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作物学报 ›› 2013, Vol. 39 ›› Issue (12): 2145-2153.doi: 10.3724/SP.J.1006.2013.02145

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆谷氨酰胺合成酶基因的分类及根瘤特异表达GmGS1β2基因功能的初步分析

王晓波1,滕婉2,何雪2,童依平2,*   

  1. 1 安徽农业大学农学院,安徽合肥230036; 2 中国科学院遗传与发育生物学研究所 / 植物细胞与染色体工程国家重点实验室,北京100101
  • 收稿日期:2013-04-07 修回日期:2013-07-25 出版日期:2013-12-12 网络出版日期:2013-10-01
  • 通讯作者: 童依平, E-mail: yptong@genetics.cas.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31201226), 植物细胞与染色体工程国家重点实验室开放课题(PCCE-KF-2011-05)和安徽省自然科学基金(1308085QC49)。

Classification of Glutamine Synthetase Gene and Preliminary Functional Analysis of the Nodule-Predominantly Expressed Gene GmGS1β2 in Soybean

WANG Xiao-Bo1,TENG Wan2,HE Xue2,TONG Yi-Ping2,*   

  1. 1 School of Agronomy, Anhui Agricultural university, Hefei 230036, China; 2 State Key Laboratory of Plant Cell and Chromosome Engineering / Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
  • Received:2013-04-07 Revised:2013-07-25 Published:2013-12-12 Published online:2013-10-01

摘要:

Phytozome数据库中获得包括大豆在内的12种植物的谷氨酰胺合成酶(glutamine synthetase, GS)氨基酸序列,利用MEGA5.10软件进行多序列比对、构建进化树。进化分析表明,植物GS可以分成胞质型(GS1)和质体型(GS2)两大类,GS1可进一步分成分5个亚类,包括双子叶植物为主的IIIIII亚类、低等植物类(IV)和单子叶植物类(V)。这5亚类中,第II类是豆科植物特有的一类,大豆的4GS1 (GmGS1β1/2GmGS1γ1/2)属于该亚类;利用qPCR在大豆盛花期分析GS1基因的组织表达特异性,结果表明不同类型GmGS1基因在表达部位和表达丰度上存在较大差异,而同一类基因之间具有相似的表达规律;4个豆科植物特有的GS1基因在大豆根瘤中都有较高的表达量,其中位于大豆第18染色体上的GmGS1β2基因表达丰度最高;利用原核表达系统体外表达GmGS1β2蛋白,诱导出分子量大小与理论预测值一致的目标蛋白,酶活性分析表明GmGS1β2可以与底物发生催化反应,具有谷氨酰胺合成酶活性,推测该基因在大豆根瘤氮素同化代谢中具有重要作用。

关键词: 大豆, GmGS1基因, 组织特异表达, GS酶活性

Abstract:

The purpose of this study was to analyze the evolution, classification and tissue expression specificity of soybean cytosolic glutamine synthetase genes. Glutamine synthetase (GS) proteins from 12 species were downloaded from phytozome database, and used for phylogenetic analysis. The GSs were divided into two groups, cytosolic glutamine synthetase (GS1) and plastidic glutamine synthetase (GS2) based on the maximum likelihood of MEGA 5.10 software. The GS1 group was further divided into five subgroups, including three dicotyledons subgroups (I, II, and III), lower plants subgroup (IV) and monocotyledons subgroup (V). All GSs in subgroup II were mainly derived from leguminous plants including four GmGS1 proteins (GmGS1β1/2 and GmGS1γ1/2) in soybean. Real-time RT-PCR analysis of GmGS1 genes indicated that soybean GS duplicated genes had the same tissue specificity of expression, while the four subgroup II GmGS1 genes presented higher expression level in soybean nodules than in other tissues. Among the six GmGS1 genes, GmGS1β2 located on chromosome 18 had the highest expression level and predominantly expressed in developing nodules. GmGS1β2 was then ligated into pGEX4T-1 vector and transformed into BL21 (DE3) for prokaryotic expression. GmGS1β2 recombinant protein was purified by 4B-Beads and tested for GS activity by spectrophotometer. Prokaryotic expression result showed that the molecular weight of GmGS1β2 was 39 kD, which is consistent with previous theoretical prediction. Catalyzing reaction showed that the recombinant protein had glutamine synthetase activity, which means GmGS1β2 encoded a functional GmGS1β2 glutamine synthetase. These results provide useful information for further functional research of GS1 proteins involved in nitrogen assimilation in soybean nodule.

Key words: Glycine max (L.) Merr, GmGS1 genes, Tissue specific expression, Glutamine synthetase activity

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