草地早熟禾NADH-GOGAT基因的克隆及表达分析

doi:10.11733/j.issn.1007-0435.2019.02.026

草地学报 ›› 2019, Vol. 27 ›› Issue (2): 459-465.DOI: 10.11733/j.issn.1007-0435.2019.02.026

草地早熟禾NADH-GOGAT基因的克隆及表达分析

陈阳¹, 孙华山², 王玉书¹, 张学通¹, 师冉¹, 熊良兵¹, 金一锋¹

1. 齐齐哈尔大学生命科学与农林学院, 抗性基因工程与寒地生物多样性保护黑龙江省重点实验室, 黑龙江齐齐哈尔 161006;
2. 东北农业大学园艺园林学院, 黑龙江哈尔滨 150030

收稿日期:2018-12-03 修回日期:2019-03-14 出版日期:2019-04-15 发布日期:2019-05-30
通讯作者: 金一锋
作者简介:陈阳(1986-),女,黑龙江齐齐哈尔人,讲师,博士研究生,研究方向为园林植物逆境,E-mail:chenyang8368215@126.com
基金资助:
国家自然科学基金项目（31501785，31701958）；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）；黑龙江省省属高等学校基本科研业务费科研项目（135209314）；黑龙江省大学生创新创业项目（201810232019）资助

Cloning and Expression Analysis of a NADH-GOGAT Gene in Kentucky Bluegrass

CHEN Yang¹, SUN Hua-shan², WANG Yu-shu¹, ZHANG Xue-tong¹, SHI Ran¹, XIONG Liang-bing¹, JIN Yi-feng¹

1. Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, College of Life Sciences, Agriculture and Forestry, Qiqihar University, Qiqihar, Heilongjiang Province 161006, China;
2. College of Horticulture and Garden, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China

Received:2018-12-03 Revised:2019-03-14 Online:2019-04-15 Published:2019-05-30

摘要/Abstract

摘要： 氮素是制约草坪草生长发育的重要因素之一，GS/GOGAT循环是植物氮同化的主要途径，谷氨酸合酶（glutamine：2-oxoglutarate amidotransferase，glutamate synthase，GOGAT）在植物氮素转化过程中发挥着重要的催化作用。本研究以草地早熟禾（Poa pratensis）为试材，基于二代测序RNA-seq相关数据，克隆得到草地早熟禾NADH-GOGAT基因，并进行生物信息学分析。结果表明：草地早熟禾NADH-GOGAT基因序列为3 236 bp，完整的开放阅读框（Open Reading Frame Finder，ORF）为1 338 bp，编码445个氨基酸残基组成的蛋白；预测NADH-GOGAT蛋白的分子量为49.89KD，等电点（isoelectric point，pI）为6.11，无信号肽，含有1个Glu_syn_central结构域，属于Glu_syn_central超级家族；二级结构以无规则卷曲、α-螺旋、延伸链和β-转角为主；同源进化分析结果表明草地早熟禾NADH-GOGAT与硬粒黑小麦氨基酸序列相似度为97%。草地早熟禾NADH-GOGAT基因在叶部的相对表达量高于根与茎，低氮浓度更有利于该基因的表达。NADH-GOGAT在干旱胁迫和水氮互作中表达差异显著（P<0.05）。草地早熟禾NADH-GOGAT基因的克隆，及相关序列分析和基因表达，对进一步研究该基因的功能和草地早熟禾GS/GOGAT循环的调控过程具有一定意义。

关键词: 草地早熟禾, 谷氨酸合酶, 基因克隆, 氮素调控

Abstract: Nitrogen nutrition is one of the most important factors restricting the growth and development of turfgrass. GS/GOGAT cycle is the main pathway of plant nitrogen assimilation,and GOGAT (glutamate synthase) plays an important catalytic role in plant nitrogen transformation. Based on transcriptome sequencing data,NADH-GOGAT gene of Kentucky bluegrass was cloned and analyzed by bioinformatics. The results showed that the NADH-GOGAT sequence of Kentucky bluegrass was 3 236 bp,and the complete ORF region was 1 338 bp,encoding a protein composed of 445 amino acid residues. It is predicted that the protein has a molecular weight of 49.89KD,an isoelectric point of 6.11,a signal-free peptide,and a Glu-syn-central domain,belonging to the Glu_syn_central superfamily. The secondary structure is mainly composed of irregular curl,alpha helix,extended chain and beta corner. Homology analysis showed that the highest similarity was 97% with the Secale cereale×Triticum turgidum subsp. durum of NADH-GOGAT amino acid sequence. The relative expression of NADH-GOGAT gene in leaves of Kentucky bluegrass was higher than that of roots and stems. Low nitrogen concentration was more conducive to the expression of the gene. Cloning sequence characteristics and expression analysis of the NADH-GOGAT gene in Kentucky bluegrass have a certain significance for further study of the function,of this gene and the regulation of GS/GOGAT cycle.

Key words: Kentucky bluegrass, GOGAT, Molecular cloning, Nitrogen management

中图分类号:

S543

陈阳, 孙华山, 王玉书, 张学通, 师冉, 熊良兵, 金一锋. 草地早熟禾NADH-GOGAT基因的克隆及表达分析[J]. 草地学报, 2019, 27(2): 459-465.

CHEN Yang, SUN Hua-shan, WANG Yu-shu, ZHANG Xue-tong, SHI Ran, XIONG Liang-bing, JIN Yi-feng. Cloning and Expression Analysis of a NADH-GOGAT Gene in Kentucky Bluegrass[J]. Acta Agrestia Sinica, 2019, 27(2): 459-465.

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草地早熟禾NADH-GOGAT基因的克隆及表达分析

Cloning and Expression Analysis of a NADH-GOGAT Gene in Kentucky Bluegrass

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