【作者】 路群；

【导师】 周根余； 明凤；

【作者基本信息】 上海师范大学 ， 遗传学， 2005， 硕士

【摘要】 磷是高等植物生长必需的大量元素,主要由根系从土壤中吸收获得。水稻是世界的重要粮食作物,全球有二分之一左右人口以大米为主食。水稻土壤磷肥短缺现象,是全世界农业生产中限制水稻产量的一个重要因素。在大多数植物中,磷吸收系统由低亲和力和高亲和力两个部分组成;磷缺乏时,高亲和力转运系统是植物根中吸收磷的主要机制。目前,已经从很多高等植物如番茄、拟南芥等中分离出高亲和力磷酸盐转运蛋白,但有关水稻磷酸盐转运蛋白基因和功能的研究工作还很少,仍处于初级阶段。本研究从水稻中分离到了磷酸盐转运蛋白基因OrLPT1,并对其表达进行研究。研究结果如下:1. 利用RT-PCR 方法,从受低磷胁迫(Pi 2.82 μM)处理5 天的水稻(Oryza sativa L.)叶片cDNA 中克隆到一个全长为1605bp 的磷酸盐转运蛋白基因(Genbank accession number:AF335588),命名为OrLPT1;2. OrLPT1 ORF 编码长为535 个氨基酸(分子量57.6 KDaltons)的蛋白,与其 他植物物种磷酸盐转运蛋白的同源性在64%以上;多肽链的疏水性分析表 明,OrLPT1 具有12 个疏水的跨膜区及高亲和力磷酸盐转运蛋白中保守的3 个磷酸化作用位点:蛋白激酶C作用位点(T-A-R)、N端糖基化部位(N-A-T-T) 和酪蛋白激酶II 作用部位(S-L-E-E);3. 提取水稻材料基因组,酶切,以OrLPT1 ORF 部分片段(459bp)为探针进行 Southern 杂交分析,结果表明该基因以三个拷贝存在于水稻基因组中,杂交 结果与水稻基因组序列分析完全吻合;4. 采用MEGA 软件(version2.01)自展法对OrLPT1 与其他磷酸盐转运蛋白的 系统进化关系进行分析,OrLPT1 包括在单子叶植物转运蛋白基因组成的大的 分支内,并与OsPT6 的进化关系非常近;5. 在大肠杆菌中进行OrLPT1 的原核表达,并以抗GFP 抗体为一抗作Western blot 检测,结果显示蛋白条带与预计大小一致;6. RT-PCR 方法分析了OrLPT1 器官表达的特异性,低磷(2.82μM)和适磷 (282μM)营养液处理下,叶片中表达都很强;低磷胁迫条件下,根中OrLPT1 的表达显著提高,此表达方式与马铃薯的StPT1 类似;更多还原

【Abstract】 Phosphorus (Pi) is one of the essential macronutrients required by the roots ofplants. Rice is a major crop that feeds about one half of the world’s population.Phosphorus deficiency is a major limiting factor for rice production of the agricultureof world. Pi uptake systems consist of low-affinity and high-affinity transportcomponents. Under Pi deficiency, the high-affinity transport system is considered tobe the major mechanism for Pi uptake by roots. Genes encoding high affinity Pitransporters have been isolated from many plant species including Lycopersiconesculentum, Arabidopsis thaliana and so on. But there are little study on the genes andfunction of rice Pi transporters. In this paper, OrLPT1, a novel Pi transporter from ricewas cloned and its expression under both Pi sufficiency and deficiency was analyzed.The main results as following:1. A full-length cDNA clone, OrLPT1, encoding rice phosphate transporter was isolated from the total RNA of phosphate-starved leaves. The open reading frame of OrLPT1 is 1605 bp in length (Genbank accession number:AF335588).2. OrLPT1 ORF encods a polypeptide with 535 amino acid residues (molecular mass of 57.6KDaltons), which shares more than 64% amino acid identity with some other high-affinity Pi transporters of plants. Hydropathy analysis of the deduced polypeptides showed that the OrLPT1 is highly hydrophobic and contains 12 putative membrane-spanning regions. The phosphorylation sites for protein kinase C(TAR), casein kinase II(SLEE) and the N-glycosylation site(NATT) are conserved among high affinity Pi transporters from other eukaryotes.3. Southern blot with rice genomic DNA digested by different restriction enzymes was performed by using a fluorescein labeled probe of the 459bp cDNA region of OrLPT1. The probe hybridized with three bands indicating the existence of more related sequences in the rice genome.4. To estimate the phylogenetic relationship between OrLPT1 and other Pi transporters in plants, we used MEGA software (version 2.01) and bootstrap更多还原