马铃薯抗逆基因家族StDGK的功能及其上下游调控机理研究

Plant DGK(Diacylglycerol kinases) is one of the hubs of cell signaling related to plant development and responses to environmental stimuli and stresses. DGK is also the pivotal signaling kinase that yields phosphatidic acid (PA) in response to biotic and abiotic stresses. In all plant genomes explored, researchers were able to find DGK representatives, with well-conserved sequences and structural domains. So far, plant DGK isoforms all have diacylglycerol kinase accessory domain (DGKa) and diacylglycerol kinase catalytic domain (DGKc), which play a key role on the biosynthesis of PA. Plant DGK are widespread in higher plants and many of them have been well characterized. However, no functional identification has been done in potato StDGK till now. In our previous research, we identified three potato StDGK gene candidates, with conserved DGKa and DGKc domain, from potato DM genome. The expression profiling data showed that these three StDGKs all had individual tissue specific expression profile and were able to be rapidly in response to certain time exposures of biotic, abiotic stresses and hormones. These suggested that StDGKs may mediate signals triggered by these stresses or elements. Intriguingly, by looking at the homologous gene AtDGK7 in Arabidopsis, we were able to get some ethyl methanesulfonate (EMS) mutagenesis in which the AtDGK7 promoter activity had been significantly decreased or enhanced. Identification of these EMS mutagenesis can display the transcriptional regulation mechanism of AtDGK7 and is thought to underlie the potato StDGK gene transcription regulation research. In this proposal, relying on luciferase reporter mediated plant transient expression system, CRISPR/Cas、RNAi、RNA-seq etc. technologies, we will be able to clarify the potato StDGK gene function, the regulation mechanism at the StDGK gene translational level and downstream target genes. This study will ultimately reveal the molecular mechanism of StDGK in response to biotic and abiotic stresses, and also provide applicable genetic resources and theoretical basis for improving the potato stress tolerance.

DGK激酶是高等植物抗逆相关信号传导途径中重要的节点基因之一。DGK在多种环境胁迫下上调表达，通过其蛋白激酶催化产物信号分子PA来调控下游多个靶蛋白的活性，最终增强植物的抗逆性。DGK存在于所有已公开的高等植物基因组中，结构和功能高度保守，而马铃薯StDGK尚无详细报道。前期通过分析马铃薯DM基因组和转录组数据，确认马铃薯至少有3个高丰度表达，结构完整，与其他植物DGK高度同源的StDGK蛋白。其基因表达有组织特异和胁迫响应特征，预测其基因上游启动子包含胁迫相关转录因子的结合区。此外，筛选到拟南芥的可显著调控StDGK同源基因启动子活性的突变体。本项目拟利用植物生物发光活体成像、CRISPR/Cas、RNAi、RNA-seq等技术，解析StDGK的基因功能、上游基因转录调控和下游蛋白激酶调控机制，构建以StDGK基因为中心的马铃薯抗逆机理模型，为马铃薯抗逆品种的选育提供理论指导和基因资源。

高温干旱等逆境胁迫严重影响马铃薯的产量。而DGK作为高等植物中功能和序列均高度保守的重要第二信使和抗逆节点基因，可在多种环境胁迫下上调表达，通过激活下游多个靶基因的活性，最终增强植物的抗逆性。马铃薯StDGK基因是否能影响马铃薯的抗逆能力尚未有深入研究。.本项目围绕马铃薯StDGKs多基因家族的功能以及上下游调控机制进行初步研究。首先，通过基因克隆获得6个StDGK成员的全长，并利用单细胞测序等技术筛选出马铃薯DGK家族中的主要功能基因StDGK5和7。.其次，通过分析马铃薯全基因组开放染色质数据库（DNase I hypersensitive sites，简称 DHS），发现StDGK基因家族的启动子共有5个DHS区域。利用植物活体成像、瞬时表达和转基因等技术对这5个DHS进行增强子（enhancer）和启动子功能检测。结果发现，其中StDGK5具有两个增强子DHS，且其中一个DHS还兼具强启动子活性。同时，结合拟南芥同源基因AtDGK7启动子DHS的活性验证，发现AtDGK7基因的DHS同样具有类似的增强子功能，该DHS还对干旱胁迫表现出敏感。拟南芥转基因实验再次证明StDGK5的DHS是该基因上调表达的核心增强子区。.最后，通过马铃薯遗传转化，获得马铃薯StDGK5的Crispr-Cas9敲除转基因株系。转基因阳性株系表型鉴定发现，马铃薯转基因株系呈现明显的生长迟缓，而拟南芥AtDGK7的转基因株系同样出现了生长和开花均延迟的表型。耐热性实验显示，StDGK5基因的敲除导致马铃薯的耐热性明显下降。RNA-seq转录组测序显示DGK的表达的降低导致植株信号传导和多个代谢相关的调控网络均受到显著影响。综上，StDGK5是马铃薯的关键抗逆基因，其转录水平受到基因上游的两个核心DHS区域的协同调控作用，并对干旱胁迫敏感。DGK基因可调控下游信号传导和多个代谢网络，并影响植株的耐热性和生长发育。.本项目初步阐明了马铃薯DGK抗逆家族在植物抗逆方面的功能和上下游调控机制。该研究为马铃薯DGK介导的抗逆分子机制研究，良种选育提供一定的理论支持与资源。