SUMO E3连接酶ZmSIZ1及其环状RNA在玉米根系应答干旱胁迫中的功能与分子机制研究

Drought is one of the major disasters in maize production. To help survive in continuous environmental challenges, plants have evolved numerous mechanisms to detect stress and mitigate the damage inflicted. The plant SUMO E3 ligase SIZ1 plays an important role in abiotic stress signal transduction and abiotic stress resistance. We identified a gene regulatory module that significantly associated with maize plant survival rate under drought stress and centered by ZmSIZ1 and its CircRNA (CircSIZ1). The biological functions of maize ZmSIZ1 in drought stress response is rarely reported, especially the function of its CircRNA, generated from the exons of ZmSIZ1 and significantly increasing in expression under drought stress, is still largely unknown. In this study, the full cDNA length of ZmSIZ1 and CircSIZ1, will be cloned and vectors for functional research, including the overexpression, RNA interference and CRISPR/Cas9 knockout vectors, will be constructed. Then the genetic transformation will be performed in maize. Different types of transgenic plants will be obtained and their phenotypic variations under drought stress will be observed. In addition, the relevant physiological and biochemical experiments will be carried out to find out the differences among different transgenic plants and wild type. On the other hand, sequences specific probes for CircSIZ1 will be designed to pull down proteins that interact with CircSIZ1 by using RNA Pull down. To obtain direct evidence for translation of CircSIZ1, sucrose density gradient centrifugation will be carried out to isolate polysome-bound RNAs for CircSIZ1 quantitative study. On the basis of confirmed translation potential of CircSIZ1, the interaction proteins for CircSIZ1, as well as its downstream regulated genes could be identified by CoIP and ChIP-seq assays. Thus, the circRNA mediated maize drought response mechanism could be revealed both from transcript and translation levels. The results from this study will unveil the ZmSIZ1 centered molecular regulatory network for maize drought tolerance and provide better strategy to maize abiotic stress resistant breeding.

植物SUMO E3连接酶SIZ1在干旱等非生物胁迫信号转导中具有重要作用。前期研究表明，以ZmSIZ1及其表达的环状RNA（CircSIZ1）为中心节点的调控模块与玉米干旱胁迫下植株存活率紧密关联，但其相关功能和作用机制尚不清楚。本研究拟克隆ZmSIZ1和CircSIZ1，构建过表达、RNAi和CRISPR/Cas9敲除载体，并在玉米中进行遗传转化。通过对各转基因植株耐旱性鉴定和相关生理生化指标分析，明晰ZmSIZ1和CircSIZ1在干旱胁迫应答中的生物学功能。同时，针对CircSIZ1进行RNA Pull-down等实验，发掘CircSIZ1 RNA结合蛋白。在检验CircSIZ1翻译能力基础上，利用CoIP和ChIP-seq等技术，挖掘CircSIZ1互作蛋白和靶基因，以期从RNA和蛋白不同层面揭示circRNA作用机制，解析以ZmSIZ1为中心的玉米根系应答干旱胁迫的分子调控网络。

本研究对玉米根系中ZmSIZ1 (SAP and MIZ1 domain-containing ligase1)及其环状RNA CircSIZ1分别进行全长克隆，构建circRNA环化过表达载体和其宿主基因ZmSIZ1的过表达和CRISPR/Cas9敲除载体，并在玉米中进行遗传转化。通过对玉米各转基因植株苗期和成株期进行耐旱性鉴定和相关生理生化实验分析表明，在自交系KN5585遗传背景下过表达ZmSIZ1和CircSIZ1均促进了玉米根系发育，提高玉米耐旱性。而ZmSIZ1和CircSIZ1共有外显子的敲除突变体siz1-1及ZmSIZ1内含子敲除突变体siz1-2根长和根尖数均小于野生型。在耐旱相关的C5 RIL群体中，CircSIZ1和ZmSIZ1表达量均呈显著正相关。Ribosome profiling qPCR和CircSIZ1环化载体在烟草中瞬时表达分析证明，CircSIZ1不翻译蛋白。针对CircSIZ1设计特异探针进行RNA Pull-down并联合质谱分析发现，CircSIZ1与ZmSIZ1蛋白互作，并通过EMSA证明其互作关系。利用anti-SUMO抗体检测各转基因株系中蛋白SUMO水平发现，CircSIZ1和ZmSIZ1过表达株系中蛋白SUMO修饰水平均显著增加。结合转录组测序发现，CircSIZ1和ZmSIZ1过表达株系中细胞壁合成通路基因表达显著高于野生型，通过提高干旱胁迫下Fasciclin-Like Arabinogalactan-Protein等基因表达水平，促进根系发育。综上，项目鉴定了玉米根系中环状RNA CircSIZ1，揭示了其表达特征与生物学功能，证明其与ZmSIZ1蛋白互作并影响蛋白SUMO修饰，明确了CircSIZ1在干旱胁迫下通过调控细胞壁合成通路基因表达，促进根系生长，提高玉米耐旱性。玉米中CircSIZ1及其宿主基因ZmSIZ1的功能研究对玉米耐旱分子育种及解析circRNA参与的干旱胁迫应答机制具有重要的理论意义和应用价值。