擬南芥AtNHX5和AtNHX6調(diào)節(jié)種子貯藏蛋白運(yùn)輸?shù)难芯?/H1>
發(fā)布時(shí)間：2018-05-14 16:50
本文選題：擬南芥 + Na~+ ； 參考：《蘭州大學(xué)》2016年博士論文
【摘要】：Na+,K+/H+反向轉(zhuǎn)運(yùn)體(NHX)是H+偶聯(lián)的反向轉(zhuǎn)運(yùn)蛋白,其生化活性是催化Na+,K+/H+跨膜反向轉(zhuǎn)運(yùn)。植物NHX在維持細(xì)胞離子平衡、調(diào)節(jié)p H、膜融合、滲透調(diào)節(jié)、逆境響應(yīng)、蛋白運(yùn)輸及植物生長(zhǎng)發(fā)育等過(guò)程中起著重要的作用。擬南芥NHX家族包括8個(gè)成員,根據(jù)亞細(xì)胞定位可以分為三類(lèi):質(zhì)膜NHX(At NHX7/SOS1和At NHX8),液胞NHX(At NHX1-At NHX4)和內(nèi)膜NHX(endosomal NHX)(At NHX5和At NHX6)。目前對(duì)于質(zhì)膜NHX和液胞NHX的研究較為深入,關(guān)于內(nèi)膜NHX的研究才逐漸展開(kāi)。At NHX5和At NHX6是定位于內(nèi)膜體的NHX,二者氨基酸序列高度相似。At NHX5和At NHX6分布于Golgi、TGN和PVC。研究發(fā)現(xiàn),At NHX5和At NHX6能夠調(diào)節(jié)鉀/鈉離子轉(zhuǎn)運(yùn),維持細(xì)胞p H平衡,調(diào)控蛋白運(yùn)輸。然而關(guān)于At NHX5和At NHX6調(diào)節(jié)蛋白運(yùn)輸?shù)姆肿訖C(jī)理尚不清楚。我們利用分子遺傳學(xué)、細(xì)胞生物學(xué)和分子生物學(xué)技術(shù)方法,觀察了擬南芥nhx5 nhx6雙突變體植株和種子的生長(zhǎng)發(fā)育表型,分析了種子貯藏蛋白向蛋白質(zhì)貯藏液胞(protein storage vacuoles,PSVs)的運(yùn)輸過(guò)程。我們進(jìn)一步觀察了nhx5nhx6 syp22三突變體的生長(zhǎng)表型,分析了SNARE復(fù)合體的亞細(xì)胞定位以及At NHX5和At NHX6與SNARE復(fù)合體的相互作用。我們的研究目的是闡明At NHX5和At NHX6調(diào)節(jié)蛋白運(yùn)輸?shù)姆肿訖C(jī)理。本研究取得以下成果:(1)我們首先制備nhx5 nhx6雙突變體并觀察其生長(zhǎng)發(fā)育表型。我們發(fā)現(xiàn)nhx5nhx6不僅植株矮小、發(fā)育受阻,而且角果小、種子產(chǎn)量低,表明At NHX5和At NHX6不僅調(diào)節(jié)著植株的生長(zhǎng)發(fā)育也調(diào)節(jié)著種子的生長(zhǎng)和發(fā)育。(2)觀察人工貯藏蛋白GFP-CT24向PSV的運(yùn)輸,發(fā)現(xiàn)GFP-CT24在nhx5nhx6中存在于細(xì)胞間隙,不能被運(yùn)輸?shù)絇SV中。SDS-PAGE和免疫印跡分析發(fā)現(xiàn),nhx5 nhx6存在種子貯藏蛋白12S球蛋白和2S白蛋白的前體蛋白。免疫透射電鏡顯示,nhx5 nhx6成熟種子的細(xì)胞間隙聚集12S球蛋白前體蛋白。同時(shí)發(fā)現(xiàn),在nhx5 nhx6中,液胞特異性標(biāo)記蛋白Spo:GFP不能被運(yùn)輸?shù)揭喊�。表明At NHX5和At NHX6調(diào)節(jié)著蛋白質(zhì)向液胞的運(yùn)輸過(guò)程。利用激光共聚焦顯微鏡觀察擬南芥成熟種子的PSV形態(tài)及大小,發(fā)現(xiàn)nhx5 nhx6 PSV數(shù)目增多但體積減小,表明At NHX5和At NHX6調(diào)控?cái)M南芥種子PSV的形成。(3)At NHX5和At NHX6的跨膜區(qū)存在四個(gè)保守的氨基酸殘基,分別為At NHX5的D164,E188,D193,E320和At NHX6的D165,E189,D194,E320。將以上氨基酸殘基位點(diǎn)突變后,轉(zhuǎn)化nhx5 nhx6,經(jīng)SDS-PAGE及免疫印跡分析發(fā)現(xiàn),At NHX5的D164,E188,D193及At NHX6的D165,E189,D194氨基酸殘基位點(diǎn)影響種子貯藏蛋白的運(yùn)輸,表明At NHX5和At NHX6通過(guò)其離子轉(zhuǎn)運(yùn)活性調(diào)節(jié)著種子貯藏蛋白的運(yùn)輸過(guò)程。(4)通過(guò)雜交獲得nhx5 nhx6 syp22三突變體,發(fā)現(xiàn)nhx5 nhx6 syp22表現(xiàn)出比親本植株更為嚴(yán)重的生長(zhǎng)缺陷表型,不僅植株矮化,蓮座葉小,抽薹開(kāi)花延遲,并且角果短小,種子產(chǎn)量低,種子增大,種子敗育。表明At NHX5、At NHX6和At SYP22對(duì)植株和角果的生長(zhǎng)發(fā)育起著共同的調(diào)節(jié)作用。利用激光共聚焦顯微鏡觀察nhx5 nhx6 syp22 PSV形態(tài)及大小,發(fā)現(xiàn)nhx5 nhx6syp22種子細(xì)胞PSV體積變小但數(shù)目增多,表明At NHX5,At NHX6和At SYP22可能共同調(diào)控著擬南芥種子細(xì)胞PSV的形成。SDS-PAGE及免疫印跡分析發(fā)現(xiàn),nhx5 nhx6 syp22存在大量的種子貯藏蛋白12S球蛋白和2S白蛋白的前體蛋白,表明nhx5 nhx6 syp22種子貯藏蛋白運(yùn)輸路徑受阻。(5)擬南芥原生質(zhì)體瞬時(shí)表達(dá)實(shí)驗(yàn)發(fā)現(xiàn),在nhx5 nhx6中,SNARE蛋白At VAMP727和At SYP22在PVC的亞細(xì)胞定位受到抑制;At VAMP727和At SYP22被滯留在Golgi和TGN中,表明At NHX5和At NHX6調(diào)節(jié)著SNARE復(fù)合體的亞細(xì)胞定位。擬南芥原生質(zhì)體瞬時(shí)表達(dá)實(shí)驗(yàn)發(fā)現(xiàn),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)并未檢測(cè)出At NHX5或At NHX6與SNARE蛋白SYP22、VTI11或SYP51有相互作用,表明At NHX5或At NHX6與SNARE復(fù)合體無(wú)直接的相互作用�？傊�,我們的研究表明,At NHX5和At NHX6調(diào)節(jié)種子貯藏蛋白的運(yùn)輸。At NHX5和At NHX6通過(guò)調(diào)節(jié)SNARE復(fù)合體的亞細(xì)胞定位而調(diào)控著種子貯藏蛋白向PSV的運(yùn)輸。At NHX5和At NHX6通過(guò)其離子轉(zhuǎn)運(yùn)活性調(diào)節(jié)著蛋白質(zhì)的運(yùn)輸過(guò)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943
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本文選題：擬南芥 + Na~+ ； 參考：《蘭州大學(xué)》2016年博士論文
【摘要】：Na+,K+/H+反向轉(zhuǎn)運(yùn)體(NHX)是H+偶聯(lián)的反向轉(zhuǎn)運(yùn)蛋白,其生化活性是催化Na+,K+/H+跨膜反向轉(zhuǎn)運(yùn)。植物NHX在維持細(xì)胞離子平衡、調(diào)節(jié)p H、膜融合、滲透調(diào)節(jié)、逆境響應(yīng)、蛋白運(yùn)輸及植物生長(zhǎng)發(fā)育等過(guò)程中起著重要的作用。擬南芥NHX家族包括8個(gè)成員,根據(jù)亞細(xì)胞定位可以分為三類(lèi):質(zhì)膜NHX(At NHX7/SOS1和At NHX8),液胞NHX(At NHX1-At NHX4)和內(nèi)膜NHX(endosomal NHX)(At NHX5和At NHX6)。目前對(duì)于質(zhì)膜NHX和液胞NHX的研究較為深入,關(guān)于內(nèi)膜NHX的研究才逐漸展開(kāi)。At NHX5和At NHX6是定位于內(nèi)膜體的NHX,二者氨基酸序列高度相似。At NHX5和At NHX6分布于Golgi、TGN和PVC。研究發(fā)現(xiàn),At NHX5和At NHX6能夠調(diào)節(jié)鉀/鈉離子轉(zhuǎn)運(yùn),維持細(xì)胞p H平衡,調(diào)控蛋白運(yùn)輸。然而關(guān)于At NHX5和At NHX6調(diào)節(jié)蛋白運(yùn)輸?shù)姆肿訖C(jī)理尚不清楚。我們利用分子遺傳學(xué)、細(xì)胞生物學(xué)和分子生物學(xué)技術(shù)方法,觀察了擬南芥nhx5 nhx6雙突變體植株和種子的生長(zhǎng)發(fā)育表型,分析了種子貯藏蛋白向蛋白質(zhì)貯藏液胞(protein storage vacuoles,PSVs)的運(yùn)輸過(guò)程。我們進(jìn)一步觀察了nhx5nhx6 syp22三突變體的生長(zhǎng)表型,分析了SNARE復(fù)合體的亞細(xì)胞定位以及At NHX5和At NHX6與SNARE復(fù)合體的相互作用。我們的研究目的是闡明At NHX5和At NHX6調(diào)節(jié)蛋白運(yùn)輸?shù)姆肿訖C(jī)理。本研究取得以下成果:(1)我們首先制備nhx5 nhx6雙突變體并觀察其生長(zhǎng)發(fā)育表型。我們發(fā)現(xiàn)nhx5nhx6不僅植株矮小、發(fā)育受阻,而且角果小、種子產(chǎn)量低,表明At NHX5和At NHX6不僅調(diào)節(jié)著植株的生長(zhǎng)發(fā)育也調(diào)節(jié)著種子的生長(zhǎng)和發(fā)育。(2)觀察人工貯藏蛋白GFP-CT24向PSV的運(yùn)輸,發(fā)現(xiàn)GFP-CT24在nhx5nhx6中存在于細(xì)胞間隙,不能被運(yùn)輸?shù)絇SV中。SDS-PAGE和免疫印跡分析發(fā)現(xiàn),nhx5 nhx6存在種子貯藏蛋白12S球蛋白和2S白蛋白的前體蛋白。免疫透射電鏡顯示,nhx5 nhx6成熟種子的細(xì)胞間隙聚集12S球蛋白前體蛋白。同時(shí)發(fā)現(xiàn),在nhx5 nhx6中,液胞特異性標(biāo)記蛋白Spo:GFP不能被運(yùn)輸?shù)揭喊�。表明At NHX5和At NHX6調(diào)節(jié)著蛋白質(zhì)向液胞的運(yùn)輸過(guò)程。利用激光共聚焦顯微鏡觀察擬南芥成熟種子的PSV形態(tài)及大小,發(fā)現(xiàn)nhx5 nhx6 PSV數(shù)目增多但體積減小,表明At NHX5和At NHX6調(diào)控?cái)M南芥種子PSV的形成。(3)At NHX5和At NHX6的跨膜區(qū)存在四個(gè)保守的氨基酸殘基,分別為At NHX5的D164,E188,D193,E320和At NHX6的D165,E189,D194,E320。將以上氨基酸殘基位點(diǎn)突變后,轉(zhuǎn)化nhx5 nhx6,經(jīng)SDS-PAGE及免疫印跡分析發(fā)現(xiàn),At NHX5的D164,E188,D193及At NHX6的D165,E189,D194氨基酸殘基位點(diǎn)影響種子貯藏蛋白的運(yùn)輸,表明At NHX5和At NHX6通過(guò)其離子轉(zhuǎn)運(yùn)活性調(diào)節(jié)著種子貯藏蛋白的運(yùn)輸過(guò)程。(4)通過(guò)雜交獲得nhx5 nhx6 syp22三突變體,發(fā)現(xiàn)nhx5 nhx6 syp22表現(xiàn)出比親本植株更為嚴(yán)重的生長(zhǎng)缺陷表型,不僅植株矮化,蓮座葉小,抽薹開(kāi)花延遲,并且角果短小,種子產(chǎn)量低,種子增大,種子敗育。表明At NHX5、At NHX6和At SYP22對(duì)植株和角果的生長(zhǎng)發(fā)育起著共同的調(diào)節(jié)作用。利用激光共聚焦顯微鏡觀察nhx5 nhx6 syp22 PSV形態(tài)及大小,發(fā)現(xiàn)nhx5 nhx6syp22種子細(xì)胞PSV體積變小但數(shù)目增多,表明At NHX5,At NHX6和At SYP22可能共同調(diào)控著擬南芥種子細(xì)胞PSV的形成。SDS-PAGE及免疫印跡分析發(fā)現(xiàn),nhx5 nhx6 syp22存在大量的種子貯藏蛋白12S球蛋白和2S白蛋白的前體蛋白,表明nhx5 nhx6 syp22種子貯藏蛋白運(yùn)輸路徑受阻。(5)擬南芥原生質(zhì)體瞬時(shí)表達(dá)實(shí)驗(yàn)發(fā)現(xiàn),在nhx5 nhx6中,SNARE蛋白At VAMP727和At SYP22在PVC的亞細(xì)胞定位受到抑制;At VAMP727和At SYP22被滯留在Golgi和TGN中,表明At NHX5和At NHX6調(diào)節(jié)著SNARE復(fù)合體的亞細(xì)胞定位。擬南芥原生質(zhì)體瞬時(shí)表達(dá)實(shí)驗(yàn)發(fā)現(xiàn),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)并未檢測(cè)出At NHX5或At NHX6與SNARE蛋白SYP22、VTI11或SYP51有相互作用,表明At NHX5或At NHX6與SNARE復(fù)合體無(wú)直接的相互作用�？傊�,我們的研究表明,At NHX5和At NHX6調(diào)節(jié)種子貯藏蛋白的運(yùn)輸。At NHX5和At NHX6通過(guò)調(diào)節(jié)SNARE復(fù)合體的亞細(xì)胞定位而調(diào)控著種子貯藏蛋白向PSV的運(yùn)輸。At NHX5和At NHX6通過(guò)其離子轉(zhuǎn)運(yùn)活性調(diào)節(jié)著蛋白質(zhì)的運(yùn)輸過(guò)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q943
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