INDEHISCENT 对拟南芥果实开裂的调控机理研究

Fruit dehiscence is a natural process required for release of seeds from mature dry fruits of many angiosperm species. The timing and extent of fruit dehiscence also affect the harvest and processing of crops. As a result of a developmental program involving the formation of multiple cell types and coordinated actions of these cells, fruit dehiscence occurs in specific locations within the fruit. Significant progress in understanding the dehiscence process has been made in recent years, a transcriptional network in the regulation of fruit dehiscence has been identified. It has been reported that the cell wall degrading enzyme play an important role in the regulation of fruit dehiscent, however, the mechanisms about how the cell wall-degrading enzymes lead to fruit dehiscence in Arabidopsis is not well understood, in particular, the involvement of the transcriptional factors in the regulation of cell wall degrading enzyme for dehiscence has not been reported. Here, our project aims to characterize the role of transcriptional factor INDEHISCENT (IND) in the regulation of cell differentiation and the activity of the cell wall degrading enzymes during fruit dehiscence in Arabidopsis. Our previous results suggested that the cell walls of the separation layer is first softened by cellulases, and the softened cells are then compressed by adjacent cells to form the narrow separation layer that eventually separates from the replum due to pectinases secreted from the replum border and retraction of the valve upon fruit drying, and IND is involved in the regulation of the activity of the cell wall degrading enzymes during fruit dehiscence. This model may suggest possible ways for manipulating fruit dehiscence for better retrieving crop seeds.

拟南芥（Arabidopsis thaliana (L.) Heynh.）角果的开裂是通过开裂区的分化和离层细胞的分离来实现的，离层细胞的分离与细胞壁水解酶（果胶酶，纤维素酶等）的活动密切相关。申请者的前期工作发现，果胶酶的积累与离层细胞的分离在时空上密切相关，而纤维素酶则调控开裂区的细胞形态建成；调控果实开裂区分化的转录因子INDEHISCENT (IND)可能对果胶酶和纤维素酶有重要调控作用。在拟南芥中，编码果胶酶和纤维素酶的基因分别为67个和39个，究竟哪些成员参与调控果实开裂目前还没有系统研究。为了从全基因组水平探讨细胞壁水解酶基因在果实开裂过程中的贡献，以及IND与水解酶的相互作用关系，本项目拟选择野生型和ind-6突变体为研究材料，运用基因芯片技术，RT-PCR等技术研究果实发育过程中的基因表达规律，筛选出与果实开裂最密切相关的水解酶基因，为阐明果实开裂的调控机理提供理论依据。

果实开裂是许多经济作物的一个必要发育阶段，直接影响着作物的产量。在分子生物学层面，果实开裂往往涉及到数千个基因的表达变化以及相关的生物学功能的改变，是一个非常复杂的调节过程。尽管目前已有报道细胞壁水解酶（果胶酶，纤维素酶等）在果实开裂过程中发挥重要作用，但其具体的分子调控机制尤其是如何受转录因子的调控还远未清晰。为了从全基因组水平探讨细胞壁水解酶在果实开裂过程中的调控机理，以及转录因子INDEHISCENT (IND)与水解酶的相互作用关系，本项目选择野生型拟南芥（Arabidopsis thaliana (L.) Heynh.）和果实不开裂突变体ind-6的角果作为研究材料，选取其开裂区细胞形态建成时期（阶段11，15和17早期）的角果开展了以下研究：1，本研究首先采用全基因组表达谱技术对所有基因在上述三个时期的表达进行检测，筛选了野生型和ind-6突变体的差异表达基因，从而筛选出调控离层细胞分离的关键细胞壁水解酶候选基因。我们的研究还发现，该候选基因的时空表达与果实开裂区的发育密切相关。2，利用上述基因表达数据，筛选野生型不同发育时期的差异表达基因，并对差异表达基因进行聚类分析，分别得到9个表达模式不同的基因集合，并分别进行功能富集分析，此外，还使用转录因子（TF）富集分析尝试寻找了这9个基因集合显著相关的转录因子。最终，我们发现两个转录因子（SEPALLATA3与AGL15）可显著的调控一个与细胞壁分解相关的基因集合。我们通过进一步分析这两个转录因子所调控的基因，发现大量的糖基水解酶。此外，我们的果胶酶与纤维素酶的细胞化学定位实验也发现大量的糖基水解酶显著富集到果实的开裂区。综合这些分析结果，我们认为SEPALLATA3与AGL15可能是拟南芥果实开裂过程中关键的转录因子。本研究的数据将基因，转录因子以及具体的生物学功能联系在一起，为进一步阐明果实开裂的分子调控机制做了一定的支撑。