Cloning，subcellular localization and expression analysis of RhD14 gene in rose

【Objective】Strigolactone（SLs）signal response substrate receptor，α/β folded hydrolyzed protein gene Dwarf 14（D14），was cloned from rose for bioinformatics analysis and subcellular localization in order to provide theoretical support for exploring the biological function of D14 gene and its transduction mechanism of SLs in regulating the lateral branching of Rosa hybrida Dianhong.【Method】Rosa hybrida Dianhong was the material，RhD14 gene was cloned and bioinformatics analysis was conducted. The expression levels of RhD14 in different tissues was detected by real-time quantitative PCR（qRT-PCR），and the subcellular localization of the protein encoded by RhD14 gene was performed by a tobacco transient transformation system.【Result】The cDNA sequence of the RhD14 gene（GenBank accession number OP009358）was 1094 bp，with a 1045 bp open reading frame（ORF），encoding 347 amino acid residues. The molecular formula of the protein was C₁₇₃₈H₂₇₀₃N₄₄₁O₅₁₀S₁₅，the relative molecular weight was 38.41 kD，and the theoretical isoelectric point（pI）was 5.71. α-helix and random coil were the main secondary structures of its amino acid sequence. RhD14 was a stable protein without transmembrane structure and signal peptide，which belonged to the α/β hydrolase family. The subcellular localization results showed that the encoded protein of RhD14 was located in the cytoplasmic and cytomembrane membrane. Homologous sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of RhD14（OP009358）had the highest similarity of 98.05% with that of the old garden rose，Rosa chinensis Old BlushRcD14（XP_024283944.1），followed by Fragaria vesca subsp. vesca（XP_004287076.1）of the same subfamily with the similarity for 89.75%，which were closely related. The analysis of qRT-PCR showed that RhD14 gene was expressed in all of the test tissues（roots，stems，leaves，axillary buds，nodes，and apical buds）. Taking roots as the control，the expression levels of RhD14 in stems and axillary buds were the highest，and were extremely significantly higher than that in roots （P<0.01，the same below）. The expression pattern of RhD14 gene in stems and axillary buds after topping treatment was similar，both of which were significantly up-regulated compared with that of non-topping treatment. With the extension of treatment time，the expression of RHD14 gene firstly increased and then decreased，and reached the peak at 12 h after topping treatment. In particular，the relative expression levels of RhD14 gene in axillary buds at 6，12，24 and 48 h were significantly different（P<0.05）or extremely significant different compared with those without topping treatment.【Conclusion】RhD14 gene plays a role in the cytoplasmic and cytomembrane membrane with tissue specificity，which can be highly expressed under topping induction. It is inferred that RhD14 gene participates in the process of axillary bud germination.