甘薯盐诱导高表达基因IbNAC4的分离及其耐盐机理研究

Sweetpotato (Ipomoea batatas) is the bottom line to ensure the food security in our country, but the soil salinization severely restricts its growth and yield. NAC transcription factor family has been found to be widely involved in the regulation of plants to environmental stress. However, there is no report on the salt tolerance of NAC gene in sweetpotato, here the salt tolerant cultivar Xu 22 and the salt sensitive cultivar Xu 32 were employed, and the IbNAC4 gene, induced significantly by salt stress was screened by RNA-seq and qRT-PCR. To further understand the functions and molecular mechanisms of IbNAC4 in response to salt stress, the following works will be roundly done in present project: (1) over-express and silence IbNAC4 in sweetpotato to investigate its functions in salt tolerance, and its application in improving tomato salt tolerance will be also studied for the first time by heterologous transformation; (2) identify the downstream target genes regulated by IbNAC4 via a series of molecular biological methods such as RNA-seq and EMSA; (3) screen and confirm the interaction factors of IbNAC4 by yeast two-hybrid system and BiFC to elucidate the regulatory network and molecular mechanisms of IbNAC4 in salt tolerance. This project will help to better understand the molecular mechanisms of sweetpotato to salt stress, and provide theoretical evidence and material basis for the breeding of high salt-tolerant sweetpotato.

甘薯是保证我国粮食安全的底线作物，但土壤盐碱化严重制约了其生长与产量。NAC转录因子家族被证实广泛参与了植物逆境胁迫调控，然而，目前在甘薯中对NAC基因的耐盐功能研究尚未见报道。前期研究中，我们通过RNA-seq与qRT-PCR分析从耐盐性品种徐薯22与盐敏感性品种徐薯32中筛选出一个受盐胁迫显著诱导的IbNAC4基因。本研究拟运用超表达和RNAi等反向遗传学手段探究其在甘薯中的耐盐功能，并通过异源转化首次在番茄中探索IbNAC4基因的耐盐应用价值；同时，拟利用RNA-seq及凝胶阻滞电泳（EMSA）等一系列分子生物学手段鉴定IbNAC4直接调控的下游靶基因，应用酵母双杂交筛库和双分子荧光互补（BiFC）确认IbNAC4的互作因子，进而揭示IbNAC4基因参与甘薯耐盐胁迫的调控网络和作用机理。本研究成果将有助于进一步理解甘薯耐盐的分子机制，并为培育高耐盐甘薯新品种提供理论依据和材料基础。

甘薯是保证我国粮食安全的底线作物，具有抗性强、耐贫瘠等特性，且具有投入少、产量高、稳产性好等优点。此外，甘薯还具有重要的保健功能和工业应用价值。然而，土壤盐碱等胁迫严重制约了甘薯的生长与产量，因此，深入研究甘薯的耐盐特性具有重要意义。本研究围绕IbNAC3（IbNAC4更名为IbNAC3）的筛选及其在盐胁迫响应中的功能及调控机理展开探究。结果表明：1. 利用前期实验中获得的耐盐品种徐22与盐敏感品种徐32在盐胁迫下的RNA-seq数据，筛选到12个盐胁迫响应IbNAC基因，生物信息学分析表明这些IbNAC的NAC结构域高度保守，C端序列则高度变异。qRT-PCR分析表明大多数IbNAC基因的表达显著地受多种非生物胁迫及激素处理的诱导。2. 筛选出IbNAC3基因进行功能验证和作用机理解析。结果表明IbNAC3定位于细胞核，具有独特的转录激活域：其N端前9个氨基酸以及完整的C末端是其转录激活活性所必须的。表型、生理生化及基因表达检测证实超表达IbNAC3增强了转基因拟南芥植株的耐盐性。另外，利用IP-MS技术筛选，并通过酵母双杂交、双分子荧光互补、荧光素酶互补成像实验证实IbNAC3能够与AtNAP、ANAC083、ANAC100、ANAC072及ANAC011相互作用，且证实IbNAC3蛋白的不同或相似区域分别与上述5个NAC蛋白相互作用。通过ChIP-seq筛选，并利用ChIP-qPCR、酵母单杂交及EMSA实验证实IbNAC3能够与胁迫相关的AtCHX25, AtZFP及AtRH33基因的启动子结合。双荧光素酶报告实验表明IbNAC3能够激活上述基因启动子的转录活性，且IbNAC3与ANAC072的互作增强了AtZFP基因启动子的转录激活活性。此外，通过多重实验证实IbNAC3能够直接调控并促进ABA信号转导负调控基因AtERA1的表达，从而减弱了转基因植株对ABA的敏感性。另外，我们初步获得了8株超表达IbNAC3的转基因甘薯。以上结果系统揭示了IbNAC3基因参与耐盐胁迫的调控网络和作用机理，将有助于进一步解析甘薯耐盐的分子机制。