水稻花粉結鈣激活酵素OSCK1之基因表現、蛋白胞內分布位置與基因轉殖植物分析

Abstract

Calcium-dependent protein kinase (CDPK)為一種結鈣激活酵素，在植物中發現常以多基因家族存在，且於細胞內分佈呈多樣性，並經常專一地表現於特定的組織或發育時期，故被推測在多種訊息傳導中扮演重要的角色。本實驗室藉由差異性表現基因選殖法，由水稻中篩選出一主要於成熟花粉表現的OSCK1 (Oryza sativa calcium-dependent protein kinase 1)基因。經由南方墨點法分析與水稻基因組資料庫搜查，得到一與OSCK1基因序列及結構均高度相似的基因，命名為OSCK2，以專一性引子進行RT-PCR分析可知兩個基因表現模式相同，均僅於花粉成熟後期有大量訊號，此與偵測蛋白表現模式的結果相符。為探討OSCK1基因的功能，分別構築能大量增加(pOSCK1-31)或干擾(pOSCK1-29)OSCK1表現的質體，轉殖水稻獲得獨立轉殖株，分析後不論是外表型或數個逆境誘導基因的表現而言，轉殖株均與野生株無異，轉殖株仍可正常授粉結種子。藉由超高速離心區隔法配合溶液萃取，可得知無論是花粉或轉殖株葉片大量表現的OSCK1蛋白，大多以疏水性力量附著於膜上，進而由G2A-OSCK1突變蛋白的分布分析，可證明荳蔻酸化即是OSCK1附著於胞膜的主要關鍵。

Numerous stimuli can alter the Ca2+ concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of Ca2+ signals and bring about changes in metabolism and gene expression. The abundant calcium-stimulated protein kinase activity found in plant is associated with calcium-dependent protein kinases (CDPKs). Using differential screens, we isolated a OSCK1 gene which is mainly expressed in the mature pollen of rice. Genomic Southern hybridization combined with the GenBank blast search revealed a gene sharing a high sequence identity and similar gene structure as OSCK1, therefore it was designated as OSCK2. Investigated by gene-specific RT-PCR, both genes were found predominantly expressed in the mature pollen. Similar results were obtained when examining the amount of OSCK1 proteins. Transgenic rice that putatively induce the silencing of OSCK1 by RNA interference (pOSCK1-29) or overproduce the OSCK1 proteins (pOSCK1-31) were generated by Agrobacteria-mediated transformation. Southern blotting confirmed the independence of each of the transgenic lines. The OSCK1 proteins were abundantly accumulated in the leaves of transgenic lines. However, no significant differences could be found between the transgenic and wild type plants in terms of phenotype observations and molecular characterizations for expressions of the stress-inducible genes. The OSCK1 proteins were mainly associated with membranes either in pollen or in leaves of transgenic plants. Differential extraction of the pelleted membranes using various chemicals revealed that OSCK1 proteins were bound to membrane through hydrophobic interactions. Moreover, the G2A-OSCK1 mutant demonstrated that OSCK1 proteins are attached to membranes via a myristoyl group.