光皮桦OFP基因在次生壁形成中的功能及调控机制

Secondary cell wall biosynthesis is a key step of wood formation, which is a highly complex biological processes. Betula luminifera is not only a high-quality timber species with a priority to the promotion, but also an ideal material for forest genetic research. On the basis of digital gene expression and co-expression analysis, the BlOFP1 and BlOFP2 were cloned using RACE, which were coordinately expressed with genes encoding key enzymes involved in cellulose biosynthesis. Our previous results showed that the two BlOFPs were predominantly expressed in cambium and xylem region of lignifing stem. And BlOFP proteins could both interact with BlSTM in the yeast two-hybrid system, respectively. In B. luminifera, BlOFP2 over-expression resulted in the abnormal cell walls of xylem. We predicted that these two genes were important regulators involed in secondary cell wall synthesis. The further transformations of BlOFPs will be conducted in this study, and transgene plants will be used to analyze the functions of BlOFPs through molecular and anatomy experiments. It remains unclear how OFP genes could regulate the secondary wall biosynthesis of woody plant. So the yeast one-hybrid experiment will be applied to screen genes which regulate two BlOFPs expression from cDNA library of B. luminifera. In the other hand, the proteins interacted with BlOFPs will be screened by means of the yeast two-hybrid assay. The interactions will be further confirmed using bimolecular fluorescence complementation in B. luminifera, respectively. The implementation of this project will provide the theoretical basis for revealing the molecular mechanism of wood formation, and molecular marker deriving from these OFP transcription factors will be developed for marker-assisted selection of wood properties in the Betula luminifera breeding.

木材形成是一个高度复杂的受控生物学过程，次生细胞壁的形成是该过程的关键步骤。光皮桦既是优质珍贵用材树种，也是林木遗传研究的理想材料。在前期研究中，我们利用数字表达谱和基因共表达分析技术，挖掘到2个与光皮桦纤维素合成关键酶基因协同表达的OFP基因，其在木质化茎的形成层和木质部区域优势表达，相应表达产物均能与BlSTM蛋白互作，且超表达会导致细胞壁异常，很可能是参与次生壁形成的重要调控因子。本课题拟进一步开展光皮桦遗传转化，对转基因植株的木质部显微结构、细胞壁成分和基因表达谱进行分析，明确BlOFPs的功能；同时针对木本植物次生壁形成中OFP基因调控机制不明确的现状，运用酵母单杂交和双杂交、双分子荧光互补等技术，对BlOFPs的上游调控基因，以及与其表达产物互作的蛋白进行筛选和分析，明确其在次生壁形成中的调控机制。期望为揭示光皮桦木材形成分子机制，以及分子辅助育种提供科学依据和重要基因资源。

木材形成是一个高度复杂的受控生物学过程，次生细胞壁的形成是该过程的关键步骤。OVATE家族蛋白（OFP）是类植物特有的新型转录因子，拟南芥相关研究证实该家族部分成员调控次生壁形成。本项目首次在光皮桦基因组中鉴定到20个OFP基因；在生物信息学和表达特性分析基础上，筛选出参与次生壁发育调控的候选基因BlOFP1和BlOFP8。基于光皮桦和拟南芥的遗传转化显示，BlOFP1、BlOFP8超表达会导致拟南芥细胞伸长受阻、花序茎基部木质化延缓、束间纤维细胞壁变薄，以及光皮桦木质部纤维细胞壁变薄等，表现出一定的功能冗余；且均通过负向调控木质素、纤维素合成相关酶基因表达来参与次生壁的形成。这些表明两个BlOFPs基因与来自拟南芥的直系同源基因AtOFP1、AtOFP4具一定的功能相似性。然而，两个BlOFPs基因的调控路径与AtOFP1、AtOFP4存在一定分歧，BlOFP8蛋白能分别与TALE超家族成员BlBLH6、BlBLH7、BlKNOX5和BlKNOX7发生互作；BlOFP1只与BlBLH9蛋白间存在互作。另外，一个A20/AN1型锌指蛋白BlZFP1调控BlOFP8基因的转录。基于遗传转化、酵母单双杂交、共表达网络构建分析的结果，绘制了一张BlOFP8、BlBLHs和BlKNOXs等参与的光皮桦次生壁发育调控路径图。本项目首次在木本植物中开展OFP基因功能解析，研究结果补充了木本植物次生壁合成转录调控网络，为木材形成分子机制的深入研究提供了重要依据。此外，BlOFP1、BlOFP8及其调控的下游基因可通过分子标记技术用于光皮桦材性改良育种。