【作者】 常正尧；

【导师】 印莉萍；

【作者基本信息】 首都师范大学 ， 细胞生物学， 2006， 硕士

【摘要】 铁在生物体内具有重要的生理功能。但世界范围内约30％的可利用土地为碱性土壤，导致植物缺铁失绿。植物缺铁不仅严重影响了植物的正常生长发育，导致作物减产，还是造成人类缺铁的一个重要原因。 水稻（Oryza sativa L）是人类最重要的粮食作物之一，为全球1／2以上人口提供了主要食物。水稻缺铁的研究对于提高粮食产量，解决人类缺铁有着重要的意义。水稻基因组计划完成后，以功能基因组学方法研究水稻缺铁相关基因的表达调控成为一种有效的方法。在对缺铁水稻根进行10531个cDNA克隆的微点阵分析后，发现与膜泡相关的基因群相对转录表达率最高。利用超薄切片技术，在透射电镜下观察缺铁胁迫下水稻根尖细胞的结构变化，发现缺铁培养的水稻根尖细胞中膜泡的个数较多且明显，表明缺铁可能诱导了膜泡相关基因的高效表达。 对经FeCl₃和缺铁处理的植物凝胶培养基中培养的水稻根进行微点阵分析后，获得309个差异cDNA。通过NCBI的BLAST对其进行了初步的功能分析。 对转运体、膜泡运输相关的基因（IRT、Nramp、YSL、渗透酶基因）和Ferritin进行的Real-Time PCR分析证明缺铁诱导的IRT、Nramp、YSL、渗透酶基因等转录本在缺铁时增多。Ferritin转录本在EDTA-Fe和FeCl₃培养时增多。 通过RT-PCR的方法从缺铁培养5天水稻根中扩增得到渗透酶基因的全长cDNA，1973bp。分别构建了植物正义、反义表达载体pCAMBIA1302-permease（S／A）-GFP，使渗透酶基因与GFP基因构成融合基因，测序结果证明其序列正确。将pCAMBIA1302-permease（S／A）-GFP转入农杆菌EHA105，通过农杆菌侵染洋葱表皮和基因枪轰击洋葱表皮的方法对渗透酶进行了亚细胞定位，结果表明渗透酶定位在质膜上。利用蛋白质分析软件（ConPredⅡ softwear）和NCBI比对，对渗透酶进行了功能、疏水性和跨膜域分析，进一步证明它是一个具有12个跨膜域的质膜蛋白。可能参与黄嘌呤（xanthine）、尿嘧啶（uracil）的转运。 通过叶盘法转化烟草，初步获得MS培养基培养的具有潮霉素抗性芽。最后，初步建立了水稻悬浮系，为研究水稻细胞定位和转基因水稻提供了受体。更多还原

【Abstract】 Iron is an essential micronutrient with numerous cellular functions, and plays a critical role in important biochemical processes. Iron deficiency can be particularly pronounced in plants grown on calcareous soils, which cover approximately one-third of the earth’s surface. Iron deficiency is not only affective development of plants, if severe, can lead to reduction in crop yields and even complete crop failure, but also is an important reason for people’s iron-deficiency.Rice, one of the most important cereal crop, is the food staple of more than half the world’s population. It is an effective way to increase the yield of crop and solve the iron-deficiency problem by promoting the iron content in rice. With the completion of sequencing of the entire rice genome, the most effective strategy of studies on the expression of genes responded to Fe-deficiency in rice is functional genomic approaches. Microarray methods were employed through the use of a cDNA clones collection representing 10531 individual gene sequences for understanding deeply the strategy responded to lack iron stress. The highest ratio of -Fe/+Fe of transcripts relevant membrane vesicle were found among the whole up-regulated cDNAs. The vesicles in rice root apices were abundant and distinct, when the structural change of rice root apices under both Fe-deficiency and EDTA-Fe treatment observed with a transmission electron microscope, using the technique of ultrathin sections, which indicated that genes relevant to membrane vesicle may be expressed highly in Fe-deficiency.Microarray analysis was used to characterize the expression profile of rice at transcriptional levels in response to iron deficiency and FeC13 cultured in phytagel medium. There were total 309 distinguish cDNA spots, each gene among them was analyzed in the function through using the BLAST （basic local alignment search tool） of NCBI （national center of biological information）.The analysis of the genes related to transporter （irt、 nramp、 ysl、 Permease） or vesicle transporting and Ferritin gene by Real-Time PCR indicated that the transcription of irt, nramp, ysl, Permease all increase in iron deficient. Whereas the transcription of Ferritin increase under EDTA-Fe and FeCl₃ conditions.Permease gene with the full length of cDNA 1973bp has been cloned by RT-PCR method from iron-stressed rice root for 5days. Then two new plant expression vectors both sence and antisence pCAMBIA1302- Permease（S/A）-GFP were constructed respectively. Fused protein was transferred into onion epidermal cell by agrobacterium- mediated transformation or particle gun bombardment. Sub-cellular localization of permease protein was analyzed through GFP observed under scanning fluresent confocal microscopy. The results showed that permease protein was located in plasma membrane. Function .Hydropathy and transmembrane domain were analized through ConPredII softwear and NCBI and proved that permease span 12 transmembrane domain.It may may be tansport xanthine and uracil.Plant expression vectors had been transformed into tobacoo using leaf disc transformation , and obtained hygromycin-resistant buds in the MS medium and created rice suspension for researching cellular location in rice and transgenic rice.更多还原