StABI5基因调控马铃薯干旱胁迫应答的分子机制研究

Drought is the most devastative abiotic stress that severely affects the growth and development of potato. In our previous research, we cloned a drought-response gene StABI5, which strongly enhanced transgenic Arabidopsis drought tolerance. However, the biological function and the molecular mechanism of StABI5 are still not clear in potato. In current proposal, we want to acquire two kinds of transgenic potato plants, one can over-express the target genes, the other carries an CRISPR/Cas9 plasmid, which could knock out target gene specifically. Afterwards, we could quantify the phenotype of both transgenic potato plants, as well as determine various physiological indexes that indicates the ability of drought tolerance. This would help us to clarify the ability for drought tolerance of the target gene in potato. Moreover, the difference of transcriptome between drought-tolerant transgenic and control potato plants will be determined with high-throughput sequencing technology, which would facilitate to identify potential key regulatory genes. Furthermore, yeast two-hybrid, together with BIFC technology will be applied to identify the protein that can directly interact with StABI5. Taken together, the strategy in present research will help us to acquire a modest comprehensive picture of the molecular mechanism for the drought tolerance function for StABI5 in potato. The present research will have a great practical effect on creating drought-tolerant potato breeds for breeding programs in future.

干旱是影响马铃薯生长发育最主要的逆境因子之一。项目组从马铃薯中克隆了一个干旱胁迫响应基因StABI5，过量表达该基因可显著增强转基因拟南芥抗旱性。但该基因在马铃薯中的生物学功能以及分子机制目前还不清楚。本研究拟创制StABI5的过量表达和敲除的转基因株系，分析转基因马铃薯的抗旱表型、测定抗旱相关的生理生化指标，以明确StABI5基因的生物学功能。同时，利用转录组测序技术，分析转基因植株与对照植株的差异表达基因，揭示StABI5基因所参与干旱胁迫的信号通路；利用酵母双杂交、BIFC等技术筛选、鉴定StABI5的互作蛋白，解析马铃薯中StABI5响应干旱胁迫的分子机制。本项目的开展丰富了抗旱分子机制的研究，对于培育抗旱马铃薯新品种具有重要的指导意义和实际应用价值。

马铃薯作为我国重要的粮食作物之一，常年受干旱、高温等环境因子影响。bZIP转录因子家族基因在调控植物非生物胁迫响应等方面发挥着重要的作用，很多植物中bZIP基因生物学功能也逐步被鉴定。到目前为止关于马铃薯中bZIP基因功能的研究报道还相对较少，因此对马铃薯中bZIP基因的功能研究及机制解析具有重要意义。本项目从马铃薯中分离得到一个bZIP基因StABI5，荧光定量PCR分析表明该基因在马铃薯各个组织中均有表达，且受外源ABA、干旱和盐胁迫诱导表达。亚细胞定位分析显示StABI5蛋白定位在细胞核中。构建StABI5基因过表达载体和CRISPR敲除载体并转化马铃薯。功能分析表明StABI5过表达转基因植株对干旱更加的敏感，转基因植株的丙二醛含量和相对电导率在干旱处理后要显著高于野生型植株。相反地，stabi5敲除突变株对干旱的抗性增强，突变植株的丙二醛含量和相对电导率在干旱处理后要低于野生型植株。转录组测序结果表明过表达StABI5基因改变了马铃薯中抗逆相关基因的表达。荧光定量PCR也进一步证实了StCM44、StMAPK6、StHSP18.2等6个基因在转基因植株与野生型植株中的表达发生了改变。此外BIFC和Pulldown实验证实StABI5能够与8个14-3-3家族蛋白相结合。综合分析结果表明StABI5能够结合14-3-3家族成员在马铃薯干旱胁迫应答途径中发挥负调控作用。该项目成果为研究马铃薯干旱胁迫应答调控机制提供理论基础，为马铃薯创建抗旱优质材料提供新基因资源。