拟南芥C3H14和C3H15基因调控次生细胞壁形成的分子机制研究

Plants have evolved to posses secondary cell walls that not only provide mechanical support for their growing body and serve as a conduit for long-distance transport of water and solutes，but also contain the main compoments,partically in trees.Therfore，it's important to understand and modify secondary cell wall regulatory mechanism.In model plant Arabidopsis, secondary cell wall formation is coordinatedly regulated by multiple gene families and forms a complex network.However,it is not clear for its secondary cell wall regulatory mechanism, specially at the posttranscriptional level to date. Recently, we functionally characterized two CCCH zinc finger proteins C3H14 and C3H15 and found that the two genes were highly expressed in stems and flowers, their proteins were able to bind to Ribohomopolymers and redudantly regulated secondary cell wall formation with predominantly in stems for C3H14 gene and anthers for C3H15 gene. Based on the aboved results, this project aims to futher analyze the biological functions of C3H14 and C3H15 genes, use RIP-Chip and REMSA to identify downstream target genes involved in cell wall formation, respectively, and finally clarify the molecular regulatory mechanism of C3H14 and C3H15 in secondary cell wall formation at the posttranscriptional level. The results in this project may provide the the theoretical basis for genetic engineering plant secondary cell walls.

植物次生细胞壁不仅在水分、营养的长距离运输和机械支撑中起重要作用，而且也是纤维生物质储存的主要场所，因而对次生细胞壁形成机制的研究具有重要意义。在模式植物拟南芥中，次生细胞壁的形成受多个基因家族的调控，形成一个复杂的调控网络，但目前其分子调控机制，特别是转录后调控机制知之甚少。我们前期研究表明，拟南芥两个CCCH锌指蛋白基因C3H14和C3H15均在茎和花中大量表达，编码的蛋白具有RNA结合活性，冗余调控次生细胞壁的形成，且分别在茎秆和花药次生细胞壁中起主导作用。以此为基础，本项目拟通过免疫定位、RIP-Chip、REMSA等技术，进一步深入分析C3H14和C3H15基因在次生细胞壁形成中的生物学功能，鉴定其参与细胞壁形成的下游各自作用靶标，揭示其在转录后水平上调控次生细胞壁形成的分子机制，为未来利用基因工程手段调控植物次生细胞壁的形成奠定基础。

植物次生细胞壁不仅在水分、营养的长距离运输和机械支撑中起重要作用，而且也是纤维生物质（特别是林木）储存的主要场所，因而对次生细胞壁形成机制的研究具有重要意义。拟南芥CCCH锌指蛋白C3H14和C3H15定位于细胞质和细胞核，具有转录激活活性，能够特异结合DNA和RNA。编码的基因在基部茎和花中大量表达，冗余正调控茎秆次生细胞壁的形成和花粉发育，而且C3H14和C3H15分别在细胞壁形成和花粉发育中起偏好性作用；qRT-PCR和免疫组化分析表明过表达和双突变C3H14/15基因分别增高和降低茎秆纤维生物质三种主要组分（纤维素、半纤维素和木质素）的含量；基因芯片分析表明C3H14/15通过调控多个基因（包括一些MYBs转录因子）的表达参与茎秆次生细胞壁的形成。特别是，REMSA结果显示C3H14蛋白能够直接结合MWL-2 RNA 3’UTR的ARE，抑制其表达，意味着C3H14可能在转录后水平调控MWL-2参与茎秆次生细胞壁的形成；除了既定研究目标，我们也鉴定了拟南芥C3H14在杨树（次生细胞壁研究的模式树种）中同源基因PdC3H17/18的功能。遗传分析表明这两个基因均能正调控杨树茎秆木质部分化和细胞壁增厚，而且是木材（次生细胞壁）形成二级调控枢纽PdMYB3/21的直接靶基因，意味着C3H14/15，PdC3H17/18在草本植物和木本植物中调控茎秆细胞壁形成的分子途径保守；为了鉴定C3H14/PdC3H17下游木材形成相关的基因，我们系统研究了杨树194个R2R3-MYB转录因子，结果表明，这些基因形成81个基因对，有5种不同的进化命运，其中PdMYB10/128过表达促进茎秆细胞壁增厚，PdMYB90/167、PdMYB92/125、PdMYB161/221过表达抑制茎秆细胞壁增厚，意味着PdC3H17通过调控下游功能相反的PdMYBs参与木材的形成。基于以上结果，我们目前已发表学术论文5篇（IF>5 SCI收录4篇），参编学术专著1部，授权专利1个，培养青年骨干4名、研究生2名，超额完成了预期目标。而且在本项目研究工作基础上，本人中标国家自然科学基金面上项目（31670606）。