【作者】 陈洁；

【导师】 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2006， 硕士

【摘要】 鞘脂普遍存在于包括植物在内的各种真核生物的生物膜中。鞘脂及其衍生物在生物体内参与了极重要的细胞功能,它们可以作为第二信使调控细胞生长、分化、存活、增殖和程序性死亡等。近年来,1-磷酸鞘氨醇作为细胞外递质和胞内第二信使的功能受到关注。细胞内的SPP水平高低是由其合成、分解和转运到胞外这三者相互作用调控,其中鞘氨醇激酶催化鞘氨醇形成1-磷酸鞘氨醇,1-磷酸鞘氨醇磷酸酶催化1-磷酸鞘氨醇还原为鞘氨醇,同时1-磷酸鞘氨醇裂解酶将1-磷酸鞘氨醇分解。为探讨SPP在植物抗逆反应中的作用,我们克隆得到水稻中编码鞘氨醇激酶的基因,OsSPK1,并通过转基因烟草分析了OsSPK1在抗病反应中的功能。 以水稻cDNA文库为模板,PCR扩增得到了水稻SPK基因OsSPK1全长cDNA。OsSPK1全长cDNA序列为2910bp,含有一个2247bp的ORF区域,预测编码一个757个氨基酸组成的SPK蛋白,推测的分子量为83.27kDa,等电点为7.4。OsSPK1基因位于水稻10号染色体上,含有8外显子和7个内含子。根据水稻OsSPK1基因氨基酸序列与人类、老鼠、酵母、线虫和植物中的SPKs家族的氨基酸序列进行比对分析,我们发现都包含一个125aa左右的保守DAGK催化结构域。同样,OsSPK1氨基酸序列还含有保守的C1～C4保守结构域。在C1结构域中的GXGX基序是这类鞘脂激酶的ATP结合位点。 为了进一步明确OsSPK1在水稻抗病性中的作用,我们将OsSPK1基因编码区置于植物双元载体CHF3pp2p212中CaMV 35S组成型强表达启动子的下游,运用根癌农杆菌介导的叶盘法转化烟草,经卡那霉素抗性筛选和PCR检测获得14株OsSPK1转基因植株。RT-PCR分析表明,转基因T₁代烟草植株都正常表达OsSPK1基因。抗病性测定表明,过量表达OsSPK1的转基因烟草植株提高了对烟草黑胫病菌（Phytophthora parasitica var.nicotianae）和烟草野火病（Pseudomonas syringae pv.tabaci）的抗病性。更多还原

【Abstract】 Sphingolipids are ubiquitous constituents of membrane systems in all eukaryotic organims. There is now substantial evidence that sphingolipids and their metabolic products are involved in the regulation of cell growth, differentiation, survival, proliferation and programmed cell death via their participation as second messengers in specific signal-transduction pathways. As a novel class of sphingolipid metabolites, sphingisine-1-phosphate (SPP) functions as a second messenger and as ligands for cell surface receptors. Cellular levels of SPP are largely mediated by its formation from sphingosine by the activity of sphingosine kinase (SPK) and to a lesser extent by its degradation by SPP lyase and SPP phosphatase activities. In order to elucidate the biological function of SPP in plant responses to abiotic and biotic stresses, we cloned and identified a rice SPK gene, OsSPKl, and studied its function in disease resistance response by overexpressing in transgenic tobacco.The cDNA of OsSPKl was amplified by PCR using phage DNA prepared from a rice cDNA library as template. The full-length cDNA of the OsSPKl is 2910 bp with a predicted open reading frame of 2247 bp in length, which predicted to encode a putative SPK protein with 757 amino acid residues with a predicted molecular mass of 83.27 kDa and pI of 7.4. The OsSPKl gene was located on chromosome 10 of the rice genomes and is composed of 8 exons and 7 introns. Alignment of OsSPKl with other SPKs from human, mouse, yeast, Caenorhabditis elegans and plants showed that OsSPKl has a conserved diacylglycerol kinase catalytic domain of ~125 amino acid residues in length. Also, OsSPKl contains an extra conserved C1-C4 regions, including GXGX motif in the C1 domain, which may be involved in ATP binding.To better understand the function of OsSPKl in disease resistance response, we cloned the OsSPKl coding region into a plant binary vector, CHF3pp2p212, under control of the CaMV 35S promoter and transformed tobacco leaf discs by Agrobacterium-mediated transformation. A total of fourteen independent OsSPKl transgneic tobacco lines were obtained and confirmed by PCR detection. RT-PCR analysis indicated that OsSPKl gene was transcribed in the transgenic tobacco plants. We evaluated disease resistance of the transgenic lines and found that the transgenic plants showed enhanced disease resistance against Phytophthora parasitica var nicotianae and Phytophthora parasitica var nicotianae. Our preliminary results indicate that OsSPKl-mediated SPP signaling may play a role in disease resistance response in plants.更多还原