本文關(guān)鍵詞：OsNOA1調(diào)控葉綠體蛋白合成的機理研究　出處：《華南農(nóng)業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： 水稻 低溫 NO NOA1

【摘要】：一氧化氮(NO)是一種重要的信號物質(zhì)。在動物體中,NO已被證明與多種代謝途徑有關(guān):包括神經(jīng)傳遞、免疫以及血管平滑肌的松弛過程等。在植物體中,NO也被證明參與到呼吸作用、光形態(tài)建成、種子萌發(fā)、根葉的生長發(fā)育、氣孔運動的調(diào)節(jié)、衰老等多種生理過程中。動物主要通過L-精氨酸依賴的NOS途徑來合成體內(nèi)的NO。而關(guān)于植物體中NO合成途徑的研究目前還存在諸多爭議,但研究者們普遍接受在植物體中普遍存在兩種互不關(guān)聯(lián)的NO合成途徑:即硝酸/亞硝酸還原酶依賴的NO合成途徑以及類似于動物的以L-精氨酸為底物的一氧化氮合酶(NOS)依賴的NO合成途徑。2003年,Guo等報道了擬南芥中的一氧化氮合酶—AtNOS1。然而,后續(xù)的多項相關(guān)研究都表明AtNOS1并不具有NOS活性,它只是間接影響了擬南芥細胞內(nèi)部NO的含量。At NOS1也由此被重新命名為AtNOA1,意為一氧化氮合成相關(guān)蛋白1。盡管NOA1并不具有NO合酶的活性,但NOA1的缺失突變體仍然因為具有較低的NO水平而存在研究價值。本研究室的前期研究發(fā)現(xiàn):OsNOA1的干涉植株在22℃條件下生長時表現(xiàn)出黃化表型;而在30℃條件下生長時,干涉植株與野生型水稻卻無明顯表型差異�；谝陨蟽牲c,本文以干涉植株(OsNOA1-RNAi)與超表達植株(OsNOA1-Ox)為材料研究了OsNOA1轉(zhuǎn)基因水稻低溫敏感表型的內(nèi)在機理及低溫敏感表型與轉(zhuǎn)基因植株中NO水平的關(guān)系。主要研究結(jié)果如下:1.干涉與超表達OsNOA1轉(zhuǎn)基因水稻的比較分析通過表型觀察、葉綠素與Rubisco含量測定等實驗,我們發(fā)現(xiàn)Os NOA1超表達株系表現(xiàn)出與干涉植株相類似的低溫敏感黃化表型。并且在低溫條件下,超表達植株的葉綠素與Rubisco含量隨著OsNOA1表達量的提高而逐漸降低。葉綠素與Rubisco代謝途徑相關(guān)基因的定量RT-PCR分析結(jié)果表明:低溫處理條件下,干涉植株與OsNOA1上調(diào)較多的超表達株系(OsNOA1-Ox line22;OsNOA1-Ox line45)中葉綠素與蛋白的合成代謝都受到了不同程度的影響,而在常溫條件下轉(zhuǎn)基因植株中這些代謝途徑受到的影響不明顯。通過溫度轉(zhuǎn)換實驗證明,OsNOA1對葉綠體蛋白的合成起到了調(diào)控作用。定量蛋白質(zhì)組學分析結(jié)果表明,干涉植株與超表達植株中顯著差異蛋白的生物學過程極為相似,進一步說明顯著下調(diào)或者上調(diào)OsNOA1對水稻蛋白合成機制所造成的影響是類似的。對蛋白質(zhì)表達譜進行的GO分析也表明OsNOA1與葉綠體蛋白質(zhì)的合成密切相關(guān)。2.OsNOA1轉(zhuǎn)基因植株的低溫敏感表型與NO的關(guān)系在低溫條件下(22℃),向木村B培養(yǎng)基中添加外源NO供體SNP并不能挽救OsNOA1干涉植株的黃化表型,以Rubisco大小亞基為代表的葉綠體蛋白在SNP處理前后也無明顯變化。因此,OsNOA1轉(zhuǎn)基因植株的低溫敏感表型與NO含量之間并無直接聯(lián)系。半定量RT-PCR鑒定到OsNOA1干涉植株中積累了大量的葉綠體16S rRNA前體,表明OsNOA1與葉綠體30S核糖體小亞基的功能相關(guān),葉綠體核糖體功能受損可能是造成OsNOA1轉(zhuǎn)基因植株低溫敏感表型的直接原因。3.OsNOA1互作蛋白的分辨使用STRING蛋白互作數(shù)據(jù)庫預測了OsNOA1的互作蛋白。結(jié)果表明:核糖體蛋白很可能是OsNOA1的互作對象。本文利用多種方法篩查了OsNOA1的互作蛋白,并通過體外His-tag pull down實驗成功篩選到了與OsNOA1互作的靶蛋白,其中包含葉綠體30S核糖體蛋白S2等多種葉綠體核糖體蛋白。上述結(jié)果表明OsNOA1的功能確實與葉綠體核糖體相關(guān)。4.葉綠體逆行信號在OsNOA1調(diào)控葉綠體蛋白合成過程中的作用通過熒光HPLC測定了低溫下生長的OsNOA1干涉植株與野生型水稻中鎂原卟啉IX(Mg-Proto IX)的含量。結(jié)果表明:干涉植株中的Mg-Proto IX大量積累。因此,OsNOA1有可能通過Mg-Proto IX介導的葉綠體逆行信號通路來調(diào)控細胞核編碼的葉綠體基因的表達。同時,通過基于Cre-loxP重組體系的多基因干涉系統(tǒng),構(gòu)建并轉(zhuǎn)化了OsNOA1與OsGUN1的雙基因干涉載體,獲得了OsNOA1與OsGUN1表達量同時下調(diào)的雙基因干涉水稻植株。但我們發(fā)現(xiàn)下調(diào)OsGUN1的表達量并不能挽救OsNOA1表達量下調(diào)所導致的低溫敏感表型,意味著OsNOA1不是通過OsGUN1介導的逆行信號通路來實現(xiàn)對核編碼的葉綠體基因的調(diào)控。綜合上述結(jié)果,表明OsNOA1可能通過影響葉綠體核糖體的功能實現(xiàn)對葉綠體自編碼蛋白的直接調(diào)控,繼而通過鎂原卟啉IX介導的葉綠體逆行信號通路將信號傳至細胞核,進一步影響到核基因編碼的葉綠體蛋白的表達。其調(diào)控過程與植物體內(nèi)的NO含量無關(guān)。
[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.

【學位授予單位】：華南農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q943.2

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相關(guān)期刊論文 前2條

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本文編號：1408399