草地早熟禾硝酸盐转运蛋白<i>NRT2.4</i>基因序列的克隆及表达分析

doi:10.7668/hbnxb.20191685

华北农学报 ›› 2021, Vol. 36 ›› Issue (2): 1-8. doi: 10.7668/hbnxb.20191685

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草地早熟禾硝酸盐转运蛋白NRT2.4基因序列的克隆及表达分析

陈阳^1,2, 金一锋^1,2, 金忠民¹, 王玉书¹, 蔡荣建¹, 邵竹林¹, 王杰¹

1. 齐齐哈尔大学生命科学与农林学院, 黑龙江齐齐哈尔 161006;
2. 黑龙江省抗性基因工程与寒地生物多样性保护重点实验室, 黑龙江齐齐哈尔 161006

收稿日期:2020-12-24 出版日期:2021-04-28
通讯作者: 金一锋(1984-),男,黑龙江鹤岗人,讲师,在读博士,主要从事园林植物逆境相关研究。
作者简介:陈阳(1986-),女,黑龙江齐齐哈尔人,副教授,博士,主要从事园林植物逆境相关研究。
基金资助:
国家自然科学基金项目（31701958）；黑龙江省自然科学基金项目（QC2017026）；黑龙江省普通高等学校青年创新人才培养计划项目（UNPYSCT-2018102）；黑龙江省教育厅基本科研业务费科研项目（135309365）；齐齐哈尔大学大学生创新创业项目（202010232161；202010232170）

Cloning and Expression Analysis of NRT2.4 Gene in Kentucky Bluegrass

CHEN Yang^1,2, JIN Yifeng^1,2, JIN Zhongmin¹, WANG Yushu¹, CAI Rongjian¹, SHAO Zhulin¹, WANG Jie¹

1. College of Life Science and Agro-Forestry, Qiqihar University, Qiqihar 161006, China;
2. Heilongjiang Province Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, Qiqihar 161006, China

Received:2020-12-24 Published:2021-04-28

摘要/Abstract

摘要： 植物硝酸盐转运蛋白（Nitrate transporter，NRT）可有效调节与转运NO₃^-，提升氮素利用效率。分析草地早熟禾硝酸盐转运蛋白NRT2.4基因在不同氮素浓度下的表达规律，旨在揭示硝酸盐转运蛋白NRT2.4基因在氮素调控中扮演的角色。以草地早熟禾为材料，首先设计特异引物，利用RT-PCR技术克隆得到草地早熟禾NRT2.4基因cDNA序列，其长度为1 694 bp，包含一个1 257 bp的CDS区，编码418个氨基酸序列，属于Nitrate/nitrite transporter NarK超级家族1，与二穗短柄草、节节麦NRT2.4基因高度同源。第6，7个跨膜区之间有一段MFS家族所特有的序列特征：G-X3-D-X2-G-X-R，第4个跨膜区有典型的NO₃^-/NO₂^-转运载体特征序列：A-G-W-G/A-N-M-G，第8个跨膜区之后含有保守的蛋白激酶C识别基序（S/T-X-R/K）：SKR。利用qRT-PCR方法，测定草地早熟禾NRT2.4基因表达水平存在组织特异性，叶部表达量最多，硝态氮利于NRT2.4基因的表达，氮饥饿和高浓度NaNO₃水培液处理均利于NRT2.4基因的表达。上述结果为草地早熟禾硝酸盐转运蛋白NRT2.4在草坪草抗逆基因工程中的应用提供理论依据。

关键词: 草坪草, 草地早熟禾, NRT2.4, 氮素营养胁迫, 克隆, 表达

Abstract: Nitrate transporter (NRT) in plants can effectively regulate and transport NO₃^- and improve nitrogen utilization efficiency. In order to investigate the relationship between Poa pratensis L. NRT2.4 and nitrogen stress, Poa pratensis L. was selected as the material for the cloning and bioinformatics analysis of NRT2.4 gene, and the regulation of this gene expression was observed under treatment by nitrogen nutrient solution. The cloned NRT2.4 gene of Poa pratensis L.belonged to the Nitrate/nitrite transporter NarK superfamily, and was highly homologous to that of Brachypodium distachyon (L.) Beauv. and Aegilops tauschii Coss. Between the 6th and 7th transmembrane domain (TMD), there was a sequence with signature of G-X3-D-X2-G-X-R unique to the MFS family. The 4th TMD had a typical NO₃^-/NO₂^- transporter signature sequence of A-G-W-G/A-N-M-G. There was a conservative protein kinase C motif (S/T-X-R/K):SKR after the 8th TMD.The expression level of Poa pratensis L.NRT2.4 gene was tissue-specific, with the most expressed in leaves. Nitrate nitrogen was conducive to the expression of NRT2.4 gene, both nitrogen starvation and the treatment by nitrogen nutrient solution with a high NaNO₃ were beneficial to the expression of NRT2.4 gene. The results above have provided a theoretical bad for the application of Poa pratensis L.NRT2.4 in stress-tolerance gene engineering in turfgrass.

Key words: Turf grass, Kentucky bluegrass, NRT2.4 gene, Nitrogen nutrition deficiency, Clone, Expression

中图分类号:

陈阳, 金一锋, 金忠民, 王玉书, 蔡荣建, 邵竹林, 王杰. 草地早熟禾硝酸盐转运蛋白NRT2.4基因序列的克隆及表达分析[J]. 华北农学报, 2021, 36(2): 1-8. doi: 10.7668/hbnxb.20191685.

CHEN Yang, JIN Yifeng, JIN Zhongmin, WANG Yushu, CAI Rongjian, SHAO Zhulin, WANG Jie. Cloning and Expression Analysis of NRT2.4 Gene in Kentucky Bluegrass[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(2): 1-8. doi: 10.7668/hbnxb.20191685.

http://www.hbnxb.net/CN/Y2021/V36/I2/1

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草地早熟禾硝酸盐转运蛋白NRT2.4基因序列的克隆及表达分析

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草地早熟禾硝酸盐转运蛋白NRT2.4基因序列的克隆及表达分析

Cloning and Expression Analysis of NRT2.4 Gene in Kentucky Bluegrass

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