叶绿体伴侣蛋白Hsp70在抗旱响应中的功能研究

Drought is one of the major problem for the ecosystem. Chloroplast is involved in the drough resistance process by retrograde signaling, which plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins. To date, the identity of retrograde signaling regulation has remained elusive. Chloroplast stroma located molecular chaperone Hsp70 is the major motor responsible for protein import across chloroplast membrane in Physcomitrella patens. Our previous work showed that overexpression Hsp70 plants had increased drought resistance, but the molecular mechanism is unknown. The proposed project aim to investigate the detailed molecular mechanims that Hsp70 mediated drought resistance by retrograde signaling. Specific research aims are proposed: 1) Determine what is the upstream regulators for Hsp70 by promoter analysis; 2) Identify Hsp70-interacting proteins and their roles in Hsp70 mediated retrograde signaling; 3) investigate Hsp70 regulated nuclear genes and chloroplast genes by RNA-seq analysis in Hsp70 mediated retrograde signaling; 4) Investigate biology mechanisms mediated by Hsp70 and its ATPase activity mutants. This project is of major relevance for choroplast mediated drought resistance in plants and will contribute to the plant genetic engineering of stress resistance improvement.

干旱是目前生态系统面临的主要问题之一。植物叶绿体通过退化信号途径（Retrograde signaling）调控细胞核基因表达参与抵御干旱的过程，但人们对其信号传递途径和分子机理认识十分有限。定位于小立碗藓叶绿体基质的伴侣蛋白Hsp70是负责蛋白质向叶绿体运输的动力马达。我们的前期工作证明超表达Hsp70转基因植株抗旱能力增强，但尚不清楚Hsp70参与叶绿体调控抗旱的分子机制。本项目旨在研究Hsp70在抗旱调控中的具体分子机制，主要开展以下工作：1）分析Hsp70启动子调控区域，揭示其转录水平上接受上游信号调控的分子机理；2）免疫共沉淀得到干旱复水处理后与Hsp70互作的蛋白质，揭示蛋白水平上调控抗旱相关因子的机制；3）RNA-seq分析Hsp70介导抗旱的退化途径中细胞核基因和叶绿体基因；4）利用我们已构建的Hsp70蛋白质ATPase活性改变突变体分析Hsp70调控抗旱的生物学基础。

本项目系统研究了小立碗藓叶绿体蛋白Hsp70在抗旱调控中的分子作用机制。1）上游调控方面，对小立碗藓Hsp70基因功能保守元件进行分析后发现，含有-500bp顺式元件的区段特异性响应干旱处理，该位点含有数个热激元件HSEa和HSEt；2）调控网络和机制方面，RNA-seq分析发现Hsp70基因以前期响应的记忆表达模式参与到了小立碗藓的干旱复水响应中，与定位于叶绿体的苔藓特异新基因互作调控细胞核内抗逆响应基因的表达和分子信号传递；3）蛋白互作方面，免疫共沉淀发现Hsp70蛋白水平上与Hsp93和TOC/TIC复合体互作形成异源六聚体转运叶绿体光合蛋白，以确保干旱处理后恢复时小立碗藓叶绿体膜结构完整；4）水稻中异源表达PpHsp70能激活水稻叶片抗逆通路。在此研究过程中为解析PpHsp70和调控网络基因家族功能开发的CRISPR/LbCPF1多靶标基因编辑，是苔藓植物中实现的第一例高效多靶标基因组编辑技术，为解析陆生起源基因家族的功能进化奠定了基础。在本执行期内，共发表7篇与项目相关的通信作者文章，申报7个与项目相关的专利 (其中3个已获得授权)。