Isolation And Functional Confirmation Of DhPRP39and DhFVE Of A Doritaenopsis Hybird Involved In The Regulation Of Flowering

Doritaenopsis, an intergeneric hybrid between the Phalaenopsis and Doritis, is a kind of perennialherbaceous flowers and belongs to Orchidaceae. These Phalaenopsis hybrids are becoming increasinglypopular in the domestic and international flower market for their ornate flower shapes, and longerflowering period than the traditional Phalaenopsis. Doritaenopsis which has a high ornamental valueand economic value is one of the most widely cultivated and the most popular Phalaenopsis hybridtypes.In previous study, a cDNA library was established by suppression subtractive hybridization andmany EST sequences were obtained. Comparative analysis showed that two sequences had separatelyhigh identity with PRP39gene and FVE gene in the Arabidopsis thaliana. Seven-month-oldDoritaenopsis ‘Tinny Tender’(Doritaenopsis Happy smile×Happy valentine) cloned seedings wereused as test materials. Rapid amplification of cDNA end (RACE) was performed to isolate the fulllength cDNA of the two genes, and transformation of Arabidopsis thaliana was used to verify the genesfunction. The main research results were as follows:1. The full-length of the two genes were isolated from Doritaenopsis using RACE. The full-lengthDhPRP39cDNA was2,962bp long with a2,472bp open reading frame and encodes823amino acids.GenBank Accession No. is JF508182. The full-length DhFVE cDNA was1,856bp long with a1,407bpopen reading frame and encodes468amino acids. GenBank Accession No. is JF508181.2. Roots, stems, leaves, pedicels, buds, and floral organs (petals, sepals, lips, and columns) werecollected from different periods of Doritaenopsis. Real-time RT-PCR was conducted to investigate thedevelopmental and tissue-specific expression of DhPRP39and DhFVE. The results indicated thatDhPRP39and DhFVE might be involved in the initial stage of the Doritaenopsis from vegetativegrowth to reproductive growth. These two genes might play an important role in inflorescence initiation.3. Plant expression vector pCAMBIA1301and intermediate vector pMD20-T were used toconstruct PC1301-DhPRP39and PC1301-DhFVE sense expression vectors.4. Arabidopsis thaliana transformation was performed through the floral dipping method.Transgenic plants were selected by50mg/L hygromycin B, and the phenotypic changes were observed.The results indicated that flowering time of the DhPRP39and DhFVE transgenic plants have differentlevels of delay. The expression of the two genes can be detected in the transgenic plants at DNA andRNA levels. Similar results had been reported from a previous study. DhPRP39and DhFVE gene mayalso involve in the regulation of flowering through the the autonomous pathway in Doritaenopsis.