Expression Analysis Of DoWOX And DoSERK In Dendrobium Officinale During Protocorm Development

Dendrobium officinale belongs to Orchidaceae,is a kind of precious Chinese medicinal materials.The seed germination of Orchidaceae must pass through the middle structure which called protocorm to form the seedlings,protocorm development is the combine of embryo development with seed germination.There have a large number of differentially expressed genes in the P1 and P2 stages for the protocorm development.The main pathways involved are: amino acid biosynthesis,hormone signal transduction,plant and pathogen interaction.WOX genes play a important role in the maintenance of meristem stem cells,lateral root development and embryo development.In this study,11 DoWOX genes were isolated and identified from the genome of Dendrobium officinale by homologous comparison.Among them,DoWOX1,DoWOX2,DoWOX3,DoWOX4,DoWOX5 and DoWOX11 belong to modern clade;DoWOX6,DoWOX7 and DoWOX10 belong to intermediate clade;DoWOX8and belong to ancient clade.Protein DoWOX3,DoWOX4 and DoWOX11 have typical WUS-box motif in there C-terminal.The consistency of Homeodomain(HD)for DoWOX is 61.4%,contains 68-69 amino acid residues,and its common sequence is X4-RW-X-P-X3-Q-X3-L-X7-G-X3-P-X4-L-X3-G-X6-V-X2-WFQN-X3-R-X7(X is inconsistent amino acid residue).Homology-modelling results show that HD for all DoWOX proteins have a common spatial structure which is "helix-loop-helix-turn-helix".The quantitative expression results showed that all DoWOX genes were differentially expressed in protocorm except DoWOX7 during the development of protocorm.Among them,DoWOX1,DoWOX2,DoWOX3,DoWOX4,DoWOX6,DoWOX9,DoWOX10 and DoWOX11 these eight genes have highest expression in the embryo activation period.DoWOX5 was expressed only in the degeneration stage of the protocorm and seedling stage,suggest that DoWOX5 may not play a role in the protocorm development,but only in mature plants.The highest expression level of DoWOX8 is in the apical meristem formation stage,and indicate it may be related to the development of apical meristem.Somatic embryogenesis receptor-like kinase(SERK)is widely found in plants and has avariety of biological functions.Two SERK genes were identified from the genomic scaffold data by BLAST alignment in Dendrobium officinale,named DoSERK1 and DoSERK2.DoSERK1 has been reportedencoding 633 amino acids;but DoSERK2 is a new SERKgene.The Coding sequence(CDS)lengthof DoSERK2 is 1877 bp,encoding 628 amino acids;DoSERK1 and DoSERK2 all have the typical structure of SERK protein;Which has five leucine-rich repeat(LRR)regions,one transmembrane region,one SPP motif and one intracellular kinase area.Conserved domains analysis finds that active site of the intracellular kinase for DoSERK2 is in agrooveby three-dimensional structure.And DoSERK1 have a similar grooves corresponding to their location.The 3 'end of DoSERK2 was clonedand found that its 3'-UTR area long 178 bp.The quantitative expression showed that the expression of DoSERK1 increased gradually in the early periodof protocorm,and the expression level reached to highest in P4 period(apical meristem formation period).And then the expression levelwas gradually decreasing,but the expression ofP7 period(the formation period of the root meristem)was the lowest.It can be speculated that DoSERK1 may play an important role in the formation of apical meristem and leaf.DoSERK2 expresses in all ninestages of protocorm development,and have a highest expression level in the first stage;The expression level of P2-P9 period was basically the same.Tissue expression analysis showed that the DoSERK2 expression was highest in flower,but in other tissues and organs it also can be detected.The gene expression profile indicating that DoSERK2 may play an important role in the earlyembryodevelopment and reproductive process.This paper provides a foundation for the expound of molecular mechanism of protocorm development in Dendrobium officinale,and provides a foundation for the study of biological functions of DoWOX and DoSERK.