bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Ogura cytoplasmic male sterility (CMS) in Chinese cabbage (<i>Brassica rapa</i>) is characterized by complete pollen abortion, wherein stamens fail to produce viable pollen while pistils retain normal fertility. This maternally inherited trait is valuable for hybrid breeding. This study employed integrated analysis of miRNA, transcriptome, and degradome sequencing data aligned to the Chinese cabbage reference genome to elucidate the molecular function of bra-miR9569 in Ogura CMS pollen fertility and explore its associated pathways. Subsequently, a bra-miR9569 overexpression vector was constructed and transformed into <i>Arabidopsis thaliana</i>. Phenotypic characterization of transgenic Arabidopsis lines, combined with anther viability assessment and quantification of ATP content and reactive oxygen species (ROS) levels in Chinese cabbage, was performed to analyze the effects of bra-miR9569. Our findings demonstrate that mutation of the mitochondrial gene <i>orf138</i> in Ogura CMS lines leads to upregulation of bra-miR9569. This microRNA negatively regulates the expression of the ATP-related gene <i>AHA6</i>, resulting in reduced H⁺-ATPase activity. The consequent energy deficiency triggers cellular content degradation, ultimately causing failure of pollen wall formation and pollen abortion.