蒺藜苜蓿bZIP转录因子MtbZIP29的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0482

摘要/Abstract

摘要：

目的克隆蒺藜苜蓿（Medicago truncatula）MtbZIP29基因，研究bZIP29转录因子转录自激活、亚细胞定位及表达特征等，为后续阐明MtbZIP29基因参与蒺藜苜蓿生长发育、内源激素信号转导提供一定的理论研究基础。方法从野生型苜蓿‘R108’中克隆MtbZIP29基因，构建试验所需的表达载体，通过瞬时表达融合蛋白观察其亚细胞定位，利用酵母表达分析其自激活活性；对MtbZIP29基因进行生物信息学分析，包括蛋白质理化性质、启动子顺式作用元件、蛋白质结构等；利用RT-qPCR方法对不同组织中及不同激素（ABA、SA、6-BA、IAA、MeJA）处理条件下的MtbZIP29进行表达分析；利用农杆菌介导法，获得转基因烟草植株，分析MtbZIP29的功能。结果成功克隆MtbZIP29基因，基因编码区全长1 518 bp，编码506个氨基酸，分子量55.830 kD，理论等电点为6.82，不稳定系数63.60，为不稳定亲水性蛋白；二级结构预测α螺旋占比25.89%，延伸链为0.59%，无规则卷曲为73.52%；亚细胞定位结果表明，该蛋白定位于细胞核；酵母实验表明，该基因编码蛋白具有转录自激活活性；表达特征分析显示，MtbZIP29基因在叶片中的表达水平最高，而茎和荚果中的表达水平明显低于其他组织，同时MtbZIP29基因的表达水平明显受到不同激素处理的影响。对转基因和野生型植株对比发现，转基因烟草的根系更加发达，同时根茎处具有明显的膨大。结论 MtbZIP29基因参与根系生长发育，对不同激素均有响应，可能通过整合激素信号参与植物根系形态建成及逆境适应性的调控过程。

关键词: 蒺藜苜蓿, MtbZIP29转录因子, 表达特征, 自激活, 细胞核定位, 烟草, 农杆菌转化, 启动子分析

Abstract:

Objective Clone the MtbZIP29 gene from Medicago truncatula to study the self-activation, subcellular localization, and expression characteristics of the bZIP29 transcription factor, providing a theoretical research foundation for elucidating the involvement of the MtbZIP29 gene in the growth and development of M. truncatula and endogenous hormone signaling transduction. Method The MtbZIP29 gene was cloned from the wild-type M. truncatula 'R108', and expression vectors were constructed for experimental purposes. Subcellular localization was observed using a transient fusion protein assay, and using yeast to analyze its self-activation activity. Bioinformatics analyses were conducted on the MtbZIP29 gene, including protein physicochemical property analysis, prediction of promoter cis-acting elements, and prediction of secondary and tertiary protein structures. Additionally, RT-qPCR was employed to analyze the expression patterns of MtbZIP29 across different tissues and under treatments with various hormones (ABA, SA, 6-BA, IAA, and MeJA). Transgenic tobacco plants were obtained using Agrobacterium-mediated method, and the function of MtbZIP29 was analyzed. Result The MtbZIP29 gene was successfully cloned, with a coding sequence (CDS) length of 1,518 bp encoding 506 amino acids. The encoded protein had molecular weight of 55.830 kD, a theoretical isoelectric point (pI) of 6.82, an instability index of 63.60, and is classified as an unstable hydrophilic protein. Secondary structure prediction revealed that α-helixes accounted for 25.89%, extended strands for 0.59%, and the remaining 73.52% as random coils. Subcellular localization indicated the protein was localized to the nucleus. Yeast assays demonstrated the encoded protein has transcriptional self-activation activity. Expression profiling showed that MtbZIP29 transcripts were the most abundant in the leaves, significantly lower in the stems and pods compared to other tissues and markedly influenced by different hormone treatments. Analysis of MtbZIP29-overexpressing plants showed that the rhizomes of transgenic tobacco were significantly enlarged compared with wild-type tobacco. Conclusion The MtbZIP29 gene is involved in root growth and development, responding to different hormones, and may participate in the regulatory processes of root morphology construction and stress adaptability by integrating hormone signals.

Key words: Medicago truncatula, MtbZIP transcription factor, expression pattern, self-activation, nuclear localization, tobacco, agrobacterium transformation, promoter analysis

张驰昊, 刘晋囡, 晁跃辉. 蒺藜苜蓿bZIP转录因子MtbZIP29的克隆及功能分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0482.

ZHANG Chi-hao, LIU Jin-nan, CHAO Yue-hui. Cloning and Functional Analysis of a bZIP Transcription Factor MtbZIP29 from Medicago truncatula[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0482.

图/表 10

参考文献 38

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引物名称 Primer name	序列 Sequence（5′-3′）	用途 Usage
MtbZIP29-F	GATAAGCGCCTGCTCCTT	克隆基因 Cloning gene
MtbZIP29-R	ACGACCTCCAATCCAACC
M13-47	CAGGGTTTTCCCAGTCACG
M13-48	GAGCGGATAACAATTTCACAC
3302-bZIP29-F	CATGGTAGATCTGACTAGTATGGAAGATACTGAAGCAGCTTCATCTTTCAAAATG	构建亚细胞定位载体 Construction of subcellular localization vector
3302-bZIP29-R	GGCCTCCTAGGTACACGCGTTTGCTTTAACTCATTCTTTGAGTTGGCATT	构建亚细胞定位载体 Construction of subcellular localization vector
BDbZIP29-F	TCAGAGGAGGACCTGCATATGGAAGATACTGAAGCAGCT	构建酵母表达载体 Construction of yeast expression vector
BD-bZIP29-R	CGCTGCAGGTCGACGGATCCCTATTGCTTTAACTCATTCTTTGAGTTG	构建酵母表达载体 Construction of yeast expression vector
BD-bZIP29-U-F	GCCATGGAGGCCGAATTCCCGATGGAAGATACTGAAGCAGC	构建酵母表达载体（N端） Construction of yeast expression vector （N-terminus）
BD-ZIP29-U-R	CTGCAGGTCGACGGATCCCCTTACACAACTTCACCTTCCGGCG
BD-bZIP29-D-F	GCCATGGAGGCCGAATTCCCGGATGATCTTTTTTCTGCTTA	构建酵母表达载体（C端） Construction of yeast expression vector （C-terminus）
BD-bZIP29-D-R	CTGCAGGTCGACGGATCCCCCTATTGCTTTAACTCATTCT
MtbZIP29 qRT-F	TGGGAATGGTGAGTTTAG	基因表达分析 Gene expression analysis
MtbZIP29 qRT-R	CAGAAAGTGTAGTGGCTTC	基因表达分析 Gene expression analysis
MtActin-F	TGATCTGGCTGGTCGTGACCTTA	蒺藜苜蓿内参基因表达分析 Expression analysis of Medicago truncatula internal reference gene
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
Nt-bZIP29-F	ATCAGATCCCGGTTTCAC	转基因烟草PCR检测 PCR analysis of transgenic tobacco
Nt-bZIP29-R	CTATCATCAGTAGCCGCGT	转基因烟草PCR检测 PCR analysis of transgenic tobacco

引物名称 Primer name	序列 Sequence（5′-3′）	用途 Usage
MtbZIP29-F	GATAAGCGCCTGCTCCTT	克隆基因 Cloning gene
MtbZIP29-R	ACGACCTCCAATCCAACC
M13-47	CAGGGTTTTCCCAGTCACG
M13-48	GAGCGGATAACAATTTCACAC
3302-bZIP29-F	CATGGTAGATCTGACTAGTATGGAAGATACTGAAGCAGCTTCATCTTTCAAAATG	构建亚细胞定位载体 Construction of subcellular localization vector
3302-bZIP29-R	GGCCTCCTAGGTACACGCGTTTGCTTTAACTCATTCTTTGAGTTGGCATT	构建亚细胞定位载体 Construction of subcellular localization vector
BDbZIP29-F	TCAGAGGAGGACCTGCATATGGAAGATACTGAAGCAGCT	构建酵母表达载体 Construction of yeast expression vector
BD-bZIP29-R	CGCTGCAGGTCGACGGATCCCTATTGCTTTAACTCATTCTTTGAGTTG	构建酵母表达载体 Construction of yeast expression vector
BD-bZIP29-U-F	GCCATGGAGGCCGAATTCCCGATGGAAGATACTGAAGCAGC	构建酵母表达载体（N端） Construction of yeast expression vector （N-terminus）
BD-ZIP29-U-R	CTGCAGGTCGACGGATCCCCTTACACAACTTCACCTTCCGGCG
BD-bZIP29-D-F	GCCATGGAGGCCGAATTCCCGGATGATCTTTTTTCTGCTTA	构建酵母表达载体（C端） Construction of yeast expression vector （C-terminus）
BD-bZIP29-D-R	CTGCAGGTCGACGGATCCCCCTATTGCTTTAACTCATTCT
MtbZIP29 qRT-F	TGGGAATGGTGAGTTTAG	基因表达分析 Gene expression analysis
MtbZIP29 qRT-R	CAGAAAGTGTAGTGGCTTC	基因表达分析 Gene expression analysis
MtActin-F	TGATCTGGCTGGTCGTGACCTTA	蒺藜苜蓿内参基因表达分析 Expression analysis of Medicago truncatula internal reference gene
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
Nt-bZIP29-F	ATCAGATCCCGGTTTCAC	转基因烟草PCR检测 PCR analysis of transgenic tobacco
Nt-bZIP29-R	CTATCATCAGTAGCCGCGT	转基因烟草PCR检测 PCR analysis of transgenic tobacco

用途 Usage	网址 Website
启动子及顺式作用元件预测 Promoter and cis-acting element prediction	PlantCARE， http：//bioinformatics.psb.ugent.be/webtools/plantcare/html
系统进化树分析 Phylogenetic tree analysis	NCBI， https：//www.ncbi.nlm.nih.gov/
蛋白质结构域分析 Protein structure domain analysis	UniProt， https：//www.uniprot.org/
蛋白质二级结构预测 Protein secondary structure prediction	SOPMA， https：//npsa.lyon.inserm.fr/cgibin/npsa_automat.pl page=/NPSA/npsa_sopma.html
蛋白质三级结构预测 Protein tertiary structure prediction	Swissmodel， https：//swissmodel.expasy.org/
蛋白质理化性质分析 Analysis of physicochemical properties of proteins	Expasy-ProtParam， http：//web.expasy.org/protparam Expasy-ProtScale， http：//web.expasy.org/protscale

用途 Usage	网址 Website
启动子及顺式作用元件预测 Promoter and cis-acting element prediction	PlantCARE， http：//bioinformatics.psb.ugent.be/webtools/plantcare/html
系统进化树分析 Phylogenetic tree analysis	NCBI， https：//www.ncbi.nlm.nih.gov/
蛋白质结构域分析 Protein structure domain analysis	UniProt， https：//www.uniprot.org/
蛋白质二级结构预测 Protein secondary structure prediction	SOPMA， https：//npsa.lyon.inserm.fr/cgibin/npsa_automat.pl page=/NPSA/npsa_sopma.html
蛋白质三级结构预测 Protein tertiary structure prediction	Swissmodel， https：//swissmodel.expasy.org/
蛋白质理化性质分析 Analysis of physicochemical properties of proteins	Expasy-ProtParam， http：//web.expasy.org/protparam Expasy-ProtScale， http：//web.expasy.org/protscale