沙漠极端耐干藓类齿肋赤藓A-5类DREB转录因子调控耐干性分子机制研究

“Drying without dying” is specially described to the desiccation tolerance ability of resurrection plants, which have passing from a moribund dry state to a healthy active life as it rehydrates. The mechanism of plant desiccation tolerance has been paid more and more attention and become a research hotspot. Syntrichia caninervis is a widespread desert moss species in biological soil crusts which showed exterme desiccation tolerance and rapid recovery ability upon rehydration, is considered as an excellent model for understanding plant desiccation tolerance mechanism and a good plant source for identification of stress-realted genes. Based on our previous work, a novel A-5 type DREB gene ScDREB10 which showed strong and multiple stress tolerances and play important role in responding to desiccation stress in S.caninervis were selected as our target gene. The aim of this proposed research is to reveal the molecular mechanism of desiccation tolerance of ScDREB10. Specifically, 1) on the basis of the transcriptome comparison analysis among wild type, over-expression and inhibit expression transgenic Arabidopsis and Chromatin Immunoprecipitation (Chip) analysis, the cis-elements and target genes of ScDREB10 will be identified ; 2) using Co-Immunoprecipitation (Co-IP) and BIFC analysis, the proteins interacted with ScDREB10 are confirmed; and 3) according to the Immunoprecipitation and modification specific proteomics technology, the effect of post-translational modification of ScDREB10 in desiccation tolerance are decided. Through the above research, we aim to illustrate the molecular mechanism of the desiccation tolerances mediated by ScDREB10 gene. Our research may also helpful for understanding the regulatory mechanism of A-5 type DREB gens, as well as revealing the new regulatory mechanism of transcriptional factor and posttranslational protein modification in plant desiccation tolerance.

植物“干儿不死，死而复生”的耐干特性是非常特殊的自然现象，成为抗逆生物学研究的热点之一。齿肋赤藓因其超强的耐干特性和快速的复水能力已成为耐干研究新的模式种。本项目在明确了A-5类DREB基因ScDREB10能显著提高齿肋赤藓耐干性的基础上，拟从转录因子转录调控、转录因子蛋白互作、转录因子翻译后修饰三个层面展开研究，以系统揭示ScDREB10调控齿肋赤藓耐干的分子机理。即通过转录组分析差异表达基因结合Chip-seq分析，鉴定ScDREB10识别的顺式作用元件和调控的下游靶基因；通过免疫共沉淀和双分子荧光互补技术，明确ScDREB10的互作蛋白，通过免疫沉淀和蛋白质修饰组学分析，明晰翻译后修饰在ScDREB10耐干调控中的作用。本项目不仅能揭示ScDREB10调控齿肋赤藓耐干的分子机理，丰富A-5类DREB蛋白的转录调控机制，还有望在转录因子与翻译后修饰协同调控耐干性作用方面产生创新性认识。

植物“干儿不死，死而复生”的耐干特性是非常特殊的自然现象，成为抗逆生物学研究的热点之一。齿肋赤藓因其超强的耐干特性和快速的复水能力已成为耐干研究新的模式种。本项目的目的是鉴定ScDREB10直接和间接调控的下游靶基因、明确与ScDREB10互作的蛋白及明晰翻译后修饰蛋白磷酸化在耐干调控通路中的作用。获得的主要结果：转ScDREB10能够提高植株的抗逆性，从转ScDREB10基因植株的转录组数据出发，发现在渗透胁迫下的差异基因数量明显增多，进一步的GO和KEGG通路富集分析发现苯丙烷代谢通路中的木质素合成相关通路显著富集，qRT-PCR实验表明，过表达ScDREB10基因能上调一系列木质素合成相关基因的表达，研究进一步通过元件富集、酵母单杂交和EMSA实验表明，ScDREB10主要通过结合RAV1元件直接调控木质素合成相关通路上的PAL1和CAD4基因来提高抗逆性；本研究构建了齿肋赤藓干燥-复水下的酵母cDNA文库，使用ScDREB10无自激活区域作为诱饵，初步通过酵母双杂交系统筛选得到30个与ScDREB10互作的候选蛋白，选定6个靶标蛋白和ScDREB10进行一对一的酵母互作和双分子荧光互补验证，最终鉴定出ScDREB10的互作蛋白为ATG8和bZIP44；干燥-复水过程的磷酸化蛋白组共鉴定了12286个独特的磷酸肽，涉及3228个蛋白质和8367个磷酸化位点，46个磷酸化基序。磷酸化水平差异最为显著的转录因子有C3H、MYB，此外，在磷酸化蛋白组中共鉴定到23个转录因子，其中12个转录因子磷酸化水平有显著变化，bZIP转录因子是差异磷酸化水平最多的家族，包括2个PosF21和1个RF2a。本项目揭示了ScDREB10调控齿肋赤藓耐干的分子机理，丰富A-5类DREB蛋白的转录调控机制，同时，也明确了干燥-复水过程中关键的差异磷酸化水平转录因子，在转录因子与翻译后修饰协同调控耐干性作用方面产生创新性认识。