Cloning, Prokaryotic Expression And Transformation Of Plant Overexpression And RNAi Vector Of FT-like Gene Of Populus Xiaohei

After vegetative growth in juvenility, trees usually turn into the period of reproductive growth of anthesis through the response to a series of internal and external factors (such as vernalization, etc), and vegetative reproduction of trees will slow down and stagnate.In plant, FT gene, encoding a MADS-box protein which is activated by CO, SOC1, AGL24 and other gene and suppressed by FLC, can promote by activating the floral meristem specific genes API and LFY. So in the case of the expression level of FT gene could decline in populus, the flowering of the populus will be delayed and maintain in vegetative growth period (juvenile phase) to greatly increase the timber production. On the contrary, if FT gene could overexpress in populus, the anthesis will be promoted to accelerate the process and shorten the cycle of breeding of populus.Total RNA has been extracted form the leaves of Populus X xiaohei, and then a 535bp cDNA sequence has been cloned and compared the homology between AtFT gene of Arabidopsis thaliana with a 70% similarity. We named it PnFT1. We obtained the PnFT1-pET-52b recombinant vector by the insertion of PnFT1 into the MCS on pET-52b prokaryotic expression vector. Because of the predictive analysis of the E. coli rare codons of PnFTl gene showing 24.5% probability, we selected the RosettaTM of E. coli as express strains. After the induction of The RosettaTM of E. coli express strains carrying PnFT1-pET-52b recombinant vector by IPTG, we have found a the 28KD PnFTl recombinant inclusion protein by the SDS-PAGE detection analysis of the extraction products of total protein of thallus. The purification PnFT1 recombinant protein has reverted to the solvable and activated condition through the extraction of inclusion, the purification of electroelution, dialysis and precipitation by acetone. The protein sample with a lOug/ul concentration and 90% purity didn't contain the materials which are not beneficial to the preparation of the polyclonal antibody such as SDS, urea, CBB(Coomassie brilliant blue) and so on. The PnFT1 protein has absolutely reaching the standard of the sample for the preparation of the polyclonal antibody and been sent to Abmart company to finish the preparation. We have transformed the plant overexpression and RNAi expression vectors of PnFT1 gene by In-fusion and traditional method to research the functional genomics of PnFTl gene using the reverse genetics and finally obtained the PnFT1-pROKâ…¡å’ŒPnFT1(F)-pFGC5941-PnFT1(R) recombinant vectors.In this study, we have transformed the PnFT1 recombinant vectors of prokaryotic expression, plant overexpression and RNAi expression and obtained the PnFT1 recombinant protein and polyclonal antibody. The results of this study will laid the foundation for the further research on the function of PnFTl gene and the molecular regulate network of flowering and reveal the molecular mechanism of the floral development in populus, important to the cultivation of poplar varieties of populus.