Construction Of Suppression Subtractive Hybridization Cdna Library And Trapping "Temperature-sensitive" Genes Expressed During Flowering In Doritaenopsis Hybrid

Doritaenopsis hybrid was originally from subtropics, for"temperature-sensitive"is a common phenomenon during flowering of Doritaenopsis hybrid, which is a special requirement for the bud differentiation. Regional distribution of production and sales has been formed in china. Seeking the best combining method of reducing producing cost with artificial regulation of flowering is the inevitable choice and trend of developing Doritaenopsis hybrid industry. This study used the materials of clone plantlet leaves of Doritaenopsis'Tinny Tender'under different temperature treatments (high temperature, low temperature).The systematic research revealed several physiological and molecular influences on the growth and metabolism of Doritaenopsis'Tinny Tender'under the condition of different temperatures by using physiological detection and suppression subtractive hybridization (SSH). The main research results were as follows:1. The influences on the growth and metabolism of Doritaenopsis'Tinny Tender'under the conditions of different temperatures: The changes of areas of leaves, chlorophyll fluorescence parameter (Fv/Fm,Fv/ Fo), carbohydrates content, CAT activity and SOD activity in leaves of Doritaenopsis'Tinny Tender'were different under different temperatures (20℃/18℃, 25℃/23℃, 30℃/25℃) during floral induction. The results showed that the areas of leaves increased continuously in 30℃/ 25℃treated group, leaves were in the vegetative growth stage, SOD activity in 30℃/25℃treatment group was high position in most of treatment stages, while the starch content was increasing in 30℃/25℃treated group during the treatment time, the condition of 30℃/25℃treatment was a disadvantage to bud induction. Fv/Fm in 20℃/18℃treated group was low before 35d treatment, and Fv/ Fo decreased rapidly in earlier stage; CAT activity of the three treatment groups were rising in later stage of floral induction; Total sugar content were decreased after 28d treatment, but reducing sugar content changed smoothly in 20℃/18℃and 25℃/23℃treatment groups, Starch content was downtrend in 20℃/18℃treated group before 42d treatment, the condition of 20℃/18℃treatment was in favour of bud induction.2. Doritaenopsis'Tinny Tender'suppression subtractive hybridization cDNA library was constructed using leaves of low temperature (20℃/18℃)(Tester)and high temperature(30℃/25℃)(Driver)after 40 days treatment with the purpose of enriching"temperature-sensitive"genes: The SSH cDNA library contained 2373 positive clones, The recombination rate is 95%, including 1540 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 GI gene, genes related ubiquitin pathogenesis, zinc finger binding protein and sucrose synthase.The analysis information were collected in data base. Primary screening of SSH cDNA library showed that the cDNA library contained candidate genes related"temperature-sensitive"phenomenon of Doritaenopsis'Tinny Tender'. The results provided studying basis and technology platform for the mechanism of"temperature-sensitive"in flowering of Doritaenopsis'Tinny Tender'.