Molecular Cloning And Expression Analysis Of FCA And FT Homologous(DiFCA&DiFT) In Davidia Involucrata Baill

Davidia involucrata Baill. also known as the dove tree, is a tertiary ancient relic species of tropical plants, It has high ornamental value, medicinal value and scientific value.Davidia involucrata need strict survival environment, mature seedlings reproduction rate is low. Davidia involucrata is a perennial tree, sexual generation cycle is long. reproductive growth is valuable, flowering induction process is the key process.FCA (FLOWERING CONTROL LOCUS A) gene and FT (FLOWERING LOCUS T) gene are important regulatory genes of photoperiod pathway and autonomous pathway.the study got a gene fragments form Davidia involucrata seeds,do a homologous comparison and RACE to obtain Davidia involucrata FCA full-length sequence gene and the proportion of the FT.Then use qRT-PCR to analysis the FCA and FT expression pattern, for the flowering gene function and transgenic flowering mechanism research.Due to the material acquisition is early stage of this experiment in the Davidia involucrata and is difficult to obtain,,so we did a certain seed experiment, to make sure the late reaserch.This research main consists and conclusion as follows:1.Tissue culture and rapid propagation of Davidia involucrata:Using green fruit with 100% alcohol burning, take the embryo with endosperm, vaccination in WPM basic culture medium without hormone.remove perisperm while growthing aFTer a certain time, complete and fast growth. Davidia involucrata embryo on medium as Browning degree is high, cause the Davidia involucrata low survival rate of seed.with root, stem, leaf and are difficult to cultivate callus.This study Provides methods to get the dovetree seedling quickly and efficiently, breaking the dovetree seed natural dormant and avoid the tissue culture of root, stem and leaf regeneration difficulty, high rooting rate and low cost. For subsequent experimental material for genetic transformation and transgenic somaclone system and to laid a solid foundation.2.Davidia involucrata FCA and FT gene cloningUsing the RACE and ordinary PCR cloning technology to get the Davidia involucrata DiFCA complete ORF sequence which size 1596 bp, and coding the conservative function structure domain (RRM2, WW-protein interactions), encoding 507 amino acids.The DiFCA genes of Bioinformatics analysis for the Davidia involucrata is FCA family genes of transcription regulation, and the protein as hydrophilic protein,the protein is mainly located in the body and nucleus.then cloning the Davidia involucrata DiFT (Davidia involucrata FT) part ORF sequence which is 321 bp, its analysis and own PEBP family conservative structure with D-D-A-P-P,86H-(histidine residues), as well as the substrate binding site 86 H-and conservative sites Y 84-FT and family (coning amino acid residues), speculated that it belongs to the Davidia involucrata FT gene.3. Expression analysis of DiFCA and DiFT geneWe confirmed the temporal and spatial expression pattern by qRT-PCR.1) Starting flowering period in Davidia involucrata, DiFCA expressed the most in flower bud tissue organ (spend), in big bract and small bract expression more, Then in the old leaves, green leaves expressed in the least;DiFT in old leaves is higher than any other organization, at least in the green leaves.2) In the same tree and flowering period, DiFCA and DiFT show the same trend as the flowering process, along with the changes in the flowering period from rising to down, but the expression of DiFT’s peak is early than DiFCA.DiFCA gene expression slightly higher in the inflorescence than leaves;The DiFT gene expression further higher in old leaves than inflorescence;In the same blade organization, with the flowering period, autonomous pathway DiFCA expression of peak fit to the Davidia involucrata flower bloom degree, the expression peak of DiFT is slightly earlier than blossom, reflects the way the sensitivity to the light.3) Flowering period in different altitude Davidia involucrata:within 900-1300 meters, when the it increase altitude, the expression of DiFCA quantity gradually decline, the expression of DiFT is down after rising first.In flowering starting date (April 27),flower get a optimal environment in the 900 meters, go up flower state delay.At low altitudes (no flowering Davidia involucrata), DiFCA expression quantity low, and they did not detect the expression of DiFT.4) In the annual Tissue culture Davidia involucrata:there are slightly expression of DiFCA in hypocotyl and crown, while DiFT has not detected expression;In the uncopletely leaf and true leaf has not detected with DiFCA and DiFT.