Objective Expression of LOB domain-containing transcription factors in response to Epulorhiza sp. inoculation and molecular characteristics of LOB1 in Dendrobium officinale were studied.
Method Based on the previously obtained data on the root transcriptome in the symbiotic system of D. officinale and Epulorhiza sp., members and conserved structures of the LOB domain-containing (LBD) family were identified. Differential expressions of LBDs in the roots with and without Epulorhiza sp. infection were determined using qRT-PCR. Gene cloning, prokaryotic expression, phylogeny, and subcellular localization of the significantly responsive DoLOB1 was analyzed.
Result Twenty-three LBDs were identified from the D. officinale root transcriptome that included 20 Class I members and 3 Class II DoLBD38, DoLBD39, and DoLBD40. They all contained the characteristic conserved LOB domain but were different in the compositions of GAS, leucine zipper-like, and other conserved motifs. The qRT-PCR analysis showed 11 of the 23 LBDs significantly differentially expressed by the Epulorhiza sp. inoculation. DoLOB1 had an ORF of 681 bp in length and encoded 226 amino acids with a molecular weight of 24.7 kDa and a theoretical isoelectric point of 6.21. The prokaryotic expression produced a recombinant protein of approximately 26 kDa. Phylogenetically, it clustered with LOBs of monocotyledonous plants and closely related with 82.35% sequence identity to Canna indica. Exhibiting a pronounced nuclear localization pattern, the DoLOB1-GFP fusion protein was distributed in the nucleus, plasma membrane, and cytoplasm.
Conclusion Multiple DoLBDs showed significant expressions in response to Epulorhiza inoculation. Among them, DoLOB1 had the characteristic structural and subcellular localization features of LOB proteins and might participate in the mycorrhizal symbiosis of the medicinal plant.
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