Abstract: 【Objective】Hippophae rhamnoides subsp. sinensis Rousi is a plant of substantial economic value and ecological significance, attracting sustained research interest. The ACBPs gene family is integral to plant biology, playing a critical role in normal plant growth and development. This gene family is deeply involved in cellular material and energy metabolism and plays a vital role in plants’ adaptation to abiotic stress and biomembrane repair, particularly in response to heavy metal stress. Information on the ACBPs gene family in H. rhamnoides remains scarce. This study aims to accurately and comprehensively identify the members of the ACBPs gene family in H. rhamnoides using bioinformatics tools, and to investigate the gene expression analysis of these genes under heavy metal (lead) stress through molecular biology approaches.【Methods】In this study, the ACBPs gene family in Hippophae rhamnoides subsp. sinensis Rousi was identified using the hidden Markov model (PF00887) and the HMMER search script. Physicochemical properties and subcellular localization were analyzed via the ExPASy and WoLF PSORT websites. Multiple sequence alignment and gene structure analyses were conducted using MEGA software and the GSDS website. Phylogenetic and motif analyses were performed using the TBtools FastTree plugin and the MEME website. Chromosome localization and cis-acting element analyses were conducted using Mapchart software and the PlantCARE website. Intra- and interspecific gene collinearity analyses were conducted via the TBtools Advanced Circos and One Step MCScanX plugins. Protein-protein interaction networks and three-dimensional structure prediction analyses were conducted using the STRING database and the SWISS-MODEL website. Additionally, expression profiles of ACBPs gene were visualized using heatmaps based on FPKM values from RNA-Seq data, and relative gene expression levels were validated through qRT-PCR experiments.【Results】A comprehensive search of the Hippophae rhamnoides subsp. sinensis Rousi proteome identified eight ACBPs gene, named HrLACBPs1 – HrLACBPs8. Physicochemical analysis revealed that most translated protein sequences were rich in acidic amino acids, with only one being basic, and most family proteins were localized in the nucleus. Multiple sequence alignment identified two main motifs, YKQA and KWDAW, responsible for acyl-CoA binding in the ACBPs gene family of H. rhamnoides Chromosomal mapping indicated that HrLACBPs4 and HrLACBPs5 were on chromosome 4, while the other six genes were on chromosomes 1, 2, 3, 5, 11, and 12. Phylogenetic analysis with the ACBPs gene families of Arabidopsis, Elaeagnus mollis Diels and Helianthus annuus divided the HrLACBPs family into three groups: Groups 1 with three members, Group 2 with one member, Group 3 and 4 each with two members. Gene structure and motif analysis showed conserved gene structures and minimal motif differences within each group, with Group 3 containing the most exons and motif sequences. Cis-acting element analysis, focusing on hormone regulation, meristem expression, and stress response elements, revealed that HrLACBPs members generally contained three or more of these elements. Collinearity analysis demonstrated no collinearity between HrLACBPs4 and the other seven genes, but HrLACBPs2, HrLACBPs3, and HrLACBPs5 exhibited mutual collinearity on chromosomes 2, 3, and 4. Nine collinearities were identified between the H. rhamnoides ACBPs gene family and the Arabidopsis ACBPs gene family, and seventeen with the Elaeagnus mollis Diels ACBPs gene family. Protein interaction network analysis suggested that HrLACBPs1 in H. rhamnoides may functionally resemble ACBP6 in Arabidopsis. Protein 3D structure predictions revealed that Group 3 proteins were the most complex with high model similarity; Group 1 proteins were simpler but also similar; Group 4 proteins showed low similarity and large differences. Transcriptome analysis and qRT-PCR validation indicated that, except for HrLACBPs1, the other genes had lower expression levels at 2000 mg·kg-1 lead compared to controls, while their expression increased at 5000 mg·kg-1 lead. 【Conclusion】In Hippophae rhamnoides subsp. sinensis Rousi, eight ACBPs gene were identified, with Group 1 and 3 members exhibiting conserved patterns in gene structure, motif sequences, and three-dimensional protein structures. Group 3 contained the most exons, increasing the likelihood of alternative splicing and enhancing functional diversity. The gene family’s promoter regions contained various cis-acting elements responsive to plant hormones, meristematic activity, and stress stimuli, suggesting that HrLACBPs genes have multiple biological functions. Protein interaction network analysis highlighted AtACBP6, from the Arabidopsis ACBP family, as a core protein that enhances plant cold tolerance when overexpressed under cold stress. Low-temperature stress response elements were also identified in HrLACBPs1, suggesting its role in enhancing plant cold tolerance. Transcriptome analysis and qRT-PCR validation showed that most ACBPs gene in H. rhamnoides were significantly downregulated at 1000 mg·kg-1 lead, with upregulation occurring at 5000 mg·kg-1. This may occur because low lead concentrations cause less oxidative stress, allowing plants to enhance antioxidant defenses by upregulating gene expression to mitigate lead damage. HrLACBPs1 expression, localized in the endoplasmic reticulum, decreased at 500 mg·kg-1 lead compared to controls, then gradually increased at 2000 and 5000 mg·kg-1. Genes in the endoplasmic reticulum may reduce expression to alleviate protein synthesis burden at low lead stress and increase expression to address protein damage and accumulation at higher stress levels. These findings provide a solid theoretical basis for further research on H. rhamnoides’s response to heavy metal lead stress.
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