玉米铁还原酶基因ZmFRO2的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0281

摘要/Abstract

摘要：

Fe是植物生长发育过程中最重要的微量元素之一,植物缺铁会导致叶片黄化,进而影响产量。植物吸收转运铁共有两种机理,即机理I和机理II,相应的植物称为机理I植物和机理II植物,机理I植物主要包括双子叶植物和非禾本科单子叶植物,机理II植物主要包括禾本科植物。铁还原酶基因FRO（Ferric reduction oxidase）作为机理I植物中的关键基因已在多种植物中被发现与研究,包括禾本科植物水稻。为了解该基因在玉米中的功能,对玉米铁还原酶基因ZmFRO2进行了基因功能的分析,包括生物信息学分析、表达模式分析及亚细胞定位分析,证明了ZmFRO2具有铁还原酶的结构基础且与OsFRO1、MtFRO2、AtFRO6和AtFRO7具有较近的亲缘关系,ZmFRO2定位在质膜与胞质,该基因主要在叶片中大量表达,且该基因的表达受缺铁先诱导后抑制。另外,通过农杆菌介导的方法获得了过表达ZmFRO2玉米植株,对其进行铁还原酶活力及锌铁含量测定发现过表达ZmFRO2能够提高玉米根部铁还原酶活力,根部、叶片和籽粒的铁含量均有不同程度的升高。通过这些分析和测定为之后基因功能的深入研究奠定基础,进而为提高玉米铁含量提供新的功能基因和新途径。

关键词: 铁还原酶基因, ZmFRO2, 功能验证, 缺铁胁迫

Abstract:

Fe is one of the most important micronutrients in plant growth and development,and iron deficiency in plant leads to leaves yellowing,which in turn affects yield. There are two mechanisms for plant absorption and transport of iron,that is,mechanism I and mechanism II,the corresponding plant called mechanism I plant and mechanism II plant. Dicots and non-graminaceous monocots employ the mechanism I,and graminaceous plants adopt mechanism II. As the key gene in mechanism I plant,gene FRO（ferric reduction oxidase）has been found and studied in a variety of plants,including graminaceous plant rice. In order to understand the function of FRO in maize,functional analysis of ZmFRO2,a ferric reductase gene in maize,was done,including bioinformatics analysis,expression pattern and subcellular localization. The results indicated that ZmFRO2 had the structural basis of iron reductase and had a closer kinship with OsFRO1,MtFRO1,AtFRO6 and AtFRO7. ZmFRO2 was located in the plasma membrane and cytoplasm. ZmFRO2 gene was expressed in large quantities in leaves and the expression of it was first induced and then inhibited by iron deficiency. In addition,transgenic maize plants with over-expression of ZmFRO2 were obtained using the method of agrobacterium-mediated infection. The iron reductase activity and Zn/Fe content determination of them showed that the over-expression of ZmFRO2 increased the iron reductase activity in maize roots and the iron content in the root,leaf and grain all increased at varied degrees. Through these analyses and determinations,the foundation for the further study of gene function verification is laid,and a new functional gene and a new way for improving iron content of maize will be provided.

Key words: iron reductase gene, ZmFRO2, function verification, iron deficiency stress

乔孟欣, 李素贞, 陈景堂. 玉米铁还原酶基因ZmFRO2的功能分析[J]. 生物技术通报, 2020, 36(11): 9-20.

QIAO Meng-xin, LI Su-zhen, CHEN Jing-tang. Functional Analysis of Ferric Reductase Gene ZmFRO2 in Maize[J]. Biotechnology Bulletin, 2020, 36(11): 9-20.

图/表 8

图1 ZmFRO2与其他FRO的氨基酸序列比对

图2 ZmFRO2与其他FRO的系统进化树分析

图3 土壤条件下ZmFRO2在玉米各部位的表达模式

图4 不同供铁条件下ZmFRO2在玉米幼苗叶片和根部的表达模式误差线代表标准差（n=3）。星号代表差异显著性,* 代表P<0.05;** 代表P<0.01;*** 代表P<0.001

图5 ZmFRO2在玉米叶肉原生质体中的定位

图6 ZmFRO2在转基因玉米中的表达 A：pCAMBIA3301-ZmFRO2过表达载体图谱;B：ZmFRO2在转基因玉米OX-1,OX-2的相对表达量;误差线代表标准差（n=3）。星号代表差异显著性,* 代表P<0.05;** 代表P<0.01;*** 代表P<0.001

图7 ZmFRO2过表达玉米根部铁还原酶活力误差线代表标准差（n=3）,* 代表P<0.05;** 代表P<0.01;*** 代表P<0.001

图8 ZmFRO2过表达玉米各部位锌铁含量 A：幼苗叶片的锌铁含量;B：幼苗根部的锌铁含量;C：成熟叶片的锌铁含量;D：籽粒的锌铁含量。误差线代表标准差（n=3）。* 代表P<0.05;** 代表P<0.01;*** 代表P<0.001

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