Aux/IAA和ARF蛋白特异性互作调控山核桃嫁接成活的分子机制

The specific interactions between Aux/IAA and ARF proteins play important roles in auxin signaling pathway as well as growth and development of plants. Currently, related studies are focusing on the regulatory roles of specific interactions between Aux/IAA and ARF proteins in the normal growth and development in model plants like Arabidopsis and Populus, but the underlying mechanism of the specific interactions in tree grafting is little studied. In this project, based on the data from genome sequencing and transcriptome sequencing during the grafting process of Chinese hickory, members in the Aux/IAA and ARF gene family in Chinese hickory will be cloned; the specific interactions between members in Aux/IAA and ARF family will be identified by techniques of yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP); the expression levels and locations of specific interacting Aux/IAA and ARF proteins during the grafting process of Chinese hickory will be detected by in situ hybridization (ISH) and immune colloidal gold techniques; the dynamic changes of auxin during the grafting process will also be determined. This project aims to reveal the molecular mechanism mediated by the specific interactions between Aux/IAA and ARF proteins in successful grafting of Chinese hickory. Results from this project will not only provide theoretical references for understanding the mechanism of auxin in regulating the grafting process of trees, but also lay the foundation for the extensive application of grafting technique in production practice.

Aux/IAA和ARF蛋白通过特异性互作在植物生长素信号转导和生长发育中起重要调节作用。目前相关研究主要集中于Aux/IAA和ARF蛋白特异性互作调控拟南芥、杨树等模式植物的正常生长发育，在木本植物嫁接研究领域少见报道。本项目拟在山核桃基因组和嫁接成活过程转录组测序的基础上克隆山核桃Aux/IAA和ARF基因家族成员，利用酵母双杂交、双分子荧光互补和免疫共沉淀技术鉴定山核桃Aux/IAA和ARF蛋白的特异性互作，利用原位杂交和免疫胶体金技术检测特异互作的Aux/IAA和ARF蛋白在山核桃嫁接成活过程中的表达量及定位，结合嫁接过程生长素含量测定，解析Aux/IAA和ARF蛋白特异性互作调控山核桃嫁接成活的分子机制，为揭示生长素调控木本植物嫁接成活的作用机制提供理论参考，为嫁接技术在生产中的广泛应用奠定实践基础。

山核桃（Carya cathayensis Sarg.）是我国特有的优质干果和木本油料树种，产业化过程中面临树体高大、抗性差、良种化程度低等问题。揭示山核桃嫁接成活的机制，可为山核桃产业的转型升级提供理论和实践参考。前期研究发现生长素在山核桃嫁接成活过程中发挥着重要调控作用。在生长素信号通路中，Aux/IAA和ARF家族蛋白通过相互作用协同调控生长素响应基因的表达。然而，山核桃Aux/IAA与ARF蛋白家族成员具有怎样的互作关系，它们是否参与对嫁接成活的调控尚未见报道。基于本项目研究获得研究结果如下：（1）克隆获得25个CcARFs全长CDS序列，其编码蛋白含394~1123个氨基酸，外显子数量为1~15。CcARFs蛋白为亲水性不稳定蛋白，与薄壳山核桃（Carya illinoinensis）同源蛋白有更近的亲缘关系。CcARFs家族基因启动子上存在多个与激素相关的顺式作用元件。（2）发现CcARFs与CcIAAs蛋白大部分定位于细胞核；但CcARF19a、CcARF19b定位于细胞质，CcARF7a、CcIAA16、CcIAA28定位于细胞核和细胞膜。（3）对无自激活的15个CcIAAs与25个CcARFs进行基于Y2H技术的互作分析，结果显示113个CcARFs-CcIAAs组合有明显的互作，14个CcARFs-CcIAAs组合互作较弱；4个CcARFs和6个CcIAAs分别可以与超过10个CcIAAs和CcARFs存在互作关系。利用BiFC技术验证了部分CcARFs-CcIAAs组合的互作，发现与Y2H结果一致。（4）CcARFs和CcIAAs在嫁接过程中的表达量及表达变化趋势不同。5个CcARFs基因和5个CcIAAs基因被鉴定为嫁接过程中的差异表达基因，其中CcIAAs的表达量下调、CcARFs的表达量上调。在差异表达基因中，CcARF2d-CcIAA27a1、CcARF4a-CcIAA27a1、CcARF4a-CcIAA4、CcARF6a-CcIAA27a1和CcARF16d-CcIAA4 5个组合存在蛋白互作关系。CcIAA27a1和CcARF6a主要在茎部表达、在嫁接过程中表达水平高、在蛋白水平相互作用，推测在山核桃嫁接成活中发挥重要调控作用。