转录因子AtIDD4、AtIDD5、AtIDD6和赤霉素在拟南芥花丝伸长中的互作研究

AtIDD4, AtIDD5 and AtIDD6 belong to zinc finger protein IDD family distributed in Arabidopsis thaliana. The three genes formed a group in the family. Our previous study showed that over expression of AtIDD4, AtIDD5 or AtIDD6 gene alone in Arabidopsis resulted in filaments development of stamen inhibition. The filaments remained short and were unable to pollinate when anthesis. The plant which overexpression of gene IDD4 has defect in filament length and this phenotype can be reversed by exogenous GA treatment. The deletion of AtIDD4, AtIDD5 or AtIDD6 alone mutation did not cause abnormal filaments. Meanwhile the filaments of a triple mutant idd4 idd5 idd6 were longer than those in the wild-type, while the expression level of IDD4, IDD5 and IDD6 were obviously decreased at same time. These results suggest that the three genes may be involved in regulating development of Arabidopsis filaments and linked to plant hormone gibberellin (GA). Thus, in this project, we plan to research the expressional pattern of these three genes to obtain the information of their distribution in tissues involved in stamen development process through qRT-PCR and GUS report gene system. It will be detected that the levels of GA1, GA3 and GA4 in the flowers of different gene-type plants and in the filament and anther of 35S::IDD4 plants. And the genes which are possibly regulated by IDD4, IDD5 and IDD6 will be screened by using RNA-Swq system. Based on the results, the chromatin immunoprecipitation and CRISPR-Cas9 gene knockout method will be performed to detect the downstream target gene(s) which IDD4 directly bound. In summery, the roles of AtIDD4, AtIDD5 and AtIDD6 played in regulation of filament development through GA in Arabidopsis and their possible mechanisms will be revealed. Our work will provide the data for further study molecular mechanisms of filament development in plants.

AtIDD4、AtIDD5和AtIDD6属于拟南芥锌指蛋白IDD家族，并形成一个组。我们发现分别过量表达这三个基因的拟南芥雄蕊花丝伸长受阻，不能完成授粉。外施GA可使35S::IDD4植株花丝短小的缺陷表型得到恢复。AtIDD4/5/6的单独缺失突变并未造成花丝异常，而三个基因表达量均下降的idd4idd5idd6三突变体的花丝长度比野生型的长，推测这三个基因协同调控花丝的伸长，且与GA相关。因此，本项目拟用qRT-PCR和报告基因系统，研究三个基因表达的时空性质。用LC–MS/MS法检测不同基因型的花、35S::IDD4的花丝和花药中不同形式的GA水平，用转录组测序法分析三个基因所调控的基因。在此基础上，用染色质免疫共沉淀和基因敲出法鉴定IDD4蛋白直接结合的靶基因。通过综合分析，拟揭示AtIDD4/5/6协同通过GA途径调控拟南芥花丝发育的机制，为进一步研究花丝发育的分子机制奠定基础。

拟南芥花发育到第13至14期时，花丝在约24 小时迅速伸长以确保授粉完成，若此过程受阻就会影响育性。我们的前期研究发现AtIDD4、AtIDD5和AtIDD6（IDD4/5/6）编码锌指蛋白，各自表达量上调后均导致雄蕊数目减少、花丝生长受阻。本研究对单、双和三基因缺失突变体做了观察，进一步证明IDD4/5/6功能冗余。用qRT-PCR和GUS染色检测了IDD4/5/6在野生型和表达量上调植株中的表达模式。结果三个基因均为组成型表达，过量表达后表达模式没有改变。融合GPF蛋白定位显示IDD4/5/6分布于细胞核，酵母双杂交显示IDD4/5/6均有转录活性，符合转录因子特性。超薄切片实验显示花丝细胞变短是由细胞纵向生长受阻所致。HPLC-MS测定发现，IDD4/5/6过表达花中GA和IAA含量显著低于野生型。外施GA3使花丝伸长受抑制表型恢复，说明GA3含量下降是花丝缩短的重要原因。用RNA-Seq技术测定了IDD4-OE、tIDD5-OE、IDD6-OE和idd4 idd5 idd6的花在13-14期的转录组变化，用RT-PCR检测验证了部分结果。筛选到四个材料与野生型之间的差异表达基因（DEGs）。对四个材料的各种组合的DEGs进行GO和KEGG分析。用ChIP_Seq检测筛选到36个IDD6结合的靶基因。综合分析转录组和ChIP数据，揭示了关键靶基因如GA20OX2、GASA、SAUR、FAD、WRKY家族等与发育、防卫、GA合成和信号转导相关。本研究表明IDD6的表达量下降突变体idd6 对真菌病原物灰葡萄孢菌的抗性提高，而过量表达植物IDD6-OE的抗性则下降，IDD6负调控植物的抗性。本研究揭示了AtIDD4、AtIDD5和AtIDD6协同调控GA合成和信号转导，从而调控拟南芥雄蕊花丝的伸长。本研究为IDD基因家族在GA合成和转导中的功能研究提供了重要依据，在人工调控植物雄性育性方面有潜在应用价值。