水稻低氮胁迫和细菌侵染应答基因OsSPL11和OsRP-1的功能研究

发布时间：2018-01-15 23:31

  本文关键词：水稻低氮胁迫和细菌侵染应答基因OsSPL11和OsRP-1的功能研究　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

  更多相关文章： OsSPL11 低氮胁迫 生长发育 OsRP-1 OsAHP2 蛋白互作 水稻黄单胞菌抗病性

【摘要】：氮素是植物正常生长发育过程中所必需的矿质元素。在低氮胁迫下,植物进化出一套微妙的调节机制来减轻低氮胁迫所带来的伤害。之前的研究已表明,水稻编码泛素E3连接酶的OsSPL11(Spotted leaf 11,Spl11)(LOC_Os12g38210)受低氮胁迫的调控,而OsSPL11也是植物防御信号和细胞死亡的负调控因子。为了鉴定不同氮素条件下OsSPL11在水稻生长发育过程中的功能,spill突变体及其过表达植株分别在不同含氮量的液体培养液中培养。研究结果显示OsSPL11在水稻整个植株中都能被诱导表达；与正常氮条件下相比,低氮胁迫强烈诱导了OsSPL11在OsSPL11过表达植株根部的表达；与野生型相比,splll突变体的根密度在低氮条件下降低,而根和茎的生物量在低氮和正常氮处理下均下降。本研究中,我们证明了OsSPL11调控低氮胁迫诱导的叶片衰老。与野生型水稻相比,低氮胁迫下的splll突变体植株出现早期衰老。此外,在低氮胁迫下,早期衰老影响了splll突变体植株根的发育及对氮素的吸收。与野生型相比,在低氮胁迫下OsNRT1.1,OsNRT1.2, OsNRT2.1和OsNAR2.1在spill突变体植株根中的表达降低,而在OsSPL11-OX植株中的表达增强。同时,低氮胁迫下,典型衰老相关基因OsSGR,OsNAP和OsNYC1在splll突变体植株中显著上调表达,这一结果表明OsSPL11延缓了水稻的衰老。splll突变体的病斑表型在低氮胁迫下受到抑制,同时防御和细胞死亡相关的基因表达降低。以上结果表明OsSPL11作为正调控因子参与了水稻对低氮胁迫的适应过程。水稻白叶枯病是由黄单胞菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的世界范围内最具毁灭性的病害之一。前期研究发现Xoo侵染诱导了水稻的抗性基因OsRP-1 (LOC_Os08g42700)下调表达。本研究显示OsRP-1预测编码蛋白含有CC-NB-LRR结构域蛋白,该结构域在N端具有重复的激酶1a,2和3a单元。系统进化树显示OsRP-1与稻瘟病菌(Magnaporthe oryzae)中抗性相关蛋白Pib(LOC-Os08g47010)同源,也和乌拉尔图小麦(Triticum urartu)中的RPM1非常相近。我们之前的研究发现水稻在接种Xoo6小时后,OsRP-1下调表达。时空表达分析显示OsRP-1在叶片和茎上的表达水平要高于根部,且在叶片上的表达水平最高。OsRP-1在空白对照和Xoo接种下均下调表达。为了研究OsRP-1与其下游信号分子的互作,我们利用酵母双杂交方法,一共筛选得到137个潜在的互作蛋白。利用GO注释分类分析,137个假定蛋白被分入32类生物过程,19类分子功能组分和20类细胞组分。为了得到阳性的互作分子,我们采用变换分析法排除假阳性互作蛋白,并最终得到11个蛋白,其中4个蛋白OsAHP2(LOC_Os09g39400),OsPsaD (LOC_Os08g44680),OsMYB(LOC_Os09g12750)和OsAAA-ATPase(LOC_Os11g47970)在酵母双杂交体系中能与OsRP-1互作。利用GST pull-down方法,验证了一个假定的组氨酸磷酸转移蛋白OsAHP2与OsRP-1的互作。同时,我们采用荧光素酶互补图像技术确定了OsRP-1和OsAHP2在植物体内的互作,该相互作用较弱且是非特异性的。进一步的GST pull-down结果表明OsRP-1的N端结构域与OsAHP2发生了互作。为了研究OsAHP2在抵抗水稻白叶枯病菌中的作用,对1月苗龄的OsAHPs-RNAi水稻植株接种黄单胞菌,结果显示OsAHPs-RNAi水稻植株的病斑长度明显短于野生型水稻,这表明OsAHPs负调控了水稻对Xoo抗性。此外,防卫相关基因OsPR1a, OsPRlb和OsPBZ1在接种后6h被强烈诱导表达。以上结果表明OsRP-1与OsAHP2的互作调控了水稻抗白叶枯病菌的信号途径。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
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本文编号：1430591