miR840a与WHIRLY3-PPRs介导的RNA剪辑调控植物衰老机理的研究

Plant senescence is controlled by genes and affected by internal and external environmental factors or signals including delivered from organelle, and it plays an important role in the whole life cycle of plants. Understanding its regulatory mechanism of plant senescence initiation and processes has important biological significance and theoretical and application value on increasing crop biomass, improving crop agronomic traits. Our previous results showed that miR840a is a natural antisense miRNA (nat-miRNA), which was expressed from the opposite strand of its predicted target gene, WHIRLY3 (WHY3), and within the annotated 3´ untranslated region (3´UTR) of a PPR (PPRs) mRNA. MiR840a is developmentally expressed at the onset of plant senescence, its overexpression or knockdown results in a opposite leaf senescent phenotype as compared to the WT plants. Using oemiR840a and kdmiR840a transgenic plants, the results of quantitative RT-PCR and immunodetection reveal that miR840a repress PPRs transcription by targeting and cleaving mRNA or inhibit WHY3 protein accumulation by targeting 3’UTR and interference its translation, but does not synergistically change WHY3 transcription, indicating that miR840a independently affect PPRs at transcriptional level and WHY3 at translational level. However, single mutation neither of pprs nor why3 could result in leaf senescent phenotype; and the double mutations of pprs why3 partly achieved senescence phenotype. The expression of senescence related genes that are differentially expressed in oemiR840a transcriptome show mostly consistent expression in the pprs why3 double mutants. It demonstrates that miR840a affected both targeted genes WHY3 and PPRs independently, which both synergistically co-regulated leaf senescence events. Based on existing knowledge, WHY3 functions as a cofactor of WHY1 for double strand break (DSBs) related DNA damage repair in plastids, for RNA processing, and for DNA binding protein of kenesin gene in the nucleus; The typical PPR protein have high consensus function domain, is targeted to mitochondria or chloroplasts in plants, binds one or several organelle transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. The PPRs is an E subclass of PPR protein, is targeted to chloroplast. Most PPR proteins have functions in one of the four processes, RNA editing, splicing, maturation, and translation activation. All the editing functions reported so far are associated with either PPR-DYW or PPR-E type proteins. Based on this and our previous research, this project is intended to use the identified miR840a, why3, pprs homozygous single mutant, double mutant and triple mutants in Arabidopsis, by comprehensively using CHIP-seq, bulk cDNA sequencing, RNA-CoIP-seq, MALDI-MS, CoIP, BIFC, Y2H etc techniques and bioinformatics methods, it is explored that : (1) how miR840a targets, cleavages, inhibits or modifies target genes both WHY3 and PPRs transcription, translation, or expression; (2) how WHY3，PPRs and both effect on downstream targeted RNA processing including RNA recognized site, RNA editing, splicing, maturation, and translation activation, (3) how the plastid target genes of both miR840a and WHIRLYY3-PPRs mediated RNA processing affect the incidence of plant aging. It will illustrate the function and genetic interaction of miR840a with two anti-orientation target genes WHY3 and PPRs, reveal the regulatory mechanism of miR840a and WHY3-PPRs mediated RNA splicing and editing on plant senescence, and demonstrate the action of plastid gene RNA editing on the nuclear events of plant aging.

已表明miR840a结合在双反向靶基因WHIRLY3和PPRs的3’UTR上，过表达和敲除miR840a促进和延缓拟南芥叶片衰老和改变衰老相关基因的表达，双突变whirly3 pprs具有和过表达miR840a相同的早衰表型。基于此，本项目利用拟南芥miR840a/WHIRLY3/PPRs的单、双和三敲除和过表达纯合突变体，综合CHIP-seq、bulk cDNA sequencing、RIP-seq和MALDI-MS等技术，探索miR840a如何剪切、抑制或修饰WHIRLY3和PPRs的转录、翻译，WHIRLY3和PPRs及其互作如何对靶基因的转录本进行剪辑，miR840a和WHY3-PPRs RNA剪辑的靶基因如何调控植物衰老的发生；阐明miR840a对靶基因WHIRY3和PPRs的作用机制，揭示miR840a与WHIRLY3-PPRs共同介导的靶基因的RNA剪辑调控植物衰老的分子机理

植物细胞衰老是植物衰老的具体体现，以往的研究大多应用芯片技术系统分析与鉴定和植物衰老有关的基因，以及验证与衰老相关转录因子家族在调控衰老中的作用，也包括激素与环境因子影响信号传导的 细胞死亡的 研究， 近几年对植物衰老的分子机理， 随着分子遗传学的发展 开始 从植物个体细胞精细水平入手，在转录与表观遗传包括组蛋白修饰和小 RNA 等的调控上有了初略的报道，但对更深层的分子调控 还远不清楚。本项目发现一类natmiRNA miR840能参与植物衰老的调控，利用拟南芥miR840/WHIRLY3/PPR的单、双和三敲除和过表达纯合突变体解析了miR840以非经典的位点模式剪切和抑制PPR和WHIRLY3的转录和翻译，阐明miR840对靶基因WHIRY3和PPR的作用机制，揭示miR840a与WHIRLY3-PPR共同介导的靶基因的RNA剪辑调控植物衰老的分子机理。通过水稻的WHIRLY蛋白CRISPR/Cas9编辑突变体，阐明WHIRLY1 蛋白通过改变细胞内的ROS水平和自身在不同细胞器的分布调控靶基因的转录本的剪辑，进而调控植物白化和叶片衰老的发生；同时解析了质体编辑核心蛋白MORF9的生物学功能及其对糖信号的响应作用。尤其是单链DNA/RNA结合蛋白WHIRLY家族的剪切和编辑功能在叶绿体和线粒体基因组稳定性和作为逆行信号调控植物叶片衰老、叶绿体降解、果荚发育以及根生长中的作用机理的深化，在细胞生物学，分子遗传学和植物生长发育领域具有重要生物学意义，为调控水稻叶片衰老和产量、品质和对环境胁迫响应都具有重要的应用前景。

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