Cloing And Functional Identification Of Constans In Rosa

Rosa chinensis originated from China,is one of ten traditional flowers of China and the four major cut flowers of the world.It is popularized worldwide because of its frangance,abundant colors and continuous flowering.In the present study,rose CONSTANS gene was cloned,and its function was identified by genetic transformation.The present study lays the foundation for dissecting the mechanism of continuous flowering of rose.The main findings are stated below:1.The primer of CONSTANS was designed by homology analysis according to the published transcriptome of Rosa.The full lengths of CONSTANS of Rosa chinensis ’old blush’ and Rosa chinensis ’Viridiflorawerewere’ were 1853bp,merely different in 3 bases,containing 2 exons and 1 intron.The cds region of Rosa chinensis ’old blush’ and Rosa chinensis ’Viridiflorawerewere’ were 1188bp,encoding an identity polypeptide containing 395 amino acids.And two BBX motifs were also found in the sequence.2.Protein cluster analysis showed that RoCO belongs to BBX1 as known as CONSTANS with highhomology to 32 BBX in Arabidopsis thaliana.The analysis showed that RoCO was closest to AtCO,which also meant that the nomination of CONSTANS in Rosa was solid.3.Rose seedlings in vitro were transferred to long day(16h light;8h dark)and short day(8h light;16h dark)conditions after tissue culturing.The rose grown in long day condition gave flowering earlier than those in short day.Furthermore,the flowering time was positively correlated with the expression of CO and FT.This result suggested that flowering time of rose is sensitive to the photoperiod,and the flowering time is regulated by photoperiod.4.To identify the function of RoCO in details,plant expression vector 35S::CO was constructed by using recombinated vector pFast-R05,and was transferred into wild type(gl-1)and co mutant of A.thaliana by using Agrobacterium tumefaciens-mediated inflorescence dipping method.Two kinds of transgenic A.thaliana in different background were obtained successfully.Both two kinds of transgenic seedlings with overexpression of RoCO gave flowering earlier than their background in long day.The flowering time of transgenic co mutant was similar to wild type(gl-1),suggesting that overexpression RoCO could recover the delayed flowering phenotype in co mutant.RT-PCR showed that,the expression of RoCO had a positive correlation with the expression of endogenous AtFT,indicating that RoCO had the similar function to AtCO in flowering regulation through activating its downstream gene AtFT,as well as promoting flowering time in Arabidopsis thaliana by responding to photoperiod.5.35S::CO vectors were transformed into rose adventitious buds by using Agrobacterium tumefaciens-mediated vacuum infiltration method.The transgenic rose shoots were obtained by antibiotic gradient screening and PCR identification.The indentification of flowering phenotype is still in processing because of the long life cycle of rose.