小麦干旱记忆相关miRNA的鉴定及其生物学功能研究

Drought memory, which is a conservative stress resistance mechanism, is developed during long-term evolution of plants under abiotic stresses. Understanding the mechanisms of wheat drought memory (drought exercise) and its functional traits is crucial for improving wheat drought resistance and ensuring wheat production. However, little information is available regarding which miRNAs are activated by memory functions during the drought-rehydration-drought process for wheat, and what are the main miRNAs participate in regulatory networks in response to drought stresses? Furthermore, what are the biological functions of candidate miRNAs, and do these miRNAs improved the drought resistance of crops? These questions are also need to be addressed. Here, this study will be performed with the following three objectives: (1) to systematically identify wheat miRNAs related to drought memory and its target genes by using high-throughput sequencing. Moreover, key miRNAs will be screened on the base of target genes and co-expression regulatory networks; (2) to explore the spatial and temporal expression patterns of miRNAs and target genes by using Northern blot and qPCR techniques; (3) to investigate biological functions of the key miRNAs by constructing the STTM(over-expression vector). Collectively, the goal of this study is to elucidate the molecular mechanism of drought memory in wheat. Additionally, the results are of important significance and will provide scientific evidences for improving the drought resistance of wheat.

干旱记忆是植物在长期进化过程中形成的保守性抗逆机制，深入理解小麦干旱记忆(干旱锻炼)的分子机制和生物学功能，对提高小麦的抗旱性、保障小麦生产具有重要科学意义。然而小麦干旱-复水-干旱过程中，干旱记忆功能激活了哪些miRNAs的表达，且它们又参与了哪些调控网络去响应干旱胁迫这一过程？以及小麦干旱记忆响应关键miRNAs具有哪些生物学功能，能否显著提高作物的抗旱能力？这些问题都有待回答。鉴于此，本项目拟首先采用高通量测序技术鉴定小麦干旱记忆相关miRNAs及其靶基因，并基于靶基因和共表达调控网络获得关键miRNAs；其次采用Northern blot和qPCR技术验证及明确候选miRNAs在小麦干旱胁迫记忆过程中的表达调控方式；最后构建STTM/过表达载体来研究小麦干旱记忆关键miRNAs的生物学功能。旨在阐明miRNAs参与调控小麦干旱记忆的作用机制，为小麦抗旱遗传改良提供科学依据。

干旱记忆是植物在长期进化过程中形成的保守性抗逆机制，深入理解小麦干旱记忆的分子机制和生物学功能，对提高小麦的抗旱性、保障小麦生产具有重要科学意义。本研究首先对中国春小麦在干旱记忆、直接干旱过程中脯氨酸含量和Phi2效率进行测定，结果表明小麦经过干旱胁迫后，干旱记忆可以提高其抗旱性；在此基础上，采用小RNA测序对中国春小麦在干旱记忆、直接干旱过程中的miRNA表达谱进行分析，共鉴定到1481个miRNAs，包括644个小麦已知的miRNAs、837个保守的miRNAs和146个新的miRNA；通过差异表达模式分析获得228个直接干旱响应miRNA和195个干旱记忆响应miRNA，降解组测序验证64个干旱记忆响应miRNA的靶基因。进一步，解析干旱响应miRNA及靶基因的调控网络，获得42个显著富集的干旱记忆响应miRNA的靶基因，基于关联分析发现干旱记忆表现出转录应激记忆，它参与调节生长素信号转导通路、钙转运、重金属转运、谷胱甘肽生物合成来提高小麦的抗旱性；此外，这些关键的干旱记忆响应miRNA可以调节多种植物激素和葡萄糖代谢途径，间接调节脯氨酸稳态，进一步提高小麦的抗旱性。同时，选取10个干旱记忆响应miRNA及18个靶基因在小麦干旱记忆过程中的表达特性进行分析，7个miRNA在直接干旱和干旱记忆中具有相反的表达模式。最后，分别构建了miRNA531及其靶基因wcold410的过表达载体并转化了拟南芥和小麦，获得T3代阳性转基因拟南芥植株和T1代阳性转基因小麦植株，T3代纯合转基因拟南芥株系的芽期和苗期抗逆性鉴定发现过表达miR531株系能显著提高其抗旱性，过表达Wcor410b株系能显著提高其耐寒性，初步证实了miR531和Wcor410b的生物学功能。本研究在干旱记忆过程中miRNA及靶基因进行了初步的鉴定和挖掘，这为深入揭示小麦干旱记忆的分子机理奠定理论基础，也为小麦抗旱遗传改良提供科学依据。