本文關(guān)鍵詞：水稻低氮脅迫和細(xì)菌侵染應(yīng)答基因OsSPL11和OsRP-1的功能研究　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： OsSPL11 低氮脅迫 生長(zhǎng)發(fā)育 OsRP-1 OsAHP2 蛋白互作 水稻黃單胞菌抗病性

【摘要】：氮素是植物正常生長(zhǎng)發(fā)育過程中所必需的礦質(zhì)元素。在低氮脅迫下,植物進(jìn)化出一套微妙的調(diào)節(jié)機(jī)制來減輕低氮脅迫所帶來的傷害。之前的研究已表明,水稻編碼泛素E3連接酶的OsSPL11(Spotted leaf 11,Spl11)(LOC_Os12g38210)受低氮脅迫的調(diào)控,而OsSPL11也是植物防御信號(hào)和細(xì)胞死亡的負(fù)調(diào)控因子。為了鑒定不同氮素條件下OsSPL11在水稻生長(zhǎng)發(fā)育過程中的功能,spill突變體及其過表達(dá)植株分別在不同含氮量的液體培養(yǎng)液中培養(yǎng)。研究結(jié)果顯示OsSPL11在水稻整個(gè)植株中都能被誘導(dǎo)表達(dá)；與正常氮條件下相比,低氮脅迫強(qiáng)烈誘導(dǎo)了OsSPL11在OsSPL11過表達(dá)植株根部的表達(dá)；與野生型相比,splll突變體的根密度在低氮條件下降低,而根和莖的生物量在低氮和正常氮處理下均下降。本研究中,我們證明了OsSPL11調(diào)控低氮脅迫誘導(dǎo)的葉片衰老。與野生型水稻相比,低氮脅迫下的splll突變體植株出現(xiàn)早期衰老。此外,在低氮脅迫下,早期衰老影響了splll突變體植株根的發(fā)育及對(duì)氮素的吸收。與野生型相比,在低氮脅迫下OsNRT1.1,OsNRT1.2, OsNRT2.1和OsNAR2.1在spill突變體植株根中的表達(dá)降低,而在OsSPL11-OX植株中的表達(dá)增強(qiáng)。同時(shí),低氮脅迫下,典型衰老相關(guān)基因OsSGR,OsNAP和OsNYC1在splll突變體植株中顯著上調(diào)表達(dá),這一結(jié)果表明OsSPL11延緩了水稻的衰老。splll突變體的病斑表型在低氮脅迫下受到抑制,同時(shí)防御和細(xì)胞死亡相關(guān)的基因表達(dá)降低。以上結(jié)果表明OsSPL11作為正調(diào)控因子參與了水稻對(duì)低氮脅迫的適應(yīng)過程。水稻白葉枯病是由黃單胞菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的世界范圍內(nèi)最具毀滅性的病害之一。前期研究發(fā)現(xiàn)Xoo侵染誘導(dǎo)了水稻的抗性基因OsRP-1 (LOC_Os08g42700)下調(diào)表達(dá)。本研究顯示OsRP-1預(yù)測(cè)編碼蛋白含有CC-NB-LRR結(jié)構(gòu)域蛋白,該結(jié)構(gòu)域在N端具有重復(fù)的激酶1a,2和3a單元。系統(tǒng)進(jìn)化樹顯示OsRP-1與稻瘟病菌(Magnaporthe oryzae)中抗性相關(guān)蛋白Pib(LOC-Os08g47010)同源,也和烏拉爾圖小麥(Triticum urartu)中的RPM1非常相近。我們之前的研究發(fā)現(xiàn)水稻在接種Xoo6小時(shí)后,OsRP-1下調(diào)表達(dá)。時(shí)空表達(dá)分析顯示OsRP-1在葉片和莖上的表達(dá)水平要高于根部,且在葉片上的表達(dá)水平最高。OsRP-1在空白對(duì)照和Xoo接種下均下調(diào)表達(dá)。為了研究OsRP-1與其下游信號(hào)分子的互作,我們利用酵母雙雜交方法,一共篩選得到137個(gè)潛在的互作蛋白。利用GO注釋分類分析,137個(gè)假定蛋白被分入32類生物過程,19類分子功能組分和20類細(xì)胞組分。為了得到陽性的互作分子,我們采用變換分析法排除假陽性互作蛋白,并最終得到11個(gè)蛋白,其中4個(gè)蛋白OsAHP2(LOC_Os09g39400),OsPsaD (LOC_Os08g44680),OsMYB(LOC_Os09g12750)和OsAAA-ATPase(LOC_Os11g47970)在酵母雙雜交體系中能與OsRP-1互作。利用GST pull-down方法,驗(yàn)證了一個(gè)假定的組氨酸磷酸轉(zhuǎn)移蛋白OsAHP2與OsRP-1的互作。同時(shí),我們采用熒光素酶互補(bǔ)圖像技術(shù)確定了OsRP-1和OsAHP2在植物體內(nèi)的互作,該相互作用較弱且是非特異性的。進(jìn)一步的GST pull-down結(jié)果表明OsRP-1的N端結(jié)構(gòu)域與OsAHP2發(fā)生了互作。為了研究OsAHP2在抵抗水稻白葉枯病菌中的作用,對(duì)1月苗齡的OsAHPs-RNAi水稻植株接種黃單胞菌,結(jié)果顯示OsAHPs-RNAi水稻植株的病斑長(zhǎng)度明顯短于野生型水稻,這表明OsAHPs負(fù)調(diào)控了水稻對(duì)Xoo抗性。此外,防衛(wèi)相關(guān)基因OsPR1a, OsPRlb和OsPBZ1在接種后6h被強(qiáng)烈誘導(dǎo)表達(dá)。以上結(jié)果表明OsRP-1與OsAHP2的互作調(diào)控了水稻抗白葉枯病菌的信號(hào)途徑。
[Abstract]:Nitrogen is the normal growth of plant mineral elements required in the process. In the under low nitrogen stress, plants have evolved a subtle regulatory mechanism to reduce the damage caused by low nitrogen stress. Previous studies have shown that rice encoding ubiquitin E3 ligase OsSPL11 (Spotted leaf 11, Spl11) (LOC_Os12g38210) regulated by low nitrogen stress, while OsSPL11 is a negative regulator of plant defense signaling and cell death. In order to process identification of different nitrogen OsSPL11 in rice growth and development function of spill mutants and overexpressing plants respectively in different nitrogen content in liquid culture medium. The results showed that OsSPL11 could be induced to express in the whole rice plant; compared with normal nitrogen under low nitrogen stress induced OsSPL11 expression strongly in OsSPL11 overexpressing plants roots; compared with the wild type, splll mutant root Density under low nitrogen condition decreased, while the biomass of root and stem were decreased under low nitrogen and normal nitrogen treatment. In this study, we demonstrate that OsSPL11 regulates leaf senescence induced by low nitrogen stress. Compared with the wild type rice, splll mutant plants under low nitrogen stress in the early age. In addition, under the low nitrogen stress, early aging effects of the splll mutant plant root growth and nitrogen uptake. Compared with the wild type under low nitrogen stress in OsNRT1.1, OsNRT1.2, OsNRT2.1 and OsNAR2.1 expression decreased in spill mutant plants in the roots, and the expression in OsSPL11-OX plants enhanced. At the same time, under the low nitrogen stress typically, senescence associated genes OsSGR, OsNAP and OsNYC1 expression was significantly up-regulated in splll mutant plants, the results showed that OsSPL11 postponing aging of the.Splll mutant phenotype of the lesion was inhibited by low nitrogen stress and defense And cell death related gene expression decreased. These results suggest that OsSPL11 is a positive regulatory factor involved in the process of adaptation to low nitrogen stress of rice. Rice Bacterial Blight by Xanthomonas (Xanthomonas oryzae, pv.oryzae, Xoo) caused a worldwide one of the most destructive diseases. Previous study found that resistance gene OsRP-1 Xoo infection induced rice (LOC_Os08g42700) expression. This study shows that the prediction of OsRP-1 protein encoding a protein containing CC-NB-LRR domain, the domain with repeat kinase 1A on the N side, and 2 3A units. The phylogenetic tree showed that OsRP-1 and Magnaporthe grisea (Magnaporthe oryzae) in resistance related protein Pib (LOC-Os08g47010) homologous, and Ural wheat (Triticum Urartu) in RPM1 is very similar. Our previous study found that rice in Xoo6 hours after inoculation, the down-regulation of OsRP-1 expression. The spatial and temporal expression analysis Shows the expression level of OsRP-1 in leaves and stems than in roots, and leaves on the highest expression level of.OsRP-1 in blank control and Xoo inoculation were down regulated. In order to research on the interaction between OsRP-1 and its downstream signal molecules, we use yeast two hybrid screening method, a total of 137 to a potential protein interaction. Classification analysis using the GO annotation, 137 putative proteins are divided into 32 kinds of biological process, 19 kinds of molecular components and 20 kinds of cell components. In order to get a positive interaction between molecules, we analysis method is adopted to eliminate false positive interaction protein transformation, and finally got 11 protein, 4 protein OsAHP2 (LOC_Os09g39400), OsPsaD (LOC_Os08g44680), OsMYB (LOC_Os09g12750) and OsAAA-ATPase (LOC_Os11g47970) could interact with OsRP-1 in yeast two hybrid system. Using GST pull-down method, verified a putative histidine phosphate transfer protein The interactions between OsAHP2 and OsRP-1. At the same time, we used luciferase complementary imaging technology to determine the OsRP-1 and OsAHP2 in the interaction of plants, the interaction is weak and nonspecific. Further GST pull-down results showed that OsRP-1 N terminal domain of OsAHP2 interacts with OsAHP2. In order to study the resistance in rice leaf the role of blight, seedling rice plants inoculated with OsAHPs-RNAi January Xanthomonas, showed that rice plants OsAHPs-RNAi lesion length was significantly shorter than the wild type rice, suggesting that OsAHPs negatively regulates the rice resistance to Xoo. Moreover, defense related genes OsPR1a, OsPRlb and OsPBZ1 in 6h after inoculation was strongly induced expression. The above results suggest that OsRP-1 and OsAHP2 interaction regulation signal pathway of rice resistance to bacterial blight.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.111.47
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本文編號(hào)：1430591