Isolation And Characterization Of A Doritaenopsis Hybrid GIGANTEA Gene, Which Possibly Involved In Inflorescence Initiation At Low Temperatures

Doritaenopsis, abbreviated Dtps. in the horticultural trade, is an intergeneric hybrid between the orchid genera Doritis and Phalaenopsis. These hybrids are becoming more and more popular for their multiple flowering spikes, longer flowering period and more color than the traditional parents. For most grown orchid species and hybrids, flowering is influenced by temperature and light. However, compared to photoperiod, low temperature is a more important environmental signal than other factors induced inflorescence in orchids. The requirement for low temperature in inflorescence initiation of the orchid species increases the cost of their industrial production. Therefore, to determine the best method to reduce production cost of artificial regulation of flowering, understanding the physiological and molecular mechanism of flowering is necessary. In this paper, clone plantlets of Doritaenopsis'Tinny Tender'had been studied under different temperature treatments (high temperature, low temperature). The main research results were as follows:1. In our study, it was found that low temperature treatment had a significant effect on flowering initiation in Doritaenopsis hybrid plants. After the low temperature treatment for 4 weeks, the peduncles were initiated in some plants of the low temperature-treated group (LTG) (22/18°C), and after 8 weeks, some flower buds were formed. After 5 months, 100% of the plants were in flower. By contrast, the plants maintained vegetative growth and none of the plants had initiated an inflorescence in high temperature-treated group (HTG) (30/25°C) .2. Doritaenopsis'Tinny Tender'suppression subtractive hybridization cDNA library was constructed using leaves of LTG(22℃/18℃, Tester)and HTG(30℃/25℃, Driver)with the purpose of enriching"temperature-sensitive"genes. The SSH cDNA library contained 2,373 positive clones (recombination rate is 95%), including 1,540 high quality ESTs. Blast search and analysis of the sequences showed that 17.7% unigenes were involved in metabolism, 9% unigenes participated in perception and response to stimuli; 7.65% involved in information pathways; 2.4% in transport ; and 1.71% in developmental processes; 1.1% in localization; Unigenes were obtained by GO function analysis which induced by"temperature-sensitive"phenomenon and related to flower formation, such as GIGANTEA (GI) gene, genes related ubiquitin pathogenesis, Zinc finger binding protein and sucrose synthase. The analysis information were collected in database. 3. To understand the molecular mechanism of flowering, the orchid GI gene, DhGI1, was isolated and characterized by using the rapid amplification of cDNA ends (RACE) PCR technique. Sequence analysis showed that the full-length cDNA is 4,022 bp with a major open reading frame of 3,483 bp, and the amino acid sequence showed high similarity to GI proteins in Zea mays, Oryza sativa, Arabidopsis and others.4. Semi-quantitative RT-PCR revealed that DhGI1 was expressed throughout development and can be detected in roots, stems, leaves, peduncles and flower buds. The expression level of DhGI1 was much higher when the plants were flowering at low temperature (22/18°C day/night) than other growth stages. Further analysis indicated that the accumulation of DhGI1 transcripts were significantly increased at low temperature, and concomitantly, initiation of the peduncle was observed. However, DhGI1 levels were low under high temperature (30/25°C) conditions, and the initiation of flower was inhibited.These results indicated that the expression of DhGI1 was regulated by low temperature and there may be a link through DhGI1 between inflorescence initiation and low temperature in the Doritaenopsis hybrid. Further investigation, that overexpression or knockout of DhGI1, would help to confirm the function of DhGI1 in transition from vegetative to reproductive growth at low temperature of Doritaenopsis, and elucidate the complicated mechanism of flowering in orchid plants. In industrial production, the application of DhGI1 through gene engineering would be another way for promoting inflorescence initiation, which would greatly reduced the production cost of Doritaenopsis hybrid or even other orchid plants.