolerance.