毛白杨PtoRabE GTPases 介导的高尔基-细胞壁膜运输对细胞壁沉积的影响

Wood is a product of plant cell wall polymers and wood formation is the consequences of plant cell wall deposition. The necessities of ecological values and wood supply are the two major challenges that we are facing in forest plantation. Although the application of fast-growing tree species increased the timber production to some extent, the improvement of wood quality is poorly achieved. The bottleneck of wood quality improvement is due to the unclearness of key regulatory factors and the mechanisms of these regulations during wood formation..Various plant cell wall synthesis-related enzymes and metabolites have been shown to be mediated by membrane trafficking during wood formation. Studies on model plants and animals revealed that Rab GTPases are members of the ras superfamily of regulatory GTPases and are emerging as key regulatory factors of targeting specificity in eukaryotic membrane traffic. Based on the limited previous studies, we have found that PtoRabE GTPases mediate secretion from Golgi to cell wall. Extracellular secretory protein markers secGFP and cell wall formation related protein AtCTL1-GFP are largely retained in Golgi when co-expressed with nuclleotide-free mutant version of PtoRabE1a, which means Golgi-plasma membrane secretion is at least partially blocked by the expression of PtoRabE1a(NI) mutant version. Whether PtoRabE GTPases systematically or specifically affect the deposition of plant cell wall matrix and the transport of cell wall enzymes and metabolic products remains unclear...This project is intended to use the main planted poplar species Populus tomemtosa, which already has plenty of genomic resources and experiences vigorous cell wall deposition during wood formation, as research materials to study the possible relationship between PtoRabE GTPases and cell wall deposition. The mechanism of the specificity of PtoRabE GTPases on regulated cargoes, espicially on cell wall related enzymes and products et al., will provide us a new way to systematically regulate the cell wall deposition during wood formation through PtoRabE GTPases-mediated membrane trafficking.

)：木材是植物细胞壁沉积的结果。多种与植物细胞壁合成相关的酶类和产物的运输已被证实由膜系统运输介导。模式动植物中大量研究表明Rab GTPases是调控膜系统运输的一类关键因子。前期实验发现PtoRabE GTPases介导了从高尔基到细胞壁的分泌过程，并且胞外分泌蛋白标记secGFP及细胞壁形成相关蛋白AtCTL1-GFP在与毛白杨PtoRabE1a非核苷酸结合性失活突变体共表达时，大量停留在高尔基体上，说明从高尔基到细胞膜的运输至少部分受阻。但PtoRabE GTPases是否系统地影响细胞壁沉积相关酶类和产物还有待进一步证实。本研究试图揭示PtoRabE GTPases介导的高尔基-细胞壁膜运输与细胞壁形成之间的关系，鉴定出PtoRabE GTPases所控制的运输物质种类,特别是与细胞壁沉积相关的酶类和产物，为人工系统地调控木材形成开辟一条新的途径。

植物高尔基体-细胞膜/壁的膜系统运输参与了植物细胞壁的合成。Rab GTPases是属于RAS超级家族的一大类调控GTPases，其介导的囊泡运输是迄今为止发现的从高尔基体到细胞膜的最主要的运输方式。本项目运用生物信息学方法鉴定了PtRab基因家族的成员、基因结构、染色体定位、启动子顺式作用元件，发现杨树基因组中共有67个PtRab基因，不均匀的分布在杨树19条染色体上。对其基因结构的分析发现不同亚家族间外显子/内含子数目存在明显差异，而同一亚家族中的成员基因结构基本相同。利用基因芯片数据对杨树PtRab在不同组织中的表达模式分析，发现大部分PtRabE基因在木质部和韧皮部中高表达。为了进一步研究PtRabE在木材形成中的作用，我们制备了超表达持续活性型PtRabE1b的转基因植株PtRabE1bQL。对转基因植株的细胞壁单糖含量分析表明，PtRabE1bQL中葡萄糖和木糖的含量显著升高。材性分析表明，PtRabE1bQL中木质素含量明显上调。对PtRab基因家族成员启动子顺式作用元件分析预测，发现这些基因启动子中包含大量与胁迫响应有关的元件。因此我们分析了PtRabE1bQL的耐盐性，发现其在NaCl胁迫初期（2小时）和持续胁迫（12小时）条件下均高表达。通过基因共表达网络分析，初步探讨了PtRabE1b调控植物耐盐性的分子机理。此外，我们分析了另一类重要的小G蛋白ROP的系统进化、基因结构及基因表达特性，鉴定出在杨树次生茎中特异表达的ROP基因为PtROP7、PtROP9和PtROP13，并构建了PtROP的功能基因网络。本研究为进一步揭示PtRabE 和PtROP基因在木材形成中的功能奠定了基础。