ABSTRACT
We induced embryogenic calli (EC) and non-embryogenic calli (NEC) from flower filaments of Vitis vinifera L. cv. Chardonnay about 10 days before full bloom. The callus were sub-cultured, observed and verified by somatic embryo induction. PCR primers for VvSUC12 and VvSCU27 were designed according to the corresponding sequences in GenBank. After RNA extraction with RNAplant for EC and NEC cell lines, we synthesized the 1st strand DNA for semi quantitative RT-PCR, and normalized the density of the bands against house-keeping gene Actin. The results of 31 cycles semi-quantitative RT-PCR showed that VvSUC12 was highly expressed in both EC and NEC, with higher expression intensity in NEC than in EC, but not reached the significant level; while the expression of VvSUC27 was only detected in EC, and the expression level was significantly lower than that of VvSUC12. We increased the semi-quantitative RT-PCR cycle number to 35 and found that VvSUC27 gene was weakly expressed in NEC, in EC the intensity of the band was increased comparing with 31 cycles, and the expression level was higher than that of NEC. The paper discussed the differential expression of the two sucrose transporters and their relationship with the sucrose in the tissue culture medium.
Subject(s)
Culture Techniques , Methods , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Membrane Transport Proteins , Genetics , Plant Proteins , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Vitis , Embryology , Genetics , MetabolismABSTRACT
We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-beta-D-thiogalactoside) at 28 degrees C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.