关键词: 花生, nrt2, AhNRT2.7a, 氮素效率, 氮代谢相关酶

Abstract: 【Objective】Nitrogen (N) plays a key role in determining biomass and yield in crop production. NRT2s, the high affinity nitrate transporter genes, are mainly activated under low nitrogen stress condition and have been implicated in nitrate absorption and remobilization. This study will screen NRT2 gene family responding to low-nitrogen condition (1/20 of the normal level) and conduct a preliminary functional analysis of AhNRT2.7a in order to provide target genes for breeding new peanut varieties with higher nitrogen utilization efficiency (NUE)，which will help to achieve the goal of to improve crop production with less N fertilizer demand and environmental degradation. 【Method】The spatio-temporal expression patterns under normal and low-nitrogen conditions of five peanut NRT2 genes, AhNRT2.4, AhNRT2.5b, AhNRT2.5c, AhNRT2.7a and AhNRT2.7b, were investigated. Using the cDNA of Huayu6309 as template, full length of AhNRT2.7a CDS was cloned and bioinformatic analyzed. Subcellular localization of AhNRT2.7a was conducted by construction of transient expression vector and transformation of Arabidopsis protoplasts. In order to explore the gene function of AhNRT2.7a, heterologous overexpression of the AhNRT2.7a gene in Arabidopsis were performed. Transgenic plants were used to determine chlorophyll content, nitrogen accumulation and the enzymatic activities of glutamine synthetase (GS), glutamate synthetase (GOGAT), nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) under normal and low-nitrogen conditions.【Result】Four NRT2 genes of peanut were highly expressed in response to low nitrogen stress, and AhNRT2.7a was highly expressed in the stems and leaves. The total length of 1 380 bp was obtained, encoding a 459-amino acid protein with a molecular weight of 49.35 kD. The total of 12 typical transmembrane protein domains with hydrophobic regions was predicted. Bioinformatics analysis showed that the amino acid sequence had 99.56% sequence similarity with the cultivated peanut (Arachis hypogaea L.), followed by the wild-parents AA (A. duranensis) and BB (A. ipaensis). Subcellular localization analysis revealed that AhNRT2.7a was located in the cell membrane. Transgenic Arabidopsis plants for over-expressing AhNRT2.7a were conducted. Relative content of chlorophyll in mature and young leaves was significantly higher than that in wild-type Arabidopsis under different nitrogen supply. Meanwhile, the activity of five enzymes involved in nitrogen metabolism were examined. Furthermore, uptake, assimilation and re-mobilization of N, concentration of phosphorus and potassium were determined. The results have revealed that the activity of the two nitrogen metabolizing enzymes (NR and GS) and nitrogen accumulation in transgenic plants were significantly higher than in wild-type Arabidopsis.【Conclusion】These results indicated that AhNRT2.7a could enhance the nitrogen use efficiency (NUE) in plants, and also improve carbon metabolism. AhNRT2.7a seems promising as a candidate gene in breeding new peanut varieties with higher NUE.

Key words: peanut,  , nrt2,  , AhNRT2.7a,  , nitrogen efficiency, enzymes related to nitrogen metabolism