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標題: Characterization and functional study of rice Ose705 protein
作者: 林威廷
Lin, Wei-Ting
關鍵字: Ose705 gene;水稻胚特異性基因;gene silencing;eGFP;基因靜默法;綠色螢光蛋白
出版社: 分子生物學研究所
本研究之目的為探討 Ose705 (Oryza sativa embryogenesis) 基因在水稻中的特性及其功能。Ose705 是一個水稻胚特有表現的基因,但其功能卻尚未被了解,本實驗再次確認 Ose705 基因只有在水稻授粉後的胚才會表現, Ose705 蛋白會於授粉後 11 天至 14 天時表現量特別大,在其他部位及時期並無法偵測到Ose705 mRNA 及蛋白的存在。本研究利用 RNAi 的方式,將含有正反向 Ose705 基因片段的 RNAi 表現載體送入水稻基因組後,觀察這些 Ose705 RNAi 轉殖植株與台農 67 號之差異。比較結果發現,RNAi 轉殖株的穗重有比較輕的現象,每穗粒數較少,而且35S-705i 轉殖植株的稔實率相當低,但在 705-705i 中並無此現象發生,這與 35S-705i 轉殖植株穗重比 705-705i 轉殖植株輕有其相關性,但在穗長、種子的外觀與重量則沒有太大的差異。另外,在兩個 RNAi 轉殖株已經結穗並且採收種子後,將其播種至 MS 培養基中發現其發芽的情形比野生株還要慢,同時影響了種子長芽與發根的速度,由 Ose705 表現的部位及時期推測可能是胚發育時缺少 Ose705 蛋白而造成缺陷導致水稻在發芽時受到了影響,但在後期會慢慢的趕上,而且並不影響轉殖植株後期的成長情況。研究中也針對 Ose705 基因序列以及蛋白序列以生物資訊法的方式來預測其可能之功能,雖然無法預測到相關之 domain 或是具有功能之序列,但卻預期出 Ose705 蛋白可能具有一個 signal peptide ,因此將Ose705 基因序列接上增強型綠色螢光基因序列,預期能形成一個螢光融合蛋白。將這表現載體送入水稻基因組後,利用西方墨點法確定此轉殖水稻確實有 Ose705-eGFP 蛋白的生成也初步利用螢光顯微鏡觀察轉殖植株根部確實有螢光產生,但無法確認此融合蛋白是否分泌於細胞間隙中。本研究結果推論 Ose705 是一個胚特異表現的基因,可能與胚成熟相關,而且此基因之表現會影響子代種子之發芽能力。

Ose705 has been characterized as one of the rice embryo-specific genes, however its functional role in the embryo development has not been well characterized. To better understand the functional role of this gene, RNA interference and other approaches were performed. In this study, the expression of Ose705 was further confirmed that it expressed only in the embryo at approximately 11 to 14 days after pollination and no expression was detected in any other developmental stages and tissues. In order to investigate the function of the Ose705 gene, a gene expression knock-down approach using RNA interference (RNAi) was carried out and 11 to 12 independent 35S-705i and 705-705i RNAi transgenic lines were obtained. The 35S-705i transgenic lines showed no different to TNG 67 during vegetative growth, and in the panicle length and grain weight, but had very low fertility (15% to 30%) and reduced grain number in each panicle. In comparison, the 705-705i transgenic lines showed no difference to the wild type (TNG67) in all aspects. Retarded germination of 35S-705i and 705-705i RNAi transgenic lines were also observed when sowing transgenic seeds in MS medium and further shooting and rooting were also affected. The results suggest that lacking Ose705 proteins during development of embryos affects germination of seeds in TNG67. Through bioinformatics analysis, a 28-amino acids signal peptide was predicted in Ose705. Further investigation utilizing eGFP was employed to localize the Ose705 protein in the sub-cellular level. The presence of Ose705-eGFP fused proteins were confirmed in the transgenic rice lines using Western Blotting and observed in fluorescent microscope analysis. These preliminary observations suggest that the Ose705 is an embryo-specific gene and it might involved in regulating germination of seeds in TNG67 rice.
Appears in Collections:分子生物學研究所

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