白桦NAC类转录因子BpVND7基因调控木材发育的机理研究

The NAC genes are specific transcription factors in plants, which showed various biological functions. The VND gene is a member of NAC and mainly related to plant secondary growth. In this research, The PROK2-BpVND7 overexpression vector，suppress expression vector and PBI121-GFP-BpVND7 vector was respectively transformated into Betula platyphylla. In order to understand the function and regulation mechanism of BpVND7 in the wood development. Firstly,The frozen section and scanning electron microscope were tested among transgenic lines and WT. Then the downstream regulators of BpVND7 will analyzed by comparison the transcriptomes of transgenic and WT lines. Meanwhile, ChIP libraries were respectively built from GFP transgenic B.platyphylla. ChIP-Seq were further performed to generate the DNA fragments bound by BpVND7. The conserved motifs among these DNA fragments were identified, which may be recognized by BpVND7 and will be further confirmed by yest one hybrid analysis. The transcriptoms were assembled with the sequences generated from ChIP-Seq, and these motifs contained in which genes will be revealed by the assembled sequences. Based on these studies, the motifs recognzied by BpVND7 protein and the wood development pathways these motifs involved will be revealed.

NAC是植物所特有的一类转录因子，具有多种多样的生物学功能。其家族成员VND与植物次生生长密切相关。我们将从白桦中克隆到的BpVND7基因的过表达、抑制表达和GFP荧光定位表达载体转入到白桦基因组中。以野生型为对照，通过冷冻切片和扫描电镜，研究白桦BpVND7基因在木材发育过程中的功能；通过转录组比较转基因白桦和野生型白桦的基因表达差异，确定BpVND7所调控的下游靶基因；对荧光定位表达载体的白桦转基因植株进行染色质免疫共沉淀研究，建立ChIP文库，进行ChIP-Seq高通量测序，获得BpVND7结合的DNA片段序列，进行序列的保守元件分析，结合转录组，鉴定出BpVND7可能结合的元件，酵母单杂交验证；同时，通过转录组与ChIP序列的深度拼接，研究所鉴定的不同顺式作用元件所调控的基因，通过分析阐明在木材发育过程中BpVND7所参与的调控机理。

木材是工业生产和能源生产中广泛使用的可再生原料，次生细胞壁（SCW）是木材的主要成分。 VND（Vascular-related NAC domain）家族基因是次生壁生物合成的关键转录因子，在调控植物次生生长、木材形成中扮演着重要角色。然而只有少数VND基因的功能被鉴定，本项目探讨了BpVND7基因调控白桦生长及材性的功能及分子机制。首先从白桦中克隆一条VND基因，该蛋白定位在细胞核中。然后对该基因的功能进行详细的研究，发现BpVND7过表达转基因白桦木质部纤维细胞壁增厚、木质素含量增加；而BpVND7抑制表达转基因白桦的生长和生物量提高、综纤维素含量增加、纤维长度增加、纤维长宽比增加。最后研究了BpVND7所调控的下游基因及调控木材发育的分子机制，确定BpVND7能够直接调控MYB54、MYB83、CCR1、CCoAOMT1和4CL1这5个与次生壁合成相关的基因，能够识别SNBE元件；阐明BpVND7是通过调控次生壁合成相关基因及转录因子的表达，进而调控白桦次生细胞壁的生物合成。本项目系统研究了BpVND7基因在生长及次生细胞壁发育过程中的功能及调控机制，为白桦遗传改良及木材形成的调控机理研究提供了理论基础。本项研究共发表SCI论文2篇,但研究仍在深入，目前已投稿或在准备中的论文3篇，预期这些论文将在2021年底前完成发表。申请专利1件，培养博士研究生1名。