Cloning and Freezing Tolerance Analysis of the MsCZF29 Gene in Alfalfa

doi:10.13560/j.cnki.biotech.bull.1985.2026-0186

Abstract

Abstract:

Objective CZF zinc finger proteins (ZFPs) play important regulatory roles in plant responses to biotic and abiotic stresses. Cloning the MsCZF29 gene from alfalfa and analyzing its freezing tolerance function lays a foundation for revealing the regulatory mechanism of this gene in freezing tolerance of Arabidopsis. Method Using Arabidopsis thaliana as the experimental model, we identified the freezing stress-responsive gene MsCZF29 based on previous transcriptomic analysis of cold tolerance in Medicago sativa. The gene was cloned and designated as MsCZF29, and bioinformatics analysis and phenotypic characterization were conducted on it. An overexpression vector was constructed and introduced into plants via Agrobacterium-mediated transformation to generate transgenic lines (OE2-1, OE2-2, and OE6-2). Under freezing stress conditions, we evaluated T₃ generation survival rates, measured key physiological and biochemical parameters, and quantified expression levels of relevant cold-responsive genes. Transcriptional autoactivation activity was verified using yeast assays. Result The MsCZF29 coding sequence (CDS) spans 1 554 bp, encoding 517 amino acids that form a protein which is both unstable and hydrophilic. Subcellular localization confirmed that the protein is localized in the nucleus, while yeast autoactivation assays demonstrated its transcriptional activation capability. Heterologous overexpression of MsCZF29 significantly enhanced the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in Arabidopsis lines under freezing stress, while simultaneously reducing relative electrical conductivity (REL) and malondialdehyde (MDA) content. Transgenic lines exhibited markedly higher survival rates and superior growth status compared to wild-type plants under freezing stress conditions. Conclusion We successfully cloned MsCZF29 gene, demonstrating that its overexpression exerts a positive regulatory effect during freezing stress responses, thereby enhancing plant freezing tolerance.

Key words: Alfalfa, MsCZF29 gene, freezing stress, freezing resistance, physiological indicators

FAN Yu-xin, REN Nai-peng, XUAN Yi-fu, ZANG Hui, LIU Xiang-ping. Cloning and Freezing Tolerance Analysis of the MsCZF29 Gene in Alfalfa[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0186.

Figures/Tables 7

Fig. 1 Subcellular localization analysis of MsCZF29 in tobacco leaves

Fig. 2 Activity validation of pGBKT7-MsCZF29 in transformed Y2H yeast strains

Fig. 3 Screening and molecular identification of A. thaliana positive Seedlings

Fig. 4 Phenotypic analysis of wild-type and MsCZF29-transgenic A. thaliana under freezing stressA: Phenotypes of wild-type and transgenic A. thaliana seedlings under untreated conditions. B: Phenotypes of wild-type and transgenic A. thaliana seedlings under freezing treatment. C: Changes in the phenotypes of A. thaliana potted plants before and after freezing treatment. D: Survival rates of wild-type and transgenic A. thaliana after freezing

Fig. 5 Cell membrane permeability analysis of wild-type and MsCZF29-transgenic A. thaliana under freezing stress

Fig. 6 Analysis of antioxidant enzyme activity in wild-type and MsCZF29-transgenic A. thaliana under freezing stress

Fig. 7 Expression analysis of the cold-responsive genes in overexpressing MsCZF29 A. thaliana

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