Cloning, Prokaryotic Expression And Antibody Preparation Of GPP And IMP Gene From Apple Fruit

L-Ascorbic acid (AsA) is one of the most important antioxidants and cofactor for many enzymes, it is the main source of natural AsA to people. Apple fruit is a kind of favorite fruit, but it contains lower AsA than other fruit, which seriously affected its quality. Studying the AsA biosynthesis of plant and improving the content of AsA in plant tissues are significant for plant themselves and human health. Studying the function of AsA biosynthesis and metabolism genes which involved in apple fruit is the basis of apple fruit AsA accumulation mechanism. This experiment focused on the L-galactose-1-P phosphatase(GPP) and Myo-inositol-1-P phosphatase(IMP) which involved in AsA biosynthesis and the results are useful for further study of function of GPP and IMP gene, and they also provide the theoretical basis of the molecular mechanism of synthesis and accumulation of AsA in apple fruit.The complete GPP and IMP gene sequences were amplificated by RT-PCR method, and the bioinformatic tools were used to analyze the sequences. In order to further understand the function of the two genes, expression plasmids of GPP and IMP gene were constructed with pET-32a and expressed in E.coli BL21, IPTG was added to induce the fusion protein expression,then analyzed the characteristics of the fusion proteins. His-GPP and His-IMP fusion proteins were used to immunolize rabbits to prepare Polyclonal antibody, then the specific recognition were detected by Western blot experiment, and the expression of GPP, IMP gene in apple different tissues were analysised by the same method. The main results were as follows:1. The full-length cDNA of GPP is 1121 bp and contains a complete open reading frame of 813 bp, which encoding 270 amino acids. And the full-length cDNA of IMP is 1201 bp and contains a complete open reading frame of 813 bp, which encoding 335 amino acids. Homology analysis of their sequences showed high homology with the cDNA from other plants, respectively. Through the bioinformatic analyze, we found the secondary structure and tertiary structure of GPP are similar to IMP's, maybe it is the explanation of the similar function between GPP and IMP gene. 2. We signed other two pairs of specific primers based on the sequences of GPP, IMP gene and pET-32a, then GPP and IMP gene were subcloned into the expression vector pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were digested by BamHI and SalI and the results show that GPP and IMP were correctly linked to the pET-32a. The recombinant expression vectors pET-GPP and pET-IMP were transformed into E.coli BL21 cells and then induced by IPTG, the molecular weights of the cells specifically expressed proteins are 49 KD and 57 KD based on SDS-PAGE gel results, respectively. They are identical to the known His-GPP and His-IMP fusion proteins, so the GPP and IMP gene were expressed in E.coli BL21. We also fixed the best expression condition by compare the expression result of different IPTG density and cell induced culture times. The result shows that the best expression condition for His-GPP induced is 0.1 mmol?L-1 IPTG at 28℃for 5 h, and the best expression condition for His-IMP induced is 0.01 mmol?L-1 IPTG at 37℃for 4 h. Through solublity analysis we found His-GPP and His-IMP fusion proteins were almost expressed in inclusion body.3. Inclusion body of His-GPP and His-IMP from E.coli induced by IPTG were used as antigens, then injected into rabbit to prepare the antibodies. The validity of polyclonal antibodies were confirmed by western blot and the results show that they had specific antigen-antibody recognition to the His-GPP and His-IMP.4. Total Proteins were extracted from different tissues of apple and separated by SDS-PAGE,then transferred to a PVDF membrane. Western blot was Performed using antibodies against GPP and IMP, respectively. Specific bands about 33 KD and 39 KD were found in Western blot immune images. The reason of the appearance of these bands are the expression signals of GPP and IMP gene in apple tissues were detected.