||Research on characteristics and utilization of storage protein in Curcuma alismatifolia Gagnep.
94 , 2016-03-23 , 新潟大学
学位の種類: 博士（農学）. 報告番号: 甲第4196号. 学位記番号: 新大院博（農）甲第157号. 学位授与年月日: 平成28年3月23日
Curcuma alismatifolia Gagnep. consisted of two underground storage organs that are a rhizome and storage roots. Both organs support the initial growth of Curcuma plants, and become senescent at the end of growth. A rhizome is a major organ of N storage, and the storage roots mainly reserve carbohydrate with relatively low concentration of storage protein. N stored in the rhizomes and storage roots are used for initial growth and development after planting (Ruamrungsri et al., 2001). From previous research, Khuankaew et al. (2010) reported that most of absorbed N was translocated to the leaves, and original N from old rhizomes and old storage roots were transported to aboveground parts to support growth and development. After that a new rhizome formed and N continuously accumulated in this organ. When the Curcuma senesced both of N from the nutrient solution and also N accumulated in the previous organ was assimilated and remobilized to new storage organs and mainly stored in the new rhizome. Therefore, the study on storage protein profiles in the rhizomes and storage roots of Curcuma is important to understand the characteristics of nitrogen storage and nitrogen use after planting in Curcuma cultivation. To success the objective of this study, the experiments were divided into three parts. First, localizations of protein in storage organs of Curcuma were observed by using plant micro-techniques. Second, experiment was studied on the changes of soluble and insoluble nitrogenous compounds in storage organs at five growth stages of Curcuma for understanding N utilization and gave the basic knowledge to investigate change of storage protein. Third parts focused on the protein characteristics in storage organ of Curcuma in each growth stage and protein identification particular storage protein. I. Localization of protein in storage organs of C. alismatifolia From the first study, it was confirmed that the rhizome of C. alismatifolia is a greater source of N storage than storage roots, while the storage roots mainly store starch that demonstrated by using freezing microtome and free-hand section techniques together with CBB and KI-I_2 staining. The intensity of CBB staining of tissues was stronger in rhizomes than in storage roots. Protein staining was present in the cytosol and in the cell walls in rhizomes, and was intense in small particle-like protein bodies in the cytosol.
II. Changes of nitrogenous compound in storage organs of C. alismatifolia Freeze-dried powder of old rhizomes and old storage roots harvested at 5 stages were extracted with dilute phosphate buffer (PBS), and the N concentrations of PBS-soluble and PBS-insoluble fractions are analyzed. PBS soluble fraction contains water-soluble compounds, such as soluble proteins and amino acid. On the other hand, PBS-insoluble fraction contains mainly insoluble protein, nucleic acid and lipids. At every growth stage, the amount of N in the PBS-insoluble fraction was 3-4 times higher than the PBS-soluble fraction in both rhizomes and storage roots. Along with growth stages, both PBS-soluble and PBS-insoluble N in rhizome and storage roots were decreased sharply when plants become to dormancy, which indicates that most of N stored in rhizome and storage roots are a kind of storage proteins supplying N for plant growth. PBS-soluble fractions are further fractionated into trichloroacetic acid (TCA) soluble and insoluble fractions. TCA-insoluble fraction contains proteins, and TCA-soluble fraction contains amino acids and low molecular weight compounds. From planting to sprouting stage, the N in the TCA-insoluble tentatively increased both in rhizomes and storage roots, suggesting that protein synthesis was active using amino acids in this stage for degradation of storage compounds such as starch and storage nitrogen. III. Protein characteristics in storage organs of C. alismatifolia Protein profiles in the PBS soluble fraction of rhizomes and storage roots were further analyzed by SDS-PAGE. The results of protein profiles showed that peptides of 10.6kDa and 12.0kDa were the two major soluble proteins in rhizomes and were different from the proteins in storage roots. The major polypeptide separated by SDS-PAGE from storage roots was found only a 14.3kDa. In rhizomes, the 10.6kDa and 12.0kDa bands contained five peptides and one peptide, respectively, when separated by 2D-PAGE. The amino acid sequences of these six peptides were not homologous to any known proteins using BLASTP or FASTA analysis. The N-terminal amino acid sequences of these two polypeptides did not start with methionine, indicating that they had undergone posttranslational modification. Knowledge of the major storage proteins in the rhizomes and storage roots of C. alismatifolia may provide information to improve N-fertilization.