Cloning And Functional Verification Of A Key Gene DhCOL Of Doritaenopsis Involved In The Regulation Of Flowering

Doritaenopsis, an intergeneric hybrid general designation between Phalaenopsis and Doritis,compared with the traditional Phalaenopsis orchid, which has higher ornamental value and economicvalue. The full-length cDNA open reading frame (ORF) region of DhCOL was cloned fromDoritaenopsis ‘Tinny Tender'. And the plant expression vector of DhCOL was constructed andtransformed into Arabidopsis thaliana to verify its functions. Besides, DhCOL and DhGI1relativeexpression leveles were detected and analyzed by realtime fluores-cence quantitative PCR (RTFQ PCR)separately under LD (16h/8h, day/night) and SD (8h/16h, day/night). Its aim is to further study thefunctions of DhCOL in floral network pathway. The results were as follows:1. The novel cDNA with an ORF of834bp, encoding277amino acids with a molec-ularweight of29.3527kDa and a theoretical isoelectric point of5.34. The genomic clone containedone exon at the conserved site. DhCOL had two B-box zinc finger motifs and a CCT domain.Its protein was unstable and nucleus-localized. The analysis based on amino acid sequence alignment showed that DhCOL shared a high identity with PhalCOL (98%) from Phalaenopsis hybrid, OnCOL (84%) from Oncidium hybrid cultivar, DlCOL (83%) from Dendrobium loddigesiiand CeCOL4(80%) from Cymbidium ensifolium respectively.2. RTFQ PCR analysis showed that the rhythmic cycle of DhCOL expression is almost to8h under LD, and It has an expression peak in a short transition period of light to dark or dark to light; DhGI1has an expression after light treatment for7h either under LD or SD, but it has a low expression when light turnd into darkness, and its expression in both conditions ishigher then DhCOL. DhCOL and DhGI1expressions are also higher in leaves of Doritaenopsis,when its pedicel is less than4cm under LD. But the expression of DhGI1is highest in SDwhen the pedicel is almost7cm-10cm. DhCOL and DhGI1expressions are higher in larger buds both under LD and SD, and the expression is higher in LD. DhCOL and DhGI1expressionsare highest in stems either under LD or SD. And DhCOL expression in tissues is higher underLD than SD. But the DhGI1expression are higher in stems and flowers under SD than LD.DhCOL and DhGI1were expressed at higher levels in the lip under LD, but they were sexpressed at higher levels in the pollen block under SD.3. The plant expression vector pCAMBIA1301were used to construct the positive PC1301-DhCOL plant expression vector. And which were verified correct by restriction enzyme digestion and sequencing. The floral dipping method was performed to Arabidopsis thaliana transformation. Transgenic plants were screen out by hygromycin B and the expression of the two geneswere detected on genome and transcriptional level. The results indicated that The over-expressio n of DhCOL in Arabidopsis thaliana caused an delay-flowering phenotype and no seeds gained.These results indicated that DhCOL may play an important role in initiating buds and promoting flowering.