Studies on Rice Prolamins : Purification and characterization of 10 kDa prolamin and expression of prolamin gene RP5

• Main Authors: Luo, Ia-Uen, 羅雅文
• Other Authors: Chen, Ching-San
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/08354666915414697237

碩士 === 國立海洋大學 === 水產生物技術研究所 === 88 === Rice prolamins consist of three polypeptide groups, 10, 13 and 16 kDa, which all accumulate in protein body I (PB-I). Recently, several studies on maize, barley, and wheat suggest a model in which prolamins are organized into PBs, forming a spherical concentric ring structures by specific interaction between sulfur-rich and sulfur-poor prolamins. The 10 kDa prolamin was shown to be sulfur-rich, containing about 20% of methionine, 10% of cysteine, and high contents of glutamine and hydrophobic amino acids. Considering the staining mechanism in electron micrography, the dark dotted area is suggested to be the deposition site of reducing substances. Thus, it is suggested that 10 and/or 16 kDa prolamin is abundant in the dark area. To study the role of sulfur-rich prolamin in rice seed development, we purified and characterized the 10 kDa rice prolamin. Prolamins extracted from ground rice seeds (Oryza sativa L. cv Tainung 67) were separated into three polypeptide groups by SDS-PAGE: 10, 14 and 16 kDa. N-terminal amino acid sequence analysis indicated that the 10 kDa polypeptide was sulfur-rich prolamin. The purified 10 kDa prolamin was used for preparation of antibody. The antibody was used for investigating the time course of 10 kDa prolamin formation during rice seed development. Localization of 10 kDa prolamin in rice endosperm, and involvement of 10 kDa prolamin in formation of PB-I will be investigated using the anti-10 kDa prolamin antiserum. We are also interested in the physiological function of prolamins in rice plant. One of the rice prolamin genes isolated in our laboratory, RP5, is employed for this purpose because RP5 has a strong promoter. Three RP5 expression vectors RP5G (RP5p：：RP5-sense：：RP5-ter), RP5OAS (RP5p：：RP5-antisense：：nos-ter) and RP5OT (35S：：RP5-sense：：RP5-ter) were constructed for overexpression or antisense suppression of RP5 in rice plants. These three constructs were used to transform rice plants using Agrobacterium transformation system. Some transgenic rice plants were obtained. The effect of RP5 expression on the development of transgenic rice seeds will be studied by correlating the RP5 expression level with the maturation of transgenic rice seeds.