拟南芥AtNHX5和AtNHX6调节种子贮藏蛋白运输的研究

发布时间：2018-05-14 16:50

本文选题：拟南芥 + Na~+　；参考：《兰州大学》2016年博士论文

【摘要】：Na+,K+/H+反向转运体(NHX)是H+偶联的反向转运蛋白,其生化活性是催化Na+,K+/H+跨膜反向转运。植物NHX在维持细胞离子平衡、调节p H、膜融合、渗透调节、逆境响应、蛋白运输及植物生长发育等过程中起着重要的作用。拟南芥NHX家族包括8个成员,根据亚细胞定位可以分为三类:质膜NHX(At NHX7/SOS1和At NHX8),液胞NHX(At NHX1-At NHX4)和内膜NHX(endosomal NHX)(At NHX5和At NHX6)。目前对于质膜NHX和液胞NHX的研究较为深入,关于内膜NHX的研究才逐渐展开。At NHX5和At NHX6是定位于内膜体的NHX,二者氨基酸序列高度相似。At NHX5和At NHX6分布于Golgi、TGN和PVC。研究发现,At NHX5和At NHX6能够调节钾/钠离子转运,维持细胞p H平衡,调控蛋白运输。然而关于At NHX5和At NHX6调节蛋白运输的分子机理尚不清楚。我们利用分子遗传学、细胞生物学和分子生物学技术方法,观察了拟南芥nhx5 nhx6双突变体植株和种子的生长发育表型,分析了种子贮藏蛋白向蛋白质贮藏液胞(protein storage vacuoles,PSVs)的运输过程。我们进一步观察了nhx5nhx6 syp22三突变体的生长表型,分析了SNARE复合体的亚细胞定位以及At NHX5和At NHX6与SNARE复合体的相互作用。我们的研究目的是阐明At NHX5和At NHX6调节蛋白运输的分子机理。本研究取得以下成果:(1)我们首先制备nhx5 nhx6双突变体并观察其生长发育表型。我们发现nhx5nhx6不仅植株矮小、发育受阻,而且角果小、种子产量低,表明At NHX5和At NHX6不仅调节着植株的生长发育也调节着种子的生长和发育。(2)观察人工贮藏蛋白GFP-CT24向PSV的运输,发现GFP-CT24在nhx5nhx6中存在于细胞间隙,不能被运输到PSV中。SDS-PAGE和免疫印迹分析发现,nhx5 nhx6存在种子贮藏蛋白12S球蛋白和2S白蛋白的前体蛋白。免疫透射电镜显示,nhx5 nhx6成熟种子的细胞间隙聚集12S球蛋白前体蛋白。同时发现,在nhx5 nhx6中,液胞特异性标记蛋白Spo:GFP不能被运输到液胞中。表明At NHX5和At NHX6调节着蛋白质向液胞的运输过程。利用激光共聚焦显微镜观察拟南芥成熟种子的PSV形态及大小,发现nhx5 nhx6 PSV数目增多但体积减小,表明At NHX5和At NHX6调控拟南芥种子PSV的形成。(3)At NHX5和At NHX6的跨膜区存在四个保守的氨基酸残基,分别为At NHX5的D164,E188,D193,E320和At NHX6的D165,E189,D194,E320。将以上氨基酸残基位点突变后,转化nhx5 nhx6,经SDS-PAGE及免疫印迹分析发现,At NHX5的D164,E188,D193及At NHX6的D165,E189,D194氨基酸残基位点影响种子贮藏蛋白的运输,表明At NHX5和At NHX6通过其离子转运活性调节着种子贮藏蛋白的运输过程。(4)通过杂交获得nhx5 nhx6 syp22三突变体,发现nhx5 nhx6 syp22表现出比亲本植株更为严重的生长缺陷表型,不仅植株矮化,莲座叶小,抽薹开花延迟,并且角果短小,种子产量低,种子增大,种子败育。表明At NHX5、At NHX6和At SYP22对植株和角果的生长发育起着共同的调节作用。利用激光共聚焦显微镜观察nhx5 nhx6 syp22 PSV形态及大小,发现nhx5 nhx6syp22种子细胞PSV体积变小但数目增多,表明At NHX5,At NHX6和At SYP22可能共同调控着拟南芥种子细胞PSV的形成。SDS-PAGE及免疫印迹分析发现,nhx5 nhx6 syp22存在大量的种子贮藏蛋白12S球蛋白和2S白蛋白的前体蛋白,表明nhx5 nhx6 syp22种子贮藏蛋白运输路径受阻。(5)拟南芥原生质体瞬时表达实验发现,在nhx5 nhx6中,SNARE蛋白At VAMP727和At SYP22在PVC的亚细胞定位受到抑制;At VAMP727和At SYP22被滞留在Golgi和TGN中,表明At NHX5和At NHX6调节着SNARE复合体的亚细胞定位。拟南芥原生质体瞬时表达实验发现,At NHX5或At NHX6和At SYP22和At VAMP727共定位于PVC之中;但是免疫共沉淀(Co-IP)并未检测出At NHX5或At NHX6与SNARE蛋白SYP22、VTI11或SYP51有相互作用,表明At NHX5或At NHX6与SNARE复合体无直接的相互作用。总之,我们的研究表明,At NHX5和At NHX6调节种子贮藏蛋白的运输。At NHX5和At NHX6通过调节SNARE复合体的亚细胞定位而调控着种子贮藏蛋白向PSV的运输。At NHX5和At NHX6通过其离子转运活性调节着蛋白质的运输过程。
[Abstract]:Na+, K+/H+ reverse transporter (NHX) is a H+ coupled reverse transporter, whose biochemical activity is catalyzed by Na+ and K+/H+ transmembrane reverse transport. Plant NHX plays an important role in maintaining cell ion balance, regulating P H, membrane fusion, osmotic regulation, adversity response, protein transport and plant growth. The Arabidopsis NHX family consists of 8 members, Subcellular localization can be divided into three types: plasma membrane NHX (At NHX7/SOS1 and At NHX8), NHX (At NHX1-At NHX4) and intima NHX (endosomal NHX). The highly similar sequence of acid sequences.At NHX5 and At NHX6 distributed in Golgi, TGN and PVC. studies found that At NHX5 and At NHX6 can regulate the transport of potassium / sodium ions, maintain cell p balance and regulate protein transport. However, the molecular mechanism of the transport of proteins is not clear. The growth and development phenotype of nhx5 nhx6 double mutant plants and seeds of Arabidopsis thaliana were observed. The transport process of seed storage protein to protein storage vacuoles (PSVs) was analyzed. We further observed the growth phenotype of the nhx5nhx6 syp22 three mutant and analyzed the subcell of the SNARE complex. The interaction between At NHX5 and At NHX6 and SNARE complex. Our aim is to elucidate the molecular mechanism of At NHX5 and At NHX6 regulated protein transport. The following results are obtained: (1) we first prepared nhx5 nhx6 double mutants and observed their growth phenotypes. We found that nhx5nhx6 is not only small, but also hindered, and the development is hindered. At NHX5 and At NHX6 not only regulate the growth and development of the plants, but also regulate the growth and development of the seeds. (2) observe the transport of the artificial storage protein GFP-CT24 to PSV, and find that GFP-CT24 exists in the space of nhx5nhx6 in nhx5nhx6, and can not be transported to PSV in.SDS-PAGE and Western blot analysis, and nhx5 nhx6 exists. The precursor protein of seed storage protein 12S globulin and 2S albumin. Immunological transmission electron microscopy showed that the intercellular space of nhx5 nhx6 mature seeds aggregated 12S globulin precursor protein. At the same time, in nhx5 nhx6, the cell specific marker protein Spo:GFP could not be transported to the cell. It showed that At NHX5 and At NHX6 regulate the transport of protein to the cell. The PSV morphology and size of the mature seeds of Arabidopsis thaliana were observed by laser confocal microscopy, and the number of nhx5 nhx6 PSV increased but the volume decreased, indicating that At NHX5 and At NHX6 regulate the formation of PSV in Arabidopsis seeds. (3) there are four conservative amino acid residues in the trans membrane region of At NHX5 and At. 0 and At NHX6 D165, E189, D194, E320. mutated the above amino acid residues and transformed nhx5 nhx6. It was found by SDS-PAGE and immunoblotting. The transport process of substorage protein. (4) the nhx5 nhx6 syp22 three mutant was obtained by hybridization. It was found that nhx5 nhx6 syp22 showed a more serious growth defect phenotype than the parent plant, which not only dwarfed the plants, the lotus leaves were small, the bolting and flowering were delayed, and the seeds were short, the seed yield was low, the seeds were enlarged, and the seeds were aborted. It indicated that At NHX5, At NHX6 and At S. YP22 plays a common role in regulating the growth and development of plant and cones. The morphology and size of nhx5 nhx6 syp22 PSV were observed by laser confocal microscopy. It was found that the PSV volume of nhx5 nhx6syp22 seed cells became smaller but the number increased, indicating At NHX5. Western blot analysis showed that nhx5 nhx6 syp22 had a large number of seed storage protein 12S globulin and 2S albumin precursor protein, indicating that the transport path of nhx5 nhx6 syp22 seed storage protein was blocked. (5) the transient expression of Arabidopsis protoplasts was found to be in nhx5 nhx6. Inhibition, At VAMP727 and At SYP22 were detained in Golgi and TGN, indicating that At NHX5 and At NHX6 regulate the subcellular localization of the SNARE complex. The interaction between the E protein SYP22, VTI11 or SYP51 indicates that At NHX5 or At NHX6 has no direct interaction with the SNARE complex. In conclusion, our study shows that At NHX5 and At regulators regulate the transport of seed storage proteins by regulating the subcellular localization of the seed storage proteins. X5 and At NHX6 regulate the transport process of protein through its ion transport activity.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943

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