花生高亲和硝酸盐转运蛋白基因AhNRT2.7a响应低氮胁迫的功能研究

doi:10.3864/j.issn.0578-1752.2022.22.003

摘要/Abstract

摘要：

【目的】 氮素在作物生产中对生物量和产量起关键作用。高亲和硝酸盐转运蛋白基因NRT2在植物响应低氮胁迫时被激活并具有维持氮吸收和转运的作用。通过筛选花生低氮耐受相关的NRT2基因并解析其生物学功能，为培育高氮素利用率的花生新品种提供理论参考，最终有助于实现节氮、高效的绿色生产目标。【方法】 检测正常氮浓度及1/20正常氮浓度（15 mmol·L^-1）条件下5个具有典型跨膜结构的花生NRT2基因（AhNRT2.4、AhNRT2.5b、AhNRT2.5c、AhNRT2.7a和AhNRT2.7b）的时空表达情况。以花育6309的cDNA为模板，对AhNRT2.7a进行克隆和生物信息学分析，并通过亚细胞定位确定AhNRT2.7a的表达部位。进一步构建异源过表达AhNRT2.7a的拟南芥株系，分别在正常以及低氮胁迫条件下测定其叶绿素含量、氮积累量以及谷氨酰胺合成酶（GS）、谷氨酸合成酶（GOGAT）、硝酸还原酶（NR）、亚硝酸还原酶（NiR）、谷氨酸脱氢酶（GDH）5个氮代谢关键酶的活性。【结果】 5个NRT2基因中有4个NRT2基因在花生响应低氮胁迫条件下大量表达。其中，AhNRT2.7a能够响应低氮胁迫，并在花生茎和叶中高表达。获得AhNRT2.7a的cDNA序列，全长为1 380 bp，编码459个氨基酸。蛋白结构分析显示其为具有12个典型跨膜结构域的膜蛋白。该氨基酸序列与栽培种花生（Arachis hypogaea L.）相似性高达99.56%，其次是野生亲本AA和BB基因组花生。亚细胞定位显示其主要定位于细胞质膜上。构建异源过表达AhNRT2.7a的转基因拟南芥植株，在不同供氮条件下，成熟叶和幼叶叶片的叶绿素相对含量均显著高于野生型拟南芥。同时，对上述5个氮代谢相关酶活性以及氮、磷、钾积累量测定显示，转基因植株中2个氮代谢相关酶（GS和NR）的酶活性以及氮积累量均比野生型拟南芥有显著升高。【结论】 花生中4个NRT2基因响应低氮胁迫，其中AhNRT2.7a能够提高植物氮代谢过程的氮素利用率。AhNRT2.7a的表达在促进氮代谢过程的同时，促使碳代谢作用的进一步加强。因此，AhNRT2.7a适合作为花生氮素高效利用为目的的候选基因。

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

王娟,陈皓宁,石大川,于天一,闫彩霞,孙全喜,苑翠玲,赵小波,牟艺菲,王奇,李春娟,单世华. 花生高亲和硝酸盐转运蛋白基因AhNRT2.7a响应低氮胁迫的功能研究[J]. 中国农业科学, 2022, 55(22): 4356-4372.

WANG Juan,CHEN HaoNing,SHI DaChuan,YU TianYi,YAN CaiXia,SUN QuanXi,YUAN CuiLing,ZHAO XiaoBo,MOU YiFei,WANG Qi,LI ChunJuan,SHAN ShiHua. Functional Analysis of AhNRT2.7a in Response to Low-Nitrogen in Peanut[J]. Scientia Agricultura Sinica, 2022, 55(22): 4356-4372.

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基因名称 Gene name	正向引物序列 F-primer (5′-3′)	反向引物序列 R-primer (5′-3′)
AhNRT2.4	TGCCCTTTTGTATGCAACATTT	CTCATACCAAACAACCGGGC
AhNRT2.5b	TCTGTGTTTGTTCAAGCTGC	CCTCCTCCTGTCATTCCTGA
AhNRT2.5c	CTCTGTGTTTGTTCAAGCTG	TCCTCCTGTCATTCCTGATA
AhNRT2.7a	ACGGTCAAGATCTCCCTTCT	CACGCACCACAACAACAAAT
AhNRT2.7b	GGTTCTGGGACTGCTGTATG	ACCCCGAACCTGTCATAGAA
Actin	TTGGAATGGGTCAGAAGGATGC	AGTGGTGCCTCAGTAAGAAGC