玉米氮胁迫响应GLK基因的鉴定及ZmGLK4的功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2026-0400

摘要/Abstract

摘要：

目的 GOLDEN2-LIKE（GLK）转录因子属于GARP转录因子家族，在植物生长发育以及非生物和生物胁迫响应过程中发挥重要作用。鉴定玉米中响应氮素的GLK基因，并分析ZmGLK4的功能，为阐明GLK转录因子调控作物生长发育和氮素吸收的分子机制提供理论依据。方法基于玉米转录组数据，筛选响应氮素的差异表达ZmGLK基因，并分析其保守结构域、保守基序和启动子顺式作用元件。克隆氮素响应最显著的ZmGLK4，进行表达模式分析、亚细胞定位和转录活性检测，并通过转化拟南芥进行过表达功能验证。结果共获得9个响应氮素的ZmGLK基因，均含有典型的MYB-DNA结合结构域，其启动子区域均包含多个植物逆境响应元件。ZmGLK4编码区长度为1 380 bp，编码460个氨基酸，与拟南芥AtNIGT1.2同源性最高。该蛋白定位于细胞核，并具有转录激活活性。实时荧光定量PCR结果表明，ZmGLK4在根、花丝、叶片和茎组织中表达量较高，低氮处理显著抑制其在根和叶片中的表达。过表达ZmGLK4抑制拟南芥的生长，导致株高降低、产量下降，同时抑制根的生长和氮素的吸收。此外，过表达ZmGLK4显著抑制了低氮和正常氮条件下拟南芥AtNRT1.1、AtNRT1.5、AtNRT2.1和AtNRT2.4基因的表达。结论 ZmGLK4对玉米植株生长发育和氮素吸收具有负调控作用。

关键词: 玉米, ZmGLK4, 过表达, 氮素吸收, 功能分析

Abstract:

Objective GOLDEN2-LIKE (GLK) transcription factors, which belong to the GARP transcription factor family, play crucial roles in plant growth, development, and responses to both abiotic and biotic stresses. This study aims to identify nitrogen-responsive GLK genes in maize and analyze the function of ZmGLK4, thereby providing a theoretical basis for elucidating the mechanisms through which GLK transcription factors regulate crop growth, development, and nitrogen uptake. Methods Using maize transcriptome data, we screened for differentially expressed ZmGLK genes responsive to nitrogen, analyzing their conserved domains, conserved motifs, and promoter cis-acting elements. The most nitrogen-responsive gene, ZmGLK4, was cloned for expression pattern analysis, subcellular localization, and transcriptional activity detection. Its function was further verified through overexpression in Arabidopsis thaliana via transformation. Results We identified nine nitrogen-responsive ZmGLK genes, all of which contain typical MYB-DNA binding domains, and their promoter regions include multiple plant stress-responsive elements. The coding region of ZmGLK4 is 1380 bp in length, encoding 460 amino acids, with the highest homology to ArabidopsisAtNIGT1.2. This protein localizes to the nucleus and exhibits transcriptional activation activity. Real-time quantitative PCR results showed that ZmGLK4 is highly expressed in root, filament, leaf, and stem tissues, while low nitrogen treatment significantly inhibited its expression in roots and leaves. Overexpression of ZmGLK4 inhibited the growth of Arabidopsis, resulting in reduced plant height and yield, and suppressed root growth and nitrogen uptake. Furthermore, overexpression of ZmGLK4 significantly inhibited the expression of AtNRT1.1, AtNRT1.5, AtNRT2.1, and AtNRT2.4 genes under both low-nitrogen and normal-nitrogen growth conditions. Conclusion ZmGLK4 negatively regulates the growth and nitrogen uptake of maize plants.

Key words: maize, ZmGLK4, overexpression, nitrogen uptake, functional analysis

于本帅, 郑荣繁, 谭桐菲, 王玉斌. 玉米氮胁迫响应GLK基因的鉴定及ZmGLK4的功能验证[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0400.

YU Ben-shuai, ZHENG Rong-fan, TAN Tong-fei, WANG Yu-bin. Identification of Nitrogen Stress-responsive GLK Genes in Maize and Functional Characterization of ZmGLK4[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0400.

图/表 9

图1 ZmGLK保守序列和基因结构分析A：蛋白质系统发育树；B：保守结构域；C：保守基序；D：基因结构；E：保守结构域序列

Fig. 1 Conserved motifs and gene structure analysis of the ZmGLKA: Protein phylogeny; B: conserved motif; C: conserved domain; D: gene structure; E: conserved domain sequence

图2 ZmGLK4基因的表达模式分析A： ZmGLK4在不同组织中的表达；B： ZmGLK4在不同低氮胁迫处理时间下根中的表达；C： ZmGLK4在不同低氮胁迫处理时间下叶片中的表达

Fig. 2 Analysis of the expression pattern of ZmGLK4A: Expression of ZmGLK4 in different tissues; B: expression of ZmGLK4 in roots under different low-nitrogen stress durations; C: expression of ZmGLK4 in leaves under different low-nitrogen stress durations

图3 ZmGLK4转录活性分析

Fig. 3 Transcriptional activity analysis of ZmGLK4

图4 烟草叶片和玉米原生质体中ZmGLK4亚细胞定位A：GFP 空载体和ZmGLK4 -GFP 融合蛋白在烟草叶片中的瞬时表达，Bar=20 µm；B：GFP 空载体和ZmGLK4-GFP 融合蛋白在玉米原生质体中的瞬时表达，Bar=10 µm。从左到右分别为 GFP 绿色荧光蛋白、RFP荧光蛋白、明场和叠加图

Fig. 4 Subcellular localization of ZmGLK4 in tobacco leaves and maize protoplastsA: Transient expression of the empty GFP vector and ZmGLK4-GFP fusion protein in tobacco leaves, bar=20 µm. B: Transient expression of the empty GFP vector and ZmGLK4-GFP fusion protein in maize protoplasts, bar=10 µm. From left leaf to right, GFP fluorescent protein, RFP fluorescent protein, bright field, and merged image

图5 ZmGLK4过表达株系的定量PCR检测WT表示野生型拟南芥，OE1-OE14分别表示独立的ZmGLK4过表达转基因拟南芥株系

Fig. 5 RT-qPCR detection of ZmGLK4-overexpressing linesWT represents wild-type Arabidopsis plants, and OE1-OE14 represents independent transgenic Arabidopsis lines overexpressing ZmGLK4

图6 ZmGLK4过表达株系的表型鉴定A-C：ZmGLK4转基因拟南芥和野生型拟南芥在正常条件下生长30 d 的表型；D：ZmGLK4转基因拟南芥和野生型拟南芥成熟期表型；E-G：ZmGLK4转基因拟南芥和野生型拟南芥成熟期株高、分枝数量和产量

Fig. 6 Phenotypic identification of ZmGLK4 transgenic ArabidopsisA-C: Phenotypes of ZmGLK4 transgenic Arabidopsis and wild-type Arabidopsis at 30 days after growth under normal conditions; D: phenotypes of ZmGLK4 transgenic Arabidopsis and wild-type Arabidopsis at maturity stage; E-G: plant height, branch number, and yield of ZmGLK4 transgenic Arabidopsis and wild-type Arabidopsis at maturity stage

图7 低氮胁迫下ZmGLK4转基因拟南芥的生长发育情况与根长、叶绿素含量统计A：正常氮培养基生长6 d后，分别转移至低氮和正常氮培养基上4 d后ZmGLK4转基因拟南芥和野生型拟南芥的表型； B：鲜重；C：根长；D：叶绿素含量

Fig. 7 Growth and development of ZmGLK4 transgenic Arabidopsis under low nitrogen stress, along with statistics of root length and chlorophyll contentA: Phenotypes of ZmGLK4 transgenic Arabidopsis and wild-type Arabidopsis after 6 days of growth on normal nitrogen medium followed by 4 days of transfer to low nitrogen and normal nitrogen media, respectively; B: fresh weight; C: root length; D: Chlorophyll content

图8 低氮胁迫下ZmGLK4转基因拟南芥的表型鉴定A：低氮和正常氮处理30 d后ZmGLK4转基因拟南芥和野生型拟南芥的表型； B：鲜重；C：硝酸盐含量

Fig. 8 Phenotypic identification of ZmGLK4 transgenic Arabidopsis under low nitrogen stressA: Phenotypes of ZmGLK4 transgenic Arabidopsis and wild-type Arabidopsis after 30 d of low nitrogen and normal nitrogen treatment. B: Fresh weight. C: Nitrate content

图9 ZmGLK4过表达株系中AtNRTs基因定量PCR检测正常氮培养基上生长6 d后，分别转移至低氮和正常氮培养基上4 d后ZmGLK4转基因拟南芥和野生型拟南芥根系

Fig. 9 RT-qPCR validation of AtNRTs genes in ZmGLK4-overexpressing transgenic plantsRoots of ZmGLK4 transgenic and wild-type Arabidopsis after 6-day growth on normal-N medium, then 4-day transfer to low-N and normal-N media, respectively

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