【作者】 李文；

【导师】 杨代常；

【作者基本信息】 武汉大学 ， 遗传学， 2014， 博士

【摘要】 水稻胚乳是植物繁衍后代时储藏氮源和碳源的主要场所,也是分子医药农业最为理想的宿主之一。在水稻胚乳中存在着四种储藏蛋白,谷蛋白glutelin,球蛋白globulin,醇溶蛋白prolamin和清蛋白albumin,它们总共占胚乳细胞内总蛋白的90%以上。亚细胞定位研究显示谷蛋白和球蛋白在细胞中定位于蛋白贮藏囊泡(Protein Storage Vacuole, PSV),在水稻中也被称为蛋白体II(Protein Body II),而醇溶蛋白则定位于蛋白体Ⅰ。目前对于蛋白质在细胞中的定向转运这一过程的机理,普遍认为是通过存在于蛋白质一级结构中的一小段氨基酸序列组成囊泡转运信号(Vacuolar Sorting Determinant, VSD)或者是基于高级结构而产生的信号介导的,并且这一过程也需要相对应的受体参与。而目前为止,还没有任何关于这些蛋白的囊泡转运信号的报道,储藏蛋白在水稻胚乳细胞中转运机理的研究也较少。我们利用谷蛋白作为研究对象,通过序列缺失和定点突变,在水稻胚乳细胞稳定表达和烟草悬浮细胞系中瞬时表达的方法,研究了谷蛋白在水稻胚乳细胞中定向转运机理。另外,通过同重标签相对绝对定量(isobaric Tags for Relative and Absolute Quantitation, iTRAQ)的方法,我们对表达重组蛋白PDI-IGF的未成熟水稻胚乳细胞的总蛋白以及膜蛋白进行了蛋白组学的研究。主要结果如下：1、通过MacVector软件对谷蛋白β亚基氨基酸序列进行疏水结构的分析,氨基酸序列被分成了三个片段GlutN, GlutM和GlutC,并连接上GFP构建成三个融合蛋白的载体GlutN:GFP, GlutM:GFP和GlutC:GFP。通过农杆菌侵染得到了稳定表达植株后,经两代繁殖后都得到了纯合子。用荧光显微镜观察开花后7天左右的胚乳切片,发现GlutN和GlutM定位到蛋白体Ⅰ中,而GlutC则定位在蛋白体Ⅱ中。2、再将GlutC通过疏水性进一步分为三个片段GlutC-1, GlutC-2和GlutC-3,并构建了三个融合蛋白载体GlutC-1:GFP, GlutC-2:GFP和GlutC-3:GFP,经过转化后得到三个稳定表达的纯合子系。荧光显微镜观察,发现GlutC-1:GFP和GlutC-2:GFP融合蛋白可以定位到蛋白体Ⅱ中,而GlutC-3:GFP定位到蛋白体Ⅰ中。经过分析发现GlutC-1和GlutC-2共同拥有一段六个氨基酸QRLKHN的序列。将QRLKHN融合到GFP蛋白的N端,并在胚乳细胞中稳定表达后,我们发现这六个氨基酸序列可以指导GFP定位到蛋白体Ⅱ中,而单独表达GFP则定位在细胞质中。因此这六个氨基酸QRLKHN被定义为OsVSD (Oryza sativa Vacuolar Sorting Determinant)。3、另外,通过分子排阻色潽法(Size Exclusion Chromatography, SEC),我们发现GlutC:GFP融合蛋白存在于一个680KDa的复合体中,而GlutM:GFP则不存在复合体。并且我们发现水稻的RMR蛋白也存在于这个680kDa的复合体中。经过Co-IP验证,我们发现确实OsRMR3与GlutC:GFP是存在于同一复合体中的。通过BiFC鉴定,我们发现OsRMR3可以与GlutC或Glutelinβ亚基Glut相互作用,而不与GlutM相互作用。4、为了研究OsRMR3是否在含有OsVSD的蛋白转运中起作用,我们在烟草BY-2悬浮细胞系中表达了一系列的载体进行研究。结果显示在OsRMR3存在的情况下,Glut:GFP和GlutC:GFP都能定位在MVB (Multivesicular Body)中,而不定位在TGN中。GlutM:GFP无论是否与OsRMR3共表达都不能定位在MVB,而会定位在TGN中。因此OsVSD是蛋白转运进入MVB的前提,而OsRMR3则能提高这一转运的效率。5、通过对OsVSD六个氨基酸中的亮氨酸和精氨酸定点突变,我们发现两者对OsVSD的功能都很重要,而亮氨酸起到的功能更强大一些。6、经过以上结果我们最终总结出了水稻胚乳细胞中蛋白质转运的三条途径,第一条是依赖于VSD的转运途径,蛋白将通过此途径进入到蛋白体Ⅱ中。第二条是不依赖于VSD的默认转运途径,蛋白会从内质网合成后进入内质网膜出芽生成的蛋白体工。最后一条途径是非储藏蛋白的途径,这条途径经常在外源重组蛋白表达的时候观察到。7、通过对表达重组蛋白PDI-IGF的未成熟水稻胚乳细胞亚细胞结构的观察,我们发现在高表达量系中细胞由于受到的内质网压力较大,形态结构发生了较大的变化。8、通过对未成熟的水稻胚乳细胞的总蛋白进行iTRAQ蛋白组学分析,我们发现表达量最高的系中的显著差异蛋白是11个,上调6个,下调5个。中等表达量系中显著差异蛋白数量是12个,上调8个,下调4个。在表达量最低的系中,显著差异蛋白数量是18个,上调5,下调13个。9、对未成熟的水稻胚乳细胞的膜蛋白分析后,在高表达量系中,显著差异蛋白数量是20个,上调11个,下调9个；中等表达量系中,显著差异蛋白数目是18个,上调8个,下调10个；低表达量系中显著差异蛋白数目是23个,上调6个,下调17个。10、通过对转基因胚乳的研究,我们发现大量的外源蛋白表达对细胞的亚细胞结构会有很大影响,少量的表达则影响很小。而外源蛋白的表达对蛋白组没有大的影响,仅仅有个别分子伴侣和转录相关的蛋白会有较大的上下调。这为分子医药农业的转基因植株安全性提供了一些证据。更多还原

【Abstract】 The rice endosperm is the major sources of the nitrogen and carbon that are necessary for plant growth in subsequent generations and a favorite biopharming host for the recombinant protein expression. Four types of proteins, i.e., glutelin, globulin, prolamin, and albumin, account for90%of the storage proteins were found in the rice endosperm. In the rice endosperm, glutelin and globulin localize to the protein storage vacuole (PSV) named protein body Ⅱ (PB-Ⅱ), while prolamin is stored in protein body Ⅰ. The mechanism of intracellular protein sorting was considered to depend on the vacuolar sorting determinant (VSD) or the signals created by tertiary structure, and their corresponding receptors. Neither vacuolar sorting determinant has been reported for those storage proteins, nor the mechanism of trafficking route in rice endosperm cells. By sequential deletion and site-directed mutation, we studied the mechanism of glutelin sorting and trafficking in rice endosperm cells and tobacco B Y-2cells. Besides, the membrane and global proteomics of transgenic rice endosperm cells expressing recombinant PDI-IGF was investigated. The results show as below.1, The glutelin oteins, i.e., glutelin, globulin, prolamin, and albumin, account for90%of the storage proteins were found in the rice endosperm. In the rice endosperm, glutelin and globulin localize to tsed in rice endosperm cells. GlutN and GlutM were found in protein body Ⅰ, while the GlutC in protein body Ⅱ.2, The GlutC was further divided into three fragments GlutC-1, GlutC-2and GlutC-3. Then three fusion protein GlutC-1:GFP, GlutC-2:GFP and GlutC-3:GFP were expressed in rice endosperm cells. The microscopy results show the GlutC-1:GFP and GlutC-2:GFP were sorted into the protein body Ⅱ,but the GlutC-3:GFP into the protein body Ⅰ.A six amino acids sequence was found both in GlutC-1and GlutC-2.3, By size exclusion chromatography method, the GlutC:GFP and OsRMR3were found in a680kDa sorting complex, while no complex in the GlutM.GFP extracts. By Co-IP assay, it was confirmed the OsRMR3and GlutC:GFP were in the same complex. Furthermore, OsRMR3was found to interact with the GlutC or Glut, but not GlutM by BiFC assay.4, To investigate the function of OsRMR3in OsVSD-containing protein sorting, transient expression in BY-2cells was employed. The fusion protein was colocalized in the MVB but not TGN in the cells expressing Glut:GFP or GlutC:GFP when co-expressed with OsRMR3. However, GlutM:GFP was found in the TGN but not MVB, when whether co-expressed with OsRMR3or not. Thus, the OsVSD would be the prerequisite for sorting into MVB and the presence of OsRMR3could significantly increase the trafficking efficiency.5, The leucine or arginine replaced by glycine would lead to the sorting function polished, while leucine play a more critical role on that, indicating the function of those two residues for OsVSD function.6, Based on the results, three routes in rice endosperm cells were proposed. The first one is the VSD-dependent pathway, in this route the protein would be sorted into protein body II; The other pathway is VSD-independent pathway, protein synthesized in ER would be sorted into the ER derived protein body I. The last trafficking route could be independent of the storage vacuole pathway. In this route, endogenous or recombinant proteins are sorted into the intracellular space in storage organs in plant cell. This trafficking route has frequently been observed in transgenic endosperm cells overexpressing recombinant proteins7, In the rice endosperm cells highly expressing PDI-IGF, the subcellular structure changed greatly, which was caused by the ER stress.8, By the iTRAQ proteomics analysis to the total protein of immature rice endosperm cells,11significant different proteins were found in highly expression line, including six up-regulated and five down-regulated proteins; In the middle expression level line, there were12significant different proteins, while eight proteins were up-regulated, four proteins were down-regulated; And18proteins were significantly changed in the line with lowest expression level, with five proteins up-regulated and13down-regulated. 9, For the membrane proteins of immature rice endosperm cells,20proteins were significantly changed in the highly expression line, with11proteins up-regulated and nine down-regulated; In the middle expression line,18proteins were significantly changed, with eight proteins up-regulated and10down-regulated; And in the lowest expression line, the significant different protein, up-regulated and down-regulated protein are23, six and17, respectively.10, By investigating the transgenic rice expressing the recombinant proteins, we found the high level expressing protein would greatly affect the subcellular organelle structure, while low expressing protein hardly have effect in rice endosperm cells. Besides, the exogenous proteins lead to slight changes to the proteome, just by changing a few chaperones and translation factors. This result can be an evidence for safety genetic modified cereal for biopharming.更多还原