OsNOA1调控叶绿体蛋白合成的机理研究

发布时间：2018-01-11 06:10

本文关键词：OsNOA1调控叶绿体蛋白合成的机理研究　出处：《华南农业大学》2016年博士论文　论文类型：学位论文

【摘要】：一氧化氮(NO)是一种重要的信号物质。在动物体中,NO已被证明与多种代谢途径有关:包括神经传递、免疫以及血管平滑肌的松弛过程等。在植物体中,NO也被证明参与到呼吸作用、光形态建成、种子萌发、根叶的生长发育、气孔运动的调节、衰老等多种生理过程中。动物主要通过L-精氨酸依赖的NOS途径来合成体内的NO。而关于植物体中NO合成途径的研究目前还存在诸多争议,但研究者们普遍接受在植物体中普遍存在两种互不关联的NO合成途径:即硝酸/亚硝酸还原酶依赖的NO合成途径以及类似于动物的以L-精氨酸为底物的一氧化氮合酶(NOS)依赖的NO合成途径。2003年,Guo等报道了拟南芥中的一氧化氮合酶—AtNOS1。然而,后续的多项相关研究都表明AtNOS1并不具有NOS活性,它只是间接影响了拟南芥细胞内部NO的含量。At NOS1也由此被重新命名为AtNOA1,意为一氧化氮合成相关蛋白1。尽管NOA1并不具有NO合酶的活性,但NOA1的缺失突变体仍然因为具有较低的NO水平而存在研究价值。本研究室的前期研究发现:OsNOA1的干涉植株在22℃条件下生长时表现出黄化表型;而在30℃条件下生长时,干涉植株与野生型水稻却无明显表型差异。基于以上两点,本文以干涉植株(OsNOA1-RNAi)与超表达植株(OsNOA1-Ox)为材料研究了OsNOA1转基因水稻低温敏感表型的内在机理及低温敏感表型与转基因植株中NO水平的关系。主要研究结果如下:1.干涉与超表达OsNOA1转基因水稻的比较分析通过表型观察、叶绿素与Rubisco含量测定等实验,我们发现Os NOA1超表达株系表现出与干涉植株相类似的低温敏感黄化表型。并且在低温条件下,超表达植株的叶绿素与Rubisco含量随着OsNOA1表达量的提高而逐渐降低。叶绿素与Rubisco代谢途径相关基因的定量RT-PCR分析结果表明:低温处理条件下,干涉植株与OsNOA1上调较多的超表达株系(OsNOA1-Ox line22;OsNOA1-Ox line45)中叶绿素与蛋白的合成代谢都受到了不同程度的影响,而在常温条件下转基因植株中这些代谢途径受到的影响不明显。通过温度转换实验证明,OsNOA1对叶绿体蛋白的合成起到了调控作用。定量蛋白质组学分析结果表明,干涉植株与超表达植株中显著差异蛋白的生物学过程极为相似,进一步说明显著下调或者上调OsNOA1对水稻蛋白合成机制所造成的影响是类似的。对蛋白质表达谱进行的GO分析也表明OsNOA1与叶绿体蛋白质的合成密切相关。2.OsNOA1转基因植株的低温敏感表型与NO的关系在低温条件下(22℃),向木村B培养基中添加外源NO供体SNP并不能挽救OsNOA1干涉植株的黄化表型,以Rubisco大小亚基为代表的叶绿体蛋白在SNP处理前后也无明显变化。因此,OsNOA1转基因植株的低温敏感表型与NO含量之间并无直接联系。半定量RT-PCR鉴定到OsNOA1干涉植株中积累了大量的叶绿体16S rRNA前体,表明OsNOA1与叶绿体30S核糖体小亚基的功能相关,叶绿体核糖体功能受损可能是造成OsNOA1转基因植株低温敏感表型的直接原因。3.OsNOA1互作蛋白的分辨使用STRING蛋白互作数据库预测了OsNOA1的互作蛋白。结果表明:核糖体蛋白很可能是OsNOA1的互作对象。本文利用多种方法筛查了OsNOA1的互作蛋白,并通过体外His-tag pull down实验成功筛选到了与OsNOA1互作的靶蛋白,其中包含叶绿体30S核糖体蛋白S2等多种叶绿体核糖体蛋白。上述结果表明OsNOA1的功能确实与叶绿体核糖体相关。4.叶绿体逆行信号在OsNOA1调控叶绿体蛋白合成过程中的作用通过荧光HPLC测定了低温下生长的OsNOA1干涉植株与野生型水稻中镁原卟啉IX(Mg-Proto IX)的含量。结果表明:干涉植株中的Mg-Proto IX大量积累。因此,OsNOA1有可能通过Mg-Proto IX介导的叶绿体逆行信号通路来调控细胞核编码的叶绿体基因的表达。同时,通过基于Cre-loxP重组体系的多基因干涉系统,构建并转化了OsNOA1与OsGUN1的双基因干涉载体,获得了OsNOA1与OsGUN1表达量同时下调的双基因干涉水稻植株。但我们发现下调OsGUN1的表达量并不能挽救OsNOA1表达量下调所导致的低温敏感表型,意味着OsNOA1不是通过OsGUN1介导的逆行信号通路来实现对核编码的叶绿体基因的调控。综合上述结果,表明OsNOA1可能通过影响叶绿体核糖体的功能实现对叶绿体自编码蛋白的直接调控,继而通过镁原卟啉IX介导的叶绿体逆行信号通路将信号传至细胞核,进一步影响到核基因编码的叶绿体蛋白的表达。其调控过程与植物体内的NO含量无关。
[Abstract]:Nitric oxide (NO) is a kind of important signal substances. In animal body, NO has been shown to be associated with a variety of metabolic pathways including neurotransmission, immunity and vascular smooth muscle relaxation. In plants, NO has also been shown to participate in respiration, photomorphogenesis, seed germination, root and leaf. The growth and development, regulation of stomatal movement, the physiological process of aging animal. Mainly through the NOS pathway of L- arginine dependent synthesis of NO. in vivo and Research on plant NO synthesis pathway is still controversial, but the researchers generally accepted generally there are two kinds of unrelated NO synthesis way in plants: nitrate / NO pathway dependent nitrite reductase and similar to the animal with L- arginine as a substrate of nitric oxide synthase (NOS) on the NO synthesis pathway.2003, Guo reported that a quasi oxygen in Arabidopsis NO synthase AtNOS1. however, on a number of related follow-up showed that AtNOS1 does not have NOS activity, it is only indirectly affect the Arabidopsis cell internal NO content of.At NOS1 was renamed to AtNOA1 for nitric oxide synthesis related protein 1., although NOA1 does not have the activity of NO synthase, but deletion mutant NOA1 because of still having lower NO levels and research value. In our previous study found that interference plant growth of OsNOA1 under the condition of 22 DEG C showed chlorosis phenotype; and growth under the condition of 30 DEG C, interference plants and wild rice had no obvious phenotypic differences. Based on the above two points. In this paper, interference plants (OsNOA1-RNAi) and the over expression lines (OsNOA1-Ox) on the level of NO and the intrinsic mechanism of transgenic plants and low temperature sensitive phenotype of OsNOA1 transgenic rice low temperature sensitive phenotype in turn for materials . the main results are as follows: 1. interference and overexpression of OsNOA1 transgenic rice by comparing phenotypic observation, chlorophyll and Rubisco content determination experiments, we found that Os NOA1 overexpression lines exhibited low temperature sensitive phenotype similar to plant chlorosis interference. And under the condition of low temperature, chlorophyll content of plants with overexpression of Rubisco with the expression of OsNOA1 was increased and gradually decreased. The results of quantitative RT-PCR analysis of chlorophyll related metabolic pathways of Rubisco gene showed that the low temperature condition, interference plants with the upregulation of OsNOA1 more overexpression lines (OsNOA1-Ox line22; OsNOA1-Ox line45) in the synthesis and metabolism of chlorophyll and protein are affected by different degrees, and affected by these metabolic pathways in transgenic plants under normal temperature conditions in the not obvious. Through the temperature conversion experiment proved that OsNOA1 of chloroplast protein Synthesis plays a role in regulation. Quantitative proteomic analysis results show that the interference of plants and biological processes over expression significant difference in plant protein is very similar, further significantly reduced or influence caused by up regulation of OsNOA1 on the synthesis mechanism of rice protein is similar. Also shows that the temperature sensitive phenotype and NO OsNOA1 and chloroplast protein synthesis of.2.OsNOA1 is closely related to the relationship between the transgenic plants under low temperature conditions on GO spectrum analysis of protein expression (22 C), medium supplemented with exogenous NO donor SNP and OsNOA1 interference can save the disease phenotype to Kimura B culture, chloroplast protein size in Rubisco subunit is represented in the SNP before and after treatment and no obvious changes. Therefore, OsNOA1 transgenic plants cold sensitive phenotype and NO content is not directly linked. Semi quantitative RT-PCR to identify OsNOA1 interference accumulation in plants A large number of chloroplast 16S rRNA precursor, OsNOA1 and 30S showed that the chloroplast small subunit ribosome function, chloroplast ribosomal dysfunction may be the direct cause of.3.OsNOA1 OsNOA1 transgenic plants of low temperature sensitive phenotype of the protein interaction resolution interaction database predicted protein interaction with the use of OsNOA1 STRING protein. The results showed that the ribosomal protein is may be the interaction of object OsNOA1. The screening of OsNOA1 interacting proteins using a variety of methods, and through the His-tag pull down in vitro experiments successfully screened the target protein interaction with OsNOA1, which contains the chloroplast 30S ribosomal protein S2 and other chloroplast ribosomal protein. The results showed that the role of the function of OsNOA1 is positively correlated with chloroplast ribosomes.4. chloroplast retrograde signals in OsNOA1 regulation of chloroplast protein synthesis process by fluorescence determination of HPLC under low temperature life Long OsNOA1 interference plants and wild type rice in magnesium protoporphyrin IX (Mg-Proto IX) content. The results show that the interference in Mg-Proto IX plants accumulated. Therefore, the expression of chloroplast genes of chloroplast OsNOA1 signaling pathway may be retrograde through the Mg-Proto IX mediated regulation of nucleus encoding. At the same time, through the interference system multi gene Cre-loxP recombinant system based on double OsNOA1 gene was constructed and transformed with OsGUN1 vector to obtain OsNOA1 and OsGUN1 double gene expression was cut at the same time interference in rice plants. But we found that the down-regulation of OsGUN1 expression and OsNOA1 expression of cold sensitive phenotype can save the reduction caused by means of retrograde signaling OsNOA1 is not mediated by OsGUN1 to realize the control of the nuclear gene encoding the chloroplast. The results show that, OsNOA1 may influence the chloroplast ribosomes. Realize the function of direct regulation of chloroplast protein encoding from the chloroplast retrograde signaling pathway by magnesium protoporphyrin IX mediated signal will be transmitted to the nucleus, further affect the expression of nuclear genes encoding chloroplast proteins. The independent regulation process and plant NO content.

【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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