组蛋白乙酰化调控应拉木中木质素单体基因表达的调控机制

Lignin, one of the major component of wood, makes the wood recalcitrant to the process for ethanol production. Tension wood is a specialized wood developed in angiosperm tree stems or branches to counteract mechanical stress, and has lower lignin contents. The knowledge of the mechanism controlling tension wood formation is still unknown. In this proposed project, we will use Populus trichocarpa as a model system to study the regulation of lignin biosynthesis during tension wood formation. Using RNA-seq and ChIP-seq, we identified the expression of two genes, PtrC4H2 and PtrHCT1, involved in monolignol biosynthesis pathway are regulated by the histone acetylation marker, H3K9ac. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) control the level of histone acetylation through the protein-protein interaction with transcription factors (TFs). We will construct a TF library containing all xylem preferentially expressed TFs for Y1H to screen for the TF directly regulating PtrC4H2 and PtrHCT1. To verify the regulation of these TFs, we will use our recently developed SDX protoplasts system to test the direct binding of these TFs to PtrC4H2 and PtrHCT1 promoters and their transactivation ability. Comparing to the protein sequence of HAT and HDAC in Arabidopsis, we identified total 44 orthologs in P. trichocarpa, and these genes will be used for Y2H to identify the protein-protein interaction between HAT/HDAC and the verified TFs. The regulatory mechanism of PtrC4H2 and PtrHCT1 will be revealed by the transgenics overexpressing the TFs identified from both Y1H and Y2H. This research will provide accurate information for gene modification to improve wood property for low lignin contents to increase the efficiency of the ethanol production.

木质素是木材中的主要成份之一， 但木质素使木材难以被转化成为酒精。 应拉木有较低的木质素含量， 主产生于开花木本植物的树干或是枝条在受到物理压力时。 然而应拉木形成的机制目前尚不清楚。 本研究将以木本模式植物毛果杨进行应拉木形成过程中木质素合成机制的研究。我们将会建立一个木质部专一表达转录因子文库， 并且使用酵母单杂交技術篩選出与木质素单体合成基因启动子直接结合的转录因子。 并且使用木质部原生质体系统验证这些转录因子是否能直接结合且调控木质素单体合成基因。本团队鉴定出木质素单体合成基因(C4H2和HCT1)在应拉木的形成过程中受到组蛋白乙酰化修饰 的调控，并且在毛果杨中鉴定出44个拟南芥組蛋白乙酰轉移酶 和组蛋白去乙酰化酶的同源基因， 我们将使用酵母双杂交筛选与这44个基因互作的转录因子并验证功能。本研究將指导遗传改良木材提高使酒精的生产效率。

木材是地球上最丰富的陆地生物量，是用材和纸浆及其他生物质化学品生产的基本原料。与普通木材相比，应拉木产生的木材纤维素含量增加、木质素含量降低，提高了生物燃料生产的纸浆产量和酶糖化。在本项目书中，我们以应拉木形成过程中，基因表达和乙酰化修饰均下调的木质素单体合成酶基因出发，通过酵母单杂交技术由下向上筛选，得到在应拉木形成过程中直接结合并抑制木质素单体合成酶基因的关键转录因子PtrbHLH81和PtrbZIP44。通过酵母双杂交和BiFC技术筛选得到与PtrbHLH81和PtrbZIP44相互作用的组蛋白去乙酰化酶PtrHDA15。利用原生质体系统对PtrbHLH81/ PtrHDA15和PtrbZIP44/ PtrHDA15复合体，对木质素单体合成酶基因的调控作用进行分析。同时，我们以应拉木形成过程中差异表达最显著的转录因子出发，由上向下挑选机械压力处理下表达量差异最显著的两个转录因子PtrMYB092和PtrHSFB3-1，通过正向遗传学和反向遗传学分析显示，植物正常生长情况下，PtrHSFB3-1和PtrMYB092直接结合并激活木质素单体合成酶基因的表达。当植物受到机械压力后，PtrHSFB3-1和PtrMYB092的表达受到抑制，从而抑制了下游木质素单体合成酶基因的表达，导致木质素含量下降，改变了木材的组分。以上研究揭示了应拉木形成过程中，转录因子直接调控毛果杨木质素生物合成，为林木分子育种角度提供了新思路。