油菜抗旱基因高通量克隆及功能分析

Rapeseed, as an important oil crop, is inclined to be subjected drought injury due to its relatively sensitive to drought stress among the primary crops. Using transcriptome sequencing (RNA-Seq) with comparative genomic analysis, 68 genes were screened out maybe involving in drought stress response in Brassica napus. these 68 genes were intrduced into Arabidopsis to construct rape FOX Arabidopsis mutant library by Gateway and FOX hunting system, including 378 FOX strains. In this study, take these rape FOX Arabidopsis mutants as materials, select drought-tolerant rape FOX transformants by high throughput far-infrared thermal imaging technology. Combining with drought-resistant phenotypic, physiological and cytological analysis, isolate candidate genes related drought stress from rape FOX Arabidopsis plants by PCR and sequencing. By gene expression analysis and according to the model plant Arabidopsis thaliana drought stress signal transduction networks, primarily explore key 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 oilseed rape, preliminary elucidate the molecular mechanisms of these genes and collaborative regulatory mechanisms in drought-resistance in canola, and provide important gene resources for rapeseed drought genetic engineering.

油菜是我国重要的油料作物，同时又是耐旱性较差的作物，干旱严重制约其生产的发展。本课题组运用转录组测序（RNA-Seq）结合比较基因组学分析，筛选到68个可能参与油菜干旱胁迫响应的基因，并运用高通量的Gateway技术和FOX hunting system将68个基因全长cDNA转入拟南芥，获得包含378个转基因株系的油菜FOX拟南芥突变体库。本研究以这些油菜FOX植株为材料，运用高通量的远红外热成像技术筛选抗旱的油菜FOX突变株，结合抗旱表型、生理及细胞学分析，克隆获得相应的油菜干旱相关基因。通过基因表达分析并借助油菜近缘模式植物拟南芥干旱胁迫信号转导网络，发掘油菜干旱响应的关键节点基因。

本研究运用转录组测序（RNA-Seq）结合比较基因组学分析，筛选到68个可能参与油菜干旱胁迫响应的基因，并运用高通量的Gateway技术和FOX hunting system将68个基因全长cDNA转入拟南芥，两批次共获得包含727个转基因株系的油菜FOX拟南芥突变体库。以这些油菜FOX植株为材料，运用高通量的远红外热成像技术，结合抗旱表型、生理及细胞学分析，克隆获得20个油菜干旱相关基因，并全部转入油菜，正在进行转基因油菜纯合体鉴定和抗逆性分析。对其中7个基因进行了组织特异性表达分析和逆境诱导表达分析，结果表明，这7个基因均在子叶和根中高表达，并受干旱和盐的诱导表达。其中2个基因的亚细胞定位结果表明它们定位于细胞核中，是一类型转录因子。由于油菜生长周期长，项目执行期只有两年，进一步的机制分析仍在进行中。发表论文2篇，其中SCI（IF=5.228）1篇；育成杂交油菜新品种1个：开油1208，已通过河南省农作物品种审定委员会审定；申请专利2项；参与项目的毕业研究生2个、在读研究生3名。在研期间主持人由副教授晋升为教授，一名骨干成员由实验师晋升为高级实验师。