Functional Analysis Of Flower-Related Gene EFS1 In Arabidopsis Thaliana

Flowering is the most important result of a plant developmental process that controls the transition from vegetative maturity to the reproductive stage.It plays a key role in the phase and quality of development.Investigation of flowering controlling molecular mechanism is important to regulate development and propagation,improve crop's output and quality.In previous research,we had got a mutant from T-DNA insert library named efs1(early flowering in short mutant 1),which had identified based on Genome Walking and bio-information to be a candidate gene involved with Arabidopsis thaliana flowering regulation.To investigate the function and role in the regulation pathway,we designed the complementation and overexpression to confirm that EFS1 certainly belongs to the flowering controlling gene.Semi-quality RT-PCR and GUS histochemical staining were used to confirm the expression level and tissue localization.RT-PCR and Real-Time PCR were used to confirm the location of EFS1 in the regulating pathway.We have provided a foundation to the further regulation mechanism research.1.EFS1 was constructed to the pCAMBIA1300 vector,complementation and overexpression were obtained via Agrobacterium-mediated transformed to efs1 mutant and Col-0.The EFS1 was certainly integrated to the efs1 mutant as multiple copies based on the PCR and Southern blotting.The RT-PCR analysis showed that in the reverse mutant, EFS1 had a higher expression than the wild type.In the complementation,the flowering time was similar to the wild type Col-0,and also the flowering time was positively correlated to the expression level of EFS1.This result showed that the phenotypic reversion was caused by the expression of exogenous EFS1.2.RT-PCR was used to analyze the expression of EFS1.The results showed that the EFS1 might be a constitutive expression in the different tissues such as rosette leaf,stem, cauline leaf and flower.It had a highest expression in the flower.Binary vector pCAMBIA1300-ProEFS1::GUS-NOS was constructed to transform to Nicotiana tabacum and Arabidopsis thaliana to get the tissue localization analysis,EFS1 promoter-driven GUS expression was detectable in rosette leaf,stem,cauline leaf and flower of six-week seedings,and in the whole one-week seedings,this result is consistent with RT-PCR.3.Expressed pattern of some important genes regulated in flowering were detected, such as SPY,FLC,GI,CO,FT and LFY.The results indicated that the expression of LFY was increased,the expression of SPY,FLC,GI,CO and FT was decreased.So a conclusion was determined that the EFS1 might suppresse flowering.4.According to the results,the flowering model of EFS1 was determined.The reduced expression of EFS1 may promote flowering by repressing the expression of SPY via gibberellin pathway,at the same time,the expression of FLC of vernalization pathway and the expression of GI,CO and FT of photoperiod pathway was also decreased.