菊花耐涝关键基因挖掘及其调控机制

Chrysanthemum, one of the top ten traditional famous flowers in China and one of the four cut flowers in the world, is of great ornamental and economic values. Chrysanthemum is readily sensitive to waterlogging which greatly hampers its growth, yield and quality. We previously found that the expression levels of CmACS1/6/7 encoding ethylene biosynthesis rate limiting enzyme ACC synthases (ACS) and CmMBF1c as the homolog of multiprotein bridging factor 1c (MBF1c) were both induced by waterlogging, and were higher in waterlogging tolerant chrysanthemum cultivars than that in sensitive ones, the promoter of CmMBF1c is more sensitive to hypoxia by transient assay, furthermore the CmMBF1c could interact with ET signaling member of CmHRE2. We suppose that CmACS1/6/8 and the CmMBF1c may be involved in the ET signaling waterlogging tolerance in chrysanthemum, however, the mechanisms remained unknown. Here, we attempt to clone CmACS1/6/8 and CmMBF1c, and perform functional analysis of both genes, and aimed to discover those transcription factors regulating CmACS genes transcription with yeast one-hybrid and the key regulatory and interacting genes of CmMBF1c involved in ethylene signaling to waterlogging, to identify key genes’ functions and regulatory traits via generating corresponding RNA interfering and overexpression transformants, and mining downstream differential gene expression profiles in the transformants by transcriptome sequencing, to disclose the regulatory roles of regulatory and interacting genes in regulating pathway by using EMSA, BiFC, CoIP and ChIP. In sum, present proposal will discover a number of key genes involved in ethylene mediated waterlogging response and molecular mechanisms underlying waterlogging tolerance, which in turn will give aid to genetic manipulation of chrysanthemum waterlogging tolerance by providing candidate genes and theoretic guidance.

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值高。涝渍胁迫是其主要胁迫因子之一，乙烯（ET）是涝渍早期响应信号。我们发现，菊花耐涝品种中ET合成基因CmACS1/6/7、乙烯相关基因CmMBF1c表达水平及CmMBF1c启动子响应低氧活性较高；CmMBF1c与ET信号成员CmHRE2互作，推测菊花存在CmACS1/6/7和CmMBF1c介导ET应答涝渍胁迫的新机制。为此，本项目拟以耐涝和不耐涝品种为材料，进行CmACS1/6/7和CmMBF1c的克隆与功能鉴定，采用酵母单杂交筛选CmACS1/6/7的调控蛋白，使用酵母双/单杂交筛选CmMBF1c的互作蛋白/调控蛋白；克隆筛选的关键基因，运用超表达或干扰技术获得转基因植株，采用转录组测序发掘下游调控基因，从而解析以CmACS1/6/7和CmMBF1c为核心的菊花涝渍胁迫应答信号路径，为菊花耐涝分子改良提供基因资源和理论依据。

菊花具有极高的观赏、经济和文化价值，在花卉产业中占有重要地位。涝渍胁迫是制约菊花产业健康发展的要因之一。针对目前菊花耐涝关键基因挖掘及分子机制研究滞后问题，本研究以通过RNA-seq筛选出来的响应菊花淹水胁迫的乙烯合成酶基因CmACSs和多蛋白桥梁因子CmMBF1c基因为核心展开深入研究。结果发现，CmACS6基因负调控菊花耐涝性；利用DNA affinity trapping技术筛选到CmACS6潜在的上游调控因子CmHRE2-like，通过Y1H、LUC、EMSA和ChIP-qPCR等实验证实CmHRE2-like可以直接结合在CmACS6启动子上的GCC-box元件，并激活CmACS6的表达；通过转基因功能验证揭示了CmHRE2-like负调控菊花的耐涝性；在CmHRE2-like过表达株系中瞬时沉默CmACS6基因可以缓解淹水胁迫对菊花造成的损伤，表明CmHRE2-like调控菊花应答淹水胁迫依赖于CmACS6。对CmMBF1c转基因菊花进行耐涝性鉴定发现，过表达CmMBF1c可以显著增强不耐涝菊花品种‘清露’的耐涝性，而人工干扰CmMBF1c的表达可以显著降低耐涝菊花品种‘南农雪峰’的耐涝性；通过生理指标测定推测CmMBF1c主要通过增强ROS清除酶活性，降低植株体内ROS水平，正调控菊花耐涝性；采用酵母单杂交筛库方法筛选到CmMBF1c潜在的上游调控因子CmTEM1，通过酵母单杂交、烟草LUC和菊花原生质体实验证实CmTEM1可以直接结合CmMBF1c启动子，并抑制CmMBF1c的表达；异源过表达CmTEM1可以显著降低拟南芥的耐涝性。另外，通过酵母双杂交和BiFC实验验证了CmMBF1c与CmHRE2蛋白互作，并发现CmHRE2异源过表达拟南芥株系表现出淹水耐受表型。本研究鉴定了多个菊花耐涝关键基因，阐明了CmACS6和CmMBF1c调控菊花耐涝性的分子机制，为菊花耐涝性分子改良奠定了理论基础，也可为其他植物耐涝分子机理的研究提供借鉴。项目研究成果在Plant Biotechnology Journal、Plant Physiology、Journal of Experimental Botany、Horticulture Research等刊物发表论文14篇，获国家技术发明奖二等奖、神农中华农业科技奖优秀创新团队奖、江苏省科学技术奖一等奖等奖励。