植物组蛋白脱甲基酶IBM1在木质素合成途径中的功能及其表观遗传机制研究

Lignin has negative effect on utilizing poplars as the resource plant in the paper making and cellulosic biofuel production. Regulating the lignin ingredient and content of poplars through gene engineering to get new transgenic poplars plays important roles in environment protection and economy. Most of the key enzymes in the lignin biogenesis pathway are identified and chracterazed in plant.Also, people regnized that,the expression of these eyzymes could be trascriptionally regulated by some TFs,as members of MYB. However, whether the epigentic mechanism involved in the regulation of lignin biogensis is still not clear. INCREASE IN BONSAI METHYLATION 1(IBM1) is a conserved JmjC family histone demethylase in Arabidopsis. By high-throughput sequencing, we found that loss of IBM1in Arabidopsis lead to obviously decrease of CAD1 expression, as well as reductions in CCR, F5H, COMT, CCoAOMT. It suggests the involvement of IBM1 with lignin ingredient and content in plant. For validating the functional relation between IBM1 and lignin, a series of experiments are designed and processed to analyze lignin ingredient and content and the expression levels of marker genes, in both of IBM1 loss-of-function mutant and overexpresstion lines. At the same time, the details of epgenetic regulation of lignin biosythesis will be descriped by Biochemical and molecular ways, like ChIP, Bisulphite Sequencing and IP. By blast of amino acid sequence, 6 of IBM1 homologues (also call as PtoIBMs), are identified in pupulus genome. The JmjC domain at C-terminal of these protein suggest their conserved function in polars. The overall objective of this proposal is to investigate bio-functions of PtoIBMs in biosynthesis of lignin in Populus.To accomplish this reaserch, the transgenic plants are constructed for overexpression of PtoIBMs. On another hand, the RNAi specific to PtoIBMs also constructed and transformed in plants. Finally,we will figure out the molecular mechenism of epigentic regulation in lignin biosythesis pathway.

木质素是植物体中主要的化学成分，如何有选择性地调控植物中木质素的含量及组分，对于更好地开发利用资源植物具有十分重要的意义。但植物体内木质素合成的调控机制，尤其是表观遗传学调控目前尚不清楚。我们在前期研究中发现，组蛋白脱甲基化酶IBM1在拟南芥中影响木质素合成途径上关键酶基因的表达，本项目拟获得IBM1突变体和超表达转基因植株，分别测定其木质素含量和组分；通过ChIP方法检测这些基因染色质上的组蛋白修饰水平，以Bisulphite 测序分析关键酶基因的DNA甲基化水平，利用IP技术和转基因技术验证IBM1蛋白在靶基因染色质上的结合，解析IBM1调控木质素合成的表观遗传机制；进一步克隆杨树中同源基因PtoIBMs，解剖学观察转基因植株次生壁，同时检测木质素含量及组分，分析木质素合成关键酶基因的表达，搞清PtoIBMs的具体功能，最终阐明PtoIBMs在杨树木质素合成途径中的表观遗传调控机制。

IBM1是拟南芥JmjC家族组蛋白脱甲基化酶，可特异去除H3K9位点的甲基化修饰。我们研究发现，IBM1缺失导致植物莲座叶中花青素积累和相关基因的显著上调，且响应强光的照射。分析发现植物细胞内光信号的转导受到IBM1的影响，其中SPA家族因子被IBM1所抑制。ChIP实验发现4个SPA基因的染色质的H3K9me2水平在ibm1突变体中被上调，H3K4me3水平被下调；同时DNA的CHG甲基化水平也被上调。我们进一步证明了IBM1可以直接结合在SPA基因的染色质上，并通过调控SPA的转录水平来影响花青素的合成和植物发育中对光信号的响应。我们的研究揭示了表观遗传机制在花青素合成中的作用和分子机制。