与SHORT-ROOT和SCARECROW发育途径相关的IDD家族基因的确定和功能研究

This research aims to identify the IDD genes that act together with SHORT-ROOT (SHR) and SCARECROW (SCR) in the control of stem cell renewal and endodermal cell fate specification in the Arabidopsis root. Although SHR and SCR are key regulators of stem cell renewal and endodermis specification, they do not bind to DNA and thus requires other DNA-binding factors to regulate downstream genes. Evidence suggests that IDD genes are the missing links in the SHR-SCR developmental pathway and therefore are the focus of this research. In preliminary studies, we have identified a number of IDD genes preferentially expressed in the root meristem or the endodermis. Next, we will characterize the function of these genes through analysis of multiple mutants, which would overcome the potential issue of gene redundancy. Meanwhile, physical interactions between these IDD proteins and SHR/SCR will be examined using such methods as yeast two-hybrid, Bimolecular Fluorescence Complementation (BiFC), and immunoprecipitation. Last, downstream targets of those IDD genes with a role in stem cell renewal or endodermis specification will be determined by RNA-seq and ChIP-seq, and the function of these genes will be inferred through bioinformatics analyses. These results will not only further our understanding of the mechanisms controlling root growth and development, but also provide guidance for crop improvement aimed at increased efficiency of nutrient uptake and utilization through genetic modification of root architecture in crop plants.

将对拟南芥IDD家族基因进行系统分析，以确定与SHORT-ROOT (SHR) 和SCARECROW (SCR) 共同作用的、控制根生长和发育的那些基因。虽然SHR和SCR是控制植物根干细胞延续和内皮层分化的重要因子，但有证据表明它们不具备结合DNA的能力，而是通过IDD这类转录因子调控下游基因的表达。本课题组在前期工作中已发现了多个在根尖或内皮层特异表达的IDD基因。下一步将根据这些基因表达的组织特异性，构建多重突变体，并分析其对干细胞延续或内皮层分化的影响。同时，将利用酵母双杂交、双分子荧光和蛋白免疫共沉淀等方法检测它们与SHR和SCR的互作。最后，将用转录组和ChIP-seq方法确定这些IDD基因的下游基因，并通过生物信息学分析揭示这些基因的功能。这些结果不但可以加深我们对根发育机制的了解，同时还将为通过改变根系结构来提高作物营养吸收和利用效率这一途径进行作物遗传改良提供理论依据。

根的生长取决于位于根尖的分生组织细胞的持续分裂和伸长区细胞的纵向伸长，而分生组织的维持依赖于处于其顶端的干细胞（QC）。SHORT-ROOT和SCARECROW即是维持QC的重要因子，也对内皮层的分化和皮层增生具有重要作用。虽然经过多年研究，对SHR和SCR的功能已经有了较深入的了解，但对于它们如何维持QC、促进根的生长和皮层增生仍不十分清楚。..SHORT-ROOT和SCARECROW自身不能结合DNA，而是通过IDD这类转录因子调控下游基因的表达。本研究对IDD家族基因进行了系统分析。首先构建了启动子- GUS转基因植物，通过分析GUS表达模式确定了所有成员的表达模式。结果表明，很多IDD基因在根尖都有表达。然后，通过过表达转基因植株的构建和分析，发现大多数成员，特别是IDD4、5和6对根的生长均有显著抑制作用；同时发现，IDD4、5和6的过表达还促进皮层增生。由于IDD4、5和6亲缘关系很近，且单基因突变体没有表型，所以构建了它们的三重突变体。idd456三重突变体不但影响了中皮层的产生，还对种子萌发率产生了严重影响。进一步实验发现，IDD4、5和6的功能可能与其调控生长素代谢和信号传导有关，且都与SHR和SCR有不同程度的互作。..对控制根生长和中皮层产生的机制的新发现，以及对某些IDD基因在种子发育和萌发中的作用的发现，不但深化了对植物生长和发育机制的理解，还为作物改良提供了工具。

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