甘薯转录因子IbC3H18调控耐盐抗旱性的分子机制研究

Arid and semi-arid land covers 52.5% of the total land area in China, among which 540 million mu arable land is saline, accounting for about 30% of the cultivated land. With the global warming, salinity, drought and other natural disasters are becoming more and more serious. As one of important food and energy crops, sweetpotato enjoys the advantages of high and stable production on comparatively barren soil conditions, which makes China the largest sweetpotato producer in the world.Based on the analysis of salt-tolerant sweetpotato RNA-Seq data in the previous studies funded by National Natural Science Foundation, a salt and drought tolerance related transcription factor IbC3H18 was screened. IbC3H18 was cloned and its expression was significantly up-regulated under salt and drought stress. In this study, the salt and drought tolerant function of IbC3H18 will be identified using over-expression and RNA interference technology in sweetpotato. The yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and Co-Immunoprecipitation (Co-IP) technologies will be used to screen and identify the proteins that interact with IbC3H18. The upstream and downstream genes of IbC3H18 will be screened by yeast one hybrid (Y1H) and ChIP-seq technologies and their functions will be further identified, respectively. Moreover, the molecular mechanism involved in salt and drought tolerance of this transcription factor will be well characterized. The success of this research will not only provide important gene material for breeding sweetpotato varieties with salt and drought tolerance, but also provide the theoretical basis for the study of gene regulatory network about salt and drought tolerance of sweetpotato.

我国干旱、半干旱地区面积占国土面积的52.5%，其中盐渍化耕地达5.4亿亩，占耕地面积的约30%。随着全球气温升高，盐碱和干旱等自然灾害越来越严重。我国是世界上最大的甘薯生产国，甘薯作为重要的粮食和能源作物之一可以种植于滩涂瘠薄土地。通过上一个国家自然科学基金项目的甘薯耐盐转录组分析，筛选克隆了耐盐抗旱相关的转录因子IbC3H18，该转录因子在盐旱胁迫下显著上调表达。本项目在此基础上，将IbC3H18导入甘薯，鉴定过表达及干扰表达甘薯植株的耐盐性和抗旱性，确认IbC3H18的功能；用酵母双杂筛选互作蛋白，并用双分子荧光互补和免疫共沉淀技术对其进行验证；用酵母单杂筛选上游基因、ChIP-seq筛选下游基因并进行鉴定，分析IbC3H18调控耐盐抗旱性的分子机理。研究结果不仅为甘薯耐盐抗旱育种提供重要的基因及材料，同时也为揭示甘薯耐盐抗旱性的调控网络提供理论依据。

项目通过分析甘薯耐盐转录组，筛选克隆到转录因子IbC3H18，该转录因子在盐旱胁迫下显著上调表达。通过农杆菌介导法，将IbC3H18导入甘薯，获得IbC3H18的过表达及RNAi沉默甘薯转基因植株，系统的离体、水培及盆栽鉴定结果表明，过表达该基因能够显著增强转基因甘薯的耐盐、抗旱及抗氧化性，而RNAi沉默植株的抗性显著降低。对盐胁迫下的过表达及RNAi沉默甘薯株系进行了RNA-seq和ChIP-seq分析，结果表明IbC3H18能够结合在氧清除相关基因CCS、ABA受体基因PYL8及抗逆相关基因SOS5的启动子上激活这些基因的表达。结合RNA-seq差异基因富集结果及生理生化指标测定结果，过表达IbC3H18通过激活ROS清除系统和ABA信号途径，增加光合活性，维持离子渗透平衡来提高甘薯的抗逆性。同时通过酵母双杂交、荧光素酶互补（LCI）、双分子荧光互补（BiFC）及免疫共沉淀（Co-IP）技术获得并验证了IbC3H18的互作蛋白IbPR5。过表达IbPR5能够通过激活ROS清除系统提高转基因烟草的抗逆性。该研究阐明了甘薯抗逆转录因子IbC3H18调控甘薯逆境应答的分子机制。研究结果发表于New Phytologist杂志。这不仅为甘薯耐盐抗旱育种提供重要的基因及材料，同时也为揭示甘薯耐盐抗旱性的调控网络提供理论依据。. 此外，通过耐盐抗旱的研究，我们发掘到甘薯蔓割病抗性基因IbBBX24， 过表达IbBBX24基因能够显著提高转基因甘薯的茉莉酸含量，并增强其蔓割病抗性且不引起产量下降，而RNAi沉默植株的抗性显著降低。IbBBX24可以在转录水平及蛋白水平上调控茉莉酸信号转导途径的关键信号分子，从而通过茉莉酸途径增强甘薯的抗病性。研究结果发表于The Plant Cell杂志。. 本项目研究圆满完成了研究目标。研究结果发表于国际著名的New Phytologist和The Plant Cell等杂志，共发表论文6篇，其中SCI论文5篇 ，会议论文1篇；受权国家发明专利3项；培养了1名博士和5名硕士。