Isolation And Functional Confirmation Of DhEFL4 And Promotor Of A Doritaenopsis Hybrid Involved In The Regulation Of Flowering

Doritaenopsis,an intergeneric hybrid between the Phalaenopsis and Doritis,is a kind of worldwide Phalaenopsis.It's popular in the domestic and flower market for their ornate flower shapes,and longer flowering period.Doritaenopsis has a high ornamental value and economic value.In Hong-Hwa Chen's study,a cDNA library which named OrchidBase was established and many EST sequences were obtained.Comparative analysis showed that two sequences had separately high identity with ELF4 gene in the Zea mays.Seven-month-old Doritaenopsis 'Tinny Tender'(Doritaenopsis Happy smile x Happy valentine)cloned seedings were used as test materials.Rapid amplification of cDNA end(RACE)was performed to isolate the cDNA of the gene,and transformation of Arabidopsis thaliana was used to verify the gene function.At the same time,Genome Walking was performed to clone the promoter of DhEFL4.The main research results were as follows:1.The gene was isolated from Doritaenopsis using RACE.593bp of DhEFL4 cDNA was gotten,and contain 345bp open reading frame and encodes 114 amino acids.2.Roots,stems,leaves,pedicels,buds,and floral organs(petals,sepals,lips,and columns)were collected from Doritaenopsis at the moment of changing from light to dark.Real-time RT-PCR was conducted and the bud is the highest expression tissure,so the gene must have some relationship with flowering.At the same time,the Doritaenopsis's leaves which were separately treated in short and long light period,and the leaves were collected in every two hours in a constant 26hours.Real-time RT-PCR was separately conducted for the short and long period leaves to investigate the circadian rhythm of DhEFL4 gene.The results indicated that DhEFL4 have an obvious circadian rhythm and there are some difference between short and long light period which means light will influence the flowering of Doritaenopsis,although Doritaenopsis is insensitive to light.3.Plant expression vector pCAMBIA1301 and intermediate vector pMD20-T were used to construct PC1301-DhEFL4 sense expression vectors.4.Arabidopsis thaliana transformation was performed through the floral dipping method.Transgenic plants were selected by 50 mg/L hygromycin B,and the phenotypic changes were observed.The results indicated that flowering time of the DhEFL4 transgenic plants have delayed and the number of pedicel have increased.The expression of the gene can be detected in the transgenic plants at DNA and RNA levels.Similar results had been reported from a previous study.DhEFL4 gene may also involve in the regulation of flowering through the the photoperiodic pathway in Doritaenopsis.