Molecular Cloning And Characteristic Analysis Of NAC Family Genes And Somatic Embryogenesis In Dendrobium Candidum Wall. Ex Lindl.

At the beginning, somatic embryogencsis of Dendrobium candidum Wall ex. Lindl. was described in this paper. Based on our observations, the somatic embryogenesis in D.candidum is a two-step process:first step is the transition of embryonic callus cell to initial somatic embryo cell, and second these cells become somatic embryos. A typical initial somatic embryo cell has a dense cytoplasm and a clear nucleus. We also noted there are two pathways for somatic embryo origin. One is the single-cell-derived somatic embryo pathway:the somatic embryo is generated from an inner initial somatic embryo cell in embryonic callus and developed into a globular somatic embryo, which is similar to zygotic embryogenesis. The other is multiple-cell-derived somatic embryo pathway, the somatic embryo comes from peripheral grouped initial somatic cells in embryonic calli and directly forms globular embryo or multicellular somatic proembryo, in this pathway it lacks the typical early stages of embryogenesis.Then we accomplished the molecular cloning of NAC family genes from Dendrobium candidum Wall ex. Lindl.31 full-length NAC genes were cloned in which we screened nine specific NAC members during the formation process of SAM by utilizing real-time PCR, namely DcNAC1、DcNAC3、DcNACH、DcNAC113、 DcNAC23、DcNAC30、DcNAC40、DcNAC44 and DcNAC59.Finally, sub-localization and transcriptional activity analysis of the nine genes were carried out. DcNAC1, DcNAC8, DcNAC13, DcNAC23 and DcNAC44 only show nucleic localization and the rest, DcNAC3, DcNAC30, DcNA40、DcNAC59 show both nuclear and cytoplasmic signal. Especially, signal of DcNAC59 spreads full of the whole cell.In the yeast single hybridization assay, only the protein of DcNACl, DcNAC3. DcNACl3, DcNAC30 and DcNAC44 show activation activity. The reason why the DcNAC40 do not have transcription activity may due to lack of C-terminal and the reasons why products of DcNAC8, DcNAC23 and DcNAC59 do not show transcriptional activity need further study.This study not only shed light into the ontogenesis of D. cundidum, but also helps to improve the techniques of propagation and preservation in D. cuundidum. And this research lays the foundations for investigating the mechanisms underlying orchid embryogenesis and the process of protocorm development in D. cundidum.