碳离子束诱导的百脉根花瓣衰老延迟性状关联基因的挖掘及功能验证

The plant appearance quality and the economic value can be greatly promoted by slowing down the speed of petal senescence. It is one of the most environmentally friendly, and efficiently means to extend the lifespan of flower by mutation breeding of new varieties. Carbon-ion beam irradiation, as a powerful physical mutagen, has been widely used in plant breeding and functional genomics research in the post-genome era due to its unique physical and biological advantages. However, there is few study about its improvement effect on petal senescence, meanwhile, mining the key gene resources that are responsible for the mutation traits is relatively weak. Lotus japonicus (MG20), as a potential grass variety is widely used in virescence beautification, and animal husbandry. In our previous work, a novel stable mutant of Lotus japonicus numbered as C416 showing delayed petal senescence was induced by carbon ions accelerated by HIFRL-CSR. Based on the primary association analysis, we found that there were partially overlapping information for mutant genes detected by whole genome resequencing and transcriptome sequencing. Taking this as the breakthrough point, screening and identification of the mutant gene that responsible for the delayed petal senescence of C416 will be performed in this project, as well as the functional verification of the candidate genes by constructing the RNAi and overexpression lines. And then making a deeply excavation of transcriptome data to reveal the genetic mechanism of delayed petal senescence of C416. This study will provide theoretic foundation for research of petal aging in Lotus japonicus or other leguminous plants, as well as to set up an effective method to mine the associated genetic resources of mutation traits that induced by heavy ions radiation.

延缓花瓣衰老速度可极大地提升植物外观品质及经济价值，新品种诱变选育是延长花寿命最为环保经济的手段之一。重离子束辐射是植物诱变育种的重要手段，但其对花瓣衰老性状的改良鲜有报道，性状关联基因资源的挖掘也相对薄弱。前期研究中，利用碳离子束辐射诱变集观赏饲用价值为一体的优质草种百脉根，获得了花瓣衰老延迟突变体C416。基因组和转录组测序结果提示，花瓣衰老相关变异基因信息在二者之间存在重叠。以此为切入点，本项目拟以C416为研究对象，结合前期研究结果，开展性状关联基因的筛选，通过构建候选基因过表达及RNAi沉默株系进行功能验证，以期明确控制C416花瓣衰老延迟性状的关键基因；结合转录组测序在网络调控研究中的优势，系统揭示碳离子束辐射诱导的花瓣衰老延迟性状遗传机制，为豆科植物花器官衰老研究提供理论基础，同时建立起一套行之有效的手段服务于重离子束辐射诱导的突变性状关联基因资源的挖掘。

花寿命与花粉在植物中的传播和繁殖密切相关，同时花期也决定了观赏植物的商业价值。花期长短改变的突变体解析以上特性的有利工具。重离子束辐照在改善植物花形花色方面已有大量报道，但其对花瓣衰老性状的改良鲜有报道，性状关联基因资源的挖掘也相对薄弱。利用中能碳离子束辐照诱变技术，获得了百脉根花器官衰老延迟的突变体C416，其盛花期长达野生型植株的8.03倍。遗传分析表明，C416花延迟衰老为单基因显性遗传，该基因位于第III号染色体4616611 ~ 5331876 Mb之间。建立多样本基因组测序数据分选策略，并进行可行性验证。基于此策略，将候选基因锁定在CUFF.40834，该基因在其他模式植物中表现出与ETR1（乙烯受体）高度一致的特性。同时，生理实验表明C416对乙烯前体不敏感。与前人研究中通过转基因技术引入外源ETR1显性突变基因引起的花衰老延迟突变体不同，C416是首次报道的内源性ETR1突变体。基于不同发育阶段花瓣转录组分析，以植物激素为切入点，系统研究了激素调控网络导致C416花瓣衰老延迟的分子机制。简言之，乙烯、茉莉酸和水杨酸信号通路在C416中呈负调控模式，油菜素内酯和细胞分裂素信号通路呈正调控模式，生长素对花器官衰老表现出双重作用模式。脱落酸信号通路在C416中受到正向调控，表征当乙烯信号通路被阻断时，ABA可能是促进C416花器官衰老的主要激素因子。项目的执行为鉴定百脉根花器官衰老基因奠定了基础，同时建立起一套行之有效的手段服务于重离子束辐射诱导的突变性状关联基因资源的挖掘，也为重离子束在提高豆科植物花寿命的应用提供了新视角。