水稻OsJmjC714调控生长素合成基因表达的分子基础

In plant, JMJC proteins play key roles in regulating the growth and development process as well as stress response through affecting histone demethylation and gDNA demethylation. Our previous research evidenced that JMJC protein JMJ714 affectd histone demethylation of H3K9 in in vitro, and in in vivo of the transgenic rice overexpressing JMJ714. Moreover, JMJ714 affected the lateral root initiation and sensitivity to high salt in transgenic rice, while the application of auxin recovered the effect on root elongation, implying that JMJ714 is involved in mediating auxin biosynthesis, subsequently influencing the initiation of lateral root and salt response. However, the molecular mechanism of JMJ714 in the regulation of auxin biosynthesis is unclear. In the present project, we plan to conduct the following reseearch. Through analyzing the relationship of lateral root initiation with auxin between different rice materials, and investigating the expression of JMJ714 in response to hormones and in different tisssues and developmental stages, and the effects of JMJ714 on histone demethylation at different Lys sites, we will reveal the regulatory relationship of JMJ714 on histone demethylation of JMJ714 and the expression of auxin synthesis genes, combined with the whole genome based RNA-Sequencing transcriptional regulation of JMJ714 among various JMJ714 trnasgenic rice. Based on the results of RNA-Sequencing, we will identify numerous key genes targeted by JMJ714 through qRT-PCR, and focus on the further identify the transcriptional modulation of JMJ714 in auxin biosynthesis genes using ChIP assay. Our aim is to elucidate how JMJ714 protein regulates the expression of key genes involved in auxin biosynthesis through affecting the demethylation of histone in rice. The complition of the research will provide the evidence that JMJC protein mediates the modification histone to affect the expression of auxin biosynthesis-related genes, consequently resulting in the initiation of lateral root.

JMJC蛋白通过影响组蛋白脱甲基化和DNA甲基化修饰调节植物生长发育及抗逆性反应。我们前期研究发现水稻JMJC蛋白JMJ714具有组蛋白H3K9脱甲基化酶活性，参与组蛋白脱甲基化修饰；转JMJ714基因水稻抑制侧根发生和高盐敏感性，生长素处理恢复这种抑制效应,表明JMJ714可能通过影响组蛋白脱甲基化调控生长素合成进而调控水稻侧根发生和耐盐性，但不清楚JMJ714如何调控控生长素合成。本项目拟在比较JMJ714超表达和基因沉默水稻侧根发生与生长素效应的相关性、分析该基因的时空与响应激素的表达特性以及探讨其对组蛋白不同赖氨酸位点的脱甲基化修饰，结合转录组测序系统分析转基因水稻中全基因组基因表达的差异，剖析JMJ714对生长素合成基因的表达调控；并进一步运用ChIP鉴定JMJ714对生长素合成基因表达的调控功能，以期阐明JMJC蛋白通过调控组蛋白脱甲基化影响生长素合成基因表达的分子基础。

组蛋白赖氨酸的甲基化是一个动态的、可逆的过程，组蛋白赖氨酸甲基转移酶和组蛋白去甲基化酶协同调节该过程的动态平衡。组蛋白去甲基化酶分成两大类，一类是以FAD为辅因子的特异性赖氨酸去甲基化酶1，另一类是催化组蛋白去甲基化的JmjC蛋白。水稻基因组中至少有20个编码JMJC蛋白的基因，我们前期研究证明，JMJ714蛋白能催化H3K9me1/2/3的去甲基化，并且影响侧根的发。本研究对该基因的基本特性、生理功能及其分子作用基础进行了探讨。. 我们首先通过荧光定量PCR、GUS染色和JMJ714-GFP融合表达的方法分析了JMJ714的诱导表达特性、组织表达模式及其蛋白的亚细胞定位。实验结果表明，JMJ714受干旱和20% PEG诱导表达；在盐、ABA、6-BA和NAA诱导条件下，JMJ714的表达水平下调。而且该基因在水稻不同发育阶段和组织器官中表现出不同程度的表达水平，尤其在生命活动旺盛的幼嫩组织部位表达水平较高；洋葱内表皮瞬时表达证明JMJ714主要定位在细胞核中。. 我们证实JMJ714对根系发育的影响主要体现在侧根和冠根的长度及数量，胚根的的形态及向地性方面。JMJ714过表达材料表现出侧根和冠根变短并且数量减少的现象，同时胚根的向地性还受到了影响；JMJ714干扰材料则表现出侧根和冠根变长并且数量增多的相反趋势，胚根发生卷曲。另外，我们还发现过表达JMJ714会降低根系对a-NAA的敏感性，干扰JMJ714的表达会提高根系对a-NAA的敏感性。目前的研究表明，生长素在调控冠根和侧根的形成过程起主导作用，生长素水平过高往往导致胚根卷曲，胚根向地性的改变通常与生长素合成或代谢等方面的异常相关。因此，我们推测 JMJ714可能影响水稻根系中生长素的含量，从而调控根系的发育。. 我们测量了JMJ714转基因材料中生长素的含量，并通过qPCR检测了生长素代谢相关基因的表达，结果表明，JMJ714正调控生长素代谢相关基因GH3的表达，并降低了根系中生长素的含量，干扰JMJ714的表达使根系中生长素含量明显升高。综上所述，我们证明了JMJ714通过正调控生长素代谢相关基因的表达，促进生长素的代谢作用，降低了水稻根系中生长素含量，从而影响水稻根系形态建成。