MeCPK9与MeDI19互作通过抗氧化系统调控木薯耐旱性的分子机制

Cassava is a typical drought-tolerant crop. Abscisic acid (ABA) signaling plays crucial roles in regulating cassava tolerance to drought stress. However, the mechanism underlying this regulatory role is unknown. In our previous work, MeCPK9 protein kinase, an important regulatory factor of ABA pathway, was selected as a candidate to further analyze based on cassava genome sequencing, drought tolerance analyses of three genotypes, and RNA-sequencing. Further studies suggested that MeCPK9 could confer drought tolerance through activating antioxidative system and interact with MeDI19. Moreover, both MeCPK9 and MeDI19 were induced by drought and ABA treatments in the drought tolerant genotype. According to these results, we put forward a hypothesis that both MeCPK9 and MeDI19 is involved in cassava tolerant to drought. In this work, we will study the action sites of MeCPK9 phosphorylating MeDI19, the targets of MeDI19, and the function and regulatory pathway related to antioxidative system of MeCPK9 and MeDI19 in cassava responding to drought stress by using biochemical and genetic approaches. This study will deepen the understanding of molecular mechanism about ABA regulating drought tolerance of cassava and supply theory evidences for further studies on drought-tolerant mechanism and molecular breeding of crops.

木薯是一种典型的耐旱作物。脱落酸(ABA)信号在调控木薯耐旱性上起着重要作用，然而其作用机制尚不清楚。前期研究在木薯全基因组测序、三个种质的耐旱性评价及转录组测序的基础上选择ABA信号途径重要调控因子MeCPK9蛋白激酶为研究对象，进一步研究发现MeCPK9通过抗氧化系统提高植物耐旱能力，能够与MeDI19相互作用，且MeCPK9与MeDI19都受干旱和ABA诱导。据此，提出MeCPK9与MeDI19协同调控木薯耐旱性的假说。本项目拟采用蛋白磷酸化、遗传转化、染色质免疫共沉淀、凝胶迁移等技术鉴定MeCPK9磷酸化MeDI19的作用位点，挖掘MeDI19直接作用的靶基因，揭示MeCPK9和MeDI19在木薯应答干旱胁迫中的功能及其对抗氧化系统的调控方式，最终解释MeCPK9和MeDI19如何参与木薯干旱胁迫应答，从而加深对ABA调控木薯耐旱分子机制的理解，为作物抗旱分子育种提供科学依据。

木薯是一种典型的耐旱作物。脱落酸(ABA)信号在调控木薯耐旱性上起着重要作用，然而其作用机制尚不清楚。前期研究在木薯全基因组测序、三个种质的耐旱性评价及转录组测序的基础上选择ABA信号途径重要调控因子MeCPK9蛋白激酶为研究对象，进一步研究发现MeCPK9通过抗氧化系统提高植物耐旱能力，能够与MeDI19相互作用，且MeCPK9与MeDI19都受干旱和ABA诱导。据此，推测MeCPK9与MeDI19协同调控木薯耐旱性。本项目采用蛋白结构域拆分、酵母双杂交、磷酸化反应等方法证实了MeDI19 C端具有转录激活活力且能与MeCPK9互作，进一步明确了MeCPK9能够磷酸化MeDI19；采用木薯和拟南芥遗传转化及生理分析揭示了MeCPK9和MeDI19能够通过激活SOD和POD等抗氧化酶活性正调控木薯和拟南芥耐旱性，且MeCPK9过表达株系对ABA敏感；采用酵母单杂交、染色质免疫共沉淀、凝胶迁移、双荧光素酶等技术发现了MeDI19能够作为转录因子能与MePOD10启动子区域-286~ -281bp 核心元件（TACAAT）互作，且二者的相互作用可在正常和脱水处理下显著激活MePOD10表达。综合以上结果，本研究表明MeCPK9可磷酸化MeDI19，MeDI19能与MePOD10核心元件互作激活抗氧化系统进而正调控木薯耐旱性。本研究解释了MeCPK9和MeDI19如何参与木薯干旱胁迫应答，从而加深对ABA途径关键组分调控木薯耐旱分子机制的理解，为作物抗旱分子育种提供了科学依据。