Mechanism Of NAC Family On Protocorm Development In Dendrobium Candidum Wall Ex Lindl And Bioinformatic Analysis Of PIN-formed Family

In this thesis, emphasis was put on the NAC family and their role in embryogenesis, by homologous cloning based on PCR, the NAC family in the Dendrobium candidum Wall ex Lindl genome was isolated, and the potential role of the found NAC gene was analyzed by the expression profiles implemented by RT-PCR and whole mounts in situ hybridization. To validate the suggested role of NAC genes, full length of the interest NAC genes was obtained by RACE technology; expression cassettes which fuse the genes of interest and eGFP were constructed to evaluate the effect of the gene. The main results were as follows:1.57members of NAC gene family were cloned in the genome of Dendrobium candidum Wall ex Lindl, the exon-intron structures were much the same, indicating the gene structure is conservative in the NAC gene family. However, there do have some exceptions, in some cases, there is simply no intron in the fragment.2. Not all the NAC family members express during the embryogenesis process, showing diversity role of NAC genes. Also the genes expressed during the embryogenesis have different expression profile:DcNAC1are found to express in a constitutively manner, while others show different expression lever in this process. These results suggest that the NAC genes may play different roles in embryogenesis.3. Whole mount in situ hybridization results indicates the DcNAC genes may only express in some particular area in the protocorm, take DcNAC1for example, on the top where scale leaf germinate, showing that these NAC may play important role in the morphogenesis of protocorm.4. By RACE technology, five full length cDNA of NAC genes were amplified, also, the genome sequences of them were isolated. Analysis of the sequences shows that nearly all the NAC genes encode protein products with about320amino acids. Based on the similarity between sequences, the NAC proteins can be divided into two distinguish domains, a conserved N-terminus and a much more varied C-terminus. Blast results show that the conserved N-terminus served as a DNA binding domain while the C-terminus can activate transcription. Agrobacterium infiltration assay shows that the fluorescent signal can only be detected in nucleic of onion epidermal cells, i.e., DcNAC2proteins locate in the nucleic, confirming the hypothesis the NAC acting as a transcription factor.In conclusion, by homologous cloning,57putative NAC genes were isolated, and RACE technology confirmed the reliability of this method. A series of assay can be used to get more information of the cloned NA C genes.