Abstract: 【Objective】In order to explore functional genes related to low temperature stress tolerance of cotton, the characteristics of cotton dehydrin gene and its expression patterns responsed to low temperature stress in cotton were analyzed, thus providing a theoretical basis for the application of dehydrin gene in cotton chilling tolerant breeding. 【Method】 In this study, based on the upland cotton genome sequence, specific primers were designed by Primer 5 software and the dehydrin gene was cloned from the upland cotton variety Yu2067, named GhDHN1. Bioinformatics analysis was conducted to analyze the properties, amino acids content, functional domains and evolutionary relationships of the gene. Plant expression vector pBI121::GFP at XbaⅠand SmaⅠ restriction sites was constructed with double enzyme digestions and the transient expression vector pBI121-GhDHN1::GFP was constructed by In-Fusion connection technology. And the subcellular localization of GhDHN1 was studied by transient expression analysis of onion epidermal cells. Combined with the transcriptome sequencing data of chilling-resistant cotton variety Yu2067, real-time fluorescent quantitative PCR expression of leaves, stems, and roots in chilling-resistant Yu2067 and chilling-sensitive variety Hengmian No.3 under low temperature stress treatments (4℃, 24 h) at trefoil stage was performed to study the function of GhDHN1. The expression of the gene in the leaves of two different cold resistant varieties was compared. The dynamic expression of GhDHN1 gene in leaves and roots of Yu2067 was detected under 4℃ low temperature treatment. 【Result】 A cotton dehydrin gene was cloned and the sequencing analysis showed that the cDNA of the dehydrin gene was 726 bp, and the gene encoded 211 amino acids with a predicted molecular weight of about 23.79 kD and the isoelectric point was 5.04. Amino acid sequence analysis indicated that the GhDHN1 was rich in glutamic acid content (26.10%) and lysine amino acid content (19.40%) with a half-life of 30 hours. GhDHN1 was acidic and negatively charged, of which 60 percent were negatively charged residues. The second structure analysis showed that the alpha helix of GhDHN1 contained 116 amino acid residues, accounting for 54.98% of the protein composition and random coil contained 87 amino acid residues. GhDHN1 gene was located on Dt_chr9 chromosome of AD genome. GhDHN1 gene contained a 90 bp intron at 259-348 position of cDNA and two exons in lengths of 258 bp and 378 bp, respectively. SMART and CDD analysis showed that GhDHD1 includes two conserved lysine-rich K fragments, a conserved serine-rich S fragment and a dehydrin functional domain pfam00257 and therefore was sorted into K2S subfamily of dehydrins. Phylogenetic analysis showed that GhDHN1 of cotton had the closest relationship with DHN1 of cacao. The analysis of the transient expression in onion epidermal cells showed that GhDHN1 was mainly localized near the cell plasma membrane. Transcriptome analysis showed that GhDHN1 gene was up-regulated in leaves and roots of cotton after low temperature treatments at the trefoil stage. qRT-PCR analysis showed that GhDHN1 was up-regulated in leaves, stems and roots after low temperature. The expression fold in leaves was higher than that in stems and roots. There were two expression peaks in leaves under low temperature treatment for 4 h and 24 h, and there were also two peaks in roots of the low temperature treatment for 6 h and 12 h. The leaf expression of the gene in cold resistant varieties was 2.47 times the expression in the cold sensitive varieties. The results showed that GhDHN1 gene may be involved in the adaptability regulation of low temperature. 【Conclusion】 GhDHN1 belonged to the member of K2S subfamily of dehydrins. Phylogenetic analysis showed that GhDHN1 had the closest relationship with DHN1 of cacao. GhDHN1 gene was induced by low temperature stress. Significant expression difference of GhDHN1 between the cold-resistance variety and cold-sensitive variety was found, and the expression of GhDHN1 was positively correlated with cotton cold-resistance. The expression of GhDHN1 can be thus employed as a marker of cotton chilling resistance. At the same time GhDHN1 gene can also serve as an important candidate gene in cotton cultivation of cold-resistant materials.

Key words: cotton, dehydrin, sub-cellular localization, low temperature stress, transcriptional expression, real-time fluorescent quantitative PCR