| Invertase,also called β-D-fructofuranosidase (EC3.2.1.26), exists widely in higher plants, which catalyzes the cleavage of sucrose into the two monosaccharides(glucose and fructose) in an irreversible way. In plants, invertase functions in a variety of biological processes, including phloem loading and unloading, growth and development, biotic and abiotic stress responses, and so on. Invertase plays a critical role in regulation of carbohydrate partitioning, and is also the key enzyme that determined the output of the crop. In rubber tree, sucrose is the primary material for rubber biosynthesis occured in the laticifers, and the efficientcy of its utilization forms a critical facter determining rubber productivity. Physiological and biochemical evidence has demonstrated that the invertases of neutral or alkaline type(N/A-Inv)were located in the C-serum of latex, and the key enzymes in the regulation of sucrose catabolism and rubber production. However, due to its vulnerability to inactivation, little information is available to its protein expression and enzymatic characteristics, and the molecular characters for the purified latex invertase is not known yet.In this paper, the neutral/alkaline type of invertase was purified from the C-serum of Hevea latex, characterized for its enzymatic properties, identified by mass spectrometry for its encoding gene and prepared for its polyclonal antisera. By western blotting, the expression patterns were analyzed under various experimental conditions.The results laid groundwork for unraveling the mechanisms underlying the involvement of N/A-Inv in the regulation of sucrose catabolism and latex productivity. Following are the major research results.1. N/A-Inv has been purified from latex C-serum of rubber tree. The latex C-serum was obtained by high speed centrifugation, and subjected to sequential steps of purification for N/A-Inv:ammonium sulfate fractionation, Sephacryl S-300gel filtration chromatography and DEAE-Sephacel ion exchange column chromatography, The ultimate invertase sample was purified by up to29folds and the target protein band was identifiable by SDS-PAGE, but the enzymatic activity recovery was only0.5%.2. The major invertase isozyme and its basic molecular characters have been made clear. Through tandem mass spectrometry, the expected protein band fractionated by SDS-PAGE was identified to be N/A-Inv and encoded by HbNIN2, which furher confirmed our previous work done in our group. The purified N/A-Inv has an apparent molecular weight of69kDa and lacks glycoconjugate.3. The enzymatic kinetics and properties of purfied latex invertatse have been characterized. The temperature and pH optima for the purified N/A-Inv were45℃and7.6, respectively. The Km of the N/A-Inv for sucrose is18.69mmol/L and the Vmax is1.33μmol/min. The inhibition constants Ki value for fructose and glucose were38mmol/L and74mmol/L, respectively.4. The enzymatic activities of purified invertase have been found to be affected by a number of metal ions and biochemical reagents. Pyridoxine hydrochloride and pyridoxal hydrochloride activate N/A-Inv, whereas the heavy metal ions including Cu2+, Hg2+, Ag+, Zn2+, Co2+, Fe3+and Mn2+and PMSF and EDTA strongly inhibit its activity.5. Preliminary study on the expression characteristics of the latex invertase isozymes has been made. The polyclonal antibody of the HbNIN2with higher specificity were purified by ammonium sulphate frationation and DEAE-Sephacel ion exchange column chromatography and then used for protein expression analysis through western blotting. In the latex of virgin rubber trees, it was showed that the HbNIN2expression and activity were decreased first and then raised with the process of consecutive tappings, and were markedly up-regulated by wounding. The treatments of different phytohormones posed varied effects on HbNIN2expression and enzymatic activity, depressed by jasmonic acid, stimulated by Ethrel and cytokinin, depressed first then up-regulated by gibberellin, and not affected by salicylic acid, auxin (2,4-D) and abscisic acid. Among the three large recommendation rubber clones, PR107, Reyan7-33-97and Reyan8-79, HbNIN2protein was abundantly expressed in Reyan7-33-97, then in Reyan8-79and the least in PR107. Among the tisses tested, the expression of HbNIN2is the strongest in the latex, then in leaves and the least in the bark. In combination with our previous results of HbNIN2gene expression, we found that under a variety of experimental conditions the protein levels and activity of HbNIN2did not have an obvious correlation with the levels of HbNIN2transcripts, indicating that posttranscriptional regulation may play a critical role in the regulation of HbNIN2gene expression. |
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