| Rape (Brassica napus L.) bee pollen, the male reproductive spores of rape collected by honeybee, is usually mixed with added nectar and bee secretions. It is of high nutritional value, containing proteins, nucleic acid, carbohydrates, lipids, vitamins, minerals and physiologically active substances. Reversibly glycosylated polypeptide (RGP) is a kind of water-soluble protein found in monocots and dicots. Previous studies confirmed that it is associated with the synthesis of polysaccharide and the formation of cell wall. We found that the content of RGP is high and there is little information about the property and biological function in rape bee pollen. In this paper, the rape bee pollens RGP (BnRGP) was isolated, purified, characterized and localized by the analysis of the water-soluble protein in rape bee pollen. The results are as follows.(1) The method of ammonium sulfate precipitation was used to extract the water-soluble protein in rape bee pollen. The result showed that, the protein concentration was higher when the ammonium sulfate saturation were 20?40%?40?60%?60?80%, and their contents were 736.70,748.05 and 516.88 ?g/g, respectively. Through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different water-soluble proteins in rape bee pollen, a variety of proteins were found and their molecular weight ranged from 25?90 kDa.(2) The dialyzed crude protein from the 20?40% saturation fraction of the rape bee pollen was further purified by ion-exchange column chromatography (EEC) in a DEAE-Sepharose Fast Flow and Sepharose CL-6B gel filtration chromatography (GFC) and two kinds of proteins were obtained with molecular weight of ?40 kD and ?42 kDa, respectively. The two proteins were characterized by the MALDI-tof-MS and LC-MS using SDS-PAGE bands spots. After homology comparison through MASCOT software, the two proteins finally were identified as reversibly glycosylated polypeptide and we named them BnRGP1 and BnRGP2.(3) We mainly focused on the properties of BnRGP2 in this part. The precise molecular weight of BnRGP2 is 42388 Da determined by MALDI-tof-MS. The isoelectric point (pI) of BnRGP2 is 5.46, indicating BnRGP2 is an acidic protein. High performance liquid chromatography (HPLC) was applied to determine the amino acids composition of BnRGP2, and the results showed that BnRGP2 is rich in amino acids, containing 17 kinds of amino acids and the content of aspartic acid is the highest (71.56 mg/g). Immunological histological chemistry (IHC) was used to identify the localization of BnRGP2 in rape anther by transmission electron microscope (TEM), and the result showed that BnRGP2 exist in extine and intine of anther cells of rape flower. The subcellular localization result showed that BnRGP2 appeared around the Golgi apparatus in cytoplasm. |
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