【作者】 程彦伟；

【导师】 张炜；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 以粳稻推广品种徐稻三号为实验材料,通过两相分配法提取根尖细胞高纯度质膜,并利用IEF/SDS-PAGE双向电泳和SDS-PAGE单向电泳对盐胁迫下质膜蛋白质组进行了比较研究,结合MALDI-TOF/TOF和LC-MS/MS质谱分析,鉴定了质膜蛋白质组中的胁迫响应蛋白；然后利用实时定量PCR对鉴定的蛋白质在转录水平的变化进行了分析；在此基础上,对其中一个LRR型受体蛋白激酶N末端片段进行原核表达,制备了该蛋白的多克隆抗体,通过免疫印迹(Western blot)、免疫组化(Immunohistochemistry)、免疫电镜(Immunoelectron microscopy)和免疫共沉淀(Immunoprecipitation)对该蛋白的功能进行了分析,主要内容如下：1.盐胁迫下水稻根尖质膜蛋白质组和相关转录组研究150mM NaCl处理水稻水培根尖发现盐胁迫48h显著抑制根的生长。取盐胁迫48h水稻根尖,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度接近90%的质膜组分,IEF/SDS-PAGE双向电泳分析,发现34个蛋白点在盐处理后的表达有显著差异(>1.5倍),其中16个蛋白点上调,18个蛋白点下调。MALDI-TOF/TOF质谱鉴定表明这些差异蛋白大多数是膜相关蛋白,涉及很多重要的生理过程：(1)膜稳定性,包括参与细胞和膜骨架形成的remorin;细胞壁骨架连接的肌球蛋白重链相关蛋白；参与跨膜物质运输的α-可溶性NSF附着蛋白等。(2)离子动态平衡,包括参与了液泡膜电化学梯度和离子运输的V-ATPase E亚基和putative YLP;参与钾离子通道控制的超敏诱导蛋白；调节质膜H+-ATPase活性和离子通道(K+外排)控制的14-3-3蛋白等。(3)信号转导,例如参与Ca2+信号转导途径的钙调素和DREPP蛋白；参与环境胁迫信号转导的LRR(Leucine Rich Repeat)型受体蛋白激酶等。为了分析质膜上多次跨膜蛋白,利用高盐(0.6 M KC1)和温和去污剂(15 mM CHAPS)对纯化的质膜进行洗涤,以降低质膜组分的复杂程度,然后通过SDS-PAGE单向电泳分离和LC-MS/MS质谱分析,鉴定了多个疏水性较强的多次跨膜蛋白：如参与细胞水分调控的水通道蛋白aquaporinPIP2.2,参与膜结构稳定性维持和膜运输的annexin,参与质膜跨膜电势形成和物质运输的H+-ATPase和参与囊泡运输的Rab11C等。在此基础上,利用实时荧光定量PCR对双向电泳和SDS-PAGE分离鉴定的部分蛋白进行转录水平的研究,发现有5种蛋白转录水平和蛋白水平变化一致(putative remorin 1 protein, putative receptor proteinkinase, hypersensitive-induced response protein,14-3-3 like protein和cytochrome b5)，3种蛋白转录水平和蛋白水平变化不一致(Calmodulin, putative alpha-soluble NSF attachment protein和putative YLP).研究结果表明高等植物中基因表达存在转录水平和翻译水平的差异,推测这种差异和mRNA、蛋白质各自的修饰及稳定性有关。2.水稻LRR型受体蛋白激酶胞外区的原核表达、抗体制备和生物功能分析上述组学研究发现了一个新的水稻LRR型受体蛋白激酶OsRPK1。OsRPK1蛋白利用SMART软件分析其保守结构域,该蛋白含有一个跨膜域,N端存在信号肽,胞外区具有6个亮氨酸拉链富集区(LRR); C端含有蛋白激酶保守结构域。OsRPK1和水稻OsRLK1、OsLRK1、OsBRI1、OsXa21、拟南芥AtRPK1、玉米ZmRLK和胡萝卜DcPSKR等植物中的LRR型受体蛋白激酶序列同源性分别25.31%、22.7%、21.3%、23.07%、22.68%、20.47%和25.05%。构建系统进化树分析表明OsRPK1和玉米中ZmRLK同源关系较近。为了进一步研究该激酶的生理功能,利用DNAStar Protean软件对水稻受体激酶OsRPK1勺抗原指数分布进行了分析,选择抗原性和特异性均较高的Aa 154-350片段作为抗原,通过反转录PCR得到cDNA片段,并亚克隆至pET29a原核表达载体,在大肠杆菌BL21中实现了高表达,表达量约为细胞总蛋白的30%。重组蛋白经SDS-PAGE分离,染色切胶收集后,作为抗原免疫新西兰家兔,分离抗血清,经纯化和Western blot检测,得到1：20000效价的多克隆抗体。该抗体能特异识别在原核表达系统内表达的抗原,以及水稻根尖细胞质膜组分中的LRR型受体蛋白激酶。利用该制备抗体通过免疫电镜直接验证了OsRPK1的亚细胞定位是细胞质膜。通过western blot研究了盐、干旱、冷和ABA等非生物胁迫条件下OsRPK1的表达情况,发现盐胁迫12h OsRPK1的表达显著上调,至胁迫48h表达仍维持在较高的水平,OsRPK1免疫组化分析进一步证实了上述结果；干旱胁迫3h和12h OsRPK1的表达显著上调,48h后又恢复到正常水平；OsRPK1对冷不敏感,胁迫48h表达量只有轻微增加；ABA处理下,OsRPK1表达持续上升。研究结果表明,OsRPK1表达主要由渗透胁迫引起,因为在植物中盐胁迫和干旱胁迫比冷胁迫能更直接引起渗透胁迫；由于这三种胁迫都和ABA相关,所以外源ABA处理能快速引起OsRPK1表达。更多还原

【Abstract】 A highly purified plasma membrane fraction from rice Xudao 3 (oryza sativa) root tips was isolated by two phase partition system. Then we studied the changes of plasma membrane proteome under salt stress with IEF/SDS-PAGE two dimensional gel electrophoresis and separated the multiple span membrane strongly hydrophobic proteins with SDS-PAGE. The stress response proteins in plasma membrane were identified by MALDI-TOF/TOF and LC-MS/MS. Furthermore, the identified proteins were confirmed on mRNA level by real-time PCR analysis under salt stress. One of the identified proteins was a LRR Receptor Protein kinase. The functions of this protein were analyzed by Prokaryotic Expression of the N-terminal fragment, production of Polyclonal Antibody, western blot,immunohistochemistry, immunoelectron microscopy and immunoprecipitation in rice root tips under abiotic stress (such as salt, drought, cold and ABA treatment). The main contents of this dissertation are as followings:1. Research of plasma membrane comparative proteome and relative transcriptome in rice root tips under salt stress.The root growth of rice seedling was significantly inhibited by the 150mM NaCl stress for 48h. With or without 150 mM NaCl exposure for 48 h, a highly purified (90%) plasma membrane fraction from root tip cells of rice were isloated by two phase partition system (Dextran-PEG). Solubilized plasma membrane fractions were then analyzed by IEF/SDS-PAGE. Comparative analysis between three independent biological replicates revealed that the expressions of 34 proteins were changed after salt treatment, of which 16 were up-regulated and 18 were down-regulated, respectively. Mass spectrometry analysis indicated that most of them were membrane associated proteins which were involved in important physiological processes, (1) Membrane stabilization. It includes that remorin which possibly participate in the formation of membrane skeleton and cytoskeleton, Myosin heavy chain protein which is link protein in cell wall skeleton and promotes the synthesis of membrane callose by signal reception from cell wall, Solubleα-NSF attachment protein which possibly participate in transmembrane transport. (2) Ion homeostasis. It includes that E-subunit of V-Atpase and putative YLP which participate in the establishment of electrochemical gradient and iron transportation across the vacuolar membrane, HIR (putative hypersensitive-induced response protein) which participates in the control of K+channel,14-3-3 protein which regulates plasma membrane H+-ATPase activity and controls iron channel (e.g. K+efflux). (3) Signal transduction. It includes that CaM (Calmodulin) and DREPP which participates in Ca2+signal transduction, OsRPK1 which possibly participates in the signal transduction of environmental stress. High-salt solutions (0.6 MKC1) and gentle detergents (15 mMCHAPS) were used to wash plasma membrane proteome in order to reduce the complexity of the fraction. According to the results of the SDS-PAGE and ESI-MS/MS, many mostly hydrophobic transmembrane proteins were identified. For example, aquaporin PIP2.2 regulates water status in cell; annexin participates in transmembrane transport and maintaining the stability of membrane structure; plasma membrane ATPase participates in the establishment of electrochemical gradient across membrane; RabllC participates in vesicle transportation, etc. The research on mRNA levels for the proteins identified by the two-dimensional gel electrophoresis and SDS-PAGE was carried out by Real-time quantitative PCR. It was found that the changes of 5 proteins (putative remorin 1 protein, putative receptor protein kinase, hypersensitive-induced response protein,14-3-3 like protein, and cytochrome b5) were consistent between on the level of transcription and translation while 3 proteins (calmodulin, putative alpha-soluble NSF attachment protein, and putative YLP) were not as that. These results suggested that the changes on transcriptional level did not completely reflect the changes on translational level, which probably caused by the modification and stabilization of mRNA and protein.2.Function Analysis, Prokaryotic Expression and Polyclonal Antibody Preparation of the Extracellular Domain of Rice LRR Receptor-like Protein Kinase OsRPKl2D-PAGE study indicated a new LRR receptor-like protein kinase OsRPKl whose expression could be induced by salt stress in root tips of rice. Sequence analysis by the SMART software showed that OsRPK1 consist of three domains:signal peptide and six leucine-zipper extracellular domain, transmembrane domain, and cytoplasmic kinase domain. The Amino Acid sequence homology of OsRPK1 is 25.31%、22.7%、21.3%、23.07%、22.68%、20.47%and 25.05%with that of OsRLK1、OsLRK1、OsBRI1、OsXa21、AtRPKl、ZmRLK and DcPSKR. Phylogenetic tree of these receptor-like protein kinases shows that OsRPKl and ZmRLK have a more close relationship.In order to investigate the function of OsRPK1, we analyzed the antigenicity of OsRPKl by DNAStar Protean. The 154-350 Aa extracellular fragment of OsRPKl obtained by RT-PCR that has high antigenicity and specificity was subcloned into pET29a. The recombinant plasmid pET29a-OsRPK1 was transformed into E. coli BL21 (DE3) for over-expression in host strain. The expression level was about 30% of the total cellular proteins. After separated by SDS-PAGE, the target band excised from the gel was used as an antigen to raise the antibody in New Zealand rabbits. After separation, purification and Western blot of antiserum, we successfully prepared the anti-OsRPK1 polyclonal antibody with the titers of 1:20000. The results of western blot analysis showed that the antibody could specifically recognize the expressed fragment in E. coli and OsRPKl protein in root tips of rice. OsRPKl was proved to locate on the plasma membrane by Immunoelectron microscopy study. The expression of rice LRR-receptor protein kinase OsRPK1 were analyzed by using the obtained antibody under salt, drought, cold and ABA stress. The results showed that there was a significant increase in OsRPK1 expression after 12h of salt treatment as compared to the control, and the expression of OsRPK1 still remained a relatively high level until 48h after salt treatment, this result was confirmed by immunohistochemical analysis of the expression of the OsRPK1 in the root tips of rice seedlings under salt stress. Under drought stress, OsRPK1 expression kept a significant increase at 3h and 12h, then it returned to the control level at 48h. OsRPK1 was not sensitive to cold, which was only a slight increase after 48h of cold stress. Finally, ABA treatment induced significant increase of OsRPK1 expression. The research shows that: OsRPK1 is induced by osmotic stress, since drought and salt treatment cause osmotic stress in plants more directly than cold treatment; and OsRPK1 is involved in ABA signaling, because all three stresses are related to ABA production, and extracellular ABA induced OsRPK1 expression rapidly.更多还原