Identification and Expression Analysis of MADS-Box Gene Family in Pinus koraiensis and Overexpression of PkMADS9 Promoting Early Flowering in Transgenic Arabidopsis

Korean pine (<i>Pinus koraiensis</i>) is a vital woody oil tree species native to Northeast Asia, with its pine nuts serving as the primary global source of edible pine nuts globally due to their rich nutritional content. Currently, seed yield from Korean pine is low and unstable, failing to meet the market demand. The limited number of female cones is the primary factor restricting its yield. MADS-box family members are crucial in regulating the initiation, differentiation, and morphogenesis of floral organs. However, systematic identification and characterization of MADS-box proteins in Korean pine have not been reported. This study utilized transcriptome data from reproductive and vegetative buds during the flower bud differentiation stage of Korean pine to comprehensively identify MADS-box family members through bioinformatics analysis and molecular biology approaches. A total of 37 <i>PkMADS-box</i> genes were identified, including 6 type I and 31 type II (MIKC) genes, which were classified into 8 subfamilies. The physicochemical properties, conserved domains, conserved motifs, protein structures, gene expression profiles, and protein-protein interaction networks of these genes were analyzed. Key genes associated with physiological differentiation (flower induction) and sexual organogenesis were identified based on expression patterns during flower bud differentiation and flower organ development. Among these, <i>PkMADS4</i> and <i>PkMADS26</i> are likely involved in positively regulating flower induction, while <i>PkMADS9</i> plays a role in the morphological differentiation of sexual organs in a dose-dependent manner and overexpression of <i>PkMADS9</i> promoting early flowering in transgenic <i>Arabidopsis</i>. These genes were also identified as key candidates for regulating reproductive phase changes and strobilus development. This study provides a theoretical foundation for further investigation of <i>MADS-box</i> genes in reproduction and offers insights into genetic improvements aimed at enhancing the seed yield of Korean pine.