Cloning and Functional Analysis of a bZIP Transcription Factor MtbZIP29 from Medicago truncatula

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

Abstract

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

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.

Figures/Tables 10

References 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