【作者】 洪芳；

【导师】 谈家桢； 杨金水；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 真核细胞中，在基因的表达转录后调控的过程中涉及到多种蛋白质的参与，其中最重要的是RNA结合蛋白。已知的RNA结合蛋白中，研究的较多的是具有RNA识别基序的结构域(RNA recognition motif，RRM)、富含亚精氨的结构域(the arginine motif，ARM)和KH结构域(K homology motif，KH)的这几类RNA结合结构域(RNA binding domain，RBD)。RRM结构域是迄今研究得最多的一类RNA结合结构域，在真核生物基因表达调控中发挥着重要的功能。在拟南芥自主开花过程中，基因FCA编码的蛋白起着非常重要的作用，FCA蛋白具有两个RRM结构域和一个WW蛋白互作域。我们已报道克隆的水稻rFCA基因与拟南芥FCA基因同源，编码两个RRM结构域。为了研究RRM结构域的功能，将RRM1、RRM2片段分别克隆到植物表达载体pCAMIBIA 1304、pBY520，通过基因枪转化法转化籼稻9311、粳稻中花11。结果显示，RRM1、RRM2过表达转基因阳性植株表型都发生了相似的改变，与对照植株相比较，生育期延迟两周左右、转基因植株的叶片较对照植株叶片增厚、坚硬、加宽且呈深绿色；转基因水稻种子体积增大，千粒重增加；转基因植株花粉、柱头细胞较对照植株细胞增大。由此，推测水稻rFCA基因编码的两个RRM结构域，RRM1、RRM2在转基因水稻中行使相似的生物功能。参与调控转基因水稻的生育期：导致转基因水稻细胞增大；并且对水稻种子大小这一受多基因控制的重要的产量性状产生影响。为了进一步研究RRM结构域的过表达在转基因水稻中的生物功能，选取了粒型较籼稻9311、粳稻中花11要大的水稻品种南京大粒稻作为转化受体，构建了RRM1∶GFP融合蛋白植物表达载体，通过基因枪法转化水稻品种南京大粒稻。实验初步检测到转基因南京大粒稻的种子体积增大、千粒重增加，表明RRM的过表达影响转基因水稻种子的发育进程。RRMs结构域的过表达在不同的水稻转化受体籼稻9311、粳稻中花11以及南京大粒稻中呈现出相似的表型变化，说明水稻rFCA基因编码的两个RRM结构域，RRM1、RRM2所行使的生物功能的分子机制相似，而且这种分子机制在不同的遗传背景下相对稳定。更多还原

【Abstract】 In eukaryotic cells, regulation of gene expression at the post-transcriptional level is mainly achieved by proteins, which containing well defined sequence motifs involved in RNA binding. Most RNA-binding proteins contain one or several conserved domains, such as the RNA recognition motif (RRM), the K-homology (KH) motif, RGG (Arg-Gly-Gly) boxes, and double-stranded RNA-binding domains (dsRBDs)RNA recognition motifs as important regulators of gene expression are highly conserved in animals and plants. The FCA floral promotion gene in Arabidopsis encodes a protein, containing two RNA recognition motifs (RRM) and a WW protein interaction domain. Here we isolated rFCA cDNA from rice. FCA in rice (rFCA) was homologous to FCA-gamma of Arabidopsis and contained conserved two RRM domains. To investigate the function of RRM domain, fragment RRM1 and RRM2 of rFCA were introduced into rice subspecies Oryza sativa L. subsp. Indica var. 9311 and another rice subspecies Oryza sativa L. subsp. Japonica var. zhonghua11 transformation. We examined whether RNA recognition motif domains, rFCA-RRM1 and rFCA-RRM2 would cause any similar effect on the development of the transgenic plants by regulating the rice gene expression. In transgenic lines, pCAM-RRM1 and pBY-RRM2, the vegetative stage and growth of transgenic rice plants were retarded about two weeks compared with the wild type rice. Transgenic plants showed relatively dark green and thicker leaves. It was indicated that the two transgenic lines plants had larger seeds and pollen. Two transgenic lines exhibited similar phenotypes, flowering time delay, seed size and cell volume of transgenic plants was increased. These results showed that constitutive overexpression of RRM domains could regulate transgenic rice cellular size. The patterns of overexpression of two RRM domains and their similar morphologies indicate they may play a similar role. To test the function of RRM domain in increasing the transgenic rice seed mass, RRM1: GFP was introduced into another rice subspecies Nanjing Dalidao, and the similar phenotypes were obtained. We also found that the weight of seed increased in the transgenic rice. Thus, the overexpression of RRM1 may have involved into regulate seed development.These results indicated that the same phenotypic characteristics of late flowering and increased rice seed mass, were associated with the overexpression of the rFCA RRMs.更多还原