转录因子CsERF025-like在硅诱导黄瓜抗盐性中的作用机理研究

Salt stress decrease plant production and quality. Application of exogenous silicon could to some extent mitigate the inhibiting effect of salt stress on plant growth and development. In previous study, we identified an ERF transcription factor CsERF025-like through transcriptome analysis (RNA-seq) that could be regulated by both NaCl and silicon treatment. Previous study also proved that silicon could regulate ethylene metabolism under salt stress. Since ERF (ethylene response factor) located in the downstream of ethylene signal pathway, it could be speculated that silicon may mediated ERF transcription factor CsERF025-like through regulating ethylene metabolism and thus alleviated salt stress. Based on the previous study, we will construct over-expression and RNAi vectors of CsERF025-like gene, make transgenic tomato plants, analyze their phenotypic changes under both NaCl and NaCl plus silicon treatment, from which the regulation effect of silicon in CsERF025-like could be confirmed. To determine the regulation mechanism of silicon in CsERF025-like, we will investigate the effect of exogenous ethylene precursor and ethylene biosynthesis inhibitor application on the expression pattern of CsERF025-like and physiology and biochemical index of cucumber seedlings. Moreover, abscisic acid (ABA) and salicylic acid (SA) content，the activities of ethylene and ABA synthesis enzymes and their genes expression patterns will be determined, which will help to reveal the relationship between the regulation effect of silicon in CsERF025-like and plant hormone ABA and SA. This study will help to shed light on the molecular mechanisms of silicon-induced stress tolerance and provide some scientific basis on the application of silicon fertilizers in agriculture.

盐胁迫严重影响作物的产量和品质。外源加硅可在一定程度上缓解盐胁迫对植物生长发育的抑制。申请人前期通过黄瓜转录组测序筛选到一个同时响应盐胁迫和硅处理的乙烯响应因子CsERF025-like。此外，前期研究证明硅可调控乙烯代谢，而乙烯信号调控下游ERF转录因子，暗示硅可通过调节乙烯信号通路来调控CsERF025-like的表达。以此为基础，本研究拟通过CsERF025-like超表达和沉默转基因材料的制备，结合生理生化指标测定，系统说明该基因在硅增强植物抗盐能力中的作用；通过提高或抑制乙烯合成，结合基因表达模式及相关指标测定，阐明硅通过乙烯调控CsERF025-like的作用机制；通过对赤霉素和水杨酸含量、关键酶活性和基因表达水平的分析，阐明硅对CsERF025-like的调控和逆境激素间的关系。本研究将有助于进一步揭示硅抗盐的分子机制，为硅肥在生产中的应用提供理论依据。

通过前期转录组分析，筛选得到一个受盐胁迫诱导的转录因子CsERF025-like。通过系统发育分析、转录因子激活活性分析以及亚细胞定位分析，明确了CsERF025-like为定位在细胞核，具有转录激活活性的转录因子。qPCR分析结果表明，加硅在不同处理时间点可以显著上调CsERF025-like的表达水平。随后构建CsERF025-like基因的超表达载体，通过农杆菌介导转化黄瓜和拟南芥，并筛选获得基因超表达的稳定转基因株系。针对野生型植株的研究结果表明，盐胁迫下加硅可以显著改善黄瓜植株的生长以及植株的水分状况。在盐胁迫下，黄瓜幼苗根系MDA、H2O2和电解质渗透率显著上升，加硅显著缓解了盐胁迫造成的MDA、H2O2和电解质渗透率的升高，缓解盐胁迫造成的膜脂过氧化。此外，同单独盐胁迫相比，加硅显著提高CAT活性、非蛋白巯基和抗坏血酸含量，降低POD和SOD活性，从而缓解了盐胁迫造成的氧化损伤。同时，逆境激素和脯氨酸代谢研究结果表明，加硅可以调节盐胁迫下脯氨酸和细胞分裂素代谢，并且这一调控作用和脯氨酸代谢相关酶活性以及细胞分裂素代谢关键基因表达，包括CKX和IPT等，从而调节脯氨酸和细胞分裂素在根系中的积累。烟草瞬时过表达CsERF025-like可通过调节活性氧迸发、气孔导度和水分散失提高烟草叶片的抗盐性和抗旱性，并且参与调控植物抗病响应基因的表达。无论加硅与否，拟南芥中稳定过表达CsERF025-like可显著提高植株抗盐性，且加硅可进一步提高转基因植株的抗盐性，并且这一抗性提高和脯氨酸以及细胞分裂素代谢密切相关。