油菜BnWRKY类转录因子参与干旱胁迫应答反应的功能分析

WRKY-type transcription factors (WRKY TFs) plays an important role indrought and salt stress responses in plants. The mechanism for WRKY TFs mediated stress response is still largely unknown. There are 277 BnWRKY-type genes in Brassica napus L.. Among them, 69 genes’ exprssion were sinifficantly regulated by drought stress, and 31 genes’ expression levels were more than 4 times differentially between drought stress and normal water condition by RNA-Seq. This project aims to identify thekey BnWRKY-type genes determining drought tolerance in Brassica napus. The key genes will be screened out through the large scale genes expression analysis with high throughput transformants identification. The biological functions of the key genes will be studied through the analysis on the morphorlogical, cytolaogical, biophysiological and biochemical traits of overexpressing transgenic plants under drought stress. By use of gene expression profiling and EMSA technology, the target genes of the key BnWRKY-type proteins with activity in enhancing drought tolerance will be identified and analyzed. According to the drought stress signal transduction networks of model plant Arabidopsis thaliana, primarily explore the key BnWRKY genes for drought stress responce in Brassica napus. Then use the genome editing techniques -CRISPR/Cas9 system and multi-gene over expression technology -MISSA system for the key genes functional verification in Brassica napus, preliminary elucidate the functions of these genes and collaborative regulatory mechanisms in drought-tolerance in Brassica napus, and provide important gene resources for crop drought-resistance improving.

已知WRKY类转录因子参与植物抗旱、耐盐等逆境反应，但其机制尚需探索。我们分析发现油菜基因组中有277个BnWRKY类家族成员。RNA-Seq分析表明，其中69个基因明显受干旱诱导表达，31个基因在干旱胁迫前后表达差异达4倍以上。本项目旨在探索这些转录因子在油菜耐受干旱过程中的作用机制。在大规模表达分析和高通量的转基因植物抗旱性筛选的基础上，初步鉴定响应干旱胁迫反应的BnWRKY基因；通过研究这些转基因植株形态、细胞和生理生化特征，分析这些候选基因的生物学功能；运用基因表达谱分析、EMSA等技术分析候选基因的下游靶基因及其互作。并借助拟南芥干旱胁迫信号转导网络，发掘参与油菜干旱应答的关键BnWRKY基因，进而运用CRISPR/Cas9系统和MISSA多基因组装技术在油菜中进行功能验证，初步阐明这些转录因子的耐旱反应机制及功能，为作物抗旱遗传改良提供新的基因资源。

WRKY类转录因子在植物抗旱、耐盐等逆境应答过程中发挥着重要的作用，序列分析发现油菜中有278个BnWRKY类转录因子家族成员。RNA-Seq分析表明，其中68个基因明显受干旱诱导表达，31个基因在干旱胁迫前后表达差异达4倍以上。本项目通过大规模表达分析和高通量的转基因植物筛选，从这些干旱胁迫响应的基因中挖掘和克隆了4个与抗旱密切相关的BnWRKY基因BnWRKY40、BnWRKY157、BnWRKY184和BnWRKY255；通过研究这些转基因植株形态、细胞和耐旱相关生理生化特征，探究其生物学功能；通过基因表达谱分析、互作分析挖掘它们的下游靶基因及其互作模式，初步阐明了这些关键BnWRKY基因在植物抗旱中的功能及调控机制，为作物抗旱遗传改良提供了新的基因资源，为利用BnWRKY基因进行作物抗旱遗传改良提供理论依据。. 项目自立项实施以来，以通讯作者和第一单位发表论文6篇，授权发明专利3项，育成油菜新品种3个，转让新品种1个。培养青年科技骨干教师3名，培养研究生7名，其中5名已毕业。