硝酸盐诱导的GARP类转录因子AhNIGT1.2调控花生结瘤的功能分析

Functional analysis of a nitrate-induced GARP transcription factor AhNIGT1.2 in peanut nodulation

摘要:

花生是重要的经济及油料作物，富含丰富的蛋白质和油脂等，在我国广泛种植。花生能与根瘤菌共生形成根瘤，根瘤的固氮酶能将空气中N₂转化为自身可吸收利用的氨态氮。解析结瘤固氮对避免化肥过度施用、发展可持续农业具有积极意义。本研究通过生物信息学分析鉴定到一个主要在花生根瘤中表达的NIGT家族成员AhNIGT1.2。进一步的时空表达分析表明AhNIGT1.2在根瘤中高表达，且显著响应高氮、低氮、高磷、低磷和根瘤菌处理，组化染色表明该基因主要在发育的根瘤中表达，并在成熟根瘤中和花生根系连接处表达。亚细胞定位表明35S::AhNIGT1.2-GFP融合蛋白定位于烟草表皮细胞的细胞核中。进一步的功能研究发现AhNIGT1.2-OE显著提高了花生的结瘤数量，AhNIGT1.2-RNAi显著抑制花生根瘤数量，表明AhNIGT1.2正向调控花生结瘤。对AhNIGT1.2-OE根中硝酸盐转运和信号通路中的基因表达进行分析发现，NRT1.2、NRT2.4、NLP1和NLP7的表达量显著降低，表明AhNIGT1.2可能通过影响硝酸盐转运和NLP1相关基因的表达进而调控花生的结瘤。对转化的AhNIGT1.2-OE和对照根进行转录组分析，发现过表达AhNIGT1.2会显著富集硝酸盐响应、结瘤因子信号通路、四萜类生物合成酶和类胡萝卜素生物合成等途径的差异表达基因。综上所述，AhNIGT1.2可能通过影响硝酸盐转运、硝酸盐响应等通路调控花生的结瘤过程。本研究对解析氮磷营养调控豆科植物结瘤固氮的分子机制和创制在高氮环境高效结瘤固氮的豆科作物具有重要意义。

关键词:花生;AhNIGT1.2;硝酸盐转运;结瘤;转录组

Abstract:

Peanut, a major economic and oil crop known for the high protein and oil content, is extensively cultivated in China. Peanut plants have the ability to form nodules with rhizobia, where the nitrogenase converts atmospheric nitrogen into ammonia nitrogen that can be utilized by the plants. Analysis of nodule fixation is of positive significance for avoiding overapplication of chemical fertilizer and developing sustainable agriculture. In this study, AhNIGT1.2, a member of the NIGT family predominantly expressed in peanut nodules, was identified by bioinformatics analysis. Subsequent spatiotemporal expression analysis revealed that AhNIGT1.2 was highly expressed in nodules and showed significant responses to high nitrogen, low nitrogen, high phosphorus, low phosphorus, and rhizobia treatments. Histochemical staining indicated that the gene was primarily expressed in developing nodules and at the connection region between mature nodules and peanut roots. The fusion protein AhNIGT1.2-GFP was located in the nucleus of tobacco epidermal cells. The AhNIGT1.2-OE significantly increased the number of peanut nodules, while AhNIGT1.2-RNAi reduced the number of nodules, which suggested a positive regulatory role of AhNIGT1.2 in peanut nodulation. The AhNIGT1.2-OE in roots down-regulated the expression levels of NRT1.2, NRT2.4, NLP1, and NLP7, which indicated that AhNIGT1.2 influenced peanut nodulation by modulating nitrate transport and the expression of NLP genes. The transcriptome analysis of AhNIGT1.2-OE and control roots revealed that overexpressing AhNIGT1.2 significantly enriched the differentially expressed genes associated with nitrate response, nodulation factor pathway, enzymes for triterpene biosynthesis, and carotenoid biosynthesis. These findings suggest that AhNIGT1.2 play a key role in peanut nodulation by regulating nitrate transport and response and other related pathways. This study gives insights into the molecular mechanisms of nitrogen and phosphorus in regulating legume nodulation and nitrogen fixation, and sheds light on the development of legume crops that can efficiently fix nitrogen in high nitrogen environments.

Key words:peanut; AhNIGT1.2;nitrate transport;nodulation;transcriptome