关键词: 紫花苜蓿, MsCEP基因家族, CEP2, 外施多肽, 根系生长发育

Abstract:

【Objective】CEP (C-terminal encoded peptides) is a gene that encodes hormone-like peptides secreted by roots and serves as a key regulator of plant root growth and development. To provide a molecular theoretical basis for further elucidating the function of MsCEP genes in root growth and development, members of the Medicago sativa MsCEP gene family, basic characteristics, expression differences in different tissues, and their roles in root growth were identified and analyzed. 【Method】Based on the genomic information of the alfalfa cultivar Xinjiang Daye, the MsCEP gene family members of alfalfa were accurately identified using local Blast analysis in TBtools and feature domain by referring to MtCEP family protein of Medicago truncatula sequence. The fundamental genetic and protein characteristics and the phylogenetic relationship of the MsCEP genes were analyzed by bioinformatics methods. The expression patterns of alfalfa MsCEP gene family members in various tissues were assessed using transcriptome data and real-time fluorescence quantitative PCR. The functional roles of mature MsCEP peptides in root growth and development were analyzed by exogenous application experiments. 【Result】A total of 35 MsCEP family members were identified in the genome of alfalfa Xinjiang Daye, and these genes are distributed across 18 chromosomes, lack introns, and all possess an N-terminal signal peptide and one or two conserved domains of the CEP family. The MsCEP members displayed predicted amino acid length ranging from 59 to 150, with molecular weights spanning 6.7 to 16.2 kDa, the isoelectric points varying from 5.80 to 10.41, instability indices ranging from 30.63 to 89.93, aliphatic indices ranging from 54.41 to 134.88, and the grand average of hydropathicity ranging from -1.110 to 0.377. Subcellular localization predictions indicated that the MsCEP protein predominantly localizes to the nucleus, plasma membrane, chloroplast, and Golgi apparatus. Cluster analysis delineated three distinct branches within the family, aligning with counterparts from Arabidopsis thaliana and Medicago truncatula. The largest branch encompassed 48 CEP members. Collinearity analysis highlighted a collinear relationship between the MsCEP genes in alfalfa and those in Arabidopsis thaliana and Medicago truncatula. Tissue expression analysis revealed that members of the MsCEP family exhibit distinct tissue-specific expression patterns, with higher expression levels in roots and lower or no expression detected in leaves. Among them, 22 members exhibited higher expression levels in roots compared to other tissues. The exogenous application of synthetic mature MsCEP2 peptide suppressed the growth of primary and lateral roots, reduced the number of lateral roots, and decreased the density of lateral roots. 【Conclusion】In conclusion, our investigation identified a total of 35 MsCEP members from the alfalfa 'Xinjiangdaye' genome database, which are revealed to be highly conserved. The MsCEP genes are primarily expressed in roots, and the exogenous application of synthetic mature MsCEP peptides can regulate root morphology, indicating that MsCEP peptides play important roles in root growth and development of alfalfa.

Key words: alfalfa, MsCEP gene family, CEP2, exogenous application of peptide, root growth and development